Zhen Yan

Recent studies have shed light on the mechanisms by which stress and glucocorticoids affect glutamate transmission in the prefrontal cortex and the hippocampus. Sanacora and colleagues review these studies and discuss the relevance of these mechanisms for normal brain functioning and for the pathophysiology and potential new treatments of stress-related neuropsychiatric disorders. Mounting evidence suggests that acute and chronic stress, especially the stress-induced release of glucocorticoids, induces changes in glutamate neurotransmission in the prefrontal cortex and the hippocampus, thereby influencing some aspects of cognitive processing. In addition, dysfunction of glutamatergic neurotransmission is increasingly considered to be a core feature of stress-related mental illnesses. Recent studies have shed light on the mechanisms by which stress and glucocorticoids affect glutamate transmission, including effects on glutamate release, glutamate receptors and glutamate clearance and metabolism. This new understanding provides insights into normal brain functioning, as well as the pathophysiology and potential new treatments of stress-related neuropsychiatric disorders.

In recent years, the distribution of dopamine receptor subtypes among the principal neurons of the neostriatum has been the subject of debate. Conventional anatomical and physiological approaches have yielded starkly different estimates of the extent to which D1 and D2 class dopamine receptors are colocalized. One plausible explanation for the discrepancy is that some dopamine receptors are present in physiologically significant numbers, but the mRNA for these receptors is not detectable with conventional techniques. To test this hypothesis, we examined the expression of DA receptors in individual neostriatal neurons by patch-clamp and RT-PCR techniques. Because of the strong correlation between peptide expression and projection site, medium spiny neurons were divided into three groups on the basis of expression of mRNA for enkephalin (ENK) and substance P (SP). Neurons expressing detectable levels of SP but not ENK had abundant mRNA for the D1a receptor. A subset of these cells (approximately 50%) coexpressed D3 or D4 receptor mRNA. Neurons expressing detectable levels of ENK but not SP had abundant mRNA for D2 receptor isoforms (short and long). A subset (10-25%) of these neurons coexpressed D1a or D1b mRNAs. Neurons coexpressing ENK and SP mRNAs consistently coexpressed D1a and D2 mRNAs in relatively high abundance. Functional analysis of neurons expressing lower abundance mRNAs revealed clear physiological consequences that could be attributed to these receptors. These results suggest that, although colocalization of D1a and D2 receptors is limited, functional D1 and D2 class receptors are colocalized in nearly one-half of all medium spiny projection neurons.

Chronic stress could trigger maladaptive changes associated with stress-related mental disorders; however, the underlying mechanisms remain elusive. In this study, we found that exposing juvenile male rats to repeated stress significantly impaired the temporal order recognition memory, a cognitive process controlled by the prefrontal cortex (PFC). Concomitantly, significantly reduced AMPAR- and NMDAR-mediated synaptic transmission and glutamate receptor expression were found in PFC pyramidal neurons from repeatedly stressed animals. All these effects relied on activation of glucocorticoid receptors and the subsequent enhancement of ubiquitin/proteasome-mediated degradation of GluR1 and NR1 subunits, which was controlled by the E3 ubiquitin ligase Nedd4-1 and Fbx2, respectively. Inhibition of proteasomes or knockdown of Nedd4-1 and Fbx2 in PFC prevented the loss of glutamatergic responses and recognition memory in stressed animals. Our results suggest that repeated stress dampens PFC glutamatergic transmission by facilitating glutamate receptor turnover, which causes the detrimental effect on PFC-dependent cognitive processes.

The prefrontal cortex (PFC), a key brain region controlling cognition and emotion, is strongly influenced by stress. While chronic stress often produces detrimental effects on these measures, acute stress has been shown to enhance learning and memory, predominantly through the action of corticosteroid stress hormones. We used a combination of electrophysiological, biochemical, and behavioral approaches in an effort to identify the cellular targets of acute stress. We found that behavioral stressors in vivo cause a long-lasting potentiation of NMDAR- and AMPAR-mediated synaptic currents via glucocorticoid receptors (GRs) selectively in PFC pyramidal neurons. This effect is accompanied by increased surface expression of NMDAR and AMPAR subunits in acutely stressed animals. Furthermore, behavioral tests indicate that working memory, a key function relying on recurrent excitation within networks of PFC neurons, is enhanced by acute stress via a GR-dependent mechanism. These results have identified a form of long-term potentiation of synaptic transmission induced by natural stimuli in vivo, providing a potential molecular and cellular mechanism for the beneficial effects of acute stress on cognitive processes subserved by PFC.

Calorie restriction has been regarded as the only experimental regimen that can effectively lengthen lifespan in various animal models, but the actual mechanism remains controversial. The gut microbiota has been shown to have a pivotal role in host health, and its structure is mostly shaped by diet. Here we show that life-long calorie restriction on both high-fat or low-fat diet, but not voluntary exercise, significantly changes the overall structure of the gut microbiota of C57BL/6 J mice. Calorie restriction enriches phylotypes positively correlated with lifespan, for example, the genus Lactobacillus on low-fat diet, and reduces phylotypes negatively correlated with lifespan. These calorie restriction-induced changes in the gut microbiota are concomitant with significantly reduced serum levels of lipopolysaccharide-binding protein, suggesting that animals under calorie restriction can establish a structurally balanced architecture of gut microbiota that may exert a health benefit to the host via reduction of antigen load from the gut.

Synaptic spines are dynamic structures that regulate neuronal responsiveness and plasticity. We examined the role of the schizophrenia risk factor DISC1 in the maintenance of spine morphology and function. We found that DISC1 anchored Kalirin-7 (Kal-7), regulating access of Kal-7 to Rac1 and controlling the duration and intensity of Rac1 activation in response to NMDA receptor activation in both cortical cultures and rat brain in vivo. These results explain why Rac1 and its activator (Kal-7) serve as important mediators of spine enlargement and why constitutive Rac1 activation decreases spine size. This mechanism likely underlies disturbances in glutamatergic neurotransmission that have been frequently reported in schizophrenia that can lead to alteration of dendritic spines with consequential major pathological changes in brain function. Furthermore, the concept of a signalosome involving disease-associated factors, such as DISC1 and glutamate, may well contribute to the multifactorial and polygenetic characteristics of schizophrenia.

Spinophilin, a protein that interacts with actin and protein phosphatase-1, is highly enriched in dendritic spines. Here, through the use of spinophilin knockout mice, we provide evidence that spinophilin modulates both glutamatergic synaptic transmission and dendritic morphology. The ability of protein phosphatase-1 to regulate the activity of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and N-methyl-D-aspartate (NMDA) receptors was reduced in spinophilin knockout mice. Consistent with altered glutamatergic transmission, spinophilin-deficient mice showed reduced long-term depression and exhibited resistance to kainate-induced seizures and neuronal apoptosis. In addition, deletion of the spinophilin gene caused a marked increase in spine density during development in vivo as well as altered filopodial formation in cultured neurons. In conclusion, spinophilin appears to be required for the regulation of the properties of dendritic spines.

Corticosteroid stress hormones have a strong impact on the function of prefrontal cortex (PFC), a central region controlling cognition and emotion, though the underlying mechanisms are elusive. We found that behavioral stressor or short-term corticosterone treatment in vitro induces a delayed and sustained potentiation of the synaptic response and surface expression of N-methyl-D-aspartic acid receptors (NMDARs) and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) in PFC pyramidal neurons through a mechanism depending on the induction of serum- and glucocorticoid-inducible kinase (SGK) and the activation of Rab4, which mediates receptor recycling between early endosomes and the plasma membrane. Working memory, a key function relying on glutamatergic transmission in PFC, is enhanced in acutely stressed animals through an SGK-dependent mechanism. These results suggest that acute stress, by activating glucocorticoid receptors, increases the trafficking and function of NMDARs and AMPARs through SGK/Rab4 signaling, which leads to the potentiated synaptic transmission, thereby facilitating cognitive processes mediated by the PFC.

Studies over the past decade have enunciated silent synapses as prominent cellular substrates for synaptic plasticity in the developing brain. However, little is known about whether silent synapses can be generated postdevelopmentally. Here, we demonstrate that highly salient in vivo experience, such as exposure to cocaine, generates silent synapses in the nucleus accumbens (NAc) shell, a key brain region mediating addiction-related learning and memory. Furthermore, this cocaine-induced generation of silent synapses is mediated by membrane insertions of new, NR2B-containing N-methyl-D-aspartic acid receptors (NMDARs). These results provide evidence that silent synapses can be generated de novo by in vivo experience and thus may act as highly efficient neural substrates for the subsequent experience-dependent synaptic plasticity underlying extremely long-lasting memory.

The density of GABA(A) receptors (GABA(A)Rs) at synapses regulates brain excitability, and altered inhibition may contribute to Huntington's disease, which is caused by a polyglutamine repeat in the protein huntingtin. However, the machinery that delivers GABA(A)Rs to synapses is unknown. We demonstrate that GABA(A)Rs are trafficked to synapses by the kinesin family motor protein 5 (KIF5). We identify the adaptor linking the receptors to KIF5 as the huntingtin-associated protein 1 (HAP1). Disrupting the HAP1-KIF5 complex decreases synaptic GABA(A)R number and reduces the amplitude of inhibitory postsynaptic currents. When huntingtin is mutated, as in Huntington's disease, GABA(A)R transport and inhibitory synaptic currents are reduced. Thus, HAP1-KIF5-dependent GABA(A)R trafficking is a fundamental mechanism controlling the strength of synaptic inhibition in the brain. Its disruption by mutant huntingtin may explain some of the defects in brain information processing occurring in Huntington's disease and provides a molecular target for therapeutic approaches.

Haploinsufficiency of the Shank3 gene, which encodes a scaffolding protein at glutamatergic synapses, is a highly prevalent and penetrant risk factor for autism. Using combined behavioral, electrophysiological, biochemical, imaging, and molecular approaches, we find that Shank3-deficient mice exhibit autism-like social deficits and repetitive behaviors, as well as the significantly diminished NMDA receptor (NMDAR) synaptic function and synaptic distribution in prefrontal cortex. Concomitantly, Shank3-deficient mice have a marked loss of cortical actin filaments, which is associated with the reduced Rac1/PAK activity and increased activity of cofilin, the major actin depolymerizing factor. The social deficits and NMDAR hypofunction are rescued by inhibiting cofilin or activating Rac1 in Shank3-deficient mice and are induced by inhibiting PAK or Rac1 in wild-type mice. These results indicate that the aberrant regulation of synaptic actin filaments and loss of synaptic NMDARs contribute to the manifestation of autism-like phenotypes. Thus, targeting actin regulators provides a strategy for autism treatment.

Parkinson's disease (PD) is defined by the degeneration of nigral dopaminergic (DA) neurons and can be caused by monogenic mutations of genes such as parkin. The lack of phenotype in parkin knockout mice suggests that human nigral DA neurons have unique vulnerabilities. Here we generate induced pluripotent stem cells from normal subjects and PD patients with parkin mutations. We demonstrate that loss of parkin in human midbrain DA neurons greatly increases the transcription of monoamine oxidases and oxidative stress, significantly reduces DA uptake and increases spontaneous DA release. Lentiviral expression of parkin, but not its PD-linked mutant, rescues these phenotypes. The results suggest that parkin controls dopamine utilization in human midbrain DA neurons by enhancing the precision of DA neurotransmission and suppressing dopamine oxidation. Thus, the study provides novel targets and a physiologically relevant screening platform for disease-modifying therapies of PD.

Dysregulation of oxidative phosphorylation is associated with increased mitochondrial reactive oxygen species production and some of the most prevalent human diseases including obesity, cancer, diabetes, neurodegeneration, and heart disease. Chemical 'mitochondrial uncouplers' are lipophilic weak acids that transport protons into the mitochondrial matrix via a pathway that is independent of ATP synthase, thereby uncoupling nutrient oxidation from ATP production. Mitochondrial uncouplers also lessen the proton motive force across the mitochondrial inner membrane and thereby increase the rate of mitochondrial respiration while decreasing production of reactive oxygen species. Thus, mitochondrial uncouplers are valuable chemical tools that enable the measurement of maximal mitochondrial respiration and they have been used therapeutically to decrease mitochondrial reactive oxygen species production. However, the most widely used protonophore uncouplers such as carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) and 2,4-dinitrophenol have off-target activity at other membranes that lead to a range of undesired effects including plasma membrane depolarization, mitochondrial inhibition, and cytotoxicity. These unwanted properties interfere with the measurement of mitochondrial function and result in a narrow therapeutic index that limits their usefulness in the clinic. To identify new mitochondrial uncouplers that lack off-target activity at the plasma membrane we screened a small molecule chemical library. Herein we report the identification and validation of a novel mitochondrial protonophore uncoupler (2-fluorophenyl){6-[(2-fluorophenyl)amino](1,2,5-oxadiazolo[3,4-e]pyrazin-5-yl)}amine, named BAM15, that does not depolarize the plasma membrane. Compared to FCCP, an uncoupler of equal potency, BAM15 treatment of cultured cells stimulates a higher maximum rate of mitochondrial respiration and is less cytotoxic. Furthermore, BAM15 is bioactive in vivo and dose-dependently protects mice from acute renal ischemic-reperfusion injury. From a technical standpoint, BAM15 represents an effective new tool that allows the study of mitochondrial function in the absence of off-target effects that can confound data interpretation. From a therapeutic perspective, BAM15-mediated protection from ischemia-reperfusion injury and its reduced toxicity will hopefully reignite interest in pharmacological uncoupling for the treatment of the myriad of diseases that are associated with altered mitochondrial function.

Haploinsufficiency of the SHANK3 gene is causally linked to autism spectrum disorder (ASD), and ASD-associated genes are also enriched for chromatin remodelers. Here we found that brief treatment with romidepsin, a highly potent class I histone deacetylase (HDAC) inhibitor, alleviated social deficits in Shank3-deficient mice, which persisted for ~3 weeks. HDAC2 transcription was upregulated in these mice, and knockdown of HDAC2 in prefrontal cortex also rescued their social deficits. Nuclear localization of β-catenin, a Shank3-binding protein that regulates cell adhesion and transcription, was increased in Shank3-deficient mice, which induced HDAC2 upregulation and social deficits. At the downstream molecular level, romidepsin treatment elevated the expression and histone acetylation of Grin2a and actin-regulatory genes and restored NMDA-receptor function and actin filaments in Shank3-deficient mice. Taken together, these findings highlight an epigenetic mechanism underlying social deficits linked to Shank3 deficiency, which may suggest potential therapeutic strategies for ASD patients bearing SHANK3 mutations. Qin et al show that autism-like social deficits in Shank3-deficient mice arise from β-catenin-mediated transcriptional upregulation of histone deacetylase 2 (HDAC2) and are persistently alleviated by brief treatment with HDAC inhibitor romidepsin.

More than a normal neurotransmitter The molecular mechanisms underlying the persistence of addiction remain largely unclear. Lepack et al. found that, with cocaine exposure, there is an intracellular accumulation of dopamine in neurons of a brain region called the ventral tegmental area (see the Perspective by Girault). Dopamine associates with chromatin to initiate a previously unknown form of epigenetic regulation called dopaminylation. This modification has an impact on ventral tegmental area function and, consequently, on dopaminergic action potentials. The result is aberrant dopamine signaling in the ventral striatum during periods of drug seeking. Science , this issue p. 197 ; see also p. 134

The prefrontal cortex (PFC) serves as the chief executive officer of the brain, controlling the highest level cognitive and emotional processes. Its local circuits among glutamatergic principal neurons and GABAergic interneurons, as well as its long-range connections with other brain regions, have been functionally linked to specific behaviors, ranging from working memory to reward seeking. The efficacy of synaptic signaling in the PFC network is profundedly influenced by monoaminergic inputs via the activation of dopamine, adrenergic, or serotonin receptors. Stress hormones and neuropeptides also exert complex effects on the synaptic structure and function of PFC neurons. Dysregulation of PFC synaptic transmission is strongly linked to social deficits, affective disturbance, and memory loss in brain disorders, including autism, schizophrenia, depression, and Alzheimer's disease. Critical neural circuits, biological pathways, and molecular players that go awry in these mental illnesses have been revealed by integrated electrophysiological, optogenetic, biochemical, and transcriptomic studies of PFC. Novel epigenetic mechanism-based strategies are proposed as potential avenues of therapeutic intervention for PFC-involved diseases. This review provides an overview of PFC network organization and synaptic modulation, as well as the mechanisms linking PFC dysfunction to the pathophysiology of neurodevelopmental, neuropsychiatric, and neurodegenerative diseases. Insights from the preclinical studies offer the potential for discovering new medical treatments for human patients with these brain disorders.

In Huntington's disease (HD), mutation of huntingtin causes selective neurodegeneration of dopaminoceptive striatal medium spiny neurons. Transgenic HD model mice that express a portion of the disease-causing form of human huntingtin develop a behavioral phenotype that suggests dysfunction of dopaminergic neurotransmission. Here we show that presymtomatic mice have severe deficiencies in dopamine signaling in the striatum. These include selective reductions in total levels of dopamine- and cAMP-regulated phosphoprotein, M(r) 32 kDA (DARPP-32) and other dopamine-regulated phosphoprotein markers of medium spiny neurons. HD mice also show defects in dopamine-regulated ion channels and in the D(1) dopamine/DARPP-32 signaling cascade. These presymptomatic defects may contribute to HD pathology.

Yan, Zhen, Wen-Jie Song, and D. James Surmeier. D 2 dopamine receptors reduce N-type Ca 2+ currents in rat neostriatal cholinergic interneurons through a membrane-delimited, protein-kinase-C-insensitive pathway. J. Neurophysiol. 77: 1003–1015, 1997. Dopamine has long been known to regulate the activity of striatal cholinergic interneurons and the release of acetylcholine. Yet, the cellular mechanisms by which this regulation occurs have not been elucidated. One way in which dopamine might act is by modulating voltage-dependent Ca 2+ channels. To test this hypothesis, the impact of dopaminergic agonists on Ca 2+ channels in neostriatal cholinergic interneurons was studied by combined whole cell voltage-clamp recording and single-cell reverse transcription–polymerase chain reactions. Cholinergic interneurons were identified by the presence of choline acetyltransferase mRNA. Nearly all interneurons tested (90%, n = 17) coexpressed D 2 (short and long isoforms) and D 1b (D 5 ) dopamine receptor mRNAs. D 1a receptor mRNA was found in only a small subset (20%) of the sample and D 3 and D 4 receptor mRNAs were undetectable. D 2 receptor agonists rapidly and reversibly reduced N-type Ca 2+ currents. D 1b /D 1a receptor activation had little or no effect on Ca 2+ currents. The D 2 receptor antagonist sulpiride blocked the effect of D 2 agonists. Dialysis with guanosine-5′-O-(2-thiodiphosphate) or brief exposure to the G protein (G i/o ) alkylating agent N-ethylmaleimide also blocked the D 2 modulation. The reduction in N-type currents was neither accompanied by kinetic slowing nor significantly reversed by depolarizing prepulses. The D 2 receptor effects were mediated by a membrane-delimited pathway, because the modulation was not seen in cell-attached patches when agonist was applied to the bath and was not disrupted by perturbations in cytosolic signaling pathways known to be linked to D 2 receptors. Activation of M2 muscarinic receptors occluded the D 2 modulation, suggesting a shared signaling element. However, activation of protein kinase C attenuated the M2 modulation without significantly affecting the D 2 modulation. Taken together, our results suggest that activation of D 2 dopamine receptors in cholinergic interneurons reduces N-type Ca 2+ currents via a membrane-delimited, G i/o class G protein pathway that is not regulated by protein kinase C. This signaling pathway may underlie the ability of D 2 receptors to reduce striatal acetylcholine release.

Interactions between dopamine and N-methyl-D-aspartate receptors (NMDARs) in prefrontal cortex (PFC) and other brain regions are believed to play an important role in normal mental function and neuropsychiatric disorders. In this study, we examined the regulation of NMDAR currents by the dopamine D1 receptor in PFC pyramidal neurons. Application of the D1 receptor agonist SKF81297 caused a prominent increase of the steady-state NMDA-evoked current in acutely isolated PFC pyramidal neurons. The D1 effect on NMDARs was independent of protein kinase A or protein phosphatase 1, but was abolished by incubation of neurons in Ca2+-free medium. Intracellular application of the Ca2+ chelator, calmodulin, or calmodulin inhibitors largely prevented the D1 modulation of NMDAR currents. Moreover, inhibiting PKC activity or disrupting PKC association with its anchoring protein also significantly reduced the D1 effect on NMDAR currents. This upregulation of NMDAR activity by dopamine D1 receptors and the previous finding on up-regulation of dopamine D1 receptors by NMDAR activation provide a cellular mechanism for the reciprocal interactions between D1 and NMDARs. These interactions may play an important role in modulating synaptic plasticity and thus in cognitive and emotional processes.

Dopamine, by activating D(1)- and D(2)-class receptors, plays a significant role in regulating gene expression. Although much is known about D(1) receptor-regulated gene expression, there has been far less information on gene regulation mediated by D(2) receptors. In this study, we show that D(2) receptors can activate the mitogen-activated protein kinase (MAPK) and the cAMP response element-binding protein (CREB) in neurons. Treatment of brain slices with the D(2) receptor agonist quinpirole induced rapid phosphorylation of MAPK and CREB. The neuroleptic drug eticlopride, a highly selective D(2) receptor antagonist, blocked the quinpirole-induced phosphorylation of MAPK and CREB. D(2) receptor-induced MAPK phosphorylation depended on intracellular Ca(2+) elevation, protein kinase C activation, and MAPK kinase activation, but not on the protein tyrosine kinase Pyk2, even though quinpirole stimulated Pyk2 phosphorylation. D(2) receptor-induced CREB phosphorylation was mediated by activation of protein kinase C and Ca(2+)/calmodulin-dependent protein kinase, but not MAPK. The dopamine and cAMP-regulated phosphoprotein DARPP-32 also was required for the regulation of MAPK and CREB phosphorylation by D(2) receptors. Our results suggest that MAPK and CREB signaling cascades are involved in the regulation of gene expression and other long-term effects of D(2) receptor activation.

Recent linkage studies have identified a significant association of the neuregulin gene with schizophrenia, but how neuregulin is involved in schizophrenia is primarily unknown. Aberrant NMDA receptor functions have been implicated in the pathophysiology of schizophrenia. Therefore, we hypothesize that neuregulin, which is present in glutamatergic synaptic vesicles, may affect NMDA receptor functions via actions on its ErbB receptors enriched in postsynaptic densities, hence participating in emotional regulation and cognitive processes that are impaired in schizophrenia. To test this, we examined the regulation of NMDA receptor currents by neuregulin signaling pathways in prefrontal cortex (PFC), a prominent area affected in schizophrenia. We found that bath perfusion of neuregulin significantly reduced whole-cell NMDA receptor currents in acutely isolated and cultured PFC pyramidal neurons and decreased NMDA receptor-mediated EPSCs in PFC slices. The effect of neuregulin was mainly blocked by application of the ErbB receptor tyrosine kinase inhibitor, phospholipase C (PLC) inhibitor, IP3 receptor (IP3R) antagonist, or Ca2+ chelators. The neuregulin regulation of NMDA receptor currents was also markedly attenuated in cultured neurons transfected with mutant forms of Ras or a dominant-negative form of MEK1 (mitogen-activated protein kinase kinase 1). Moreover, the neuregulin effect was prevented by agents that stabilize or disrupt actin polymerization but not by agents that interfere with microtubule assembly. Furthermore, neuregulin treatment increased the abundance of internalized NMDA receptors in cultured PFC neurons, which was also sensitive to agents affecting actin cytoskeleton. Together, our study suggests that both PLC/IP3R/Ca2+ and Ras/MEK/ERK (extracellular signal-regulated kinase) signaling pathways are involved in the neuregulin-induced reduction of NMDA receptor currents, which is likely through enhancing NR1 internalization via an actin-dependent mechanism.

The signaling pathways mediating the muscarinic modulation of Ca2+ currents in neostriatal cholinergic interneurons were studied by combined patch-clamp recording and single-cell reverse transcription- PCR. Cholinergic interneurons were identified by the presence of choline acetyltransferase mRNA. These neurons expressed Q-, N-, L-, P-, and R-type Ca2+ currents and the mRNA for the alpha1 subunits believed to form the channels underlying these currents (classes A, B, C, D, and E). Of the interneurons tested, nearly all expressed M2-class (m2, m4) receptor mRNAs, whereas m1 receptor mRNA was found in only a subset (approximately 30%) of the sample. The muscarinic agonist oxotremorine methiodide produced a dose-dependent reduction of N- and P-type Ba2+ currents through Ca2+ channels that was antagonized by atropine. N- ethylmaleimide eliminated the modulation, as did preincubation with pertussis toxin. The onset and offset of the modulation were rapid and dose-dependent. The modulation was also attenuated by strong depolarizing prepulses and was not observed in cell-attached membrane patches. Taken together, our results suggest that activation of M2- class muscarinic receptors in cholinergic interneurons reduces N- and P- type Ca2+ currents through a membrane-delimited pathway using a Gi/o- class G-protein. This signaling pathway provides a cellular mechanism for hetero- and homosynaptic control of interneuronal activity and acetylcholine release in the striatum.

Serotonergic neurotransmission in prefrontal cortex (PFC) has long been known to play a key role in regulating emotion and cognition under normal and pathological conditions. However, the cellular mechanisms by which this regulation occurs are unclear. In this study, we examined the impact of serotonin on GABA(A) receptor channels in PFC pyramidal neurons using combined patch-clamp recording, biochemical, and molecular approaches. Application of serotonin produced a reduction of postsynaptic GABA(A) receptor currents. Although multiple 5-HT receptors were coexpressed in PFC pyramidal neurons, the serotonergic modulation of GABA-evoked currents was mimicked by the 5-HT(2)-class agonist (-)-2,5-dimethoxy-4-iodoamphetamine and blocked by 5-HT(2) antagonists risperidone and ketanserin, indicating the mediation by 5-HT(2) receptors. Inhibiting phospholipase C blocked the 5-HT(2) inhibition of GABA(A) currents, as did dialysis with protein kinase C (PKC) inhibitory peptide. Moreover, activation of 5-HT(2) receptors in PFC slices increased the in vitro kinase activity of PKC toward GABA(A) receptor gamma2 subunits. Disrupting the interaction of PKC with its anchoring protein RACK1 (receptor for activated C kinase) eliminated the 5-HT(2) modulation of GABA(A) currents, suggesting that RACK1-mediated targeting of PKC to the vicinity of GABA(A) receptors is required for the serotonergic signaling. Together, our results show that activation of 5-HT(2) receptors in PFC pyramidal neurons inhibits GABA(A) currents through phosphorylation of GABA(A) receptors by the activation of anchored PKC. The suppression of GABAergic signaling provides a novel mechanism for serotonergic modulation of PFC neuronal activity, which may underlie the actions of many antidepressant drugs.

