NMDA Receptor Currents Research Articles

Chelerythrine inhibits NR2B NMDA receptor independent of PKC activity

N-methyl-D-aspartate receptors (NMDAR), the ligand ion glutamate receptor channels, mediate major excitatory neurotransmission in central nervous system (CNS). They highly express in CNS and involve in multiple physiological processes. Many studies implicated that NMDAR plays a crucial role in number of neurological disorders, including ischemia, dementia, and pain, indicating its potential as a therapeutic target for treatments. Chelerythrine (CHE) is a benzo-phenanthridine alkaloid extracted from Chelidonium majus with many biological activities including anti-inflammatory, anticancer effect, and antidiabetic effect. But the mechanism of CHE is not well understood. The aim of this study was to investigate the effect of CHE on the NMDAR. The results demonstrated that CHE effectively suppressed NMDA-induced currents in primary cultured cortical neurons. To elucidate the underlying mechanism, we expressed NMDARs in HEK293 cells and found that CHE and some of its structural analogues inhibited NMDAR currents and facilitated the desensitization of GluN2B NMDARs. Notably, these effects were independent of protein kinase C activity, suggesting that the effect of CHE on GluN2B-containing NMDAR may occur through a mechanism of directly interaction with NMDAR. Moreover, the inhibitory effect of CHE on GluN2B NMDARs is pH-dependent. Molecular docking prediction in conjunction with mutagenesis analysis revealed that the M3 α-helical segment of the NMDAR in close proximity to the GluN2B Thr647 amino acid plays an important role in CHE inhibition of GluN2B. This study revealed a novel function of CHE and its structural analogues in inhibiting the NMDARs and promoting GluN2B-mediated desensitization by obstructing the receptor at the channel pore region.

The Role of Calcium-Dependent Desensitization in the Potentiation by Gne-9278 of NMDA Receptor Currents in Rat Cortical Neurons in vitro

The Role of Calcium-Dependent Desensitization in the Potentiation by Gne-9278 of NMDA Receptor Currents in Rat Cortical Neurons in vitro

Characterization of Mice Carrying a Neurodevelopmental Disease-Associated GluN2B(L825V) Variant.

N-Methyl-d-aspartate receptors (NMDARs), encoded by GRIN genes, are ionotropic glutamate receptors playing a critical role in synaptic transmission, plasticity, and synapse development. Genome sequence analyses have identified variants in GRIN genes in patients with neurodevelopmental disorders, but the underlying disease mechanisms are not well understood. Here, we have created and evaluated a transgenic mouse line carrying a missense variant Grin2bL825V , corresponding to a de novo GRIN2B variant encoding GluN2B(L825V) found in a patient with intellectual disability (ID) and autism spectrum disorder (ASD). We used HEK293T cells expressing recombinant receptors and primary hippocampal neurons prepared from heterozygous Grin2bL825V/+ (L825V/+) and wild-type (WT) Grin2b+/+ (+/+) male and female mice to assess the functional impact of the variant. Whole-cell NMDAR currents were reduced in neurons from L825V/+ compared with +/+ mice. The peak amplitude of NMDAR-mediated evoked excitatory postsynaptic currents (NMDAR-eEPSCs) was unchanged, but NMDAR-eEPSCs in L825V/+ neurons had faster deactivation compared with +/+ neurons and were less sensitive to a GluN2B-selective antagonist ifenprodil. Together, these results suggest a decreased functional contribution of GluN2B subunits to synaptic NMDAR currents in hippocampal neurons from L825V/+ mice. The analysis of the GluN2B(L825V) subunit surface expression and synaptic localization revealed no differences compared with WT GluN2B. Behavioral testing of mice of both sexes demonstrated hypoactivity, anxiety, and impaired sensorimotor gating in the L825V/+ strain, particularly affecting males, as well as cognitive symptoms. The heterozygous L825V/+ mouse offers a clinically relevant model of GRIN2B-related ID/ASD, and our results suggest synaptic-level functional changes that may contribute to neurodevelopmental pathology.

