Quantitative autoradiographic characterization of l-[3H] glutamate binding sites in rat vestibular nuclei

Summary Quantitative autoradiography has been used to characterize l-[³H] glutamate binding sites and to describe their distribution in frozen sections of rat vestibular nuclei. Scatchard plots and Hill coefficients of glutamate binding suggest that glutamate interacts with a single population of sites having a K_D of about 126 nM and a capacity of 2.5 pmol/mg of protein. Although the level of glutamate binding was not very high compared to the highest levels described for some other brain regions, it was nonetheless substantial. The sites were distributed unevenly in the four vestibular nuclei and their distribution correlated well with the projection areas of the vestibular nerve, which has been described as a glutamate-mediated pathway. The highest numbers of glutamate binding sites were observed in the medial vestibular nuclei. This technique provides a very sensitive assay for characterizing the pharmacological subtypes of glutamate binding in the vestibular nuclei and for analyzing changes in these sites during development or after deafferentation of the vestibular nuclei.     

L-[3H]glutamate labels the metabotropic excitatory amino acid receptor in rodent brain

A quantitative autoradiographic assay for a novel L-[3H]glutamate binding site in rodent brain has been developed. Binding to this site was distinguished by its high affinity for quisqualate (QA), ibotenate, glutamate and trans-1-amino-cyclopentyl-1,3-dicarboxylic acid (trans-ACPD), but low affinity for [RS]-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), kainate and N-methyl-D-aspartate (NMDA). 'AMPA-insensitive, QA-sensitive [3H]glutamate binding' (AiQsGB) had a heterogeneous distribution in rat brain with high levels observed in molecular layer of cerebellum, striatum, and lateral septum. AiQsGB was reduced in molecular layer of cerebellum in mice lacking Purkinje cells. AiQsGB appears to represent binding to the 'metabotropic' neuronal excitatory amino acid receptor linked to phosphoinositide metabolism.     

Dynorphin displaces binding at the glycine site of the NMDA receptor in the rat striatum

Binding of dynorphin A (1-17 and 2-17) to NMDA receptors in the rat striatum was studied by displacing radioactive ligands for the receptor's polyamine ([3H]-Ifenprodil), glutamate ([3H]-CGP-39653), dizocilpine ([3H]-MK-801) and glycine ([3H]-MDL105,519) sites with the neuropeptide. Dynorphin A selectively displaced [3H]-MDL105,519 and none of the other ligands. Opioid antagonists did not affect displacement. Thus, in the striatum dynorphin may regulate NMDA receptor function via the glycineB site through non-opioid mechanisms. This may contribute to the long-term changes in behavioral responsiveness seen after dopamine depletion and treatment with dopaminomimetics which are associated with substantial changes in striatal dynorphin metabolism.     

Excitatory amino acid receptors in the human cerebral cortex: a quantitative autoradiographic study comparing the distributions of [3H]TCP, [3H]glycine, L-[3H]glutamate, [3H]AMPA and [3H]kainic acid binding sites

The excitatory amino acids are probably the major neurotransmitters in the cerebral cortex, and they act through at least three receptors: the N-methyl-D-aspartate, the quisqualate and the kainic acid receptors. Under the appropriate conditions, [3H]1-(1-(2-thienyl)-cyclohexyl)piperidine [( 3H]TCP), [3H]glycine and L-[3H]glutamate label different sites on the N-methyl-D-aspartate receptor, [3H]-alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid [( 3H]AMPA) labels the quisqualate receptor and [3H]kainic acid the kainic acid receptor. The anatomical localizations of these binding sites were studied in sections of blocks removed from the cerebral cortices of eight post-mortem human brains. The results showed that, in the human cerebral cortex, [3H]TCP, [3H]glycine and L-[3H]glutamate binding sites had congruent distributions, with [3H]AMPA binding sites showing a similar distribution. In the hippocampus, these four ligands had high binding site densities in the CA1 region and the dentate gyrus molecular layer. With the exception of the striate cortex, in the neocortex, a tri-laminar pattern was seen consisting of a high density across laminae I-III, a layer of low density corresponding to the region of lamina IV, and a band of moderate density across laminae V and VI, except for [3H]AMPA where the middle zone of low density was usually wider. [3H]Kainic acid showed a binding pattern which was generally complementary to that of the other four ligands. There were low levels of [3H]kainic acid binding sites in the CA1 region of the hippocampus with higher levels in the CA3 region, the hilus, and the inner third of the dentate gyrus molecular layer. In the neocortex there was a band of high density corresponding to laminae V and VI, with a thin band of moderate binding corresponding to lamina I and the outer region of lamina II. An exception was the motor cortex where the highest level of [3H]kainic acid binding was in laminae I and II. The high degree of congruence between the binding patterns of [3H]TCP, [3H]glycine and L-[3H]glutamate (using conditions appropriate for the N-methyl-D-aspartate receptor) supports data indicating that these ligands bind to different regions of the same receptor complex. The similar distribution of [3H]AMPA binding sites, with the exception of the striate cortex, supports observations made in rodents that N-methyl-D-aspartate receptors and quisqualate receptors have similar distributions and perform different but related functions in excitatory transmission.(ABSTRACT TRUNCATED AT 400 WORDS)     

