Inhibition of Food Intake by PACAP in the Hypothalamic Ventromedial Nuclei is Mediated by NMDA Receptors

Central injections of pituitary adenylate cyclase-activating polypeptide (PACAP) into the ventromedial nuclei (VMN) of the hypothalamus produce hypophagia that is dependent upon the PAC1 receptor; however, the signaling downstream of this receptor in the VMN is unknown. Though PACAP signaling has many targets, this neuropeptide has been shown to influence glutamate signaling in several brain regions through mechanisms involving NMDA receptor potentiation via activation of the Src family of protein tyrosine kinases. With this in mind, we examined the Src-NMDA receptor signaling pathway as a target for PACAP signaling in the VMN that may mediate its effects on feeding behavior. Under nocturnal feeding conditions, NMDA receptor antagonism prior to PACAP administration into the VMN attenuated PACAP-mediated decreases in feeding suggesting that glutamatergic signaling via NMDA receptors is necessary for PACAP-induced hypophagia. Furthermore, PACAP administration into the VMN resulted in increased tyrosine phosphorylation of the GluN2B subunit of the NMDA receptor, and inhibition of Src kinase activity also blocked the effects of PACAP administration into the VMN on feeding behavior. These results indicate that PACAP neurotransmission in the VMN likely augments glutamate signaling by potentiating NMDA receptors activity through the tyrosine phosphorylation events mediated by the Src kinase family, and modulation of NMDA receptor activity by PACAP in the hypothalamus may be a primary mechanism for its regulation of food intake

Treatment resistance NMDA receptor pathway polygenic score is associated with brain glutamate in schizophrenia

Dysfunction of glutamate neurotransmission has been implicated in the pathophysiology of schizophrenia and may be particularly relevant in severe, treatment-resistant symptoms. The underlying mechanism may involve hypofunction of the NMDA receptor. We investigated whether schizophrenia-related pathway polygenic scores, composed of genetic variants within NMDA receptor encoding genes, are associated with cortical glutamate in schizophrenia. Anterior cingulate cortex (ACC) glutamate was measured in 70 participants across 4 research sites using Proton Magnetic Resonance Spectroscopy (1H-MRS). Two NMDA receptor gene sets were sourced from the Molecular Signatories Database and NMDA receptor pathway polygenic scores were constructed using PRSet. The NMDA receptor pathway polygenic scores were weighted by single nucleotide polymorphism (SNP) associations with treatment-resistant schizophrenia, and associations with ACC glutamate were tested. We then tested whether NMDA receptor pathway polygenic scores with SNPs weighted by associations with non-treatment-resistant schizophrenia were associated with ACC glutamate. A higher NMDA receptor complex pathway polygenic score was significantly associated with lower ACC glutamate (β = −0.25, 95 % CI = −0.49, −0.02, competitive p = 0.03). When SNPs were weighted by associations with non-treatment-resistant schizophrenia, there was no association between the NMDA receptor complex pathway polygenic score and ACC glutamate (β = 0.05, 95 % CI = −0.18, 0.27, competitive p = 0.79). These results provide initial evidence of an association between common genetic variation implicated in NMDA receptor function and ACC glutamate levels in schizophrenia. This association was specific to when the NMDA receptor complex pathway polygenic score was weighted by SNP associations with treatment-resistant schizophrenia

Pathologically Activated Neuroprotection via Uncompetitive Blockade of \u3cem\u3eN\u3c/em\u3e-Methyl-d-aspartate Receptors with Fast Off-rate by Novel Multifunctional Dimer Bis(propyl)-cognitin

