Actions of excitatory amino acid agonists and antagonists on the primary afferents of the vestibular system of the axolotl (Ambystoma mexicanum)

In order to determine the nature of the transmitter in the synapse between hair cells and primary afferent fibers, both resting and evoked spike activity of vestibular system afferents were recorded. Excitatory amino acid agonists and antagonists were applied by micro perfusion. Excitatory amino acid agonists consistently increased the firing rate of these afferents. The rank order in potencies of the agonists tested was:kainatequisqualate>d-aspartatel-glutamatel-aspartate> N-methyld-aspartate. Blockade of synaptic transmission with high-Mg²⁺ and low-Ca²⁺ solutions did not seem to affect the responses to the excitatory amino acid agonists indicating their postsynaptic action. Excitatory amino acid antagonists inhibit both resting and physiologically evoked activity. The rank order of inhibitory potency was:kynurenate>l-glutamate diethyl ester>d,l-2-amino-4-phosphono-butyrate>d-α-amino adipate>d,l-2-amino-5-phosphonovalerate. This findings suggest that an amino acid-related compound may be transmitter at this synapse. The relative potencies of agonists and antagonists tested provide evidence that the transmitter released from the hair cell's basal pole in the axolotl vestibular system interacts with postsynaptic kainic/quisqualic type receptors.     

Pharmacological aspects of excitatory synaptic transmission to second-order vestibular neurons in the frog

Synaptic excitation of second-order vestibular neurons is mediated by two principal afferents: vestibular afferents projecting into the brain via the VIIIth cranial nerve and commissural afferents from the contralateral vestibular nuclear complex. The shape of the excitatory postsynaptic potentials (EPSPs) generated by selectively activating these two inputs differs qualitatively, such that ipsilateral VIIIth nerve afferents generate a faster-rising EPSP than do the commissural afferents. We have investigated the synaptic pharmacology of these two inputs in the isolated, intact medulla of the frog in order to determine the nature of the transmitter substances released by the afferents and the nature of the subsynaptic receptors with which these transmitters interact. Electrical stimulation of the ipsilateral VIIIth cranial nerve evokes in the region of the vestibular nuclear complex a field potential that exhibits a presynaptic (afferent volley) and a postsynaptic (slow negativity) component. Bath application of glutamate receptor antagonists, such as kynurenic acid (KENYA), blocks the postsynaptic component of this field potential in a dose-dependent manner, without affecting the presynaptic volley, suggesting that the VIIIth nerve afferent releases glutamate and/or similar substances as its neurotransmitter. A comparison of the actions of various glutamate receptor antagonists to block this postsynaptic negativity gives a rank order of effectiveness such that KENYA greater than gamma-D-glutamylglycine (gamma DGG) = gamma-D-glutamylaminomethylsulfonic acid (GAMS) greater than gamma-D-glutamyltaurine (gamma DGT) much greater than gamma-D-glutamylaminomethylphosphonic acid (GAMP) greater than D-2-amino-5-phosphonovaleric acid (D-APV) greater than D,L-APV greater than D-2-amino-7-phosphonoheptanoic acid (APH). This rank order of effectiveness suggests that the VIIIth nerve transmitter activates second-order neurons through kainate (KA)/quisqualate (QUIS) synaptic receptors. Intracellular studies support these conclusions. Chemically mediated EPSPs evoked from ipsilateral VIIIth nerve stimulation are completely blocked by high concentrations of KENYA (greater than or equal to 1 mM). Occasionally an extremely short-latency, probably electrically mediated, component to these EPSPs persists in the presence of KENYA. The slower-rising EPSPs evoked from contralateral VIIIth nerve or contralateral vestibular nuclear complex stimulation are also completely blocked by KENYA, suggesting that the transmitter released by the commissural afferents is also glutamate and/or related compounds.(ABSTRACT TRUNCATED AT 400 WORDS)     

