Glutamate Receptor Binding Research Articles

Polysynaptic regulation of glutamate receptors and mitochondrial enzyme activities in the basal ganglia of rats with unilateral dopamine depletion

After nigrostriatal dopaminergic denervation, the output nuclei of the basal ganglia, the medial globus pallidus and substantia nigra pars reticulata (Snr), become overactive, in part, because of increased activity of excitatory afferents from the subthalamic nucleus (STN). Because STN uses glutamate as a transmitter, we examined whether there are regulatory changes in glutamate receptor binding in the basal ganglia. Rats received unilateral 6-hydroxydopamine lesions of the medial forebrain bundle and substantia nigra pars compacta that were confirmed by apomorphine-induced rotation and 3H-GBR-12935 binding. As an indirect index of relative synaptic activity, succinate dehydrogenase and cytochrome oxidase activities were assayed histochemically in sections adjacent to those used for receptor binding. There were increases in enzymatic activity in entopeduncular nucleus (EP; the rodent homolog of medial globus pallidus), SNr, and globus pallidus (GP, the rodent homolog of lateral globus pallidus) in the lesioned hemisphere, suggesting increased synaptic activity, perhaps due to increased firing of the STN. Ipsilateral to the lesion, and postsynaptic to the STN, there were profound decreases in the binding of 3H-AMPA (alpha-amino-3-hydroxy-5-methylisoxazole propionic acid) in EP and SNr (45% and 30%, respectively); there were no alterations in the striatum, globus pallidus, or STN, and binding throughout the unlesioned hemisphere was equivalent to that in unlesioned control animals. In contrast, 3H-MK-801 binding to the NMDA receptor ion channel was not reduced in SNr, and was too low to be measured reliably in EP and STN. 3H-MK-801 binding was reduced by 6% in striatum and 39% in globus pallidus.(ABSTRACT TRUNCATED AT 250 WORDS)

Distribution and ontogeny of 1S,3R-1-aminocyclopentane-1,3-dicarboxylic acid-sensitive and quisqualate-insensitive [3H]glutamate binding sites in the rat brain.

Displacement of [3H]glutamate by 1S,3R-1-aminocyclopentane-1,3-dicarboxylic acid and quisqualate (in the presence of saturating concentrations of ionotropic glutamate receptor agonists) was used to characterize optimal ionic conditions, distribution, and the ontogeny of glutamate receptor binding sites in rat brain. Using rat forebrain membranes or receptor autoradiography, optimal 1S,3R-1-aminocyclopentane-1,3-dicarboxylic acid-sensitive [3H]glutamate binding was found in the presence of 100 mM bromide ions and in the absence of calcium ions. Under these conditions, [3H]glutamate binding was relatively quisqualate insensitive. In regions of the neonatal (11-day-old) and adult rat brain, this [3H]glutamate binding was highest in forebrain (striatum, cerebral cortex, and hippocampus) and hypothalamus/mid-brain but was lower in the cerebellum, olfactory bulb, and pons/medulla regions. 1S,3R-1-aminocyclopentane-1,3-dicarboxylic acid-sensitive and quisqualate-insensitive [3H]glutamate binding was present in the rat forebrain at 1 day of age and gradually increased more than twofold by day 50 (adult). Thus, in the presence of bromide ions and in the absence of calcium ions, [3H]glutamate labels a subpopulation of metabotropic glutamate receptors that are sensitive to 1S,3R-1-aminocyclopentane-1,3-dicarboxylic acid but insensitive to quisqualate. Expression of [3H]glutamate binding under these conditions was both regionally and developmentally regulated in rat brain, suggesting that [3H]glutamate is labeling a distinct population of metabotropic glutamate receptors.

