Duality of Glutamatergic and Gabaergic Control of PulsatileGnRH Secretion by Rat Hypothalamic Explants: II. ReducedNR2C- and GABAA-Receptor-Mediated Inhibition atInitiation of Sexual Maturation

N-methyl-D-aspartate (NMDA) receptors and γ-aminobutyric acid (GABA) receptors are involved in the mechanism of pulsatile gonadotrophin-releasing hormone (GnRH) secretion. The aim of this study was to elucidate the role of those receptors in the acceleration of pulsatile GnRH secretion seen at onset of puberty. Using hypothalamic explants from prepubertal (15 days), early pubertal (25 days) and adult (50 days) male rats, we studied the effects of pharmacological antagonists and antisense oligodeoxynucleotides on GnRH release evoked by NMDA and GABA receptor agonists as well as the interval between spontaneous GnRH secretory pulses. At the three studied ages, the muscimol-evoked release of GnRH is similarly inhibited by the GABA_A receptor antagonist bicuculline. In contrast, the frequency of pulsatility is stimulated by bicuculline as indicated by a reduction of the mean GnRH interpulse interval from 60 to 40 min and such an effect is seen at 15 days only. The GnRH interpulse interval is also reduced by GABA_A receptor antisense oligodeoxynucleotides at 15 days while no effects are seen at 25 days. At the three studied ages, the NMDA-evoked release of GnRH and the GnRH interpulse interval are similarly inhibited by 100 or 500 μM of the NMDA receptor antagonist 7-chlorokynurenic acid (7CK). These effects are consistent with the increase of GnRH interpulse interval caused by NR2A antisense oligodeoxynucleotides at 15 days (86 vs 64 min in controls) as well as 25 days (44 vs 36 min). A low (5 μM) concentration of 7CK does not result in any effect except a reduction of GnRH interpulse interval which is seen at 15 days only. A similar reduction of GnRH interpulse interval is obtained using NR2C antisense oligodeoxynucleotides at 15 days (50 vs 64 min in controls) while no effects are seen at 25 days (35 vs 36 min). At 25 days, muscimol can prevent the developmental increase in frequency of pulsatile GnRH secretion. In summary, pulsatile GnRH secretion by the prepubertal hypothalamus characteristically involves an inhibition mediated through GABA_A receptors and the NR2C subunit of NMDA receptors. Based on these data, we propose a model for the mechanism of the onset of puberty which involves the disappearance or inactivation of GABAergic neurons located in the retrochiasmatic hypothalamus and expressing the NR2C subtype of NMDA receptors.     

Amygdala gene expression of NMDA and GABAA receptors in patients with mesial temporal lobe epilepsy

Temporal lobe epilepsy (TLE) is the most common form of partial epilepsy and affects 40% of the patients. Seizures arising from the mesial temporal lobe structures (i.e., amygdala and hippocampus) are common, whereas neocortical seizures are rare. In recent years, many studies aimed to identify the pattern of gene expression of neurotransmitters involved in molecular mechanisms of epilepsy. We used real‐time PCR to quantify the expression of GABA_A (subunits α1, β1, β2) and NMDA (subunits NR1, NR2A, and NR2B) receptor genes in amygdalae of 27 patients with TLE and 14 amygdalae from autopsy controls. The NR1 subunit was increased in patients with epilepsy when compared with controls. No differences were found in expression of NMDA subunits NR2A and NR2B or in α1, β1, and β2 subunits of GABA_A receptors. Our results suggest that the NR1 subunit of NMDA receptors is involved in the amygdala hyperexcitability in some of the patients with TLE. © 2010 Wiley Periodicals, Inc., Inc.     

Synapse-specific localization of NMDA and GABAA receptor subunits revealed by antigen-retrieval immunohistochemistry

