Activation Of Ionotropic Glutamate Receptors Research Articles

Reverse Na+/Ca2+Exchange Contributes to Glutamate-Induced Intracellular Ca2+Concentration Increases in Cultured Rat Forebrain Neurons

Activation of ionotropic glutamate receptors causes increases in intracellular Ca2+ concentration ([Ca2+]i) and intracellular Na+ concentration in neurons. It has been suggested that reversal of the plasma membrane Na+/Ca2+ exchanger (NCE) may account in part for the rise in [Ca2+]i. Recently, KB-R7943 (2-[2-[4-(4-nitrobenzyloxy)phenyl]ethyl]isothiourea methanesulfonate) was reported to selectively inhibit the reverse mode of the NCE in non-neuronal cells. We investigated the effects of KB-R7943 on glutamate-stimulated increases in [Ca2+]i. In cultured rat forebrain neurons loaded with indo-1 acetoxymethyl ester, KB-R7943 inhibited the reverse mode of NCE (IC50 = 0.7 microM). When tested against kainate- (100 microM), N-methyl-D-aspartate- (30 microM), glutamate- (3 microM), or KCl- (50 mM) induced [Ca2+]i transients (15 sec, in the presence of Na+ and Ca2+), KB-R7943 inhibited these transients with IC50 values of 6. 6, 8.2, 5.2, and 2.9 microM, respectively. [Ca2+]i increases caused by a higher concentration of glutamate (100 microM) also were inhibited by KB-R7943 (10 microM). However, KB-R7943 had no effect on peak [Ca2+]i changes caused by prolonged application of glutamate and did not inhibit glutamate-induced neuronal injury. KB-R7943 did not inhibit N-methyl-D-aspartate- or kainate-induced whole-cell currents, nor did it substantially inhibit voltage-sensitive Ca2+ currents, excluding a direct inhibition of these ion channels. These results suggest that reverse NCE contributes to the immediate rise in [Ca2+]i resulting from glutamate receptor activation. However, reverse NCE becomes less important as the stimulus time is increased, and Ca2+ entry by this route is not critical for the expression of excitotoxic injury.

Modulation of glutamate receptor pathways in the search for new neuroprotective agents

Excessive stimulation of excitatory amino acid (EAA) receptors is responsible for a wide variety of acute and chronic neurological impairments. A separate line of investigation has focused on oxidative stress as one of the main reasons for several of these degenerative disorders. Current evidence has confirmed that activation of both ionotropic and metabotropic glutamate receptors can also result in either neuroprotection or neurodegeneration according to the role played by oxidative stress mechanisms. An outline of this research, together with our recent results aimed at the discovery of new subtype selective modulators of the central nervous system pathways as well as new classes of free radical scavengers, is presented.

Effects of over- and under-expression of Cu,Zn-superoxide dismutase on the toxicity of glutamate analogs in transgenic mouse striatum

Considerable evidence suggests that reactive oxygen species mediate the neurotoxic effects of ionotropic glutamate receptor activation. Accordingly, we have examined neuronal degeneration resulting from intrastriatal injection of quinolinic acid, an NMDA receptor agonist, and kainic acid in gene targeted and transgenic mice that under- or over-express copper, zinc superoxide dismutase (Cu,Zn-SOD; SOD-1). Elevated SOD-1 activity significantly protects against quinolinic acid and kainic acid neurotoxicity in the mouse striatum whereas reduced activity appears to potentiate neurotoxicity. Thus a `gene-dose' effect of SOD-1 has been demonstrated with regard to excitotoxic mechanisms.

AMPA/Kainate Receptor Activation Inhibits Neuronal Delayed Rectifier K+Current via Na+Entry in Rat Cortical Neurons

In the present study we evaluated the modulation of neuronal delayed rectifier K+current (IK) by activation of ionotropic glutamate receptors. In whole-cell voltage-clamp experiments, an external application of 10-100 μM kainate suppressed the amplitude of IKfollowing an inward shift of holding current. The effect of kainate on IKwas eliminated by CN QX, an AMPA/kainate receptor antagonist, indicating that the receptor-mediated cation entry caused IKsuppression. When external Na+was completely replaced by equimolar choline+or N-methyl-D-glucamine, kainate-induced IKsuppression was abolished. Our results suggest that in cultured rat cortical neurons, AMPA/kainate receptor activation leads to an intracellular Na+increase which blocks delayed rectifier K+channels. This contributes to feed-forward excitation of neuronal cells in glutaminergic responses.