Uncompetitive N-methyl-d-aspartate (NMDA) receptor antagonists with fast off-rate (UFO) may represent promising drug candidates for various neurodegenerative disorders. In this study, we report that bis(propyl)-cognitin, a novel dimeric acetylcholinesterase inhibitor and γ-aminobutyric acid subtype A receptor antagonist, is such an antagonist of NMDA receptors. In cultured rat hippocampal neurons, we demonstrated that bis(propyl)-cognitin voltage-dependently, selectively, and moderately inhibited NMDA-activated currents. The inhibitory effects of bis(propyl)-cognitin increased with the rise in NMDA and glycine concentrations. Kinetics analysis showed that the inhibition was of fast onset and offset with an off-rate time constant of 1.9 s. Molecular docking simulations showed moderate hydrophobic interaction between bis(propyl)-cognitin and the MK-801 binding region in the ion channel pore of the NMDA receptor. Bis(propyl)-cognitin was further found to compete with [3H]MK-801 with a Ki value of 0.27 μm, and the mutation of NR1(N616R) significantly reduced its inhibitory potency. Under glutamate-mediated pathological conditions, bis(propyl)-cognitin, in contrast to bis(heptyl)-cognitin, prevented excitotoxicity with increasing effectiveness against escalating levels of glutamate and much more effectively protected against middle cerebral artery occlusion-induced brain damage than did memantine. More interestingly, under NMDA receptor-mediated physiological conditions, bis(propyl)-cognitin enhanced long-term potentiation in hippocampal slices, whereas MK-801 reduced and memantine did not alter this process. These results suggest that bis(propyl)-cognitin is a UFO antagonist of NMDA receptors with moderate affinity, which may provide a pathologically activated therapy for various neurodegenerative disorders associated with NMDA receptor dysregulation

A Site of Alcohol Action at the NMDA Receptor M3-M4 Domain Interface

The N-methyl-D-aspartate (NMDA) glutamate receptor is a major target of ethanol in the brain. Previous studies have identified positions in the third and fourth membrane-associated (M) domains of the NMDA receptor GluN1 and GluN2A subunits that influence alcohol sensitivity. The structural model of the NMDA receptor, predicted from the structure of the related GluA2 subunit, indicates a close apposition of the alcohol-sensitive positions in M3 and M4 between the two subunit types. We investigated possible interactions between the M3 and M4 domain positions of the two subunit types affecting the ethanol sensitivity of the receptor by using dual substitution mutants. In an initial screen of single-substitution mutants, we found that a position in both subunits adjacent to one previously identified, GluN1(G638) and GluN2A(F636), can strongly regulate ethanol sensitivity. Significant interactions affecting ethanol inhibition were observed at four pairs of positions in GluN1/GluN2A: G638/M823, F639/L824, M818/F636, and L819/F637. Two of these interactions involve a position in M4 of both subunits, GluN1(M818) and GluN2A(L824), that does not by itself alter ethanol sensitivity, and one of the previously identified positions affecting ethanol sensitivity, GluN2A(A825), did not appear to interact with any other position tested. These results also indicate a shift by one position of the predicted alignment of the GluN1 M4 domain. These findings have allowed for the refinement of the NMDA receptor M domain structure, and support the existence of four sites of alcohol action on the NMDA receptor at the M3-M4 domain intersubunit interfaces. These studies were supported by grants R01 AA015203-01A1 and AA015203-06A1 from the NIAAA to R.W.P

Chimeric glutamate receptor subunits reveal the transmembrane domain is sufficient for NMDA receptor pore properties but some positive allosteric modulators require additional domains

NMDA receptors are ligand-gated ion channels that underlie transmission at excitatory synapses and play an important role in regulating synaptic strength and stability. Functional NMDA receptors require two copies of the GluN1 subunit coassembled with GluN2 (and/or GluN3) subunits into a heteromeric tetramer. A diverse array of allosteric modulators can upregulate or downregulate NMDA receptor activity. These modulators include both synthetic compounds and endogenous modulators, such as cis-unsaturated fatty acids, 24(S)-hydroxycholesterol, and various neurosteroids. To evaluate the structural requirements for the formation and allosteric modulation of NMDA receptor pores, we have replaced portions of the rat GluN1, GluN2A, and GluN2B subunits with homologous segments from the rat GluK2 kainate receptor subunit. Our results with these chimeric constructs show that the NMDA receptor transmembrane domain is sufficient to account for most pore properties, but that regulation by some allosteric modulators requires additional cytoplasmic or extracellular domains. SIGNIFICANCE STATEMENT Glutamate receptors mediate excitatory synaptic transmission by forming cation channels through the membrane that open upon glutamate binding. Although many compounds have been identified that regulate glutamate receptor activity, in most cases the detailed mechanisms that underlie modulation are poorly understood. To identify what parts of the receptor are essential for pore formation and sensitivity to allosteric modulators, we generated chimeric subunits that combined segments from NMDA and kainate receptors, subtypes with distinct pharmacological profiles. Surprisingly, our results identify separate domain requirements for allosteric potentiation of NMDA receptor pores by pregnenolone sulfate, 24(S)-hydroxycholesterol, and docosahexaenoic acid, three endogenous modulators derived from membrane constituents. Understanding where and how these compounds act on NMDA receptors should aid in designing better therapeutic agents