Characterization and regional distribution of strychnine-insensitive [3H]glycine binding sites in rat brain by quantitative receptor autoradiography

Recent evidence suggests that a strychnine-insensitive glycine modulatory site is associated with the N-methyl-D-aspartate receptor-channel complex. A quantitative autoradiographic method was used to characterize the pharmacological specificity and anatomical distribution of strychnine-insensitive [3H]glycine binding sites in rat brain. [3H]Glycine binding was specific, saturable, reversible, pH and temperature-sensitive and of high affinity. [3H]Glycine interacted with a single population of sites having a KD of approximately 200 nM and a maximum density of 6.2 pmol/mg protein (stratum radiatum, CA1). Binding exhibited a pharmacological profile similar to the physiologically defined strychnine-insensitive glycine modulatory site. Binding was stereoselective; the rank order of potency of simple amino acids as displacers of binding was: glycine greater than D-serine greater than D-alanine greater than L-serine greater than L-alanine greater than L-valine greater than D-valine. Binding was not altered by the inhibitory glycine receptor ligand, strychnine, by the glutamate agonists, quisqualate and kainate, or by GABA receptor selective ligands. Most competitive agonists or antagonists of the N-methyl-D-aspartate recognition site were ineffective displacers of glycine binding. The exceptions were the aminophosphono series of antagonists, D-alpha-aminoadipate, gamma-D-glutamyglycine and beta-D-aspartylaminomethylphosphonic acid. However, the inhibition of [3H]glycine binding produced by the aminophosphono compounds could be accounted for by the level of glycine contamination present in these compounds. The non-competitive NMDA receptor-channel blockers, phencyclidine, its thienyl derivative, and MK-801 did not alter glycine binding. Kynurenate, glycine methylester, L-serine-O-sulfate, L-homocysteic acid, and several glycine-containing dipeptides were effective displacers of glycine binding. Structure-activity relations of agonists and antagonists of the strychinine-insensitive glycine binding site are discussed. The distribution of strychnine-insensitive [3H]glycine binding was heterogeneous with the following rank order of binding densities: hippocampus greater than cerebral cortex greater than caudate-putamen greater than or equal to thalamus greater than cerebellum greater than brain stem. This distribution of binding was correlated with N-methyl-D-aspartate-sensitive [3H]glutamate binding (r2 = 0.77; P less than 0.001; Pearson product-moment) and [3H]thienylcyclohexylpiperidine binding (r2 = 0.72; P less than 0.001). These observations are consistent with the hypothesis that the strychnine-insensitive glycine binding site is closely associated with the N-methyl-D-aspartate receptor-channel complex.(ABSTRACT TRUNCATED AT 400 WORDS)     

Distribution of [H]kainic acid and binding sites in the rat brain: in vivo and in vitro receptor autoradiography

Brain sections incubated in vitro with a-[³H]kainic acid (KA; spec. act. 62.5 Ci/mmol), reveal a heterogenous distribution of low and high affinity KA binding sites in the brain. The highest density of KA binding sites was localised to the hippocampus CA₃ region and to superficial layers of the entorhinal cortex (3.8 6.0 μCi/g tissue). Intravenous injection of [³H]KA (1 μCi/g) reveals limited overall penetration of [³H]KA across the blood-brain barrier. However, a dense labelling of the hippocampus, entorhinal cortex and lateral septal regions (2.5–3.8 μCi/g tissue) was observed. Behaviourally, these rats exhibited mild limbic seizure activity possibly as a result of a direct action of KA in the hippocampus or entorhinal cortex.     