Cholinergic interneurons have been implicated in striatally mediated associative learning. In classical conditioning paradigms, conditioned stimuli trigger a transient suppression of neuronal activity that is dependent upon an intact dopaminergic innervation. Our hypothesis was that this suppression reflected dopaminergic enhancement of sensory-linked GABAergic input. As a test, the impact of dopamine on interneuronal GABAA receptor function was studied by combined patch-clamp recording and single-cell reverse transcription PCR. Activation of D5 dopamine receptors reversibly enhanced a Zn2+-sensitive component of GABAA currents. Although dependent upon protein kinase A (PKA) activation, the modulation was blocked by protein phosphatase 1 (PP1) inhibition, suggesting it was dependent upon dephosphorylation. These results establish a novel mechanism by which intrastriatally released dopamine mediates changes in GABAergic signaling that could underlie the initial stages of associative learning.

The efficacy of synaptic inhibition depends on the number of gamma-aminobutyric acid type A receptors (GABA(A)Rs) expressed on the cell surface of neurons. The clathrin adaptor protein 2 (AP2) complex is a critical regulator of GABA(A)R endocytosis and, hence, surface receptor number. Here, we identify a previously uncharacterized atypical AP2 binding motif conserved within the intracellular domains of all GABA(A)R beta subunit isoforms. This AP2 binding motif (KTHLRRRSSQLK in the beta3 subunit) incorporates the major sites of serine phosphorylation within receptor beta subunits, and phosphorylation within this site inhibits AP2 binding. Furthermore, by using surface plasmon resonance, we establish that a peptide (pepbeta3) corresponding to the AP2 binding motif in the GABA(A)R beta3 subunit binds to AP2 with high affinity only when dephosphorylated. Moreover, the pepbeta3 peptide, but not its phosphorylated equivalent (pepbeta3-phos), enhanced the amplitude of miniature inhibitory synaptic current and whole cell GABA(A)R current. These effects of pepbeta3 on GABA(A)R current were occluded by inhibitors of dynamin-dependent endocytosis supporting an action of pepbeta3 on GABA(A)R endocytosis. Therefore phospho-dependent regulation of AP2 binding to GABA(A)Rs provides a mechanism to specify receptor cell surface number and the efficacy of inhibitory synaptic transmission.

The postsynaptic effects of acetylcholine in the striatum are largely mediated by muscarinic receptors. Two of the five cloned muscarinic receptors (M1 and M4) are expressed at high levels by the medium spiny neurons-the principal projection neurons of the striatum. Previous studies have suggested that M4 muscarinic receptors are found primarily in medium spiny neurons that express substance P and participate in the "direct" striatonigral pathway. This view is difficult to reconcile with electrophysiological studies suggesting that nearly all medium spiny neurons exhibit responses characteristic of M4 receptors. To explore this apparent discrepancy, the coordinated expression of M1-M5 receptor messenger RNAs in identified medium spiny neurons was assayed using single-cell reverse transcription-polymerase chain reaction techniques. Nearly all medium spiny neurons had detectable levels of M1 receptor messenger RNA. Although M4 receptor messenger RNA was detected more frequently in substance P-expressing neurons (70%), it was readily seen in a substantial population of enkephalin-expressing neurons (50%). To provide a quantitative estimate of transcript abundance, quantitative reverse transcription-polymerase chain reaction experiments were performed. These studies revealed that M4 messenger RNA was expressed by both substance P and enkephalin neurons, but was roughly five-fold higher in abundance in substance P-expressing neurons. This quantitative difference provides a means of reconciling previous estimates of M4 receptor distribution and function.

A fundamental feature of Alzheimer disease (AD) is the accumulation of beta-amyloid (Abeta), a peptide generated from the amyloid precursor protein (APP). Emerging evidence suggests that soluble Abeta oligomers adversely affect synaptic function, which leads to cognitive failure associated with AD. The Abeta-induced synaptic dysfunction has been attributed to the synaptic removal of alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptors (AMPARs); however, it is unclear how Abeta induces the loss of AMPARs at the synapses. In this study we have examined the potential involvement of Ca(2+)/calmodulin-dependent protein kinase II (CaMKII), a signaling molecule critical for AMPAR trafficking and function. We found that the synaptic pool of CaMKII was significantly decreased in cortical neurons from APP transgenic mice, and the density of CaMKII clusters at synapses was significantly reduced by Abeta oligomer treatment. In parallel, the surface expression of GluR1 subunit as well as AMPAR-mediated synaptic response and ionic current was selectively decreased in APP transgenic mice and Abeta-treated cultures. Moreover, the reducing effect of Abeta on AMPAR current density was mimicked and occluded by knockdown of CaMKII and blocked by overexpression of CaMKII. These results suggest that the Abeta-induced change in CaMKII subcellular distribution may underlie the removal of AMPARs from synaptic membrane by Abeta.

MicroRNAs play important roles in many cell processes, including the differentiation process in several different lineages. For example, microRNAs can promote differentiation by repressing negative regulators of transcriptional activity. These regulated transcription factors can further up-regulate levels of the microRNA in a feed-forward mechanism. Here we show that MyoD up-regulates miR-378 during myogenic differentiation in C2C12 cells. ChIP and high throughput sequencing analysis shows that MyoD binds in close proximity to the miR-378 gene and causes both transactivation and chromatin remodeling. Overexpression of miR-378 increases the transcriptional activity of MyoD, in part by repressing an antagonist, MyoR. The 3' untranslated region of MyoR contains a direct binding site for miR-378. The presence of this binding site significantly reduces the ability of MyoR to prevent the MyoD-driven transdifferentiation of fibroblasts. MyoR and miR-378 were anticorrelated during cardiotoxin-induced adult muscle regeneration in mice. Taken together, this shows a feed-forward loop where MyoD indirectly down-regulates MyoR via miR-378.

HBV covalently closed circular DNA (cccDNA) is the template for the transcription of HBV. HBV core protein (HBc) is a main component of the HBV cccDNA minichromosome. However, the function of HBc in cccDNA is not fully understood. In light of recent findings that HBV cccDNA may be regulated epigenetically, we analyzed the binding of HBc to cccDNA and the impact of HBc on cccDNA epigenetic profile in the liver biopsy samples of 22 patients with chronic Hepatitis B (CHB). We found that HBc binding to HBV cccDNA occurred preferentially at CpG island 2, an important region for the regulation of HBV transcription. Furthermore, the relative abundances of HBc binding to CpG island 2 were positively correlated with the ratios of relaxed circular DNA to cccDNA and the levels of serum HBV DNA in those patients. Interestingly, the relative abundances of HBc binding to CpG island 2 were associated with the binding of CREB binding protein (CBP) and with hypomethylation in CpG island 2 of HBV cccDNA minichromosomes. However, relatively higher amounts of HBc binding to CpG island 2 of cccDNA were accompanied by lower amounts of HDAC1 binding. Multivariate analysis revealed that the abundances of HBc binding to CpG island 2 of cccDNA and positive HBeAg were independent factors associated with the replication of HBV (p = 0.001 for both). Apparently, HBc is a positive regulator of HBV transcription and replication, maintaining the permissive epigenetic state in the critical region of the HBV cccDNA minichromosomes.

Converging evidence suggests that females and males show different responses to stress; however, little is known about the mechanism underlying the sexually dimorphic effects of stress. In this study, we found that young female rats exposed to 1 week of repeated restraint stress show no negative effects on temporal order recognition memory (TORM), a cognitive process controlled by the prefrontal cortex (PFC), which was contrary to the impairment in TORM observed in stressed males. Concomitantly, normal glutamatergic transmission and glutamate receptor surface expression in PFC pyramidal neurons were found in repeatedly stressed females, in contrast to the significant reduction seen in stressed males. The detrimental effects of repeated stress on TORM and glutamate receptors were unmasked in stressed females when estrogen receptors were inhibited or knocked down in PFC, and were prevented in stressed males with the administration of estradiol. Blocking aromatase, the enzyme for the biosynthesis of estrogen, revealed the stress-induced glutamatergic deficits and memory impairment in females, and the level of aromatase was significantly higher in the PFC of females than in males. These results suggest that estrogen protects against the detrimental effects of repeated stress on glutamatergic transmission and PFC-dependent cognition, which may underlie the stress resilience of females.

Many psychiatric and neurological disorders are characterized by learning and memory deficits, for which cognitive enhancement is considered a valid treatment strategy. The N-methyl-D-aspartate receptor (NMDAR) is a prime target for the development of cognitive enhancers because of its fundamental role in learning and memory. In particular, the NMDAR subunit NR2B improves synaptic plasticity and memory when overexpressed in neurons. However, NR2B regulation is not well understood and no therapies potentiating NMDAR function have been developed. Here, we show that serine 1116 of NR2B is phosphorylated by cyclin-dependent kinase 5 (Cdk5). Cdk5-dependent NR2B phosphorylation is regulated by neuronal activity and controls the receptor's cell surface expression. Disrupting NR2B-Cdk5 interaction via a small interfering peptide (siP) increases NR2B surface levels, facilitates synaptic transmission, and improves memory formation in vivo. Our results reveal a regulatory mechanism critical to NR2B function that can be targeted for the development of cognitive enhancers.

The direct conversion of fibroblasts to induced dopaminergic (iDA) neurons and other cell types demonstrates the plasticity of cell fate. The low efficiency of these relatively fast conversions suggests that kinetic barriers exist to safeguard cell-type identity. Here we show that suppression of p53, in conjunction with cell cycle arrest at G1 and appropriate extracellular environment, markedly increase the efficiency in the transdifferentiation of human fibroblasts to iDA neurons by Ascl1, Nurr1, Lmx1a and miR124. The conversion is dependent on Tet1, as G1 arrest, p53 knockdown or expression of the reprogramming factors induces Tet1 synergistically. Tet1 knockdown abolishes the transdifferentiation while its overexpression enhances the conversion. The iDA neurons express markers for midbrain DA neurons and have active dopaminergic transmission. Our results suggest that overcoming these kinetic barriers may enable highly efficient epigenetic reprogramming in general and will generate patient-specific midbrain DA neurons for Parkinson's disease research and therapy.

Serotonergic (5HT) neurons exert diverse and widespread functions in the brain. Dysfunction of the serotonergic system gives rise to a variety of mental illnesses including depression, anxiety, obsessive compulsive disorder, autism and eating disorders. Here we show that human primary fibroblasts were directly converted to induced serotonergic (i5HT) neurons by the expression of Ascl1, Foxa2, Lmx1b and FEV. The transdifferentiation was enhanced by p53 knockdown and appropriate culture conditions including hypoxia. The i5HT neurons expressed markers for mature serotonergic neurons, had Ca(2+)-dependent 5HT release and selective 5HT uptake, exhibited spontaneous action potentials and spontaneous excitatory postsynaptic currents. Application of serotonin significantly increased the firing rate of spontaneous action potentials, demonstrating the functional utility of i5HT neurons for studying serotonergic neurotransmission. The availability of human i5HT neurons will be very useful for research and drug discovery on many serotonin-related mental disorders.

Shank3, which encodes a scaffolding protein at glutamatergic synapses, is a genetic risk factor for autism. In this study, we examined the impact of Shank3 deficiency on the NMDA-type glutamate receptor, a key player in cognition and mental illnesses. We found that knockdown of Shank3 with a small interfering RNA (siRNA) caused a significant reduction of NMDAR-mediated ionic or synaptic current, as well as the surface expression of NR1 subunits, in rat cortical cultures. The effect of Shank3 siRNA on NMDAR currents was blocked by an actin stabilizer, and was occluded by an actin destabilizer, suggesting the involvement of actin cytoskeleton. Since actin dynamics is regulated by the GTPase Rac1 and downstream effector p21-activated kinase (PAK), we further examined Shank3 regulation of NMDARs when Rac1 or PAK was manipulated. We found that the reducing effect of Shank3 siRNA on NMDAR currents was mimicked and occluded by specific inhibitors for Rac1 or PAK, and was blocked by constitutively active Rac1 or PAK. Immunocytochemical data showed a strong reduction of F-actin clusters after Shank3 knockdown, which was occluded by a PAK inhibitor. Inhibiting cofilin, the primary downstream target of PAK and a major actin depolymerizing factor, prevented Shank3 siRNA from reducing NMDAR currents and F-actin clusters. Together, these results suggest that Shank3 deficiency induces NMDAR hypofunction by interfering with the Rac1/PAK/cofilin/actin signaling, leading to the loss of NMDAR membrane delivery or stability. It provides a potential mechanism for the role of Shank3 in cognitive deficit in autism.

Epigenetic dysregulation, which leads to the alteration of gene expression in the brain, is suggested as one of the key pathophysiological bases of ageing and neurodegeneration. Here we found that, in the late-stage familial Alzheimer's disease (FAD) mouse model, repressive histone H3 dimethylation at lysine 9 (H3K9me2) and euchromatic histone methyltransferases EHMT1 and EHMT2 were significantly elevated in the prefrontal cortex, a key cognitive region affected in Alzheimer's disease. Elevated levels of H3K9me2 were also detected in the prefrontal cortex region of post-mortem tissues from human patients with Alzheimer's disease. Concomitantly, H3K9me2 at glutamate receptors was increased in prefrontal cortex of aged FAD mice, which was linked to the diminished transcription, expression and function of AMPA and NMDA receptors. Treatment of FAD mice with specific EHMT1/2 inhibitors reversed histone hyper-methylation and led to the recovery of glutamate receptor expression and excitatory synaptic function in prefrontal cortex and hippocampus. Chromatin immunoprecipitation-sequencing (ChIP-seq) data indicated that FAD mice exhibited genome-wide increase of H3K9me2 enrichment at genes involved in neuronal signalling (including glutamate receptors), which was reversed by EHMT1/2 inhibition. Moreover, the impaired recognition memory, working memory, and spatial memory in aged FAD mice were rescued by the treatment with EHMT1/2 inhibitors. These results suggest that disrupted epigenetic regulation of glutamate receptor transcription underlies the synaptic and cognitive deficits in Alzheimer's disease, and targeting histone methylation enzymes may represent a novel therapeutic strategy for this prevalent neurodegenerative disorder.

Actin polymerization governs activity-dependent modulation of excitatory synapses, including their morphology and functionality. It is clear from human genetics that neuropsychiatric and neurodevelopmental disturbances are multigenetic in nature, highlighting the need to better understand the critical neural pathways associated with these disorders and how they are altered by genetic risk alleles. One such signaling pathway that is heavily implicated by candidate genes for psychiatric and neurodevelopmental disorders are regulators of signaling to the actin cytoskeleton, suggesting that its disruption and the ensuring abnormalities of spine structures and postsynaptic complexes is a commonly affected pathway in brain disorders. This review will discuss recent experimental findings that strongly support genetic evidence linking the synaptic cytoskeleton to mental disorders, such as schizophrenia and autism spectrum disorders.

Autism spectrum disorder (ASD) is a neurodevelopmental disorder characterized by social communication deficits and other behavioral abnormalities. The three-chamber social preference test is often used to assess social deficits in mouse models of ASD. However, varying and often contradicting phenotypic descriptions of ASD mouse models can be found in the scientific literature, and the substantial variability in the methods used by researchers to assess social deficits in mice could be a contributing factor. Here we describe a standardized three-chamber social preference protocol, which is sensitive and reliable at detecting social preference deficits in several mouse models of ASD. This protocol comprises three phases that can all be completed within 1 d. The test mouse is first habituated to the apparatus containing two empty cups in the side chambers, followed by the pre-test phase in which the mouse can interact with two identical inanimate objects placed in the cups. During the test phase, the mouse is allowed to interact with a social stimulus (an unfamiliar wild-type (WT) mouse) contained in one cup and a novel non-social stimulus contained in the other cup. The protocol is thus designed to assess preference between social and non-social stimuli under conditions of equal salience. The broad implementation of the three-chamber social preference protocol presented here should improve the accuracy and consistency of assessments for social preference deficits associated with ASD and other psychiatric disorders. The authors describe a standardized three-chamber social preference protocol that is sensitive and reliable at detecting social preference deficits in several mouse models of autism spectrum disorder.

Drug discovery in psychiatry has been limited to chemical modifications of compounds originally discovered serendipitously. Therefore, more mechanism-oriented strategies of drug discovery for mental disorders are awaited. Schizophrenia is a devastating mental disorder with synaptic disconnectivity involved in its pathophysiology. Reduction in the dendritic spine density is a major alteration that has been reproducibly reported in the cerebral cortex of patients with schizophrenia. Disrupted-in-Schizophrenia-1 (DISC1), a factor that influences endophenotypes underlying schizophrenia and several other neuropsychiatric disorders, has a regulatory role in the postsynaptic density in association with the NMDA-type glutamate receptor, Kalirin-7, and Rac1. Prolonged knockdown of DISC1 leads to synaptic deterioration, reminiscent of the synaptic pathology of schizophrenia. Thus, we tested the effects of novel inhibitors to p21-activated kinases (PAKs), major targets of Rac1, on synaptic deterioration elicited by knockdown expression of DISC1. These compounds not only significantly ameliorated the synaptic deterioration triggered by DISC1 knockdown but also partially reversed the size of deteriorated synapses in culture. One of these PAK inhibitors prevented progressive synaptic deterioration in adolescence as shown by in vivo two-photon imaging and ameliorated a behavioral deficit in prepulse inhibition in adulthood in a DISC1 knockdown mouse model. The efficacy of PAK inhibitors may have implications in drug discovery for schizophrenia and related neuropsychiatric disorders in general.

Almost a half century ago, regular endurance exercise was shown to improve the capacity of skeletal muscle to oxidize substrates to produce ATP for muscle work. Since then, adaptations in skeletal muscle mRNA level were shown to happen with a single bout of exercise. Protein changes occur within days if daily endurance exercise continues. Some of the mRNA and protein changes cause increases in mitochondrial concentrations. One mitochondrial adaptation that occurs is an increase in fatty acid oxidation at a given absolute, submaximal workload. Mechanisms have been described as to how endurance training increases mitochondria. Importantly, Pgc-1α is a master regulator of mitochondrial biogenesis by increasing many mitochondrial proteins. However, not all adaptations to endurance training are associated with increased mitochondrial concentrations. Recent evidence suggests that the energetic demands of muscle contraction are by themselves stronger controllers of body weight and glucose control than is muscle mitochondrial content. Endurance exercise has also been shown to regulate the processes of mitochondrial fusion and fission. Mitophagy removes damaged mitochondria, a process that maintains mitochondrial quality. Skeletal muscle fibers are composed of different phenotypes, which are based on concentrations of mitochondria and various myosin heavy chain protein isoforms. Endurance training at physiological levels increases type IIa fiber type with increased mitochondria and type IIa myosin heavy chain. Endurance training also improves capacity of skeletal muscle blood flow. Endurance athletes possess enlarged arteries, which may also exhibit decreased wall thickness. VEGF is required for endurance training-induced increases in capillary-muscle fiber ratio and capillary density.

Copy number variations (CNVs) of the human 16p11.2 genetic locus are associated with a range of neurodevelopmental disorders, including autism spectrum disorder, intellectual disability, and epilepsy. In this review, we delineate genetic information and diverse phenotypes in individuals with 16p11.2 CNVs, and synthesize preclinical findings from transgenic mouse models of 16p11.2 CNVs. Mice with 16p11.2 deletions or duplications recapitulate many core behavioral phenotypes, including social and cognitive deficits, and exhibit altered synaptic function across various brain areas. Mechanisms of transcriptional dysregulation and cortical maldevelopment are reviewed, along with potential therapeutic intervention strategies.

Exposure to prolonged stress in critical developmental periods induces heightened vulnerability to psychiatric disorders, which may have sex-specific consequences. Here we investigate the neuronal circuits mediating behavioral changes in mice after chronic adolescent social isolation stress. Escalated aggression is exhibited in stressed males, while social withdrawal is shown in stressed females. In vivo multichannel recordings of free-moving animals indicate that pyramidal neurons in prefrontal cortex (PFC) from stressed males exhibit the significantly decreased spike activity during aggressive attacks, while PFC pyramidal neurons from stressed females show a blunted increase of discharge rates during sociability tests. Chemogenetic and electrophysiological evidence shows that PFC hypofunctioning and BLA principal neuron hyperactivity contribute to the elevated aggression in stressed males, while PFC hypofunctioning and VTA dopamine neuron hypoactivity contribute to the diminished sociability in stressed females. These results establish a framework for understanding the circuit and physiological mechanisms underlying sex-specific divergent effects of stress.

Epigenetic aberration is implicated in aging and neurodegeneration. Using postmortem tissues from patients with Alzheimer's disease (AD) and AD mouse models, we have found that the permissive histone mark H3K4me3 and its catalyzing enzymes are significantly elevated in the prefrontal cortex (PFC). Inhibiting H3K4-specific methyltransferases with the compound WDR5-0103 leads to the substantial recovery of PFC synaptic function and memory-related behaviors in AD mice. Among the up-regulated genes reversed by WDR5-0103 treatment in PFC of AD mice, many have the increased H3K4me3 enrichment at their promoters. One of the identified top-ranking target genes, Sgk1, which encodes serum and glucocorticoid-regulated kinase 1, is also significantly elevated in PFC of patients with AD. Administration of a specific Sgk1 inhibitor reduces hyperphosphorylated tau protein, restores PFC glutamatergic synaptic function, and ameliorates memory deficits in AD mice. These results have found a novel epigenetic mechanism and a potential therapeutic strategy for AD and related neurodegenerative disorders.

Chronic social isolation stress during adolescence induces susceptibility for neuropsychiatric disorders. Here we show that 5-week post-weaning isolation stress induces sex-specific behavioral abnormalities and neuronal activity changes in the prefrontal cortex (PFC), basal lateral amygdala (BLA), and ventral tegmental area (VTA). Chemogenetic manipulation, optogenetic recording, and in vivo calcium imaging identify that the PFC to BLA pathway is causally linked to heightened aggression in stressed males, and the PFC to VTA pathway is causally linked to social withdrawal in stressed females. Isolation stress induces genome-wide transcriptional alterations in a region-specific manner. Particularly, the upregulated genes in BLA of stressed males are under the control of activated transcription factor CREB, and CREB inhibition in BLA normalizes gene expression and reverses aggressive behaviors. On the other hand, neuropeptide Hcrt (Hypocretin/Orexin) is among the top-ranking downregulated genes in VTA of stressed females, and Orexin-A treatment rescues social withdrawal. These results have revealed molecular mechanisms and potential therapeutic targets for stress-related mental illness.

Dopaminergic neurotransmission in the prefrontal cortex (PFC) plays an important role in regulating cognitive processes and emotional status. The dopamine D4 receptor, which is highly enriched in the PFC, is one of the principal targets of antipsychotic drugs. To understand the cellular mechanisms and functional implications of D4 receptors, we examined the impact of D4 receptors in PFC pyramidal neurons on GABAergic inhibition, a key element in the regulation of "working memory." Application of the D4 agonist N-(methyl)-4-(2-cyanophenyl)piperazinyl-3-methylbenzamide maleate caused a reversible decrease in postsynaptic GABA(A) receptor currents; this effect was blocked by the D4 antagonist 3-[(4-[4-chlorophenyl]piperazine-1-yl)methyl]-[1H]-pyrrolo[2,3-b]pyridine but not by the D2 antagonist sulpiride, suggesting mediation by D4 receptors. Application of PD168077 also reduced the GABA(A) receptor-mediated miniature IPSC amplitude in PFC pyramidal neurons recorded from slices. The D4 modulation of GABA(A) receptor currents was blocked by protein kinase A (PKA) activation and occluded by PKA inhibition. Inhibiting the catalytic activity of protein phosphatase 1 (PP1) also eliminated the effect of PD168077 on GABA(A) currents. Furthermore, disrupting the association of the PKA/PP1 complex with its scaffold protein Yotiao significantly attenuated the D4 modulation of GABA(A) currents, suggesting that Yotiao-mediated targeting of PKA/PP1 to the vicinity of GABA(A) receptors is required for the dopaminergic signaling. Together, our results show that activation of D4 receptors in PFC pyramidal neurons inhibits GABA(A) channel functions by regulating the PKA/PP1 signaling complex, which could underlie the D4 modulation of PFC neuronal activity and the actions of antipsychotic drugs.

Increasing evidence has suggested that the interaction between dopaminergic and glutamatergic systems in prefrontal cortex (PFC) plays an important role in normal mental functions and neuropsychiatric disorders. In this study, we examined the regulation of NMDA-type glutamate receptors by the PFC dopamine D4 receptor (one of the principal targets of antipsychotic drugs). Application of the D4 receptor agonist PD168077 caused a reversible decrease of the NMDA receptor (NMDAR)-mediated current in acutely isolated and cultured PFC pyramidal neurons, an effect that was blocked by selective D4 receptor antagonists. Furthermore, application of PD168077 produced a potent reduction of the amplitude (but not paired-pulse ratio) of evoked NMDAR EPSCs in PFC slices. The D4 modulation of NMDA receptors in PFC involved the inhibition of protein kinase A, activation of protein phosphatase 1 and the ensuing inhibition of active Ca2+-calmodulin-dependent kinase II (CaMKII). Moreover, PD168077 reduced the surface expression of NMDARs and triggered the internalization of NMDARs in a manner dependent on CaMKII activity. These results identify a mechanistic link between D4 and NMDA receptors in PFC pyramidal neurons, suggesting that D4 receptors may play an important role in modulating synaptic plasticity and thus cognitive and emotional processes in PFC circuits.

Dopamine is a critical determinant of neostriatal function, but its impact on intrastriatal GABAergic signaling is poorly understood. The role of D(1) dopamine receptors in the regulation of postsynaptic GABA(A) receptors was characterized using whole cell voltage-clamp recordings in acutely isolated, rat neostriatal medium spiny neurons. Exogenous application of GABA evoked a rapidly desensitizing current that was blocked by bicuculline. Application of the D(1) dopamine receptor agonist SKF 81297 reduced GABA-evoked currents in most medium spiny neurons. The D(1) dopamine receptor antagonist SCH 23390 blocked the effect of SKF 81297. Membrane-permeant cAMP analogues mimicked the effect of D(1) dopamine receptor stimulation, whereas an inhibitor of protein kinase A (PKA; Rp-8-chloroadenosine 3',5' cyclic monophosphothioate) attenuated the response to D(1) dopamine receptor stimulation or cAMP analogues. Inhibitors of protein phosphatase 1/2A potentiated the modulation by cAMP analogues. Single-cell RT-PCR profiling revealed consistent expression of mRNA for the beta1 subunit of the GABA(A) receptor-a known substrate of PKA-in medium spiny neurons. Immunoprecipitation assays of radiolabeled proteins revealed that D(1) dopamine receptor stimulation increased phosphorylation of GABA(A) receptor beta1/beta3 subunits. The D(1) dopamine receptor-induced phosphorylation of beta1/beta3 subunits was attenuated significantly in neostriata from DARPP-32 mutants. Voltage-clamp recordings corroborated these results, revealing that the efficacy of the D(1) dopamine receptor modulation of GABA(A) currents was reduced in DARPP-32-deficient medium spiny neurons. These results argue that D(1) dopamine receptor stimulation in neostriatal medium spiny neurons reduces postsynaptic GABA(A) receptor currents by activating a PKA/DARPP-32/protein phosphatase 1 signaling cascade targeting GABA(A) receptor beta1 subunits.

