Astrocytic PAR1 and mGluR2/3 control synaptic glutamate time course at hippocampal CA1 synapses.

Astrocytes play an essential role in regulating synaptic transmission. This study describes a novel form of modulation of excitatory synaptic transmission in the mouse hippocampus by astrocytic G-protein-coupled receptors (GPCRs). We have previously described astrocytic glutamate release via protease-activated receptor-1 (PAR1) activation, although the regulatory mechanisms for this are complex. Through electrophysiological analysis and modeling, we discovered that PAR1 activation consistently increases the concentration and duration of glutamate in the synaptic cleft. This effect was not due to changes in the presynaptic glutamate release or alteration in glutamate transporter expression. However, blocking group II metabotropic glutamate receptors (mGluR2/3) abolished PAR1-mediated regulation of synaptic glutamate concentration, suggesting a role for this GPCR in mediating the effects of PAR1 activation on glutamate release. Furthermore, activation of mGluR2/3 causes glutamate release through the TREK-1 channel in hippocampal astrocytes. These data show that astrocytic GPCRs engage in a novel regulatory mechanism to shape the time course of synaptically-released glutamate in excitatory synapses of the hippocampus.

GluN2D subunit-containing NMDA receptors regulate reticular thalamic neuron function and seizure susceptibility.

Thalamic regulation of cortical function is important for several behavioral aspects including attention and sensorimotor control. This region has also been studied for its involvement in seizure activity. Among the NMDA receptor subunits GluN2C and GluN2D are particularly enriched in several thalamic nuclei including nucleus reticularis of the thalamus (nRT). We have previously found that GluN2C deletion does not have a strong influence on the basal excitability and burst firing characteristics of reticular thalamus neurons. Here we find that GluN2D ablation leads to reduced depolarization-induced spike frequency and reduced hyperpolarization-induced rebound burst firing in nRT neurons. Furthermore, reduced inhibitory neurotransmission was observed in the ventrobasal thalamus (VB). A model with preferential downregulation of GluN2D from parvalbumin (PV)-positive neurons was generated. Conditional deletion of GluN2D from PV neurons led to a decrease in excitability and burst firing. In addition, reduced excitability and burst firing was observed in the VB neurons together with reduced inhibitory neurotransmission. Finally, young mice with GluN2D downregulation in PV neurons showed significant resistance to pentylenetetrazol-induced seizure and differences in sensitivity to isoflurane anesthesia but were normal in other behaviors. Conditional deletion of GluN2D from PV neurons also affected expression of other GluN2 subunits and GABA receptor in the nRT. Together, these results identify a unique role of GluN2D-containing receptors in the regulation of thalamic circuitry and seizure susceptibility which is relevant to mutations in GRIN2D gene found to be associated with pediatric epilepsy.

Glutamate delta 1 receptor regulates autophagy mechanisms and affects excitatory synapse maturation in the somatosensory cortex.

The glutamate delta family of receptors is composed of GluD1 and GluD2 and serve as synaptic organizers. We have previously demonstrated several autism-like molecular and behavioral phenotypes including an increase in dendritic spines in GluD1 knockout mice. Based on previous reports we evaluated whether disruption of autophagy mechanisms may account for these phenotypes. Mouse model with conditional deletion of GluD1 from excitatory neurons in the corticolimbic regions was utilized. GluD1 loss led to overactive Akt-mTOR pathway, higher p62 and a lower LC3-II/LC3-I ratio in the somatosensory cortex suggesting reduced autophagy. Excitatory elements were increased in number but had immature phenotype based on puncta size, lower AMPA subunit GluA1 expression and impaired development switch from predominantly GluN2B to mixed GluN2A/GluN2B subunit expression. Overactive Akt-mTOR signaling and impaired autophagy was also observed in dorsal striatum upon conditional ablation of GluD1 and in the prefrontal cortex and hippocampus in constitutive knockout. Finally, cognitive deficits in novel object recognition test and fear conditioning were observed in mice with conditional ablation of GluD1 from the corticolimbic regions. Together, these results demonstrate a novel function of GluD1 in the regulation of autophagy pathway which may underlie autism phenotypes and is relevant to the genetic association of GluD1 coding, GRID1 gene with autism and other developmental disorders.

