Immunolabeling reveals cellular localization of the N-methyl-D-aspartate receptor subunit NR2B in neurosecretory cells but not astrocytes of the rat magnocellular nuclei

Previous studies suggest that activation of N-methyl-D-aspartate (NMDA) receptors facilitates phasic firing and spike clustering displayed by magnocellular neuroendocrine cells (MNCs) of the supraoptic (SON) and paraventricular nucleus of the hypothalamus (PVN). Osmotic stimulation produces similar activity patterns which, in turn, can lead to enhanced release of vasopressin and oxytocin from MNCs. Our laboratory has shown that dehydration regulates the expression of the NMDA receptor subunits, NR1 and NR2B, in the SON and PVN, suggesting their involvement in osmoregulation. In the present study, we examined the cellular localization of NR2B, one of the glutamate-binding subunits of the NMDA receptor, with an NR2B-specific antibody. Using double-label immunohistochemistry and three different detection methods with metallic, peroxidase, and fluorescence markers, it was found that both vasopressin and oxytocin-producing MNC populations synthesize NR2B. The incidence of NR2B colocalization with vasopressin-neurophysin in the SON and lateral magnocellular PVN (PVL) was 0.95 and 0.91, respectively. For oxytocin-neurophysin, the corresponding values were 0.97 and 0.95, respectively. Furthermore, the extent of colocalization in MNCs of the SON, PVL, retrochiasmatic SON, and accessory neurosecretory nuclei was similar. Astrocytes associated with the SON, and identified with antibodies targeting glial fibrillary acidic protein (GFAP) or vimentin, were not colabeled with NR2B. Our results demonstrate that NR2B protein is expressed by almost all MNCs and that it is equally prevalent in vasopressinergic and oxytocinergic populations of various magnocellular neuroendocrine nuclei supporting a role of NMDA receptors in MNC-mediated neurosecretory processes. Although NR2B may form part of functional NMDA receptors on MNCs, it is probably not present on astrocytes associated with nearby MNCs.     

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

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

Osmotic activation of the hypothalamo-neurohypophysial system reversibly downregulates the NMDA receptor subunit, NR2B, in the supraoptic nucleus of the hypothalamus.

NMDA receptor activation produces a characteristic pattern of neuronal firing in magnocellular neuroendocrine cells (MNCs) of the supraoptic nucleus of the hypothalamus (SON) which has been associated with greater hormone release in vivo and in vitro. In addition, i.c.v. administered NMDA receptor blockers suppress the dehydration-induced rise in plasma vasopressin and drinking. To investigate the role of NMDA receptor subunits in the neuroendocrine functions of the magnocellular neuroendocrine cells of the hypothalamus, we examined the effects of osmotic stimulation on the protein expression of the NMDA receptor subunits, NR1 and NR2B, important in binding glycine and glutamate, respectively. Homogenates of SON, paraventricular nucleus of the hypothalamus (PVN), cortex and lateral hypothalamus from control rats and rats given 2% saline water to drink for 4-10 days were subjected to SDS-PAGE and Western blot analysis. This saline water drinking regimen produced a significant rise in plasma osmolality levels. NR1 and NR2B immunoreactivity was detected in SON, PVN, lateral hypothalamus and cortex but not in liver homogenates using subunit-specific polyclonal antibodies and quantified using computer-assisted densitometry. Mean NR2B immunoreactivity was significantly lower in SON (29%) and PVN homogenates (23%) from saline-treated rats than in those from control rats. In addition, the effect of dehydration on NR2B was regionally specific since no significant changes in NR2B expression were observed in homogenates of cortex and lateral hypothalamus. Rehydration allowed recovery of plasma osmolality as well as NR2B protein levels in the SON. These results suggest that changes in NMDA receptor subunit expression contribute to the plasticity manifested by in magnocellular neuroendocrine cells in response to osmotic activation of the hypothalamo-neurohypophysial system. In addition, our results indicate that NMDA receptors on SON and PVN MNCs may contribute to neuroendocrinological functions associated with body fluid homeostasis.     