Subanesthetic ketamine treatment promotes abnormal interactions between neural subsystems and alters the properties of functional brain networks

Acute treatment with subanesthetic ketamine, a non-competitive N-methyl-D-aspartic acid (NMDA) receptor antagonist, is widely utilized as a translational model for schizophrenia. However, how acute NMDA receptor blockade impacts on brain functioning at a systems level, to elicit translationally relevant symptomatology and behavioral deficits, has not yet been determined. Here, for the first time, we apply established and recently validated topological measures from network science to brain imaging data gained from ketamine-treated mice to elucidate how acute NMDA receptor blockade impacts on the properties of functional brain networks. We show that the effects of acute ketamine treatment on the global properties of these networks are divergent from those widely reported in schizophrenia. Where acute NMDA receptor blockade promotes hyperconnectivity in functional brain networks, pronounced dysconnectivity is found in schizophrenia. We also show that acute ketamine treatment increases the connectivity and importance of prefrontal and thalamic brain regions in brain networks, a finding also divergent to alterations seen in schizophrenia. In addition, we characterize how ketamine impacts on bipartite functional interactions between neural subsystems. A key feature includes the enhancement of prefrontal cortex (PFC)-neuromodulatory subsystem connectivity in ketamine-treated animals, a finding consistent with the known effects of ketamine on PFC neurotransmitter levels. Overall, our data suggest that, at a systems level, acute ketamine-induced alterations in brain network connectivity do not parallel those seen in chronic schizophrenia. Hence, the mechanisms through which acute ketamine treatment induces translationally relevant symptomatology may differ from those in chronic schizophrenia. Future effort should therefore be dedicated to resolve the conflicting observations between this putative translational model and schizophrenia

Gene expression of NMDA receptor subunits in the cerebellum of elderly patients with schizophrenia

To determine if NMDA receptor alterations are present in the cerebellum in schizophrenia, we measured NMDA receptor binding and gene expression of the NMDA receptor subunits in a post-mortem study of elderly patients with schizophrenia and non-affected subjects. Furthermore, we assessed influence of genetic variation in the candidate gene neuregulin-1 (NRG1) on the expression of the NMDA receptor in an exploratory study. Post-mortem samples from the cerebellar cortex of ten schizophrenic patients were compared with nine normal subjects. We investigated NMDA receptor binding by receptor autoradiography and gene expression of the NMDA receptor subunits NR1, NR2A, NR2B, NR2C and NR2D by in situ hybridization. For the genetic study, we genotyped the NRG1 polymorphism rs35753505 (SNP8NRG221533). Additionally, we treated rats with the antipsychotics haloperidol or clozapine and assessed cerebellar NMDA receptor binding and gene expression of subunits to examine the effects of antipsychotic treatment. Gene expression of the NR2D subunit was increased in the right cerebellum of schizophrenic patients compared to controls. Individuals carrying at least one C allele of rs35753505 (SNP8NRG221533) showed decreased expression of the NR2C subunit in the right cerebellum, compared to individuals homozygous for the T allele. Correlation with medication parameters and the animal model revealed no treatment effects. In conclusion, increased NR2D expression results in a hyperexcitable NMDA receptor suggesting an adaptive effect due to receptor hypofunction. The decreased NR2C expression in NRG1 risk variant may cause a deficit in NMDA receptor function. This supports the hypothesis of an abnormal glutamatergic neurotransmission in the right cerebellum in the pathophysiology of schizophrenia

