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 Grin2b+/+ (+/+) male and female mice to assess the functional impact of the variant. Whole-cell NMDAR currents were reduced in neurons prepared from L825V/+ compared to +/+ mice. Peak amplitude of NMDAR-mediated evoked excitatory postsynaptic currents (NMDAR-eEPSC) was not changed, but NMDAR-eEPSCs in L825V/+ neurons had faster deactivation compared to +/+ 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 to wild-type 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.Significance statement Variants in genes for subunits of N-methyl-D-aspartate receptors (NMDARs), a subtype of ionotropic glutamate receptors, are associated with neurodevelopmental disorders. Here we have generated a transgenic mouse model of a de-novo missense GRIN2B gene variant, identified in a patient with intellectual disability and autism, that introduces a single amino acid substitution (L825V) in the NMDAR GluN2B subunit. Di- and triheteromeric NMDARs containing the GluN2B(L825V) subunit have a reduced channel open probability. Synaptic NMDAR currents in neurons from heterozygous L825V/+ mice have accelerated deactivation and reduced ifenprodil sensitivity, suggesting synaptic loss of GluN2B function. L825V/+ mice show increased anxiety, impaired sensorimotor gating, and cognitive deficits, consistent with patient symptoms. Our study describes a clinically relevant mouse model of GRIN2B-related neurodevelopmental pathology.

Disease-associated nonsense and frame-shift variants resulting in the truncation of the GluN2A or GluN2B C-terminal domain decrease NMDAR surface expression and reduce potentiating effects of neurosteroids.

N-methyl-D-aspartate receptors (NMDARs) play a critical role in normal brain function, and variants in genes encoding NMDAR subunits have been described in individuals with various neuropsychiatric disorders. We have used whole-cell patch-clamp electrophysiology, fluorescence microscopy and in-silico modeling to explore the functional consequences of disease-associated nonsense and frame-shift variants resulting in the truncation of GluN2A or GluN2B C-terminal domain (CTD). This study characterizes variant NMDARs and shows their reduced surface expression and synaptic localization, altered agonist affinity, increased desensitization, and reduced probability of channel opening. We also show that naturally occurring and synthetic steroids pregnenolone sulfate and epipregnanolone butanoic acid, respectively, enhance NMDAR function in a way that is dependent on the length of the truncated CTD and, further, is steroid-specific, GluN2A/B subunit-specific, and GluN1 splice variant-specific. Adding to the previously described effects of disease-associated NMDAR variants on the receptor biogenesis and function, our results improve the understanding of the molecular consequences of NMDAR CTD truncations and provide an opportunity for the development of new therapeutic neurosteroid-based ligands.

Ventral posterolateral and ventral posteromedial thalamocortical neurons have distinct physiological properties.

Somatosensory information is propagated from the periphery to the cerebral cortex by two parallel pathways through the ventral posterolateral (VPL) and ventral posteromedial (VPM) thalamus. VPL and VPM neurons receive somatosensory signals from the body and head, respectively. VPL and VPM neurons may also receive cell type-specific GABAergic input from the reticular nucleus of the thalamus. Although VPL and VPM neurons have distinct connectivity and physiological roles, differences in their functional properties remain unclear as they are often studied as one ventrobasal thalamus neuron population. Here, we directly compared synaptic and intrinsic properties of VPL and VPM neurons in C57Bl/6J mice of both sexes aged P25-P32. VPL neurons showed greater depolarization-induced spike firing and spike frequency adaptation than VPM neurons. VPL and VPM neurons fired similar numbers of spikes during hyperpolarization rebound bursts, but VPM neurons exhibited shorter burst latency compared with VPL neurons, which correlated with larger sag potential. VPM neurons had larger membrane capacitance and more complex dendritic arbors. Recordings of spontaneous and evoked synaptic transmission suggested that VPL neurons receive stronger excitatory synaptic input, whereas inhibitory synapse strength was stronger in VPM neurons. This work indicates that VPL and VPM thalamocortical neurons have distinct intrinsic and synaptic properties. The observed functional differences could have important implications for their specific physiological and pathophysiological roles within the somatosensory thalamocortical network.NEW & NOTEWORTHY This study revealed that somatosensory thalamocortical neurons in the VPL and VPM have substantial differences in excitatory synaptic input and intrinsic firing properties. The distinct properties suggest that VPL and VPM neurons could process somatosensory information differently and have selective vulnerability to disease. This work improves our understanding of nucleus-specific neuron function in the thalamus and demonstrates the critical importance of studying these parallel somatosensory pathways separately.