Crocin antagonizes ethanol inhibition of NMDA receptor-mediated responses in rat hippocampal neurons

We have previously found that crocin (crocetin di-gentiobiose ester) antagonizes the inhibitory effect of ethanol on long-term potentiation in the rat hippocampus in vivo and in vitro. To explore mechanisms underlying the antagonism of crocin against ethanol, we investigated the effects of ethanol and crocin on synaptic potentials mediated by N-methyl-d-aspartate (NMDA) receptors in the dentate gyrus of rat hippocampal slices. Synaptic potential mediated by non-NMDA receptors was recorded in normal medium (1.3 mM Mg²⁺), while NMDA receptor-mediated synaptic potential was isolated in low (0.13 mM) Mg²⁺ medium containing the non-NMDA receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (10 μM). Crocin (10 μM) alone did not affect synaptic potentials mediated by non-NMDA nor NMDA receptors. Non-NMDA response was slightly inhibited by 100 mM ethanol, while NMDA response was selectively inhibited by lower concentrations (10–50 mM) of ethanol. Crocin (10 μM) did not affect the inhibition of non-NMDA response by 100 mM ethanol, but significantly blocked the inhibition of NMDA response by 10–50 mM ethanol. In addition, we performed whole-cell patch recording with primary cultured rat hippocampal neurons, and confirmed that crocin blocked ethanol inhibition of inward currents evoked by application of NMDA. These results suggest that crocin specifically antagonizes the inhibitory effect of ethanol on NMDA receptor-mediated responses in hippocampal neurons.     

Evidence forN-methyl-d-aspartic acid receptor-mediated modulation of the commissural input to central vestibular neurons of the frog

We have investigated the role ofN-methyl-d-asparte (NMDA) receptors in the excitatory synaptic transmission to central vestibular neurons in the isolated superfused brainstem of the frog. In superfusate containing 1 mM Mg²⁺ field potentials in the vestibular nuclei evoked by electrical stimulation of either the ipsi- or the contralateral VIIIth nerve were not affected by bath-appliedd-2-amino-5-phosphonovaleric acid (D-APV, 25–50 μM), a selective NMDA antagonist. In a low Mg²⁺ solution postsynaptic field potential components were larger than control but still unaffected by D-APV. Ipsi- and contralaterally evoked excitatory postsynaptic potentials (EPSPs) differed in their shape parameters as well as their pharmacological sensitivity. Ipsilaterally evoked EPSPs were not affected by D-APV and had a rise time that was faster than that of contralaterally evoked EPSPs. The peak amplitude of the latter was reduced by D-APV (25–50 μM) to about 65% of the control value in the presence of 1 mM Mg²⁺. During bath application of NMDA (100 μM) an increased input resistance and repetitive de- and hyperpolarizing membrane potential shifts were observed. Similar events were observed during a reduction of the Mg²⁺ concentration. Bath application of NMDA (0.1–1 μM) resulted in an enhanced size of the recorded EPSPs. Dendritic and somatic EPSPs were stimulated on a computer with the assumption of a constant NMDA receptor activation and a pulse-like non-NMDA receptor activation. The results of these stimulations are consistent with the hypothesis that the efficacy of non-NMDA-mediated vestibular commissural synaptic transmission is modulated through tonically activated NMDA receptors.     

Evidence for functional native NMDA receptors activated by glycine or d‐serine alone in the absence of glutamatergic coagonist

