Localization of high affinity [H]glycine transport sites in the cerebellar cortex

A study was made of [³H]glycine uptake sites in a preparation greatly enriched in large pieces of the cerebellar glomeruli (glomerulus particles) and in morphologically well preserved slices of rat cerebellum. Electron microscopic autoradiography revealed that of the neurones in the cerebellar cortex only Golgi cells transported [³H]glycine at the low concentration used. Glial cells also took up [³H]glycine but to a lesser extent than the Golgi neurons. It was also confirmed that under comparable conditions Golgi cells transport [³H]GABA. Kinetic studies utilizing the Golgi axon terminal-containing glomerulus particles showed that glycine is a weak non-competitive inhibitor of [³H]GABA uptake (K_i over 600 μM vs theK_t of about 20 μM) and that GABA is an even weaker inhibitor of [³H]glycine uptake. These observations indicated that glycine and GABA do not share the same carrier. Quantitative electron microscopic autoradiography showed that the uptake of the two amino acids, in terms of the unit area of labelled Golgi axon terminals, was not additive. In contrast, their uptake in terms of unit protein was strictly additive. These observations, the first relating to unit volume and the latter to the total volume of Golgi terminals, are consistent with the view that there are two biochemically separate populations of Golgi neurons, one transporting glycine the other GABA. Saturable [³H]strychnine binding was detected in the preparations of glomerulus particles, but in comparison with those from the spinal cord the affinity was lower and [³H]strychnine was not displaced by glycine. Available information on glycine receptors, however, suggest that this should not exclude the possibility of strychnine resistant glycine receptors in the rat cerebellum.     

Neurotransmitters of the cerebellar glomeruli: uptake and release of labeled gamma-aminobutyric acid, glycine, serotonin and choline in a purified glomerulus fraction and in granular layer slices

We have studied some properties of the uptake and release of labeled gamma-aminobutyric acid (GABA), glycine, serotonin and choline in a purified fraction of glomeruli and in slices of the granular layer of the rat cerebellum. The uptake of both GABA and glycine into the glomerulus particles was dependent on the presence of Na+ in the medium. In contrast, the uptake of both serotonin and choline was Na+-independent. In slices of the granular layer also a slight Na+-dependence was observed for both serotonin and choline uptake; imipramine and hemicholinium partially inhibited the uptake of serotonin and choline, respectively. Choline uptake into the glomerulus particles showed two components, with apparent Km values of 16.8 and 102 microM. GABA release was stimulated by K+-depolarization about 100% (peak stimulation) and this value was reduced to 50% when Ca2+ was omitted. The release of glycine was stimulated more rapidly and notably than GABA (200%) and this stimulation was completely abolished in the absence of Ca2+. Serotonin release from the glomerulus particles was only slightly stimulated by depolarization, but this stimulation was strictly Ca2+-dependent. In slices of the granular layer, this stimulation was considerably larger (about 40%) and it was also almost totally dependent on Ca2+. In contrast, after loading with labeled choline the release of radioactivity from both the glomerulus particles and the cerebellar slices was not stimulated at all by K+-depolarization, either in the presence or in the absence of Ca2+. Most of the radioactivity released spontaneously corresponded to choline, and only a small proportion (8-14%) to acetylcholine. From the results of the release experiments and taking into account the pertinent data from the literature, it is concluded that GABA and glycine are probably the transmitters of different populations of Golgi axon terminals, whereas serotonin might be the transmitter of at least a certain population of the mossy fiber giant terminals, in the rat cerebellar glomeruli. In contrast, acetylcholine does not seem to have any transmitter role in the synaptic structures of the glomeruli.     

Transmitter histochemistry of the rat olfactory bulb III. Autoradiographic localization of [3H]GABA.