Emerging evidence has suggested that glycogen synthase kinase 3 (GSK-3) is a key regulatory kinase involved in a plethora of processes in the nervous system, including neuronal development, mood stabilization, and neurodegeneration. However, the cellular mechanisms underlying the actions of GSK-3 remain to be identified. In this study, we examined the impact of GSK-3 on the N-methyl-D-aspartate (NMDA) receptor channel, a central player involved in cognitive and emotional processes. We found that application of various structurally different GSK-3 inhibitors caused a long-lasting reduction of NMDA receptor-mediated ionic and synaptic current in cortical pyramidal neurons. Cellular knockdown of GSK-3beta in neuronal cultures with a small interfering RNA led to smaller NMDA receptor current and loss of its regulation by GSK-3 inhibitors. The NR2B subunit-containing NMDA receptor was the primary target of GSK-3, but the GSK-3 modulation of NMDAR current did not involve the motor protein kinesin superfamily member 17-based transport of NR2B-containing vesicles along microtubules. Combined electrophysiological, immunocytochemical, and biochemical evidence indicated that GSK-3 inhibitors induced the down-regulation of NMDAR current through increasing the Rab5-mediated and PSD-95-regulated NMDAR internalization in a clathrin/dynamin-dependent manner.

Cyclin-dependent kinase 5 (Cdk5) is a multifunctional neuronal protein kinase that is required for neurite outgrowth and cortical lamination and that plays an important role in dopaminergic signaling in the neostriatum through phosphorylation of Thr-75 of DARPP-32 (dopamine and cAMP-regulated phosphoprotein, molecular mass 32 kDa). Casein kinase 1 (CK1) has been implicated in a variety of cellular functions such as DNA repair, circadian rhythm, and intracellular trafficking. In the neostriatum, CK1 has been found to phosphorylate Ser-137 of DARPP-32. However, first messengers for the regulation of Cdk5 or CK1 have remained unknown. Here we report that both Cdk5 and CK1 are regulated by metabotropic glutamate receptors (mGluRs) in neostriatal neurons. (S)-3,5-dihydroxyphenylglycine (DHPG), an agonist for group I mGluRs, increased Cdk5 and CK1 activities in neostriatal slices, leading to the enhanced phosphorylation of Thr-75 and Ser-137 of DARPP-32, respectively. The effect of DHPG on Thr-75, but not on Ser-137, was blocked by a Cdk5-specific inhibitor, butyrolactone. In contrast, the effects of DHPG on both Thr-75 and Ser-137 were blocked by CK1-7 and IC261, specific inhibitors of CK1, suggesting that activation of Cdk5 by mGluRs requires CK1 activity. In support of this possibility, the DHPG-induced increase in Cdk5 activity, measured in extracts of neostriatal slices, was abolished by CK1-7 and IC261. Treatment of acutely dissociated neurons with DHPG enhanced voltage-dependent Ca(2+) currents. This enhancement was eliminated by either butyrolactone or CK1-7 and was absent in DARPP-32 knockout mice. Together these results indicate that a CK1-Cdk5-DARPP-32 cascade may be involved in the regulation by mGluR agonists of Ca(2+) channels.

We have studied the regulation of AMPA (α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid) receptor channels by serotonin signaling in pyramidal neurons of prefrontal cortex (PFC). Application of serotonin reduced the amplitude of AMPA-evoked currents, an effect mimicked by 5-HT1Areceptor agonists and blocked by 5-HT1A antagonists, indicating the mediation by 5-HT1A receptors. The serotonergic modulation of AMPA receptor currents was blocked by protein kinase A (PKA) activators and occluded by PKA inhibitors. Inhibiting the catalytic activity of protein phosphatase 1 (PP1) also eliminated the effect of serotonin on AMPA currents. Furthermore, the serotonergic modulation of AMPA currents was occluded by application of the Ca2+/calmodulin-dependent kinase II (CaMKII) inhibitors and blocked by intracellular injection of calmodulin or recombinant CaMKII. Application of serotonin or 5-HT1Aagonists to PFC slices reduced CaMKII activity and the phosphorylation of AMPA receptor subunit GluR1 at the CaMKII site in a PP1-dependent manner. We concluded that serotonin, by activating 5-HT1A receptors, suppress glutamatergic signaling through the inhibition of CaMKII, which is achieved by the inhibition of PKA and ensuing activation of PP1. This modulation demonstrates the critical role of CaMKII in serotonergic regulation of PFC neuronal activity, which may explain the neuropsychiatric behavioral phenotypes seen in CaMKII knockout mice. We have studied the regulation of AMPA (α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid) receptor channels by serotonin signaling in pyramidal neurons of prefrontal cortex (PFC). Application of serotonin reduced the amplitude of AMPA-evoked currents, an effect mimicked by 5-HT1Areceptor agonists and blocked by 5-HT1A antagonists, indicating the mediation by 5-HT1A receptors. The serotonergic modulation of AMPA receptor currents was blocked by protein kinase A (PKA) activators and occluded by PKA inhibitors. Inhibiting the catalytic activity of protein phosphatase 1 (PP1) also eliminated the effect of serotonin on AMPA currents. Furthermore, the serotonergic modulation of AMPA currents was occluded by application of the Ca2+/calmodulin-dependent kinase II (CaMKII) inhibitors and blocked by intracellular injection of calmodulin or recombinant CaMKII. Application of serotonin or 5-HT1Aagonists to PFC slices reduced CaMKII activity and the phosphorylation of AMPA receptor subunit GluR1 at the CaMKII site in a PP1-dependent manner. We concluded that serotonin, by activating 5-HT1A receptors, suppress glutamatergic signaling through the inhibition of CaMKII, which is achieved by the inhibition of PKA and ensuing activation of PP1. This modulation demonstrates the critical role of CaMKII in serotonergic regulation of PFC neuronal activity, which may explain the neuropsychiatric behavioral phenotypes seen in CaMKII knockout mice. prefrontal cortex α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid Ca2+/calmodulin-dependent kinase II 5-carboxamidotryptamine excitatory synaptic current spontaneous EPSC miniature EPSC okadaic acid okadaic acid methyl ester analysis of variance Kolmogorov-Smirnov N-methyl-d-aspartic acid protein phosphatases 1, 2A, and 2B protein kinase A protein kinase C postsynaptic density tetrodotoxin 8-hydroxy-DPAT 1-(2-methoxyphenyl)-4-(4-[2-phthalimido]butyl)-piperazine Sp-adenosine 3′,5′-cyclic monophosphothioate adenosine 3′,5′-cyclic monophosphothioate CNQX, 6-cyano-7-nitroquinoxaline-2,3-dione 2,5-dimethoxy-4-iodoamphetamine γ-amino-n-butyric acid The serotonergic system in prefrontal cortex (PFC)1 is being realized as a major player in controlling emotion and cognition under normal and pathological conditions (1Buhot M.C. Curr. Opin. Neurobiol. 1997; 7: 243-254Crossref PubMed Scopus (273) Google Scholar, 2Davidson R.J. Putnam K.M. Larson C.L. Science. 2000; 289: 591-594Crossref PubMed Scopus (1501) Google Scholar). Changes in serotonin receptors, the serotonin transporter, and serotonin release have been found in PFC from subjects with schizophrenia and depression (3Sumiyoshi T. Stockmeier C.A. Overholser J.C. Dilley G.E. Meltzer H.Y. Brain Res. 1996; 708: 209-214Crossref PubMed Scopus (156) Google Scholar, 4Simpson M.D. Lubman D.I. Slater P. Deakin J.F. Biol. Psychiatry. 1996; 39: 919-928Abstract Full Text PDF PubMed Scopus (101) Google Scholar, 5Gurevich E.V. Joyce J.N. Biol. Psychiatry. 1997; 42: 529-545Abstract Full Text PDF PubMed Scopus (163) Google Scholar). Disturbed serotonergic neurotransmission as well as altered activity of PFC are considered as characteristic features of neuropsychiatric disorders (6Breier A. Schizophr. Res. 1995; 14: 187-202Crossref PubMed Scopus (189) Google Scholar, 7Dubovsky S.L. Thomas M. J. Clin. Psychiatry. 1995; 56 Suppl. 2: 38-48PubMed Google Scholar, 8Stockmeier C.A. Ann. N. Y. Acad. Sci. 1997; 836: 220-232Crossref PubMed Scopus (110) Google Scholar). Many effective drugs that constitute a major advance in the treatment of mental disorders primarily target the serotonin system (9Fuxe K. Ogren S.O. Agnati L.F. Benfenati F. Fredholm B. Andersson K. Zini I. Eneroth P. Neuropharmacology. 1983; 22: 389-400Crossref PubMed Scopus (131) Google Scholar, 10Meltzer H.Y. Clin. Neurosci. 1995; 3: 64-75PubMed Google Scholar, 11Busatto G.F. Kerwin R.W. J. Psychopharmacol. 1997; 11: 3-12Crossref PubMed Scopus (72) Google Scholar). In addition to serotonin, it has been found that glutamate receptors are altered in selective brain regions of schizophrenia patients (12Harrison P.J. McLaughlin D. Kerwin R.W. Lancet. 1991; 337: 450-452Abstract PubMed Scopus (212) Google Scholar, 13Sokolov B.P. J. Neurochem. 1998; 71: 2454-2464Crossref PubMed Scopus (156) Google Scholar), and chronic administration of antipsychotic drugs causes the change of glutamate receptors (14Fitzgerald L.W. Deutch A.Y. Nestler E.J. J. Neurosci. 1995; 15: 2453-2461Crossref PubMed Google Scholar), implying a role for cortical glutamatergic dysfunction in mental diseases (15Bachus S.E. Kleinman J.E. J. Clin. Psychiatry. 1996; 57 Suppl. 11: 72-83PubMed Google Scholar). These studies suggest that one important target of serotonin could be the postsynaptic glutamate receptors and dysregulation of glutamatergic transmission by altered serotonin system may be responsible for the pathophysiology of neuropsychiatric disorders. The pleiotropic functions of serotonin are afforded by the concerted actions of multiple serotonin receptor subtypes. The 5-HT receptors are composed of several families that can be grouped on the basis of conserved structures and signaling mechanisms (16Martin G.R. Eglen R.M. Hamblin M.W. Hoyer D. Yocca F. Trends Pharmacol. Sci. 1998; 19: 2-4Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). Multiple G-protein-coupled 5-HT receptors have been identified in glutamatergic PFC pyramidal neurons (17Goldman-Rakic P.S. Lidow M.S. Gallager D.W. J. Neurosci. 1990; 10: 2125-2138Crossref PubMed Google Scholar, 18Feng J. Cai X. Zhao J.H. Yan Z. J. Neurosci. 2001; 21: 6502-6511Crossref PubMed Google Scholar). Although previous studies have shown that serotonin can play both inhibitory and excitatory roles in neuronal networks through the coupling of different 5-HT receptors to distinct ion channels (19Andrade R. Ann. N. Y. Acad. Sci. 1998; 861: 190-203Crossref PubMed Scopus (74) Google Scholar), it remains unclear how these receptors may regulate postsynaptic glutamatergic signaling in PFC. The fast excitatory synaptic transmission is mediated primarily by AMPA-type glutamate receptors in PFC. The AMPA receptor is an oligomeric complex composed of four subunits GluR1–4 (20Hollmann M. Heinemann S. Annu. Rev. Neurosci. 1994; 17: 31-108Crossref PubMed Scopus (3686) Google Scholar). Changes in postsynaptic AMPA receptors have been implicated in synaptic plasticity (21Bliss T.V.P. Collingridge G.L. Nature. 1993; 361: 31-39Crossref PubMed Scopus (9728) Google Scholar), and one important mechanism is the alteration of the phosphorylation state of AMPA receptors (22Roche K.W. Tingley W.G. Huganir R.L. Curr. Opin. Neurobiol. 1994; 4: 383-388Crossref PubMed Scopus (166) Google Scholar). Multiple protein kinases and phosphatases have been shown to modulate AMPA receptor functions (23Greengard P. Jen J. Nairn A.C. Stevens C.F. Science. 1991; 253: 1135-1138Crossref PubMed Scopus (387) Google Scholar, 24McGlade-McCulloh E. Yamamoto H. Tan S.E. Brickey D.A. Soderling T.R. Nature. 1993; 362: 640-642Crossref PubMed Scopus (342) Google Scholar, 25Barria A. Muller D. Derkach V. Griffith L.C. Soderling T.R. Science. 1997; 276: 2042-2045Crossref PubMed Scopus (896) Google Scholar, 26Yan Z. Hsieh-Wilson L. Feng J. Tomizawa K. Allen P.B. Fienberg A.A. Nairn A.C. Greengard P. Nat. Neurosci. 1999; 2: 13-17Crossref PubMed Scopus (248) Google Scholar, 27Lee H.K. Barbarosie M. Kameyama K. Bear M.F. Huganir R.L. Nature. 2000; 405: 955-959Crossref PubMed Scopus (910) Google Scholar). Here we report that serotonin, by activating 5-HT1A receptors, produced a reduction of AMPA-evoked currents in PFC pyramidal neurons. This regulation of postsynaptic AMPA receptors is through a CaMKII-mediated mechanism, which is operated by activated PP1. Given the critical role of glutamatergic transmission in controlling synaptic plasticity and neuronal activity, our results may provide a molecular and cellular mechanism for 5-HT1A/CaMKII regulation of PFC functions. PFC neurons from young adult (3–5 weeks postnatal) rats were acutely dissociated using procedures similar to those described previously (28Yan Z. Surmeier D.J. J. Neurosci. 1996; 16: 2592-2604Crossref PubMed Google Scholar). All experiments were carried out with the approval of the State University of New York at Buffalo Animal Care Committee. In brief, rats were anesthetized by inhaling 2-bromo-2-chloro-1,1,1-trifluoroethane (1 ml/100 g, Sigma) and decapitated; brains were quickly removed, iced, and then blocked for slicing. The blocked tissue was cut in 400-μm slices with a Vibrotome while bathed in a low Ca2+ (100 μm), HEPES-buffered salt solution (in mm: 140 isethionic acid sodium salt, 2 KCl, 4 MgCl2, 0.1 CaCl2, 23 glucose, 15 HEPES, 1 kynurenic acid, pH 7.4, 300–305 mosm). Slices were then incubated for 1–6 h at room temperature (20–22 °C) in a NaHCO3-buffered saline bubbled with 95% O2, 5% CO2 (in mm: 126 NaCl, 2.5 KCl, 2 CaCl2, 2 MgCl2, 26 NaHCO3, 1.25 NaH2PO4, 10 glucose, 1 pyruvic acid, 0.05 glutathione, 0.1 NG-nitro-l-arginine, 1 kynurenic acid, pH = 7.4, 300–305 mosm). All reagents were obtained from Sigma. Slices were then removed into the low Ca2+ buffer, and regions of the PFC were dissected and placed in an oxygenated Cell-Stir chamber (Wheaton, Inc., Millville, NJ) containing protease (1.2–1.4 mg/ml, Sigma) in HEPES-buffered Hanks' balanced salt solution (Sigma) at 35 °C. After 30 min of enzyme digestion, tissue was rinsed three times in the low Ca2+, HEPES-buffered saline and mechanically dissociated with a graded series of fire-polished Pasteur pipettes. The cell suspension was then plated into a 35-mm Lux Petri dish, which was then placed on the stage of a Nikon inverted microscope. Freshly dissociated neurons were precipitated on poly-l-lysine-coated coverslips. After 10 min, they were fixed in 4% paraformaldehyde in phosphate-buffered saline for 20 min and were permeabilized with 0.3% Triton X-100 for 5 min. Following 1-h incubation with 10% bovine serum albumin to block nonspecific staining, the cells were incubated with the polyclonal GluR1 antibody (Upstate Biotechnology Inc., 1:1000) and monoclonal MAP2 antibody (Upstate Biotechnology Inc., 1:1000) at 4 °C overnight. After washing off the primary antibodies, the cells were incubated with a fluorescein-conjugated and a rhodamine-conjugated secondary antibody (Sigma, 1:200) for 50 min at room temperature. For the staining of F-actin, the cells were incubated with rhodamine-conjugated phalloidin (Molecular Probes, 1:5000) for 20 min at room temperature. After washing in phosphate-buffered saline for three times, the coverslips were mounted on slides with VECTASHIELD mounting media (Vector Laboratories, Inc., Burlingame, CA). Fluorescent images were obtained using a Bio-Rad confocal microscope with a 100× oil lens. Whole-cell recordings of currents employed standard voltage clamp techniques (29Hamill O.P. Marty A. Neher E. Sakmann B. Sigworth F.J. Pfluegers Arch. 1981; 391: 85-100Crossref PubMed Scopus (15419) Google Scholar, 30Yan Z. Surmeier D.J. Neuron. 1997; 19: 1115-1126Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar). Electrodes were pulled from Corning 7052 glass and fire-polished prior to use. The internal solution consisted of (in mm): 180N-methyl-d-glucamine, 40 HEPES, 4 MgCl2, 0.5 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA), 12 phosphocreatine, 2 Na2ATP, 0.2 Na3GTP, 0.1 leupeptin, pH 7.2–3, 265–270 mosm. The external solution consisted of (in mm): 135 NaCl, 20 CsCl, 1 MgCl2, 10 HEPES, 0.001 TTX, 5 BaCl2, 10 glucose, pH 7.3, 300–305 mosm. Recordings were obtained with an Axon Instruments 200B patch clamp amplifier that was controlled and monitored with a IBM PC running pCLAMP (version 8) with a DigiData 1320 series interface (Axon instruments, Union City, CA). Electrode resistances were typically 2–4 MΩ in the bath. After seal rupture, series resistance (4–10 MΩ) was compensated (70–90%) and periodically monitored. Care was exercised to monitor the constancy of the series resistance, and recordings were terminated whenever a significant increase (>20%) occurred. The cell membrane potential was held at −70 mV. The application of AMPA (100 μm) or glutamate (1 mm) evoked a fast desensitizing inward current. Peak and steady-state values were measured for generating the plot as a function of time and drug application. AMPA or glutamate was applied for 1 or 2 s every 30 s to minimize desensitization-induced decrease of current amplitude. Drugs were applied with a gravity-fed “sewer pipe” system. The array of application capillaries (approximately 150-μm inner diameter) was positioned a few hundred microns from the cell under study. Solution changes were effected by the SF-77B fast-step solution stimulus delivery device (Warner Instrument Co., Hamden, CT). Serotonin receptor ligands serotonin, R(+)-8-OH-DPAT, NAN-190, 5-carboxamidotryptamine (5-CT), WAY-100635, 2,5-dimethoxy-4- iodoamphetamine (DOI), 5-methoxytryptamine, ketanserin, and SDZ205557 (Sigma/RBI, St. Louis, MO), as well as second messenger reagents Sp-cAMPS, Rp-cAMPS, PKI-(5–24), okadaic acid (OA), okadaic acid methyl ester (OAE), KN-93, KN-92, KN-62, calmodulin, recombinant CaMKII α subunit, and calcineurin autoinhibitory peptide (Calbiochem, San Diego, CA) were made up as concentrated stocks and stored at −20 °C. Stocks were thawed and diluted immediately prior to use. Serotonin was prepared freshly for every experiment. The amino acid sequence for the phosphorylated I-1 peptide pThr35I-1-(7–39) was: PRKIQFTVPLLEPHLDPEAAEQIRRRRP(pT)PATL. Data analyses were performed with AxoGraph (Axon Instruments Inc.), Kaleidagraph (Albeck Software, Reading, PA), and Statview (Abacus Concepts, Inc., Berkeley, CA). Box plots were used for graphic presentation of the data because of the small sample sizes (31Tukey J.W. Exploratory Data Analysis. Addison-Wesley, Menlo Park, CA1977Google Scholar). The box plot represents the distribution as a box with the median as a central line and the hinges as the edges of the box (the hinges divide the upper and lower distributions in half). The inner fences run to the limits of the distribution. For analysis of statistical significance, Mann-Whitney U tests were performed to compare the current amplitudes in the presence or absence of various agonists. ANOVA tests were performed to compare the differential degrees of current modulation between groups subjected to different treatment. The whole-cell voltage-clamp technique was used to evaluate the regulation of spontaneous and miniature excitatory synaptic currents by 5-HT1A receptors in PFC slices. Patch electrodes (5–9 megohms) were filled with the following internal solution (in mm): 130 cesium methanesulfonate, 10 CsCl, 4 NaCl, 10 HEPES, 1 MgCl2, 5 EGTA, 12 phosphocreatine, 5 MgATP, 0.2 Na3GTP, 0.1 leupeptin, pH 7.2–7.3, 265–270 mosm. The slice (300 μm) was placed in a perfusion chamber attached to the fixed-stage of an upright microscope (Olympus) and submerged in continuously flowing oxygenated artificial cerebrospinal fluid. It takes about 1 min to change solutions completely in the perfusion chamber. Cells were visualized with a 40X water-immersion lens and illuminated with near infrared (IR) light and the image was detected with an IR-sensitive charge-coupled device camera. Multiclamp 700A amplifier was used for these recordings. Tight seals (2–10 gigohms) from visualized pyramidal neurons were obtained by applying negative pressure. The membrane was disrupted with additional suction, and the whole cell configuration was obtained. The access resistances ranged from 13 to 18 megohms. Cells were held at −70 mV for the continuous recording of EPSCs. To evaluate the regulation of miniature excitatory synaptic currents by 5-HT1A receptors in PFC cultures, the whole-cell voltage-clamp technique was used. PFC primary cultures were prepared as previously described (32Hayashi K. Shirao T. J. Neurosci. 1999; 19: 3918-3925Crossref PubMed Google Scholar). In brief, frontal cortical neurons from E18 rats were dissociated and plated onto culture dishes. After 6–8 daysin vivo, cells were used for recording. The same internal and external solutions as those for dissociated cell recordings were used. The membrane potential was held at −70 mV. Synaptic activities were analyzed with Mini Analysis Program (Synaptosoft, Leonia, NJ). All quantitative measurements (1 min of events) were taken 3–4 min (slice) or 1–2 min (culture) after drug application. Statistical comparisons of the amplitude of synaptic currents (mean ± S.E.) were made using the Kolmogorov-Smirnov (K-S) test. After incubation, slices were transferred to boiling 1% SDS and homogenized immediately. Insoluble material was removed by centrifugation (13,000 × g for 10 min), and the protein concentration for each sample was measured. Equal amounts of protein from slice homogenates were separated on 7.5% acrylamide gels and transferred to nitrocellulose membranes. The blots were blocked with 5% nonfat dry milk for 1 h at room temperature. Then the blots were incubated with primary antibodies for 1 h at room temperature. Antibodies used include: anti-Thr286-phosphorylated CaMKII (Promega, 1:2000), anti-CaMKII (Upstate Biotechnology Inc., 1:2000), anti-Ser831-phosphorylated GluR1 (Upstate Biotechnology Inc., 1:500), and anti-GluR1 (Upstate Biotechnology Inc., 1:2000). After being rinsed, the blots were incubated with horseradish peroxidase-conjugated anti-rabbit antibodies (Amersham Biosciences, 1:2000) for 1 h at room temperature. Following three washes, the blots were exposed to the enhanced chemiluminescence substrate. Quantitation was obtained from densitometric measurements of immunoreactive bands on films. Data correspond to the mean ± S.E. values and were analyzed by ANOVA tests for statistical significance. To test the potential impact of serotonin on postsynaptic AMPA receptors in PFC, we first examined the effect of serotonin on AMPA receptor-mediated currents in dissociated pyramidal neurons located in the intermediate and deep layers (III–VI) of the rat PFC. Acutely isolated PFC pyramidal neurons were readily distinguished from GABAergic interneurons by their distinct morphological features: a pyramidal-shaped soma and a prominent apical dendrite (18Feng J. Cai X. Zhao J.H. Yan Z. J. Neurosci. 2001; 21: 6502-6511Crossref PubMed Google Scholar). The confocal image of double-immunocytochemical labeling with GluR1 (an AMPA receptor subunit) and MAP2 (a dendritic marker) in a representative dissociated PFC pyramidal neuron is shown in Fig.1 A. It is evident that the dissociated neuron has not only elaborate dendritic processes but also densely located spines, where the majority of glutamatergic inputs is received. GluR1 is highly enriched in dendritic spines and shafts in the dissociated PFC pyramidal neuron, confirming that the subcellular localization of AMPA receptors is well preserved. Given the enrichment of F-actin in dendritic spines, we also co-stained the dissociated PFC pyramidal neurons with F-actin and MAP2, to confirm that the small protrusions along dendritic arbors were indeed spines (Fig.1 A, inset). Double staining of GluR1 and F-actin indicated that they were highly co-localized (data not shown). Application of AMPA (100 μm) elicited an inward current that consisted of two major components: a fast-desensitizing peak and a non-desensitizing sustained part (Fig. 1 B). The AMPA-evoked current was completely blocked by the non-NMDA antagonist CNQX (10 μm, n = 3), indicating that it is mediated primarily by AMPA receptors. Serotonin decreased both the transient peak and the steady-state AMPA current (Fig. 1,B–D). The modulation was reversible and took 1–3 min to stabilize. In 87.5% of PFC pyramidal neurons we tested (n = 104), bath application of 5-HT (20 μm) caused a significant reduction in the amplitude of AMPA-evoked currents (peak, 15.1 ± 0.9%; steady-state, 17.9 ± 0.9%; mean ± S.E., n = 90, p < 0.01, Mann-Whitney U test). The desensitization kinetics of AMPA currents was not significantly altered by 5-HT. Serotonin also reduced the amplitude of glutamate (1 mm)-evoked currents that are mainly mediated by AMPA receptor activation (peak, 14.0 ± 1.5%; steady-state, 14.3 ± 0.5%; n = 22, p < 0.01, Mann-Whitney U test). Since multiple 5-HT receptors are simultaneously expressed in individual PFC pyramidal neurons (18Feng J. Cai X. Zhao J.H. Yan Z. J. Neurosci. 2001; 21: 6502-6511Crossref PubMed Google Scholar), we next used subtype-specific agonists and antagonists to examine which 5-HT receptor was involved in the modulation of AMPA currents. 5-HT1A, 5-HT2Aand 5-HT4 are among the most prominent serotonin receptor subtypes expressed in PFC pyramidal neurons, so we first examined the potential role of these receptors in the modulation of AMPA currents. In a sample of PFC pyramidal neurons we tested, AMPA currents were not significantly affected by the 5-HT2 receptor agonist DOI (20 μm, 1.0 ± 0.6%, mean ± S.E.,n = 5, p > 0.05, Mann-WhitneyU test) or the 5-HT4 receptor agonist 5-methoxytryptamine (20 μm, 2.7 ± 1.9%,n = 8, p > 0.05, Mann-WhitneyU test), suggesting that the serotonergic effect on AMPA receptors was not mediated by 5-HT2 or 5-HT4receptors. On the other hand, application of the 5-HT1Areceptor agonist 8-OH-DPAT (20 μm) significantly reduced the amplitude of AMPA currents (peak, 15.7 ± 1.8%; steady-state, 13.8 ± 1.0%; mean ± S.E., n = 34,p < 0.01, Mann-Whitney U test) in ∼90% of the PFC pyramidal neurons tested (an example is shown in Fig. 1,B–D), mimicking the inhibitory effect of 5-HT. As summarized in Fig. 1 E, 5-HT and 8-OH-DPAT decreased the amplitude of both peak and steady-state AMPA currents to similar extents. To verify that 5-HT1A receptors were mediating the modulation seen with 8-OH-DPAT or 5-HT, the ability of 5-HT1A antagonist NAN-190 to prevent the action of 8-OH-DPAT or 5-HT was examined. As shown in Fig.2, NAN-190 (20 μm) almost completely eliminated the effects of 5-HT (Fig. 2 A) or 8-OH-DPAT (Fig. 2 B). Removing the antagonist restored the ability of 5-HT or 8-OH-DPAT to modulate AMPA currents. Fig. 2 C summarized the effects of 5-HT1A agonists on AMPA currents in the absence or presence of various antagonists. The median reduction of peak AMPA currents by 8-OH-DPAT was 17.1% (n = 10), similar to the 5-HT effect (median reduction: 16.4%, n = 9). In the presence of NAN-190, the inhibition of AMPA currents by 5-HT (n = 9) or 8-OH-DPAT (n = 10) was both significantly blocked (median reduction: 1.3% and 1.9%, respectively,p < 0.01, ANOVA). However, in the presence of the 5-HT2A antagonist ketanserin (20 μm,n = 5) or the 5-HT4 antagonist SDZ205557 (20 μm, n = 7), 5-HT reduction of AMPA currents was not altered (median reduction of peak currents: 16.5% and 13.2%, respectively, data not shown). Another 5-HT1Areceptor agonist 5-CT (20 μm) also decreased the amplitude of AMPA currents (median reduction of peak currents: 11.4%,n = 7), and this effect was significantly attenuated in the presence of another 5-HT1A antagonist WAY-100635 (20 μm, median reduction of peak currents: 1.0%,n = 5, p < 0.01, ANOVA). These results suggest that the serotonergic effect on AMPA receptor channels is mediated by 5-HT1A receptors, consistent with the abundant expression of 5-HT1A receptors in almost all PFC pyramidal neurons detected with the single cell mRNA profiling method (18Feng J. Cai X. Zhao J.H. Yan Z. J. Neurosci. 2001; 21: 6502-6511Crossref PubMed Google Scholar). To understand the impact of 5-HT1A receptors on glutamatergic synaptic transmission, we examined the effect of 8-OH-DPAT on AMPA receptor-mediated excitatory synaptic currents (EPSCs). Spontaneous EPSCs (sEPSCs) and miniature EPSCs (mEPSCs) were recorded in PFC pyramidal neurons in acute slices and primary cultures. Application of CNQX (10 μm) blocked both sEPSCs and mEPSCs (n = 5), indicating that these synaptic currents are mainly mediated by AMPA receptors. As shown in Fig.3 (A and B), bath application of 8-OH-DPAT to the PFC slice reversibly reduced the sEPSC amplitude by 20.1% (p < 0.01, K-S test). In nine PFC pyramidal neurons we examined, 8-OH-DPAT decreased the mean amplitude of sEPSCs by 19.5 ± 1.8% (mean ± S.E., n = 7, p < 0.01, K-S test). The frequency of sEPSCs was also reduced by 8-OH-DPAT (57.1 ± 4.1%, n = 7,p < 0.001, K-S test), suggesting the existence of a presynaptic mechanism as well. To better isolate the postsynaptic effect of 5-HT1A receptors, we exposed PFC slices to TTX (0.5 μm), and mEPSCs were measured. Bath application of 8-OH-DPAT to the PFC slice reversibly reduced the mEPSC amplitude by 18.6% (Fig. 3, C and D, p < 0.01, K-S test). In eight PFC pyramidal neurons we examined, 8-OH-DPAT decreased the mean amplitude of mEPSCs by 15.9 ± 2.7% (mean ± S.E., n = 6, p < 0.01, K-S test). Because mEPSCs of pyramidal neurons in PFC slices often had small sizes (mean amplitude: 14.1 pA; n = 6), we further examined the modulation of mEPSC by 5-HT1A receptors in cultured PFC pyramidal neurons. A representative example is shown in Fig. 3(E and F). Bath application of 8-OH-DPAT caused a reversible reduction of the mEPSC amplitude by 22.0% in the cultured neuron (p < 0.01, K-S test). Similar to the results in PFC slices, 8-OH-DPAT decreased the mean amplitude of mEPSCs by 17.1 ± 1.9% in cultured PFC pyramidal neurons (mean ± S.E., n = 10, p < 0.01, K-S test). These results indicate that activation of 5-HT1A receptors could down-regulate AMPA receptor function by a postsynaptic mechanism. The frequency of mEPSCs recorded from PFC pyramidal neurons in slices and cultures was also reduced by 8-OH-DPAT (slice: 23.9 ± 3.1%,n = 6, p < 0.01, K-S test; culture: 53.1 ± 6.1%, n = 9, p < 0.001, K-S test), confirming that 5-HT1A receptors could also regulate excitatory transmission by a presynaptic mechanism. We next examined the signal transduction pathways mediating the modulation of AMPA currents by 5-HT1A receptors. A classic pathway for 5-HT1Areceptors is to couple to Gi/Go proteins to inhibit adenylate cyclase and cAMP formation (33Raymond J.R. Mukhin Y.V. Gettys T.W. Garnovskaya M.N. Br. J. Pharmacol. 1999; 127: 1751-1764Crossref PubMed Scopus (214) Google Scholar). This led us to speculate that the 5-HT1A reduction of AMPA currents is through the inhibition of PKA. If that is the case, then the effect of 5-HT1A on AMPA receptor currents should be blocked by stimulating PKA and occluded by inhibiting PKA. To test this, we applied selective PKA activators and inhibitors. As shown in Fig. 4 A, application of the membrane-permeable PKA activator Sp-cAMPS (50 μm) blocked the modulatory effect of 5-HT. Removing Sp-cAMPS restored the ability of 5-HT to modulate AMPA currents. On the other hand, in the presence of the membrane-permeable PKA inhibitor Rp-cAMPS (50 μm), application of 5-HT failed to further reduce AMPA currents (data not shown). To confirm the involvement of PKA in 5-HT modulation of AMPA currents, we dialyzed neurons with the specific PKA inhibitory peptide PKI-(5–24) (34Knighton D.R. Zheng J.H. Ten Eyck L.F. Xuong N.H. Taylor S.S. Sowadski J.M. Science. 1991; 253: 414-420Crossref PubMed Scopus (841) Google Scholar) and then examined 5-HT effects. The AMPA currents were reduced by 17.4 ± 1.2% (n = 5) during the dialysis of PKI-(5–24), which was similar to the effect of 5-HT treatment. After ∼5 min of dialysis to allow PKI-(5–24) to enter the cell to inhibit PKA activity, subsequent application of 5-HT had little effect on AMPA currents, whereas a control peptide with the scrambled sequence sPKI-(5–24) did not affect 5-HT-induced reduction of AMPA currents (Fig. 4 B). Shown in Fig. 4 C is a summary to compare the effects of 5-HT in the absence or presence of various PKA activators and inhibitors. 5-HT caused little change in AMPA currents in the presence of Sp-cAMPS (1.8 ± 0.8%, mean ± S.E., n = 12,p > 0.05, Mann-Whitney U test), or Rp-cAMPS (1.4 ± 0.8%, n = 10, p > 0.05, Mann-Whitney U test), or PKI-(5–24) (1.3 ± 0.8%,n = 8, p > 0.05, Mann-WhitneyU test), which was significantly different from the 5-HT effect in the absence of these agents (14.8 ± 1.8%,n = 22, p < 0.01, ANOVA). These results suggest that 5-HT reduction of AMPA currents depends on the inhibition of PKA. The 5-HT1A-induced inhibition of PKA could directly reduce AMPA currents through decreased phosphorylation of GluR1 subunit on the PKA site (35Roche K.W. O'Brien R.J. Mamme