Metabotropic glutamate receptors are differentially regulated during development

The postnatal expression of metabotropic glutamate receptors was studied in rat brain by in situ hybridization and autoradiographic binding techniques. The messenger RNAs encoding five metabotropic glutamate receptor subtypes named mGluR1–5 had distinct regional and temporal expression profiles. mGluR1, mGluR2 and mGluR4 messenger RNA expression was low at birth and increased during postnatal development. In contrast, mGluR3 and mGluR5 were highly expressed at birth and decreased during maturation to adult levels of expression. [ 3H]Glutamate binding competition studies in developing brain disclosed the presence of two types of binding sites with the pharmacological properties of metabotropic glutamate receptors, having high (metabotropic type-1 binding sites; K 1 = 8nM) and low affinity (metabotropic type-2 binding sites; K 1 = 50 μM) for quisqualic acid, as in adult rat brain. The densities of metabotropic binding sites changed during development in a complex, regionally specific fashion. Metabotropic type-1 binding sites were present at low levels at birth and gradually increased during the second postnatal week. In the striatum, globus pallidus and cerebellar granule layer, the increase in density of metabotropic type-1 binding sites was transient but persisted in the cerebellar molecular layer. In contrast, metabotropic type-2 binding sites were present at high densities in most regions in the first postnatal week and decreased during the second and third week, particularly in the thalamic reticular nucleus and globus pallidus. Only in the external cortex did both metabotropic type-1 and metabotropic type-2 binding sites increase during development. A striking correspondence between the temporal pattern of expression of specific metabotropic glutamate receptor transcripts and metabotropic binding sites was observed in the reticular nucleus of the thalamus (mGluR3; metabotropic type-2 binding sites) and cerebellum (mGluR1; metabotropic type-1 binding sites) suggesting early translation of these metabotropic glutamate receptor messenger RNAs into receptor proteins. In other regions the relationship between messenger RNA expression and binding sites was less direct: comparison between expression of metabotropic glutamate receptor messenger RNA and binding sites suggests both a pre- and postsynaptic location of some receptor subtypes. These data imply a functional role of mGluR3 and mGluR5 during synaptogenesis and maintenance of adult synapses and of mGluR1, mGluR2 and mGluR4 in mature synaptic transmission.

Emergence neophobia correlates with hippocampal and cortical glutamate receptor binding in rats

Previous work from our laboratory indicated that emergence neophobia is highly correlated with perforant path long-term potentiation (LTP) in rats. In the present study, we examined the relationship between hippocampal and cortical alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptors and emergence behavior in rats. Emergence neophobia was assessed in an exploratory task that provided a choice between a novel alley and a familiar nest box. Quantitative autoradiography using radiolabeled ligands specific for the AMPA subclass of glutamate receptors was performed on frozen brain sections. Both [3H]AMPA and [3H]CNQX (6-cyano-7-nitro-[3H]quinoxaline- 2,3-dione, an AMPA receptor antagonist) binding in the dentate gyrus (stratum moleculare), hippocampal area CA1 (stratum radiatum), and the parietal cortex overlying the hippocampus were significantly correlated with emergence behavior. The correlations indicated that neophobic rats, which had longer latencies to enter the novel alley, made fewer entries into the alley, and spent less time in the novel alley during a 10-min test than their neophilic counterparts, had higher levels of AMPA receptor binding. These results suggest that individual differences in specific hippocampal AMPA receptors reflect variability in a specific class of hippocampal-dependent behaviors.

Glutamate receptor binding in juvenile and adult Rana pipiens CNS.

Autoradiographic methods were used to map NMDA- and quisqualate-sensitive glutamate binding sites in the brain of mature and juvenile Rana pipiens frogs. NMDA- and quisqualate-sensitive sites were consistently co-localized in the CNS. The highest glutamate binding occurred in the telencephalon, hypothalamus, and cerebellum. Glutamate binding sites were also specifically localized in visual pathways, including the superficial neuropil of the optic tectum, consistent with glutamate being the retinal ganglion cell neurotransmitter. The distribution of glutamate binding sites in the brain of juvenile postmetamorphic frogs was similar to that in adults. In general, Quis binding increased about twofold in adults compared to juveniles, whereas NMDA binding did not show a comparable developmental increase. To test whether glutamate binding sites are located on retinal axon terminals or on tectal cell dendrites in the optic tectum, juvenile postmetamorphic frogs were enucleated unilaterally, and receptor binding was performed following 1, 3, 7, and 14 days survival. The denervated tectal neuropil showed a delayed decrease in NMDA- and quisqualate-sensitive binding, consistent with the receptors being located on postsynaptic tectal cell dendrites.