The distribution of [3H]gamma-aminobutyric acid (GABA) labeled elements in rat olfactory bulb was studied by light and electron microscopic autoradiography. [3H]GABA was strongly taken up into glial cells and pericytes in all layers of the bulb. The neuronal uptake of [3H]GABA was mainly seen in certain types of nerve terminals. About one-third of the granule dendritic terminals, some nerve endings of short axon cells, and certain nerve endings of extrabulbar origin showed a strong labeling. Labeling was seen in a small population of the periglomerular, short axon and granule cell bodies. Most cell bodies of these 3 types as well as the mitral cells did not, however, accumulate any appreciable amo9nt of [3H]GABA. The labeling pattern seen after injection of [3H]glycine and [3H]leucine was clearly different from the pattern seen after [3H]GABA injection. The labeling was more uniformly distributed over the components of the neuropil with a considerably higher activity over certain cell somata such as the mitral cells. The present results demonstrate that neuronal uptake and accumulation of [3H]GABA occur into populations of olfactory bulb cells and processes, which from neurophysiological and/or immunohistochemical studies are supposed to use GABA as a neurotransmitter.     

Transmitter histochemistry of the rat olfactory bulb. III. Autoradiographic localization of [H]GABA

The distribution of [³H]γ-aminobutyric acid (GABA) labeled elements in rat olfactory bulb was studied by light and electron microscopic autoradiography. [³H]GABA was strongly taken up into glial cells and pericytes in all layers of the bulb. The neuronal uptake of [³H]GABA was mainly seen in certain types of nerve terminals. About one-third of the granule dendritic terminals, some nerve endings of short axon cells, and certain nerve endings of extrabulbar origin showed a strong labeling. Labeling was seen in a small population of the periglomerular, short axon and granule cell bodies. Most cell bodies of these 3 types as well as the mitral cells did not, however, accumulate any appreciable amount of [³H]GABA. The labeling pattern seen after injection of [³H]glycine and [³H]leucine was clearly different from the pattern seen after [³H]GABA injection. The labeling was more uniformly distributed over the components of the neuropil with a considerably higher activity over certain cell somata such as the mitral cells. The present results demonstrate that neuronal uptake and accumulation of [³H]GABA occur into populations of olfactory bulb cells and processes, which from neurophysiological and/or immunohistochemical studies are supposed to use GABA as a neurotransmitter.     

GABAergic structures in the chick telencephalon: GABA immunocytochemistry combined with light and electron microscope autoradiography, and Golgi impregnation

The distribution of gamma-aminobutyric acid (GABA)-ergic elements in 3 forebrain regions (medial mid-telencephalic hyperstriatum ventrale; paleostriatum augmentatum; lobus parolfactorius) of two-day-old domestic chicks was investigated using (1) light and electron microscope autoradiography following [3H]GABA uptake in vitro in combination with pre-embedding GABA immunocytochemistry and (2) Golgi impregnation and 'gold-toning' combined with postembedding GABA immunocytochemistry. In both the paleostriatal regions and the medial (mid-telencephalic) hyperstriatum ventrale, GABA immunolabelling was demonstrated with the pre-embedding technique. Radiolabelling with [3H]GABA was also shown in these regions, co-localised in many cases with the immunolabelling. In the paleostriatal regions, the majority of perikaryal labelling was found in ovoid, elongated or fusiform cell bodies of 6-7 micron diameter whereas in the medial (mid-telencephalic) hyperstriatum ventrale, larger (10-15 micron) multipolar and smaller (5-6 micron) bipolar neurons were found labelled. In the latter region, Golgi impregnated neurons of similar morphology were found to be immunopositive to GABA using the postembedding technique. The ultrastructure of [3H]GABA accumulating cells is characterised by pale or moderately granular nuclei with small invaginations, few mitochondria and a prominent Golgi apparatus. Astrocytes and ependymal cells are also labelled with [3H]GABA. GABA-labelled axon terminals represent 29-36% of the total in the 3 brain regions studied. They appear as electron-lucent boutons with few and often scattered synaptic vesicles and in most cases they form symmetrical axo-dendritic junctions.     