Pharmacology of the vestibular hair cell-afferent fiber synapse in the frog

The isolated, intact, membranous labyrinth of the frog (Rana temporaria) has been investigated electrophysiologically in vitro to determine the nature of the transmitter substance at the synapse between the vestibular hair cells and afferent fibers. Spontaneous synaptic activity can be monitored with intra-axonal recordings from the afferents. Increased K+ in the bath results in an increase in frequency of presynaptic release, as indicated by an increased frequency of spontaneous synaptic potentials. Adding Mg2+ and lowering Ca2+ results in a decrease in synaptic potential frequency (often to zero) with no change in their mean amplitude, indicating pre-synaptic blockade. Extracellular recordings from individual vestibular afferents indicate that bath-applied glutamate and related acidic amino acids consistently increase the firing rates of these afferents in a dose-dependent manner with no evidence of desensitization. In the presence of presynaptic blockade (high Mg2+/low Ca2+), bath application of glutamate and its agonists results in a reversible depolarization of vestibular afferents, suggesting a postsynaptic action of these substances. 2-Amino-5-phosphonovaleric acid, kynurenic acid, and other acidic amino acid antagonists reversibly decrease the amplitudes of spontaneously occurring synaptic potentials without affecting their frequency, indicating subsynaptic blockade. These antagonists also block the postsynaptic depolarizations due to glutamate and its agonists. GABA and its agonists and antagonists have no consistent effect upon afferent activity. These findings suggest that glutamate, aspartate, or a related compound is the transmitter at this synapse. However, the antagonists used, or the receptors themselves, are not selective enough to discriminate adequately between the agonists. Therefore, which of these glutamate agonists are actually involved in synaptic transmission remains to be determined.     

Heterogeneity of sodium-dependent excitatory amino acid uptake mechanisms in rat brain

The pharmacologic and kinetic characteristics of sodium-dependent uptake of [3H]L-glutamate, [3H]D-aspartate, and [3H]L-aspartate into crude synaptosomal preparations of rat corpus striatum and cerebellum have been examined in vitro. In cerebellum the apparent Kts and Vmax for the three excitatory amino acids were identical whereas in striatal synaptosomes, the Vmax for [3H]L-glutamate was 30% greater (P less than or equal to .001) than for [3H]D-aspartate and 50% greater (P less than or equal to .001) than for [3H]L-aspartate. L-Amino adipic acid inhibited the uptake of the three amino acids in both regions of brain was 15- to 20-fold more potent in cerebellum than in striatum. In contrast, dihydrokainic acid inhibited transport processes in the corpus striatum but was without activity in cerebellar preparations. The neurotoxin kainic acid blocked only a portion (60%) of [3H]L-glutamate and [3H]D-aspartate uptake in cerebellum while completely inhibiting amino acid transport in corpus striatum. Three days post kainic acid lesion, [3H]D-aspartate uptake was attenuated more than [3H]L-glutamate uptake in the corpus striatum; destruction of corticostriatal afferents reduced [3H]L-glutamate to a greater extent than [3H]D-aspartate. Various lesions of the cerebellum affected excitatory amino acid transport processes to a similar extent. These results suggest that excitatory amino acid transport systems are pharmacologically distinct in different brain regions and may be heterogeneous within a single region.     

Structure - function relationships for gamma-substituted glutamate analogues on dentate granule cells

We previously demonstrated in the Schaffer collateral-CA1 region of the hippocampus that bath-applied agonists could be distinguished from antagonists among a group of acidic amino acid analogues by extracellular recording techniques. Here we report the use of the extracellular signs of agonist activity for discerning agonists and antagonists among several gamma-substituted glutamate analogues tested in the perforant path. The two-pathway composition of the perforant path offers the advantage over CA1 in that pathway-specificity, a postulated characteristic of antagonists, may be tested. By extracellular recording, D- and L-homocysteic acid, L-serine-O-sulfate, and L-2-amino-4-(5-tetrazolyl)-butanoic acid [L-glutamate tetrazole] were identified as agonists, and all 4 analogues were more potent than L-glutamate for inhibiting synaptic field potentials. Two previously identified antagonists, L-2-amino-4-phosphonobutyric acid and L-O-phosphoserine, exhibited the pathway-specificity and inhibitory kinetics consistent with properties expected for antagonists; both compounds detected 3 perforant path components with the same rank in sensitivity, suggesting that they are acting on the same set of receptors.     