P-212 - Negative regulation of rat cerebellar GABAergic inhibitory transmission via activation of metabotropic and ionotropic glutamate receptors

P-212 - Negative regulation of rat cerebellar GABAergic inhibitory transmission via activation of metabotropic and ionotropic glutamate receptors

Metabotropic glutamate receptors in the plasticity of excitatory responses in the hippocampus. Clinical impact.

Excitatory connections in the hippocampal network have been shown to exhibit plasticity when subjected to specific modes of activation. These include short term and long term potentiation (STP and LTP, respectively) as well as long term depression (LTD). Ionotropic glutamate receptor (iGluR) activation, especially that mediated via NMDA receptors, is key in regulating many of these processes and is discussed in more detail in other chapters in this text. This chapter will demonstrate the participation of metabotropic glutamate receptors (mGluRs) in the plastic processes of the hippocampal circuit and will emphasize the possible clinical relevance of these processes. I will first provide a review of the receptor classification and the actions of the various mGluR subgroups. I will briefly summarize the work to date demonstrating the different ways in which each of these subgroups participates in synaptic plasticity. I will discuss ways in which mGluR-mediated activities may influence clinical conditions such as epilepsy. And finally, I will present the results of my studies on the role of these receptors in the production of epilep-tiform activities in the in vitro hippocampus, focusing on their unique capacity for autopotentiation, providing a potential mechanism for epileptogenesis.

Glutamate-induced release of the nitric oxide precursor, arginine, from glial cells.

Arginine, the nitric oxide precursor, is predominantly localized in glial cells, whereas the constitutive nitric oxide synthase is mainly found in neurons. Therefore, a transfer of arginine from glial cells to neurons is needed to replenish the neuronal precursor pool. This is further supported by the finding that arginine is released upon selective pathway stimulation both in vitro and in vivo. We investigated the mechanism underlying this glial-neuronal interaction by analysing the effect of glutamate receptor agonists on the extracellular [3H]arginine level in cerebellar and cortical slices and in cultures of either cortical astroglial cells or neurons. We present data indicating that arginine is released from cerebellar and cortical slices and astroglial cell cultures upon activation of ionotropic non-NMDA glutamate receptors. Glutamate had no effect on the extracellular [3H]arginine level in neuronal cultures. Moreover, the effect of glutamate in cerebellar slices was tetrodotoxin-insensitive, and the calcium ionophore A23187 evoked the release of [3H]arginine from astroglial cell cultures. Thus, nitric oxide synthesis and nitric oxide transmission may be based on the glial-neuronal transfer of arginine which is induced by activation of excitatory amino acid receptors on glial cells.

Acute ethanol alters calcium signals elicited by glutamate receptor agonists and K + depolarization in cultured cerebellar Purkinje neurons

The effect of acute ethanol on Ca 2+ signals evoked by ionotropic (iGluR) and metabotropic (mGluR) glutamate receptor (GluR) activation and K + depolarization was examined in cultured rat cerebellar Purkinje neurons to assess the ethanol sensitivity of these Ca 2+ signaling pathways. Mature Purkinje neurons ∼3 weeks in vitro were studied. iGluRs were activated by ( RS)-α-amino-3-hydroxyl-5 methyl-4-isoxazolepropionic acid (AMPA; 1 and 5 μM) and domoate (5 μM). mGluRs were activated by (1 S,3 R)-1-aminocyclopentane-1,3-dicarboxylic acid (ACPD; 300 μM) and ( R, S)-3,5-dihydroxyphenylglycine (DHPG; 200 μM). These agents and K + (150 mM) were applied from micropipettes by brief (1 s) microperfusion pulses. Ca 2+ levels were monitored at 2–3 s intervals during pre- and post-stimulus periods using microscopic digital imaging and the Ca 2+ sensitive dye fura-2. iGluR and mGluR agonists and K + produced abrupt increases in intracellular Ca 2+ that slowly recovered to baseline resting levels. Acute exposure to ethanol at 33 mM (150 mg%) and 66 mM (300 mg%) significantly reduced the amplitude of the Ca 2+ signals to iGluR agonists and K + with little or no effect on Ca 2+ signals to mGluR agonists. In contrast, acute ethanol at 10 mM (45 mg%) had no effect on the Ca 2+ signals to the iGluR agonist AMPA but significantly enhanced the Ca 2+ signals to the mGluR agonist DHPG. These results show that ethanol modulates Ca 2+ signaling linked to GluR activation in a receptor subtype specific manner, and suggest that Ca 2+ signaling pathways linked to GluR activation and membrane depolarization may be important mechanisms by which ethanol alters the transduction of excitatory synaptic signals at glutamatergic synapses and thereby affects intercellular and intracellular communication in the CNS.

