Synaptic NMDA Receptor Activation Research Articles

Tiam1-mediated maladaptive plasticity underlying morphine tolerance and hyperalgesia.

Opioid pain medications, such as morphine, remain the mainstay for treating severe and chronic pain. Prolonged morphine use, however, triggers analgesic tolerance and hyperalgesia (OIH), which can last for a long period after morphine withdrawal. How morphine induces these detrimental side effects remains unclear. Here, we show that morphine tolerance and OIH are mediated by Tiam1-coordinated synaptic structural and functional plasticity in the spinal nociceptive network. Tiam1 is a Rac1 GTPase guanine nucleotide exchange factor (GEF) that promotes excitatory synaptogenesis by modulating actin cytoskeletal dynamics. We found that prolonged morphine treatment activated Tiam1 in the spinal dorsal horn and Tiam1 ablation from spinal neurons eliminated morphine antinociceptive tolerance and OIH. At the same time, the pharmacological blockade of Tiam1-Rac1 signaling prevented the development and reserved the established tolerance and OIH. Prolonged morphine treatment increased dendritic spine density and synaptic NMDA receptor (NMDAR) activity in spinal dorsal horn neurons, both of which required Tiam1. Furthermore, co-administration of the Tiam1 signaling inhibitor NSC23766 was sufficient to abrogate morphine tolerance in chronic pain management. These findings identify Tiam1-mediated maladaptive plasticity in the spinal nociceptive network as an underlying cause for the development and maintenance of morphine tolerance and OIH and provide a promising therapeutic target to reduce tolerance and prolong morphine use in chronic pain management.

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.

Brain α2δ-1-Bound NMDA Receptors Drive Calcineurin Inhibitor-Induced Hypertension.

Calcineurin is highly enriched in immune T cells and the nervous system. Calcineurin inhibitors, including cyclosporine and tacrolimus (FK506), are the cornerstone of immunosuppressive regimens for preserving transplanted organs and tissues. However, these drugs often cause persistent hypertension owing to excess sympathetic outflow, which is maintained by N-methyl-D-aspartate receptor (NMDAR)-mediated excitatory input to the hypothalamic paraventricular nucleus (PVN). It is unclear how calcineurin inhibitors increase NMDAR activity in the PVN to augment sympathetic vasomotor activity. α2δ-1 (encoded by the Cacna2d1 gene), known colloquially as a calcium channel subunit, is a newly discovered NMDAR-interacting protein. In this study, we determined whether α2δ-1 plays a role in calcineurin inhibitor-induced synaptic NMDAR hyperactivity in the PVN and hypertension development. Immunoblotting and coimmunoprecipitation assays were used to quantify synaptic protein levels and the physical interaction between GluN1 (the obligatory NMDAR subunit) and α2δ-1. Whole-cell patch-clamp recordings of retrogradely labeled, spinally projecting PVN were conducted in perfused brain slices to measure presynaptic and postsynaptic NMDAR activity. Radio-telemetry was implanted in rodents to continuously record arterial blood pressure in conscious states. Prolonged treatment with FK506 in rats significantly increased protein levels of α2δ-1, GluN1, and the α2δ-1-GluN1 complex in PVN synaptosomes. These effects were blocked by inhibiting α2δ-1 with gabapentin or interrupting the α2δ-1-NMDAR interaction with an α2δ-1 C-terminus peptide. Treatment with FK506 potentiated the activity of presynaptic and postsynaptic NMDARs in spinally projecting PVN neurons; such effects were abolished by gabapentin, Cacna2d1 knockout, or α2δ-1 C-terminus peptide. Furthermore, microinjection of α2δ-1 C-terminus peptide into the PVN diminished renal sympathetic nerve discharges and arterial blood pressure that had been increased by FK506 treatment. Remarkably, concurrent administration of gabapentin prevented the development of FK506-induced hypertension in rats. Additionally, FK506 treatment induced sustained hypertension in wild-type mice but not in Cacna2d1 knockout mice. α2δ-1 is essential for calcineurin inhibitor-induced increases in synaptic NMDAR activity in PVN presympathetic neurons and sympathetic outflow. Thus, α2δ-1 and α2δ-1-bound NMDARs represent new targets for treating calcineurin inhibitor-induced hypertension. Gabapentinoids (gabapentin and pregabalin) could be repurposed for treating calcineurin inhibitor-induced neurogenic hypertension.

Brainstem astrocytes control homeostatic regulation of caloric intake.