Amino acid neurotransmitter candidates in rat cerebellum: Selective effects of kainic acid lesions

Kainic acid injections directly into the cerebellum destroy Purkinje, stellate, basket and Golgi II cells selectively with much less damage to granule cells. We have utilized such kainic acid lesions to evaluate the disposition of amino acid transmitter candidates in different neuronal populations of the cerebellum. Kainic acid lesions produce a 65-70% decrease in high affinity [3H]GABA uptake into synaptosomal fractions and a similar decrease in glutamic acid decarboxylase with a 50% reduction in endogenous GABA. Synaptosomal accumulation of [3H]glutamate and [3H]-aspartate is reduced 25-30% following such lesions while no decline in uptake of numerous other amino acids is observed. No significant changes are found in endogenous levels of glycine and serine are elevated following such lesions. These findings are consistent with the possibility that glutamate is the transmitter of granule cells and that GABA is the transmitter of the other cell types in the cerebellum.     

A new function for glycine GlyT2 transporters: Stimulation of γ‐aminobutyric acid release from cerebellar nerve terminals through GAT1 transporter reversal and Ca2+‐dependent anion channels

Glycine GlyT2 transporters are localized on glycine‐storing nerve endings. Their main function is to accumulate glycine to replenish synaptic vesicles. Glycine was reported to be costored with γ‐aminobutyric acid (GABA) in cerebellar interneurons that may coexpress glycine and GABA transporters, and this is confirmed here by confocal microscopy analysis showing coexpression of GAT1 and GlyT2 transporters on microtubule‐associated protein‐2‐positive synaptosomes. It was found that GABA uptake elicited glycine release from cerebellar nerve endings by various mechanisms. We investigated whether and by what mechanisms activation of glycine transporters could mediate release of GABA. Nerve endings purified from cerebellum were prelabeled with [³H]GABA and exposed to glycine. Glycine stimulated [³H]GABA release in a concentration‐dependent manner. The glycine effect was insensitive to strychnine or to 5,7‐dichlorokynurenate but it was abolished when GlyT2 transporters were blocked. About 20% of the evoked release was dependent on external Ca²⁺ entered by reversal of plasmalemmal Na⁺/Ca²⁺exchangers. A significant portion of the GlyT2‐mediated release of [³H]GABA (about 50% of the external Ca²⁺‐independent release) occurred by reversal of GABA GAT1 transporters. Na⁺ ions, reaching the cytosol during glycine uptake through GlyT2, activated mitochondrial Na⁺/Ca²⁺ exchangers, causing an increase in cytosolic Ca²⁺, which in turn triggered a Ca²⁺‐induced Ca²⁺ release process at inositoltrisphosphate receptors. Finally, the increased availability of Ca²⁺ in the cytosol allowed the opening of anion channels permeable to GABA. In conclusion, GlyT2 transporters not only take up glycine to replenish synaptic vesicles but can also mediate release of GABA by reversal of GAT1 and permeation through anion channels. © 2013 Wiley Periodicals, Inc.     

Carrier-mediated release of GABA from retinal horizontal cells

H1 horizontal cells in goldfish retina are probably GABAergic and receive excitatory synaptic input from red cones. This input should affect the synaptic release of GABA from H1 cells. We studied the uptake and release of [3H]GABA from the isolated goldfish retina by use of autoradiography. When retinas were incubated in the light for 15 min in 0.72 microM [3H]GABA, heavy label was found over the somata (HS) and axon terminals (HAT) of H1 horizontal cells, and over pyriform amacrine cell bodies and their processes in sublamina b of the IPL. Postincubation of retinas, preloaded with [3H]GABA, in 0.5-10 mM L-glutamate or 0.1-10 mM L-aspartate, resulted in a dose-dependent and selective loss of [3H]GABA from HS and very little loss from HAT. This loss was not due to an efflux of metabolites of [3H]GABA or to any calcium-dependent vesicular release of [3H]GABA from HS. The glutamate-evoked release of [3H]GABA by H1 cells was sodium dependent, sensitive to substitution of lithium for sodium, and inhibited by nipecotic acid. In addition, [3H]GABA was released from HS by 0.1 mM ouabain but not by 50 mM potassium chloride. Our results suggest that the chemically evoked release of [3H]GABA from HS is mediated by a sodium-dependent transport carrier which may be responsible for the high affinity uptake of [3H]GABA by H1 cells as well. Since synaptic vesicles are not found at presumed synaptic release sites in H1 cells, we suggest that the GABA which is released synaptically from H1 cells may derive from a cytoplasmic pool of GABA and is released by means of a transport carrier. This carrier appears to depend primarily on the sodium concentration gradient across the H1 cell membrane rather than on the membrane potential of the H1 cell for its action. The relevance of the carrier-mediated release of GABA from HS in regard to the synaptic function of H1 cells is discussed.     

