N-methyl-D-aspartate Receptors In Mice Research Articles

Melatonin attenuated the behavioral despair induced by acute neurogenic stress through blockade of N-methyl D-aspartate receptors in mice

It has been well documented that administration of melatonin could reveal antidepressant-like effect in rodents. However, the protective effect of melatonin on stress-induced depression/anxiety and its underlying mechanism is yet to be understood. In this regard, in the current study, acute foot-shock stress (FSS) was used to evaluate the antidepressant-like effect of melatonin on neurogenic stress-induced depression in mice. Behavioral evaluation was done by using the forced swimming test (FST) and Open-field test (OFT). Melatonin, MK-801, and ketamine (NMDA receptor antagonists), and NMDA (NMDA receptor agonist) were used to elucidate any association between melatonin and NMDA pathway in behavioral despair induced by acute-FSS. Applying acute-FSS to mice significantly induced depressant-like behavior in FST without any significant impact on locomotor activity in the OFT. We observed that melatonin (dose-dependently) significantly improved the depressant-like effect of FSS, but it did not impact the locomotion in animals. Acute injection of MK-801 at sub-effective doses (0.01 mg/kg) or ketamine (0.1 mg/kg) potentiated the antidepressant-like effect of a sub-effective dose of melatonin. However, the sub-effective dose of NMDA (30 mg/kg) abolished the protective effect of melatonin on the behavioral profile of stressed animals. Our results could reflect the antidepressant-like effect of melatonin on neurogenic stress-induced depressive behaviors in mice. Also, our results showed that NMDA receptors could be involved in the antidepressant-like effect of melatonin.

Involvement of NMDA receptors in ryanodine receptor expression in dopaminergic neurons in the ventral tegmental area of mice with intermittent methamphetamine treatment

Excitatory synapses on dopaminergic neurons of the ventral tegmental area (VTA) represent an important role in psychostimulant-induced rewarding effect. This study investigated the regulation of ryanodine receptor (RyR) and N-methyl-D-aspartate (NMDA) receptor expression in mice under intermittent methamphetamine (METH) treatment using a place preference procedure. RyR-1 and -2 significantly increased in the VTA of mice with METH-induced place preference, whereas RyR-3 showed no changes. In addition, the levels of NR1, NR2A, and NR2B subunits were increased in the VTA. The METH-induced place preference was inhibited by intracerebroventricular pretreatment with MK-801, a noncompetitive NMDA receptor antagonist, and ifenprodil, a selective NR2B subunit-containing NMDA receptor antagonist, in a dose-dependent manner. Under these conditions, the increase of RyR-1 and -2 in the VTA was significantly blocked by ifenprodil. The immunohistochemical analysis revealed the colocalization of RyR-1 and -2 with NR2B subunits in dopaminergic neurons in the mouse VTA. These findings suggest that RyRs could be involved in the development of METH-induced place preference and that NR2B subunit-containing NMDA receptors in mice showing METH-induced place preference play an important role in expression of RyRs.

Modulation of ethanol state-dependent learning by dorsal hippocampal NMDA receptors in mice

The possible role of N-methyl- d-aspartate (NMDA) receptors of dorsal hippocampus on ethanol state-dependent learning was studied in adult male mice (Pasteur Institute, Iran). As a model of memory, a single-trial step-down passive avoidance task was used. All animals were bilaterally implanted with cannulae into the CA1 regions of dorsal hippocampi. Results show that intraperitoneal (i.p.) administration of ethanol (0.5 and 1 g/kg) 30 min before training impaired memory performance in animals when tested 24 h later. Pretest administration of the same doses of ethanol-induced state-dependent retrieval of the memory acquired under pretraining ethanol (1 g/kg, i.p.) influence. Pretest intra-CA1 microinjection of NMDA (0.001, 0.01, and 0.1 μg/mouse) by itself had no effect on memory retrieval and ethanol-induced amnesia. However, pretest intra-CA1 administration of the same doses of NMDA with an ineffective dose of ethanol (0.25 g/kg, i.p.) significantly restored the retrieval and potentiated ethanol state-dependent learning. On the other hand, pretest administration of a competitive NMDA receptor antagonist d-AP5 ( d-(−)-2-Amino-5-phosphonopentanoic acid) (0.01, 0.1, and 1 μg/mouse, intra-CA1) or a noncompetitive NMDA receptor antagonist MK-801 maleate [(5S, 10R)-(+)-5-Methyl-10, 11-dihydro-5H-dibenzo [a, d] cyclohepten-5, 10-imine maleate] (0.25, 0.5, and 1 g/mouse, intra-CA1) 5 min before the administration of ethanol (1 g/kg, i.p.) significantly inhibited ethanol state-dependent learning. Intra-CA1 pretest administration of d-AP5 (0.01, 0.1, and 1 μg/mouse) or MK-801 maleate [5S, 10R)-(+)-5-Methyl-10, 11-dihydro-5H-dibenzo [a, d] cyclohepten-5, 10-imine maleate] (0.25, 0.5, and 1 μg/mouse) alone did not affect memory retention. It may be concluded that dorsal hippocampal NMDA receptors are involved in mediating ethanol state-dependent learning.