Prolonged high-fat diet (HFD) exposure is associated with hyperphagia, excess caloric intake and weight gain. After initial exposure to a HFD, a brief (24-48h) period of hyperphagia is followed by the regulation of caloric intake and restoration of energy balance within an acute (3-5day) period. Previous studies have demonstrated this occurs via a vagally mediated signalling cascade that increases glutamatergic transmission via activation of NMDA receptors located on gastric-projecting neurons of the dorsal motor nucleus of the vagus (DMV). The present study used electrophysiological recordings from thin brainstem slice preparations, in vivo recordings of gastric motility and tone, measurement of gastric emptying rates, and food intake studies to investigate the hypothesis that activation of brainstem astrocytes in response to acute HFD exposure is responsible for the increased glutamatergic drive to DMV neurons and the restoration of caloric balance. Pharmacological and chemogenetic inhibition of brainstem astrocytes reduced glutamatergic signalling and DMV excitability, dysregulated gastric tone and motility, attenuated the homeostatic delay in gastric emptying, and prevented the decrease in food intake that is observed during the period of energy regulation following initial exposure to HFD. Understanding the mechanisms involved in caloric regulation may provide critical insights into energy balance as well as into the hyperphagia that develops as these mechanisms are overcome. KEY POINTS: Initial exposure to a high fat diet is associated with a brief period of hyperphagia before caloric intake and energy balance is restored. This period of homeostatic regulation is associated with a vagally mediated signalling cascade that increases glutamatergic transmission to dorsal motor nucleus of the vagus (DMV) neurons via activation of synaptic NMDA receptors. The present study demonstrates that pharmacological and chemogenetic inhibition of brainstem astrocytes reduced glutamatergic signalling and DMV neuronal excitability, dysregulated gastric motility and tone and emptying, and prevented the regulation of food intake following high-fat diet exposure. Astrocyte regulation of glutamatergic transmission to DMV neurons appears to involve release of the gliotransmitters glutamate and ATP. Understanding the mechanisms involved in caloric regulation may provide critical insights into energy balance as well as into the hyperphagia that develops as these mechanisms are overcome.

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.

HDAC2 in Primary Sensory Neurons Constitutively Restrains Chronic Pain by Repressing α2δ-1 Expression and Associated NMDA Receptor Activity.

α2δ-1 (encoded by the Cacna2d1 gene) is a newly discovered NMDA receptor-interacting protein and is the therapeutic target of gabapentinoids (e.g., gabapentin and pregabalin) frequently used for treating patients with neuropathic pain. Nerve injury causes sustained α2δ-1 upregulation in the dorsal root ganglion (DRG), which promotes NMDA receptor synaptic trafficking and activation in the spinal dorsal horn, a hallmark of chronic neuropathic pain. However, little is known about how nerve injury initiates and maintains the high expression level of α2δ-1 to sustain chronic pain. Here, we show that nerve injury caused histone hyperacetylation and diminished enrichment of histone deacetylase-2 (HDAC2), but not HDAC3, at the Cacna2d1 promoter in the DRG. Strikingly, Hdac2 knockdown or conditional knockout in DRG neurons in male and female mice consistently induced long-lasting mechanical pain hypersensitivity, which was readily reversed by blocking NMDA receptors, inhibiting α2δ-1 with gabapentin or disrupting the α2δ-1-NMDA receptor interaction at the spinal cord level. Hdac2 deletion in DRG neurons increased histone acetylation levels at the Cacna2d1 promoter, upregulated α2δ-1 in the DRG, and potentiated α2δ-1-dependent NMDA receptor activity at primary afferent central terminals in the spinal dorsal horn. Correspondingly, Hdac2 knockdown-induced pain hypersensitivity was blunted in Cacna2d1 knockout mice. Thus, our findings reveal that HDAC2 functions as a pivotal transcriptional repressor of neuropathic pain via constitutively suppressing α2δ-1 expression and ensuing presynaptic NMDA receptor activity in the spinal cord. HDAC2 enrichment levels at the Cacna2d1 promoter in DRG neurons constitute a unique epigenetic mechanism that governs acute-to-chronic pain transition.SIGNIFICANCE STATEMENT Excess α2δ-1 proteins produced after nerve injury directly interact with glutamate NMDA receptors to potentiate synaptic NMDA receptor activity in the spinal cord, a prominent mechanism of nerve pain. Because α2δ-1 upregulation after nerve injury is long lasting, gabapentinoids relieve pain symptoms only temporarily. Our study demonstrates for the first time the unexpected role of intrinsic HDAC2 activity at the α2δ-1 gene promoter in limiting α2δ-1 gene transcription, NMDA receptor-dependent synaptic plasticity, and chronic pain development after nerve injury. These findings challenge the prevailing view about the role of general HDAC activity in promoting chronic pain. Restoring the repressive HDAC2 function and/or reducing histone acetylation at the α2δ-1 gene promoter in primary sensory neurons could lead to long-lasting relief of nerve pain.

Synaptic NMDA receptor activity at resting membrane potentials.

NMDA receptors (NMDARs) are crucial for glutamatergic synaptic signaling in the mammalian central nervous system. When activated by glutamate and glycine/D-serine, the NMDAR ion channel can open, but current flux is further regulated by voltage-dependent block conferred by extracellular Mg2+ ions. The unique biophysical property of ligand- and voltage-dependence positions NMDARs as synaptic coincidence detectors, controlling a major source of synaptic Ca2+ influx. We measured synaptic currents in layer 2/3 neurons after stimulation in layer 4 of somatosensory cortex and found measurable NMDAR currents at all voltages tested. This NMDAR current did not require concurrent AMPAR depolarization. In physiological ionic conditions, the NMDAR current response at negative potentials was enhanced relative to ionic conditions typically used in slice experiments. NMDAR activity was also seen in synaptic recordings from hippocampal CA1 neurons, indicating a general property of NMDAR signaling. Using a fluorescent Ca2+ indicator, we measured responses to stimulation in layer 4 at individual synaptic sites, and Ca2+ influx could be detected even with AMPARs blocked. In current clamp recordings, we found that resting membrane potential was hyperpolarized by ∼7 mV and AP firing threshold depolarized by ∼4 mV in traditional compared to physiological ionic concentrations, and that NMDARs contribute to EPSPs at resting membrane potentials. These measurements demonstrate that, even in the presence of extracellular Mg2+ and absence of postsynaptic depolarization, NMDARs contribute to synaptic currents and Ca2+ influx.