In this study we have examined the effects of N-methyl-d -aspartate (NMDA) receptor activation on the release of cholecystokinin and somatostatin from rat neocortical nerve endings. The release of cholecystokinin-like immunoreactivity (CCK-LI) and of somatostatin-like immunoreactivity (SRIF-LI) elicited by 12 mm K⁺ from superfused synaptosomes, but not the spontaneous release, was increased by NMDA in a concentration-dependent manner. The effects of NMDA could be prevented by antagonists selective for the glutamate recognition site, the receptor channel and the glycine site of the NMDA receptor. In the absence of NMDA, glycine increased on its own and in a concentration-dependent manner the depolarization-evoked release of both CCK-LI and SRIF-LI. This effect of glycine was strychnine-insensitive and could be mimicked by d -serine, a stereoselective agonist at the NMDA receptor glycine site. Antagonists selective for the glycine site or for the NMDA receptor channel prevented the effects of glycine/d -serine; these effects were, however, insensitive to blockade of the glutamate recognition site of the NMDA receptor, suggesting that glutamate released from synaptosomes or present as contaminant was not involved. The neuropeptide release elicited by d -serine was strongly inhibited by ifenprodil (0.3 μm ) and by Zn²⁺ ions (50 nm ), selective ligands at the NR2B and NR2A subunits of NMDA receptors, respectively. It is concluded that nerve terminals of CCK- and SRIF-releasing neurons possess non-conventional NMDA receptors whose channels can be operated by glycine or d -serine without apparent activation of the glutamatergic coagonist site. These receptors may display the triple subunit combination NR1/NR2A/NR2B.     

Characterization of the single-channel properties of NMDA receptors in laminae I and II of the dorsal horn of neonatal rat spinal cord

The single-channel properties of native NMDA receptors in laminae I and II of the dorsal horn of the neonatal rat spinal cord were studied using outside-out patch-clamp techniques. These receptors were found to have several features that distinguish them from native NMDA receptors elsewhere in the CNS. Single-channel currents activated by NMDA (100 nm) and glycine (10 microm) exhibited five distinct amplitude components with slope-conductance values of 19.9 +/- 0.8, 32.9 +/- 0.6, 42.2 +/- 1.1, 53.0 +/- 1.0 and 68.7 +/- 1.5 pS. Direct transitions were observed between all conductance levels but transitions between 69-pS openings and 20-, 33- and 42-pS openings were rare. There was no significant difference in the frequency of direct transitions from 42- to 20-pS compared to 20- to 42-pS transitions. The Kb (0 mV) for Mg2+ was 89 microm. The Mg2+ unblocking rate constant was similar to other reported values. However, the Mg2+ blocking rate constant was larger than other reported values, suggesting an unusually high sensitivity to Mg2+. The NR2B subunit-selective antagonist, ifenprodil, had no significant effect on overall channel activity but significantly decreased the mean open time of 53-pS openings. These results suggest neonatal laminae I and II NMDA receptors are not simply composed of NR1 and NR2B subunits or NR1 and NR2D subunits. It is possible that these properties are due to an as yet uninvestigated combination of two NR2 subunits with the NR1 subunit or a combination of NR3A, NR2 and NR1 subunits.     

Modulatory action of PACAP27 on NMDA receptor channel activity in cultured chick cortical neurons

The modulatory effect of PACAP₂₇ on NMDA receptor channel activity in cultured chick cortical neurons was investigated using the outside-out recording mode of the patch clamp technique. Channel opening frequency elicited by 20 μM NMDA, or 20 μM NMDA plus 1 μM glycine, was potentiated in the presence of 100 nM PACAP₂₇ and inhibited with 1000 nM PACAP₂₇. These effects were reversible on washout and reduced when glycine concentration was increased to 10 μM, but were not affected by the PACAP antagonist PACAP_6-27 (1 μM) or the GTP inhibitor GDP-β-S (100 μM). It is suggested that PACAP₂₇ may exert its modulatory action on NMDA receptor channel activity through the glycine site(s).     

Synaptic inputs and action potentials of magnocellular neuropeptidergic cells: Intracellular recording and staining in slices of rat hypothalamus