Scorpion venom heat-resistant synthesized peptide ameliorates epileptic seizures and imparts neuroprotection in rats mediated by NMDA receptors

Finding new and effective natural products for designing antiepileptic drugs is highly important in the scientific community. The scorpion venom heat-resistant peptide (SVHRP) was purified from Buthus martensii Karsch scorpion venom, and subsequent analysis of the amino acid sequence facilitated the synthesis of a peptide known as scorpion venom heat-resistant synthesis peptide (SVHRSP) using a technique for peptide synthesis. Previous studies have demonstrated that the SVHRSP can inhibit neuroinflammation and provide neuroprotection. This study aimed to investigate the antiepileptic effect of SVHRSP on both acute and chronic kindling seizure models by inducing seizures in male rats through intraperitoneal administration of pentylenetetrazole (PTZ). Additionally, an N-methyl-D-aspartate (NMDA)-induced neuronal injury model was used to observe the anti-excitotoxic effect of SVHRSP in vitro. Our findings showed that treatment with SVHRSP effectively alleviated seizure severity, prolonged latency, and attenuated neuronal loss and glial cell activation. It also demonstrated the prevention of alterations in the expression levels of NMDA receptor subunits and phosphorylated p38 MAPK protein, as well as an improvement in spatial reference memory impairment during Morris water maze (MWM) testing in PTZ-kindled rats. In vitro experiments further revealed that SVHRSP was capable of attenuating neuronal action potential firing, inhibiting NMDA receptor currents and intracellular calcium overload, and reducing neuronal injury. These results suggest that the antiepileptic and neuroprotective effects of SVHRSP may be mediated through the regulation of NMDA receptor function and expression. This study provides new insight into therapeutic strategies for epilepsy.

Mitochondrial Calcium Uniporter (MCU) is Involved in an Ischemic Postconditioning Effect Against Ischemic Reperfusion Brain Injury in Mice

The phenomenon of ischemic postconditioning (PostC) is known to be neuroprotective against ischemic reperfusion (I/R) injury. One of the key processes in PostC is the opening of the mitochondrial ATP-dependent potassium (mito-KATP) channel and depolarization of the mitochondrial membrane, triggering the release of calcium ions from mitochondria through low-conductance opening of the mitochondrial permeability transition pore. Mitochondrial calcium uniporter (MCU) is known as a highly sensitive transporter for the uptake of Ca2+ present on the inner mitochondrial membrane. The MCU has attracted attention as a new target for treatment in diseases, such as neurodegenerative diseases, cancer, and ischemic stroke. We considered that the MCU may be involved in PostC and trigger its mechanisms. This research used the whole-cell patch-clamp technique on hippocampal CA1 pyramidal cells from C57BL mice and measured changes in spontaneous excitatory post-synaptic currents (sEPSCs), intracellular Ca2+ concentration, mitochondrial membrane potential, and N-methyl-d-aspartate receptor (NMDAR) currents under inhibition of MCU by ruthenium red 265 (Ru265) in PostC. Inhibition of MCU increased the occurrence of sEPSCs (p = 0.014), NMDAR currents (p < 0.001), intracellular Ca2+ concentration (p < 0.001), and dead cells (p < 0.001) significantly after reperfusion, reflecting removal of the neuroprotective effects in PostC. Moreover, mitochondrial depolarization in PostC with Ru265 was weakened, compared to PostC (p = 0.004). These results suggest that MCU affects mitochondrial depolarization in PostC to suppress NMDAR over-activation and prevent elevation of intracellular Ca2+ concentrations against I/R injury.Graphical

A novel NMDA receptor modulator: the antidepressant effect and mechanism of GW043.