This Week in The Journal

Lulu Yao, Yi Rong, Xiaoyan Ma, Haifu Li, Di Deng, et al. (see pages [3066–3079][1]) Activation of synaptic NMDA receptors (NMDARs) in excitatory neurons has a well established role in synaptic plasticity. Activation of extrasynaptic NMDARs in excitatory neurons can lead to synapse removal and

N-methyl-d-aspartate Receptor-mediated Preconditioning Mitigates Excitotoxicity in Human Induced Pluripotent Stem Cell-derived Brain Organoids

Studies in rodent models of acute and chronic neurodegenerative disorders have uncovered that glutamate-induced excitotoxic cell death is mediated primarily by extrasynaptic N-methyl-d-aspartate receptors (NMDARs). Rodent neurons can also build up in an activity-dependent manner a protective shield against excitotoxicity. This form of acquired neuroprotection is induced by preconditioning with low doses of NMDA or by activation of synaptic NMDARs triggered by bursts of action potentials. Whether NMDARs in human neurons have similar dichotomous actions in cell death and survival is unknown. To investigate this, we established an induced pluripotent stem cell (iPSC)-derived forebrain organoid model for excitotoxic cell death and explored conditions of NMDAR activation that promote neuronal survival when applied prior to a toxic insult. We found that glutamate-induced excitotoxicity in human iPSC-derived neurons is mediated by NMDARs. Treatment of organoids with high concentrations of glutamate or NMDA caused the typical excitotoxicity pathology, comprising structural disintegration, neurite blebbing, shut-off of the transcription factor CRE binding protein (CREB), and cell death. In contrast, bath-applied low doses of NMDA elicited synaptic activity, a robust and sustained increase in CREB phosphorylation as well as function, and upregulation of immediate-early genes, including neuroprotective genes. Moreover, we found that conditions of enhanced synaptic activity increased survival of human iPSC-derived neurons if applied as pre-treatment before toxic NMDA application. These results revealed that both toxic and protective actions of NMDARs are preserved in human neurons. The experimental platform described in this study may prove useful for the validation of neuroprotective gene products and drugs in human neurons.

Long Noncoding RNA Expression Profiles of Rat Extrasynaptic and Synaptic Neurons Expressing the N-methyl-D-Aspartate Receptor Revealed by Microarray Analysis

To study the 24-hour expression of long noncoding RNAs (lncRNAs) in synaptic and extrasynaptic neurons expressing N-methyl-D-aspartate receptor (NMDAR), and normal neuronal cultures, via microarray analysis. Cortical neurons from embryonic (day E18) Sprague-Dawley rats were used for primary neuronal culture. NMDAR activation was blocked and the cells were then incubated for 6 hours. Total RNA was extracted, quantified, and analyzed for purity and integrity. Double-stranded cDNA was synthesized, followed by quantile normalization, quantitative polymerase chain reaction validation, and data analysis. The interactions between transcription factors and lncRNAs were analyzed by Pearson correlation. The lncRNA profiles were obtained after synaptic and extrasynaptic NMDAR activation of rat cortical neuron cultures for 24 hours. In total, 251 lncRNAs were consistently upregulated, and 335 were downregulated, after extrasynaptic NMDAR activation compared with normal neurons. After synaptic NMDAR activation, only 9 lncRNAs were upregulated and 2 were downregulated. Differential expression of lncRNAs after synaptic and extrasynaptic NMDAR activation suggests that lncRNAs may be responsible for extrasynaptic NMDAR-induced neurodegeneration.

DMV extrasynaptic NMDA receptors regulate caloric intake in rats.

Acute high-fat diet (aHFD) exposure induces a brief period of hyperphagia before caloric balance is restored. Previous studies have demonstrated that this period of regulation is associated with activation of synaptic N-methyl-D-aspartate (NMDA) receptors on dorsal motor nucleus of the vagus (DMV) neurons, which increases vagal control of gastric functions. Our aim was to test the hypothesis that activation of DMV synaptic NMDA receptors occurs subsequent to activation of extrasynaptic NMDA receptors. Sprague-Dawley rats were fed a control or high-fat diet for 3–5 days prior to experimentation. Whole-cell patch-clamp recordings from gastric-projecting DMV neurons; in vivo recordings of gastric motility, tone, compliance, and emptying; and food intake studies were used to assess the effects of NMDA receptor antagonism on caloric regulation. After aHFD exposure, inhibition of extrasynaptic NMDA receptors prevented the synaptic NMDA receptor–mediated increase in glutamatergic transmission to DMV neurons, as well as the increase in gastric tone and motility, while chronic extrasynaptic NMDA receptor inhibition attenuated the regulation of caloric intake. After aHFD exposure, the regulation of food intake involved synaptic NMDA receptor–mediated currents, which occurred in response to extrasynaptic NMDA receptor activation. Understanding these events may provide a mechanistic basis for hyperphagia and may identify novel therapeutic targets for the treatment of obesity.