Dexamethasone potentiates NMDA receptor-mediated neuronal injury in the postnatal rat

The present study investigated the effect of a synthetic glucocorticoid, dexamethasone, on excitatory amino acid receptor-mediated neurotoxicity in the 7-day-old rat. Pretreatment with dexamethasone (0.7 mg/kg i.p.) 1 h prior to unilateral intrastriatal injection of excitotoxin enhanced damage resulting from N- methyl- D- aspartate (NMDA), but not α-amino-3-hydroxy-5- methyl-4-isoxazolepropionate (AMPA), or kainate receptor activation. The glucocorticoid-induced enhancement of NMDA toxicity was dose dependent. The time of dexamethasone administration appeared critical since only treatment 1 h prior to, but not 24 h prior to, simultaneously with, or 1 h after NMDA injection, affected toxicity. The administration of the adrenal mineralocorticoid aldosterone, or the phospholipase A 2 inhibitor mepacrine, did not affect excitotoxicity. Quantitative receptor autoradiography was performed to assess the effect of dexamethasone on NMDA-sensitive [ 3H]glutamate receptor binding. Neither pretreatment in vivo nor the addition of dexamethasone in vitro affected NMDA-sensitive binding in the striatum. Possible explanations for these observations are discussed.

Increased hypothalamic glutamate receptors induced by water deprivation.

To examine the role of receptor changes in the adaptive response to physiological stimulation, the density and distribution of excitatory amino acid receptors within the hypothalamus and other brain regions were examined in rats deprived of water for 2 days. Membrane binding assay revealed an increase in glutamate receptor density and a small shift in the affinity of glutamate for the receptor. Regional analysis of these changes by receptor autoradiography specific for NMDA, non-NMDA or metabotropic glutamate receptor binding indicated that NMDA and metabotropic receptor densities are increased in the brain. Regional increases were found principally for the NMDA receptor binding within the supraoptic nucleus, anterior hypothalamus, caudate-putamen and globus pallidus with no significant changes in 24 other brain regions. No significant changes were found in any brain regions for AMPA receptors. Metabotropic and kainate receptors tended to parallel the NMDA receptor changes, although few regions reached statistical significance. These changes indicate that brain regions associated with water balance regulation show selective adaptive increases in NMDA receptors during water deprivation which may facilitate prolonged activation of these cells.

Postsynaptic factors in the expression of long-term potentiation (LTP): increased glutamate receptor binding following LTP induction in vivo.

Several lines of evidence indicate that LTP in the hippocampus is associated with a change in the properties of postsynaptic glutamate receptors. In the present study, we used quantitative autoradiography to examine the binding properties of the alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionate (AMPA) and N-methyl-D-aspartate subclasses of glutamate receptors in frozen brain sections obtained from rats in which perforant-path LTP was induced in vivo. Induction of LTP resulted in a selective increase in [3H]AMPA binding in those hippocampal subfields receiving perforant-path axons. Increases in [3H]AMPA binding in dentate gyrus (stratum moleculare) were highly correlated with the magnitude of LTP recorded in this structure. Scatchard analyses of [3H]AMPA and 6-cyano-7-nitro-[3H]quinoxaline-2,3-dione (an AMPA receptor antagonist) binding in the dentate gyrus indicated that LTP induction resulted in an increase in the number of AMPA receptor binding sites. No changes in the binding of 3H-labeled N-[1-(thienyl)cyclohexyl]piperidine (an N-methyl-D-aspartate receptor antagonist) were observed in any hippocampal subfield. These results suggest that a modification in postsynaptic AMPA receptors plays a role in the expression of synaptic enhancement following LTP induction in the hippocampus.

Brain extracts containing a Huntington disease antigen inhibit [3H]kainate binding and block synaptosomal amino acid transport.