Metabotropic glutamate receptor mRNA expression in rat spinal cord.

It has recently become clear that, analogous to the situation observed in the brain, glutamatergic neurotransmission in the spinal cord relies upon the activation of ionotropic and metabotropic glutamate receptors (mGluRs). Although electrophysiological studies have emphasized the relevance of mGluR activation in the modulation of spinal neuron responses to glutamate, a detailed study of the molecular forms of receptors belonging to the mGluR family in the spinal cord is still lacking. Using a reverse transcriptase-polymerase chain reaction technique with primers specifically recognizing each cloned mGluR subtype, we found that rat adult spinal cord specifically expresses high levels of mRNA encoding mGluR1a but not mGluR1b. The expression of mRNAs for mGluRs 5a and 5b was also found at levels roughly comparable to that observed in mGluR5-positive brain areas. mGluR2, mGluR3, mGluR4 and mGluR7 mRNAs appeared to be expressed at lower levels, while mGluR6 and mGluR8 mRNAs were not detectable.

Na+/Ca2+ exchanger modulates kainate-triggered Ca2+ signaling in Bergmann glial cells in situ.

The role of sodium-calcium exchanger in calcium homeostasis in Bergmann glial cells in situ was investigated by monitoring cytoplasmic calcium ([Ca2+]i) and sodium ([Na+]i) concentrations. The [Ca2+]i and [Na+]i transients were measured either separately by using fluorescent indicators fura-2 and SBFI, respectively, or simultaneously using the indicators fluo-3 and SBFI. Since the removal of extracellular Na+ induced a relatively small (approximately 50 nM) elevation of [Ca2+]i, the Na+/Ca2+ exchanger seems to play a minor role in regulation of resting [Ca2+]i. In contrast, kainate-triggered [Ca2+]i increase was significantly suppressed by lowering of the extracellular Na+ concentration ([Na+]o). In addition, manipulations with [Na+]o dramatically affected the recovery of the kainate-induced [Ca2+]i transients. Simultaneous recordings of [Ca2+]i and [Na+]i revealed that kainate-evoked [Ca2+]i transients were accompanied with an increase in [Na+]i. Moreover, kainate induced significantly larger [Ca2+]i and smaller [Na+]i transients under current-clamp conditions as compared to those recorded when the membrane voltage was clamped at -70 mV. The above results demonstrate that the Na(+)-Ca2+ exchanger is operative in Bergmann glial cells in situ and is able to modulate dynamically the amplitude and kinetics of [Ca2+]i signals associated with an activation of ionotropic glutamate receptors.

Glutamate-evoked currents in acutely dissociated neurons from the rat medial preoptic nucleus

Membrane currents evoked by glutamate were investigated in acutely dissociated neurons from the medial preoptic nucleus (MPN) of rat. Rapid application of glutamate induced a fast current component in all neurons studied. In addition, in >50% of the neurons, a slow current component was elicited. The fast and the slow current components were selectively blocked by the AMPA-receptor antagonist NBQX and by the NMDA-receptor channel blocker MK-801, respectively. Rapid application of AMPA induced, in all neurons tested, currents with properties similar to the fast component of the glutamate-evoked currents whereas rapid application of NMDA induced, in ≈75% of the neurons, currents similar to the slow component of the glutamate-evoked currents. The NMDA-evoked currents showed a marked outward rectification that was attributed to a potential-dependent block by extracellular Mg 2+. The NMDA-evoked currents also required the presence of extracellular glycine in the micromolar range. In conclusion, the results show that MPN neurons respond to glutamate with currents that can be attributed to activation of ionotropic glutamate receptors of the AMPA-receptor type as well as of the NMDA-receptor type.