Stimulation of Neurite Outgrowth in Cerebrocortical Neurons by Sodium Channel Activator Brevetoxin-2 Requires Both N-Methyl-D-aspartate Receptor 2B (GluN2B) and p21 Protein (Cdc42/Rac)-Activated Kinase 1 (PAK1).

N-methyl-D-aspartate (NMDA) receptors play a critical role in activity-dependent dendritic arborization, spinogenesis, and synapse formation by stimulating calcium-dependent signaling pathways. Previously, we have shown that brevetoxin 2 (PbTx-2), a voltage-gated sodium channel (VGSC) activator, produces a concentration-dependent increase in intracellular sodium [Na+]I and increases NMDA receptor (NMDAR) open probabilities and NMDA-induced calcium (Ca2+) influxes. The objective of this study is to elucidate the downstream signaling mechanisms by which the sodium channel activator PbTx-2 influences neuronal morphology in murine cerebrocortical neurons. PbTx-2 and NMDA triggered distinct Ca2+-influx pathways, both of which involved the NMDA receptor 2B (GluN2B). PbTx-2-induced neurite outgrowth in day in vitro 1 (DIV-1) neurons required the small Rho GTPase Rac1 and was inhibited by both a PAK1 inhibitor and a PAK1 siRNA. PbTx-2 exposure increased the phosphorylation of PAK1 at Thr-212. At DIV-5, PbTx-2 induced increases in dendritic protrusion density, p-cofilin levels, and F-actin throughout the dendritic arbor and soma. Moreover, PbTx-2 increased miniature excitatory post-synaptic currents (mEPSCs). These data suggest that the stimulation of neurite outgrowth, spinogenesis, and synapse formation produced by PbTx-2 are mediated by GluN2B and PAK1 signaling.

Astrocytic NMDA Receptors in the Basolateral Amygdala Contribute to Facilitation of Fear Extinction.

BACKGROUND: Enhancement of N-methyl-D-aspartate (NMDA) receptor function using glycine-site agonist D-cycloserine is known to facilitate fear extinction, providing a means to augment cognitive behavioral therapy in anxiety disorders. A novel class of glycine-site agonists has recently been identified, and we have found that the prototype, AICP, is more effective than D-cycloserine in modulating neuronal function. METHODS: Using novel glycine-site agonist AICP, local infusion studies, and genetic models, we elucidated the role of GluN2C-containing receptors in fear extinction. RESULTS: We tested the effect of intracerebroventricular injection of AICP on fear extinction and found a robust facilitation of fear extinction. This effect was dependent on GluN2C subunit, consistent with superagonist action of AICP at GluN2C-containing receptors. Local infusion studies in wild-type and GluN2C knockout mice suggested that AICP produces its effect via GluN2C-containing receptors in the basolateral amygdala (BLA). Furthermore, consistent with astrocytic expression of GluN2C subunit in the amygdala, we found that AICP did not facilitate fear extinction in mice with conditional deletion of obligatory GluN1 subunit from astrocytes. Importantly, chemogenetic activation of astrocytes in the basolateral amygdala facilitated fear extinction. Acutely, AICP was found to facilitate excitatory neurotransmission in the BLA via presynaptic GluN2C-dependent mechanism. Immunohistochemical studies suggest that AICP-mediated facilitation of fear extinction involves synaptic insertion of α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptor GluA1 subunit. CONCLUSION: These results identify a unique role of astrocytic NMDA receptors composed of GluN2C subunit in extinction of conditioned fear memory and demonstrate that further development of recently identified superagonists of GluN2C-containing receptors may have utility for anxiety disorders.

Striatal glutamate delta-1 receptor regulates behavioral flexibility and thalamostriatal connectivity.