Acute High Fat Diet‐Dependent Activation of Synaptic NMDA Receptors Requires Activation of Purinergic and Metabotropic Glutamate Receptors on Rat Dorsal Motor Nucleus of the Vagus Neurons

Previous work from this lab has demonstrated that, in rats, restoration of caloric intake following acute high fat diet (aHFD) is dependent upon upregulated glutamatergic signaling in the dorsal motor nucleus of the vagus (DMV), which recruits previously silent synaptic NMDA receptors (NMDARs). We have further shown that NMDARs activation is driven by the activation of extrasynaptic NMDA receptors (NMDARex) in an astrocyte-dependent process, although the exact mechanism involved is still unknown. The current study was designed to test the hypothesis that activation of purinergic and/or metabotropic glutamate receptors (mGluR) by the gliotransmitters ATP and glutamate is responsible for activation of NMDARex and NMDARs. Whole cell patch clamp recordings were made from DMV neurons in thin brainstem slices prepared from male and female Sprague-Dawley rats fed either a control diet (14% kcal from fat) or following acute, 3-5 days, high fat diet (aHFD; 60% kcal from fat) exposure. The presence of activated NMDARs was assessed by the ability of the antagonist AP5 (25µM) to decrease the frequency of miniature excitatory synaptic currents (mEPSC) as well as action potential (AP) firing. The involvement of P2X receptors was investigated using the antagonist PPADS (10µM) and agonist αβMeATP (10µM) while the involvement of group I mGluR was investigated using the antagonist, AIDA (300µM). In aHFD DMV neurons, perfusion with AP5 decreased action potential firing in 8/9 neurons (1.2±0.1 vs 0.2±0.13 AP/sec; P<0.05) and decreased mEPSC frequency in 8/9 neurons (3.18±1.71 vs 2.11±1.12; P<0.05). Application of PPADS blocked the ability of AP5 to decrease AP firing in 6/10 neurons (1.8±0.18 vs 1.5±0.20 AP/sec; P>0.05; P<0.05 vs AP5 alone) and mEPSC frequency (2.57±2.10 vs 2.49±1.63; P>0.05; P<0.05 vs AP5 alone). In contrast, in control DMV neurons, application of the agonist, αβMeATP, uncovered the ability of AP5 to decrease AP firing (1.09±1.31 vs 1.56±1.12 AP/sec; P<0.05) although it did not uncover actions to decrease mEPSC frequency (1.94±0.82 vs 2.17±0.91 P>0.05). In contrast, application of the group 1 mGluR antagonist, AIDA, blocked the ability of AP5 to decrease mEPSC frequency in 7/10 aHFD DMV neurons (1.88±1.45 vs 2.18±1.42; P>0.05), although it failed to block the actions of AP5 to inhibit AP firing in 7/9 neurons (0.74±0.71 vs 0.39±0.53; P<0.05). Taken together, these results show that purinergic and metabotropic glutamatergic signaling are involved in activation of NMDARs on DMV neurons following aHFD exposure. The current study provides a potential mechanism by which caloric balance is restored following aHFD exposure. Specifically, aHFD-dependent release of the brainstem gliotransmitters ATP and glutamate are required for the activation of DMV extrasynaptic and synaptic NMDA receptors. This upregulates NTS-DMV glutamatergic signaling and alters the excitability of central vagal neurocircuits involved in the modulation of gastrointestinal functions including feeding behavior.

High Salt Intake Recruits Tonic Activation of NR2D Subunit-Containing Extrasynaptic NMDARs in Vasopressin Neurons.