Excitatory postsynaptic potentials (EPSPs) and action potentials of magnocellular neuropeptidergic cells (MNCs) in the paraventricular (PVN) and supraoptic nuclei (SON) were studied with intracellular recording in coronal slices of rat hypothalamus. The fluorescent dye Lucifer Yellow (LY) was injected intracellularly and the cells were subsequently identified as magnocellular (somata >15 × 15 μm). These cells generally had a large cytoplasm-to-nucleus ratio. In PVN it was frequently possible to trace filled dendrites to the ependyma of the third ventricle, and occasionally dendritic spines could be seen. Electrical stimuli in areas dorsolateral and ventrolateral to the fornix column evoked EPSPs in some anatomically identified MNCs of PVN, which indicates that presynaptic fibers innervating MNCs approach PVN from this region. Short-latency (<1 msec) spikes could be evoked in many MNCs of PVN by stimulation near SON, which is consistent with the known projection to the neurohypophysis of many MNCs. Action potentials in MNCs of PVN and SON had significantly longer durations at one-third spike height (mean ± S.D. = 2.06 ± 0.6 msec) than hippocampal CA1 pyramidal cells (1.17 ± 0.29 msec). This suggests that neuroendocrine cells in mammals and some lower vertebrates and invertebrates are similar in this regard.     

A quantitative description of excitatory amino acid neurotransmitter responses on cultured embryonic Xenopus spinal neurons

We have performed a quantitative analysis of excitatory amino acid neurotransmitter receptors on cultured embryonic Xenopus spinal neurons using the whole-cell patch-clamp technique. Neuroblasts and underlying mesodermal cells isolated from spinal regions of neural plate-stage embryos were placed into dissociated cell culture, and responses were studied soon after the appearance of neurites on embryonic neurons. Glutamate (Glu) receptors were separated into two general classes based on responses to the characteristic agonists quisqualate (Quis), kainate (Ka) and N-methyl-d-aspartate (NMDA); these were NMDA receptors (those activated by NMDA) and non-NMDA receptors (those activated by Ka and Quis). Half-maximal responses to Glu and other agonists on NMDA and non-NMDA receptors were determined from Hill analysis of dose response relations. The order of sensitivities observed was: Glu_NMDA(ED₅₀ = 5.1 μM) >Glu_non-NMDA(ED₅₀ = 28 μM), and for Glu receptor agonists, Quis (ED₅₀ = 1.5 μM) >NMDA(ED₅₀ = 41 μM) >Ka(ED₅₀ = 58 μM). The order of response amplitudes recorded at concentrations near the appropriate ED₅₀s was Glu_NMDA > Glu_non-NMDA, and Ka > NMDA > Quis. A 10-fold decrease in external [Na⁺] shifted the reversal potentials for Glu_non-NMDA, Ka, and Quis to more negative voltages. Increasing external [Ca²⁺] shifted the reversal potential for NMDA responses to more positive potentials, an observation consistent with Ca²⁺ permeation of the embryonic NMDA-activated channel. NMDA-evoked currents could not be recorded in nominally glycine (Gly)-free media. Addition of Gly to external solutions potentiated NMDA responses (ED₅₀ = 644nM). NMDA responses were blocked by dl-2-amino-5-phosphonovaleric acid (APV;ED₅₀ = 1.9 μM) and by Mg²⁺ at negative potentials. In their sensitivities to agonists and antagonists, and ionic dependences, amino acid neurotransmitter responses on embryonic Xenopus neurons closely resembled those previously observed for mature Xenopus and mammalian central neurons. The Glu_NMDA receptors present on these immature neurons were sufficiently sensitive to be activated by endogenous concentrations of extracellular Glu, suggesting a possible role for receptor activation in modulating early neural development.     

Glial Regulation of Extrasynaptic NMDA Receptor‐Mediated Excitation of Supraoptic Nucleus Neurones During Dehydration

Magnocellular neurosecretory cells (MNCs) of the supraoptic nucleus (SON) project to the posterior pituitary gland where they release the hormones, vasopressin and oxytocin into the circulation to maintain plasma osmolality. Hormone release is proportionate to SON MNC action potential (spike) firing rate. When activated by ambient extracellular glutamate, extrasynaptic NMDA receptors (eNMDARs) mediate a tonic (persistent) depolarisation to increase the probability of action potential firing. In the present study, in vivo single‐unit electrophysiological recordings were made from urethane‐anaesthetised female Sprague–Dawley rats to investigate the impact of tonic eNMDAR activation on MNC activity. Water deprivation (for up to 48 h) caused an increase in the firing rate of SON MNCs that was associated with a general increase in post‐spike excitability. To determine whether eNMDAR activation contributes to the increased MNC excitability during water deprivation, memantine, which preferentially blocks eNMDARs, was administered locally into the SON by microdialysis. Memantine significantly decreased the firing rate of MNCs recorded from 48‐h water‐deprived rats but had no effect on MNCs recorded from euhydrated rats. In the presence of the glial glutamate transporter‐1 (GLT‐1) blocker, dihydrokainate, memantine also reduced the MNC firing rate in euhydrated rats. Taken together, these observations suggest that GLT‐1 clears extracellular glutamate to prevent the activation of eNDMARs under basal conditions and that, during dehydration, eNMDAR activation contributes to the increased firing rate of MNCs.     