Site of Action of Brain Neurosteroid Pregnenolone Sulfate at the N-Methyl-D-Aspartate Receptor.

N-methyl-D-aspartate receptor (NMDAR) hypofunction has been implicated in several neurodevelopmental disorders. NMDAR function can be augmented by positive allosteric modulators, including endogenous compounds, such as cholesterol and neurosteroid pregnenolone sulfate (PES). Here we report that PES accesses the receptor via the membrane, and its binding site is different from that of cholesterol. Alanine mutagenesis has identified residues that disrupt the steroid potentiating effect at the rat GluN1 (G638; I642) and GluN2B (W559; M562; Y823; M824) subunit. Molecular dynamics simulation indicates that, in the absence of PES, the GluN2B M1 helix residue W559 interacts with the M4 helix residue M824. In the presence of PES, the M1 and M4 helices of agonist-activated receptor rearrange, forming a tighter interaction with the GluN1 M3 helix residues G638 and I642. This stabilizes the open-state position of the GluN1 M3 helices. Together, our data identify a likely binding site for the NMDAR-positive allosteric modulator PES and describe a novel molecular mechanism by which NMDAR activity can be augmented.SIGNIFICANCE STATEMENT There is considerable interest in drugs that enhance NMDAR function and could compensate for receptor hypofunction associated with certain neuropsychiatric disorders. Positive allosteric modulators of NMDARs include an endogenous neurosteroid pregnenolone sulfate (PES), but the binding site of PES on the NMDAR and the molecular mechanism of potentiation are unknown. We use patch-clamp electrophysiology in combination with mutagenesis and in silico modeling to describe the interaction of PES with the NMDAR. Our data indicate that PES binds to the transmembrane domain of the receptor at a discrete group of residues at the GluN2B membrane helices M1 and M4 and the GluN1 helix M3, and that PES potentiates NMDAR function by stabilizing the open-state position of the GluN1 M3 helices.

Preferential Inhibition of Tonically over Phasically Activated NMDA Receptors by Pregnane Derivatives.

Postsynaptic N-methyl-d-aspartate receptors (NMDARs) phasically activated by presynaptically released glutamate are critical for synaptic transmission and plasticity. However, under pathological conditions, excessive activation of NMDARs by tonically increased ambient glutamate contributes to excitotoxicity associated with various acute and chronic neurological disorders. Here, using heterologously expressed GluN1/GluN2A and GluN1/GluN2B receptors and rat autaptic hippocampal microisland cultures, we show that pregnanolone sulfate inhibits NMDAR currents induced by a prolonged glutamate application with a higher potency than the NMDAR component of EPSCs. For synthetic pregnanolone derivatives substituted with a carboxylic acid moiety at the end of an aliphatic chain of varying length and attached to the steroid skeleton at C3, the difference in potency between tonic and phasic inhibition increased with the length of the residue. The steroid with the longest substituent, pregnanolone hemipimelate, had no effect on phasically activated receptors while inhibiting tonically activated receptors. In behavioral tests, pregnanolone hemipimelate showed neuroprotective activity without psychomimetic symptoms. These results provide insight into the influence of steroids on neuronal function and stress their potential use in the development of novel therapeutics with neuroprotective action. SIGNIFICANCE STATEMENT: Synaptic activation of N-methyl-d-aspartate receptors (NMDARs) plays a key role in synaptic plasticity, but excessive tonic NMDAR activation mediates excitotoxicity associated with many neurological disorders. Therefore, there is much interest in pharmacological agents capable of selectively blocking tonically activated NMDARs while leaving synaptically activated NMDARs intact. Here, we show that an endogenous neurosteroid pregnanolone sulfate is more potent at inhibiting tonically than synaptically activated NMDARs. Further, we report that a novel synthetic analog of pregnanolone sulfate, pregnanolone hemipimelate, inhibits tonic NMDAR currents without inhibiting the NMDAR component of the EPSC and shows neuroprotective activity in vivo without inducing psychomimetic side effects. These results suggest steroids may have a clinical advantage over other known classes of NMDAR inhibitors.