In addition to producing a classical excitatory postsynaptic current via activation of synaptic NMDA receptors (NMDARs), glutamate in the brain also induces a tonic NMDAR current (INMDA) via activation of extrasynaptic NMDARs (eNMDARs). However, since Mg2+ blocks NMDARs in nondepolarized neurons, the potential contribution of eNMDARs to the overall neuronal excitatory/inhibitory (E/I) balance remains unknown. Here, we demonstrate that chronic (7 d) salt loading (SL) recruited NR2D subunit-containing NMDARs to generate an Mg2+-resistant tonic INMDA in nondepolarized [Vh (holding potential) -70 mV] vasopressin (VP; but not oxytocin) supraoptic nucleus (SON) neurons in male rodents. Conversely, in euhydrated (EU) and 3 d SL mice, Mg2+-resistant tonic INMDA was not observed. Pharmacological and genetic intervention of NR2D subunits blocked the Mg2+-resistant tonic INMDA in VP neurons under SL conditions, while an NR2B antagonist unveiled Mg2+-sensitive tonic INMDA but not Mg2+-resistant tonic INMDA In the EU group VP neurons, an Mg2+-resistant tonic INMDA was not generated by increased ambient glutamate or treatment with coagonists (e.g., d-serine and glycine). Chronic SL significantly increased NR2D expression but not NR2B expression in the SON relative to the EU group or after 3 d under SL conditions. Finally, Mg2+-resistant tonic INMDA selectively upregulated neuronal excitability in VP neurons under SL conditions, independent of ionotropic GABAergic input. Our results indicate that the activation of NR2D-containing NMDARs constitutes a novel mechanism that generates an Mg2+-resistant tonic INMDA in nondepolarized VP neurons, thus causing an E/I balance shift in VP neurons to compensate for the hormonal demands imposed by a chronic osmotic challenge.SIGNIFICANCE STATEMENT The hypothalamic supraoptic nucleus (SON) consists of two different types of magnocellular neurosecretory cells (MNCs) that synthesize and release the following two peptide hormones: vasopressin (VP), which is necessary for regulation of fluid homeostasis; and oxytocin (OT), which plays a major role in lactation and parturition. NMDA receptors (NMDARs) play important roles in shaping neuronal firing patterns and hormone release from the SON MNCs in response to various physiological challenges. Our results show that prolonged (7 d) salt loading generated a Mg2+-resistant tonic NMDA current mediated by NR2D subunit-containing receptors, which efficiently activated nondepolarized VP (but not OT) neurons. Our findings support the hypothesis that NR2D subunit-containing NMDARs play an important adaptive role in adult brain in response to a sustained osmotic challenge.

D-Serine Signaling and NMDAR-Mediated Synaptic Plasticity Are Regulated by System A-Type of Glutamine/D-Serine Dual Transporters.

D-serine is a physiologic coagonist of NMDA receptors (NMDARs) required for synaptic plasticity, but mechanisms that terminate D-serine signaling are unclear. In particular, the identity of unidirectional plasma membrane transporters that mediate D-serine reuptake has remained elusive. We report that D-serine and glutamine share the same neuronal transport system, consisting of the classic system A transporters Slc38a1 and Slc38a2. We show that these transporters are not saturated with glutamine in vivo and regulate the extracellular levels of D-serine and NMDAR activity. Glutamine increased the NMDAR-dependent long-term potentiation and the isolated NMDAR potentials at the Schaffer collateral-CA1 synapses, but without affecting basal neurotransmission in male mice. Glutamine did not increase the NMDAR potentials in slices from serine racemase knock-out mice, which are devoid of D-serine, indicating that the effect of glutamine is caused by outcompeting D-serine for a dual glutamine-D-serine transport system. Inhibition of the system A reduced the uptake of D-serine in synaptosomes and neuronal cultures of mice of either sex, while increasing the extracellular D-serine concentration in slices and in vivo by microdialysis. When compared with Slc38a2, the Slc38a1 transporter displayed more favorable kinetics toward the D-enantiomer. Biochemical experiments with synaptosomes from Slc38a1 knock-down mice of either sex further support its role as a D-serine reuptake system. Our study identifies the first concentrative and electrogenic transporters mediating D-serine reuptake in vivo In addition to their classical role in the glutamine-glutamate cycle, system A transporters regulate the synaptic turnover of D-serine and its effects on NMDAR synaptic plasticity.SIGNIFICANCE STATEMENT Despite the plethora of roles attributed to D-serine, the regulation of its synaptic turnover is poorly understood. We identified the system A transporters Slc38a1 and Slc38a2 as the main pathway for neuronal reuptake of D-serine. These transporters are not saturated with glutamine in vivo and provide an unexpected link between the serine shuttle pathway, responsible for regulating D-serine synaptic turnover, and the glutamine-glutamate cycle. Our observations suggest that Slc38a1 and Slc38a2 have a dual role in regulating neurotransmission. In addition to their classical role as the glutamine providers, the system A transporters regulate extracellular D-serine and therefore affect NMDAR-dependent synaptic plasticity. Higher glutamine export from astrocytes would increase extracellular D-serine, providing a feedforward mechanism to increase synaptic NMDAR activation.

Calcineurin Inhibition Causes α2δ-1-Mediated Tonic Activation of Synaptic NMDA Receptors and Pain Hypersensitivity.