Developmental changes in localization of NMDA receptor subunits in primary cultures of cortical neurons

Immunoblot analysis, using antibodies against distinct N-methyl-d -aspartic acid (NMDA) receptor subunits, illustrated that the NR2A and NR2B subunit proteins have developmental profiles in cultured cortical neurons similar to those seen in vivo. NR1 and NR2B subunits display high levels of expression within the first week. In contrast, the NR2A subunit is barely detectable at 7 days in vitro (DIV) and then gradually increased to mature levels at DIV21. Immunocytochemical analysis indicated that NMDA receptor subunits cluster in the dendrites and soma of cortical neurons. Clusters of NR1 and NR2B subunits were observed as early as DIV3, while NR2A clusters were rarely observed before DIV10. At DIV18, NR2B clusters partially co-localize with those of NR2A subunits, but NR2B clusters always co-localize with those of NR1 subunits. Synapse formation, as indicated by the presence of presynaptic synaptophysin staining, was observed as early as 48–72 h after plating. However, in several neurons at ages less than DIV5 where synapses were scarce, NR2B and NR1 clusters were abundant. Furthermore, while NR2B subunit clusters were seen both at synaptic and extrasynaptic sites, NR2A clusters occurred almost exclusively in front of synaptophysin-labelled boutons. This result was supported by electrophysiological recording of NMDA-mediated synaptic activity [NMDA-excitatory postsynaptic currents (EPSCs)] in developing neurons. At DIV6, but not at DIV12, CP101, 606, a NR1/NR2B receptor antagonist, antagonized spontaneously occurring NMDA-EPSCs. Our data indicate that excitatory synapse formation occurs when NMDA receptors comprise NR1 and NR2B subunits, and that NR2A subunits cluster preferentially at synaptic sites.     

NMDA Receptor Properties in Rat Supraoptic Magnocellular Neurons: Characterization and Postnatal Development

Hypothalamo-neurohypophysial magnocellular neurons display specific electrical activities in relation to the mode of release of their hormonal content (vasopressin or oxytocin). These activities are under strong glutamatergic excitatory control. The implication of NMDA receptors in the control of vasopressinergic and oxytocinergic neurons is still a matter of debate. We here report the first detailed characterization of functional properties of NMDA receptors in voltage-clamped magnocellular neurons acutely dissociated from the supraoptic nucleus. All cells responded to NMDA with currents that reversed polarity around 0 mV and were inhibited by D-2-amino-5-phosphonovalerate (d-APV) and by 100 μM extracellular Mg²⁺ (at -80 mV). Sensitivity to the co-agonist glycine (EC₅₀, 2 μM) was low compared with most other neuronal preparations. The receptors displayed low sensitivity to ifenprodil, were insensitive to glycine-independent potentiation by spermine, and had a unitary conductance of 50 pS. No evidence was found for two distinct cell populations, suggesting that oxytocinergic and vasopressinergic neurons express similar NMDA receptors. Characterization of NMDA receptors at different postnatal ages revealed a transient increase in density of NMDA currents during the second postnatal week. This was accompanied by a specific decrease in sensitivity to d-APV, with no change in NMDA sensitivity or any other properties studied. Supraoptic NMDA receptors thus present characteristics that strikingly resemble those of reconstituted receptors composed of NR1 and NR2A subunits. Understanding the functional significance of the development of NMDA receptors in the supraoptic nucleus will require further knowledge about the maturation of neuronal excitability, synaptic connections and neurohormone release mechanisms.     