The N-methyl-D-aspartate (NMDA) receptor (NMDAR) has been proven to be strongly correlated with rapid antidepressant effects. Here, GW043, as a new compound targeting NMDAR, we explored its antidepressant effects and its mechanism of action. Our study utilized electrophysiological techniques to confirm the effect of GW043 on NMDAR currents. Additionally, we assessed the selectivity of GW043 through high-throughput receptor-ligand binding experiments. The antidepressant properties of GW043 were examined using rodent behavioral models including the Forced Swim Test (FST), Tail Suspension Test (TST), and Chronic Unpredictable Mild Stress (CUMS). Mechanistic insight into GW043's onset was gained through western blot analysis, BrdU staining, Golgi staining, and electrophysiological techniques. Electrophysiological studies indicated that GW043 acts as a partial agonist of NMDAR. Behavioral experiments confirmed the antidepressant effect of GW043 in rodents. Mechanistic investigations revealed that GW043 modulates synaptic plasticity through the LTP and BDNF-mTOR pathways, consequently leading to an increase in the number of newborn neurons and subsequent antidepressant effects. Our findings disclose that GW043, as a partial agonist of NMDAR, can reverse depression-like behaviors in rats by modulating synaptic plasticity, indicating its potential as an antidepressant agent.

Plasticity in the Functional Properties of NMDA Receptors Improves Network Stability during Severe Energy Stress.

Brain energy stress leads to neuronal hyperexcitability followed by a rapid loss of function and cell death. In contrast, the frog brainstem switches into a state of extreme metabolic resilience that allows them to maintain motor function during hypoxia as they emerge from hibernation. NMDA receptors (NMDARs) are Ca2+-permeable glutamate receptors that contribute to the loss of homeostasis during hypoxia. Therefore, we hypothesized that hibernation leads to plasticity that reduces the role of NMDARs within neural networks to improve function during hypoxia. To test this, we assessed a circuit with a large involvement of NMDAR synapses, the brainstem respiratory network of female bullfrogs, Lithobates catesbeianus Contrary to our expectations, hibernation did not alter the role of NMDARs in generating network output, nor did it affect the amplitude, kinetics, and hypoxia sensitivity of NMDAR currents. Instead, hibernation strongly reduced NMDAR Ca2+ permeability and enhanced desensitization during repetitive stimulation. Under severe hypoxia, the normal NMDAR profile caused network hyperexcitability within minutes, which was mitigated by blocking NMDARs. After hibernation, the modified complement of NMDARs protected against hyperexcitability, as disordered output did not occur for at least one hour in hypoxia. These findings uncover state-dependence in the plasticity of NMDARs, whereby multiple changes to receptor function improve neural performance during metabolic stress without interfering with their normal role during healthy conditions.

Tonic NMDAR Currents of NR2A-Containing NMDARs Represent Altered Ambient Glutamate Concentration in the Supraoptic Nucleus.

NMDA receptors (NMDARs) modulate glutamatergic excitatory tone in the brain via two complementary modalities: a phasic excitatory postsynaptic current and a tonic extrasynaptic modality. Here, we demonstrated that the tonic NMDAR-current (I NMDA) mediated by NR2A-containing NMDARs is an efficient biosensor detecting the altered ambient glutamate level in the supraoptic nucleus (SON). I NMDA of magnocellular neurosecretory cells (MNCs) measured by nonselective NMDARs antagonist, AP5, at holding potential (V holding) -70 mV in low concentration of ECF Mg2+ ([Mg2+]o) was transiently but significantly increased 1-week post induction of a DOCA salt hypertensive model rat which was compatible with that induced by a NR2A-selective antagonist, PEAQX (I PEAQX) in both DOCA-H2O and DOCA-salt groups. In agreement, NR2B antagonist, ifenprodil, or NR2C/D antagonist, PPDA, did not affect the holding current (I holding) at V holding -70 mV. Increased ambient glutamate by exogenous glutamate (10 mM) or excitatory amino acid transporters (EAATs) antagonist (TBOA, 50 mM) abolished the I PEAQX difference between two groups, suggesting that attenuated EAATs activity increased ambient glutamate concentration, leading to the larger I PEAQX in DOCA-salt rats. In contrast, only ifenprodil but not PEAQX and PPDA uncovered I NMDA at V holding +40 mV under 1.2 mM [Mg2+]o condition. I ifenprodil was not different in DOCA-H2O and DOCA-salt groups. Finally, NR2A, NR2B, and NR2D protein expression were not different in the SON of the two groups. Taken together, NR2A-containing NMDARs efficiently detected the increased ambient glutamate concentration in the SON of DOCA-salt hypertensive rats due to attenuated EAATs activity.