Functional support of glutamate as a vestibular hair cell transmitter in an amniote

Although hair cells in the cochlea in the vestibular endorgans of anamniotes are thought to release glutamate or a similar compound as their transmitter, there is little evidence in amniotes (which, unlike anamniotes, possess both type I and II hair cells) as to the nature of the hair cell transmitters in the vestibular labyrinth. We have recorded extracellularly from sigle semicircular canal afferents in the turtle labyrinth maintained in vitro and have bath-applied a number of transmitter agonists and antagonists to relate the effects of these substances to the actions of the endogenous transmitter subtstances. Both glutamate and aspartate strongly excite the afferents while GABA and carbachol have negligible or weak effects. In contrast to its lack of effect on afferent activity in some anamniotes, N-methyl-d-aspartate (NMDS) was also found to excite these afferents. Kynurenic acid reversibly reduced the resting firing rates of the afferents and the increases in firing due to the application of glutamate and aspartate. These findings provide preliminary support for the hypothesis that glutamate (or a related compound) is also a vestibular hair cell transmitter in amniotes.     

Pharmacological characterization of voltage-clamped catfish rod horizontal cell responses to kainic acid

Excitatory amino acid-induced currents were examined in voltage-clamped rod horizontal cells dissociated from the catfish retina. The cells responded to glutamate (GLU) and the GLU analogues kainate (KA), quisqualate (QA), and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), while N-methyl-D-aspartate (NMDA) produced inconsistent responses. Of the effective agonists, only KA produced large, concentration-dependent current responses. While QA, AMPA, GLU, and NMDA were poor agonists, these compounds were able to block rod horizontal cell responses to KA. The rank order potency for this inhibition was: QA greater than AMPA greater than or equal to L-GLU much greater than D-GLU = NMDA. Several excitatory amino acid receptor antagonists were also able to inhibit rod horizontal cell responses to KA. The rank order potency for the inhibition by the compounds tested was: kynurenate greater than cis-piperidine-dicarboxylic acid much greater than D,L-alpha-amino-adipate. Comparison of the potency of several ligands to inhibit rod and cone horizontal cell responses to KA suggested similarities in the KA binding sites of both cell types.     

Differential effects of N-methyl-D-aspartic acid and L-homocysteic acid on cerebellar Purkinje neurons

The effects of N-methyl-D-aspartic acid (NMDA) and L-homocysteic acid (LH) were measured on cerebellar Purkinje neurons. In urethane-anesthetized rats, iontophoretic application of NMDA elicited 3 different effects on the spontaneous activity of Purkinje cells: excitation, inhibition and biphasic responses consisting of excitation followed by inhibition. On the other hand, LH elicited excitation, only, regardless of the actions of NMDA on the same neurons. We also examined the effects of various excitatory amino acid antagonists on NMDA- and LH-mediated responses. Excitatory effects of NMDA were antagonized effectively by D.L-2-amino-5-phosphonovalerate (APV), ketamine, gamma-D-glutamylglycine (DGG), D,L-2-amino-7-phosphonoheptanoate (APH), and were not influenced significantly by L-glutamate diethylester (GDEE). Inhibitory responses of NMDA were antagonized by APV, APH and ketamine. LH-mediated excitations were influenced significantly by DGG and ketamine whereas GDEE, APV and APH failed significantly to attenuate the effects of LH. Based on the differential actions of LH and NMDA and the selectivity of NMDA antagonists for NMDA rather than LH-mediated excitations, it appears that the major actions of LH may not be mediated through NMDA receptor sites, at least in the cerebellum.     