Gp120 can revert antagonism at the glycine site of NMDA receptors mediating GABA release from cultured hippocampal neurons

The effects of the human immunodeficiency virus type 1 envelope protein gp120 on the release of GABA elicited by N-methyl-D-aspartate (NMDA) from rat hippocampal neurons in primary culture has been investigated. NMDA (1–300 μM) increased in a concentration-dependent manner (EC₅₀ = 37.9 ± 12 μM) the release of [³H]-GABA. The effect of 100 μM NMDA was prevented by 30 μM of the GABA transport inhibitor N-(4,4-diphenyl-3-butenyl)guvacine (SKF 100330A). Glycine (10 μM) or gp120 (0.01 μM) affected neither the basal nor the NMDA-evoked [³H]-GABA release. The NMDA (100 μM)-evoked release was prevented by 5,7-dichloro-kynurenic acid (5,7-DCKA), a selective antagonist at the glycine site of the NMDA receptor, in a concentration-dependent manner (IC₅₀ ≃ 0.3 μM). Glycine (3–10 μM) or gp120 (0.003–0.01 μM) produced reversal of the 5,7-DCKA antagonism in a way that suggested competition at a same site; gp120 was at least 3 orders of magnitude more potent than glycine. It is suggested that gp120 may mimic glycine at NMDA receptors. J. Neurosci. Res. 49:732–738, 1997. © 1997 Wiley-Liss, Inc.     

Patch-Clamp Analysis of Direct Steroidal Modulation of Glutamate Receptor-Channels

Steroid hormones regulate the neuroendocrine and behavioral functions of the brain by using a number of diverse cellular mechanisms. Many steroids exert rapid electrophysiological effects on neurons, involving specific interactions with membrane components, such as neurotransmitter receptors. Previous studies suggest that the steroids, estrogen and pregnenolone sulfate (PS), might directly modulate glutamate receptors. The present experiments utilized patch-clamp recording of glutamate receptor-channels in excised membrane patches to test for direct modulation by these steroids. Characteristic single-channel activity from N-methyl-D-aspartate (NMDA) receptors could be elicited in both inside-out and outside-out patches excised from acutely dissociated hippocampal neurons. PS, but not 17ß-estradiol, increased the open probability of NMDA channel activity in inside-out and outside-out patches. The PS-induced increase in open probability could be attributed to an increase in both frequency of opening and mean open time of the NMDA receptor, though the effect on frequency of opening was more prominent. The non-NMDA agonist, kainate, induced continuous shifts and increased noise in the baseline current of outside-out patches, but rarely activated clearly resolvable single-channel openings. 17ß-estradiol and PS had no apparent effect on the kainate-induced currents. These findings suggest that some steroids can directly modulate glutamate receptors, but other steroids may utilize indirect mechanisms for regulating glutamatergic synaptic transmission.     

Evidence for NMDA receptor in the afferent synaptic transmission of the vestibular system

This study aimed to define the pharmacology and physiological role of the N-methyl-d-aspartate (NMDA) receptor in the synapse between the hair cells and primary afferent neurons in the vestibular system. The spontaneous and mechanically evoked spike discharges of vestibular nerve fibers were extracellularly recorded in isolated inner ear from the axolotl (Ambystoma tigrinum). Pressure ejection of NMDA (10⁻⁶ to 10⁻³ M) elicited a dose-dependent increase of the basal spike discharge from the vestibular nerve fibers. Extracellular magnesium antagonized the NMDA effect in a dose-dependent manner.d(-)-2-amino-5-phosphonovaleric acid (AP5, 10⁻⁵ to 10⁻³ M) and 7-chloro-kynurenic acid (7ClKyn, 10⁻⁶ to 10⁻³ M) inhibited the basal activity of the vestibular nerve fibers. 7ClKyn also diminished the responses elicited by the mechanical stimulation of the preparation. Glycine (10⁻⁹ to 10⁻⁶ M) applied by bath substitution enhanced the NMDA responses, and the glycine agonistd-serine partially reversed the 7ClKyn inhibitory action. These results suggest that NMDA receptors participate in the generation of the basal spike discharge of vestibular system primary afferent neurons, but its activation is not critical for the response to brief mechanical stimuli.     