The expression of excitatory amino acid binding sites during neuritogenesis in the developing rat cerebellum

The present study has examined excitatory amino acid transmitter binding sites as measured autoradiographically in cryostat sections prepared from developing rat cerebella during the period of granule cell neuritogenesis. The external germinal layer (EGL) and molecular layer (ML), which during development contain granule cells at early stages of axon growth, contained only low levels of NMDA-displaceable L-[3H]glutamate binding sites. Similarly, [3H]glycine binding to the NMDA receptor linked binding site was not enriched in the EGL. Radioligand binding to the NMDA receptor was always greater in the granular layer (GL) than in the ML. The developmental increases in NMDA-displaceable L-[3H]glutamate and in [3H]glycine binding to the GL were similar but NMDA displaceable L-[3H]glutamate binding density increased before [3H]glycine binding sites. Glycine increased NMDA-displaceable L-[3H]glutamate binding only in the adult cerebellum. These results suggest that NMDA stimulation of neuritogenesis in granule cell cultures may reflect stimulation of dendritogenesis in the developing glomerulus rather than a stimulation of axon growth in the EGL. Also, NMDA receptors may be present in an immature form during cerebellar development and have different properties to the adult receptor. Binding sites for [3H]kainate and [3H]AMPA were present in both the GL and ML and increased during development. At all times the amount of binding sites for [3H]kainate were highest in the GL whereas those for [3H]alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate were highest in the ML.     

Substrate specificity and developmental aspects of a presynaptic GABA receptor regulating glutamate release in the rat cerebellum

In order to better characterize the presynaptic GABA receptors regulating glutamate release in the cerebellum [Levi and Gallo, 1981], a number of GABA agonists and GABA transport inhibitors were tested for their ability to potentiate the depolarization-induced release of the glutamate analog D-[3H]aspartate from superfused cerebellar synaptosomes. Of all the compounds tested, only those which are known to interact specifically with GABA receptors were effective when tested on synaptosomal preparations. The order of effectiveness found was the following: muscimol congruent to 3-APS greater than or equal to P4S greater than isoguvacine greater than THIP. GABA uptake inhibitors were unable to enhance D-[3H]aspartate evoked release from synaptosomes, but were effective when tested in cerebellar slices; in the latter case, the activation of the GABA receptors may be achieved indirectly, through an increase of the extracellular GABA concentrations. The substrate specificity of the presynaptic GABA receptors regulating cerebellar acidic amino acid release appears to be similar to that reported for GABA receptors in radioligand binding studies and for GABA autoreceptors. Studies on synaptosomes from immature cerebella suggested that the presence of the potentiating effect on the acidic amino acid release by GABA agonists is correlated with the development of the parallel fiber terminals, which are believed to be the main site from which glutamate is released in the adult cerebellum.     