Impaired behavioral flexibility and repetitive behavior is a common phenotype in autism and other neuropsychiatric disorders, but the underlying synaptic mechanisms are poorly understood. The trans-synaptic glutamate delta (GluD)-Cerebellin 1-Neurexin complex, critical for synapse formation/maintenance, represents a vulnerable axis for neuropsychiatric diseases. We have previously found that GluD1 deletion results in reversal learning deficit and repetitive behavior. In this study, we show that selective ablation of GluD1 from the dorsal striatum impairs behavioral flexibility in a water T-maze task. We further found that striatal GluD1 is preferentially found in dendritic shafts, and more frequently associated with thalamic than cortical glutamatergic terminals suggesting localization to projections from the thalamic parafascicular nucleus (Pf). Conditional deletion of GluD1 from the striatum led to a selective loss of thalamic, but not cortical, terminals, and reduced glutamatergic neurotransmission. Optogenetic studies demonstrated functional changes at thalamostriatal synapses from the Pf, but no effect on the corticostriatal system, upon ablation of GluD1 in the dorsal striatum. These studies suggest a novel molecular mechanism by which genetic variations associated with neuropsychiatric disorders may impair behavioral flexibility, and reveal a unique principle by which GluD1 subunit regulates forebrain circuits.

Antipsychotic-like profile of CIQ isomers in animal models of schizophrenia.

Earlier, we have shown the efficacy of racemic (±) CIQ, a positive allosteric modulator of GluN2C/2D receptor against MK-801 induced impairment of prepulse inhibition as well as working memory. The present study investigated the antipsychotic-like profile of different CIQ (±, +, -) isomers against schizophrenia-like symptoms in series of behavioural animal models like apomorphine climbing, social isolation behaviour and NMDA receptor antagonist MK-801 induced cognitive deficits. Further, we also tested CIQ (±, +, -) isomers in neurodevelopmental model against MK-801induced deficits using open field test, Y-maze test and novel object recognition test. CIQ (±, +, -) isomers decreased climbing behaviour, increased social interaction and improved the MK-801 induced deficits in working memory in Y-maze. Further, CIQ (±, +) but not CIQ (-) improved the recognition memory in novel object recognition test as well as reduced hyperlocomotion and stereotyped behaviour. We conclude that CIQ (±, +) but not CIQ (-) exhibit the significant antipsychotic-like profile.

A mouse model of seizures in anti-N-methyl-d-aspartate receptor encephalitis.

OBJECTIVE: Seizures develop in 80% of patients with anti-N-methyl-d-aspartate receptor (NMDAR) encephalitis, and these represent a major cause of morbidity and mortality. Anti-NMDAR antibodies have been linked to memory loss in encephalitis; however, their role in seizures has not been established. We determined whether anti-NMDAR antibodies from autoimmune encephalitis patients are pathogenic for seizures. METHODS: We performed continuous intracerebroventricular infusion of cerebrospinal fluid (CSF) or purified immunoglobulin (IgG) from the CSF of patients with anti-NMDAR encephalitis or polyclonal rabbit anti-NMDAR IgG, in male C57BL/6 mice. Seizure status during a 2-week treatment was assessed with video-electroencephalography. We assessed memory, anxiety-related behavior, and motor function at the end of treatment and assessed the extent of neuronal damage and gliosis in the CA1 region of hippocampus. We also performed whole-cell patch recordings from the CA1 pyramidal neurons in hippocampal slices of mice with seizures. RESULTS: Prolonged exposure to rabbit anti-NMDAR IgG, patient CSF, or human IgG purified from the CSF of patients with encephalitis induced seizures in 33 of 36 mice. The median number of seizures recorded in 2 weeks was 13, 39, and 35 per mouse in these groups, respectively. We observed only 18 brief nonconvulsive seizures in 11 of 29 control mice (median seizure count of 0) infused with vehicle (n = 4), normal CSF obtained from patients with noninflammatory central nervous system (CNS) conditions (n = 12), polyclonal rabbit IgG (n = 7), albumin (n = 3), and normal human IgG (n = 3). We did not observe memory deficits, anxiety-related behavior, or motor impairment measured at 2 weeks in animals treated with CSF from affected patients or rabbit IgG. Furthermore, there was no evidence of hippocampal cell loss or astrocyte proliferation in the same mice. SIGNIFICANCE: Our findings indicate that autoantibodies can induce seizures in anti-NMDAR encephalitis and offer a model for testing novel therapies for refractory autoimmune seizures.