Pharmacological characterization and differential expression of NMDA receptor subunits in the chicken vestibular system during development

Previous studies demonstrated that in vitro preparations of the isolated vestibular system of diverse animal species still exhibit stable resting electrical activity and mechanically evoked synaptic transmission between hair cells and primary afferent endings. However, there are no reports related to their neurodevelopment. Therefore, this research aimed to examine whether NMDA receptors mediate these electrical signals in an isolated preparation of the chicken vestibular system at three developmental stages, E15, E18, and E21. We found that the spontaneous and mechanically evoked discharges from primary afferents of the posterior semicircular canal were modulated by agonists NMDA and glycine, but not by the agonist d -serine applied near the synapses. Moreover, the individually applied by bath perfusion of three NMDA receptor antagonists (MK-801, ifenprodil, and 2-naphthoic acid) or high Mg²⁺ decreased the resting discharge rate, the NMDA response, and the discharge rate of mechanically evoked activity from these primary afferents. Furthermore, we found that the vestibular ganglion shows a stage-dependent increase in the expression of NMDA receptor subunits GluN1, GluN2 (A-C), and GluN3 (A-B), being greater at E21, except for GluN2D, which was inversely related to the developmental stage. However, in the crista ampullaris, the expression pattern remained constant throughout development. This could suggest the possible existence of presynaptic NMDA receptors. Our results highlight that although the NMDA receptors are functionally active at the early embryonic stages of the vestibular system, NMDA and glycine reach their mature functionality to increase NMDA responses close to hatching (E21).     

Involvement of excitatory amino acids in neurotransmission of inspiratory drive to spinal respiratory motoneurons

The role of excitatory amino acids in the transmission of bulbospinal respiratory drive to spinal motoneurons was investigated in the in vitro and in vivo spinal cord of the rat. In vitro studies were performed with a preparation of neonatal rat brain stem and spinal cord that spontaneously generates rhythmic respiratory drive to spinal respiratory motoneurons. This in vitro system allowed examination of the effects of pharmacological agents on spinal motoneuron activity, without perturbing the activity of bulbospinal neurons transmitting the respiratory drive. The amplitude of spontaneous motor discharge in spinal ventral roots containing phrenic and intercostal motor axons was reduced in a dose-dependent manner by antagonists to excitatory amino acids acting at NMDA receptors [D,L-2-amino-5-phosphonovaleric acid (D,L-AP5)] and non-NMDA receptors [kynurenic acid, gamma-D-glutamylglycine, gamma-D-glutamyltaurine, and L- and D,L-2-amino-4-phosphonobutyric acid (L-AP4,D,L-AP4)]. The order of potency of the antagonists for complete block of the motor output was L-AP4 greater than D,L-AP4 greater than kynurenic acid greater than gamma-D-glutamylglycine greater than D,L-AP5 greater than or equal to gamma-D-glutamyltaurine. Amino acid uptake inhibitors augmented the spontaneous motoneuron activity, further confirming the involvement of endogenous excitatory amino acids in transmission of respiratory drive. The results obtained in vitro with AP4, kynurenic acid, and amino acid uptake inhibitors were confirmed in vivo by bathing segments of the rat spinal cord in situ with solutions containing antagonists and uptake inhibitors. The present results suggest that an important component of the neurotransmission of bulbospinal respiratory drive involves endogenous excitatory amino acids acting at AP4-sensitive sites and other non-NMDA (quisqualate/kainate) receptors. The bulbospinal-spinal respiratory motoneuron synapse may provide a convenient model synapse in the spinal cord for detailed analysis of mechanisms underlying excitatory amino acid-mediated synaptic transmission of motor drive.     

Localization of L-glutamate and glutamate-like receptors at the squid giant synapse.