The Neuroactive Steroid Pregnenolone Sulfate Stimulates the Release of Gonadotropin-Releasing Hormone from GT1-7 Hypothalamic Neurons, throughN-Methyl-d-Aspartate Receptors

Immortalized hypothalamic GT1-7 neurons represent a good model system to investigate the control of GnRH secretion. Using these cells, we observed that the neuroactive steroid, pregnenolone sulfate (PREGS), is able to stimulate the release of GnRH in a dose-dependent manner through N-methyl-D-aspartate (NMDA) receptors, because its action is completely blocked by a specific NMDA receptor antagonist and magnesium. GT1-7 neurons express mRNAs for various mouse NMDA receptor subunits (zeta,1, epsilon3, epsilon4, and epsilon2, corresponding to the NR1, NR2C, NR2D, and NR2B rat subunits) and increase their spontaneous release of GnRH when incubated in the presence of exogenous glutamate or NMDA. In addition, we found that these neurons are able to release and synthesize glutamate, as demonstrated by the presence of glutamate accumulated in the defined incubation medium of the neurons, during the experiment and the expression of mRNA coding for vesicular glutamate transporter 2, a specific marker of glutamatergic neurons. The potentiating effect of PREGS on the secretion of GnRH induced by glutamate is consistent with the role of the steroid as a positive allosteric modulator of NMDA receptors. Together these results point to a novel mechanism by which the neuroactive steroid PREGS may potentiate an autocrine excitatory loop in GnRH neurons.

Weaver cerebellar granule neurons show altered expression of NMDA receptor subunits bothin vivo andin vitro

Biochemical, immunocytochemical, and molecular biological techniques were used to investigate the expression of N-methyl-D-aspartate (NMDA) receptor subunits in migration-deficient weaver mouse cerebellum in vivo and in primary cultures of the vermal weaver granule neurons with or without a rescue by verapamil. We found that both NMDAR1(zeta1) message and protein were expressed by the weaver granule neurons in situ. Immunocytochemical and biochemical analyses indicated that granule neurons of the weaver cerebellum expressed R1(zeta1) and R2A(epsilon1) subunits but showed little expression of the R2B(epsilon2) subunit. In weaver cerebellum, the R2B(epsilon2) subunit was primarily expressed in nerve fibers of the internal granule cell layer and white matter. Reverse-transcriptase-polymerase chain reaction followed by sequence analysis of the R1(zeta1) subunit indicated that the zeta1 subunit amplicons of both normal and weaver cerebella were identical, and that splice variants with exon 22 (1-2) and with or without exon 5 (a/b) or exon 21 (1-4) were detectable. The R2A(epsilon1), and R2B(epsilon2) subunits of the normal and weaver mouse cerebellum revealed no primary structural differences between the normal and weaver NMDA receptor subunits or the cloned mouse NMDA receptor subunits. In vermal cultures, normal granule neurons expressed all three NMDA receptor subunits (zeta1, epsilon1, and epsilon2), whereas the weaver neurons failed to express the epsilon2 subunit. Rescue of the weaver neurons by verapamil induced expression of the epsilon2 protein along the granule neuronal surfaces. The present results suggest that lack of the epsilon2 subunit in the weaver cerebellum may relate to the lack of functional NMDA receptors and/or to the migratory failure of the weaver granule neurons. Our data further suggest that NMDA receptor-mediated neurotoxicity is an unlikely mediator of neuronal death of the weaver granule neurons. In fact, down-regulation of the NMDA receptor expression and function may be a protective measure of the weaver granule neurons to reduce calcium entry via these receptors.

Weaver cerebellar granule neurons show altered expression of NMDA receptor subunits both in vivo and in vitro