Cholesterol modulates open probability and desensitization of NMDA receptors.

NMDA receptors (NMDARs) are glutamate-gated ion channels that mediate excitatory neurotransmission in the CNS. Although these receptors are in direct contact with plasma membrane, lipid-NMDAR interactions are little understood. In the present study, we aimed at characterizing the effect of cholesterol on the ionotropic glutamate receptors. Whole-cell current responses induced by fast application of NMDA in cultured rat cerebellar granule cells (CGCs) were almost abolished (reduced to 3%) and the relative degree of receptor desensitization was increased (by seven-fold) after acute cholesterol depletion by methyl-ß-cyclodextrin. Both of these effects were fully reversible by cholesterol repletion. By contrast, the responses mediated by AMPA/kainate receptors were not affected by cholesterol depletion. Similar results were obtained in CGCs after chronic inhibition of cholesterol biosynthesis by simvastatin and acute enzymatic cholesterol degradation to 4-cholesten-3-one by cholesterol oxidase. Fluorescence anisotropy measurements showed that membrane fluidity increased after methyl-ß-cyclodextrin pretreatment. However, no change in fluidity was observed after cholesterol enzymatic degradation, suggesting that the effect of cholesterol on NMDARs is not mediated by changes in membrane fluidity. Our data show that diminution of NMDAR responses by cholesterol depletion is the result of a reduction of the open probability, whereas the increase in receptor desensitization is the result of an increase in the rate constant of entry into the desensitized state. Surface NMDAR population, agonist affinity, single-channel conductance and open time were not altered in cholesterol-depleted CGCs. The results of our experiments show that cholesterol is a strong endogenous modulator of NMDARs.

Activity-regulated RNA editing in select neuronal subfields in hippocampus.

RNA editing by adensosine deaminases is a widespread mechanism to alter genetic information in metazoa. In addition to modifications in non-coding regions, editing contributes to diversification of protein function, in analogy to alternative splicing. However, although splicing programs respond to external signals, facilitating fine tuning and homeostasis of cellular functions, a similar regulation has not been described for RNA editing. Here, we show that the AMPA receptor R/G editing site is dynamically regulated in the hippocampus in response to activity. These changes are bi-directional, reversible and correlate with levels of the editase Adar2. This regulation is observed in the CA1 hippocampal subfield but not in CA3 and is thus subfield/celltype-specific. Moreover, alternative splicing of the flip/flop cassette downstream of the R/G site is closely linked to the editing state, which is regulated by Ca(2+). Our data show that A-to-I RNA editing has the capacity to tune protein function in response to external stimuli.

Steric antisense inhibition of AMPA receptor Q/R editing reveals tight coupling to intronic editing sites and splicing.

Adenosine-to-Inosine (A-to-I) RNA editing is a post-transcriptional mechanism, evolved to diversify the transcriptome in metazoa. In addition to wide-spread editing in non-coding regions protein recoding by RNA editing allows for fine tuning of protein function. Functional consequences are only known for some editing sites and the combinatorial effect between multiple sites (functional epistasis) is currently unclear. Similarly, the interplay between RNA editing and splicing, which impacts on post-transcriptional gene regulation, has not been resolved. Here, we describe a versatile antisense approach, which will aid resolving these open questions. We have developed and characterized morpholino oligos targeting the most efficiently edited site--the AMPA receptor GluA2 Q/R site. We show that inhibition of editing closely correlates with intronic editing efficiency, which is linked to splicing efficiency. In addition to providing a versatile tool our data underscore the unique efficiency of a physiologically pivotal editing site.

RNA-interacting proteins act as site-specific repressors of ADAR2-mediated RNA editing and fluctuate upon neuronal stimulation.