Stimulation of the ventral subiculum of the hippocampus evokes glutamate receptor-mediated changes in dopamine efflux in the rat nucleus accumbens.

The effects of electrical stimulation of the ventral subiculum/CA1 region of the hippocampus on changes in dopamine oxidation current (corresponding to dopamine efflux) in the nucleus accumbens were examined using in vivo chronoamperometry with stearate-graphite paste electrodes in urethane-anaesthetized rats. Burst-patterned monophasic pulses (10-100 Hz/burst delivered at 0.8-4 Hz) evoked a three-component change in dopamine efflux in the nucleus accumbens with an initial transient increase in the dopamine signal above baseline, followed by an immediate decrease below baseline, and thereafter by a prolonged increase in the dopamine signal above baseline. 6-Hydroxydopamine lesions of the mesoaccumbens dopamine pathway or transection of the fimbria-fornix blocked all of the evoked changes in the dopamine signal. Both the first and third components of enhanced dopamine efflux were blocked by microinfusion into the nucleus accumbens of the ionotropic glutamate receptor antagonists (+/-)-2-amino-5-phosphonopentanoic acid, 6,7-dinitroquinoxaline-2,3-dione and kynurenate. Burst stimulation-evoked decreases in the dopamine signal were abolished following microinfusions into the nucleus accumbens of the metabotropic glutamate receptor antagonist (+)-alpha-methyl-4-carboxyphenylglycine. These results suggest that ventral subiculum/CA1 glutamatergic inputs to the nucleus accumbens may presynaptically modulate dopamine efflux by synaptic activation of both ionotropic and metabotropic glutamate receptors in the nucleus accumbens. These glutamate-dopamine interactions may constitute part of the mechanisms by which hippocampal signals are integrated through selective modulation of dopamine release in the nucleus accumbens in both physiological and pathological conditions.

Chronic administration of a partial agonist at strychnine-insensitive glycine receptors: a novel experimental approach to the treatment of ischemias.

During the past decade, converging lines of evidence have linked the abnormal release or leak of excitatory amino acids to the neurodegeneration associated with a wide range of pathologies including cerebral ischemias, Huntington's disease, and AIDS dementia (Coyle and Robinson, 1987; Lipton, 1994; Meldrum, 1994). Pharmacological studies indicate that activation of both ionotropic and metabotropic glutamate receptors can substantially contribute to excitotoxic cell damage (Choi, 1992; Pizzi et al., 1993; Sheardown et al., 1993; Xue et al., 1994). Based on these findings, therapeutic strategies based on blunting or blocking glutamatergic transmission may be useful in treating a variety of neurodegenerative disorders.

Two forms of hippocampal long-term depression, the counterpart of long-term potentiation.

In the hippocampus there are two distinct forms of long-term depression (LTD) of excitatory synaptic transmission. In the CA1 region, prolonged low-frequency stimulation induces LTD by activating postsynaptic NMDA receptors, which causes a moderate rise in Ca2+ concentrations. In mossy fiber synapses of the CA3 region, similar low-frequency stimulation also gives rise to LTD. However, this form of LTD (mossy fiber LTD) does not require activation of NMDA receptors, but is mediated by activation of presynaptic metabotropic glutamate receptors. Induction of mossy fiber LTD is not dependent on postsynaptic depolarization or activation of postsynaptic ionotropic glutamate receptors, thus it is likely to be mediated by purely presynaptic mechanisms. This conclusion is confirmed by the analysis of mutant mice lacking presynaptic mGluR2, in which mossy fiber LTD is almost absent. Since long-term potentiation at mossy fiber synapses is also induced presynaptically, the synaptic efficacy may be regulated through common mechanisms bidirectionally, which may contribute to neural information processing in the hippocampus.