Selective inhibitor of sodium-calcium exchanger, SEA0400, affects NMDA receptor currents and abolishes their calcium-dependent block by tricyclic antidepressants.

The open-channel block of N-methyl-D-aspartate receptors (NMDARs) and their calcium-dependent desensitization (CDD) represent conventional mechanisms of glutamatergic synapse regulation. In neurotrauma, neurodegeneration, and neuropathic pain the clinical benefits of cure with memantine, ketamine, Mg2+, and some tricyclic antidepressants are often attributed to NMDAR open-channel block, while possible involvement of NMDAR CDD in the therapy is not well established. Here the effects of selective high-affinity sodium-calcium exchanger (NCX) isoform 1 inhibitor, SEA0400, on NMDA-activated whole-cell currents and their block by amitriptyline, desipramine and clomipramine recorded by patch-clamp technique in cortical neurons of primary culture were studied. We demonstrated that in the presence of extracellular Ca2+, 50nM SEA0400 caused a reversible decrease of the steady-state amplitude of NMDAR currents, whereas loading neurons with BAPTA or the removal of extracellular Ca2+ abolished the effect. The decrease did not exceed 30% of the amplitude and did not depend on membrane voltage. The external Mg2+ block and 50nM SEA0400 inhibition of currents were additive, suggesting their independent modes of action. In the presence of Ca2+ SEA0400 speeded up the decay of NMDAR currents to the steady state determined by CDD. The measured IC50 value of 27nM for SEA0400-induced inhibition coincides with that for NCX1. Presumably, SEA0400 effects are induced by an enhancement of NMDAR CDD through the inhibition of Ca2+ extrusion by NCX1. SEA0400, in addition, at nanomolar concentrations could interfere with Ca2+-dependent effect of tricyclic antidepressants. In the presence of 50nM SEA0400, the IC50s for NMDAR inhibition by amitriptyline and desipramine increased by about 20 folds, as the Ca2+-dependent NMDAR inhibition disappeared. This observation highlights NCX1 involvement in amitriptyline and desipramine effects on NMDARs and unmasks competitive relationships between SEA0400 and these antidepressants. Neither amitriptyline nor desipramine could affect NCX3. The open-channel block of NMDARs by these substances was not affected by SEA0400. In agreement, SEA0400 did not change the IC50 for clomipramine, which acts as a pure NMDAR open-channel blocker. Thus, NCX seems to represent a promising molecular target to treat neurological disorders, because of the ability to modulate NMDARs by decreasing the open probability through the enhancement of their CDD.

CCK-sensitive C fibers activate NTS leptin receptor-expressing neurons via NMDA receptors.

The hormone leptin reduces food intake through actions in the peripheral and central nervous systems, including in the hindbrain nucleus of the solitary tract (NTS). The NTS receives viscerosensory information via vagal afferents, including information from the gastrointestinal tract, which is then relayed to other central nervous system (CNS) sites critical for control of food intake. Leptin receptors (lepRs) are expressed by a subpopulation of NTS neurons, and knockdown of these receptors increases both food intake and body weight. Recently, we demonstrated that leptin increases vagal activation of lepR-expressing neurons via increased NMDA receptor (NMDAR) currents, thereby potentiating vagally evoked firing. Furthermore, chemogenetic activation of these neurons was recently shown to inhibit food intake. However, the vagal inputs these neurons receive had not been characterized. Here we performed whole cell recordings in brain slices taken from lepRCre × floxedTdTomato mice and found that lepR neurons of the NTS are directly activated by monosynaptic inputs from C-type afferents sensitive to the transient receptor potential vanilloid type 1 (TRPV1) agonist capsaicin. CCK administered onto NTS slices stimulated spontaneous glutamate release onto lepR neurons and induced action potential firing, an effect mediated by CCKR1. Interestingly, NMDAR activation contributed to the current carried by spontaneous excitatory postsynaptic currents (EPSCs) and enhanced CCK-induced firing. Peripheral CCK also increased c-fos expression in these neurons, suggesting they are activated by CCK-sensitive vagal afferents in vivo. Our results indicate that the majority of NTS lepR neurons receive direct inputs from CCK-sensitive C vagal-type afferents, with both peripheral and central CCK capable of activating these neurons and NMDARs able to potentiate these effects.