Fractions isolated from mammalian brain which had previously been shown to inhibit the rate of migration of peripheral blood leukocytes taken from Huntington disease cases, and also to inhibit [3H]kainic acid binding, were characterized further. By use of repeated ultrafiltration onto a 1000D MW cutoff filter, and by the isolation and extensive washing of an enriched ammonium sulfate fraction, their activity was shown not to be due to the presence of endogenous glutamate, and to be relatively selective for brain glutamate receptor binding sites. Inhibitory activity at [3H]GABA, 5-[3H]hydroxytryptamine 5HT1 and dopamine D1 or D2 binding sites was much weaker or absent. Factor extracts were also shown to act as non-competitive inhibitors of synaptosomal amino acid transport: increasing concentrations of the factor had no significant effect on the KM for the uptake of either [3H]glutamate or [3H]GABA, but at a final concentration of 66 micrograms protein x ml-1 had reduced the VMAX for [3H]glutamate uptake to approximately 20% of control, and the VMAX for [3H]GABA uptake to approximately 40% of control. This may enhance the factor's potential excitotoxicity.

Glutamate Receptor Binding Sites in MPTP-Treated Mice

Changes in excitatory amino acid (EAA) neurotransmission are thought to play an important role int he development of parkinsonian symptoms. We examined EAA receptor binding sites in substantia nigra, striatum, globus pallidus, and cortex at 2 weeks and 2 months after MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) injection in C57b16 mice. At 2 weeks striatal dopamine content in MPTP-treated mice was reduced to 7% of control and N-methyl- d-aspartate (NMDA)-sensitive [ 3H]glutamate and [ 3H]α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) binding sites were decreased in substantia nigra to 57 and 76% of control, respectively. In globus pallidus only [ 3H]AMPA binding sites were decreased to 80% of control; no significant changes were found in striatum or cortex. [ 3H]Kainate binding sites remained unchanged. At 2 months striatal dopamine content was reduced to 31% and no changes in EAA binding sites could be detected in any of the structures examined. [ 3H]Mazindol binding to striatal monoamine-uptake sties was decreased to 17% of control at 2 weeks versus 37% at 2 months. Our data indicate that modulation of NMDA and AMPA binding sites in substantia nigra and globus pallidus, the major projection areas of the subthalamic nucleus, takes place only after severe impairment of the nigrostriatal system.

Localisation of glutamate receptor binding sites and mRNAS to the dorsal horn of the rat spinal cord

The autoradiographic distribution of binding sites for [ 3H]AMPA, [ 3H]kainate, [ 3H]MK-801 and [ 3H] L-glutamate and the in situ hybridization of GluR-A, GluR-B, GluR-C and GluR-D in rat spinal cord sections were determined. All of the radioligands had similar localisations with the dorsal horn being the most intensely labelled region. In the in situ hybridization experiments only the GluR-B mRNA was detected at high levels in the spinal cord with predominant expression in the substantia gelatinosa. These results support electrophysiological evidence for the presence of glutamate activated neurones in the spinal cord and suggest that these excitatory pathways are confined mainly to the dorsal horn.

Quantitative Autoradiographic Demonstration of Changes in Binding to NMDA-sensitive [3H]Glutamate and [3H]MK801, but not [3H]AMPA Receptors in Chick Forebrain 30 min After Passive Avoidance Training.