Roles of metabotropic glutamate receptors in glial function and glial-neuronal communication.

The amino acid glutamate plays a key role in brain function. One of the major roles of glutamate is to mediate fast excitatory neurotransmission via activation of ionotropic glutamate receptors (iGluRs). More recently, however, it has become clear that glutamate also serves a regulatory function through activation of receptors coupled to modulation of second messenger systems [metabotropic glutamate receptors (mGluRs)]. A body of evidence suggests that mGluRs regulate neuronal function through modulation of ion channels and enzymes to modulate cellular excitability and synaptic transmission. Interestingly, it has become clear that in addition to activation of neuronal receptors, glutamate can activate both iGluRs and mGluRs on glia. A growing body of evidence suggests that the mGluRs on glia play important roles in both glial function and mediation of intercellular signaling.

Activity of ionotropic glutamate receptors in retinal cells: effect of ascorbate/Fe(2+)-induced oxidative stress.

The effect of oxidative stress induced by the oxidant pair ascorbate/Fe2+ on the activity of ionotropic glutamate receptors was studied in cultured chick retina cells. The release of [3H]GABA and the increase of the intracellular free Na+ concentration ([Na+]i), evoked by glutamate receptor agonists, were used as functional assays for the activity of the receptors. The results show that the maximal release of [3H]GABA evoked by kainate (KA; approximately 20% of the total) or AMPA (approximately 11% of the total) was not different in control and peroxidized cells, whereas the EC50 values determined for peroxidized cells (33.6 +/- 1.7 and 8.0 +/- 2.0 microM for KA and AMPA, respectively) were significantly lower than those determined under control conditions (54.1 +/- 6.6 and 13.0 +/- 2.2 microM for KA and AMPA, respectively). The maximal release of [3H]GABA evoked by NMDA under K+ depolarization was significantly higher in peroxidized cells (7.5 +/- 0.5% of the total) as compared with control cells (4.0 +/- 0.2% of the total), and the effect of oxidative stress was significantly reduced by a phospholipase A2 inhibitor or by fatty acid-free bovine serum albumin. The change in the intracellular [Na+]i evoked by saturating concentrations of NMDA under depolarizing conditions was significantly higher in peroxidized cells (8.9 +/- 0.6 mM) than in control cells (5.9 +/- 1.0 mM). KA, used at a subsaturating concentration (35 microM), evoked significantly greater increases of the [Na+]i in peroxidized cells (11.8 +/- 1.7 mM) than in control cells (7.1 +/- 0.8 mM). A saturating concentration (150 microM) of this agonist triggered similar increases of the [Na+]i in control and peroxidized cells. Accordingly, the maximal number of binding sites for (+)-5-[3H]methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10- imine 11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine maleate ([3H]MK-801) was increased after peroxidation, whereas the maximal number of binding sites for [3H]KA was not affected by oxidative stress. These data suggest that under oxidative stress the activity of the ionotropic glutamate receptors is increased, with the NMDA receptor being the most affected by peroxidation.

Differences in agonist and antagonist activities for two indices of metabotropic glutamate receptor‐stimulated phosphoinositide turnover