Astrocyte β-Adrenergic Receptor Activity Regulates NMDA Receptor Signaling of Medial Prefrontal Cortex Pyramidal Neurons.

Glutamate spillover from the synapse is tightly regulated by astrocytes, limiting the activation of extrasynaptically located NMDA receptors (NMDAR). The processes of astrocytes are dynamic and can modulate synaptic physiology. Though norepinephrine (NE) and β-adrenergic receptor (β-AR) activity can modify astrocyte volume, this has yet to be confirmed outside of sensory cortical areas, nor has the effect of noradrenergic signaling on glutamate spillover and neuronal NMDAR activity been explored. We monitored changes to astrocyte process volume in response to noradrenergic agonists in the medial prefrontal cortex of male and female mice. Both NE and the β-AR agonist isoproterenol (ISO) increased process volume by ∼20%, significantly higher than changes seen when astrocytes had G-protein signaling blocked by GDPβS. We measured the effect of β-AR signaling on evoked NMDAR currents. While ISO did not affect single stimulus excitatory currents of Layer 5 pyramidal neurons, ISO reduced NMDAR currents evoked by 10 stimuli at 50 Hz, which elicits glutamate spillover, by 18%. After isolating extrasynaptic NMDARs by blocking synaptic NMDARs with the activity-dependent NMDAR blocker MK-801, ISO similarly reduced extrasynaptic NMDAR currents in response to 10 stimuli by 18%. Finally, blocking β-AR signaling in the astrocyte network by loading them with GDPβS reversed the ISO effect on 10 stimuli-evoked NMDAR currents. These results demonstrate that astrocyte β-AR activity reduces extrasynaptic NMDAR recruitment, suggesting that glutamate spillover is reduced.

STIM2 regulates NMDA receptor endocytosis that is induced by short-term NMDA receptor overactivation in cortical neurons

Recent findings suggest an important role for the dysregulation of stromal interaction molecule (STIM) proteins, activators of store-operated Ca2+ channels, and the prolonged activation of N-methyl-D-aspartate receptors (NMDARs) in the development of neurodegenerative diseases. We previously demonstrated that STIM silencing increases Ca2+ influx through NMDAR and STIM–NMDAR2 complexes are present in neurons. However, the interplay between NMDAR subunits (GluN1, GluN2A, and GluN2B) and STIM1/STIM2 with regard to intracellular trafficking remains unknown. Here, we found that the activation of NMDAR endocytosis led to an increase in STIM2–GluN2A and STIM2–GluN2B interactions in primary cortical neurons. STIM1 appeared to migrate from synaptic to extrasynaptic sites. STIM2 silencing inhibited post-activation NMDAR translocation from the plasma membrane and synaptic spines and increased NMDAR currents. Our findings reveal a novel molecular mechanism by which STIM2 regulates NMDAR synaptic trafficking by promoting NMDAR endocytosis after receptor overactivation, which may suggest protection against excessive uncontrolled Ca2+ influx through NMDARs.

Tonic Activation of NR2D-Containing NMDARs Exacerbates Dopaminergic Neuronal Loss in MPTP-Injected Parkinsonian Mice.