Conventional immunohistochemistry provides little evidence for the synaptic localization of ionotropic neurotransmitter receptors, suggesting that their epitopes are not readily accessible in situ. Here, we have adapted antigen retrieval procedures based on microwave irradiation to enhance the immunohistochemical staining of γ-aminobutyric acid type A (GABA_A) and N-methyl-D-aspartate (NMDA) receptor subunits in rat brain tissue. Microwave irradiation of fixed tissue produced a marked reduction of nonspecific staining, allowing an improved detection of GABA_A receptor subunits. However, staining of NMDA receptor subunits remained suboptimal. In contrast, microwave irradiation of cryostat sections prepared from fresh tissue resulted in a major enhancement of both NMDA and GABA_A receptor subunit staining. The diffuse, partially intracellular signals were largely replaced by numerous, intensely immunoreactive puncta outlining neuronal somata and dendrites, highly suggestive of synaptic receptors. In hippocampus CA1–CA3 fields, the NR2A and NR2B subunit positive puncta exhibited an extensive colocalization in the stratum oriens and radiatum, whereas pyramidal cell bodies, which receive no excitatory synapses, were unstained. In addition, the NR2A subunit, but not the NR2B subunit, was selectively detected on pyramidal cell dendrites in the stratum lucidum of CA3, suggesting a selective targeting to sites of mossy fiber input. For the GABA_A receptor subunits, the most striking change induced by this protocol was the selective staining of the axon initial segment of cortical and hippocampal pyramidal cells. The α2 subunit immunoreactivity was particularly prominent in these synapses. In control experiments, the staining of cytoskeletal proteins (neurofilaments, glial fibrillary acid protein) was not influenced by prior microwave irradiation. The enhancement of cell-surface–associated staining is therefore strongly suggestive of an ‘unmasking’ of subunit epitopes by the microwave treatment. These results reveal a remarkable specificity in the synaptic targeting of NMDA and GABA_A receptor subunits in hippocampal and neocortical neurons, suggesting that individual neurons can express multiple receptor subtypes in functionally distinct synapses. J. Comp. Neurol. 390:194–210, 1998. © 1998 Wiley-Liss, Inc.     

Functional excitatory GABAA receptors precede ionotropic glutamate receptors in radial glia-like neural stem cells

The involvement of neurotransmission in neuronal development is a generally accepted concept. Nevertheless, the precise regulation of neurotransmitter receptor expression is still unclear. To investigate the expression profiles of the most important ionotropic neurotransmitter receptors, namely GABA_A receptors (GABA_ARs), NMDA receptors (NMDARs), and AMPA receptors (AMPARs), quantitative RT-PCR, immunoblot analysis and patch clamp studies were performed in in vitro-generated neural stem cells (NSCs). This clearly defined cell line is closely related to radial glia cells, the stem cells in the neonate brain.We found functional GABA_ARs of the subunit composition α2, β3, and γ1 to be expressed. Unexpectedly, functional ionotropic glutamate receptors were absent. However, NSCs expressed the NMDAR subunits NR2A and NR3A, and the AMPAR subunit GluR4 at the protein level, and GluR3 at the mRNA level.The overexpression of functional NMDARs in NSCs led to an increased mRNA level of AMPAR subunits, indicating a role in synaptogenesis. Early neuronal markers remained unchanged. These data extend our knowledge about ionotropic neurotransmitter receptor expression during neuronal development and will aid further investigations on activity-dependent neurogenesis.     

Diazepam protects against rat hippocampal neuronal cell death induced by antisense oligodeoxynucleotide to GABAA receptor γ2 subunit

Antisense oligodeoxynucleotides (ODNs) are used for the selective inhibition of gene expression. Antisense ODNs are promising tools for the investigation of physiological implications of proteins in the central nervous system of rodents in vivo. We have previously demonstrated that a phosphorothioate antisense ODN to the GABA_A receptor γ2 subunit, but not sense or mismatch control ODNs, induces a decrease in ex vivo benzodiazepine receptor radioligand binding in rat hippocampus when infused into the hippocampus in vivo [Karle et al., Neurosci. Lett., 202 (1995) 97–100]. This effect is parallelled by a decrease in the number of GABA_A receptors and an extensive loss of hippocampal neurones. There is increasing awareness of risks of toxic `non-antisense' effects induced by ODNs, and in particular phosphorothioate ODNs. The present experiments were designed to investigate the specificity of effects induced by the γ2 subunit antisense ODN. The temporal development of changes in [³H]flunitrazepam and [³H]quinuclidinyl benzilate binding as well as in tissue protein levels supports the notion that the antisense ODN primarily acts by blocking the expression of the targeted receptor subunit protein. Furthermore, it is shown that a threshold for the elicitation of neurodegenerative changes exists. Finally, it is demonstrated that diazepam treatment of rats protects against the development of neuronal cell death induced by the antisense ODN. Collectively, the results support the hypothesis that the neurodegeneration induced by the antisense ODN is a consequence of diminished GABAergic inhibitory tonus following a selective down-regulation of γ2 subunit-containing GABA_A receptor complexes.     