1. The abilities of the four diastereoisomers of 1-aminocyclopentane-1,3-dicarboxylic acid (ACPD) to stimulate, and the metabotropic glutamate receptor (mGluR) antagonist (+/-)-alpha-methylcarboxyphenylglycine (MCPG) to inhibit, phosphoinositide turnover in neonatal rat cerebral cortex have been studied. Two indices of phosphoinositide cycle activity were assessed; inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) mass accumulation, and total inositol phosphate [3H]-InsPx accumulation (in the presence of Li+) in myo-[3H]-inositol prelabelled slices. 2. The diastereoisomers of ACPD stimulated each response with a rank order of potency of 1S, 3R > 1R, 3R > 1S, 3S >> 1R, 3S. The response to 1R, 3R-ACPD was largely prevented by pre-addition of the NMDA-receptor antagonist, MK-801, or omission of extracellular Ca2+, suggesting that this isomer acts indirectly on phosphoinositide responses through activation of NMDA-type ionotropic glutamate receptors. In contrast, the responses to 1S, 3R- and 1S, 3S-ACPD were unaffected by prior addition of MK-801, but were blocked by MCPG. 3. The concentration of 1S, 3R-ACPD required to half-maximally stimulate the Ins(1,4,5)P3 response (-log EC50 (M), -4.09 +/- 0.10) was significantly higher than that required to exert a similar effect on [3H]-InsPx accumulation (-log EC50 (M), -4.87 +/- 0.07; P < 0.01; n = 4). A similar marked 8-9 fold discrepancy between these two values was observed for the 1S, 3S isomer, which elicited similar maximal responses to those caused by 1S, 3R-ACPD. 4. Significant differences were also observed with respect to the ability of (+/-)-MCPG (1 mM) to cause a rightward shift in the concentration-response relationships for 1S, 3R-ACPD-stimulated Ins(1,4,5)P3 (5.59 +/- 0.24 fold shift) and [3H]-InsPx (3.04 +/- 0.34 fold shift; P < 0.01; n = 4) responses, giving rise to Kd values of 218 and 490 microM for (+/-)-MCPG antagonism of the respective responses. 5. The potency difference between the 1S, 3R-ACPD-stimulated Ins(1,4,5)P3 and [3H]-InsPx responses was reduced when experiments were performed in nominally calcium-free medium ([Ca2+]e = 2 - 5 microM) and EC50 values were almost identical when extracellular calcium was reduced further by EGTA addition ([Ca2+]e < or = 100 nM). Similarly, the Kd value for (+/-)-MCPG antagonism of the 1S, 3R-ACPD-stimulated [3H]-InsPx response decreased under [Ca2+]e-free conditions, approaching those obtained for the 1S, 3R-ACPD-stimulated Ins(1,4,5)P3 response in the presence of normal [Ca2+]e. 6. These data suggest that estimates of the activities of mGluR agonists and antagonists, derived by measuring phosphoinositide turnover, can differ significantly depending on whether Ins(1,4,5)P3 mass or [3H]-InsPx responses are measured. In particular, the possibility that the mGluR-mediated [3H]-InsPx response may not simply reflect direct receptor/G protein/phosphoinositidase C (PIC) activation, but may also be the consequence of stimulation of a facilitatory Ca2+-influx pathway is discussed.

Glutamate transporter mRNA expression in proliferative zones of the developing and adult murine CNS

Neuronal migration, differentiation, and synapse formation are developmental processes within the CNS significantly influenced by ionotropic and metabotropic glutamate receptor activity. Extracellular glutamate concentrations mediating this activity are regulated by transport proteins localized in neuronal and glial cell membranes. We have used in situ hybridization analysis with subtype-specific antisense-oligonucleotides to study the distribution of glia-specific excitatory amino acid transporter (mEAAT1 and mEAAT2) mRNAs during the later stages of embryogenesis and postnatal CNS development. Distinct but overlapping embryonic and postnatal patterns of localization were observed for the two transporter transcripts. Both mEAAT1 and mEAAT2 mRNAs were found during the peak period of gliogenesis (E15-E19) in the telencephalic and mesencephalic CNS proliferative zones. The overall expression of mEAAT1 mRNA diminished after the completion of cell migration, whereas mEAAT2 mRNA expression increased significantly during postnatal development. Interestingly, mEAAT2 transcript expression continued in the subventricular zone postnatally and persisted in this proliferative zone in the adult brain. From PO onward, mEAAT1 mRNA was present predominantly in the cerebellar Purkinje cell layer and at a much lower abundance in the cortex, hippocampus, basal nuclei, and septum, whereas from P7 onward, mEAAT2 mRNA expression increased throughout most of the neuraxis. Postnatally, transcripts for mEAAT1 and mEAAT2 were found in cell bodies, processes, and commissural white matter tracts of the CNS. The divergent temporal and spatial expression of EAAT subtypes and their persistence in mature fiber tracts and radial glia layers reveal that specific EAATs are likely to play multiple distinct roles in the developing and adult CNS, including the regulation of cell proliferation, axon-glia interactions, and neuronal survival.