L-glutamate specific [3H]kainic acid binding in the rat neostriatum after degeneration of the cortico-striatal pathway.

Four weeks after lesion of the cortico-striatal pathway, the specific kainic acid binding was reduced by 20% in the neostriatum. It is concluded that the binding sites are most likely not located on cortico-striatal neurons. The displacement of kainic acid by some chemical indicates that the kainic acid binding site is different from the glutamate binding site, as defined iontophoretically.     

Effects of age and visual experience on [3H] MK801 binding to NMDA receptors in the kitten visual cortex

Summary We have investigated the postnatal development of N-methyl-D-aspartate (NMDA) receptors in kitten neocortex by measuring the density of [³H] MK801 binding sites. NMDA receptor density in area 17 markedly increases between postnatal day 7 and 35, and then remains at a high level into adulthood. Similar temporal changes were observed in area 6. A brief period of binocular deprivation did not alter the density of [³H] MK801 binding sites in either brain region. However, dark rearing kittens from birth resulted in a significant decrease after 35 and 120 days in both area 17 and area 6. These results demonstrate that the density of NMDA receptors does not effectively predict the capacity for visual cortical plasticity in kitten neocortex. In addition, these results show that dark rearing produces significant effects in areas of the kitten neocortex not normally associated with visual function.     

Reduction of GABAA receptor binding of [3H]muscimol in the barrel field of mice after peripheral denervation: transient and long-lasting effects

The effect of peripheral sensory deprivation upon GABA_A receptor binding of [³H]muscimol was investigated in the barrel cortex — cortical representation of mystacial vibrissae of mice — by means of in vitro quantitative autoradiography. Unilateral lesions of all vibrissae or selected rows of whiskers were performed neonatally or in adulthood. [³H]muscimol binding was examined after various survival times up to 60 days. Both types of lesions performed in adult mice resulted in a transient decrease (10–25%) of binding values in the deafferented areas of the barrel field as compared with the unoperated control side. Sixty days after denervation [³H]muscimol binding returned to control values. Similar results were found after neonatal removal of all vibrissae. Neonatal lesion of selected rows of vibrissae, however, resulted in a decrease of [³H]muscimol binding (by about 26%) lasting up to 60 days in corresponding rows of barrels. This last result was accompanied by severe cytoarchitectonic malformation of the barrel field. The results support the hypothesis that a decrease of inhibition plays a facilitatory role in the plastic reorganization of cortical circuitry.     

Glycine-like immunoreactivity in the cerebellum of rat and Senegalese baboon,Papio papio: a comparison with the distribution of GABA-like immunoreactivity and with [3H]glycine and [3H]GABA uptake

Summary An antiserum against conjugated glycine was characterized and applied to cerebellar sections of rats and baboons that had been perfusion-fixed with glutaraldehyde. After immunosorbent purification the serum reacted with brain protein-glutaraldehyde-glycine conjugates, but did not stain similar test conjugates prepared from other amino acids, including GABA and -alanine. In the rat cerebellum the glycine antiserum selectively labelled a subpopulation of Golgi neurons. Adjacent Vibratome sections treated with an antiserum against conjugated GABA revealed an about equally large subpopulation of immunopositive Golgi cells. A proportion of the Golgi cells that were cleaved by the plane of section contained both immunoreactivities. Additional evidence for a colocalization of glycine and GABA was obtained by postembedding staining of alternate semithin sections with the GABA antiserum and glycine antiserum, respectively. The ability of the antisera to distinguish between fixed glycine and GABA was corroborated by preincubation of the antisera with glutaraldehyde-amino acid fixation complexes: glycine complexes abolished staining with the glycine antiserum but had no effect on the GABA antiserum. The opposite effects were obtained with the GABA complexes. Matching the distributions of the respective immunoreactivities, [³H]glycine uptake was restricted to glomerulus-like structures in the granule cell layer whereas [³H]GABA uptake also occurred in punctate and fibrous profiles in the molecular layer. The baboon showed a distribution of glycine-like immunoreactivity similar to that in the rat, except that a few immunopositive neurons occurred in the molecular layer. The latter neurons were interpreted as outlying Golgi neurons; however, the possibility that they represent a subpopulation of basket cells could not be excluded. The Purkinje cells were negative in both species. Glial cells were weakly stained with the glycine antiserum but were strongly immunopositive after incubation with an antiserum raised against conjugates of the structurally similar amino acid -alanine. The present data suggest that glycine and GABA occur in about equally large subpopulations of Golgi neurons. A subpopulation of the Golgi neurons appears to contain both glycine and GABA.     