Calcineurin inhibitors, such as tacrolimus (FK506) and cyclosporine, are widely used as standard immunosuppressants in organ transplantation recipients. However, these drugs can cause severe pain in patients, commonly referred to as calcineurin inhibitor-induced pain syndrome (CIPS). Although calcineurin inhibition increases NMDAR activity in the spinal cord, the underlying mechanism remains enigmatic. Using an animal model of CIPS, we found that systemic administration of FK506 in male and female mice significantly increased the amount of α2δ-1-GluN1 complexes in the spinal cord and the level of α2δ-1-bound GluN1 proteins in spinal synaptosomes. Treatment with FK506 significantly increased the frequency of mEPSCs and the amplitudes of monosynaptic EPSCs evoked from the dorsal root and puff NMDAR currents in spinal dorsal horn neurons. Inhibiting α2δ-1 with gabapentin or disrupting the α2δ-1-NMDAR interaction with α2δ-1Tat peptide completely reversed the effects of FK506. In α2δ-1 gene KO mice, treatment with FK506 failed to increase the frequency of NMDAR-mediated mEPSCs and the amplitudes of evoked EPSCs and puff NMDAR currents in spinal dorsal horn neurons. Furthermore, systemic administration of gabapentin or intrathecal injection of α2δ-1Tat peptide reversed thermal and mechanical hypersensitivity in FK506-treated mice. In addition, genetically deleting GluN1 in dorsal root ganglion neurons or α2δ-1 genetic KO similarly attenuated FK506-induced thermal and mechanical hypersensitivity. Together, our findings indicate that α2δ-1-bound NMDARs mediate calcineurin inhibitor-induced tonic activation of presynaptic and postsynaptic NMDARs at the spinal cord level and that presynaptic NMDARs play a prominent role in the development of CIPS.SIGNIFICANCE STATEMENT Calcineurin inhibitors are immunosuppressants used to prevent rejection of transplanted organs and tissues. However, these drugs can cause severe, unexplained pain. We showed that calcineurin inhibition enhances physical interaction between α2δ-1 and NMDARs and their synaptic trafficking in the spinal cord. α2δ-1 is essential for calcineurin inhibitor-induced aberrant activation of presynaptic and postsynaptic NMDARs in the spinal cord. Furthermore, inhibiting α2δ-1 or disrupting α2δ-1-NMDAR interaction reduces calcineurin inhibitor-induced pain hypersensitivity. Eliminating NMDARs in primary sensory neurons or α2δ-1 KO also attenuates calcineurin inhibitor-induced pain hypersensitivity. This new information extends our mechanistic understanding of the role of endogenous calcineurin in regulating synaptic plasticity and nociceptive transmission and suggests new strategies for treating this painful condition.

Acute High‐Fat Diet Induced Regulation of Caloric Intake is Dependent on Activation of Extrasynaptic NMDA Receptors in Dorsal Motor Nucleus of the Vagus Neurons

During HFD exposure, both humans and experimental animal models undergo a 24hr hyperphagic period before caloric balance is restored within 3–5 days. We have demonstrated previously that regulation is associated with activation of synaptic NMDA receptors (NMDA‐Rsyn) on neurons of the dorsal motor nucleus of the vagus (DMV), which increases motoneuron excitability and efferent control of gastric functions (PMID: 29368945). Previous studies have suggested that activation of extrasynaptic NMDA‐R(ex) may cause a sufficient local depolarization to remove the Mg2+ block on NMDA‐Rsyn, allowing for their activation. The purpose of this study is to test the hypothesis that, following acute HFD exposure, increased activation of DMV NMDA‐Rsyn occurs subsequent to activation of NMDA‐Rex, and is responsible for the regulation of caloric intake.Whole‐cell patch clamp recordings were made from gastric‐projecting DMV neurons in thin (300μm) brainstem slices of Sprague‐Dawley rats, 4–6 weeks of age. Rats were fed a control or HFD (14%, 60% kcal from fat, respectively) for 3–5 days prior to experimentation. The effects of the NMDA‐Rex antagonist, memantine (30μM), and the glutamate uptake inhibitor, dihydrokainate (DHK; 30μM), on NMDAsyn‐mediated miniature excitatory postsynaptic currents (mEPSCs) and DMV neuronal excitability (action potential firing; AP) were assessed. The ability of 4th ventricular application of memantine or DHK (50 and 20pmol/2μL, respectively) to attenuate or uncover the inhibitory effects of the glutamate antagonist, kynurenic acid (Kyn A, 100pmol/60nL) on gastric tone and motility were recorded in anesthetized rats. Implantation of indwelling 4th ventricular cannulae allowed for chronic administration of memantine (50pmol/2mL); food intake was measured twice daily for 10 days prior to, and throughout, HFD exposure in freely moving rats, to assess the role of NMDA‐Rex on the homeostatic regulation of caloric intake.Following acute HFD, memantine prevented the inhibitory actions of the NMDA‐Rsyn antagonist AP5 (25mM) on mEPSC frequency and AP firing rate in DMV neurons. In control rats, however, increasing extracellular glutamate concentration with DHK uncovered this inhibitory effect pf AP5. Brainstem microinjection of memantine attenuated the glutamate‐mediated decrease in gastric tone and motility observed following HFD exposure, while DHK uncovered this effect in control rats. Chronic application of memantine attenuated the homeostatic regulation of caloric intake observed following acute HFD exposure (for all data and statistics, see Table 1).These data suggest that the acute HFD‐induced increase in NMDA‐Rsyn activation occurs following NMDA‐Rex activation, although the source of this glutamate remains to be elucidated. This mechanism is also responsible for the restoration of caloric balance. Understanding these mechanisms and, importantly, how they are lost following chronic HFD exposure, may provide a mechanistic understanding hyperphagia and weight gain as well as identify potential therapeutic targets for the treatment of obesity.Support or Funding InformationNIH DK111667 to KNB NIH F31 118833 to CC Memantine + AP5 in HFD DHK + AP5 in CTL In vitro mEPSC Frequency 96±6.2% of baseline, N=6 73±6.7% of baseline *, N=13 AP firing rate 111±11.0% of baseline, N=9 73±10.9% of baseline *, N=15 Memantine+ KynA in HFD DHK + KynA in CTL Antrum Corpus Antrum Corpus In vivo Gastric motility −4±8.2% of baseline, N=6 −1±7.5% of baseline, N=6 −53±4.6% of baseline *, N=6 −44±9.2% of baseline *, N=6 Gastric tone −6±10.8 mg, N=6 −15±18.0 mg, N=6 −86±17.3 mg *, N=6 −123±34.23mg *, N=6 4th Ventricular Memantine (AUC) 4th Ventricular PBS (AUC) Caloric Intake 251±22.9 *, N=6 178±16.2, N=6 *P&lt;0.05 compared to controls or baseline from Students T‐Test