In the Telencephalon, GluN2C NMDA Receptor Subunit mRNA is Predominately Expressed in Glial Cells and GluN2D mRNA in Interneurons.

N-methyl-D-aspartate receptors (NMDARs) are widely distributed in the brain with high concentrations in the telencephalon where they modulate synaptic plasticity, working memory, and other functions. While the actions of the predominate GluN2 NMDAR subunits, GluN2A and GluN2B are relatively well understood, the function of GluN2C and GluN2D subunits in the telencephalon is largely unknown. To better understand the possible role of GluN2C subunits, we used fluorescence in situ hybridization (FISH) together with multiple cell markers to define the distribution and type of cells expressing GluN2C mRNA. Using a GluN2C-KO mouse as a negative control, GluN2C mRNA expression was only found in non-neuronal cells (NeuN-negative cells) in the hippocampus, striatum, amygdala, and cerebral cortex. For these regions, a significant fraction of GFAP-positive cells also expressed GluN2C mRNA. Overall, for the telencephalon, the globus pallidus and olfactory bulb were the only regions where GluN2C was expressed in neurons. In contrast to GluN2C, GluN2D subunit mRNA colocalized with neuronal and not astrocyte markers or GluN2C mRNA in the telencephalon (except for the globus pallidus). GluN2C mRNA did, however, colocalize with GluN2D in the thalamus where neuronal GluN2C expression is found. These findings strongly suggest that GluN2C has a very distinct function in the telencephalon compared to its role in other brain regions and compared to other GluN2-containing NMDARs. NMDARs containing GluN2C may have a specific role in regulating L-glutamate or D-serine release from astrocytes in response to L-glutamate spillover from synaptic activity.

Essential Roles for ARID1B in Dendritic Arborization and Spine Morphology of Developing Pyramidal Neurons.

De novo truncating mutations in ARID1B, a chromatin-remodeling gene, cause Coffin-Siris syndrome, a developmental disorder characterized by intellectual disability and speech impairment; however, how the genetic elimination leads to cognitive dysfunction remains unknown. Thus, we investigated the neural functions of ARID1B during brain development. Here, we show that ARID1B regulates dendritic differentiation in the developing mouse brain. We knocked down ARID1B expression in mouse pyramidal neurons using in utero gene delivery methodologies. ARID1B knockdown suppressed dendritic arborization of cortical and hippocampal pyramidal neurons in mice. The abnormal development of dendrites accompanied a decrease in dendritic outgrowth into layer I. Furthermore, knockdown of ARID1B resulted in aberrant dendritic spines and synaptic transmission. Finally, ARID1B deficiency led to altered expression of c-Fos and Arc, and overexpression of these factors rescued abnormal differentiation induced by ARID1B knockdown. Our results demonstrate a novel role for ARID1B in neuronal differentiation and provide new insights into the origin of cognitive dysfunction associated with developmental intellectual disability. SIGNIFICANCE STATEMENT: Haploinsufficiency of ARID1B, a component of chromatin remodeling complex, causes intellectual disability. However, the role of ARID1B in brain development is unknown. Here, we demonstrate that ARID1B is required for neuronal differentiation in the developing brain, such as in dendritic arborization and synapse formation. Our findings suggest that ARID1B plays a critical role in the establishment of cognitive circuitry by regulating dendritic complexity. Thus, ARID1B deficiency may cause intellectual disability via abnormal brain wiring induced by the defective differentiation of cortical neurons.

Glutamate Delta-1 Receptor Regulates Metabotropic Glutamate Receptor 5 Signaling in the Hippocampus.