Biochemical, immunocytochemical, and molecular biological techniques were used to investigate the expression of N-methyl-D-aspartate (NMDA) receptor subunits in migration-deficient weaver mouse cerebellum in vivo and in primary cultures of the vermal weaver granule neurons with or without a rescue by verapamil. We found that both NMDAR1(ζ1) message and protein were expressed by the weaver granule neurons in situ. Immunocytochemical and biochemical analyses indicated that granule neurons of the weaver cerebellum expressed R1(ζ1) and R2A(ϵ1) subunits but showed little expression of the R2B(ϵ2) subunit. In weaver cerebellum, the R2B(ϵ2) subunit was primarily expressed in nerve fibers of the internal granule cell layer and white matter. Reverse-transcriptase–polymerase chain reaction followed by sequence analysis of the R1(ζ1) subunit indicated that the ζ1 subunit amplicons of both normal and weaver cerebella were identical, and that splice variants with exon 22 (1-2) and with or without exon 5 (a/b) or exon 21 (1-4) were detectable. The R2A(ϵ1), and R2B(ϵ2) subunits of the normal and weaver mouse cerebellum revealed no primary structural differences between the normal and weaver NMDA receptor subunits or the cloned mouse NMDA receptor subunits. In vermal cultures, normal granule neurons expressed all three NMDA receptor subunits (ζ1, ϵ1, and ϵ2), whereas the weaver neurons failed to express the ϵ2 subunit. Rescue of the weaver neurons by verapamil induced expression of the ϵ2 protein along the granule neuronal surfaces. The present results suggest that lack of the ϵ2 subunit in the weaver cerebellum may relate to the lack of functional NMDA receptors and/or to the migratory failure of the weaver granule neurons. Our data further suggest that NMDA receptor–mediated neurotoxicity is an unlikely mediator of neuronal death of the weaver granule neurons. In fact, down-regulation of the NMDA receptor expression and function may be a protective measure of the weaver granule neurons to reduce calcium entry via these receptors. © 1999 John Wiley & Sons, Inc. J Neurobiol 38: 441–454, 1999

Gene structure and chromosomal localization of the mouse NMDA receptor channel subunits.

Multiple espilon subunits are major determinants of the diversity of the N-methyl-D-aspartate (NMDA) receptor channel. The four epsilon subunit mRNAs exhibit distinct expression patterns in the brain. In an attempt to elucidate the molecular basis of selective and characteristic expression of the NMDA receptor channel subunits, we have isolated the gene encoding the mouse NMDA receptor epsilon 3 subunit and have determined its structural organization. The epsilon 3 subunit gene spans 17.5 kb and consists of 14 exons. The major transcription start site is 439 bp upstream of the ATG initiation codon as determined by primer extension and S1 nucleas protection analyses. Two polyadenylation sites are 397 (or 398) and 402 bp downstream of the termination codon. The 5'-flanking region of the epsilon 3 subunit gene contains GC-rich segments including consensus sequences for binding of the transcription factors Spl and EGR-1. The murine chromosomal locations of the five NMDA receptor channel subunits, the epsilon 1 (Grin2a), epsilon 2 (Grin2b), epsilon 3 (Grin2c), epsilon 4 (Grin2d) and zeta 1 (Grinl) subunits, were determined using an interspecific backcross mapping panel derived from crosses of [(C57BL/6JxM. spretus) F1xC57BL/6J] mice. Each of these genes mapped to a single chromosome location. The mapping results assigned the five loci to five different mouse autosomes, indicating that they have become well dispersed among mouse chromosomes.

Functional characterization of a heteromeric NMDA receptor channel expressed from cloned cDNAs.

The glutamate receptor (GluR) channel plays a key part in brain function. Among GluR channel subtypes, the NMDA (N-methyl-D-aspartate) receptor channel which is highly permeable to Ca2+ is essential for the synaptic plasticity underlying memory, learning and development. Furthermore, abnormal activation of the NMDA receptor channel may trigger the neuronal cell death observed in various brain disorders. A complementary DNA encoding a subunit of the rodent NMDA receptor channel (NMDAR1 or zeta 1) has been cloned and its functional properties investigated. Here we report the identification and primary structure of a novel mouse NMDA receptor channel subunit, designated as epsilon 1, after cloning and sequencing the cDNA. The epsilon 1 subunit shows 11-18% amino-acid sequence identity with rodent GluR channel subunits that have been characterized so far and has structural features common to neurotransmitter-gated ion channels. Expression from cloned cDNAs of the epsilon 1 subunit together with the zeta 1 subunit in Xenopus oocytes yields functional GluR channels with high activity and characteristics of the NMDA receptor channel. Furthermore, the heteromeric NMDA receptor channel can be activated by glycine alone.

Cloning, expression and modulation of a mouse NMDA receptor subunit

The primary structure and presence of two forms of the mouse N-methyl- d-aspartate (NMDA) receptor channel subunit ζl have been disclosed by cloning and sequencing the cDNAs. The ζl subunit shows −20% amino acid sequence identities with the rodent α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA)- or kainate-selective GluR subunits and has structural features common to neurotransmitter-gated ion channels. Functional homomeric ζl channels expressed in Xenopus oocytes by injection of the subunit specific mRNA exhibit current responses characteristics for the NMDA receptor channel such as activation by glycine, Ca 2+ permeability, blocking by Mg 2+ and activation by polyamine. It has been found that the ζl channel activity is positively modulated by treatment with 12- O-tetradecanoylphorbol 13-acetate (TPA).