Neural Mechanisms Responsible for the Regulation of Caloric Intake Following Acute High Fat Diet are Developmentally Regulated

Previous studies have shown that, in adulthood, acute high fat diet (HFD) exposure is associated with an initial period of hyperphagia before caloric intake is restored within 3–5 days. This period of homeostasis is accompanied by activation of NMDA receptors (NMDA‐R) and increased excitability of identified vagal efferent motoneurons regulating gastric motility. The mechanisms responsible regulation of caloric intake, as well as how and why these mechanisms are lost, are critical to understanding the development of obesity. Alterations in vagal neurocircuit maturation following perinatal (p)HFD exposure, has profound effects on neural control of gastric motility in adulthood, including the failure to regulate caloric intake, which may increase the predisposition to develop obesity. Our previous studies have shown that, following pHFD, inhibitory synaptic inputs to vagal efferent motoneurons are increased, which may prevent the voltage‐dependent activation of synaptic NMDA‐Rs. The purpose of this study was to test the hypothesis that pHFD exposure attenuates the homeostatic activation of DMV NMDA‐Rs following acute HFD exposure in adulthood, leading to dysregulated gastric motility.Sprague Dawley rats were exposed to a control or HFD (14% or 60% kcal from fat, respectively) during postnatal (P) days 1–10. In adulthood, (&gt;P28) rats were re‐exposed to a HFD for 3–5 days prior to experimentation. Whole‐cell patch clamp recordings were made from DMV neurons and the effects of the NMDA‐R antagonist, AP5 (25μM) was tested on miniature excitatory postsynaptic current (mEPSC) amplitude and action potential (AP) firing rates. The 13C octanoic acid breath test was used to determine the effects of pHFD (P1–10) exposure on basal gastric emptying rates. Food intake and body weight was measured twice daily for 10 days prior to, and throughout, adult exposure to HFD.pHFD (P1–10) exposure attenuated the actions of AP5 to inhibit DMV NMDA‐R activation (mEPSC amplitude: 103±5.7% vs. 35±9.4%, P&lt;0.05, N=4–6), and to decrease neuronal excitability (AP firing rate: 102±7.7% vs. 21±13.7% of baseline, P&lt;0.05; N=4–7) observed previously following acute HFD exposure in adulthood. Measurement of gastric emptying rates suggest that pHFD exposure itself significantly delayed baseline gastric emptying (73±1.4 vs. 63±4.3min, P&lt;0.05 N=8–12), and, unlike control rats, gastric emptying was not delayed further by adult dietary challenge (102±13.0% vs. 144±15.8% of baseline, respectively, P&gt;0.05, N=4–6). Additionally, rats that were exposed to a pHFD (P1–10) did not regulate their caloric intake within 3–5 days of adult HFD exposure, as observed control rats (AUC: 215±28.0 vs. 393±34.7, P&lt;0.05, N=9–16).These data suggest that pHFD (P1–10) exposure disrupts central vagal neurocircuit development, increasing the inhibitory drive to DMV neurons, delaying gastric emptying, and preventing the homeostatic regulation of gastric emptying and caloric intake in response to acute adult HFD exposure. Understanding how diet affects the development of vagal neurocircuits is critical for identifying therapeutic targets for the development of obesity.Support or Funding InformationNIH DK111667 to KNB NIH F31 118833 to CC

Chronic Stress Induces Persistent Hypertension and Increases NMDA Receptor Activity in the Paraventricular Nucleus in Borderline Hypertensive Rats

Stress activates sympathetic outflow and promotes hypertension development in humans with genetic predisposition. The enhanced N‐methyl‐D‐aspartate receptor (NMDAR) activity in the hypothalamus paraventricular nucleus (PVN) plays a critical role in controlling sympathetic output in hypertension. However, it is unclear how chronic stress affects NMDAR activity in the PVN in borderline hypertensive rats (BHR). BHR are the F1 male offspring from crossing female spontaneously hypertensive rats with male Wistars Kyoto rats (WKY). Arterial blood pressure (ABP) was monitored in free moving rats by using radiotelemetry. Using a 6‐week chronic unpredictable stress protocol, we found that stressed WKY rats had increased ABP during chronic stress but ABP returned to the normal level within a few days after stress. However, the stressed BHR showed a greater increase in ABP during stress and the elevated ABP was sustained for at least 2 weeks after discontinuing stress. Furthermore, whole‐cell voltage clamp recording in brain slices showed that the amplitude of evoked‐NMDAR currents in spinally projecting PVN neurons was significantly higher in stressed BHR than in non‐stressed BHR controls. Inhibiting α2δ‐1 with gabapentin normalized the increased synaptic NMDAR activity of spinally projecting PVN neurons in stressed BHR. In addition, the expression level of GluN1 and α2δ‐1 in the PVN was significantly greater in stressed BHR than in stressed WKY or non‐stressed BHR. These results suggest that chronic stress induces persistent hypertension in BHR, which is associated with α2δ‐1‐mediated NMDAR hyperactivity in PVN presympathetic neurons.Support or Funding InformationAcknowledgmentsSupported by NIH grants HL131161 and HL142133.