The delta family of ionotropic glutamate receptors consists of glutamate delta-1 (GluD1) and glutamate delta-2 receptors. We have previously shown that GluD1 knockout mice exhibit features of developmental delay, including impaired spine pruning and switch in the N-methyl-D-aspartate receptor subunit, which are relevant to autism and other neurodevelopmental disorders. Here, we identified a novel role of GluD1 in regulating metabotropic glutamate receptor 5 (mGlu5) signaling in the hippocampus. Immunohistochemical analysis demonstrated colocalization of mGlu5 with GluD1 punctas in the hippocampus. Additionally, GluD1 protein coimmunoprecipitated with mGlu5 in the hippocampal membrane fraction, as well as when overexpressed in human embryonic kidney 293 cells, demonstrating that GluD1 and mGlu5 may cooperate in a signaling complex. The interaction of mGlu5 with scaffold protein effector Homer, which regulates mechanistic target of rapamycin (mTOR) signaling, was abnormal both under basal conditions and in response to mGlu1/5 agonist (RS)-3,5-dihydroxyphenylglycine (DHPG) in GluD1 knockout mice. The basal levels of phosphorylated mTOR and protein kinase B, the signaling proteins downstream of mGlu5 activation, were higher in GluD1 knockout mice, and no further increase was induced by DHPG. We also observed higher basal protein translation and an absence of DHPG-induced increase in GluD1 knockout mice. In accordance with a role of mGlu5-mediated mTOR signaling in synaptic plasticity, DHPG-induced internalization of surface α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor subunits was impaired in the GluD1 knockout mice. These results demonstrate that GluD1 interacts with mGlu5, and loss of GluD1 impairs normal mGlu5 signaling potentially by dysregulating coupling to its effector. These studies identify a novel role of the enigmatic GluD1 subunit in hippocampal function.

Pharmacology and Structural Analysis of Ligand Binding to the Orthosteric Site of Glutamate-Like GluD2 Receptors.

The GluD2 receptor is a fundamental component of postsynaptic sites in Purkinje neurons, and is required for normal cerebellar function. GluD2 and the closely related GluD1 are classified as members of the ionotropic glutamate receptor (iGluR) superfamily on the basis of sequence similarity, but do not bind l-glutamate. The amino acid neurotransmitter D-Ser is a GluD2 receptor ligand, and endogenous D-Ser signaling through GluD2 has recently been shown to regulate endocytosis of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid-type iGluRs during synaptic plasticity in the cerebellum, such as long-term depression. Here, we investigate the pharmacology of the orthosteric binding site in GluD2 by examining the activity of analogs of D-Ser and GluN1 glycine site competitive antagonists at GluD2 receptors containing the lurcher mutation (GluD2(LC)), which promotes spontaneous channel activation. We identify several compounds that modulate GluD2(LC), including a halogenated alanine analog as well as the kynurenic acid analog 7-chloro-4-oxo-1H-quinoline-2-carboxylic acid (7-chlorokynurenic acid; 7-CKA). By correlating thermodynamic and structural data for 7-CKA binding to the isolated GluD2 ligand binding domain (GluD2-LBD), we find that binding 7-CKA to GluD2-LBD differs from D-Ser by inducing an intermediate cleft closure of the clamshell-shaped LBD. The GluD2 ligands identified here can potentially serve as a starting point for development of GluD2-selective ligands useful as tools in studies of the signaling role of the GluD2 receptor in the brain.

Bidirectional Effect of Pregnenolone Sulfate on GluN1/GluN2A N-Methyl-D-Aspartate Receptor Gating Depending on Extracellular Calcium and Intracellular Milieu.

Pregnenolone sulfate (PS), one of the most commonly occurring neurosteroids in the central nervous system, influences the function of several receptors. PS modulates N-methyl-D-aspartate receptors (NMDARs) and has been shown to have both positive and negative modulatory effects on NMDAR currents generally in a subtype-selective manner. We assessed the gating mechanism of PS modulation of GluN1/GluN2A receptors transiently expressed in human embryonic kidney 293 cells using whole-cell and single-channel electrophysiology. Only a modest effect on the whole-cell responses was observed by PS in dialyzed (nonperforated) whole-cell recordings. Interestingly, in perforated conditions, PS was found to increase the whole-cell currents in the absence of nominal extracellular Ca(2+), whereas PS produced an inhibition of the current responses in the presence of 0.5 mM extracellular Ca(2+). The Ca(2+)-binding DRPEER motif and GluN1 exon-5 were found to be critical for the Ca(2+)-dependent bidirectional effect of PS. Single-channel cell-attached analysis demonstrated that PS primarily affected the mean open time to produce its effects: positive modulation mediated by an increase in duration of open time constants, and negative modulation mediated by a reduction in the time spent in a long-lived open state of the receptor. Further kinetic modeling of the single-channel data suggested that the positive and negative modulatory effects are mediated by different gating steps which may represent GluN2 and GluN1 subunit-selective conformational changes, respectively. Our studies provide a unique mechanism of modulation of NMDARs by an endogenous neurosteroid, which has implications for identifying state-dependent molecules.