NR1 Knockdown Reveals CA1 Injuryduring a Developmental Period of High Seizure Susceptibility Despite Reduced Seizure Activity

NMDA receptors (NMDARs) are important for the propagation of seizures. To understand the role of NR1 subunits in the propagation of seizures we knocked down the NR1 subunit by intracranial injection of antisense deoxyoligonucleotides (NR1-AS-ODNs) into the right hippocampus during a window of maximal seizure susceptibility in development. Control missense and sense ODNs followed by focal injection of NMDA (2.5–25 nmoles) into the hippocampal CA1 and sensorimotor cortex of P15 rat pups resulted in behavioral and electrographic (EEG) seizures. After NR1 knockdown, low- and high-doses produced little or no spike activity in the hippocampus and overlying sensorimotor cortex as predicted. Despite reduced activity in the hippocampal and cortical EEG, intracranial NMDA or peripheral kainate (KA)-induced seizures led to paradoxical cell death of CA1 neurons, which is not typically observed in this age group. Histological changes were modest or absent in the cortex away from the infusion site. Signal specificity of the targeted CA1 or cortex was observed in autoradiograms, immunohistochemistry and Western blots. After knockdown, Ca²⁺ influx was suppressed as both NMDA and muscimol-stimulated Ca²⁺ permeability of the immature CA1 was blocked in ex-vivo slices measured with FURA-2AM optical dye imaging. Data suggest that certain constituent levels of NMDA receptors distributed on excitatory and/or inhibitory interneurons may be developmentally required for survival of CA1 pyramidal neurons during a critical period when ictal activity is present. Moreover, selective NR1 subunit downregulation simultaneously reduces NMDA and GABA_A receptor Ca²⁺ ion permeability properties that may contribute to a premature cell death mechanism.     

Age- and Hormone-Regulation of N-Methyl-d-Aspartate Receptor Subunit NR2b in the Anteroventral Periventricular Nucleus of the Female Rat: Implications for Reproductive Senescence

Glutamate, acting through its N‐methyl‐d ‐aspartate (NMDA) and non‐NMDA receptors in the hypothalamus, regulates reproductive neuroendocrine functions via direct and indirect actions upon gonadotrophin‐releasing hormone (GnRH) neurones. Previous studies indicate that the NMDA receptor subunit NR2b undergoes changes in protein and gene expression in the hypothalamus in general, and on GnRH neurones in particular, during reproductive ageing. In the present study, we examined whether the NR2b‐expressing cell population, both alone and in association with the NR1 subunit (i.e. the latter subunit is necessary for a functional NMDA receptor), is altered as a function of age and/or steroid hormone treatment. Studies focused on the anteroventral periventricular (AVPV) nucleus of the hypothalamus, a region critically involved in the control of reproduction. Young (3–5 months), middle‐aged (9–12 months), and aged (approximately 22 months) female rats were ovariectomised and, 1 month later, they were treated sequentially with oestradiol plus progesterone, oestradiol plus vehicle, or vehicle plus vehicle, then perfused. Quantitative stereologic analysis of NR2b‐immunoreactive cell numbers in the AVPV showed an age‐associated decrease in the density of NR2b‐immunoreactive cells, but no effect of hormone treatment. In a second study, immunofluorescent double labelling of NR2b and NR1 was analysed by confocal microscopy of fraction volume, a semi‐quantitative measure of fluorescence intensity. No effect of ageing was detected for immunofluorescent NR1 or NR2b alone, whereas the NR2b fraction volume increased in the oestradiol plus vehicle group. With ageing, the fraction volume of the NR2b/NR1‐colocalised subunits increased. Together with the stereology results, this suggests that, although fewer cells express the NR2b subunit in the ageing AVPV, a greater percentage of these subunits are co‐expressed with NR1. Our results suggest that the subunit composition of NMDA receptors in the AVPV undergo both age‐ and hormonal‐regulation, which may be related to previous observations of changes in functional responses of reproductive neuroendocrine systems to NMDA receptor modulators with ageing.     