Day-old domestic chicks (Gallus domesticus) were trained on a one-trial passive avoidance task in which the aversive stimulus was an unpleasant tasting substance, methyl anthranilate. Thirty minutes later, localization of N-methyl-d-aspartic acid (NMDA)-sensitive [3H]glutamate receptor binding sites, [3H]MK801 and [3H]AMPA binding sites in 17 regions of the forebrain of methylanthranilate-trained and control (water-trained) chicks was determined using quantitative receptor autoradiography. Significant differences in binding to both MK801- and NMDA-sensitive glutamate receptors, but not alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors, were found in three regions of the forebrain of trained compared to control chicks; two of these regions have been implicated from previous lesion, biochemical and morphological studies as playing a key role in the process of memory formation and storage following passive avoidance training. For NMDA-sensitive [3H]glutamate receptors, significant elevations in binding were observed in two regions, the left intermediate and medial hyperstriatum ventrale (IMHV) (39%) and the lobus parolfactorius (LPO) (34%), at 30 min post-training, but a decrease (44%) occurred in binding to the lateral neostriatum. Significant increases in binding to MK801 receptors were observed in the left IMHV (19%) and right IMHV (13%), and left LPO (22%) at 30 min post-training, though there was a decrease in the right LPO (15%). These findings, coupled with those described in a previous paper from our group (Burchuladze and Rose, Eur. J. Neurosci., 4, 533 - 538, 1992), demonstrate that a glutamate receptor subtype is involved in learning and memory formation in the chick.

Glutamate receptor binding in discrete brain structures of rats under postnatal development

Glutamate receptor binding in discrete brain structures of rats under postnatal development

Prenatal ethanol exposure decreases hippocampal NMDA-sensitive [ 3H]-Glutamate binding site density in 45-day-old rats

The effect of prenatal ethanol exposure on N-methyl-D-aspartate (NMDA)-sensitive [3H]-glutamate receptor binding site density was studied in rat brain. Pregnant Sprague-Dawley rats were fed a liquid diet containing 3.35% ethanol throughout gestation. This diet produced maternal peak blood ethanol levels of about 39 mg/dl eight hours after the administration of the liquid diet. Pair-fed dams received an isocalorically matched liquid diet and an ad lib lab chow group served as control for the paired feeding technique. At 45 days of age, offspring from each of the three diet groups were sacrificed and brain NMDA-sensitive [3H]-glutamate binding site density measured using in vitro radiohistochemical techniques. NMDA-sensitive [3H]-glutamate binding site density was reduced significantly by 19 to 29% in the apical dendritic field regions of dentate gyrus, hippocampal CA1 and subiculum of dorsal hippocampal formation of fetal alcohol rats compared to pair-fed and ad lib controls. NMDA-sensitive [3H]-glutamate binding site density was not significantly different among the three groups in the ventral hippocampal formation, posterior neocortex, lateral entorhinal cortex or cerebellum. These results are consistent with our previous observations of a reduction in total [3H]-glutamate receptor binding site density in the dorsal hippocampal formation of fetal alcohol rats, as well as more recent electrophysiological observations of a decrease in the sensitivity of fetal alcohol hippocampal slices to NMDA.

Selective degeneration of cerebellar cortical neurons caused by cycad neurotoxin, L‐β‐methylaminoalanine (L‐BMAA), in rats

Both the racemate and the L-form of BMAA (beta-methylaminoalanine), when injected intraperitoneally into young rats, produced acute signs of cerebellar dysfunction and degeneration of cerebellar stellate, basket, Purkinje and Golgi cells, but not granule cells. Degenerative changes were also occasionally seen in cerebellar roof nuclei which may be secondary in nature. No other changes were found in the remainder of the central nervous system. The doses of the L-form of BMAA producing these changes were from 6 to 14 mumols/g body weight, i.e. the lower and upper levels of the dose range used by Vega and Bell (1967) and equivalent to 75 and 183 mg/rat. Doses of 1 to 4 mg/g body weight of the racemate were given to young rats less than 100 g in weight, but no changes were apparent after daily doses of the racemate of 0.5 mg/g body weight. Damage to cerebellar neurons is considered to be the result of excitotoxic activity. All cells showing degeneration are GABAergic, although not all are known to possess N-methyl-D-aspartate (NMDA) receptors. The present finding of selective cerebellar neuron damage may not conflict with the earlier findings of others, but our results suggest that L-BMAA has unusual glutamate receptor binding properties.