Intravenous recombinant cerebellin 1 treatment restores signalling by spinal glutamate delta 1 receptors and mitigates chronic pain.

BACKGROUND AND PURPOSE: Chronic pain remains a major clinical problem that needs effective therapeutic agents. Glutamate delta 1 (GluD1) receptors and the protein cerebellin 1 (Cbln1) are down-regulated in the central amygdala (CeA) in models of inflammatory and neuropathic pain. One treatment with Cbln1, intracerebroventricularly (ICV) or in CeA, normalized GluD1 and reduced AMPA receptor expression, resulting in lasting (7-10 days) pain relief. Unlike many CNS-targeting biological agents, the structure of Cbln1 suggests potential blood-brain barrier penetration. Here, we have tested whether systemic administration of Cbln1 provides analgesic effects via action in the CNS. EXPERIMENTAL APPROACH: Analgesic effects of intravenous recombinant Cbln1 was assessed in complete Freund's adjuvant inflammatory pain model in mice. GluD1 knockout and a mutant form of Cbln1 were used. KEY RESULTS: A single intravenous injection of Cbln1 mitigated nocifensive and averse behaviour in both inflammatory and neuropathic pain models. This effect of Cbln1 was dependent on GluD1 receptors and required binding to the amino terminal domain of GluD1. Time course of analgesic effect was similar to previously reported ICV and intra-CeA injection. GluD1 in both spinal cord and CeA was down -regulated in the inflammatory pain model, whereas GluD1 expression in spinal cord but not in CeA, was partly normalized by intravenous Cbln1. Importantly, recombinant Cbln1 was detected in the synaptoneurosomes in spinal cord but not in the CeA. CONCLUSIONS AND IMPLICATIONS: Our results describe a novel mechanism by which systemic Cbln1 induces analgesia potentially by central actions involving normalization of signalling by spinal cord GluD1 receptors.

Neuropathogenic role of astrocyte-derived extracellular vesicles in HIV-associated neurocognitive disorders.

Our previous findings demonstrated that astrocytic HIF-1α plays a major role in HIV-1 Tat-mediated amyloidosis which can lead to Alzheimer's-like pathology-a comorbidity of HIV-Associated Neurocognitive Disorders (HAND). These amyloids can be shuttled in extracellular vesicles, and we sought to assess whether HIV-1 Tat stimulated astrocyte-derived EVs (ADEVs) containing the toxic amyloids could result in neuronal injury in vitro and in vivo. We thus hypothesized that blocking HIF-1α could likely mitigate HIV-1 Tat-ADEV-mediated neuronal injury. Rat hippocampal neurons when exposed to HIV-1 Tat-ADEVs carrying the toxic amyloids exhibited amyloid accumulation and synaptodendritic injury, leading to functional loss as evidenced by alterations in miniature excitatory post synaptic currents. The silencing of astrocytic HIF-1α not only reduced the biogenesis of ADEVs, as well as amyloid cargos, but also ameliorated neuronal synaptodegeneration. Next, we determined the effect of HIV-1 Tat-ADEVs carrying amyloids in the hippocampus of naive mice brains. Naive mice receiving the HIV-1 Tat-ADEVs, exhibited behavioural changes, and Alzheimer's 's-like pathology accompanied by synaptodegeneration. This impairment(s) was not observed in mice injected with HIF-1α silenced ADEVs. This is the first report demonstrating the role of amyloid-carrying ADEVs in mediating synaptodegeneration leading to behavioural changes associated with HAND and highlights the protective role of HIF-1α.