Activation of gamma-aminobutyric acid GAT-1 transporters on glutamatergic terminals of mouse spinal cord mediates glutamate release through anion channels and by transporter reversal

The effects of gamma-aminobutyric acid (GABA) on the release of glutamate from mouse spinal cord nerve endings have been studied using superfused synaptosomes. GABA elicited a concentration-dependent release of [3H]D-aspartate ([3H]D-ASP; EC50= 3.76 microM). Neither muscimol nor (-)baclofen mimicked GABA, excluding receptor involvement. The GABA-evoked release was strictly Na+ dependent and was prevented by the GABA transporter inhibitor SKF89976A, suggesting involvement of GAT-1 transporters located on glutamatergic nerve terminals. GABA also potentiated the spontaneous release of endogenous glutamate; an effect sensitive to SKF89976A and low-Na+-containing medium. Confocal microscopy shows that the GABA transporter GAT-1 is coexpressed with the vesicular glutamate transporter vGLUT-1 and with the plasma membrane glutamate transporter EAAT2 in a substantial portion of synaptosomal particles. The GABA effect was external Ca2+ independent and was not decreased when cytosolic Ca2+ ions were chelated by BAPTA. The glutamate transporter blocker DL-TBOA or dihydrokainate inhibited in part (approximately 35%) the GABA (10 microM)-evoked [3H]D-ASP release; this release was strongly reduced by the anion channel blockers niflumic acid and NPPB. GABA, up to 30 microM, was unable to augment significantly the basal release of [3H]glycine from spinal cord synaptosomes, indicating selectivity for glutamatergic transmission. It is concluded that GABA GAT-1 transporters and glutamate transporters coexist on the same spinal cord glutamatergic terminals. Activation of these GABA transporters elicits release of glutamate partially by reversal of glutamate transporters present on glutamatergic terminals and largely through anion channels.     

Influence of stereotaxically injected scrapie on neurotransmitter systems of mouse cerebellum

The 22L strain of scrapie was injected stereotaxically into the cerebellum of C57BL/6J mice to determine its effect on several cerebellar neurotransmitter systems during the early clinical stages of the disease. In this model vacuolar lesions are restricted to the cerebellum with no evidence of vacuolization in other brain regions. Although vacuolar lesions develop throughout all cell layers of the cerebellum, they are most severe in the granule cell layer. Modest but significant (P less than 0.01) reductions in cerebellar weight, glutamate decarboxylase activity, and in the affinity of the N6-[adenine-2,8-3H]cyclohexyladenosine binding sites, were observed in scrapie affected mice. The densities of the high- and low-affinity adenosine receptors were unaffected. Adenosine receptors in the cerebellum are highly localized to the axon terminals of the glutamatergic, GABA receptive granule cells. GABA, benzodiazepine, glutamate, and muscarinic cholinergic receptors were not significantly altered. In addition, the high-affinity uptake of glutamate, and the activity of choline acetyltransferase were not significantly changed. GABA high-affinity uptake was slightly increased. Even though the granule cell layer of the cerebellum had undergone severe vacuolation, only modest neurotransmitter changes were apparent. Although these results suggest a tenuous relationship between scrapie pathology and the integrity of neurotransmitter systems, it is possible that compensatory neurochemical changes in uncompromised neuronal populations may have masked potentially specific neurotransmitter effects.     

Putative acidic amino acid transmitters in the cerebellum. II. Electron microscopic localization of transport sites

In structurally preserved cerebellar slices, the sites of high affinity uptake of acidic amino acids were analyzed using the non-metabolizable analogue,d-[³H]aspartate. Electron microscopic autoradiography showed the greatest accumulation of grains to be over glial structures. The labelling of the perikarya, dendrites and axonsof the putatively glutamatergic granule cells was very low. However ‘hypothetical grain’ analysis indicated that the terminals of these cells are probable sources of radioactivity even though they contained less than 9% of the total grains in the molecular layer. The resolution of the autoradiographic technique did not permit definitive conclusion, as the parallel fibre terminals are too small and are ensheathed by thin glial processes. Nevertheless, further supporting evidence for somed-[³H]aspartate uptake into parallel fibre terminals was obtained using mechanically chopped cerebellar slices in which compared with glia presynaptic structures are selectively preserved. It is concluded that, in line with hypotheses relating to the compartmentation of glutamate metabolism, the principal sites of uptake of acidic amino acids in the cerebellum are the glial cells. The results have clear implications regarding the use of high affinity uptake as a marker for glutamatergic nerve terminals.     