Functional activation of punch-cultured magnocellular neuroendocrine cells by glutamate receptor subtypes.

To provide a simple means to isolate and study the cellular functions of small groups of neurons, we developed a modified 'punch' culture procedure that facilitates acute and long-term in vitro physiological studies. Primary 'punch' cultures of magnocellular neuroendocrine cells (MNCs) from the supraoptic nucleus (SON) were established and the basic physiological effects of subtype-specific glutamate receptor agonists were characterized. MNCs from the punch cultures established a mature morphology in culture with extensive outgrowth of axons and varicosities. After 8 days, a single cultured SON punch produced an average of 10.0 +/- 2.1 pg AVP and contained an average of 222 +/- 53 vasopressin-neurophysin immunoreactive cells. Patch clamp recordings from MNCs demonstrated the presence of N-methyl-D-aspartate (NMDA)-sensitive and DL, alpha-amino-3-hydroxy-5-methylisoxazole propionic acid (AMPA)-receptors. Stimulation of metabotropic receptors with 1S,3R ACPD induced acute or gradual increases in intracellular calcium. NMDA, AMPA and metabotropic receptors all contributed to the secretion of vasopressin from the punch cultures with an agonist rank order potency of: NMDA (10 microM) > AMPA (1 microM) = 1S,3R ACPD (100 microM) > kainate (10 microM). This culture preparation should be useful for cellular studies of small groups of neuroendocrine and other cells.     

Suppression by topiramate of epileptiform burst discharges in hippocampal CA3 neurons of spontaneously epileptic rat in vitro

Topiramate, a novel antiepileptic drug, inhibits the seizures of spontaneously epileptic rat (SER), a double mutant (zi/zi, tm/tm) which exhibits both tonic convulsion and absence-like seizures from the age of 8-weeks. Hippocampal CA3 pyramidal neurons in SER show a long-lasting depolarization shift with accompanying repetitive firing when a single electrostimulation is delivered to the mossy fibers in vitro. The effects of topiramate on the excitability of CA3 pyramidal neurons in SER were examined to elucidate the mechanism underlying the antiepileptic action. Intracellular recordings were performed in 23 hippocampal slice preparations of 16 SER aged 8–17 weeks. Topiramate (10–100 μM) dose-dependently inhibited the depolarizing shifts with repetitive firing induced by mossy fiber stimulation without affecting the first spike and resting membrane potentials in hippocampal CA3 neurons of SER. Higher dose of topiramate (100 μM) sometimes inhibited the first spike, and decreased excitatory postsynaptic potentials in the SER CA3 neurons. However, topiramate up to 100 μM did not affect the single action potential elicited by the stimulation in the hippocampal CA3 neurons of age-matched Wistar rat devoid of the seizure. Application of topiramate (100 μM) did not significantly affect the firing induced by depolarizing pulse applied in the CA3 neurons of the SER. In addition, topiramate (100 μM) had no effects on the Ca²⁺ spike induced by intracellularly applied depolarizing pulse in the presence of tetrodotoxin and tetraethylammonium. In contrast, a dose-dependent inhibition of depolarization and repetitive firing induced by bath application of glutamate in CA3 pyramidal neurons was obtained with topiramate (10–100 μM). Furthermore, topiramate (100 μM) decreased the number of miniature postsynaptic potential of CA3 pyramidal neurons of SER. In patch clamp whole cell recording using acutely dissociated hippocampal CA3 neurons from SER aged 8-weeks and age-matched normal Wistar rats, there were no remarkable effects on voltage dependent Ca²⁺ current with topiramate up to 300 μM in either animal; the current was completely blocked by Cd²⁺ at a concentration of 1 mM. These findings suggest that topiramate inhibits release of glutamate from the nerve terminals and/or abnormal firing of the CA3 pyramidal neurons of SER by mainly blocking glutamate receptors in the neurons.     

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.