The regulation of the number of gamma2-subunit-containing GABA(A) receptors (GABA(A)Rs) present at synapses is critical for correct synaptic inhibition and animal behavior. This regulation occurs, in part, by the controlled removal of receptors from the membrane in clathrin-coated vesicles, but it remains unclear how clathrin recruitment to surface gamma2-subunit-containing GABA(A)Rs is regulated. Here, we identify a gamma2-subunit-specific Yxxvarphi-type-binding motif for the clathrin adaptor protein, AP2, which is located within a site for gamma2-subunit tyrosine phosphorylation. Blocking GABA(A)R-AP2 interactions via this motif increases synaptic responses within minutes. Crystallographic and biochemical studies reveal that phosphorylation of the Yxxvarphi motif inhibits AP2 binding, leading to increased surface receptor number. In addition, the crystal structure provides an explanation for the high affinity of this motif for AP2 and suggests that gamma2-subunit-containing heteromeric GABA(A)Rs may be internalized as dimers or multimers. These data define a mechanism for tyrosine kinase regulation of GABA(A)R surface levels and synaptic inhibition.

Abnormal serotonin-glutamate interaction in prefrontal cortex (PFC) is implicated in the pathophysiology of many mental disorders, including schizophrenia and depression. However, the mechanisms by which this interaction occurs remain unclear. Our previous study has shown that activation of 5-HT1A receptors inhibits N-methyl-d-aspartate (NMDA) receptor (NMDAR) currents in PFC pyramidal neurons by disrupting microtubule-based transport of NMDARs. Here we found that activation of 5-HT2A/C receptors significantly attenuated the effect of 5-HT1A on NMDAR currents and microtubule depolymerization. The counteractive effect of 5-HT2A/C on 5-HT1A regulation of synaptic NMDAR response was also observed in PFC pyramidal neurons from intact animals treated with various 5-HT-related drugs. Moreover, 5-HT2A/C stimulation triggered the activation of extracellular signal-regulated kinase (ERK) in dendritic processes. Inhibition of the β-arrestin/Src/dynamin signaling blocked 5-HT2A/C activation of ERK and the counteractive effect of 5-HT2A/C on 5-HT1A regulation of NMDAR currents. Immunocytochemical studies showed that 5-HT2A/C treatment blocked the inhibitory effect of 5-HT1A on surface NR2B clusters on dendrites, which was prevented by cellular knockdown of β-arrestins. Taken together, our study suggests that serotonin, via 5-HT1A and 5-HT2A/C receptor activation, regulates NMDAR functions in PFC neurons in a counteractive manner. 5-HT2A/C, by activating ERK via the β-arrestin-dependent pathway, opposes the 5-HT1A disruption of microtubule stability and NMDAR transport. These findings provide a framework for understanding the complex interactions between serotonin and NMDARs in PFC, which could be important for cognitive and emotional control in which both systems are highly involved. Abnormal serotonin-glutamate interaction in prefrontal cortex (PFC) is implicated in the pathophysiology of many mental disorders, including schizophrenia and depression. However, the mechanisms by which this interaction occurs remain unclear. Our previous study has shown that activation of 5-HT1A receptors inhibits N-methyl-d-aspartate (NMDA) receptor (NMDAR) currents in PFC pyramidal neurons by disrupting microtubule-based transport of NMDARs. Here we found that activation of 5-HT2A/C receptors significantly attenuated the effect of 5-HT1A on NMDAR currents and microtubule depolymerization. The counteractive effect of 5-HT2A/C on 5-HT1A regulation of synaptic NMDAR response was also observed in PFC pyramidal neurons from intact animals treated with various 5-HT-related drugs. Moreover, 5-HT2A/C stimulation triggered the activation of extracellular signal-regulated kinase (ERK) in dendritic processes. Inhibition of the β-arrestin/Src/dynamin signaling blocked 5-HT2A/C activation of ERK and the counteractive effect of 5-HT2A/C on 5-HT1A regulation of NMDAR currents. Immunocytochemical studies showed that 5-HT2A/C treatment blocked the inhibitory effect of 5-HT1A on surface NR2B clusters on dendrites, which was prevented by cellular knockdown of β-arrestins. Taken together, our study suggests that serotonin, via 5-HT1A and 5-HT2A/C receptor activation, regulates NMDAR functions in PFC neurons in a counteractive manner. 5-HT2A/C, by activating ERK via the β-arrestin-dependent pathway, opposes the 5-HT1A disruption of microtubule stability and NMDAR transport. These findings provide a framework for understanding the complex interactions between serotonin and NMDARs in PFC, which could be important for cognitive and emotional control in which both systems are highly involved. Serotonin (5-HT) 2The abbreviations used are: 5-HT, serotonin; NMDA, N-methyl-d-aspartate; NMDAR, NMDA receptor; MAP, microtubule-associated protein; MAP2, microtubule-associated protein 2; PFC, prefrontal cortex; ERK, extracellular signal-regulated kinase; ANOVA, analysis of variance; GABAA, γ-aminobutyric acid, type A; 8-OH-DPAT, 8-hydroxy-2(di-n-propylamino)-tetralin; PP2, 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine; PP3, 4-amino-7-phenylpyrazol[3,4-d]pyrimidine; siRNA, small interference RNA; GFP, green fluorescent protein; MES, 4-morpholineethanesulfonic acid; fluox, fluoxetine; ket, ketanserin; α-Me-5HT, α-methyl-5-hydroxytryptamine maleate; EPSC, excitatory postsynaptic current. is a key neuromodulator mediating diverse cognitive and emotional functions in the central nervous system (1Buhot M.C. Curr. Opin. Neurobiol. 1997; 7: 243-254Crossref PubMed Scopus (273) Google Scholar). The pleiotropic functions of serotonin are afforded by the concerted actions of multiple 5-HT receptor subtypes, 5-HT1- to 5-HT7 (2Andrade R. Ann. N. Y. Acad. Sci. 1998; 861: 190-203Crossref PubMed Scopus (74) Google Scholar, 3Martin G.R. Eglen R.M. Hamblin M.W. Hoyer D. Yocca F. Trends Pharmacol. Sci. 1998; 19: 2-4Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). Mice lacking 5-HT receptors exhibit phenotypes ranging from increased anxiety (4Gross C. Zhuang X. Stark K. Ramboz S. Oosting R. Kirby L. Santarelli L. Beck S. Hen R. Nature. 2002; 416: 396-400Crossref PubMed Scopus (767) Google Scholar), to elevated aggression (5Saudou F. Amara D.A. Dierich A. LeMeur M. Ramboz S. Segu L. Buhot M.C. Hen R. Science. 1994; 265: 1875-1878Crossref PubMed Scopus (756) Google Scholar), to antidepressant-like behaviors (6Heisler L.K. Chu H.M. Brennan T.J. Danao J.A. Bajwa P. Parsons L.H. Tecott L.H. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 15049-15054Crossref PubMed Scopus (616) Google Scholar). One of the major targets of serotonin is prefrontal cortex (PFC), a crucial brain region controlling emotion and cognition (7Goldman-Rakic P.S. Neuron. 1995; 14: 477-485Abstract Full Text PDF PubMed Scopus (1935) Google Scholar, 8Miller E.K. Neuron. 1999; 22: 15-17Abstract Full Text Full Text PDF PubMed Scopus (221) Google Scholar). Several lines of evidence have shown that 5-HT1A and 5-HT2A receptors, which are abundantly co-expressed in most of PFC pyramidal neurons (9Feng J. Cai X. Zhao J. Yan Z. J. Neurosci. 2001; 21: 6502-6511Crossref PubMed Google Scholar), often have opposing actions on common substrates. For instance, activation of 5-HT1A receptors results in neuronal inhibition by increasing potassium currents (10Araneda R. Andrade R. Neuroscience. 1991; 40: 399-412Crossref PubMed Scopus (538) Google Scholar) and decreasing calcium currents (11Penington N.J. Kelly J.S. Neuron. 1990; 4: 751-758Abstract Full Text PDF PubMed Scopus (101) Google Scholar). In contrast, 5-HT2A receptor stimulation leads to neuronal excitation by suppressing potassium currents (2Andrade R. Ann. N. Y. Acad. Sci. 1998; 861: 190-203Crossref PubMed Scopus (74) Google Scholar) and enhancing pre-synaptic glutamate release (12Aghajanian G.K. Marek G.J. Brain Res. 1999; 825: 161-171Crossref PubMed Scopus (272) Google Scholar). Moreover, elevated 5-HT1A receptors and reduced 5-HT2A receptors are found in PFC of schizophrenia patients (13Schreiber R. De Vry J. Prog. Neuropsychopharmacol. Biol. Psychiatry. 1993; 17: 87-104Crossref PubMed Scopus (151) Google Scholar, 14Burnet P.W. Eastwood S.L. Harrison P.J. Neuropsychopharmacology. 1996; 15: 442-455Crossref PubMed Scopus (268) Google Scholar), suggesting that the levels of 5-HT1A and 5-HT2A receptors are differentially altered in diseased states. One of the potential cellular targets of 5-HT receptors involved in cognitive and emotional control is the NMDA-type glutamate receptor, a ligand-gated ion channel that has been implicated in the pathophysiology of mental disorders (15Tsai G. Coyle J.T. Annu. Rev. Pharm. Toxicol. 2002; 42: 165-179Crossref PubMed Scopus (525) Google Scholar). NMDAR hypofunction caused by systemic administration of non-competitive NMDAR antagonists or knockdown of NMDAR expression produces schizophrenia-like behavioral symptoms (16Javitt D.C. Zukin S.R. Am. J. Psychiatry. 1991; 148: 1301-1308Crossref PubMed Scopus (2638) Google Scholar, 17Jentsch J.D. Roth R.H. Neuropsychopharmacology. 1999; 20: 201-225Crossref PubMed Scopus (1164) Google Scholar, 18Mohn A.R. Gainetdinov R.R. Caron M.G. Koller B.H. Cell. 1999; 98: 427-436Abstract Full Text Full Text PDF PubMed Scopus (921) Google Scholar). Moreover, it has been found that the expression of NMDARs is reduced in postmortem brains of depressed patients (19Law A.J. Deakin J.F. Neuroreport. 2001; 12: 2971-2974Crossref PubMed Scopus (192) Google Scholar), and chronic antidepressant treatment enhances NMDAR levels in mouse brains (20Boyer P.A. Skolnick P. Fossom L.H. J. Mol. Neurosci. 1998; 10: 219-233Crossref PubMed Scopus (153) Google Scholar). Our previous study has shown that activation of 5-HT1A receptors suppresses NMDAR channel function in PFC pyramidal neurons (21Yuen E.Y. Jiang Q. Chen P. Gu Z. Feng J. Yan Z. J. Neurosci. 2005; 25: 5488-5501Crossref PubMed Scopus (196) Google Scholar). It remains unknown whether 5-HT2A receptor activation has any impact on the 5-HT1A-NMDAR interaction. Here we show that activation of 5-HT1A and 5-HT2A/C receptors in PFC pyramidal neurons regulates NMDAR channels in a counteractive manner by converging on the microtubule-based transport of NMDARs that is regulated by ERK. Given the importance of NMDAR and serotonin in mental processes under normal and pathological conditions, the complex regulation of NMDAR function by 5-HT1A and 5-HT2A/C receptors may provide a molecular and cellular mechanism underlying the role of serotonin in regulating cognitive and emotional behaviors. Whole Cell Recordings—Whole cell current recordings of cultured PFC neurons employed standard voltage-clamp techniques as those we described previously (21Yuen E.Y. Jiang Q. Chen P. Gu Z. Feng J. Yan Z. J. Neurosci. 2005; 25: 5488-5501Crossref PubMed Scopus (196) Google Scholar, 22Yuen E.Y. Jiang Q. Feng J. Yan Z. J. Biol. Chem. 2005; 280: 29420-29427Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). The external solution for recording NMDAR-mediated current contained (in mm): 127 NaCl, 20 CsCl, 1 CaCl2, 10 HEPES, 5 BaCl2, 12 glucose, 0.001 tetrodotoxin, and glycine 0.02, pH 7.3–7.4, 300–305 mosm/liter. The internal solution contained (in mm): 180 N-methyl-d-glucamine, 4 MgCl2, 40 HEPES, 0.5 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid, 12 phosphocreatine, 3 Na2ATP, and 0.5 Na2GTP, 0.1 leupeptin, pH 7.2-3, 265–270 mosm/liter. Recordings were obtained with an Axon Instruments (Union City, CA) 200B patch clamp amplifier that was monitored by an IBM PC running pClamp 8 with a DigiData 1320 series interface. Electrode resistances were normally 2–4 MΩ in bath solution. Following seal rupture, series resistance (4–10 MΩ) was compensated (70–90%). Attention was applied to monitor the series resistance, and recordings were stopped when a significant increase (>20%) occurred. The whole cell NMDAR-mediated current was evoked by NMDA (100 μm) application for 2 s every 30 s with neurons held at –60 mV. Drugs were applied with a “sewer pipe” system. The array of drug capillaries was positioned a few hundred micrometers from the cell under recording. Solution changes were controlled by the SF-77B fast-step solution stimulus delivery device (Warner Instruments, Hamden, CT). Data were analyzed with AxoGraph (Axon instruments) and KaleidaGraph (Albeck Software). ANOVA was performed to compare the differential degrees of current regulation between experimental groups subjected to different drug treatment. Data are expressed as the mean ± S.E. Electrophysiological Recordings in Slices—To record NMDAR-mediated synaptic transmission, we performed the standard whole cell recording techniques in layer V PFC pyramidal neurons (21Yuen E.Y. Jiang Q. Chen P. Gu Z. Feng J. Yan Z. J. Neurosci. 2005; 25: 5488-5501Crossref PubMed Scopus (196) Google Scholar, 23Wang X. Zhong P. Gu Z. Yan Z. J. Neurosci. 2003; 23: 9852-9861Crossref PubMed Google Scholar). Patch pipettes (5–9 MΩ) were filled with the following internal solution (in mm): 130 cesium methanesulfonate, 10 CsCl, 4 NaCl, 1 MgCl2, 10 HEPES, 5 EGTA, 2.2 QX-314, 12 phosphocreatine, 5 MgATP, 0.5 Na2GTP, 0.1 leupeptin, pH 7.2–7.3, 265–270 mosm/liter. PFC slices (300 μm) were perfused at room temperature (22–24 °C), with artificial cerebrospinal fluid was bubbled with 95% O2/5% CO2 containing 6-cyano-7-nitroquinoxaline-2,3-dione (20 μm) and bicuculline (10 μm) to block α-amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid/kainite receptors and GABAA receptors, respectively. Neurons were observed with a 40× water-immersion lens and illuminated with near infrared IR light, and the image was captured with an IR-sensitive charge-coupled device camera. All recordings were performed using a Multiclamp 700A amplifier. Upon application of negative pressure, the membrane was tightly sealed with resistance (2–10 GΩ). With additional suction, the membrane was disrupted into the whole cell configuration. The access resistances ranged from 13 to 18 MΩ with 50–70% compensation. Evoked currents were generated with a pulse from a stimulation isolation unit controlled by an S48 pulse generator (Astro-Med). A bipolar stimulating electrode (Fredrick Haer Company) was positioned ∼100μm from the neuron upon recording. Prior to stimulation, neurons (clamped at –70 mV) were depolarized to +60 mV for 3 s to fully eliminate the voltage-dependent Mg2+ block of NMDAR. Age-matched saline controls were done side-by-side with drug-injected animals on each day of experiments. To minimize variations between slices, the stimulus with the same intensity was delivered by the stimulating electrode placed at the same location. Data analyses were performed with the Clampfit software (Axon Instruments). The agents we used include serotonin, 8-hydroxy-2(di-n-propylamino)tetralin (8-OH-DPAT), α-Me-5HT, (–)-2,5-dimethoxy-4-iodoamphetamine, ketanserin, benzothiazole, colchicine (Sigma), dynamin inhibitory peptide (Tocris), 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2) and 4-amino-7-phenylpyrazol[3,4-d]pyrimidine (PP3) (Calbiochem). They were made up as concentrated stocks in water or DMSO and stored at –20 °C. Stocks were thawed and diluted immediately prior to experiments. Animal Treatment—Young male rats (25–28 days old) were administered intraperitoneally with the drugs as described in the text. For stereotaxic injection, rats were anesthetized with pentobarbital sodium (50 mg/kg intraperitoneal) and then mounted into a stereotaxic apparatus (David Kopf Instruments). Fluoxetine (fluox) (2 μl, 0.34 mg/ml, dissolved in saline) were injected unilaterally into the PFC region using a Hamilton syringe (22-gauge needle) at a rate of 0.5 μl/min. The coordinates of the lateral PFC used are 1.0–1.4 mm lateral from midline, 2.2 mm anterior to Bregma, and 3.3 mm dorsal to ventral. Small Interfering RNA—To knock down the endogenous β-arrestin expression, we used the small interfering RNA (siRNA) that specifically targets β-arrestin1 or β-arrestin2 mRNA (24Jiang Q. Yan Z. Feng J. J. Neurosci. 2006; 26: 4318-4328Crossref PubMed Scopus (36) Google Scholar). The siRNA oligonucleotide sequences were: 5′-GGCGAGUCUACGUGACACUtt-3′ (for β-arrestin1) and 5′-GGACCGGAASGUGUUUGUGtt-3′ (for β-arrestin2). The siRNA (purchased from Ambion, Austin, TX) was co-transfected with enhanced GFP into cultured PFC neurons (11 days in vitro) using the Lipofectamine 2000 method. Biochemical, immunocytochemical, or electrophysiological experiments were performed in neurons after 2–3 days of transfection. Measurement of Free Tubulin—Free tubulin from PFC cultures was extracted as described previously (21Yuen E.Y. Jiang Q. Chen P. Gu Z. Feng J. Yan Z. J. Neurosci. 2005; 25: 5488-5501Crossref PubMed Scopus (196) Google Scholar). Cultured PFC neurons (2 × 105 cells/cm2, 14 days in vitro) in 3.5-cm dishes were washed twice at 37 °C with 1 ml of microtubule stabilizing buffer containing (0.1 m MES (pH 6.75), 1 mm MgSO4, 2 mm EGTA, 0.1 mm EDTA, and 4 m glycerol). Cultures were then incubated at 37 °C for 5 min in 600 μl of soluble tubulin extraction buffer (0.1 m MES (pH 6.75), 1 mm MgSO4, 2 mm EGTA, 0.1 mm EDTA, 4 m glycerol, and 0.1% Triton X-100) with the addition of protease inhibitor mixture tablets (Roche Applied Science). The soluble extract was centrifuged at 37 °C for 2 min, and the supernatant was collected. Equal amounts of total protein were analyzed by Western blotting using anti-α-tubulin (Sigma). The intensity of tubulin bands was quantitatively analyzed with Image (National Institutes of Health). Immunocytochemical Staining—The detection of surface GFP-NR2B (25Luo J.H. Fu Z.Y. Losi G. Kim B.G. Prybylowski K. Vissel B. Vicini S. Neuropharmacology. 2002; 42: 306-318Crossref PubMed Scopus (79) Google Scholar) was performed as how we described before (21Yuen E.Y. Jiang Q. Chen P. Gu Z. Feng J. Yan Z. J. Neurosci. 2005; 25: 5488-5501Crossref PubMed Scopus (196) Google Scholar, 22Yuen E.Y. Jiang Q. Feng J. Yan Z. J. Biol. Chem. 2005; 280: 29420-29427Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). Briefly, cultured PFC neurons were treated with various agents after transfection, and then fixed in 4% paraformaldehyde for 30 min at room temperature without permeabilization. After incubating in 5% bovine serum albumin to block nonspecific staining, cells were incubated with the anti-GFP antibody (1:100, Chemicon, Temecula, CA) for 1 h at room temperature. After three washes in phosphate-buffered saline, cells were incubated with a rhodamine-conjugated secondary antibody (1: 200, Sigma) for 1 h at room temperature. After washing in phosphate-buffered saline, coverslips were mounted on slides with Vectashield mounting media (Vector Laboratories, Burlingame, CA). Fluorescence images were detected using a 60× objective with a cooled charge-coupled device camera mounted on a Nikon microscope. The surface GFP-NR2B clusters were analyzed with ImageJ software. All specimens were imaged under identical conditions and analyzed with identical parameters. A 50-μm segment of dendrite was selected from the equal distance away from the soma of four to six individual neurons. To define dendritic clusters, a single threshold was selected manually. Signal was counted as clusters when its intensity was 2- to 3-fold greater than the overall fluorescence on the dendritic shaft. Three to four independent experiments were performed. Quantitative analyses were performed blindly without knowledge of experimental conditions. Biochemical Measurement of Surface Receptors—After treatment, PFC slices were incubated with artificial cerebrospinal fluid containing 1 mg/ml Sulfo-NHS-LC-Biotin (Pierce) for 20 min on ice. The slices were then rinsed three times in Tris-buffered saline to quench the biotin reaction, followed by homogenization in 300 μl of modified radioimmune precipitation assay buffer (1% Triton X-100, 0.1% SDS, 0.5% deoxycholic acid, 50 mm NaPO4, 150 mm NaCl, 2 mm EDTA, 50 mm NaF, 10 mm sodium pyrophosphate, 1 mm sodium orthovanadate, 1 mm phenylmethylsulfonyl fluoride, and 1 mg/ml leupeptin). The homogenates were centrifuged at 14,000 × g for 15 min at 4 °C. 15 μg of homogenates was removed to measure total proteins. For surface protein detection, 150 μg of homogenates was incubated with 100 μl of 50% NeutrAvidin-Agarose (Pierce) for 2 h at 4 °C, and bound proteins were resuspended in 25 μl of SDS sample buffer and boiled. Quantitative Western blots were performed on both total and biotinylated (surface) proteins using anti-NR1, anti-NR2B (Upstate Biotechnology, Lake Placid, NY), and anti-GABAAR β2/3 (Chemicon). Activation of 5-HT1A and 5-HT2A/C Receptors Regulates NMDAR-mediated Ionic and Synaptic Currents in a Counteractive Manner—We have previously found that activation of 5-HT1A receptors reduces NMDAR currents in PFC pyramidal neurons (21Yuen E.Y. Jiang Q. Chen P. Gu Z. Feng J. Yan Z. J. Neurosci. 2005; 25: 5488-5501Crossref PubMed Scopus (196) Google Scholar). To examine the potential interactions between 5-HT1A and 5-HT2A/C receptors on NMDAR functions, we tested the impact of 5-HT2 activation on 5-HT1A regulation of NMDAR currents by preincubating PFC cultures with the 5-HT2A/C agonist α-Me-5HT (20 μm, 10–30 min). Application of NMDA (100 μm) elicited an inward current that was partially desensitized and was completely eliminated by the NMDA receptor antagonist d-aminophosphonovalerate (50 μm). As shown in Fig. 1 (A and B), application of the 5-HT1A agonist 8-OH-DPAT (20 μm) reversibly reduced NMDAR currents (21.3 ± 0.7%, n = 16). However, in α-Me-5HT-treated neurons, the reduction of NMDAR current by 8-OH-DPAT was substantially attenuated (6.4 ± 0.7%, n = 8). α-Me-5HT alone did not significantly affect NMDAR currents (1 μm: 4.0 ± 0.8%, n = 5; 20 μm: 5.7 ± 1.4%, n = 7). Another 5-HT2A/C agonist DOI also had no effect on NMDAR currents at low doses (0.05 μm: 4.2 ± 1.2%, n = 5; 0.1 μm: 4.0 ± 2.5%, n = 5), which are different from the inhibitory effect of DOI shown before (26Arvanov V.L. Liang X. Russo A. Wang R.Y. Eur. J. Neurosci. 1999; 11: 3064-3072Crossref PubMed Scopus (51) Google Scholar). The discrepancy may be due to different experimental procedures in recording NMDA-induced currents. In a previous study (26Arvanov V.L. Liang X. Russo A. Wang R.Y. Eur. J. Neurosci. 1999; 11: 3064-3072Crossref PubMed Scopus (51) Google Scholar), microdrops of NMDA (1 mm, every 15 min) were applied to PFC slices, which did not allow the accurate detection of peak NMDA currents because of the slow diffusion of the ligand. Moreover, it was not a pure postsynaptic preparation like dissociated neurons, which could have indirect effects due to changes in the circuit. Our results suggest that 5-HT2A/C activation alone does not directly affect NMDAR currents but opposes 5-HT1A regulation of NMDAR currents in PFC pyramidal neurons. We further tested the influence of 5-HT2 activation on 5-HT1A regulation of NMDAR-EPSC mediated by synaptic NMDA receptors in PFC slices. As shown in Fig. 1 (C and D), application of 8-OH-DPAT potently reduced the amplitude of NMDAR-EPSC (37.1 ± 2.1%; n = 10 (Fig. 1F)). However, this reduction was significantly attenuated in the presence of α-Me-5HT (10.4 ± 0.7%; n = 13 (Fig. 1F)). To verify that 5-HT2A/C receptors were mediating this regulatory effect of α-Me-5HT, we pre-treated PFC slices with the specific 5-HT2A/C antagonist ketanserin (20 μm). As shown in Fig. 1E, α-Me-5HT failed to oppose the effect of 5-HT1A on NMDAR-EPSC in the presence of ketanserin (36.8 ± 7.1%; n = 5 (Fig. 1F)). In contrast, the reduction of NMDAR-EPSC by 8-OH-DPAT was intact in the presence of the 5-HT4 agonist benzothiazole (20 μm, 33 ± 2.9%; n = 5 (Fig. 1F)). These results indicate that 5-HT2A/C receptor activation selectively counteracts 5-HT1A regulation of NMDAR functions in PFC pyramidal neurons. We next examined what would happen when both 5-HT1A and 5-HT2A/C receptors are co-activated by serotonin. If both signals oppose each other, then blocking one receptor activation would unmask the effect of the other. Thus, we tested the effect of serotonin on NMDAR-EPSC in PFC slices treated with or without 5-HT2 antagonists. As shown in Fig. 2 (A and B), different concentrations of serotonin reduced the amplitude of NMDAR-EPSC to different extents (10 μm:21 ± 2.3%, n = 4; 40 μm: 28.5 ± 4.9%, n = 4; 100 μm: 29.4 ± 4.8%, n = 5). However, in the presence of the 5-HT2A/C antagonist ketanserin (20 μm), the reduction of NMDAR-EPSC by serotonin was greatly augmented (10 μm: 35.2 ± 2.2%, n = 7; 40 μm: 43.0 ± 2.0%, n = 6; 100 μm:47 ± 1.9%, n = 7). It suggests that 5-HT2 receptor activation in response to serotonin masks part of the inhibitory action of 5-HT1A receptors on NMDAR channels. To assess whether the serotonergic regulation of NMDARs, which we found in vitro, is also occurring in vivo, we tested whether endogenous serotonin, via the activation of 5-HT1A and 5-HT2A/C receptors, could regulate NMDAR functions in a similar manner in intact animals. First, we examined NMDAR-EPSC in PFC slices from animals intraperitoneally injected with 5-HT1A or 5-HT2A/C agonists. As shown in Fig. 3A, the amplitude of NMDAR-EPSC was significantly smaller in animals intraperitoneally injected with the 5-HT1A agonist 8-OH-DPAT (saline: 379.8 ± 8.9 pA, n = 8; DPAT: 166 ± 11.5 pA, n = 11; p < 0.001, ANOVA), whereas it was largely unaltered by injecting the 5-HT2A/C agonist α-Me-5HT (349 ± 12.2 pA, n = 10). However, the effect of 8-OH-DPAT injection was blocked by injecting α-Me-5HT (DPAT + α-Me-5HT: 335 ± 15.9 pA, n = 12). These results indicate that activation of 5-HT1A receptors in vivo down-regulates NMDAR functions, and this effect of 5-HT1A is opposed by 5-HT2A/C receptor activation in vivo. Moreover, the amplitude of α-amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid receptor-EPSC was unchanged by 8-OH-DPAT injection (saline: 113 ± 11.8 pA, n = 11; DPAT: 112 ± 9.9 pA, n = 10), suggesting that 5-HT1A activation in PFC is specifically targeting postsynaptic NMDARs rather than presynaptic glutamate release. Next, we examined NMDAR-EPSC in PFC slices from animals intraperitoneally injected with the serotonin re-uptake inhibitor fluoxetine (20 mg/kg) to elevate endogenous 5-HT levels at synapses. As shown in Fig. 3B, the amplitude of NMDAR-EPSC in fluoxetine-injected animals was significantly smaller, compared with saline controls (saline: 403.5 ± 15.8 pA, n = 11; fluox: 195.1 ± 12.4 pA, n = 14; p < 0.001, ANOVA). This effect of fluoxetine was blocked by injecting the 5-HT1A antagonist WAY-100635 (WAY: 367 ± 12.9 pA, n = 12; WAY + fluox: 390 ± 15.8 pA, n = 12), suggesting the mediation by 5-HT1A receptors. Moreover, the reducing effect of fluoxetine on NMDAR-EPSC was potentiated by injecting the 5-HT2A/C antagonist ketanserin (ket: 392 ± 12.3 pA, n = 11; ket + fluox: 59.6 ± 5.2 pA, n = 12; p < 0.001, ANOVA), revealing the counteracting effect of 5-HT2A/C on 5-HT1A regulation of synaptic NMDA responses. Moreover, the effect of bath application of 8-OH-DPAT (40 μm) on NMDAR-EPSC was occluded in animals injected with fluoxetine, as compared with saline controls (Fig. 3C, saline: 40.2 ± 1.9%, n = 5, fluox: 10.5 ± 2.5%, n = 6), confirming the common mechanism underlying the in vivo fluoxetine effect and in vitro 8-OH-DPAT effect on NMDARs. In contrast to the strong effect in PFC pyramidal neurons, fluoxetine injection failed to affect NMDAR-EPSC in striatal medium spiny neurons (saline: 375 ± 15.9 pA, n = 8; fluox: 351 ± 13.9 pA, n = 10), suggesting the specificity of serotonergic regulation of NMDARs in PFC. To further test the effect of 5-HT on NMDARs in PFC networks in vivo without affecting other circuits, we also performed stereotaxic injection of fluoxetine to PFC to elevate the local 5-HT levels. We found that the amplitude of NMDAR-EPSC in PFC pyramidal neurons localized at the proximity of injection sites was significantly smaller, compared with saline controls (saline: 401 ± 10.1 pA, n = 11; fluox: 162 ± 13.2 pA, n = 11, p < 0.001, ANOVA). The Opposing Regulation of NMDA Receptors by 5-HT1A and 5-HT2 Receptors Depends on Microtubule Stability—Next, we investigated the potential mechanism by which 5-HT2 receptors counteract the effect of 5-HT1A on NMDAR currents in PFC neurons. Previously, we have found that 5-HT1A disrupts NMDA receptor trafficking by destabilizing microtubule integrity (21Yuen E.Y. Jiang Q. Chen P. Gu Z. Feng J. Yan Z. J. Neurosci. 2005; 25: 5488-5501Crossref PubMed Scopus (196) Google Scholar). Thus, we examined whether microtubule dynamics is the convergent target of 5-HT1A- and 5-HT2A-mediated signaling. To test this, we compared the level of free (depolymerized) tubulin in PFC cultures subjected to 8-OH-DPAT treatment in the absence or presence of α-Me5HT. As shown in Fig. 4 (A and B), application of 8-OH-DPAT (40 μm, 20 min) caused a potent increase in free tubulin (1.7 ± 0.3-fold increase, n = 3, p < 0.001, ANOVA), however, this effect was significantly blocked by α-Me-5HT (20 μm, 0.6 ± 0.2-fold increase, n = 3), indicating that 5-HT2 activation could increase microtubule stability and prevent 5-HT1A-induced microtubule depolymerization. We then tested whether the counteractive effect of 5-HT2 on 5-HT1A regulation of NMDAR currents is due to the opposing regulation of microtubule dynamics by 5-HT1A and 5-HT2 receptors. As shown in Fig. 4 (C and D), bath application of the microtubule depolymerizer, colchicine (30 μm), gradually reduced NMDAR-EPSC in PFC slices (38.0 ± 2.9%, n = 5, also see Ref. 22Yuen E.Y. Jiang Q. Feng J. Yan Z. J. Biol. Chem. 2005; 280: 29420-29427Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar), mimicking and occluding the 5-HT1A effect (21Yuen E.Y. Jiang Q. Chen P. Gu Z. Feng J. Yan Z. J. Neurosci. 2005; 25: 5488-5501Crossref PubMed Scopus (196) Google Scholar). However, this inhibitory effect of colchicine was largely attenuated in the presence of α-Me-5HT (20 μm, 11.2 ± 13.4%, n = 6). These results suggest that activation of 5-HT1A and 5-HT2 receptors could regulate NMDAR currents in a counteractive manner by converging on microtubule dynamics. Activation of 5-HT2 Receptors Induces ERK Activation in Neuronal Processes—How could 5-HT2 activation increase microtubule stability? Evidence has shown that activation of ERK stabilizes microtubule integrity (27Ray L.B. Sturgill T.W. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 1502-1506Crossref PubMed Scopus (472) Google Scholar, 28Sanchez C. Diaz-Nido J. Avila J. Prog. Neurobiol. 2000; 61: 133-168Crossref PubMed Scopus (425) Google Scholar). Moreover, our previous findings have suggested that 5-HT1A reduces ERK activity, which in turn reduces MAP2 phosphorylation and microtubule stability (21Yuen E.Y. Jiang Q. Chen P. Gu Z. Feng J. Yan Z. J. Neurosci. 2005; 25: 5488-5501Crossref PubMed Scopus (196) Google Scholar). Thus, we speculate that 5-HT2 receptors might activate ERK, leading to increased