Distribution of hypertrophied locus coeruleus projection to adult cerebellum after neonatal 6-hydroxydopamine

Following treatment as neonates with a high subcutaneous dose of 6-hydroxydopamine (6-OHDA), the projections of the locus coeruleus were mapped in the brains of adult rats. This was done using the technique of unilateral lesions in the nucleus followed by simultaneous determinations of norepinephrine (NE) levels, dopamine-beta-hydroxylase (DBH) activity and synaptosomal [3H]NE uptake in various terminal areas. In particular the cerebellum was subdivided into 3 areas in order to assess any changes from normal in the distribution of the hypertrophied noradrenergic projections here. In vehicle-control rats the lesions resulted in an 80--85% loss of NE in the parietal cortex ipsilateral to the lesion and a 15--20% loss contralaterally. In the control cerebellum the locus coeruleus projection, based upon changes in all 3 markers, is distributed 2/3 ipsilaterally and 1/3 contralaterally with the same pattern present in all 3 subregions. The neonatal 6-OHDA treatment resulted in virtually complete loss of noradrenergic terminals in the cerebral cortex. Following neonatal 6-OHDA treatment cerebellar levels of NE, DBH and [3H]NE uptake increased by between 20--60%, with the smallest increases occurring in [3H]NE uptake. In these rats the locus coeruleus accounted for at least 75--80% of the cerebellar noradrenergic parameters. Unlike control rats however, the lesions in these rats produced only ipsilateral decreases in NE and DBH. On the other hand changes in [3H]NE uptake indicated a normal 2/3 ipsilateral, 1/3 contralateral pattern. It is suggested that two separable events occur in the noradrenergic projection to the cerebellum. The first is the regeneration of an increased number of nerve terminals, or sprouting, and the second is a build-up of synaptic vesicles, or collateral accumulation. The sprouting, judging from the [3H]NE uptake data, occurs with apparently normal distribution, but the accumulation of NE and DBH is confined predominantly to the ipsilaterally projecting axon terminals. This may be the consequence of collateral accumulation resulting from the degeneration of the largely ipsilateral coeruleocortical projection.     

Growth cones isolated from developing rat forebrain: uptake and release of GABA and noradrenaline

A growth cone-enriched fraction isolated from neonatal rat forebrain was shown to accumulate gamma-amino [3H]butyric acid ([3H]-GABA) and [3H]noradrenaline ([3H]NA). Uptake of both neurotransmitters was sodium- and temperature-dependent and exhibited saturation kinetics with Km values of 17.7 microM and 4.5 microM respectively and Vmax values of 114 pmol/min/mg protein and 59 pmol/min/mg protein respectively. Electron microscopic autoradiography showed that about 50% of isolated growth cones can accumulate [3H]GABA. Inhibitor studies showed that beta-alanine was a relatively weak inhibitor of [3H]GABA uptake compared to nipecotic acid and diamino-butyric acid. Growth cone fractions preloaded with [3H]GABA and [3H]NA demonstrated a K+ (25 mM) -induced release of both neurotransmitters. Of the K+-stimulated release of [3H]GABA 50% was Ca2+-dependent, whereas the release of [3H]NA was entirely Ca2+-independent.     

Colocalization of glycine-like and GABA-like immunoreactivities in Golgi cell terminals in the rat cerebellum: a postembedding light and electron microscopic study