NR2D subunit-containing NMDA receptors (NMDARs) gradually disappear during brain maturation but can be recruited by pathophysiological stimuli in the adult brain. Here, we report that 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) intoxication recruited NR2D subunit-containing NMDARs that generated an Mg2+-resistant tonic NMDA current (INMDA) in dopaminergic (DA) neurons in the midbrain of mature male mice. MPTP selectively generated an Mg2+-resistant tonic INMDA in DA neurons in the substantia nigra pars compacta (SNpc) and ventral tegmental area (VTA). Consistently, MPTP increased NR2D but not NR2B expression in the midbrain regions. Pharmacological or genetic NR2D interventions abolished the generation of Mg2+-resistant tonic INMDA in SNpc DA neurons, and thus attenuated subsequent DA neuronal loss and gait deficits in MPTP-treated mice. These results show that extrasynaptic NR2D recruitment generates Mg2+-resistant tonic INMDA and exacerbates DA neuronal loss, thus contributing to MPTP-induced Parkinsonism. The state-dependent NR2D recruitment could be a novel therapeutic target for mitigating cell type-specific neuronal death in neurodegenerative diseases.SIGNIFICANCE STATEMENT NR2D subunit-containing NMDA receptors (NMDARs) are widely expressed in the brain during late embryonic and early postnatal development, and then downregulated during brain maturation and preserved at low levels in a few regions of the adult brain. Certain stimuli can recruit NR2D subunits to generate tonic persistent NMDAR currents in nondepolarized neurons in the mature brain. Our results show that MPTP intoxication recruits NR2D subunits in midbrain dopaminergic (DA) neurons, which leads to tonic NMDAR current-promoting dopaminergic neuronal death and consequent abnormal gait behavior in the MPTP mouse model of Parkinson's disease (PD). This is the first study to indicate that extrasynaptic NR2D recruitment could be a target for preventing neuronal death in neurodegenerative diseases.

NMDA Receptor–Dependent Synaptic Potentiation via APPL1 Signaling Is Required for the Accessibility of a Prefrontal Neuronal Assembly in Retrieving Fear Extinction

BackgroundThe ventromedial prefrontal cortex has been viewed as a locus for storage and recall of extinction memory. However, the synaptic and cellular mechanisms underlying these processes remain elusive. MethodsWe combined transgenic mice, electrophysiological recording, activity-dependent cell labeling, and chemogenetic manipulation to analyze the role of adaptor protein APPL1 in the ventromedial prefrontal cortex in fear extinction retrieval. ResultsWe found that both constitutive and conditional APPL1 knockout decreased NMDA receptor (NMDAR) function in the ventromedial prefrontal cortex and impaired fear extinction retrieval. Moreover, APPL1 undergoes nuclear translocation during extinction retrieval. Blocking APPL1 nucleocytoplasmic translocation reduced NMDAR currents and disrupted extinction retrieval. We also identified a prefrontal neuronal ensemble that is both necessary and sufficient for the storage of extinction memory. Inducible APPL1 knockout in this ensemble abolished NMDAR-dependent synaptic potentiation and disrupted extinction retrieval, while chemogenetic activation of this ensemble simultaneously rescued the impaired behaviors. ConclusionsOur results indicate that a prefrontal neuronal ensemble stores extinction memory, and APPL1 signaling supports these neurons in retrieving extinction memory by controlling NMDAR-dependent potentiation.

Kinetic mechanism of NMDA receptor inhibition by the ketamine metabolite 2R,6R-hydroxynorketamine.

2R,6R-hydroxynorketamine (2R,6R-HNK) is a metabolite of the fast-acting dissociative anesthetic ketamine (KET). It is currently in clinical trials for the treatment of major depressive disorder due to its strong safety profile at larger doses. The endogenous therapeutic target of 2R,6R-HNK is unknown. We reported recently that like the parent compound ketamine, 2R,6R-HNK reduces NMDA receptor currents with an open-channel block mechanism which is voltage-dependent, pH-dependent, and competitive with Mg2+, and has ∼100-fold lower efficacy (IC50, 46 µM) than KET (IC50, 0.5 µM) at pH 7.2 and −70 mV. We used electrophysiology, kinetic analyses, and kinetic modeling to examine the effect of 2R,6R-HNK on the reaction mechanism of NMDA receptors. We recorded on-cell single-channel currents from HEK293 cells expressing GluN1/GluN2A receptors (and GFP) with pipettes containing (in mM): 150 NaCl, 2.5 KCl, 1 EDTA, 10 HEPES, pH 7.2 (NaOH), agonists glutamate (1) and glycine (0.1), and applied +100 mV (membrane potential ∼−120 mV). Relative to activity recorded in the absence of 2R,6R-HNK (control, n=7), activity recorded with 50 µM 2R,6R-HNK (n=7) displayed a lower open probability (Po), shorter openings (MOT), and longer closed durations (MCT). Fitting these data with state models allowed us to optimize rate constants for the kinetic model. In the future, we will test these models by simulating macroscopic responses and recording whole cell currents in similar conditions.