Glutamate Transport and Not Glutamate Receptor Binding Is Stimulated by Gangliosides in a Ca2+‐Dependent Manner in Rat Brain Synaptic Plasma Membranes

Crude as well as purified synaptic plasma membrane (SPM) preparations were analyzed for the influence of the ganglioside galactosyl-N-acetylgalactosaminyl-(N-acetylneuraminyl)-galactosylgluc osyl ceramide (GM1) on high-affinity binding of L-[3H]glutamate. Assayed in two different buffer systems, SPM consistently exhibited increased (40-50%) binding upon incubation with GM1 plus Ca2+, as compared to controls without GM1. Incorporation experiments with 3H-labeled GM1 proved trypsin-stable insertion of GM1 into SPM, with a maximum incorporation of four times the endogenous amount (35 nmol/mg of protein). The observed increase in glutamate binding was not due to a change in the affinity of the binding sites, but to a change in the number of binding sites, and it was absolutely dependent on the presence of Ca2+. A pharmacological profile of the GM1/Ca2+-stimulated glutamate binding is presented. The original classification of the stimulatory effect as an effect on glutamate receptor binding had to be revised to take into account the observed temperature sensitivity of the ganglioside effect, its sensitivity to high osmolarity and to ultrasonication, and the lack of binding stimulation after detergent treatment of membranes or after receptor solubilization. Vesicular space measured in both SPM preparations was found to be around 7 microliters/mg of protein, in ganglioside-treated as well as in control membranes. From the data, it is concluded that a special, Na+- and Cl- -independent form of glutamate transport into resealed membrane vesicles is stimulated by gangliosides in the presence of Ca2+.

β‐N‐Oxalylamino‐L‐Alanine Action on Glutamate Receptors

beta-N-Oxalylamino-L-alanine (L-BOAA) is a non-protein excitatory amino acid present in the seed of Lathyrus sativus L. This excitotoxin has been characterized as the causative agent of human neurolathyrism, an upper motor neuron disease producing corticospinal dysfunction from excessive consumption of the lathyrus pea. Previous behavioral, tissue-culture, and in vitro receptor binding investigations revealed that L-BOAA might mediate acute neurotoxicity through quisqualate (QA)-preferring glutamate receptors. The present study demonstrates the stereospecific action of L-BOAA on glutamate receptor binding in whole mouse brain synaptic membranes. L-BOAA was most active in displacing thiocyanate (KSCN)-sensitive specific tritiated (RS)-alpha-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) binding (i.e., QA receptor) (Ki = 0.76 microM) with a rank-order potency of QA greater than kainate greater than N-methyl-D-aspartate (NMDA). By contrast, the nonneurotoxic D-BOAA isomer (100 microM) was essentially inactive in displacing radioligands for glutamate receptors, except the NMDA site, where it was equipotent with L-BOAA. Scatchard analysis of L-BOAA displacement of specific [3H]AMPA binding indicated competitive antagonism (KD: control, 135 nM; L-BOAA, 265 nM) without a significant change in QA-receptor density, and Hill plots yielded coefficients approaching unity. Differential L-BOAA concentration-dependent decreases in specific [3H]AMPA binding were observed in synaptic membranes, indicating that the neurotoxin was more potent in displacing specific binding from frontal cortex membranes, followed by that for corpus striatum, hippocampus, cerebellum, and spinal cord. (ABSTRACT TRUNCATED AT 250 WORDS)

3H]MK-801 binding sites in neonate rat brain

[ 3H]MK-801 binding sites are present in neonate rat brain as early as 3 days after birth. Immature hippocampus and cortex contain approximately one sixth the concentration of binding sites of the adult, while brainstem concentration is twice as high as that of adult. [ 3H]MK-801 binding is stimulated by glutamate and glycine and blocked by phencyclidine and Mg 2+ both in 7-day-old neonate and adult, indicating that as early as 7 days postnatally, the N-methyl- d-asparatate-type glutamate receptor and MK-801 binding site are functionally coupled.