Effect of ifenprodil on GluN1/GluN2B N-methyl-D-aspartate receptor gating.

Ifenprodil is an allosteric inhibitor of GluN1/GluN2B N-methyl-D-aspartate receptors. Despite its widespread use as a prototype for drug development and a subtype-selective tool for physiologic experiments, its precise effect on GluN1/GluN2B gating is yet to be fully understood. Interestingly, recent crystallographic evidence identified that ifenprodil, unlike zinc, binds at the interface of the GluN1/GluN2B amino terminal domain dimer by an induced-fit mechanism. To delineate the effect of this unique binding on GluN1/GluN2B receptor gating, we recorded steady-state currents from cell-attached and outside-out patches. At pH 7.9 in cell-attached patches, ifenprodil increased the occupancy of the long-lived shut conformations, thereby reducing the open probability of the receptor with no change in the mean open time. In addition, ifenprodil selectively affected the area of shut time constants, but not the time constants themselves. Kinetic analyses suggested that ifenprodil prevents the transition of the receptor to an open state and increases its dwell time in an intrinsically occurring closed conformation or desensitized state. We found distinct differences in the action of ifenprodil at GluN1/GluN2B in comparison with previous studies on the effect of zinc on GluN1/GluN2A gating, which may arise due to their unique binding sites. Our data also uncover the potential pH-dependent action of ifenprodil on gating. At a low pH (pH 7.4), but not pH 7.9, ifenprodil reduces the mean open time of GluN1/GluN2B receptors, which may be responsible for its usefulness as a context-dependent inhibitor in conditions like ischemia and stroke, when the pH of the extracellular milieu becomes acidic.

Effect of D-cycloserine in conjunction with fear extinction training on extracellular signal-regulated kinase activation in the medial prefrontal cortex and amygdala in rat.

D-cycloserine (DCS) is currently under clinical trials for a number of neuropsychiatric conditions and has been found to augment fear extinction in rodents and exposure therapy in humans. However, the molecular mechanism of DCS action in these multiple modalities remains unclear. Here, we describe the effect of DCS administration, alone or in conjunction with extinction training, on neuronal activity (c-fos) and neuronal plasticity [phospho-extracellular signal-regulated kinase (pERK)] markers using immunohistochemistry. We found that intraperitoneal administration of DCS in untrained young rats (24-28 days old) increased c-fos- and pERK-stained neurons in both the prelimbic and infralimbic division of the medial prefrontal cortex (mPFC) and reduced pERK levels in the lateral nucleus of the central amygdala. Moreover, DCS administration significantly increased GluA1, GluN1, GluN2A, and GluN2B expression in the mPFC. In a separate set of animals, we found that DCS facilitated fear extinction and increased pERK levels in the infralimbic prefrontal cortex, prelimbic prefrontal cortex intercalated cells and lateral nucleus of the central amygdala, compared with saline control. In the synaptoneurosomal preparation, we found that extinction training increased iGluR protein expression in the mPFC, compared with context animals. No significant difference in protein expression was observed between extinction-saline and extinction-DCS groups in the mPFC. In contrast, in the amygdala DCS, the conjunction with extinction training led to an increase in iGluR subunit expression, compared with the extinction-saline group. Our data suggest that the efficacy of DCS in neuropsychiatric disorders may be partly due to its ability to affect neuronal activity and signaling in the mPFC and amygdala subnuclei.

Glutamate delta-1 receptor regulates oligodendrocyte progenitor cell differentiation and myelination in normal and demyelinating conditions.