Consecutive sections of rat cerebella were incubated with antisera raised against glycine or γ-aminobutyric acid (GABA) conjugated to protein by glutaraldehyde. The sections were subsequently processed according to the peroxidase-antiperoxidase technique (semithin sections) or treated with secondary antibody coupled to colloidal gold particles (ultrathin sections). Corroborating previous light microscopic observations based on pre-embedding immunocytochemistry²⁰, a major proportion (about 70%) of the Golgi cell bodies showed immunoreactivity for both glycine and GABA. Analyses of semithin sections further suggested that the two immunoreactivities were colocalized in the same glomeruli and even in the same Golgi cell terminals. This was confirmed by electron microscopy. Quantification of the immunogold labelling for glycine (which is assumed to play metabolic roles in addition to its presumed role as a transmitter) showed that the net gold particle density was an order of magnitude higher over Golgi cell terminals than over the other constituents of the cerebellar glomeruli (mossy fibre terminals and granule cell dendrites). The total particle density over the latter was only slightly higher than the background level (over empty resin), suggesting that the concentration of ‘metabolic’ glycine is generally low compared to the concentration of glycine in Golgi cells. The stellate and basket cell terminals (which similarly to the Golgi cell are thought to release GABA as transmitter) were immunoreactive for GABA, but (with very few exceptions) virtually unlabelled for glycine, suggesting that our results were not confounded by any crossreactivity of the glycine antiserum with fixed GABA. Direct evidence that the sera reacted selectively with fixed glycine or GABA under the conditions used was obtained by incubating the tissue sections together with test sections containing a series of different amino acid-glutaraldehyde-brain macromolecule conjugates. Adsorption tests with soluble amino acid-glutaraldehyde complexes similarly suggested that the double-labelling of the Golgi terminals indeed reflected a colocalization of glycine and GABA. The results show that two ‘classical’ transmitters, both being inhibitory and acting on Cl⁻ channels, may coexist in the same nerve terminals.     

Release of [3H]gamma-aminobutyric acid from glial (Müller) cells of the rat retina: effects of K+, veratridine, and ethylenediamine

In several neural systems, glial cells appear to take up and release gamma-aminobutyric acid (GABA) upon depolarization. We have studied the release of [3H]GABA from Müller (glial) cells in the rat retina by a double isotope-labeling technique in which Müller cells are preloaded with 3H-GABA while a population of neurons is prelabeled with [14C]glycine. By autoradiography, we have confirmed that [3H]GABA is taken up by the radially oriented Müller cells, whereas [3H]glycine is accumulated by a subset of amacrine cells (neurons). Using the double-labeling procedure, we have examined the effects of two depolarizing agents, high K+ and veratridine, and the GABA mimetic, ethylenediamine, on transmitter release from glial cells and neurons simultaneously. We found the following. (1) Depolarization with 56 mM K+ released both [3H]GABA and [14C]glycine. About 70 to 80% of this release was blocked in Ca2+-free medium. (2) Veratridine (10 microM) also released both of the transmitters. This release was strongly inhibited by 100 nM tetrodotoxin or 1mM procaine. Under Ca2+-free conditions, less than 20% isotope release was observed. (3) Ethylenediamine released [3H]GABA readily, whereas little [14C]glycine release was observed. Removal of Ca2+ had no significant effect on transmitter release. Furthermore, in Na+-free medium ethylenediamine failed to induce [3H] GABA or [14C]glycine release. These results suggest that high K+ and veratridine release [3H]GABA from Müller cells by a Ca2+-dependent process. Ethylenediamine, on the other hand, appears to induce [3H]GABA release by a Ca2+-independent, carrier-mediated exchange mechanism.     

Characterization of antagonistic activity and binding properties of SR 95531, a pyridazinyl-GABA derivative, in rat brain and cultured cerebellar neuronal cells.