Interactions Among Positions in the Third and Fourth Membrane-Associated Domains at the Intersubunit Interface of the N-Methyl-D-Aspartate Receptor Forming Sites of Alcohol Action

The N-methyl-d-aspartate (NMDA) glutamate receptor is a major target of ethanol in the brain. Previous studies have identified positions in the third and fourth membrane-associated (M) domains of the NMDA receptor GluN1 and GluN2A subunits that influence alcohol sensitivity. The predicted structure of the NMDA receptor, based on that of the related GluA2 subunit, indicates a close apposition of the alcohol-sensitive positions in M3 and M4 between the two subunit types. We tested the hypothesis that these positions interact to regulate receptor kinetics and ethanol sensitivity by using dual substitution mutants. In single-substitution mutants, we found that a position in both subunits adjacent to one previously identified, GluN1(Gly-638) and GluN2A(Phe-636), can strongly regulate ethanol sensitivity. Significant interactions affecting ethanol inhibition and receptor deactivation were observed at four pairs of positions in GluN1/GluN2A: Gly-638/Met-823, Phe-639/Leu-824, Met-818/Phe-636, and Leu-819/Phe-637; the latter pair also interacted with respect to desensitization. Two interactions involved a position in M4 of both subunits, GluN1(Met-818) and GluN2A(Leu-824), that does not by itself alter ethanol sensitivity, whereas a previously identified ethanol-sensitive position, GluN2A(Ala-825), did not unequivocally interact with any other position tested. These results also indicate a shift by one position of the predicted alignment of the GluN1 M4 domain. These findings have allowed for the refinement of the NMDA receptor M domain structure, demonstrate that this region can influence apparent agonist affinity, and support the existence of four sites of alcohol action on the NMDA receptor, each consisting of five amino acids at the M3-M4 domain intersubunit interfaces

Slow Mg²⁺ unblock and inherent voltage dependence of NMDA receptors

N-methyl-D-aspartate (NMDA) receptors are a subtype of ligand-gated ionotropic glutamate receptors that are involved in most fast, excitatory neuronal transmission in the mammalian central nervous system (CNS). NMDA receptor activity is crucial for normal brain function, and NMDA receptor dysregulation has been linked to a number of diseases of the CNS. There are several NMDA receptor subtypes. Each subtype has a unique temporal and spatial expression pattern, suggesting that different subtypes play different physiological roles in the CNS. Here, we have investigated how changes in membrane voltage impact the activity of various NMDA receptor subtypes in the absence and presence of the highly physiologically relevant channel blocker magnesium (Mg²⁺). Mg²⁺ strongly blocks all NMDA receptor subtypes at, and near, typical resting membrane potentials. Only upon depolarization is Mg²⁺ block relieved. We found that, upon depolarization, NMDA receptors containing NR2C or NR2D subunits unblock Mg2+ very rapidly (τ < 1 ms), while Mg²⁺ unblock from NMDA receptors containing the NR2A or NR2B subunit displays a prominent slow component (τ of several ms). We go on to show that the slow component of Mg²⁺ unblock from NR2A and NR2B containing NMDA receptors actually reflects inherent voltage-dependent alterations in NMDA receptor gating. In the absence of Mg²⁺, NR2A and NR2B containing NMDA receptor currents are enhanced upon membrane depolarization. Utilizing data collected in the absence of Mg²⁺, we developed kinetic models of NR2A and NR2B containing NMDA receptors that included inherent voltage sensitivity such that the receptors open more rapidly at positive membrane potentials. The NR2 subunit specific models reproduce experimentally recorded currents during changes in membrane voltage in both the absence and presence of Mg²⁺. The models also reproduce several other previously described voltage-dependent characteristics of the NMDA receptor channel. Inherent voltage dependence further emphasizes the strong link between NMDA receptor activity and neuronal depolarization