Microbial methodological artifacts in [ 3H]glutamate receptor binding assays

Incubation of radiolabeled l-glutamic acid, a putative central excitatory neurotransmitter, in 50 m m Tris-acetate buffer (pH 7.4) at 30°C in the absence of brain synaptic membranes resulted in a significant adsorption of the radioactivity to glass fiber filters routinely employed to trap the bound ligand in receptor binding assays. The adsorption was not only eliminated by the inclusion of l-isomers of structurally related amino acids, but also inhibited by that of most presumed agonists and antagonists for the brain glutamate receptors. This displaceable adsorption was a temperature-dependent, nonreversible, and saturable phenomenon. Scatchard analysis of these data revealed that the adsorption consisted of a single component with an apparent dissociation constant of 73 n m. The displaceable adsorption was significantly attenuated by a concurrent incubation with papain, pronase E, and phospholipase C. A significant amount of the radioactivity was detected in the pass-through fraction of the Dowex column following an application of the reaction mixture incubated with purified [ 3H]glutamate at 30°C for 60 min in the absence of membranous proteins added. Complete abolition of the displaceable adsorption resulted from the use of incubation buffer boiled at 100°C as well as filtered through a nitrocellulose membrane filter with a pore size of 0.45 μm immediately before use. These results suggest that the displaceable adsorption may be attributable to the radioactive metabolite of [ 3H]glutamate by microorganisms contaminating the Tris-acetate buffer. This might in part contribute to some of the controversial results with regard to receptor binding studies on acidic amino acids.

Dissociation between changes in glutamate receptor binding sites and their coupling to phosphatidylinositol metabolism following intrahippocampal colchicine injection

Intrahippocampal colchicine injection produces a rapid death of granule cells and pyramidal neurons in the hippocampus in the rat. Under the appropriate assay conditions, [ 3H]glutamate labels the N-methyl- d-aspartate type of glutamate receptors while [ 3H]α-amino-3-hydroxy-5-methylisoxazole-4-propionate labels the quisqualate type. Unilateral injection of colchicine (15 μg) in the dorsal hippocampus did not produce any change in [ 3H]glutamate and [ 3H]α-amino-3-hydroxy-5-methylisoxazole-4-propionate binding in membrane fractions from the dentate gyrus or CA1 field contralateral to the injection side, at least up to 12 days after the injection. However, it produced a progressive decrease in the binding of both ligands in dentate gyrus and CA1 of the injected hippocampus. In the dentate gyrus the changes in binding as a function of time after the injection were biphasic with a rapid exponential decrease ( t 1/2) about 8 days for both [ 3H]glutamate and [ 3H]α-amino-3-hydroxy-5-methylisoxazole-4-propionate) until 12 days after the injection followed by a much slower decrease afterwards. A similar pattern was observed in CA1 although the changes in binding were smaller and delayed by about three days as compared to the dentate gyrus. Kinetic analyses of the binding at equilibrium were performed seven days after the injection and indicated that the changes in [ 3H]glutamate binding were due to a change in the maximum number of sites but not in affinity for the ligand. For [ 3H]α-amino-3-hydroxy-5-methylisoxazole-4-propionate Scatchard plots indicated the existence of both high and low affinity sites and the effect of colchicine injection appeared to be a decrease in the maximum number of low affinity sites. Basal and quisqualate-stimulated conversion of phosphatidylinositol to inositol phosphate derivatives were also determined in hippocampal slices at various times following colchicine injection. The changes in basal phosphatidylinositol hydrolysis were biphasic with a marked increase occurring one to two days after the injection followed by a marked decrease in hydrolysis and a recovery to control values by 30 days. Quisqualate-stimulated phosphatidylinositol hydrolysis was markedly enhanced between four and eight days after the injection and was still slightly increased by 30 days. No effect of N-methyl- d-aspartate was observed at any time after colchicine injection. These results indicate that the majority of the sites labeled by [ 3H]glutamate and [ 3H]α-amino-3-hydroxy-5-methylisoxazole-4-propionate are localized mainly postsynaptically and on neuronal structures. The exponential decay of the receptor binding suggests that the number of binding sites directly reflects the number of surviving neurons on the hippocampal subfields. Finally the changes in quisqualate-stimulated phosphatidylinositol hydrolysis suggest that colchicine treatment modifies the coupling between certain types of glutamate receptors to the phosphatidylinositol system possibly as a result of the modification of cytoskeletal proteins.