The stress-related neuropeptide corticotropin-releasing factor (CRF) and the serotonin system are both critically involved in the pathophysiology of mental disorders, including anxiety and depression. To understand the potential link between them, we investigated the impact of CRF on 5-HT functions in pyramidal neurons of the prefrontal cortex (PFC), a brain region that is crucial for the control of emotion and cognition. One prominent function of serotonin in PFC is to regulate GABAergic inhibitory transmission, as indicated by a 5-HT-induced large, desensitizing (approximately 4 min) enhancement of the amplitude and frequency of spontaneous IPSCs (sIPSCs). In PFC slices exposed to CRF treatment, the regulation of sIPSCs by 5-HT was significantly prolonged (8-10 min), and this effect of CRF was blocked by treatment with the competitive CRF receptor antagonist alpha-helical CRF9-41 and with the CRF-R1-specific antagonist astressin. Inhibiting phospholipase C or protein kinase C (PKC) abolished the prolongation by CRF of the effects of 5-HT on sIPSCs. In PFC slices prepared from animals previously exposed to acute stress (forced swim or elevated platform), the regulation of sIPSCs by 5-HT was significantly prolonged, mimicking the effect of CRF treatment. The stress-induced prolongation of the effects of 5-HT on sIPSCs was diminished by alpha-helical CRF9-41 treatment, mimicked by direct activation of PKC, and reversed by short-term treatment with drugs that have anxiolytic efficacy. These results show that in response to stressful stimuli, CRF alters the serotonergic regulation of GABA transmission through a mechanism that is dependent on PKC. The interaction between CRF and 5-HT may play an important role in psychiatric disorders, in which both are highly implicated.

The <i>N</i>-methyl-d-aspartate (NMDA) receptor is a cation channel highly permeable to calcium and plays critical roles in governing normal and pathologic functions in neurons. Calcium entry through NMDA receptors (NMDARs) can lead to the activation of the Ca²⁺-dependent protease, calpain. Here we investigated the involvement of calpain in regulation of NMDAR channel function. After prolonged (5-min) treatment with NMDA or glutamate, the whole-cell NMDAR-mediated current was significantly reduced in both acutely dissociated and cultured cortical pyramidal neurons. The down-regulation of NMDAR current was blocked by bath application of selective calpain inhibitors. Intracellular injection of a specific calpain inhibitory peptide also eliminated the down-regulation of NMDAR current induced by prolonged NMDA treatment. In contrast, dynamin inhibitory peptide had no effect on the depression of NMDAR current, suggesting the lack of involvement of dynamin/clathrin-mediated NMDAR internalization in this process. Immunoblotting analysis showed that the NR2A and NR2B subunits of NMDARs were markedly degraded in cultured cortical neurons treated with glutamate, and the degradation of NR2 subunits was prevented by calpain inhibitors. Taken together, our results suggest that prolonged activation of NMDARs in neurons activates calpain, and activated calpain in turn down-regulates the function of NMDARs, which provides a neuroprotective mechanism against NMDAR overstimulation accompanying ischemia and stroke.