Age-related changes in hypothalamic gonadotropin-releasing hormone and N-methyl-D-aspartate receptor gene expression, and their regulation by oestrogen, in the female rat

During reproductive ageing, the oestrous cycles of female rats become irregular and eventually cease. The mechanisms for reproductive senescence in rodents are believed to involve changes in hypothalamic neurones, including gonadotropin-releasing hormone (GnRH) cells and their afferent inputs. In addition, effects of oestrogen on hypothalamic function may vary in animals of different ages. These issues were addressed using young (aged 4-5 months), middle-aged (12-14 months) and old (24-26 months) female Sprague-Dawley rats. Animals were ovariectomized and given oestrogen or vehicle replacement. They were killed and the preoptic area-anterior hypothalamus (POA-AH) and the medial basal hypothalamus-median eminence (MBH-ME) were dissected out, RNA extracted, and RNase protection assay used to quantify gene expression of several hypothalamic molecules. In the first experiment, GnRH RNA levels were measured in the POA-AH. No effects of ageing or oestrogen were observed on GnRH gene expression. This finding suggests that ageing and oestrogen may affect GnRH release from neuroterminals independently of de novo biosynthesis, and that this may involve other neurones that affect GnRH neurosecretory function. In the second experiment, we investigated changes in N-methyl-D-aspartate (NMDA) receptor subunit mRNA levels. These receptors play an important regulatory role in mediating effects of glutamate on GnRH function, and are themselves regulated by oestrogen and ageing. NMDA receptor subunit (NR) 1, 2a and 2b mRNA levels were quantified in the POA-AH and MBH-ME, the sites of GnRH perikarya and neuroterminals, respectively. In general, oestrogen had inhibitory effects on NR1 and NR2a, and differential effects on NR2b subunit mRNA levels. NMDA receptor subunit mRNA levels also changed during ageing: age-related decreases in NR1 mRNA occurred in the MBH-ME, and an age-related increase in NR2b mRNA occurred in the POA-AH. Taken together, these results demonstrate subunit- and region-specific changes in hypothalamic NMDA receptor subunit gene expression with oestrogen and ageing. These alterations could have implications for the physiological effects of glutamate on its NMDA receptor, and impact the regulation of reproductive and other neuroendocrine and autonomic functions by hypothalamic glutamatergic inputs.     

Concentration-dependent effects of muscimol to enhance pulsatile GnRH release from GT1–7 neurons in vitro

Immortalized GT1–7 neurons were used to characterize the effect of muscimol, a GABA_A receptor agonist, to enhance pulsatile gonadotropin-releasing hormone (GnRH) release. GT1–7 neurons were grown on Cytodex-3 beads and placed in special superfusion microchambers. The cells were superfused at a rate of 6.2 ml·h⁻¹ with Media 199 (pH 7.35) using a commercially available perfusion system. After a pre-muscimol period of 120 min, the cells were exposed for 5 min to 0.35, 1, 5 or 10 μM muscimol or 5 μM muscimol+20 μM of the GABA_A receptor antagonist, bicuculline. Following removal of the muscimol (and bicuculline, in the case of the latter experiment), the superfusion was continued for another 115 min. Sample fractions were collected at 5 min intervals throughout the perfusion. Basal GnRH release from the GT1–7 neurons was pulsatile with an average interpulse interval of 45.4±0.5 min and an average pulse amplitude of 191.5±22.6 pg·min·ml⁻¹. Our results also demonstrated that the GABA_A receptor agonist, muscimol, enhances pulsatile GnRH release from GT1–7 neurons in culture. The response to muscimol was saturable and concentration-dependent with an EC₅₀ of 0.47 μM. The effects of 5 μM muscimol to increase GnRH pulsatility were blocked by co-exposure to the GABA_A receptor antagonist, bicuculline. The average GnRH interpulse intervals were 41.7±1.8 min, 32.5±2.9 min, 30.6±0.7 min and 25.5±0.4 min in the period following exposure to 0.35, 1, 5 and 10 μM of muscimol, respectively (post-muscimol period). GnRH pulse amplitude (mean-area for each pulse) was increased during exposure to muscimol but not during the pre- or post-muscimol periods. The GABA_A receptor antagonist, bicuculline, itself had no effect on pulsatile GnRH release. These results are consistent with previously published reports suggesting that activation of the GABA_A receptor stimulates hypothalamic GnRH release in embryonic and neonatal animals.     