RNA editing by adenosine deaminases that act on RNA (ADARs) diversifies the transcriptome by changing adenosines to inosines. In mammals, editing levels vary in different tissues, during development, and also in pathogenic conditions. From a screen for repressors of editing we have isolated three proteins that repress ADAR2-mediated RNA editing. The three proteins RPS14, SFRS9 and DDX15 interact with RNA. Overexpression or depletion of these proteins can decrease or increase editing levels by 15%, thus allowing a modulation of RNA editing up to 30%. Interestingly, the three proteins alter RNA editing in a substrate-specific manner that correlates with their RNA binding preferences. In mammalian cells, SFRS9 significantly affects editing of the two substrates CFLAR and cyFIP2, while the ribosomal protein RPS14 mostly inhibits editing of cyFIP2 messenger RNA. The helicase DDX15, in turn, has a strong effect on editing in Caenorhabditis elegans. Expression of the three factors decreases during mouse brain development. Moreover, expression levels of SFRS9 and DDX15 respond strongly to neuronal stimulation or repression, showing an inverse correlation with editing levels. Colocalization and immunoprecipitation studies demonstrate a direct interaction of SFRS9 and RPS14 with ADAR2, while DDX15 associates with other helicases and splicing factors. Our data show that different editing sites can be specifically altered in their editing pattern by changing the local RNP landscape.

Analysis of Surface Expression of NMDAR Subunits in Primary Hippocampal Neurons.

The expression and activity of ionotropic glutamate receptors control signal transduction at the excitatory synapses in the CNS. The NMDAR comprises two obligatory GluN1 subunits and two GluN2 or GluN3 subunits in different combinations. Each GluN subunit consists of four domains: the extracellular amino-terminal and agonist-binding domains, the transmembrane domain, and the intracellular C-terminal domain (CTD). The CTD interaction with various classes of intracellular proteins is critical for trafficking and synaptic localization of NMDARs. Amino acid mutations or the inclusion of premature stop codons in the CTD could contribute to the emergence of neurodevelopmental and neuropsychiatric disorders. Here, we describe the method of preparing primary hippocampal neurons and lentiviral particles expressing GluN subunits that can be used as a model to study cell surface expression and synaptic localization of NMDARs. We also show a simple method of fluorescence immunostaining of eGFP-tagged GluN2 subunits and subsequent microscopy technique and image analysis to study the effects of disease-associated mutations in the CTDs of GluN2A and GluN2B subunits.

The disruption of circadian rhythmicity of gene expression in the hippocampus and associated structures in Gria2R/R mice; a comparison with C57BL/6J and Adar2-/- mice strains.

Adar2-/- mice are a widely used model for studying the physiological consequences of reduced RNA editing. These mice are viable only when the Q/R editing site of the Gria2 subunit of the AMPA receptor is constitutively mutated to the codon for arginine, and Gria2R/R mice often serve as the sole control for Adar2-/- mice. Our study aimed to investigate whether ADAR2 inactivity and the Gria2R/R phenotype affect the rhythmicity of the circadian clock gene pattern and the expression of Gria1 and Gria2 subunits in the suprachiasmatic nucleus (SCN), hippocampus, parietal cortex and liver. Our data show that Gria2R/R mice completely lost circadian rhythmicity in the hippocampus compared to Adar2-/- mice. Compared to C57BL/6J mice, the expression profiles in the hippocampus and parietal cortex of Gria2R/R mice differ to the same extent as in Adar2-/-. No alterations were detected in the circadian profiles in the livers. These data suggest that the natural gradual postnatal increase in the editing of the Q/R site of the Gria2 subunit may be important for the development of circadian clockwork in some brain structures, and the use of Gria2R/R mice as the only control to Adar2-/- mice in the experiments dependent on the hippocampus and parietal cortex should therefore be considered.

Effects of Pregnanolone Glutamate and Its Metabolites on GABAA and NMDA Receptors and Zebrafish Behavior.