Aging but not estrogen alters regional [3H]5-HT binding in the CNS of female Fischer 344 rats

Age-related changes in serotonergic regulation of neuroendocrine function were investigated in female Fischer 344 rats; serotonin ([3H]5-HT) binding sites were characterized in several brain regions. Neither the number (Bmax) nor the affinity (Kd) of [3H]5-HT sites were altered in the frontal cortex of reproductively young and senescent groups. However, a significant decline in receptor affinity was observed in the hypothalamus and midbrain dorsal raphe nucleus. An increase in the density of binding sites was also observed in the hypothalamus with advancing age. Acute 48 h exposure to estrogen failed to influence [3H]5-HT binding site characteristics in these brain regions. In summary, these results suggest that age-related changes in [3H]5-HT binding are regionally specific. Moreover, the observed changes in hypothalamic 5-HT function may underlie neuroendocrine aging events.     

Sulphur-containing excitatory amino acid-evoked Ca(2+)-independent release of D-[3H]aspartate from cultured cerebellar granule cells: the role of glutamate receptor activation coupled to reversal of the acidic amino acid plasma membrane carrier.

Sulphur-containing excitatory amino acid transmitter candidates (500 microM) stimulated the Ca(2+)-independent efflux of exogenously-supplied D-[3H]aspartate from primary cultures of cerebellar granule cells superfused continuously with HEPES-buffered saline containing CoCl2 (1 mM) in place of CaCl2. The stimulated release of D-[3H]aspartate was markedly attenuated by 200 microM 6,7-dinitroquinoxalinedione, a concentration at which the antagonist inhibits both non-N-methyl-D-aspartate and N-methyl-D-aspartate ionotropic excitatory amino acid receptors. The Ca(2+)-independent component of evoked release was also markedly attenuated and, in some cases, abolished by removing NaCl from the superfusion medium. Furthermore, when 700 microM dihydrokainate (demonstrated herein as a mixed/non-competitive inhibitor of the high-affinity dicarboxylic amino acid transporter in cultured granule cells) was included in the superfusion medium, stimulated efflux of D-[3H]aspartate was reduced by between 15-78% of the control response; the extent of inhibition varying with the agonist employed. In constrast, agents which act as competitive inhibitors of the plasma membrane carrier in granule cells, e.g. beta-methylene-D,L-aspartate, potentiated the release of D-[3H]aspartate in a synergistic manner. Taken together, these findings are consistent with a mechanism for the Ca(2+)-independent release of D-[3H]aspartate that is mediated predominantly by activation of excitatory amino acid receptors resulting in a reversal of the high-affinity dicarboxylic amino acid transport system. Although the physiological relevance of such non-vesicular release from the cytosol remains obscure and is still a matter of some debate, this mode of release may be of pathological significance.     

Uptake of [3H]Glutamic acid in excitatory nerve endings: light and electronmicroscopic observations in the hippocampal formation of the rat.

Slices of the rat hippocampal formation were incubated in vitro with 2 μM [^3H]glutamic acid in Krebs' solution at 25°C for 10 min, fixed with 5% glutaraldehyde and processed for autoradiography. The highest concentrations of autoradiographic grains were found in the target zones of axons from the pyramidal cells of CA3/4 and in the target zones of mossy fibres from the granular cells in the area dentata. The mossy fibre boutons, which can be identified by their characteristic ultrastructure, were found to have a grain density six times that of non-axonal tissue elements (dendrites, glia and intercellular space). The corresponding figure-was four for boutons in the target zone of pyramidal axons. The overall grain density was 30–60% lower in the target zones of the perforant path axons (from the entorhinal area) than in those of the mossy fibres and pyramidal cell axons, but the grains were still concentrated over nerve endings and fibres. Consideration of ‘cross-firing’ effects led to the conclusion that boutons and axons probably contained a higher proportion of the radioactivity than is reflected in the grain counts (41–65%). Thus hypothetical grain analysis of the target zone of the medial perforant path suggested that axons and boutons, which constituted 20% of the section area, accounted for some 80% of the radioactivity. In a parallel study (Storm-Mathisen, 1977) the pyramidal and perforant path axon terminals have been shown to contain glutamate uptake sites, the uptake being dramatically reduced in their target zones following axotomy. The three systems of nerve endings found to contain glutamate uptake are all recognized excitatory systems. Only 4–6% of the autoradiographic grains occurred over glial elements which constituted 6–9% of the area of tissue sections. According to the hypothetical grain analysis 13 ± 5% of the radioactivity could be ascribed in glia.The findings show that high affinity glutamate uptake sites are concentrated in excitatory nerve endings, which may be ‘glutamergic’ according to other data. In the conditions used, such uptake may therefore be useful as a marker for glutamergic nerve endings.     