Article26 November 2009free access Assembly of a β2-adrenergic receptor—GluR1 signalling complex for localized cAMP signalling Mei-ling A Joiner Mei-ling A Joiner Department of Pharmacology, University of Iowa, Iowa City, IA, USA Search for more papers by this author Marie-France Lisé Marie-France Lisé Department of Psychiatry and Brain Research Center, University of British Columbia, Vancouver, British Columbia, Canada Search for more papers by this author Eunice Y Yuen Eunice Y Yuen Department of Physiology and Biophysics, State University of New York, Buffalo, NY, USA Search for more papers by this author Angel Y F Kam Angel Y F Kam Department of Pharmacology, University of Minnesota, Minneapolis, MN, USA Search for more papers by this author Mingxu Zhang Mingxu Zhang Department of Pharmacology, University of Iowa, Iowa City, IA, USA Search for more papers by this author Duane D Hall Duane D Hall Department of Pharmacology, University of Iowa, Iowa City, IA, USA Search for more papers by this author Zulfiqar A Malik Zulfiqar A Malik Department of Pharmacology, University of Iowa, Iowa City, IA, USA Search for more papers by this author Hai Qian Hai Qian Department of Pharmacology, University of Iowa, Iowa City, IA, USA Search for more papers by this author Yucui Chen Yucui Chen Department of Pharmacology, University of Iowa, Iowa City, IA, USA Search for more papers by this author Jason D Ulrich Jason D Ulrich Department of Pharmacology, University of Iowa, Iowa City, IA, USA Search for more papers by this author Alain C Burette Alain C Burette Department of Cell and Developmental Biology, and Neuroscience Center, University of North Carolina, Chapel Hill, NC, USA Search for more papers by this author Richard J Weinberg Richard J Weinberg Department of Cell and Developmental Biology, and Neuroscience Center, University of North Carolina, Chapel Hill, NC, USA Search for more papers by this author Ping-Yee Law Ping-Yee Law Department of Pharmacology, University of Minnesota, Minneapolis, MN, USA Search for more papers by this author Alaa El-Husseini Alaa El-Husseini Department of Psychiatry and Brain Research Center, University of British Columbia, Vancouver, British Columbia, CanadaDeceased. Search for more papers by this author Zhen Yan Zhen Yan Department of Physiology and Biophysics, State University of New York, Buffalo, NY, USA Search for more papers by this author Johannes W Hell Corresponding Author Johannes W Hell Department of Pharmacology, University of Iowa, Iowa City, IA, USA Search for more papers by this author Mei-ling A Joiner Mei-ling A Joiner Department of Pharmacology, University of Iowa, Iowa City, IA, USA Search for more papers by this author Marie-France Lisé Marie-France Lisé Department of Psychiatry and Brain Research Center, University of British Columbia, Vancouver, British Columbia, Canada Search for more papers by this author Eunice Y Yuen Eunice Y Yuen Department of Physiology and Biophysics, State University of New York, Buffalo, NY, USA Search for more papers by this author Angel Y F Kam Angel Y F Kam Department of Pharmacology, University of Minnesota, Minneapolis, MN, USA Search for more papers by this author Mingxu Zhang Mingxu Zhang Department of Pharmacology, University of Iowa, Iowa City, IA, USA Search for more papers by this author Duane D Hall Duane D Hall Department of Pharmacology, University of Iowa, Iowa City, IA, USA Search for more papers by this author Zulfiqar A Malik Zulfiqar A Malik Department of Pharmacology, University of Iowa, Iowa City, IA, USA Search for more papers by this author Hai Qian Hai Qian Department of Pharmacology, University of Iowa, Iowa City, IA, USA Search for more papers by this author Yucui Chen Yucui Chen Department of Pharmacology, University of Iowa, Iowa City, IA, USA Search for more papers by this author Jason D Ulrich Jason D Ulrich Department of Pharmacology, University of Iowa, Iowa City, IA, USA Search for more papers by this author Alain C Burette Alain C Burette Department of Cell and Developmental Biology, and Neuroscience Center, University of North Carolina, Chapel Hill, NC, USA Search for more papers by this author Richard J Weinberg Richard J Weinberg Department of Cell and Developmental Biology, and Neuroscience Center, University of North Carolina, Chapel Hill, NC, USA Search for more papers by this author Ping-Yee Law Ping-Yee Law Department of Pharmacology, University of Minnesota, Minneapolis, MN, USA Search for more papers by this author Alaa El-Husseini Alaa El-Husseini Department of Psychiatry and Brain Research Center, University of British Columbia, Vancouver, British Columbia, CanadaDeceased. Search for more papers by this author Zhen Yan Zhen Yan Department of Physiology and Biophysics, State University of New York, Buffalo, NY, USA Search for more papers by this author Johannes W Hell Corresponding Author Johannes W Hell Department of Pharmacology, University of Iowa, Iowa City, IA, USA Search for more papers by this author Author Information Mei-ling A Joiner1, Marie-France Lisé2, Eunice Y Yuen3, Angel Y F Kam4, Mingxu Zhang1, Duane D Hall1, Zulfiqar A Malik1, Hai Qian1, Yucui Chen1, Jason D Ulrich1, Alain C Burette5, Richard J Weinberg5, Ping-Yee Law4, Alaa El-Husseini2, Zhen Yan3 and Johannes W Hell 1 1Department of Pharmacology, University of Iowa, Iowa City, IA, USA 2Department of Psychiatry and Brain Research Center, University of British Columbia, Vancouver, British Columbia, Canada 3Department of Physiology and Biophysics, State University of New York, Buffalo, NY, USA 4Department of Pharmacology, University of Minnesota, Minneapolis, MN, USA 5Department of Cell and Developmental Biology, and Neuroscience Center, University of North Carolina, Chapel Hill, NC, USA *Corresponding author. Department of Pharmacology, University of California, Davis, CA 95616-8636, USA. Tel.: +1 319 384 4732; Fax: +1 319 335 8930; E-mail: [email protected] The EMBO Journal (2010)29:482-495https://doi.org/10.1038/emboj.2009.344 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Central noradrenergic signalling mediates arousal and facilitates learning through unknown molecular mechanisms. Here, we show that the β2-adrenergic receptor (β2AR), the trimeric Gs protein, adenylyl cyclase, and PKA form a signalling complex with the AMPA-type glutamate receptor subunit GluR1, which is linked to the β2AR through stargazin and PSD-95 and their homologues. Only GluR1 associated with the β2AR is phosphorylated by PKA on β2AR stimulation. Peptides that interfere with the β2AR–GluR1 association prevent this phosphorylation of GluR1. This phosphorylation increases GluR1 surface expression at postsynaptic sites and amplitudes of EPSCs and mEPSCs in prefrontal cortex slices. Assembly of all proteins involved in the classic β2AR–cAMP cascade into a supramolecular signalling complex and thus allows highly localized and selective regulation of one of its major target proteins. Introduction Although cAMP can in general freely diffuse, it can also be spatially restricted and exert highly localized actions (e.g. Levitzki, 1988; Neubig, 1994; Zaccolo and Pozzan, 2002; Rebois and Hebert, 2003; Fischmeister et al, 2006; Richter et al, 2008; Dai et al, 2009). Evidence for constitutive association of G protein-coupled receptors and their downstream effectors, the trimeric Gs protein and adenylyl cyclase, with each other has been established and interactions of such complexes with downstream targets have been proposed to form the basis for spatially restricted cAMP signalling (Levitzki, 1988; Neubig, 1994; Rebois and Hebert, 2003; Dai et al, 2009). Here, we describe a complex containing the β2-adrenergic receptor (β2AR), Gs, adenylyl cyclase, PKA, and the AMPA receptor (AMPAR) GluR1 subunit that allows highly localized signalling by cAMP in neurons. A brief period of high activity can permanently enhance synaptic transmission between neurons in the hippocampus. This long-term potentiation (LTP) has been a focus of intense study, as a tractable experimental model for learning and memory. Current evidence implicates trafficking of AMPARs to the postsynaptic membrane as the primary mechanism of LTP expression. AMPARs are tetramers formed by GluR1-4 subunits, with GluR1/2 and GluR2/3 heterotetramers accounting for the majority of AMPARs in the hippocampus (Hollmann and Heinemann, 1994; Wenthold et al, 1996; Dingledine et al, 1999) and GluR1/2 constituting the functionally by far prevailing AMPAR component at postsynaptic sites under basal conditions (Lu et al, 2009). The GluR1 subunit is thought to have a special function in initiating the remodelling associated with increased synaptic expression of AMPARs on LTP. Phosphorylation of GluR1 by PKA on serine 845 (S845) is critical for activity-driven accumulation of GluR1 at postsynaptic sites (Esteban et al, 2003) and fosters surface expression of GluR1 (Ehlers, 2000; Swayze et al, 2004; Sun et al, 2005; Oh et al, 2006; Man et al, 2007). Moreover, GluR1 is physically associated with PKA and the counteracting phosphatase calcineurin/PP2B, optimizing the efficacy of this regulation (Colledge et al, 2000; Tavalin et al, 2002). Norepinephrine, released by an extensive network of fibres originating from the locus coeruleus, supports arousal and learning under novel and emotionally charged situations (Cahill et al, 1994; Nielson and Jensen, 1994; Berman and Dudai, 2001; Strange et al, 2003; Strange and Dolan, 2004; Minzenberg et al, 2008) and facilitates various forms of LTP in the dentate gyrus and CA1 region of the hippocampus (Thomas et al, 1996; Lin et al, 2003; Walling and Harley, 2004; Gelinas and Nguyen, 2005). However, the molecular basis of these noradrenergic actions is unclear. Norepinephrine acts through αAR and βAR. Stimulation of the β1AR and β2AR activates Gs, adenylyl cyclase, and PKA. The β2AR is concentrated at excitatory postsynaptic sites in pyramidal neurons (Davare et al, 2001). We now show that GluR1 forms a signalling complex with the β2AR, Gs, and adenylyl cyclase for highly localized GluR1 phosphorylation, which promotes surface expression of GluR1 and increases EPSC and mEPSC amplitudes in cortex. Results β2AR and GluR1 colocalize in hippocampal neurons Double labelling for the β2AR and GluR1 in tissue slices from adult hippocampus showed prominent colocalization of immunoreactive puncta (Figure 1A). Most of these puncta were closely associated with puncta immunoreactive for the presynaptic marker synaptophysin, suggesting that they represent bona fide synapses. Quantitative analysis indicates that >90% of the β2AR puncta associated with synaptophysin were also positive for GluR1 (Figure 1B). Furthermore, >80% of GluR1 puncta associated with synaptophysin were also immunoreactive for β2AR, implying that the large majority of GluR1-containing synapses also possess the β2AR. Figure 1.Colocalization and co-immunoprecipitation of β2AR and GluR1. (A) Rat brain sections were triple-labelled for β2AR (left panels; green in overlays), GluR1 (centre panels; red), and synaptophysin (blue in bottom right panel). GluR1 and β2AR puncta colocalize with each other (boxes) and associate with synaptophysin puncta (triple overlay, bottom right panel) in the stratum radiatum of the CA1 area. (B) Quantification of the number of β2AR, GluR1, or synaptophysin (syn)-labelled puncta that shows positive (pos.) or no (neg.) staining for one or both of the other proteins. More than 90% of puncta that are positive for the β2AR and associated with synaptophysin are also positive for GluR1 (top bar) and >80% of puncta that are positive for GluR1 and associated with synaptophysin are also positive for the β2AR (second bar; a total of 329 synapses in 15 fields in stratum radiatum 25–100 μm away from the pyramidal cell layer from three adult s.d. rats (five fields per rat) were analysed). (C) Post-embedding immunogold staining for the β2AR in PFC. (a) Three immunopositive axospinous synapses in a single field. (b) A large strongly immunolabelled synapse. Labelling is mainly over PSD, close to postsynaptic plasma membrane (n=2 brains). (D–F) GluR1, but not NMDARs, specifically co-immunoprecipitate with β2AR from rat brain lysates. Rat brain was solubilized with 1% Triton X-100 and cleared by ultracentrifugation before immunoprecipitation (500 μg protein per lysate sample) with the H-20 antibody against β2AR or a control antibody and immunoblotting with antibodies against proteins indicated on the left side. The H-20 antibody, but not control IgG, immunoprecipitated the β2AR (D; n=3) and GluR1 (E; n=6), but not NR2B (E; n=2). The β2AR did not co-precipitate with NMDARs (F; n=3; 25 μg total lysate protein were loaded where indicated). Download figure Download PowerPoint The precise localization of the β2AR was determined by post-embedding immunogold labelling in the prefrontal cortex (PFC), which receives especially prominent noradrenergic innervation (e.g. Minzenberg et al, 2008). A large fraction of gold label was synaptic, and a large fraction of asymmetric axospinous synapses were immunopositive (Figure 1C). Gold particles concentrated over the postsynaptic density close to the postsynaptic membrane. Particles could also be seen within spines, in large dendritic shafts, in which they were typically associated with microtubules and in the cytoplasm of neuronal somata (data not shown). After solubilization with Triton X-100 and removal of non-solubilized material by ultracentrifugation, immunoprecipitation of the β2AR from rat forebrain (Figure 1D) led to co-immunoprecipitation of GluR1 (Figure 1E). This co-precipitation was specific, as the NMDA-type glutamate receptor (NMDAR) subunit NR2B, which has an overall structure and cellular distribution similar to GluR1, did not co-precipitate with the β2AR, and control IgG did not immunoprecipitate GluR1 or the β2AR. In contrast, immunoprecipitation of the NMDAR–PSD-95 complex (Leonard et al, 1998; Valtschanoff et al, 2000; Lim et al, 2002, 2003) did not result in co-precipitation of the β2AR (Figure 1F). We conclude that the β2AR assembles with GluR1-containing AMPARs, but not with NMDAR into a molecular complex at synapses. GluR1 interacts with the β2AR through PSD-95 and stargazin/γ2 PSD-95 co-immunoprecipitated with the β2AR from rat brain extracts (Figure 2A). In vitro pull-down experiments indicate that this association is mediated by the C-terminus of the β2AR. The distal end of the β2AR C-terminus (DSPL), which conforms to a type 1 PDZ domain ligand, binds to the third PDZ domain of PSD-95 (Figure 2B). Stargazin (or γ2) and its homologues γ3, γ4, γ5, γ7, and γ8 (TARPs) associate with AMPARs to promote their surface expression and modulate their biophysical properties. Stargazin binds with its C-terminus to the first two PDZ domains of PSD-95, and this interaction is required for surface expression and postsynaptic targeting of AMPARs (Chen et al, 2000; Schnell et al, 2002). Direct interactions between the β2AR with PSD-95 and stargazin with both PSD-95 and GluR1 could establish a physical link between the β2AR and AMPARs (Figure 2C). Figure 2.GluR1 forms a complex with the β2AR through PSD-95 and stargazin, which also contains Gs and adenylyl cyclase. (A) Rat brains were extracted with 1% deoxycholate (left) and 1% Triton X-100 (right) before immunoprecipitation with two different antibodies against β2AR (H-20, M-20) or control IgG, followed by immunoblotting with anti-PSD-95 ('JH62092' in Sans et al (2000); n=4). Specificity of the observed co-immunoprecipitation of PSD-95 with β2AR (lanes 4,5,9,10) is indicated by a lack of PSD-95 signal in the control IgG samples (lanes 3 and 8). (B) GST fusion proteins of the PDZ1–2, PDZ2, PDZ3, SH3 and GK domains of PSD-95 and GST alone were immobilized on glutathione Sepharose and incubated with MBP fusion protein of the full cytosolic C-terminus of the human β2AR (MBP-β2AR-CT) before washing and immunoblotting with anti-MBP antibodies. MBP-β2AR-CT bound strongly and specifically to the third PDZ domain (upper panel lane 3), but not to other domains of PSD-95 or to GST alone (lane 6). Reprobing with anti-GST antibodies shows that the different GST fusion proteins were present in comparable amounts (lower panel; n=8). (C) Schematic illustration of the β2AR–GluR1 complex. Stg or one of its homologues in the AMPAR complex binds with the C-terminus to the first two PDZ domains of PSD-95 or one of its homologues. PSD-95 interacts with its third PDZ domain with the C-terminus of the β2AR. (D) Loss of stargazin dissociates GluR1 from β2AR. Triton X-100 extracts of cortices and cerebella of wild-type and litter-matched stargazer (Stg−/−) mice were cleared by ultracentrifugation before immunoprecipitation of β2AR and immunoblotting with anti-GluR1 antibodies. GluR1 co-immunoprecipitated with β2AR from both cortex and cerebellum of wild-type mice (upper left panel) and with the β2AR from cortex, but not cerebellum of stg−/− mice (upper right panel). Lower panels indicate that similar amounts of GluR1 were present in the various extracts. Similar results were obtained in three independent experiments with three wt and three stg−/− mice. (E) Association of Gs and adenylyl cyclase with the β2AR–AMPAR complex. Triton X-100 extracts of crude rat forebrain membrane fractions were cleared by ultracentrifugation before immunoprecipitation with antibodies against GluR1 (lane 1), β2AR (lane 2), or a non-specific control IgG (lane 3), followed by immunoblotting for the proteins indicated on the left side (Leonard et al, 1998; Davare et al, 2001). Anti-GluR1 and anti-β2AR precipitated PSD-95 (positive control, n=3), adenylyl cyclase (AC, n=3), Gαs (n=3), and Gβ. The latter was detected with two different antibodies that recognized the N- (BN1; n=2) and C-termini (BC1; n=3) of Gβ1−4 (left panels). PKA (here the RIIα subunit; n=2) and Stg (n=4) were also present in GluR1 and β2AR precipitates. The metabotropic glutamate receptors mGluR1 (n=3) and mGluR5 (n=3), caveolin 1(Cav1; n=3) and the NMDA receptor subunits NR2A (n=1), NR2B (n=3), and NR1 (n=1) did not co-immunoprecipitate with either GluR1 or β2AR (right panels), although all proteins were clearly detectable in lysate (lane 4). All immunoprecipitations were made from lysate samples containing 500 μg total protein; lysate aliquots containing 25 μg protein were loaded when indicated. Download figure Download PowerPoint To test whether stargazin links the β2AR to GluR1 through PSD-95 in neurons, we evaluated whether co-immunoprecipitation of GluR1 with the β2AR depends on the presence of stargazin. Stargazer mice (stg−/−) lack functional stargazin, the prevailing TARP in adult cerebellum (Chen et al, 2000; Tomita et al, 2003; Menuz and Nicoll, 2008; Menuz et al, 2008). GluR1 co-immunoprecipitated with the β2AR from cerebral cortex and cerebellum from wild-type mice (Figure 2D, top left panel). In stg−/− mice, GluR1 co-immunoprecipitated with the β2AR from cortex, but not from cerebellum (Figure 2D, top right panel). These results indicate a requirement for stargazin for association of the β2AR with GluR1 in the cerebellum, but not cortex, consistent with published evidence that other TARPs substitute for stargazin in cortex. As the β2AR forms a complex with GluR1, but not with NMDAR, that depends on stg and PSD-95 and their homologues, the interaction of the β2AR with PSD-95 or its homologues is governed by other interactions of PSD-95. In other words, it is controlled by the immediate molecular environment of PSD-95. That only certain combinations of binding partners for complex formation with PSD-95 are realized in vivo seems to be critical to avoid physiologically undesirable assemblies or, worse, chaos by random complex formation. β2AR–GluR1 complex also contains Gs and adenylyl cyclase Association of the β2AR with GluR1 could allow selective and spatially restricted signalling. If so, the trimeric Gs protein, adenylyl cyclase, and PKA must also be localized near GluR1. PKA is structurally and functionally linked to GluR1 through the A kinase anchor protein, AKAP150 (Colledge et al, 2000; Tavalin et al, 2002). AKAP150 associates with SAP97 (Colledge et al, 2000; Tavalin et al, 2002), which in turn binds directly to the C-terminus of GluR1 (Leonard et al, 1998). Figure 2E illustrates that Gs and adenylyl cyclase are also associated with GluR1: immunoprecipitation of either GluR1 or the β2AR co-precipitated Gαs, Gβ, and adenylyl cyclase in extracts from total forebrain (left upper panels) as well as specifically from PFC and cerebellum (Supplementary Figure 1A). PKA and stargazin also co-precipitated with GluR1 and the β2AR under these conditions (Figure 2E, left lower panels) as did GluR2 as tested in PFC and cerebellar extracts (Supplementary Figure 1A). As stargazin expression is low in forebrain (Tomita et al, 2003), immunoreactive signals are low in lysate and immunoprecipitates from forebrain. In contrast to these proteins, several other proteins that are likewise concentrated at the postsynaptic site did not co-immunoprecipitate with GluR1 or the β2AR, including the metabotropic glutamate receptors mGluR1 and mGluR5, and the NMDAR subunits NR1, NR2A, and NR2B (Figure 2E, right panels). The plasma membrane raft marker caveolin 1 was also absent in these immunoprecipitates. Control IgG did not precipitate PSD-95, adenylyl cyclase, Gαs, Gβ, PKA, and stargazin. Therefore, we conclude that their co-immunoprecipitation with GluR1 and the β2AR is specific. Regulation of GluR1 S845 phosphorylation by associated β2AR By phosphorylating S845 on the intracellular COOH-terminal region of GluR1 (Roche et al, 1996), PKA promotes activity-driven synaptic targeting of GluR1 (Esteban et al, 2003). To determine whether β2AR, Gs, adenylyl cyclase, PKA, and GluR1 are organized into a functional complex, primary hippocampal cultures were treated with the β-adrenergic agonist isoproterenol (ISO; 3 μM, 15 min). This treatment increased phosphorylation of S845 of GluR1 in hippocampal neurons (Figure 3A and B). The increase was attenuated with the highly selective β2AR antagonist ICI118551 (1 μM), which by itself reduced basal S845 phosphorylation in some, but not all experiments. We next tested whether the β2AR–GluR1 interaction is necessary for S845 phosphorylation, as predicted if signalling from the β2AR to the AMPAR through PKA is localized. After treatment with vehicle or ISO and extraction with Triton X-100, each sample was divided into two equal halves. One half was mock treated before immunoprecipitation of GluR1 to assess the total GluR1 population. The other half was first depleted of GluR1 that was associated with the β2AR by immunoprecipitation with anti-β2AR antibody. The resulting supernatant lacking β2AR–GluR1 complexes was then used for immunoprecipitation with anti-GluR1. As before, ISO treatment induced S845 phosphorylation in the total GluR1 pool, but no such increase in phosphorylation was observed for the GluR1 pool from which β2AR-associated GluR1 had been removed (Figure 3C and D). An analogous experiment with forskolin (10 μM, 15 min), a direct activator of adenylyl cyclase, led to increased S845 phosphorylation in the total GluR1 as well as the β2AR-depleted GluR1 populations (Figure 3E and F). These results indicate that a substantial fraction of GluR1 is available for phosphorylation by PKA on S845 on massive stimulation of cAMP production even if not associated with the β2AR. However, activation of the β2AR leads to selective phosphorylation of β2AR-linked GluR1. Figure 3.Localized regulation of GluR1 S845 phosphorylation by the β2AR. Primary hippocampal cultures (18 DIV) were treated with vehicle (VEH), 3 μM ISO, 10 μM forskolin (FSK), or 10 μM 1,9-dideoxyforskolin (DDF; inactive forskolin homologue) for 15 min. Cultures were extracted with Triton X-100 and cleared by ultracentrifugation. GluR1 was immunoprecipitated directly or after pre-immunoprecipitation of β2AR complexes (H-20) before immunoblotting with antibodies against phosphorylated S845 (top) and total GluR1 (bottom). Immunosignals were quantified by densitometry. Phospho-S845 (pS845) signals were corrected with respect to total GluR1 signals and normalized to control. (A, B) ISO significantly increased S845 phosphorylation. Pre-treatment for 15 min with 1 μM ICI118551-blocked ISO-induced phosphorylation. All lanes are from same blot and exposure, but non-relevant lanes have been removed between lane 3 and 4. (C–F) After treatments, culture extracts were split into two equal portions. In contrast to the total GluR1 population (left pairs in C, D), GluR1 remaining after depletion of GluR1–β2AR complexes by pre-immunoprecipitation with H-20 showed no ISO-induced increase in S845 phosphorylation (right pairs in C, D), although forskolin stimulated S845 phosphorylation in both the total and the GluR1–β2AR-depleted GluR1 populations (E, F). (G–J) Pre-treatment for 2 h with membrane-permeant DSPL and ESDV peptides, but not their inactive analogues DAPA and EADA (1 μM each) prevented induction of S845 phosphorylation by ISO. *P<0.05 compared with control treatments; error bars: s.e.m.; n: number of independent experiments. Download figure Download PowerPoint To further test whether the complex formation between β2AR and GluR1 is necessary for efficient signalling, cultures were pre-treated for 2 h with the membrane-permeable peptide 11R-QGRNSNTNDSPL ('DSPL'). This peptide mimics the extreme C-terminus of the β2AR, which interacts with the third PDZ domain of PSD-95, and disrupts the co-immunoprecipitation of the β2AR with GluR1 (Supplementary Figure 1B). It had little effect on the co-immunoprecipitation of PSD-95 with GluR1, as expected, because the latter engages PDZ domains 1 and 2 of PSD-95 and its homologues. Binding selectivities of the first and second PDZ domains of PSD-95 and related proteins such as SAP97 and SAP102 are very similar to each other, but differ from those of the third PDZ domains (Lim et al, 2002, 2003). Control treatments were performed with its inactive analogue 11R-QGRNSNTNDAPA ('DAPA') altered at the 0 and −2 position (in bold), which are critical for PDZ domain binding. Subsequent incubation with ISO (3 μM, 15 min) led to increased S845 phosphorylation only in samples pre-treated with DAPA, but not in those pre-treated with DSPL (Figure 3G and H). A second pair of peptides gave analogous results: 11R-VYKKMPSIESDV ('ESDV') and its inactive analogue 11R-VYKKMPSIEADA ('EADA') is based on the C-terminus of NR2A. ESDV has optimal binding affinity for PDZ1 and 2 of PSD-95 and its homologues (Lim et al, 2002) and effectively blocks PDZ1 and 2 interactions (Lim et al, 2003). The latter conclusion had been based on immunoprecipitation experiments of PSD-95 with the NMDAR and was now confirmed for the co-immunoprecipitation of PSD-95 with GluR1, which was disrupted as expected (Supplementary Figure 1B). Pre-treatment with ESDV, but not EADA, prevented the ISO-triggered increase in S845 phosphorylation (Figure 3I and J). Collectively, these peptide experiments indicate that PSD-95 and its homologues structurally and functionally connect through PDZ domains the β2AR to the GluR1–stargazin complex for S845 phosphorylation. Increased surface expression of GluR1 by the associated β2AR The amount of GluR1 is increased at postsynaptic sites on stimulation of S845 phosphorylation by PKA (Swayze et al, 2004; Sun et al, 2005; Man et al, 2007). We asked whether the classic β-adrenergic signalling pathway controls postsynaptic GluR1 accumulation in hippocampal neurons using ectopically expressed GluR1 with superecliptic pHluorin (SEP) at its extracellular N-terminus. The pH-sensitive SEP–GluR1 signal was nearly completely quenched if the extracellular pH was lowered from 7.4 to 6.0 (Supplementary Figure 2), implying that the SEP–GluR1 signal was only detectable if SEP–GluR1 was present at the surface, but not in acidic intracellular organelles. The SEP–GluR1 fluorescence was stable for several hours under our imaging paradigm (data not shown). In initial experiments, 1 μM ISO in combination with the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX; 250 μM) to enhance cAMP levels on β-adrenergic stimulation strongly increased surface expression of SEP–GluR1 at dendritic spines within 15 min, elevating both the intensity for individual puncta and the number of detectable puncta per dendritic length (Supplementary Figure 3). IBMX alone was without effect indicating that signalling was driven by ISO. The ISO effect was completely blocked if the β2AR antagonist ICI118551 (1 μM) was added immediately before monitoring SEP–GluR1 signals. Subsequent experiments illustrate that a 5 min treatment with 1 μM ISO is sufficient for the full increase in synaptic SEP–GluR1 accumulation (Figure 4). Figure 4.ISO increases SEP–GluR1 surface expression in dendritic spines. Primary hippocampal cultures were transfected with SEP–GluR1 at 5–7 DIV and imaged at 21DIV. ISO (1 μM) increased density and signal intensity of SEP–GluR1 puncta within 5 min, which otherwise remained constant under untreated control conditions. *P<0.05 compared with control; error bars: SEM. In each case, a minimum of 5 dendrites from 15 neurons in 3 different experiments were analysed. Download figure Download PowerPoint Similarly, ISO alone (10 μM, 15 min) elevated the number and intensity of GluR1-positive puncta detected by surface labelling of endogenous GluR1 with an antibody against its

The dopamine D3 receptor, which is highly enriched in nucleus accumbens (NAc), has been suggested to play an important role in reinforcement and reward. To understand the potential cellular mechanism underlying D3 receptor functions, we examined the effect of D3 receptor activation on GABAA receptor (GABAAR)-mediated current and inhibitory synaptic transmission in medium spiny neurons of NAc. Application of PD128907 [(4aR,10bR)-3,4a,4,10b-tetrahydro-4-propyl-2H,5H-[1]benzopyrano-[4,3-b]-1,4-oxazin-9-ol hydrochloride], a specific D3 receptor agonist, caused a significant reduction of GABAAR current in acutely dissociated NAc neurons and miniature IPSC amplitude in NAc slices. This effect was blocked by dialysis with a dynamin inhibitory peptide, which prevents the clathrin/activator protein 2 (AP2)-mediated GABAA receptor endocytosis. In addition, the D3 effect on GABAAR current was prevented by agents that manipulate protein kinase A (PKA) activity. Infusion of a peptide derived from GABAAR beta subunits, which contains an atypical binding motif for the clathrin AP2 adaptor complex and the major PKA phosphorylation sites and binds with high affinity to AP2 only when dephosphorylated, diminished the D3 regulation of IPSC amplitude. The phosphorylated equivalent of the peptide was without effect. Moreover, PD128907 increased GABAAR internalization and reduced the surface expression of GABAA receptor beta subunits in NAc slices, which was prevented by dynamin inhibitory peptide or cAMP treatment. Together, our results suggest that D3 receptor activation suppresses the efficacy of inhibitory synaptic transmission in NAc by increasing the phospho-dependent endocytosis of GABAA receptors.

Cyclin-dependent kinase 5 (Cdk5) regulates dopamine neurotransmission and has been suggested to serve as a homeostatic target of chronic psychostimulant exposure. To study the role of Cdk5 in the modulation of the cellular and behavioral effects of psychoactive drugs of abuse, we developed Cre/loxP conditional knock-out systems that allow temporal and spatial control of Cdk5 expression in the adult brain. Here, we report the generation of Cdk5 conditional knock-out (cKO) mice using the alphaCaMKII promoter-driven Cre transgenic line (CaMKII-Cre). In this model system, loss of Cdk5 in the adult forebrain increased the psychomotor-activating effects of cocaine. Additionally, these CaMKII-Cre Cdk5 cKO mice show enhanced incentive motivation for food as assessed by instrumental responding on a progressive ratio schedule of reinforcement. Behavioral changes were accompanied by increased excitability of medium spiny neurons in the nucleus accumbens (NAc) in Cdk5 cKO mice. To study NAc-specific effects of Cdk5, another model system was used in which recombinant adeno-associated viruses expressing Cre recombinase caused restricted loss of Cdk5 in NAc neurons. Targeted knock-out of Cdk5 in the NAc facilitated cocaine-induced locomotor sensitization and conditioned place preference for cocaine. These results suggest that Cdk5 acts as a negative regulator of neuronal excitability in the NAc and that Cdk5 may govern the behavioral effects of cocaine and motivation for reinforcement.

Accumulating evidence suggests that glycogen synthase kinase 3 (GSK-3) is a multifunctional kinase implicated in neuronal development, mood stabilization, and neurodegeneration. However, the synaptic actions of GSK-3 are largely unknown. In this study, we examined the impact of GSK-3 on AMPA receptor (AMPAR) channels, the major mediator of excitatory transmission, in cortical neurons. Application of GSK-3 inhibitors or knockdown of GSK-3 caused a significant reduction of the amplitude of miniature excitatory postsynaptic current (mEPSC), a readout of the unitary strength of synaptic AMPARs. Treatment with GSK-3 inhibitors also decreased surface and synaptic GluR1 clusters on dendrites and increased internalized GluR1 in cortical cultures. Rab5, the small GTPase controlling the transport from plasma membrane to early endosomes, was activated by GSK-3 inhibitors. Knockdown of Rab5 prevented GSK-3 inhibitors from regulating mEPSC amplitude. Guanyl nucleotide dissociation inhibitor (GDI), which regulates the cycle of Rab5 between membrane and cytosol, formed an increased complex with Rab5 after treatment with GSK-3 inhibitors. Blocking the function of GDI occluded the effect of GSK-3 inhibitors on mEPSC amplitude. In cells transfected with the non-phosphorylatable GDI mutant, GDI(S45A), GSK-3 inhibitors lost the capability to regulate GDI-Rab5 complex, mEPSC amplitude, and AMPAR surface expression. These results suggest that GSK-3, via altering the GDI-Rab5 complex, regulates Rab5-mediated endocytosis of AMPARs. It provides a potential mechanism underlying the role of GSK-3 in synaptic transmission and plasticity.

The Notch signaling pathway is a well known regulator of skeletal muscle stem cells known as satellite cells. Loss of Notch1 signaling leads to spontaneous myogenic differentiation. Notch1, normally expressed in satellite cells, is targeted for proteasomal degradation by Numb during differentiation. A homolog of Notch1, Notch3, is also expressed in these cells but is not inhibited by Numb. We find that Notch3 is paradoxically up-regulated during the early stages of differentiation by an enhancer that requires both MyoD and activated Notch1. Notch3 itself strongly inhibits the myogenic transcription factor Mef2c, most likely by increasing the p38 phosphatase Mkp1, which inhibits the Mef2c activator p38 MAP kinase. Active Notch3 decreases differentiation. Mef2c, however, induces microRNAs miR-1 and miR-206, which directly down-regulate Notch3 and allow differentiation to proceed. Thus, the myogenic differentiation-induced microRNAs miR-1 and -206 are important for differentiation at least partly because they turn off Notch3. We suggest that the transient expression of Notch3 early in differentiation generates a temporal lag between myoblast activation by MyoD and terminal differentiation into myotubes directed by Mef2c. The Notch signaling pathway is a well known regulator of skeletal muscle stem cells known as satellite cells. Loss of Notch1 signaling leads to spontaneous myogenic differentiation. Notch1, normally expressed in satellite cells, is targeted for proteasomal degradation by Numb during differentiation. A homolog of Notch1, Notch3, is also expressed in these cells but is not inhibited by Numb. We find that Notch3 is paradoxically up-regulated during the early stages of differentiation by an enhancer that requires both MyoD and activated Notch1. Notch3 itself strongly inhibits the myogenic transcription factor Mef2c, most likely by increasing the p38 phosphatase Mkp1, which inhibits the Mef2c activator p38 MAP kinase. Active Notch3 decreases differentiation. Mef2c, however, induces microRNAs miR-1 and miR-206, which directly down-regulate Notch3 and allow differentiation to proceed. Thus, the myogenic differentiation-induced microRNAs miR-1 and -206 are important for differentiation at least partly because they turn off Notch3. We suggest that the transient expression of Notch3 early in differentiation generates a temporal lag between myoblast activation by MyoD and terminal differentiation into myotubes directed by Mef2c.