The Synaptonuclear Messenger RNF10 Acts as an Architect of Neuronal Morphology.

The Ring Finger Protein 10 [RNF10] is a novel synapse-to-nucleus signaling protein that specifically links activation of synaptic NMDA receptors to modulation of gene expression. RNF10 dissociation from the GluN2A subunit of the NMDA receptor represents the first step of its synaptonuclear transport and it is followed by an importin-dependent translocation into the nucleus. Here, we have identified protein kinase C [PKC]-dependent phosphorylation of RNF10 Ser31 as a key step for RNF10 detachment from NMDA receptor and its subsequent trafficking to the nucleus. We show that pSer31-RNF10 plays a role both in synaptonuclear signaling and in neuronal morphology. In particular, the prevention of Ser31 RNF10 phosphorylation induces a decrease in spine density, neuronal branching, and CREB signaling, while opposite effects are obtained by mimicking a stable RNF10 phosphorylation at Ser31. Overall, these results add novel information about the functional and structural role of synaptonuclear protein messengers in shaping dendritic architecture in hippocampal neurons.

Acute High‐Fat Diet Induced Modulation of Glutamatergic Currents in Dorsal Motor Nucleus of the Vagus Neurons is Dependent on Activation of Extrasynaptic NMDA Receptors

Following HFD exposure, both humans and experimental animals undergo a brief period of hyperphagia (1–2 days), before caloric balance is restored (3–5 days). We showed previously that acute HFD increases glutamatergic transmission to neurons of the dorsal motor nucleus of the vagus (DMV) via activation of synaptic NMDA receptors (NMDA‐R), increasing motoneuron excitability and efferent control of gastric function (PMID: 29368945) although the mechanism responsible has not been elucidated. Previous studies have suggested, however, that activation of extrasynaptic NMDA‐Rs may cause sufficient local depolarization to remove the Mg2+ block on NMDA‐Rs, allowing for their activation. The purpose of this study is to test the hypothesis that the increased synaptic NMDA‐R activation in DMV neurons following acute HFD exposure occurs subsequent to activation of extrasynaptic NMDA‐R.Whole‐cell patch clamp recordings were made from gastric‐projecting DMV neurons in thin (300μm) brainstem slices of Sprague‐Dawley rats, 4–6 weeks of age. Rats were fed control or HFD (14% and 60% kcal from fat, respectively) for 3–5 days prior to experimentation. The effects of increasing (from 1.2mM to 2.4mM) or decreasing (to 0.6mM) extracellular levels of Mg2+, the NMDA‐R antagonist memantine (30μM), and the glutamate uptake inhibitor, dihydrokainate (DHK; 30μM), on NMDA‐mediated miniature excitatory postsynaptic currents (mEPSCs) and DMV neuronal excitability (action potential firing rate; AP) were assessed. In vivo recordings of gastric tone and motility were made in response to brainstem microinjection of DHK or 4th ventricular application of memantine (60pmol/60nL and 50pmol/2mL, respectively).Following acute HFD, the NMDA‐R antagonist AP5 (25mM) decreases mEPSC frequency and AP firing rate in DMV neurons; these actions were blocked by memantine (65.1±9% vs. 12.6±9% and 78.9±14% vs. 18.1±8%, respectively n=5–13, P&lt;0.05). In control rate, however, increasing extracellular glutamate concentration with DHK uncovered inhibitory actions of AP5 on mEPSCs and AP firing rate (115.7±12% vs. 77.2%±6% and 87.6%±14% vs. 70.5%±16% respectively, n=5–15, P&lt;0.05). Furthermore, increasing extracellular [Mg2+] blocked the effects of AP5 following acute HFD exposure (89.0±25%, n=4, p&gt;0.05) whereas decreasing the extracellular [Mg2+] uncovered synaptic NMDA‐R mediated synaptic currents in control rats (48.6±5%, n=4, P&lt;0.05). Preliminary in vivo recordings suggest that brainstem microinjection of memantine attenuated the glutamate‐dependent decrease in gastric tone and motility following acute HFD exposure, whereas DHK uncovered this effect in control rats.These data suggest that the acute HFD‐induced increase in synaptic NMDA‐R activation occurs following extrasynaptic NMDA‐R activation, although the source of this extrasynaptic glutamate remains to be elucidated. Understanding these mechanisms and, importantly, how they are lost following continued HFD exposure, may provide a mechanistic understanding of hyperphagia and weight gain as well as identify potential therapeutic targets for the treatment of obesity.Support or Funding InformationNIH DK111667 to KNBThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.