Afferents to the median raphe nucleus of the rat: retrograde cholera toxin and wheat germ conjugated horseradish peroxidase tracing, and selective D-[3H]aspartate labelling of possible excitatory amino acid inputs

Afferents to the median-paramedian raphe nuclear complex, which contains the B8 serotonergic cell group, were investigated in the rat with neuroanatomical and transmitter-selective retrograde labelling techniques. Injection of sensitive retrograde tracers, cholera toxin genoid or wheat germ agglutinin conjugated horseradish peroxidase into the median raphe resulted in labelling of neurons in a large number of brain regions. Projections from 26 of these regions are supported by available orthograde tracing data; the cingulate cortex, bed nucleus of stria terminalis, medial septum and diagonal band of Broca, ventral pallidum, medial and lateral preoptic areas, lateral hypothalamus, dorsomedial nucleus of hypothalamus, lateral habenula, interpeduncular nucleus, substantia nigra, central (periaqueductal) gray, and laterodorsal tegmental nucleus seem to represent major sources of afferents to the median-paramedian raphe complex. Retrogradely labelled cells were also observed in a number of regions for which anterograde tracing data are not available, including the perifornical hypothalamic nucleus, ventral premammillary nucleus, supramammillary and submammillothalamic nuclei and the B9 area. Possible excitatory amino acid afferents were identified with retrograde D-[3H]aspartate labelling. Microinjection of D-[3H]aspartate at a low concentration, 10(-4) M in 50 nl, resulted in retrograde labelling of a limited number of median raphe afferents. The most prominent labelling was observed in the lateral habenula and the interpeduncular nucleus, but retrogradely labelled cells were also noted in the medial and lateral preoptic areas, lateral and dorsal hypothalamus, ventral tegmental area, laterodorsal tegmental nucleus, medial parabrachial nucleus, and the pontine tegmentum. After injections of 10(-3) M D-[3H]aspartate selective labelling also appeared in more distant afferent regions, including cells in cingulate cortex, and in some regions located at shorter distances, such as the supramammillary nucleus. Injections of D-[3H]aspartate at high concentration, 10(-2) M, resulted in the appearance of weakly to moderately labelled cells in most afferent areas which were devoid of labelled cells after injections of lower concentrations, suggesting that this labelling may be non-specific. It was concluded that the median-paramedian raphe receives afferents from a large number of forebrain and hypothalamic regions, while relatively few brain stem regions project to this nuclear complex. The selectivity of retrograde labelling with D-[3H]aspartate was found to be concentration dependent, and it is suggested that the connections showing high affinity for D-[3H]aspartate may use excitatory amino acids as transmitters. Excitatory amino acid inputs from lateral habenula and interpeduncular nucleus may play predominant roles in the control of ascending serotonergic and non-serotonergic projections originating in the median and paramedian raphe nuclei.     

The effect of temperature and chloride ions on the stimulation of [3H]flunitrazepam binding by the muscimol analogues THIP and piperidine-4-sulfonic acid

THIP and piperidine-4-sulfonic acid (PSA) interact with [3H]GABA binding sites and have GABAmimetic efficacy in vivo, but fail to enhance benzodiazepine receptor binding performed at 0 degree C. However, when [3H]flunitrazepam binding is determined at elevated temperature (30 or 37 degrees C), THIP and PSA display potent chloride ion-dependent stimulatory effects. These results resolve apparent discrepancies between the properties of GABA receptors observed in vivo and in vitro, and they suggest that the modulation of benzodiazepine receptor binding investigated at physiological temperatures can be used as an experimental system for the characterization of GABA receptors.