Opposing effects on blood pressure following the activation of metabotropic and ionotropic glutamate receptors in raphe obscurus in the anaesthetized rat.

The microinjection of L-glutamate (1-6 nmol/rat) and N-methyl-D-aspartate (NMDA 1-10 nmol/rat), ionotropic glutamate receptor (iGluR) agonists, into the nucleus raphe obscurus caused a concentration -dependent increase of arterial blood pressure. In contrast, (+/-)-1-aminocyclopentane-trans-1,3-dicarboxylic acid (t-ACPD, 14-42 nmol/rat), a metabotropic glutamate receptor (mGluRs) agonist, caused a concentration-dependent decrease in blood pressure. Pretreatment with D,L-2-amino-phosphono valeric acid (2-APV, 5 nmol/rat) a selective NMDA iGluR antagonist, and (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,b] cyclohepten-5,10-imine hydrogen maleate (MK801, 0.9 nmol/rat), a noncompetitive NMDA iGluR antagonist, blocked both the glutamate and NMDA pressor responses, while pretreatment with (+)-alpha-methyl-4-carboxyphenylglycine (MCPG, 0.05 nmol/rat), a mGluR1 antagonist, increased the glutamate-induced pressor effects and blocked the fall in blood pressure induced by t-ACPD. 6-Cyano-7-nitroquinoxaline-2,3-dione (CNQX, 0.4 nmol/rat) a non-NMDA iGluR antagonist, did not affected the glutamate-induced hypertension. These observations indicate opposing roles for ionotropic and metabotropic receptors in the glutamate-induced blood pressure changes elicited from the nucleus raphe obscurus. Moreover, we suggest that the glutamate-induced hypertension may be due to the activation of NMDA ionotropic receptor subtypes and the metabotropic receptors may influence this activation through a reduction of excitability at level of synapses.

Developmental Regulation of Expression of GABAA Receptor α1 and α6 Subunits in Cultured Rat Cerebellar Granule Cells

We have studied the postnatal development of GABAA receptor α1 and α6 subunits expressed by primary cultures of cerebellar granule cells originating from 2-day-old (postnatal day 2, P2) and 10-day-old (P10) rat neonates. At these ages, the granule cells are at distinct stages of cerebellar development. In both cases, GABAA receptor α1 and α6 subunit-like immunoreactivities were detected, and displayed temporal expression profiles that were correlated with the maturity of the cerebella from which the cultured granule cells were derived. Using two different specificity anti-α1 subunit-specific antibodies, immunoreactive species with Mr 53,000 Da and 54,000 Da were detected by immunoblotting. The lower 53,000-Da band co-migrated with the α1 subunit-like immunoreactivity detected in GABAA receptors purified from adult rat forebrain by benzodiazepine affinity chromatography. This 53,000-Da α1 subunit-like immunoreactive species was detected at day 1 in vitro (1 DIV) in P10 cultures and 3–5 DIV in P2 cultures. The GABAA receptor α6 subunit-like immunoreactivity (58,000 Da) was not detected until 5–7 DIV in P10 and 9–11 DIV in P2-derived cultures. The appearance of α6 subunit-like immunoreactivity was paralleled by an up-regulation of α1 subunit expression and a concomitant increase in diazepam-insensititive (DZ-IS) [3H]Ro 15-4513 binding activity, a pharmacological characteristic of α6 and α1α6-subunit-containing GABAA receptors (Pollard et al. J. Biol. Chem., 270, 21,285–21,290, (1995)). Antagonism of both non-NMDA and NMDA subtypes of ionotropic glutamate receptors did not significantly affect the developmental profile, the level of GABAA receptor α6 subunit or the total DZ-IS or DZ-S [3H]Ro 15-4513 binding activities expressed by these neurons. These results provide further evidence that the expression of specific GABAA receptor subunit genes is subject to differential regulation. Furthermore, developmental expression of the GABAA receptor α6 subunit gene by these neurons is either a preprogrammed event or is initiated by an environmental cue that is received early in granule cell development, and it is not a result of afferent activation of ionotropic glutamate receptors. Copyright © 1996 Elsevier Science Ltd