In this study, we investigated the role of glutamate delta 1 receptor (GluD1) in oligodendrocyte progenitor cell (OPC)-mediated myelination during basal (development) and pathophysiological (cuprizone-induced demyelination) conditions. Initially, we sought to determine the expression pattern of GluD1 in OPCs and found a significant colocalization of GluD1 puncta with neuron-glial antigen 2 (NG2, OPC marker) in the motor cortex and dorsal striatum. Importantly, we found that the ablation of GluD1 led to an increase in the number of myelin-associated glycoprotein (MAG+) cells in the corpus callosum and motor cortex at P40 without affecting the number of NG2+ OPCs, suggesting that GluD1 loss selectively facilitates OPC differentiation rather than proliferation. Further, deletion of GluD1 enhanced myelination in the corpus callosum and motor cortex, as indicated by increased myelin basic protein (MBP) staining at P40, suggesting that GluD1 may play an essential role in the developmental regulation of myelination during the critical window period. In contrast, in cuprizone-induced demyelination, we observed reduced MBP staining in the corpus callosum of GluD1 KO mice. Furthermore, cuprizone-fed GluD1 KO mice showed more robust motor deficits. Collectively, our results demonstrate that GluD1 plays a critical role in OPC regulation and myelination in normal and demyelinating conditions.

GluN2D Subunit in Parvalbumin Interneurons Regulates Prefrontal Cortex Feedforward Inhibitory Circuit and Molecular Networks Relevant to Schizophrenia.

BACKGROUND: Parvalbumin interneuron (PVI) activity synchronizes the medial prefrontal cortex circuit for normal cognitive function, and its impairment may contribute to schizophrenia (SZ). NMDA receptors in PVIs participate in these activities and form the basis for the NMDA receptor hypofunction hypothesis of SZ. However, the role of the GluN2D subunit, which is enriched in PVIs, in regulating molecular networks relevant to SZ is unknown. METHODS: Using electrophysiology and a mouse model with conditional deletion of GluN2D from PVIs (PV-GluN2D knockout [KO]), we examined the cell excitability and neurotransmission in the medial prefrontal cortex. Histochemical, RNA sequencing analysis and immunoblotting were conducted to understand molecular mechanisms. Behavioral analysis was conducted to test cognitive function. RESULTS: PVIs in the medial prefrontal cortex were found to express putative GluN1/2B/2D receptors. In a PV-GluN2D KO model, PVIs were hypoexcitable, whereas pyramidal neurons were hyperexcitable. Excitatory neurotransmission was higher in both cell types in PV-GluN2D KO, whereas inhibitory neurotransmission showed contrasting changes, which could be explained by reduced somatostatin interneuron projections and increased PVI projections. Genes associated with GABA (gamma-aminobutyric acid) synthesis, vesicular release, and uptake as well as those involved in formation of inhibitory synapses, specifically GluD1-Cbln4 and Nlgn2, and regulation of dopamine terminals were downregulated in PV-GluN2D KO. SZ susceptibility genes including Disc1, Nrg1, and ErbB4 and their downstream targets were also downregulated. Behaviorally, PV-GluN2D KO mice showed hyperactivity and anxiety behavior and deficits in short-term memory and cognitive flexibility. CONCLUSIONS: These findings demonstrate that GluN2D in PVIs serves as a point of convergence of pathways involved in the regulation of GABAergic synapses relevant to SZ.

Cocaine preference and neuroadaptations are maintained by astrocytic NMDA receptors in the nucleus accumbens.

Cocaine-associated memories induce cravings and interfere with the ability of users to cease cocaine use. Reducing the strength of cue-drug memories by facilitating extinction may have therapeutic value for the treatment of cocaine addiction. Here, we demonstrate the expression of GluN1/2A/2C NMDA receptor currents in astrocytes in the nucleus accumbens core. Selective ablation of GluN1 subunit from astrocytes in the nucleus accumbens enhanced extinction of cocaine preference memory but did not affect cocaine conditioning or reinstatement. Repeated cocaine exposure up-regulated GluN2C subunit expression and increased astrocytic NMDA receptor currents. Furthermore, intra-accumbal inhibition of GluN2C/2D-containing receptors and GluN2C subunit deletion facilitated extinction of cocaine memory. Cocaine-induced neuroadaptations including dendritic spine maturation and AMPA receptor recruitment were absent in GluN2C knockout mice. Impaired retention of cocaine preference memory in GluN2C knockout mice was restored by exogenous administration of recombinant glypican 4. Together, these results identify a previously unknown astrocytic GluN2C-containing NMDA receptor mechanism underlying maintenance of cocaine preference memory.