Several GABAA receptor subunits are expressed in LHRH neurons of juvenile female rats

Gamma aminobutyric acid (GABA), the dominant inhibitory neurotransmitter in brain, is involved in the developmental regulation of LHRH secretion. Morphological studies in rodents have demonstrated that LHRH neurons are innervated by GABA-containing processes, suggesting that LHRH secretion is under direct transsynaptic GABAergic control. While GABA acts through two different receptors, GABA_A and GABA_B, to exert its effects, it appears that GABA_A receptors are able to mediate both inhibitory and stimulatory effects of GABA on LHRH neurons. GABA_A receptors are heterooligomeric ligand-gated anion channels that exhibit a diverse array of functional and pharmacological properties. This diversity is determined by the structural heterogeneity of the receptors, which are assembled from the combination of different classes of subunits with multiple isoforms. Although several studies have described the effect of GABA_A receptor stimulation on LHRH and/or gonadotropin release in prepubertal animals, nothing is known about the receptor subunits that may be expressed in LHRH neurons at this phase in development. Double immunohistofluorescence followed by confocal laser microscopy revealed that subsets of prepubertal LHRH neurons are endowed with α₁, α₂, β_2/3, and γ₂ GABA_A receptor subunits. Combined immunohistochemistry for LHRH neurons and in situ hybridization for GABA_A subunit mRNAs confirmed that the genes encoding the α₁, α₂, β₃ and γ₂ subunits, but not the γ₁ subunit, are expressed in LHRH neurons. Notwithstanding the relative insensitivity of these methods, both the immunohistochemical and hybridization histochemical approaches employed indicate that only a fraction of LHRH neurons are endowed with GABA_A receptors. This arrangement suggests that those LHRH neurons bearing the appropriate GABA_A receptors are responsible for either the entire secretory response to direct GABAergic inputs or for its initiation.     

Interaction between glutamate and GABA on H-noradrenaline release from rat hypothalamus

Glutamate has been shown to stimulate noradrenaline (NA) release from hypothalamic nerve terminals. In the present study, we evaluated the possible interaction between the excitatory amino acid glutamate and gamma-aminobutyric acid (GABA), an inhibitory transmitter, on noradrenaline (NA) release from mediobasal hypothalamus (MBH) of adult male rats. Hypothalamic slices loaded in vitro with ³H-NA were superfused and exposed to glutamate, N-methyl- -aspartic acid (NMDA), or kainate (KA). We found that ³H-NA release evoked by the excitatory amino acids glutamate and NMDA was dramatically decreased by GABA. The facilitatory effects of NMDA and KA were prevented concentration-dependently by the GABA_B receptor antagonist 2-hydroxy saclofen which restored the NMDA effect. In addition, beclofen blocked K⁺-induced ³H-NA release. Activation of GABA_A receptors by muscimol and THIP was ineffective. In conclusion, glutamate and GABA, through GABA_B receptors, may interact to modulate NA release from the rat mediobasal hypothalamus.     

The positive allosteric modulation of GABAA receptors mRNA in immature hippocampal rat neurons by midazolam affects receptor expression and induces apoptosis

Abstract

Background: Numerous experimental studies show that anesthetics are potentially toxic to the immature brain. Even though benzodiazepines are widely used in pediatric anesthesia and intensive care medicine, only a few studies examine the effects of these drugs on immature neurons.

Methods: Hippocampal neuronal cell cultures of embryonic Wistar rats (15?days in culture) were incubated with midazolam 100 or 300?nM for either 30?min or 4?h. The time course of the mRNA expression of the glutamate receptors subunits NR1, NR2A and NR2B of the NMDA receptor, the GluA-1 and A-2 subunits of the AMPA receptor as well as the alpha 1 subunit of the GABA_A receptor were examined by PCR. Apoptosis was detected using Western blot analysis for BAX, Bcl-2 and Caspase-3.

Results: Midazolam at 100 and 300?nM applied for 30?min and 100?nM for 4?h affected glutamate receptor and GABA_A receptor subunit expression. However, these effects were reversible within 72?h following washout. When 300?nM midazolam was applied for 4?h a significant increase in the NR 1 and NR 2A mRNA subunit expression could be detected. The increase in NR 2B receptor subunit expression as well as the GluA1 subunit expression was not reversible within 72?h following washout. This increase in mRNA glutamate receptor subunit expression was associated with a significant increase in neuronal apoptosis.