Experiments were performed to characterize the antagonistic activity and binding properties of SR 95531 [2-(3' carbethoxy-2'-propyl)-3-amino-6-paramethoxy-phenyl-piridazinium bromide] in rat brain. SR 95531 and bicuculline methiodide inhibited muscimol-stimulated 36Cl- uptake in cortical synaptoneurosomes in a concentration-dependent manner. The inhibitory potency of SR 95531 for the muscimol-stimulated 36Cl- uptake was 15 times higher than that of bicuculline methiodide. Scatchard plots of binding isotherms exhibited two apparent binding sites for [3H]SR 95531 in both the frontal cortex and cerebellum. The IC50 value of SR 95531 for muscimol-stimulated 36Cl- uptake into cortical synaptoneurosomes was in close agreement with the KD value of low-affinity binding sites of [3H]SR 95531 in the frontal cortex. Pretreatment of the membranes with phospholipase A2 invariably decreased [3H]SR 95531 binding in the frontal cortex and cerebellum. On the other hand, the treatment significantly increased [3H]gamma-aminobutyric acid (GABA) binding in a concentration-dependent manner in the frontal cortex. Although lower concentrations of phospholipase A2 did not affect [3H]GABA binding in the cerebellum, treatment with higher concentrations of phospholipase A2 increased the binding in this region. Specific binding of [3H]SR 95531 was also detected in cultures rich in cerebellar granule cells. Pretreatment with phospholipase A2 affected the binding of [3H]GABA and [3H]SR 95531 in these cells, as in the case of the cerebellum. These effects of phospholipase A2 on the binding of [3H]GABA and [3H]SR 95531 were partially prevented by the addition of delipidated bovine serum albumin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Evidence for the presynaptic localization of opiate binding sites on striatal efferent fibers

Using quantitative receptor autoradiography, [3H]D-Ala-D-Leu-enkephalin (DADL) and [3H]naloxone binding were studied in rat striatum and striatal projection areas (globus pallidus (GP) and substantia nigra pars reticulata (SNr] after unilateral striatal kainic acid lesions. [3H]DADL and [3H]naloxone binding were each examined by two methods. Initially, [3H]DADL binding was performed in 50 mM Tris-HCl (pH 7.4), 30 mM NaCl, 3 mM manganese acetate and 2 microM GTP; [3H]naloxone binding was carried out in 50 mM Tris-HCl (pH 7.4) and 100 mM NaCl. Subsequent studies were carried out in 150 mM Tris-HCl (pH 7.4) and either [3H]DADL plus 500 nM morphiceptin (to block [3H]DADL binding to mu receptors) or [3H]naloxone plus 10 nM delta receptor peptide (to block [3H]naloxone binding to delta receptors). At one and eight weeks in the lesioned striatum, [3H]DADL binding was reduced by 70% and 82%, respectively, when compared to the control side. [3H]Naloxone binding was reduced by 35% and 20%. In GP and SNr, [3H]DADL binding was reduced by 31% and 41%, respectively, at one week and 27% and 26% at eight weeks. [3H]Naloxone binding was reduced 19% in GP at eight weeks. A parsimonious explanation of these results is that opiate binding sites are located on presynaptic terminals of striatal efferent fibers to globus pallidus and substantia nigra pars reticulata as well as on local striatal axon collaterals. Since opiate peptides have recently been found to coexist with GABA in some striatal neurons, opiate peptides may play a role in striatal function by controlling GABA release from striatal efferent fibers. It is possible that pallidal and nigral opiate binding could be utilized as a marker for striatal terminals.     

Neuronal uptake of [3H]GABA and [3H]glycine in laminae I-III (substantia gelatinosa Rolandi) of the rat spinal cord. An autoradiographic study

[3H]GABA or [3H]glycine were injected into the subarachnoidal space of adult rats at C4-C5 level. After 10-60 min, the animals were perfused with 4% paraformaldehyde-0.5% glutaraldehyde and thick sections of the cervical spinal cord were postosmicated and Epon embedded. Light microscope autoradiographs of transverse cord sections showed numerous silver grains over the dorsal column and laminae I-III, higher grain densities occurring over lamina I for GABA and lamina III for glycine. In [3H]GABA-injected animals nerve cell bodies in lamina I or at the transition to lamina II appeared strongly labeled in light and electron microscope autoradiographs. These cells were smaller and less rich in RER than marginal cells and poor in axosomatic synaptic contacts. High grain densities appeared over axon terminals synapsing with dendrites in laminae I-II and over the light peripheral axon endings of synaptic glomeruli of laminae II-III. After [3H]glycine treatment, a number of nerve cell bodies were labeled in lamina III. It is suggested that two types of inhibitory interneurons occur in the rat gelatinosa, one GABAergic with cell body in lamina I, and another glycinergic in lamina III.