Multiple molecular targets have been identified to mediate membrane-delimited and nongenomic effects of natural and synthetic steroids, but the influence of steroid metabolism on neuroactive steroid signaling is not well understood. To begin to address this question, we set out to identify major metabolites of a neuroprotective synthetic steroid 20-oxo-5ß-pregnan-3α-yl l-glutamyl 1-ester (pregnanolone glutamate, PAG) and characterize their effects on GABAA and NMDA receptors (GABARs, NMDARs) and their influence on zebrafish behavior. Gas chromatography-mass spectrometry was used to assess concentrations of PAG and its metabolites in the hippocampal tissue of juvenile rats following intraperitoneal PAG injection. PAG is metabolized in the peripheral organs and nervous tissue to 20-oxo-17α-hydroxy-5ß-pregnan-3α-yl l-glutamyl 1-ester (17-hydroxypregnanolone glutamate, 17-OH-PAG), 3α-hydroxy-5ß-pregnan-20-one (pregnanolone, PA), and 3α,17α-dihydroxy-5ß-pregnan-20-one (17-hydroxypregnanolone, 17-OH-PA). Patch-clamp electrophysiology experiments in cultured hippocampal neurons demonstrate that PA and 17-OH-PA are potent positive modulators of GABARs, while PAG and 17-OH-PA have a moderate inhibitory effect at NMDARs. PAG, 17-OH-PA, and PA diminished the locomotor activity of zebrafish larvae in a dose-dependent manner. Our results show that PAG and its metabolites are potent modulators of neurotransmitter receptors with behavioral consequences and indicate that neurosteroid-based ligands may have therapeutic potential.

Circadian Regulation of GluA2 mRNA Processing in the Rat Suprachiasmatic Nucleus and Other Brain Structures.

The mammalian circadian system consists of a major circadian pacemaker located in the suprachiasmatic nucleus (SCN) of the hypothalamus and peripheral clocks in the body, including brain structures. The SCN depends on glutamatergic neurotransmission for transmitting signals from the retina, and it exhibits spontaneous 24-h rhythmicity in neural activity. The aim of this work was to evaluate the degree and circadian rhythmicity of AMPA receptor GluA2 subunit R/G editing and alternative flip/flop splicing in the SCN and other brain structures in Wistar rats. Our data show that the circadian rhythmicity in the SCN's GluA2 mRNA level was highest at dawn, while the circadian rhythm in R/G editing peaked at CT10 and the rhythmic flip varied with the acrophase at the late subjective night. The circadian rhythmicity was confirmed for R/G editing and splicing in the CA3 hippocampal area, and rhythmic variation of the flip isoform was also measured in the olfactory bulbs and cerebellum. The correlations between the R/G editing and alternative flip/flop splicing revealed a structure-dependent direction. In the hippocampus, the edited (G)-form level was positively correlated with the flip variant abundance, in accord with published data; by contrast, in the SCN, the flip variant was in associated more with the unedited (R) form. The edited (G) form and flop isoform also predominated in the retina and cerebellum.

Evidence for the Association between the Intronic Haplotypes of Ionotropic Glutamate Receptors and First-Episode Schizophrenia.

The heritable component of schizophrenia (SCH) as a polygenic trait is represented by numerous variants from a heterogeneous group of genes each contributing a relatively small effect. Various SNPs have already been found and analyzed in genes encoding the NMDAR subunits. However, less is known about genetic variations of genes encoding the AMPA and kainate receptor subunits. We analyzed sixteen iGluR genes in full length to determine the sequence variability of iGluR genes. Our aim was to describe the rate of genetic variability, its distribution, and the co-occurrence of variants and to identify new candidate risk variants or haplotypes. The cumulative effect of genetic risk was then estimated using a simple scoring model. GRIN2A-B, GRIN3A-B, and GRIK4 genes showed significantly increased genetic variation in SCH patients. The fixation index statistic revealed eight intronic haplotypes and an additional four intronic SNPs within the sequences of iGluR genes associated with SCH (p < 0.05). The haplotypes were used in the proposed simple scoring model and moreover as a test for genetic predisposition to schizophrenia. The positive likelihood ratio for the scoring model test reached 7.11. We also observed 41 protein-altering variants (38 missense variants, four frameshifts, and one nonsense variant) that were not significantly associated with SCH. Our data suggest that some intronic regulatory regions of iGluR genes and their common variability are among the components from which the genetic predisposition to SCH is composed.

NMDA Receptor Opening and Closing-Transitions of a Molecular Machine Revealed by Molecular Dynamics.