Conclusion: In immature neurons midazolam altered GABA and glutamate mRNA receptor subunit expression. Prolonged increase in midazolam-induced glutamate receptor expression was associated with apoptosis.     

α-Adrenergic receptor antagonism and N-methyl-d-aspartate (NMDA) induced luteinizing hormone release in female rhesus macaques

The stimulatory influence of N-methyl-d-aspartate (NMDA), a glutamate receptor agonist, on LH secretion is well established in several mammalian species including the rhesus macaque. Although the mechanism of excitation appears to involve enhanced GnRH secretion, it is unclear whether the GnRH neurons respond directly to this excitation or whether stimulatory inter-neurons are involved. This study investigated the possibility that noradrenergic afferents play a major role in mediating the response of the primate hypothalamo-pituitary reproductive axis to NMDA. In situ hybridization histochemistry, using a cRNA probe coding for the NMDAR1 receptor subunit, revealed abundant mRNA in the locus coeruleus, a brain area rich in noradrenergic neurons. Furthermore, using double-label fluorescence immunocytochemistry, the tyrosine hydroxylase immunopositive neurons of the locus coeruleus showed immunoreactivity for the NMDAR1 receptor subunit protein. A second experiment examined whether prazosin, an α₁-adrenergic receptor antagonist, could attenuate NMDA-induced stimulation of LH release. Prazosin (either 1 or 5 mg/kg b.wt., i.v.) was administered to female rhesus macaques during the luteal phase of the menstrual cycle, 40 min before administration of NMDA (10 mg/kg b.wt., i.v.). Regardless of the prazosin pre-treatment, plasma LH concentrations showed a significant increase (P<0.01) within 10 min of the administration of NMDA. Therefore, in spite of the evidence that at least some of the noradrenergic neurons of the primate hindbrain express the NMDAR1 receptor subunit, it is unlikely that noradrenergic inter-neuronal pathways alone play a major role in mediating the stimulatory action of NMDA on GnRH/LH secretion in primates. Indeed, because the GnRH neurons of the rhesus macaque are located diffusely in various regions of the hypothalamus and medial-septal/preoptic area, their net response to excitatory amino acids is likely to be more complicated, involving a combination of both stimulatory and inhibitory inter-neurons, and possibly also a direct interaction.     

Antidepressant effects on GABA-stimulated Cl influx in rat cerebral cortex are altered after treatment with GABAA receptor antisense oligodeoxynucleotides

Antidepressants act at the GABA_A receptor to inhibit GABA-stimulated ³⁶Cl⁻ influx and GABA reduction of [³⁵S]TBPS binding. This study examined how selective knock-down (via antisense oligodeoxynucleotides, aODNs) of GABA_A receptor subunits modified antidepressant activity. The specific aODNs used were for the α1, β1, β2 or γ2 subunits of the GABA_A receptor. The aODN microinjections reduced corresponding GABA_A receptor subunit mRNA levels by 30–40% as assessed by RT-PCR. The inhibitory effect of the antidepressants amitriptyline and mianserin on GABA-stimulated ³⁶Cl⁻ influx was decreased after microinjections of α1, β1, or β2 subunit aODNs but potentiated after microinjections of γ2 subunit aODNs. This pattern of aODNs effect on amitriptyline and mianserin modulation of GABA-stimulated ³⁶Cl⁻ influx was the same for both antidepressants and similar to GABA but different than that of diazepam and bicuculline. We conclude that multiple subunits of the GABA_A receptor regulate the effect of amitriptyline and mianserin on the GABA_A receptor chloride ionophore complex. However, the exact identity of the subunit mediating the direct or allosteric modulation of the antidepressant effect on GABA-stimulated ³⁶Cl⁻ influx remains unclear.     

Effects of NMDA-R1 antisense oligodeoxynucleotide administration: behavioral and radioligand binding studies