HPLC analysis of the amino acid contents of the second- and third-order giant fibres at the giant synapse in the stellate ganglion of the squid Loligo vulgaris shows that there are significantly higher amounts of L-glutamate and L-aspartate in the second-order (presynaptic) fibre than in the third-order (postsynaptic) fibre. Immunocytochemical staining of sections of the ganglion with an antibody raised against L-glutamate produces specific positive staining in the synaptic region of the second-order fibre. In contrast, staining with antibodies raised against glutamate-receptors (mammalian GluR1 with GluR2/3) produces positive staining in the third-order fibre at the postsynaptic region. These data provide further evidence for the hypothesis that L-glutamate is an excitatory transmitter at the giant synapse.     

Primary afferent excitatory transmission recorded intracellularly in vitro from rat medial vestibular neurons

Intracellular recordings were made from rat medial vestibular nucleus (MVN) neurons in transverse brain slices containing the root of the vestibular nerve (N. VIII). Electrical stimuli applied to the N. VIII tract evoked an orthodromic excitatory postsynaptic potential (EPSP) that lasted about 50 ms following a 0.5 to 1.5 ms delay between the stimulus artifact and synaptic potential. These orthodromic EPSPs were insensitive to the following antagonists: atropine, hexamethonium, diphenhydramine, and caffeine. Based on these results we conclude that the primary afferent excitatory transmitter is not acetylcholine, histamine, or adenosine, respectively. However, kynurenic acid, a general excitatory amino acid receptor antagonist, blocked the orthodromic EPSP while having no effect on the resting membrane potential, input resistance, or action potential configuration of MVN neurons. Our data suggest that an excitatory amino acid, or amino acid-like substance, is responsible for primary afferent excitatory transmission in the rat medial vestibular nucleus.     

Analysis of the effect of quisqualate, N-methyl-D- aspartate and several blockers of amino acid receptors on synaptic transmission in the ampullae of Lorenzini

Effects of bath-application of quisqualate (Q), N-methyl-D-aspartate (NMDA) and antagonists of NMDA-receptors: D-amino-adipate (AA), 2-amino-4-phosphonobutyrate (APB), 2-amino-5-phosphonovalerate (APV) and Mg2+ as well as acidic amino acid antagonist: D-glutamylglycine (DGG) on the synapse between the electroreceptor cells and afferent fibres were studied in the ampullae of Lorenzini. Q (threshold concentration 10(-8) M) and NMDA (threshold concentration 10(-5) M) strongly excited afferent fibres. Neither AA nor APB influenced the resting and evoked activities. APV blocked the synaptic transmission. Mg2+ (30-50 mM) blocked the responses to NMDA, while Q-induced responses were not affected, APV preferentially blocked NMDA-induced responses and in lesser degree--L-aspartate (L-ASP)-induced responses. DGG blocked the synaptic transmission. It is supposed that the synaptic receptor could represent a homogeneous receptor with different binding sites to the known agonists.     

Calcium channels functional roles in the frog semicircular canal

Different types of voltage-operated calcium channels have been described in hair cells; however, no clear functional role has been assigned to them. As a first functional characterization of vestibular calcium channels, we studied the effect of several calcium channel agonists and antagonists on whole nerve firing rate in an isolated frog semicircular canal preparation. Resting activity was affected by all dihydropyridines tested and by omegaconotoxin GVIA, whereas only nimodipine was able to reduce the mechanically evoked activity. These results indicate that nimodipine-sensitive channels play a major role in afferent transmitter release, and omega-conotoxin GVIA sensitive channels regulate the afferent firing (possibly on the postsynaptic side) but with a less important role.     