We report the first complete description of the molecular mechanisms behind the transition of the N-methyl-d-aspartate (NMDA) receptor from the state where the transmembrane domain (TMD) and the ion channel are in the open configuration to the relaxed unliganded state where the channel is closed. Using an aggregate of nearly 1 µs of unbiased all-atom implicit membrane and solvent molecular dynamics (MD) simulations we identified distinct structural states of the NMDA receptor and revealed functionally important residues (GluN1/Glu522, GluN1/Arg695, and GluN2B/Asp786). The role of the "clamshell" motion of the ligand binding domain (LBD) lobes in the structural transition is supplemented by the observed structural similarity at the level of protein domains during the structural transition, combined with the overall large rearrangement necessary for the opening and closing of the receptor. The activated and open states of the receptor are structurally similar to the liganded crystal structure, while in the unliganded receptor the extracellular domains perform rearrangements leading to a clockwise rotation of up to 45 degrees around the longitudinal axis of the receptor, which closes the ion channel. The ligand-induced rotation of extracellular domains transferred by LBD-TMD linkers to the membrane-anchored ion channel is responsible for the opening and closing of the transmembrane ion channel, revealing the properties of NMDA receptor as a finely tuned molecular machine.

The role of proline residues in the structure and function of human MT2 melatonin receptor.

Melatonin functions as an essential regulator of various physiological processes in all vertebrate species. In mammals, two G protein-coupled melatonin receptors (GPCR) mediate some melatonin's actions: MT1 and MT2. Transmembrane domains (TM) of most GPCRs contain a set of highly conserved proline residues that presumably play important structural and functional roles. As TM segments of MT2 receptor display several interesting differences in expression of specific proline residues compared to other rhodopsin-like receptors (rGPCRs), we investigated the role of proline residues in the structure and function of this receptor. All prolines in TM segments of MT2 receptor were individually replaced with alanine and/or glycine. In addition, the unusual NAxxY motif located in TM7 was mutated to generate highly conserved NPxxY motif found in the majority of rGPCR proteins. Following transient expression in CHO-K1 cells, binding properties of the mutant receptors and their ability to transduce signals were analyzed using (125)I-mel- and [(35)S]GTPgammaS-binding assays, respectively. The impact of the performed mutations on the receptor structure was assessed by molecular dynamic simulations of MT2 receptors embedded in the fully hydrated phospholipid bilayer. Our results indicate that residues P174, P212 and P266 are important for the ligand binding and/or signaling of the human MT2 receptor. We also show that changes within the unusual NAxxY sequence in the TM7 (mutations A305P and A305V) produce defective MT2 receptors indicating an important role of this motif in the function of melatonin receptors.

The LILI Motif of M3-S2 Linkers Is a Component of the NMDA Receptor Channel Gate.

N-methyl-D-aspartate receptors (NMDARs) mediate excitatory synaptic transmission in the central nervous system, underlie the induction of synaptic plasticity, and their malfunction is associated with human diseases. Native NMDARs are tetramers composed of two obligatory GluN1 subunits and various combinations of GluN2A-D or, more rarely, GluN3A-B subunits. Each subunit consists of an amino-terminal, ligand-binding, transmembrane and carboxyl-terminal domain. The ligand-binding and transmembrane domains are interconnected via polypeptide chains (linkers). Upon glutamate and glycine binding, these receptors undergo a series of conformational changes leading to the opening of the Ca2+-permeable ion channel. Here we report that different deletions and mutations of amino acids in the M3-S2 linkers of the GluN1 and GluN2B subunits lead to constitutively open channels. Irrespective of whether alterations were introduced in the GluN1 or the GluN2B subunit, application of glutamate or glycine promoted receptor channel activity; however, responses induced by the GluN1 agonist glycine were larger, on average, than those induced by glutamate. We observed the most prominent effect when residues GluN1(L657) and GluN2B(I655) were deleted or altered to glycine. In parallel, molecular modeling revealed that two interacting pairs of residues, the LILI motif (GluN1(L657) and GluN2B(I655)), form a functional unit with the TTTT ring (GluN1(T648) and GluN2B(T647)), described earlier to control NMDAR channel gating. These results provide new insight into the structural organization and functional interplay of the LILI and the TTTT ring during the course of NMDAR channel opening and closing.