The effects of an antisense phosphodiester oligodeoxynucleotide (ODN) directed to the NR1 subunit of the NMDA receptor mRNA and of its corresponding sense ODN were investigated in mice. Treatment with the antisense ODN significantly increased the time mice spent in the open arms of an elevated maze while the total number of arm entries was unaltered. Furthermore, seizure latencies after the administration of an ED₁₀₀ dose of NMDA (150 mg/kg) were significantly higher in antisense treated animals compared to vehicle controls. At the same time, treatment with NR1 antisense ODN significantly reduced the B_max of [³H]CGS-19755 binding (2101 fmol/mg protein) compared to both vehicle (2787 fmol/mg protein) and sense (2832±39 fmol/mg protein) controls without any significant change in K_D (33 nM). A corresponding reduction of [³H]CGP-39653 binding was also observed after treatment with NR1 antisense compared to both sense and vehicle controls. In contrast, neither antisense nor sense ODNs altered the proportion of high affinity glycine sites or the potency of glycine at either high or low affinity glycine binding sites to inhibit [³H]CGP-39653 binding. These results show that in vivo treatment with NR1 antisense ODNs to the NMDA receptor complex reduces antagonist binding at NMDA receptors and has pharmacological effects similar to those observed with some NMDA receptor antagonists. These results also suggest that treatment with antisense ODNs may provide another means to investigate allosteric modulation of receptor subtypes in vivo.     

Analysis of NMDA Receptors in the Human Spinal Cord

NMDA receptors in postmortem human spinal cord were analyzed using [³H]MK-801 ligand binding and immunoblotting with NMDA receptor subunit-specific antibodies. The averageK_Dfor [³H]MK-801 binding was 1.77 nM with aB_maxof 0.103 pmol/mg. The EC₅₀for stimulation of [³H]MK-801 binding withl-glutamate was 0.34 μM. None of these parameters were affected by postmortem intervals up to 72 h. Immunoblotting of native NMDA receptors showed that NR1, NR2A, NR2C, and NR2D subunits could all be found in the human spinal cord of which NR1 was preferentially located to the dorsal half. Immunoprecipitation of solubilized receptors revealed that NR1, NR2C, and NR2D subunits coprecipitated with the NR2A subunit, indicating that native human spinal cord NMDA receptors are heteroligimeric receptors assembled by at least three different receptor subunits. These results provide a basis for the development of drugs selectively aimed at spinal cord NMDA receptors for the future treatment of spinal cord disorders.     

Effects of Chronic NMDA‐NR2b Inhibition in the Median Eminence of the Reproductive Senescent Female Rat

Gonadotrophin‐releasing hormone (GnRH) neurones of the hypothalamic‐pituitary‐gonadal (HPG) axis drive reproductive function and undergo age‐related decreases in activation during the transition to reproductive senescence. Decreased GnRH secretion from the median eminence (ME) partially arises from attenuated glutamatergic signalling via the NMDA receptor (NMDAR) and may be a result of changing NMDAR stoichiometry to favour NR2b over NR2a subunit expression with ageing. We have previously shown that the systemic inhibition of NR2b‐containing receptors with ifenprodil, an NR2b‐specific antagonist, stimulates parameters of luteinising hormone (used as a proxy for GnRH) release in both young and middle‐aged females. In the present study, we chronically administered ifenprodil, an NR2b‐specific antagonist, at the site of GnRH terminals in the ME or at GnRH perikarya in the preoptic area, in reproductively senescent middle‐aged female rats, aiming to determine whether NR2b antagonism could restore aspects of reproductive functionality. Effects on oestrous cyclicity, serum hormones, and protein expression of GnRH, NR2b and phosphorylated NR2b (Tyr‐1472) in the ME were measured. Chronic ifenprodil treatment in the ME (but not the preoptic area) altered oestrous cyclicity by increasing the percentage of days spent in pro‐oestrus. This was accompanied by increased GnRH fluorescence intensity in the external ME zone and a greater proportion of GnRH terminals that co‐labelled with pNR2b with treatment. We also observed changes in the relationships between protein immunofluorescence, serum hormone levels and other aspects of reproductive physiology in acyclic females, as revealed by bionetwork analysis. Together, these data support the hypothesis that NMDAR‐NR2b expression and phosphorylation state play a role in reproductive senescence and highlight the ME as a major player in reproductive ageing.     

Colocalization of GABAA and NMDA receptors within the dorsal motor nucleus of the vagus nerve (DMV) of the rat

Changes in gastric motor activity are observed in response to glutamate and GABA in the DMV. We investigated the expression of GABA_A and NMDA receptors within DMV neurons projecting to the stomach using pseudorabies virus (PRV). PRV immunoreactive (PRV-IR) cells expressing GABA_A α 1-IR, also expressed NMDAR1 suggesting that NMDA and GABA_A receptors are colocalized. These results provide a neuroanatomical basis for these receptors jointly playing a role in gastric motor functions.