Disruption of Astrocyte-Dependent Dopamine Control in the Developing Medial Prefrontal Cortex Leads to Excessive Grooming in Mice

BackgroundAstrocytes control synaptic activity by modulating perisynaptic concentrations of ions and neurotransmitters including dopamine (DA) and, as such, could be involved in the modulating aspects of mammalian behavior. MethodsWe produced a conditional deletion of the vesicular monoamine transporter 2 (VMAT2) specifically in astrocytes (aVMTA2cKO mice) and studied the effects of the lack of VMAT2 in prefrontal cortex (PFC) astrocytes on the regulation of DA levels, PFC circuit functions, and behavioral processes. ResultsWe found a significant reduction of medial PFC (mPFC) DA levels and excessive grooming and compulsive repetitive behaviors in aVMAT2cKO mice. The mice also developed a synaptic pathology, expressed through increased relative AMPA versus NMDA receptor currents in synapses of the dorsal striatum receiving inputs from the mPFC. Importantly, behavioral and synaptic phenotypes were rescued by re-expression of mPFC VMAT2 and L-DOPA treatment, showing that the deficits were driven by mPFC astrocytes that are critically involved in developmental DA homeostasis. By analyzing human tissue samples, we found that VMAT2 is expressed in human PFC astrocytes, corroborating the potential translational relevance of our observations in mice. ConclusionsOur study shows that impairment of the astrocytic control of DA in the mPFC leads to symptoms resembling obsessive-compulsive spectrum disorders such as trichotillomania and has a profound impact on circuit function and behaviors.

Dually innervated dendritic spines develop in the absence of excitatory activity and resist plasticity through tonic inhibitory crosstalk

Dendritic spines can be directly connected to both inhibitory and excitatory presynaptic terminals, resulting in nanometer-scale proximity of opposing synaptic functions. While dually innervated spines (DiSs) are observed throughout the central nervous system, their developmental timeline and functional properties remain uncharacterized. Here we used a combination of serial section electron microscopy, live imaging, and local synapse activity manipulations to investigate DiS development and function in rodent hippocampus. Dual innervation occurred early in development, even on spines where the excitatory input was locally silenced. Synaptic NMDA receptor currents were selectively reduced at DiSs through tonic GABAB receptor signaling. Accordingly, spine enlargement normally associated with long-term potentiation on singly innervated spines (SiSs) was blocked at DiSs. Silencing somatostatin interneurons or pharmacologically blocking GABABRs restored NMDA receptor function and structural plasticity to levels comparable to neighboring SiSs. Thus, hippocampal DiSs are stable structures where function and plasticity are potently regulated by nanometer-scale GABAergic signaling.

α2δ-1 protein drives opioid-induced conditioned reward and synaptic NMDA receptor hyperactivity in the nucleus accumbens.

Glutamate NMDA receptors (NMDARs) in the nucleus accumbens (NAc) are critically involved in drug dependence and reward. α2δ-1 is a newly discovered NMDAR-interacting protein that promotes synaptic trafficking of NMDARs independently of its conventional role as a calcium channel subunit. However, it remains unclear how repeated opioid exposure affects synaptic NMDAR activity and α2δ-1-NMDAR interaction in the NAc. In this study, whole-cell patch-clamp recordings showed that repeated treatment with morphine in mice markedly increased the NMDAR-mediated frequency of miniature excitatory postsynaptic currents (mEPSCs) and amplitude of puff NMDAR currents in medium spiny neurons in the NAc core region. Morphine treatment significantly increased the physical interaction of α2δ-1 with GluN1 and their synaptic trafficking in the NAc. In Cacna2d1 knockout mice, repeated treatment with morphine failed to increase the frequency of mEPSCs and amplitude of puff NMDAR currents in the NAc core. Furthermore, inhibition of α2δ-1 with gabapentin or disruption of the α2δ-1-NMDAR interaction with the α2δ-1 C terminus-interfering peptide blocked the morphine-elevated frequency of mEPSCs and amplitude of puff NMDAR currents in the NAc core. Correspondingly, systemically administered gabapentin, Cacna2d1 ablation, or microinjection of the α2δ-1 C terminus-interfering peptide into the NAc core attenuated morphine-induced conditioned place preference and locomotor sensitization. Our study reveals that repeated opioid exposure strengthens presynaptic and postsynaptic NMDAR activity in the NAc via α2δ-1. The α2δ-1-bound NMDARs in the NAc have a key function in the rewarding effect of opioids and could be targeted for treating opioid use disorder and addiction.