Roles of purinergic P2X receptors as pacemaking channels and modulators of calcium-mobilizing pathway in pituitary gonadotrophs.

Anterior pituitary cells release ATP and express several subtypes of purinergic P2 receptors, but their biophysical properties and roles in spontaneous and receptor-controlled electrical activity have not been characterized. Here we focused on extracellular ATP actions in gonadotrophs from embryonic, neonatal, and adult rats. In cells from all three age groups, the Ca2+-mobilizing agonist GnRH induced oscillatory, hyperpolarizing, nondesensitizing, and slow deactivating currents. In contrast, ATP induced nonoscillatory, depolarizing, slowly desensitizing, and rapidly deactivating current, indicating that these cells express cation-conducting P2X channels but not Ca2+-mobilizing P2Y receptors. The amplitudes of P2X current response and the rates of receptor desensitization were dependent on ATP concentration. The biophysical and pharmacological properties of P2X currents were consistent with the expression of P2X2 subtype of channels in these cells. ATP-induced rapid depolarization of gonadotrophs lead to initiation of firing in quiescent cells, an increase in the frequency of action potentials in spontaneously active cells, and a transient stimulation of LH release. ATP also influenced GnRH-induced current and membrane potential oscillations and LH release in an extracellular Ca2+-dependent manner. These inositol 1,4,5-triphosphate-dependent oscillations were facilitated, slowed, or stopped, depending of ATP concentration, the time of its application, and the level of Ca2+ content in intracellular stores. These results indicate that, in gonadotrophs, P2X receptors could operate as pacemaking channels and modulators of GnRH-controlled electrical activity and secretion.

Photoperiodic regulation of histamine H3 receptor and VGF messenger ribonucleic acid in the arcuate nucleus of the Siberian hamster.

To survive winter the Siberian hamster has evolved profound physiological and behavioral adaptations, including a moult to winter pelage, regression of the reproductive axis, onset of daily torpor and increased capacity for thermogenesis. However, one of the most striking adaptations is the catabolism of intraabdominal and sc fat reserves contributing to the loss of up to 40% of body weight. These physiological and behavioral adaptations are photoperiodically driven, yet neither the site(s) in the brain nor the molecular mechanism(s) involved in the regulation of these profound adaptations is known. Here we report a dynamic regulation of gene expression in a dorsal region of the medial posterior area of the arcuate nucleus (dmpARC) of the Siberian and Syrian hamster brain in response to altered photoperiod. We show mRNA for the histamine H3 receptor is down-regulated and VGF is up-regulated in the dmpARC in hamsters switched from long- to short-day photoperiod. These data provide further evidence to support the view that the dmpARC is a major site to relay photoperiodic changes and as a site for the long-term regulation of seasonal physiology and behavior.

Circadian rhythmicity in AVP secretion and GABAergic synaptic transmission in the rat suprachiasmatic nucleus.

A variety of physiological and behavioral functions exhibit circadian changes and these circadian rhythms are driven by oscillatory expression of clock genes in the suprachiasmatic nuclei (SCN). It is still unknown how this molecular clockwork is controlled by extracellular neurohormones and neurotransmitters and which membrane receptors undergo circadian modulation. Circadian rhythm can be measured as a secretion of arginine vasopressin (AVP) in organotypic SCN culture for several weeks. Melatonin applied directly to the SCN late in the day induces a phase advance, when applied late at night or at the beginning of the day melatonin causes a phase delay. The time window for phase advance corresponds with the highest level of melatonin receptors in the SCN but the mechanism of melatonin-induced phase delay is unknown. The principal neurotransmitter on SCN synapses is gamma-aminobutyric acid (GABA), which acts at postsynaptic GABA(A) receptors. Spontaneous release of GABA from presynaptic nerve terminals, recorded as miniature inhibitory postsynaptic currents in the presence of TTX, does not change, but zinc sensitivity of exogenous GABA-induced currents varies during the day and night, possibly due to changes in subunit composition of GABA(A) receptors. We conclude that there is daily variation in the postsynaptic, but not presynaptic, function in the SCN.