Mode of synaptic transmission between vestibular afferents and neurons of the vestibular nucleus in the frog

Excitatory postsynaptic potentials (EPSPs) were evoked in vestibular neurons of the isolated frog brainstem by stimulation of the ipsilateral vestibular nerve or by direct intra-axonal activation of single vestibular fibers. Composite as well as single-fiber EPSPs usually displayed two components. A Ca2+-deficient, Mn2+-containing solution abolished the delayed chemical components of the EPSPs, but did not affect the short latency responses suggesting a dual (electrical-chemical) mode of transmission between some vestibular afferents and vestibular neurons.     

Excitatory amino acid antagonists depress transmission in hippocampal projections to the lateral septum

Antagonists of excitatory amino acid neurotransmission were tested as antagonists of septal excitatory postsynaptic potentials (EPSPs) generated by stimulation of the fimbria. Septal EPSPs were depressed in a concentration-dependent manner by kynurenic acid and P-bromobenzoyl piperazine-2,3-dicarboxylic acid but not by L-2-amino-4-phosphonobutyric acid or D-2-amino-5-phosphonopentanoic acid. These results indicate that the hippocampal projection to the lateral septum is similar to the Schaffer collateral system and is mediated by an excitatory amino acid or a similar derivative.     

Characterization of the excitatory amino acid receptor-mediated release of [H]acetylcholine from rat striatal slices

The pharmacological nature of the interaction of excitatory amino acids with striatal cholinergic neurons was investigated in vitro. Agonists of excitatory amino acid receptors evoked the release of [³H]acetylcholine from slices of rat striatum, in the presence of magnesium (1.2 mM). Removal of magnesium from the medium markedly increased the release of [³H]acetylcholine evoked by all excitatory amino acid receptor agonists tested, with the exception of kainate. In the absence but not the presence of magnesium, a clear rank order of potency was found: N-methyl-dl-aspartate = ibotenate >l-glutamate >l-aspartate cysteate > kainate = quisqualate.The excitatory amino acid receptor mediating [³H]acetylcholine release resembles the N-methyl-d-aspartate preferring (N-type) receptor, as previously characterized electrophysiologically, according to 3 criteria: (1) rank order of potency of agonists; (2) magnesium-sensitivity; and (3) antagonism by 2-amino-5-phosphonovalerate.The release of [³H]acetylcholine evoked by N-methyl-dl-aspartate was blocked by tetrodotoxin (0.5 μM). Moreover, N-methyl-dl-aspartate failed to evoke [³H]acetylcholine release from slices of hippocampus, where cholinergic afferents, rather than interneurons, are found. These results suggest that excitatory amino acids act at receptors on the dendrites of striatal cholinergic interneurons, giving rise to action potentials and release of acetylcholine from cholinergic nerve terminals.     

NMDA and AMPA receptors in the lateral nucleus of the amygdala are postsynaptic to auditory thalamic afferents

Projections from the medial geniculate body (MGB) to the lateral nucleus of the amygdala (LA) have been implicated in the conditioning of emotional reactions to acoustic stimuli. Anatomical and physiological studies indicate that this pathway uses the excitatory amino acid L-glutamate as a transmitter. Recent physiological studies have demonstrated that synaptic transmission in the thalamo-amygdala pathway requires the activation of both N-methyl-D-aspartate (NMDA) and α-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) receptors, two of the major classes of ionotrophic glutamate receptors. In order to characterize the nature of thalamo-amygdala interactions, we examined the synaptic associations between thalamic afferents and amygdala neurons that contain at least one glutamate receptor subtype. Thalamic afferents to the amygdala were identified by lesion-induced anterograde degeneration and anterograde transport of biotinylated dextran-amine, while postsynaptic glutamate receptors were labeled immunocytochemically using antisera directed the R1 subunit of the NMDA receptor and the GluR1 and GluR2/3 subunits of the AMPA receptors. Both methods demonstrated that the majority (77%) of thalamic afferents contact dendritic spines, and most (60%) of these spines express at least one glutamate receptor subtype. To a lesser extent, identified afferents also contacted small and large dendritic shafts, and many of these were immunoreactive. Thalamic afferents terminated on approximately the same proportion (60%) of immunoreactive targets for each glutamate receptor studied. These data provide morphological evidence that thalamic afferents directly synapse onto amygdala neurons that express glutamate receptors and suggest ways in which thalamic afferents activate and influence amygdala circuitry. Synapse 27:106–121, 1997. © 1997 Wiley-Liss, Inc.     