The strength of synaptic inhibition depends partly on the number of GABA A receptors (GABA A Rs) found at synaptic sites. The trafficking of GABA A Rs within the endocytic pathway is a key determinant of surface GABA A R number and is altered in neuropathologies, such as cerebral ischemia. However, the molecular mechanisms and signaling pathways that regulate this trafficking are poorly understood. Here, we report the subunit specific lysosomal targeting of synaptic GABA A Rs. We demonstrate that the targeting of synaptic GABA A Rs into the degradation pathway is facilitated by ubiquitination of a motif within the intracellular domain of the γ2 subunit. Blockade of lysosomal activity or disruption of the trafficking of ubiquitinated cargo to lysosomes specifically increases the efficacy of synaptic inhibition without altering excitatory currents. Moreover, mutation of the ubiquitination site within the γ2 subunit retards the lysosomal targeting of GABA A Rs and is sufficient to block the loss of synaptic GABA A Rs after anoxic insult. Together, our results establish a previously unknown mechanism for influencing inhibitory transmission under normal and pathological conditions.

GABAergic interneurons in prefrontal cortex (PFC) play a critical role in cortical circuits by providing feedforward and feedback inhibition and synchronizing neuronal activity. Impairments in GABAergic inhibition to PFC pyramidal neurons have been implicated in the abnormal neural synchrony and working memory disturbances in schizophrenia. The dopamine D 4 receptor, which is strongly linked to neuropsychiatric disorders, such as attention deficit–hyperactivity disorder (ADHD) and schizophrenia, is highly expressed in PFC GABAergic interneurons, while the physiological role of D 4 in these interneurons is largely unknown. In this study, we found that D 4 activation caused a persistent suppression of AMPAR-mediated synaptic transmission in PFC interneurons. This effect of D 4 receptors on AMPAR-EPSC was via a mechanism dependent on actin/myosin V motor-based transport of AMPA receptors, which was regulated by cofilin, a major actin depolymerizing factor. Moreover, we demonstrated that the major cofilin-specific phosphatase Slingshot, which was activated by calcineurin downstream of D 4 signaling, was required for the D 4 regulation of glutamatergic transmission. Thus, D 4 receptors, by using the unique calcineurin/Slingshot/cofilin signaling mechanism, regulate actin dynamics and AMPAR trafficking in PFC GABAergic interneurons. It provides a potential mechanism for D 4 receptors to control the excitatory synaptic strength in local-circuit neurons and GABAergic inhibition in the PFC network, which may underlie the role of D 4 receptors in normal cognitive processes and mental disorders.

The cAMP and cAMP-dependent protein kinase A (PKA) signaling cascade is a ubiquitous pathway acting downstream of multiple neuromodulators. We found that the phosphorylation of phosphodiesterase-4 (PDE4) by cyclin-dependent protein kinase 5 (Cdk5) facilitated cAMP degradation and homeostasis of cAMP/PKA signaling. In mice, loss of Cdk5 throughout the forebrain elevated cAMP levels and increased PKA activity in striatal neurons, and altered behavioral responses to acute or chronic stressors. Ventral striatum- or D1 dopamine receptor-specific conditional knockout of Cdk5, or ventral striatum infusion of a small interfering peptide that selectively targeted the regulation of PDE4 by Cdk5, produced analogous effects on stress-induced behavioral responses. Together, our results demonstrate that altering cAMP signaling in medium spiny neurons of the ventral striatum can effectively modulate stress-induced behavioral states. We propose that targeting the Cdk5 regulation of PDE4 could be a new therapeutic approach for clinical conditions associated with stress, such as depression.

Emerging evidence indicates that amyloid β peptide (Aβ) initially induces subtle alterations in synaptic function in Alzheimer disease. We have recently shown that Aβ binds to β2 adrenergic receptor (β2AR) and activates protein kinase A (PKA) signaling for glutamatergic regulation of synaptic activities. Here we show that in the cerebrums of mice expressing human familial mutant presenilin 1 and amyloid precursor protein genes, the levels of β2AR are drastically reduced. Moreover, Aβ induces internalization of transfected human β2AR in fibroblasts and endogenous β2AR in primary prefrontal cortical neurons. In fibroblasts, Aβ treatment also induces transportation of β2AR into lysosome, and prolonged Aβ treatment causes β2AR degradation. The Aβ-induced β2AR internalization requires the N terminus of the receptor containing the peptide binding sites and phosphorylation of β2AR by G protein-coupled receptor kinase, not by PKA. However, the G protein-coupled receptor kinase phosphorylation of β2AR and the receptor internalization are much slower than that induced by βAR agonist isoproterenol. The Aβ-induced β2AR internalization is also dependent on adaptor protein arrestin 3 and GTPase dynamin, but not arrestin 2. Functionally, pretreatment of primary prefrontal cortical neurons with Aβ induces desensitization of β2AR, which leads to attenuated response to subsequent stimulation with isoproterenol, including decreased cAMP levels, PKA activities, PKA phosphorylation of serine 845 on α-amino-2,3-dihydro-5-methyl-3-oxo-4-isoxazolepropanoic acid (AMPA) receptor subunit 1 (GluR1), and AMPA receptor-mediated miniature excitatory postsynaptic currents. This study indicates that Aβ induces β2AR internalization and degradation leading to impairment of adrenergic and glutamatergic activities. Emerging evidence indicates that amyloid β peptide (Aβ) initially induces subtle alterations in synaptic function in Alzheimer disease. We have recently shown that Aβ binds to β2 adrenergic receptor (β2AR) and activates protein kinase A (PKA) signaling for glutamatergic regulation of synaptic activities. Here we show that in the cerebrums of mice expressing human familial mutant presenilin 1 and amyloid precursor protein genes, the levels of β2AR are drastically reduced. Moreover, Aβ induces internalization of transfected human β2AR in fibroblasts and endogenous β2AR in primary prefrontal cortical neurons. In fibroblasts, Aβ treatment also induces transportation of β2AR into lysosome, and prolonged Aβ treatment causes β2AR degradation. The Aβ-induced β2AR internalization requires the N terminus of the receptor containing the peptide binding sites and phosphorylation of β2AR by G protein-coupled receptor kinase, not by PKA. However, the G protein-coupled receptor kinase phosphorylation of β2AR and the receptor internalization are much slower than that induced by βAR agonist isoproterenol. The Aβ-induced β2AR internalization is also dependent on adaptor protein arrestin 3 and GTPase dynamin, but not arrestin 2. Functionally, pretreatment of primary prefrontal cortical neurons with Aβ induces desensitization of β2AR, which leads to attenuated response to subsequent stimulation with isoproterenol, including decreased cAMP levels, PKA activities, PKA phosphorylation of serine 845 on α-amino-2,3-dihydro-5-methyl-3-oxo-4-isoxazolepropanoic acid (AMPA) receptor subunit 1 (GluR1), and AMPA receptor-mediated miniature excitatory postsynaptic currents. This study indicates that Aβ induces β2AR internalization and degradation leading to impairment of adrenergic and glutamatergic activities.

BackgroundMethylphenidate (MPH), a psychostimulant drug used to treat attention-deficit/hyperactivity disorder, produces the effects of increasing alertness and improving attention. However, misuse of MPH has been associated with an increased risk of aggression and psychosis. We sought to determine the molecular mechanism underlying the complex actions of MPH.MethodsAdolescent (4-week-old) rats were given one injection of MPH at different doses. The impact of MPH on glutamatergic signaling in pyramidal neurons of prefrontal cortex was measured. Behavioral changes induced by MPH were also examined in parallel.ResultsAdministration of low-dose (.5 mg/kg) MPH selectively potentiated N-methyl-D-aspartate receptor (NMDAR)–mediated excitatory postsynaptic currents (EPSCs) via adrenergic receptor activation, whereas high-dose (10 mg/kg) MPH suppressed both NMDAR-mediated and alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor–mediated EPSCs. The dual effects of MPH on EPSCs were associated with bidirectional changes in the surface level of glutamate receptor subunits. Behavioral tests also indicated that low-dose MPH facilitated prefrontal cortex–mediated temporal order recognition memory and attention. Animals injected with high-dose MPH exhibited significantly elevated locomotive activity. Inhibiting the function of synaptosomal-associated protein 25, a key SNARE protein involved in NMDAR exocytosis, blocked the increase of NMDAR-mediated EPSCs by low-dose MPH. In animals exposed to repeated stress, administration of low-dose MPH effectively restored NMDAR function and temporal order recognition memory via a mechanism dependent on synaptosomal-associated protein 25.ConclusionsThese results provide a potential mechanism underlying the cognitive-enhancing effects of low-dose MPH as well as the psychosis-inducing effects of high-dose MPH. Methylphenidate (MPH), a psychostimulant drug used to treat attention-deficit/hyperactivity disorder, produces the effects of increasing alertness and improving attention. However, misuse of MPH has been associated with an increased risk of aggression and psychosis. We sought to determine the molecular mechanism underlying the complex actions of MPH. Adolescent (4-week-old) rats were given one injection of MPH at different doses. The impact of MPH on glutamatergic signaling in pyramidal neurons of prefrontal cortex was measured. Behavioral changes induced by MPH were also examined in parallel. Administration of low-dose (.5 mg/kg) MPH selectively potentiated N-methyl-D-aspartate receptor (NMDAR)–mediated excitatory postsynaptic currents (EPSCs) via adrenergic receptor activation, whereas high-dose (10 mg/kg) MPH suppressed both NMDAR-mediated and alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor–mediated EPSCs. The dual effects of MPH on EPSCs were associated with bidirectional changes in the surface level of glutamate receptor subunits. Behavioral tests also indicated that low-dose MPH facilitated prefrontal cortex–mediated temporal order recognition memory and attention. Animals injected with high-dose MPH exhibited significantly elevated locomotive activity. Inhibiting the function of synaptosomal-associated protein 25, a key SNARE protein involved in NMDAR exocytosis, blocked the increase of NMDAR-mediated EPSCs by low-dose MPH. In animals exposed to repeated stress, administration of low-dose MPH effectively restored NMDAR function and temporal order recognition memory via a mechanism dependent on synaptosomal-associated protein 25. These results provide a potential mechanism underlying the cognitive-enhancing effects of low-dose MPH as well as the psychosis-inducing effects of high-dose MPH.

The lateral habenula (LHb) is a key brain region involved in the pathophysiology of depression. It is activated by stimuli associated with negative experiences and is involved in encoding aversive signals. Hyperactivity of LHb is found in both rodent models of depression and human patients with depression. However, little is known about the underlying molecular mechanisms. Here we show that in LHb neurons, p11, a multifunctional protein implicated in depression, is significantly upregulated by chronic restraint stress. Knockdown of p11 expression in LHb alleviates the stress-induced depression-like behaviors. Moreover, chronic restraint stress induces bursting action potentials in LHb neurons, which are abolished by p11 knockdown. Overexpression of p11 in dopamine D2 receptor-containing LHb neurons of control mice induces depression-like behaviors. These results have identified p11 in LHb as a key molecular determinant regulating negative emotions, which may help to understand the molecular and cellular basis of depression.

Many of the genes disrupted in autism are identified as histone-modifying enzymes and chromatin remodelers, most prominently those that mediate histone methylation/demethylation. However, the role of histone methylation enzymes in the pathophysiology and treatment of autism remains unknown. To address this, we used mouse models of haploinsufficiency of the Shank3 gene (a highly penetrant monogenic autism risk factor), which exhibits prominent autism-like social deficits. We found that histone methyltransferases EHMT1 and EHMT2, as well as histone lysine 9 dimethylation (specifically catalyzed by EHMT1/2), were selectively increased in the prefrontal cortex (PFC) of Shank3-deficient mice and autistic human postmortem brains. Treatment with the EHMT1/2 inhibitor UNC0642 or knockdown of EHMT1/2 in PFC induced a robust rescue of autism-like social deficits in Shank3-deficient mice, and restored NMDAR-mediated synaptic function. Activity-regulated cytoskeleton-associated protein (Arc) was identified as one of the causal factors underlying the rescuing effects of UNC0642 on NMDAR function and social behaviors in Shank3-deficient mice. UNC0642 treatment also restored a large set of genes involved in neural signaling in PFC of Shank3-deficient mice. These results suggest that targeting histone methylation enzymes to adjust gene expression and ameliorate synaptic defects could be a potential therapeutic strategy for autism.

Microdeletion of the human 16p11.2 gene locus has been linked to autism spectrum disorder (ASD) and intellectual disability and confers risk for a number of other neurodevelopmental deficits. Transgenic mice carrying 16p11.2 deletion (<i>16p11</i>^+/−) display phenotypes reminiscent of those in human patients with 16p11.2 deletion syndrome, but the molecular mechanisms and treatment strategies for these phenotypes remain unknown. In this study, we have found that both male and female <i>16p11</i>^+/− mice exhibit deficient NMDA receptor (NMDAR) function in the medial prefrontal cortex (mPFC), a brain region critical for high-level "executive" functions. Elevating the activity of mPFC pyramidal neurons with a CaMKII-driven Gq-DREADD (Gq-coupled designer receptors exclusively activated by designer drugs) led to the significant increase of NR2B subunit phosphorylation and the restoration of NMDAR function, as well as the amelioration of cognitive and social impairments in <i>16p11</i>^+/− mice. These results suggest that NMDAR hypofunction in PFC may contribute to the pathophysiology of 16p11.2 deletion syndrome and that restoring PFC activity is sufficient to rescue the behavioral deficits. <b>SIGNIFICANCE STATEMENT</b> The 16p11.2 deletion syndrome is strongly associated with autism spectrum disorder and intellectual disability. Using a mouse model carrying the 16p11.2 deletion, <i>16p11</i>^+/−, we identified NMDA receptor hypofunction in the prefrontal cortex (PFC). Elevating the activity of PFC pyramidal neurons with a chemogenetic tool, Gq-DREADD, led to the restoration of NMDA receptor function and the amelioration of cognitive and social impairments in <i>16p11</i>^+/− mice. These results have revealed a novel route for potential therapeutic intervention of 16p11.2 deletion syndrome.

ASH1L, a histone methyltransferase, is identified as a top-ranking risk factor for autism spectrum disorder (ASD), however, little is known about the biological mechanisms underlying the link of ASH1L haploinsufficiency to ASD. Here we show that ASH1L expression and H3K4me3 level are significantly decreased in the prefrontal cortex (PFC) of postmortem tissues from ASD patients. Knockdown of Ash1L in PFC of juvenile mice induces the downregulation of risk genes associated with ASD, intellectual disability (ID) and epilepsy. These downregulated genes are enriched in excitatory and inhibitory synaptic function and have decreased H3K4me3 occupancy at their promoters. Furthermore, Ash1L deficiency in PFC causes the diminished GABAergic inhibition, enhanced glutamatergic transmission, and elevated PFC pyramidal neuronal excitability, which is associated with severe seizures and early mortality. Chemogenetic inhibition of PFC pyramidal neuronal activity, combined with the administration of GABA enhancer diazepam, rescues PFC synaptic imbalance and seizures, but not autistic social deficits or anxiety-like behaviors. These results have revealed the critical role of ASH1L in regulating synaptic gene expression and seizures, which provides insights into treatment strategies for ASH1L-associated brain diseases.

Abstract Loss-of-function mutations of the gene Cul3 have been identified as a risk factor for autism-spectrum disorder (ASD), but the pathogenic mechanisms are not well understood. Conditional Cul3 ablation in cholinergic neurons of mice (Chat CRE Cul3 F/+ ) recapitulated ASD-like social and sensory gating phenotypes and caused significant cognitive impairments, with diminished activity of cholinergic neurons in the basal forebrain (BF). Chemogenetic inhibition of BF cholinergic neurons in healthy mice induced similar social and cognitive deficits. Conversely, chemogenetic stimulation of BF cholinergic neurons in Chat CRE Cul3 F/+ mice reversed abnormalities in sensory gating and cognition. Cortical hypofunction was also found after ChAT-specific Cul3 ablation and stimulation of cholinergic projections from the BF to the prefrontal cortex (PFC) mitigated cognitive deficits. Overall, we demonstrate that cholinergic dysfunction due to Cul3 deficiency is involved in ASD-like behavioral abnormalities, and that BF cholinergic neurons are particularly critical for cognitive component through their projections to the PFC.

Abstract Pleiotropic mechanisms have been implicated in Alzheimer’s disease (AD), including transcriptional dysregulation, protein misprocessing and synaptic dysfunction, but how they are mechanistically linked to induce cognitive deficits in AD is unclear. Here we find that the histone methyltransferase Smyd3, which catalyzes histone H3 lysine 4 trimethylation (H3K4me3) to activate gene transcription, is significantly elevated in prefrontal cortex (PFC) of AD patients and P301S Tau mice, a model of tauopathies. A short treatment with the Smyd3 inhibitor, BCI-121, rescues cognitive behavioral deficits, and restores synaptic NMDAR function and expression in PFC pyramidal neurons of P301S Tau mice. Fbxo2 , which encodes an E3 ubiquitin ligase controlling the degradation of NMDAR subunits, is identified as a downstream target of Smyd3. Smyd3-induced upregulation of Fbxo2 in P301S Tau mice is linked to the increased NR1 ubiquitination. Fbxo2 knockdown in PFC leads to the recovery of NMDAR function and cognitive behaviors in P301S Tau mice. These data suggest an integrated mechanism and potential therapeutic strategy for AD.

Roscovitine is widely used for inhibition of cdk5, a cyclin-dependent kinase expressed predominantly in the brain. A novel function of roscovitine, i.e. an effect on Ca(2+) channels and transmitter release in central neurons, was studied by whole-cell voltage-clamp recordings and time-lapse fluorescence imaging techniques. Extracellular application of roscovitine markedly enhanced the tail calcium current following repolarization from depolarized voltages. This effect was rapid, reversible and dose dependent. Roscovitine dramatically slowed the deactivation kinetics of calcium channels. The deactivation time constant was increased 3- to 6-fold, suggesting that roscovitine could prolong the channel open state and increase the calcium influx. The potentiation of tail calcium currents caused by roscovitine and by the L-channel activator Bay K 8644 was not occluded but additive. Roscovitine-induced potentiation of tail calcium currents was significantly blocked by the P/Q-channel blocker CgTx-MVIIC, indicating that the major target of roscovitine is the P/Q-type calcium channel. In mutant mice with targeted deletion of p35, a neuronal specific activator of cdk5, roscovitine regulated calcium currents in a manner similar to that observed in wild-type mice. Moreover, intracellular perfusion of roscovitine failed to modulate calcium currents. These results suggest that roscovitine acts on extracellular site(s) of calcium channels via a cdk5-independent mechanism. Roscovitine potentiated glutamate release at presynaptic terminals of cultured hippocampal neurons detected with the vesicle trafficking dye FM1-43, consistent with the positive effect of roscovitine on the P/Q-type calcium channel, the major mediator of action potential-evoked transmitter release in the mammalian CNS.

The effect of carbohydrate (CHO) ingestion on metabolic responses to exercise has been investigated. Subjects cycled at approximately 70% of maximal oxygen uptake to fatigue [135 +/- 17 (+/- SE) min] on the first occasion (control, CON) and at the same work load and duration on the second occasion but with addition of ingestion of CHO during the exercise. Biopsies were taken from the quadriceps femoris muscle before and after exercise. The sum of the hexose monophosphates (HMP), as well as lactate and alanine, in muscle was higher after CHO exercise (P less than or equal to 0.05, P less than or equal to 0.05, and P less than or equal to 0.01, respectively). Acetylcarnitine increased during exercise but was not significantly different between treatments after exercise (CON, 6.6 +/- 1.7; CHO, 10.0 +/- 1.2 mmol/kg dry wt; P = NS). The sum of the tricarboxylic acid cycle intermediates (TCAI; citrate + malate + fumarate) was increased during exercise and was higher after CHO exercise (2.34 +/- 0.32 vs. 1.68 +/- 0.17 mmol/kg dry wt; P less than or equal to 0.05). IMP was less than 0.1 mmol/kg dry wt at rest and increased to 0.77 +/- 0.26 (CON) and 0.29 +/- 0.11 mmol/kg dry wt (CHO) (P less than or equal to 0.05) during exercise. It was recently found that during prolonged exercise there is initially a rapid and large expansion of TCAI and glycogenolytic intermediates in human muscle followed by a continuous decline in TCAI and glycogenolytic intermediates [K. Sahlin, A. Katz, and S. Broberg. Am. J. Physiol. 259 (Cell Physiol. 28): C834-C841, 1990].(ABSTRACT TRUNCATED AT 250 WORDS)

Inwardly rectifying K+ (IRK) channels are critical for shaping cell excitability. Whole-cell patch-clamp and single-cell RT-PCR techniques were used to characterize the inwardly rectifying K+ currents found in projection neurons of the rat nucleus accumbens. Inwardly rectifying currents were highly selective for K+ and blocked by low millimolar concentrations of Cs+ or Ba2+. In a subset of neurons, the inwardly rectifying current appeared to inactivate at hyperpolarized membrane potentials. In an attempt to identify this subset, neurons were profiled using single-cell RT-PCR. Neurons expressing substance P mRNA exhibited noninactivating inward rectifier currents, whereas neurons expressing enkephalin mRNA exhibited inactivating inward rectifier currents. The inactivation of the inward rectifier was correlated with the expression of IRK1 mRNA. These results demonstrate a clear physiological difference in the properties of medium spiny neurons and suggest that this difference could influence active state transitions driven by cortical and hippocampal excitatory input.

Protein phosphatase 1 plays a major role in the governance of excitatory synaptic activity, and is subject to control via the neuromodulatory actions of dopamine. Mechanisms involved in regulating protein phosphatase 1 activity include interactions with the structurally related cytoskeletal elements spinophilin and neurabin, synaptic scaffolding proteins that are highly enriched in dendritic spines. The requirement for these proteins in dopamine-related neuromodulation was tested using knockout mice. Dopamine D1-mediated regulation of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate receptor activity was deficient in both striatal and prefrontal cortical neurons from neurabin knockout mice; in spinophilin knockout mice this deficit was manifest only in striatal neurons. At corticostriatal synapses long-term potentiation was deficient in neurabin knockout mice, but not in spinophilin knockout mice, and was rescued by a D1 receptor agonist. In contrast, long-term depression was deficient in spinophilin knockout mice but not in neurabin knockout mice, and was rescued by D2 receptor activation. Spontaneous excitatory post-synaptic current frequency was increased in neurabin knockout mice, but not in spinophilin knockout mice, and this effect was normalized by D2 receptor agonist application. Both knockout strains displayed increased induction of GluR1 Ser845 phosphorylation in response to D1 receptor stimulation in slices, and also displayed enhanced locomotor activation in response to cocaine administration. These effects could be dissociated from cocaine reward, which was enhanced only in spinophilin knockout mice, and was accompanied by increased immediate early gene induction. These data establish a requirement for synaptic scaffolding in dopamine-mediated responses, and further indicate that spinophilin and neurabin play distinct roles in dopaminergic signal transduction and psychostimulant response.

The effect of preexercise muscle glycogen content on the metabolic responses to exercise has been investigated. Seven men cycled at a work load calculated to elicit 75% of maximal oxygen uptake [211 +/- 17 (SE) W] on two occasions: 1) to fatigue (37.2 +/- 5.3 min) and 2) at the same work load and for the same duration as the first. Biopsies were obtained from the quadriceps femoris muscle before and after exercise. Before the first experiment, muscle glycogen was lowered by exercise and diet, and before the second experiment, muscle glycogen was elevated. In the low-glycogen condition (LG), muscle glycogen decreased from 182 +/- 15 at rest to 7 +/- 4 mmol glucosyl units/kg dry wt at fatigue, while in the high-glycogen condition (HG), glycogen decreased from 725 +/- 31 at rest to 353 +/- 53 mmol glucosyl units/kg dry wt at the end of exercise. Hexose monophosphates were not increased after LG exercise but increased approximately fivefold after HG exercise. Lactate increased more during HG exercise (LG = 16 +/- 5, HG = 61 +/- 7 mmol/kg dry wt; P less than or equal to 0.001), whereas IMP increased more during LG (LG = 2.8 +/- 0.6, HG = 0.9 +/- 0.2 mmol/kg dry wt; P less than or equal to 0.05). The increases in the sum of tricarboxylic acid cycle intermediates (TCAI; citrate+malate+fumarate) and acetylcarnitine (which is in equilibrium with acetyl CoA) were significantly greater during HG exercise (P less than or equal to 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

α-Amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA)-type glutamate receptors undergo constitutive and ligand-induced internalization that requires dynamin and the clathrin adaptor complex AP-2. We report here that an atypical basic motif within the cytoplasmic tails of AMPA-type glutamate receptors directly associates with μ2-adaptin by a mechanism similar to the recognition of the presynaptic vesicle protein synaptotagmin 1 by AP-2. A synaptotagmin 1-derived AP-2 binding peptide competes the interaction of the AMPA receptor subunit GluR2 with AP-2μ and increases the number of surface active glutamate receptors in living neurons. Moreover, fusion of the GluR2-derived tail peptide with a synaptotagmin 1 truncation mutant restores clathrin/AP-2-dependent internalization of the chimeric reporter protein. These data suggest that common mechanisms regulate AP-2-dependent internalization of pre- and postsynaptic membrane proteins.

The serotonin system in prefrontal cortex (PFC) is critically involved in the regulation of cognition and emotion. To understand the cellular mechanisms underlying its physiological actions, we investigated the role of serotonin in regulating synaptic plasticity in PFC circuits. We found that tetanic stimuli coupled to bath application of serotonin induced long‐term depression (LTD) at excitatory synapses of PFC pyramidal neurons. This effect was mediated by 5‐HT 2A/C receptors and was independent of NMDA receptor activation. A group I metabotropic glutamate receptor (mGluR) antagonist blocked the LTD induction by serotonin + tetani, and co‐application of a group I mGluR agonist and serotonin, but not application of either drug alone, induced LTD without tetani. The effect of serotonin on LTD was blocked by selective inhibitors of p38 mitogen‐activated protein kinase (MAPK), but not p42/44 MAPK. Biochemical evidence also indicated that serotonin and a group I mGluR agonist synergistically activated p38 MAPK in PFC slices. The serotonin‐facilitated LTD induction was prevented by blocking the activation of the small GTPase Rab5, as well as by blocking the clathrin‐dependent internalization of AMPA receptors with postsynaptic injection of a dynamin inhibitory peptide, while it was unaffected by manipulating the cytoskeleton. Interestingly, in animals exposed to acute stress, the LTD induction by serotonin + tetani was significantly impaired. Taken together, these results suggest that serotonin, by cooperating with mGluRs, regulates synaptic plasticity through a mechanism dependent on p38 MAPK/Rab5‐mediated enhancement of AMPA receptor internalization in a clathrin/dynamin‐dependent manner. It provides a potential mechanism underlying the role of serotonin in controlling emotional and cognitive processes that are mediated by synaptic plasticity in PFC neurons.