Restored Fyn Levels in Huntington's Disease Contributes to Enhanced Synaptic GluN2B-Composed NMDA Receptors and CREB Activity.

N-methyl-D-aspartate receptors (NMDARs) are important postsynaptic receptors that contribute to normal synaptic function and cell survival; however, when overactivated, as in Huntington’s disease (HD), NMDARs cause excitotoxicity. HD-affected striatal neurons show altered NMDAR currents and augmented ratio of surface to internal GluN2B-containing NMDARs, with augmented accumulation at extrasynaptic sites. Fyn protein is a member of the Src kinase family (SKF) with an important role in NMDARs phosphorylation and synaptic localization and function; recently, we demonstrated that Fyn is reduced in several HD models. Thus, in this study, we aimed to explore the impact of HD-mediated altered Fyn levels at post-synaptic density (PSD), and their role in distorted NMDARs function and localization, and intracellular neuroprotective pathways in YAC128 mouse primary striatal neurons. We show that reduced synaptic Fyn levels and activity in HD mouse striatal neurons is related to decreased phosphorylation of synaptic GluN2B-composed NMDARs; this occurs concomitantly with augmented extrasynaptic NMDARs activity and currents and reduced cAMP response element-binding protein (CREB) activation, along with induction of cell death pathways. Importantly, expression of a constitutive active form of SKF reestablishes NMDARs localization, phosphorylation, and function at PSD in YAC128 mouse neurons. Enhanced SKF levels and activity also promotes CREB activation and reduces caspase-3 activation in YAC128 mouse striatal neurons. This work supports, for the first time, a relevant role for Fyn protein in PSD modulation, controlling NMDARs synaptic function in HD, and favoring neuroprotective pathways and cell survival. In this respect, Fyn Tyr kinase constitutes an important potential HD therapeutic target directly acting at PSD.

Pathway-specific contribution of parvalbumin interneuron NMDARs to synaptic currents and thalamocortical feedforward inhibition

Prefrontal cortex (PFC) is a site of information convergence important for behaviors relevant to psychiatric disorders. Despite the importance of inhibitory GABAergic parvalbumin-expressing (PV+) interneurons to PFC circuit function and decades of interest in N-methyl-D-aspartate receptors (NMDARs) in these neurons, examples of defined circuit functions that depend on PV+ interneuron NMDARs have been elusive. Indeed, it remains controversial whether all PV+ interneurons contain functional NMDARs in adult PFC, which has major consequences for hypotheses of the pathogenesis of psychiatric disorders. Using a combination of fluorescent in situ hybridization, pathway-specific optogenetics, cell-type-specific gene ablation, and electrophysiological recordings from PV+ interneurons, here we resolve this controversy. We found that nearly 100% of PV+ interneurons in adult medial PFC (mPFC) express transcripts encoding GluN1 and GluN2B, and they have functional NMDARs. By optogenetically stimulating corticocortical and thalamocortical inputs to mPFC, we show that synaptic NMDAR contribution to PV+ interneuron EPSCs is pathway-specific, which likely explains earlier reports of PV+ interneurons without synaptic NMDAR currents. Lastly, we report a major contribution of NMDARs in PV+ interneurons to thalamus-mediated feedforward inhibition in adult mPFC circuits, suggesting molecular and circuit-based mechanisms for cognitive impairment under conditions of reduced NMDAR function. These findings represent an important conceptual advance that has major implications for hypotheses of the pathogenesis of psychiatric disorders.