Presynaptic actions of cholinergic agents upon the hair cell-afferent fiber synapse in the vestibular labyrinth of the frog

Spontaneous activity of semicircular canal afferents in the isolated labyrinth of the frog is altered by bath application of cholinergic agonists. Muscarinic agonists can produce an increase in action potential frequency of individual afferents. This increase develops slowly and is prolonged in the time course of its action. Nicotinic agonists can either increase (most cases) or decrease afferent activity. These effects occur rapidly and decay during the period of activation, suggesting desensitization. Muscarinic effects are blocked by prior administration of atropine and nicotinic effects (both increases and decreases in action potential frequency) by curare. Intracellular recordings reveal that the nicotinic effects on afferent action potential frequency are the result of alterations in the frequency of spontaneous synaptic potentials, indicating a presynaptic site of action on the hair cells for these compounds. This conclusion is supported by the fact that in the presence of high Mg2+/low Ca2+, which blocks hair cell release of transmitter, cholinergic agonists do not affect the resting membrane potential of the vestibular afferent. Electrical stimulation of the VIIIth cranial nerve can result in either an increase or a decrease in spontaneous synaptic potential and action potential frequency of an afferent. These effects are blocked by prior administration of curare or of nicotinic agonists. Repetitive or continuous stimulation of the VIIIth nerve results in a reversible reduction of the evoked response, suggesting desensitization. Transection of the VIIIth cranial nerve two weeks prior to recording eliminates these actions of electrical stimulation, but not the responses to cholinergic agonists, indicating that the effects of electrical stimulation are mediated by centrally arising efferents. These findings confirm that acetylcholine is probably the transmitter released from centrally arising vestibular efferents, and, in addition, demonstrate that efferent-mediated effects are predominantly expressed through nicotinic receptors. Studies comparing the effects of isolation of the semicircular canal alone versus the intact labyrinth suggest that the method of isolation may be an important factor in determining whether efferent activity results in a predominant increase or decrease in afferent activity.     

Glutamate uptake disguises neurotoxic potency of glutamate agonists in cerebral cortex in dissociated cell culture.

The pharmacological properties of glutamate agonists were compared in astrocyte-rich and astrocyte-poor cultures derived from embryonic rat cerebral cortex. The object of this investigation was to determine the extent to which glutamate uptake might influence the receptor-mediated neurotoxic actions of these compounds. In astrocyte-rich cultures, using 30 min exposures, we observed that the potencies of the poorly transported agonists NMDA (35 microM) and D-glutamate (89 microM) were higher than that of L-glutamate (205 microM). In astrocyte-poor cultures, L-glutamate was much more potent, with an EC50 of 5 +/- 4 microM (3-12 microM), for a 30 min exposure, whereas the potencies of NMDA and D-glutamate were essentially unchanged. L- and D-aspartate were also more effective in astrocyte-poor cultures, again with EC50 values of approximately 6-10 microM, as compared with 130 and 108 microM, respectively, in astrocyte-rich cultures. In other experiments, blocking sodium-dependent glutamate uptake in astrocyte-rich cultures, by using a sodium-free medium, made glutamate as potent an agonist as in astrocyte-poor cultures. Finally, we directly assessed the glutamate uptake system in astrocyte-rich and astrocyte-poor cultures and found that uptake was reduced approximately 25-fold in the astrocyte-poor cultures. These results show that in the presence of abundant astrocytes the neurotoxic potencies of L-glutamate, L-aspartate, and D-aspartate are substantially under-estimated.