Both serotonin and NMDA signaling in prefrontal cortex (PFC) are implicated in mental disorders, including depression and anxiety. To understand their potential contributions to PFC neuronal excitability, we examined the effect of co-activation of 5-HT and NMDA receptors on action potential firing elicited by depolarizing current injection in PFC pyramidal neurons. In the presence of NMDA, a low concentration of the 5-HT(1A) agonist 8-OH-DPAT substantially reduced the number of spikes, and a low concentration of the 5-HT(2A/C) agonist alpha-Me-5HT significantly enhanced it, while both agonists were ineffective when applied alone. The 8-OH-DPAT effect on firing was mediated by inhibition of protein kinase A (PKA), whereas the alpha-Me-5HT effect was mediated by activation of protein kinase C (PKC). Moreover, the extracellular signal-regulated kinase (ERK), a signaling molecule downstream of PKA and PKC, was involved in both 5-HT(1A) and 5-HT(2A/C) modulation of neuronal excitability. Biochemical evidence showed that 5-HT(1A) decreased, whereas 5-HT(2A/C) increased the activation of ERK in an NMDA-dependent manner. In animals exposed to acute stress, the enhancing effect of 5-HT(2A/C) on firing was lost, while the decreasing effect of 5-HT(1A) on firing was intact. Concomitantly, the effect of 5-HT(2A/C), but not 5-HT(1A), on ERK activation was abolished in stressed animals. Taken together, our results demonstrate that distinct 5-HT receptor subtypes, by interacting with NMDA receptors, differentially regulate PFC neuronal firing, and the complex effects of 5-HT receptors on excitability are selectively altered under stressful conditions, which are often associated with mental disorders.

Corticosterone, the major stress hormone, plays an important role in regulating neuronal functions of the limbic system, although the cellular targets and molecular mechanisms of corticosteroid signaling are largely unknown. Here we show that a short treatment of corticosterone significantly increases alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR)-mediated synaptic transmission and AMPAR membrane trafficking in pyramidal neurons of prefrontal cortex, a key region involved in cognition and emotion. This enhancing effect of corticosterone is through a mechanism dependent on Rab4, the small GTPase-controlling receptor recycling between early endosome and plasma membrane. Guanosine nucleotide dissociation inhibitor (GDI), which regulates the cycle of Rab proteins between membrane and cytosol, forms an increased complex with Rab4 after corticosterone treatment. Corticosterone also triggers an increased GDI phosphorylation at Ser-213 by the serum- and glucocorticoid-inducible kinase (SGK). Moreover, AMPAR synaptic currents and surface expression and their regulation by corticosterone are altered by mutating Ser-213 on GDI. These results suggest that corticosterone, via SGK phosphorylation of GDI at Ser-213, increases the formation of GDI-Rab4 complex, facilitating the functional cycle of Rab4 and Rab4-mediated recycling of AMPARs to the synaptic membrane. It provides a potential mechanism underlying the role of corticosteroid stress hormone in up-regulating excitatory synaptic efficacy in cortical neurons.

Dietary interventions are effective ways to extend or shorten lifespan. By examining midlife hepatic gene expressions in mice under different dietary conditions, which resulted in different lifespans and aging-related phenotypes, we were able to identify genes and pathways that modulate the aging process. We found that pathways transcriptionally correlated with diet-modulated lifespan and physiological changes were enriched for lifespan-modifying genes. Intriguingly, mitochondrial gene expression correlated with lifespan and anticorrelated with aging-related pathological changes, whereas peroxisomal gene expression showed an opposite trend. Both organelles produce reactive oxygen species, a proposed causative factor of aging. This finding implicates a contribution of peroxisome to aging. Consistent with this hypothesis, lowering the expression levels of peroxisome proliferation genes decreased the cellular peroxide levels and extended the lifespan of Drosophila melanogaster and Caenorhabditis elegans. These findings show that transcriptional changes resulting from dietary interventions can effectively reflect causal factors in aging and identify previously unknown or under-appreciated longevity pathways, such as the peroxisome pathway.

Effective inhibitory synaptic transmission requires efficient stabilization of GABA(A) receptors (GABA(A)Rs) at synapses, which is essential for maintaining the correct excitatory-inhibitory balance in the brain. However, the signaling mechanisms that locally regulate synaptic GABA(A)R membrane dynamics remain poorly understood. Using a combination of molecular, imaging, and electrophysiological approaches, we delineate a GIT1/βPIX/Rac1/PAK signaling pathway that modulates F-actin and is important for maintaining surface GABA(A)R levels, inhibitory synapse integrity, and synapse strength. We show that GIT1 and βPIX are required for synaptic GABA(A)R surface stability through the activity of the GTPase Rac1 and downstream effector PAK. Manipulating this pathway using RNAi, dominant-negative and pharmacological approaches leads to a disruption of GABA(A)R clustering and decrease in the strength of synaptic inhibition. Thus, the GIT1/βPIX/Rac1/PAK pathway plays a crucial role in regulating GABA(A)R synaptic stability and hence inhibitory synaptic transmission with important implications for inhibitory plasticity and information processing in the brain.

Emerging evidence suggests that metabotropic glutamate receptors (mGluRs) are potential novel targets for brain disorders associated with the dysfunction of prefrontal cortex (PFC), a region critical for cognitive and emotional processes. Because N-methyl-D-aspartic acid receptor (NMDAR) dysregulation has been strongly associated with the pathophysiology of mental illnesses, we examined the possibility that mGluRs might be involved in modulating PFC functions by targeting postsynaptic NMDARs. We found that application of prototypical group III mGluR agonists significantly reduced NMDAR-mediated synaptic and ionic currents in PFC pyramidal neurons, which was mediated by mGluR7 localized at postsynaptic neurons and involved the β-arrestin/ERK signaling pathway. The mGluR7 modulation of NMDAR currents was prevented by agents perturbing actin dynamics and by the inhibitor of cofilin, a major actin-depolymerizing factor. Consistently, biochemical and immunocytochemical results demonstrated that mGluR7 activation increased cofilin activity and F-actin depolymerization via an ERK-dependent mechanism. Furthermore, mGluR7 reduced the association of NMDARs with the scaffolding protein PSD-95 and the surface level of NMDARs in an actin-dependent manner. These data suggest that mGluR7, by affecting the cofilin/actin signaling, regulates NMDAR trafficking and function. Because ablation of mGluR7 leads to a variety of behavioral symptoms related to PFC dysfunction, such as impaired working memory and reduced anxiety and depression, our results provide a potential mechanism for understanding the role of mGluR7 in mental health and disorders.

Corticosteroid stress hormones exert a profound impact on cognitive and emotional processes. Understanding the neuronal circuits that are altered by chronic stress is important for counteracting the detrimental effects of stress in a brain region- and cell type-specific manner. Using the chemogenetic tool, Designer Receptors Exclusively Activated by Designer Drugs (DREADDs), which enables the remote, noninvasive and long-lasting modulation of cellular activity and signal transduction in discrete neuronal populations in vivo, we sought to identify the specific pathways that play an essential role in stress responses. We found that prolonged severe stress induced the diminished glutamatergic projection from pyramidal neurons in prefrontal cortex (PFC) to GABAergic interneurons in basolateral amygdala (BLA), leading to the loss of feedforward inhibition and ensuing hyperexcitability of BLA principal neurons, which caused a variety of behavioral abnormalities. Activating PFC pyramidal neurons with hM3D(Gq) DREADD restored the functional connection between PFC and BLA in stressed animals, resulting in the rescue of recognition memory, normalization of locomotor activity and reduction of aggressive behaviors. Inhibiting BLA principal neurons directly with hM4D(Gi) DREADD also blocked BLA hyperactivity and aggressive behaviors in stressed animals. These results have offered an effective avenue to counteract the stress-induced disruption of circuitry homeostasis.

Brain-derived neurotrophic factor (BDNF) is a potent regulator of neuronal activity, neurogenesis, and depressive-like behaviors; however, downstream effectors by which BDNF exerts these varying actions remain to be determined. Here we reveal that BDNF induces long-lasting enhancements in the efficacy of synaptic inhibition by stabilizing γ2 subunit-containing GABA(A) receptors (GABA(A)Rs) at the cell surface, leading to persistent reductions in neuronal excitability. This effect is dependent upon enhanced phosphorylation of tyrosines 365 and 367 (Y365/7) in the GABA(A)R γ2 subunit as revealed using mice in which these residues have been mutated to phenyalanines (Y365/7F). Heterozygotes for this mutation exhibit an antidepressant-like phenotype, as shown using behavioral-despair models of depression. In addition, heterozygous Y365/7F mice show increased levels of hippocampal neurogenesis, which has been strongly connected with antidepressant action. Both the antidepressant phenotype and the increased neurogenesis seen in these mice are insensitive to further modulation by BDNF, which produces robust antidepressant-like activity and neurogenesis in wild-type mice. Collectively, our results suggest a critical role for GABA(A)R γ2 subunit Y365/7 phosphorylation and function in regulating the effects of BDNF.

The complex pathogenesis of Alzheimer’s disease (AD) involves multiple contributing factors, including amyloid β (Aβ) peptide accumulation, inflammation and oxidative stress. Effective therapeutic strategies for AD are still urgently needed. Triptolide is the major active compound extracted from Tripterygium wilfordii Hook.f., a traditional Chinese medicinal herb that is commonly used to treat inflammatory diseases. The 5-month-old 5XFAD mice, which carry five familial AD mutations in the β-amyloid precursor protein (APP) and presenilin-1 (PS1) genes, were treated with triptolide for 8 weeks. We observed enhanced spatial learning performances, and attenuated Aβ production and deposition in the brain. Triptolide also inhibited the processing of amyloidogenic APP, as well as the expression of βAPP-cleaving enzyme-1 (BACE1) both in vivo and in vitro. In addition, triptolide exerted anti-inflammatory and anti-oxidative effects on the transgenic mouse brain. Triptolide therefore confers protection against the effects of AD in our mouse model and is emerging as a promising therapeutic candidate drug for AD.

Stress and the major stress hormone corticosterone induce profound influences in the brain. Altered histone modification and transcriptional dysfunction have been implicated in stress-related mental disorders. We previously found that repeated stress caused an impairment of prefrontal cortex (PFC)-mediated cognitive functions by increasing the ubiquitination and degradation of AMPA-type glutamate receptors via a mechanism depending on the E3 ubiquitin ligase Nedd4. Here, we demonstrated that in PFC of repeatedly stressed rats, active glucocorticoid receptor had the increased binding to the glucocorticoid response element of histone deacetylase 2 (HDAC2) promoter, resulting in the upregulation of HDAC2. Inhibition or knock-down of HDAC2 blocked the stress-induced impairment of synaptic transmission, AMPAR expression, and recognition memory. Furthermore, we found that, in stressed animals, the HDAC2-dependent downregulation of histone methyltransferase Ehmt2 (G9a) led to the loss of repressive histone methylation at the Nedd4-1 promoter and the transcriptional activation of Nedd4. These results have provided an epigenetic mechanism and a potential treatment strategy for the detrimental effects of chronic stress.Prolonged stress exposure can induce altered histone modification and transcriptional dysfunction, which may underlie the profound influence of stress in regulating brain functions. We report an important finding about the epigenetic mechanism controlling the detrimental effects of repeated stress on synaptic transmission and cognitive function. First, it has revealed the stress-induced alteration of key epigenetic regulators HDAC2 and Ehmt2, which determines the synaptic and behavioral effects of repeated stress. Second, it has uncovered the stress-induced histone modification of the target gene Nedd4, an E3 ligase that is critically involved in the ubiquitination and degradation of AMPA receptors and cognition. Third, it has provided the epigenetic approach, HDAC2 inhibition or knock-down, to rescue synaptic and cognitive functions in stressed animals.

Spontaneously hypertensive rats (SHR) are the most widely used animal model for the study of attention deficit hyperactivity disorder (ADHD). Here we sought to reveal the neuronal circuits and molecular basis of ADHD and its potential treatment using SHR. Combined electrophysiological, biochemical, pharmacological, chemicogenetic, and behavioral approaches were utilized. We found that AMPAR-mediated synaptic transmission in pyramidal neurons of prefrontal cortex (PFC) was diminished in SHR, which was correlated with the decreased surface expression of AMPAR subunits. Administration of methylphenidate (a psychostimulant drug used to treat ADHD), which blocks dopamine transporters and norepinephrine transporters, ameliorated the behavioral deficits of adolescent SHR and restored AMPAR-mediated synaptic function. Activation of PFC pyramidal neurons with a CaMKII-driven Gq-coupled designer receptor exclusively activated by designer drug also led to the elevation of AMPAR function and the normalization of ADHD-like behaviors in SHR. These results suggest that the disrupted function of AMPARs in PFC may underlie the behavioral deficits in adolescent SHR and enhancing PFC activity could be a treatment strategy for ADHD.

MMP13 (matrix metallopeptidase 13) plays a key role in bone metabolism and cancer development, but has no known functions in Alzheimer's disease. In this study, we used high-throughput small molecule screening in SH-SY5Y cells that stably expressed a luciferase reporter gene driven by the BACE1 (β-site amyloid precursor protein cleaving enzyme 1) promoter, which included a portion of the 5' untranslated region (5'UTR). We identified that CL82198, a selective inhibitor of MMP13, decreased BACE1 protein levels in cultured neuronal cells. This effect was dependent on PI3K (phosphatidylinositide 3-kinase) signalling, and was unrelated to BACE1 gene transcription and protein degradation. Further, we found that eukaryotic translation initiation factor 4B (eIF4B) played a key role, as the mutation of eIF4B at serine 422 (S422R) or deletion of the BACE1 5'UTR attenuated MMP13-mediated BACE1 regulation. In APPswe/PS1E9 mice, an animal model of Alzheimer's disease, hippocampal Mmp13 knockdown or intraperitoneal CL82198 administration reduced BACE1 protein levels and the related amyloid-β precursor protein processing, amyloid-β load and eIF4B phosphorylation, whereas spatial and associative learning and memory performances were improved. Collectively, MMP13 inhibition/CL82198 treatment exhibited therapeutic potential for Alzheimer's disease, via the translational regulation of BACE1.

Autism is a neurodevelopmental disorder characterized by social deficits and repetitive behaviors. Genetic screening has identified synaptic, transcriptional, and chromatin genes disrupted in autistic patients. Haploinsufficiency of Shank3, which encodes a scaffold protein at glutamatergic synapses, is causally linked to autism. Using a Shank3-deficient mouse model that exhibits prominent autism-like phenotypes, we have found that histone acetylation in the prefrontal cortex (PFC) is abnormally low, which can be reversed by MS-275 (also known as Entinostat, SNDX-275), a class I histone deacetylase (HDAC) inhibitor that is selectively potent in PFC. A brief (3-day) treatment with MS-275 (i.p.) led to the sustained (11 days) rescue of autistic social preference deficits in Shank3-deficient mice, without altering locomotion, motor coordination, anxiety, or the increased grooming. MS-275 treatment also rescued the diminished NMDAR surface expression and NMDAR function induced by Shank3 deficiency. Moreover, F-actin at synapses was restored and the transcription of actin regulators was elevated by MS-275 treatment of Shank3-deficient mice, which may contribute to the recovery of actin-based NMDAR synaptic delivery. Taken together, these results suggest that MS-275 treatment could normalize the aberrant epigenetic regulation of genes, leading to the amelioration of synaptic and social deficits associated with autism.

Estrogen-related receptor alpha (ERRα) is a transcriptional factor associated with mitochondrial biogenesis and energy metabolism. However, little is known about the role of ERRα in Alzheimer's disease (AD). Here, we report that in APP/PS1 mice, an animal model of AD, ERRα protein and mRNA were decreased in a region- and age-dependent manner. In HEK293 cells that stably express human full-length β-amyloid precursor protein (APP), overexpression of ERRα inhibited the amyloidogenic processing of APP and consequently reduced Aβ1-40/1-42 level. ERRα overexpression also attenuated Tau phosphorylation at selective sites, with the concomitant reduction of glycogen synthase kinase 3β (GSK3β) activity. Interestingly, alterations of APP processing and Tau phosphorylation induced by hydrogen peroxide were reversed by ERRα overexpression in HEK/APP cells. These results indicated that ERRα plays a functional role in AD pathology. By attenuating both amyloidogenesis and Tau phosphorylation, ERRα may serve as a potential therapeutic target for AD.

IntroductionDamage-associated molecular patterns, such as high-mobility group box 1 (HMGB1) and cell-free DNA (cfDNA), play critical roles in mediating ischemia-reperfusion injury (IRI). HMGB1 activates RAGE to exacerbate IRI, but the mechanism underlying cfDNA-induced myocardial IRI remains unknown. We hypothesized that the infarct-exacerbating effect of cfDNA is mediated by HMGB1 and receptor for advanced glycation end products (RAGE).MethodsC57BL/6 wild type mice, RAGE knockout (KO), and Toll-like receptor 9 KO mice underwent 20- or 40-minute occlusions of the left coronary artery followed by up to 60 minutes of reperfusion. Cardiac coronary perfusate was acquired from ischemic hearts without reperfusion. Exogenous mitochondrial DNA was acquired from livers of normal C57BL/6 mice. Myocardial infarct size (IS) was reported as percent risk region, as measured by 2,3,5-triphenyltetrazolium chloride and Phthalo blue (Heucotech, Fairless Hill, Pa) staining. cfDNA levels were measured by Sytox Green assay (Thermo Fisher Scientific, Waltham, Mass) and/or spectrophotometer.ResultsFree HMGB1 and cfDNA levels were increased in the ischemic myocardium during prolonged ischemia and subsequently in the plasma during reperfusion. In C57BL/6 mice undergoing 40′/60′ IRI, deoxyribonuclease I, or anti-HMGB1 monoclonal antibody reduced IS by approximately half to 29.0% ± 5.2% and 24.3% ± 3.5% (P < .05 vs control 54.3% ± 3.4%). However, combined treatment with deoxyribonuclease I + anti-HMGB1 monoclonal antibody did not further attenuate IS (29.3% ± 4.9%). In C57BL/6 mice undergoing 20′/60′ IRI, injection of 40′/5′ plasma upon reperfusion increased IS by more than 3-fold (to 19.9 ± 4.3; P < .05). This IS exacerbation was abolished by pretreating the plasma with deoxyribonuclease I or by depleting the HMGB1 by immunoprecipitation, or by splenectomy. The infarct-exacerbating effect also disappeared in RAGE KO mice and Toll-like receptor 9 KO mice. Injection of 40′/0′ coronary perfusate upon reperfusion similarly increased IS. The levels of HMGB1 and cfDNA were significantly elevated in the 40′/0′ coronary perfusate and 40′/reperfusion (min) plasma but not in those with 10′ ischemia. In C57BL/6 mice without IRI, 40′/5′ plasma significantly increased the interleukin-1β protein and messenger RNA expression in the spleen by 30 minutes after injection. Intravenous bolus injection of recombinant HMGB1 (0.1 μg/g) or mitochondrial DNA (0.5 μg/g) 5 minutes before reperfusion did not exacerbate IS (P = not significant vs control). However, combined administration of recombinant HMGB1 + mitochondrial DNA significantly increased IS (P < .05 vs individual treated groups) and this infarct-exacerbating effect disappeared in RAGE KO mice and splenectomized C57BL/6 mice. The accumulation of cfDNA in the spleen after combined recombinant HMGB1 + mitochondrial DNA treatment was significantly more elevated in C57BL/6 mice than in RAGE KO mice.Conclusions:Both HMGB1 and cfDNA are released from the heart upon reperfusion after prolonged ischemia and both contribute importantly and interdependently to post-IRI by a common RAGE-Toll-like receptor 9–dependent mechanism. Depleting either of these 2 damage-associated molecular patterns suffices to significantly reduce IS by approximately 50%.

The human 16p11.2 gene locus is a hot spot for copy number variations, which predispose carriers to a range of neuropsychiatric phenotypes. Microduplications of 16p11.2 are associated with autism spectrum disorder (ASD), intellectual disability (ID), and schizophrenia (SZ). Despite the debilitating nature of 16p11.2 duplications, the underlying molecular mechanisms remain poorly understood. Here we performed a comprehensive behavioral characterization of 16p11.2 duplication mice (16p11.2dp/+) and identified social and cognitive deficits reminiscent of ASD and ID phenotypes. 16p11.2dp/+ mice did not exhibit the SZ-related sensorimotor gating deficits, psychostimulant-induced hypersensitivity, or motor impairment. Electrophysiological recordings of 16p11.2dp/+ mice found deficient GABAergic synaptic transmission and elevated neuronal excitability in the prefrontal cortex (PFC), a brain region critical for social and cognitive functions. RNA-sequencing identified genome-wide transcriptional aberrance in the PFC of 16p11.2dp/+ mice, including downregulation of the GABA synapse regulator Npas4. Restoring Npas4 expression in PFC of 16p11.2dp/+ mice ameliorated the social and cognitive deficits and reversed GABAergic synaptic impairment and neuronal hyperexcitability. These findings suggest that prefrontal cortical GABAergic synaptic circuitry and Npas4 are strongly implicated in 16p11.2 duplication pathology, and may represent potential targets for therapeutic intervention in ASD.

Large-scale genetic screening has identified KMT5B (SUV420H1), which encodes a histone H4 K20 di- and tri-methyltransferase highly expressed in prefrontal cortex (PFC), as a top-ranking high-risk gene for autism. However, the biological function of KMT5B in the brain is poorly characterized, and how KMT5B deficiency is linked to autism remains largely unknown. Here we knocked down Kmt5b in PFC and examined behavioral and electrophysiological changes, as well as underlying molecular mechanisms. Mice with Kmt5b deficiency in PFC display social deficits, a core symptom of autism, without the alteration of other behaviors. Kmt5b deficiency also produces deficits in PFC glutamatergic synaptic transmission, which is accompanied by the reduced synaptic expression of glutamate receptor subunits and associated proteins. Kmt5b deficiency-induced reduction of H4K20me2 impairs 53BP1-mediated DNA repair, leading to the elevation of p53 expression and its target gene Ddit4 (Redd1), which is implicated in synaptic impairment. RNA-sequencing data indicate that Kmt5b deficiency results in the upregulation of genes enriched in cellular stress response and ubiquitin-dependent protein degradation. Collectively, this study has revealed the functional role of Kmt5b in the PFC, and suggests that Kmt5b deficiency could cause autistic phenotypes by inducing synaptic dysfunction and transcriptional aberration.

Alzheimer's disease is a progressive neurodegenerative disorder associated with memory loss and impaired executive function. The molecular underpinnings causing cognitive deficits in Alzheimer's disease are loosely understood. Here, we performed cross-study large-scale transcriptomic analyses of postmortem prefrontal cortex derived from Alzheimer's disease patients to reveal the role of aberrant gene expression in this disease. We identified that one of the most prominent changes in prefrontal cortex of Alzheimer's disease humans was the downregulation of genes in excitatory and inhibitory neurons that are associated with synaptic functions, particularly the SNARE-binding complex, which is essential for vesicle docking and neurotransmitter release. Comparing genomic data of Alzheimer's disease with proteomic data of cognitive trajectory, we found that many of the lost synaptic genes in Alzheimer's disease encode hub proteins whose increased abundance is required for cognitive stability. This study has revealed potential molecular targets for therapeutic intervention of cognitive decline associated with Alzheimer's disease.

Abstract Dwarfism is an agronomic trait that has substantial effects on crop yield, lodging resistance, planting density, and a high harvest index. Ethylene plays an important role in plant growth and development, including the determination of plant height. However, the mechanism by which ethylene regulates plant height, especially in woody plants, remains unclear. In this study, a 1-aminocyclopropane-1-carboxylic acid synthase (ACC) gene (ACS), which is involved in ethylene biosynthesis, was isolated from lemon (Citrus limon L. Burm) and named CiACS4. Overexpression of CiACS4 resulted in a dwarf phenotype in Nicotiana tabacum and lemon and increased ethylene release and decreased gibberellin (GA) content in transgenic plants. Inhibition of CiACS4 expression in transgenic citrus significantly increased plant height compared with the controls. Yeast two-hybrid assays revealed that CiACS4 interacted with an ethylene response factor (ERF), CiERF3. Further experiments revealed that the CiACS4–CiERF3 complex can bind to the promoters of 2 citrus GA20-oxidase genes, CiGA20ox1 and CiGA20ox2, and suppress their expression. In addition, another ERF transcription factor, CiERF023, identified using yeast one-hybrid assays, promoted CiACS4 expression by binding to its promoter. Overexpression of CiERF023 in N. tabacum caused a dwarfing phenotype. CiACS4, CiERF3, and CiERF023 expression was inhibited and induced by GA3 and ACC treatments, respectively. These results suggest that the CiACS4–CiERF3 complex may be involved in the regulation of plant height by regulating CiGA20ox1 and CiGA20ox2 expression levels in citrus.

Adverse experiences in early life can induce maladaptive responses to acute stress in later life. Chronic social isolation during adolescence is an early life adversity that can precipitate stress-related psychiatric disorders. We found that male mice after 8 weeks of adolescent social isolation (SI) have markedly increased aggression after being exposed to 2 h of restraint stress (RS), which was accompanied by a significant increase of AMPA receptor- and NMDA receptor-mediated synaptic transmission in prefrontal cortex (PFC) pyramidal neurons of SIRS males. Compared to group-housed counterparts, SIRS males exhibited a significantly decreased level of histone H3 acetylation in PFC. Systemic administration of class I histone deacetylase inhibitors, romidepsin or MS-275, ameliorated the aggressive behaviour, as well as general social interaction deficits, of SIRS males. Electrophysiological recordings also found normalization of PFC glutamatergic currents by romidepsin treatment of SIRS male mice. These results revealed an epigenetic mechanism and intervention avenue for aggression induced by chronic social isolation. KEY POINTS: Adolescent chronic social isolation can precipitate stress-related psychiatric disorders. A significant increase of glutamatergic transmission is found in the prefrontal cortex (PFC) of socially isolated male mice exposed to an acute stress (SIRS ). Treatment with class I histone deacetylase (HDAC) inhibitors ameliorates the aggressive behaviour and social interaction deficits of SIRS males, and normalizes glutamatergic currents in PFC neurons. It provides an epigenetic mechanism and intervention avenue for aberrant stress responses induced by chronic social isolation.