Immunogold electron microscopic demonstration of glutamate and GABA in normal and deafferented cerebellar cortex: correlation between transmitter content and synaptic vesicle size

Selective labeling of mossy fiber terminals and parallel fibers was obtained in rat cerebellar cortex by a glutamate antibody produced and characterized by Hepler et al. The high-resolution electron microscopic immunogold demonstration of this amino acid offered the possibility of determining the size and shape of synaptic vesicles in glutamate-positive mossy endings. Mossy terminals that stained with the glutamate antibody formed two distinct populations, one with spherical synaptic vesicles with an average diameter of 34.0 nm (more than 90% of all mossy fiber endings) and one with pleomorphic and smaller synaptic vesicles which had an average diameter of 28.5 nm. We present experimental evidence that the mossy terminals with large round vesicles are of extracerebellar origin, whereas those with small pleomorphic synaptic vesicles are endings of nucleocortical fibers. The presence of two distinct classes of gamma-aminobutyric acid (GABA)-containing axon terminals within cerebellar glomeruli has also been demonstrated; those originating from the cerebellar nuclei contain large (36.2 nm) synaptic vesicles, whereas the majority of GABA-stained axon terminals that are of local (cortical) origin contain small (29.1 nm) synaptic vesicles. It therefore appears that, at least in the case of glutamate and GABA, morphological characterization of the axon terminals based on the size and shape of synaptic vesicles is not a reliable indicator of their functional nature (i.e., whether they are excitatory or inhibitory); convincing evidence for the identity of the transmitter can be obtained only by electron microscopic immunostaining procedures. Our results also suggest the existence of both inhibitory and excitatory feedback from cerebellar nuclei to cerebellar cortex.     

Glutamine as a precursor for transmitter glutamate, aspartate and GABA in the cerebellum: a role for phosphate-activated glutaminase

Phosphate-activated glutaminase is present at high levels in the cerebellar mossy fiber terminals. The role of this enzyme for the production of glutamate from glutamine in the parallel-fiber terminals is unclear. In order to address this, we used light miroscopic immunoperoxidase and electron microscopic immunogold methods to study the localization of glutamate in rat cerbellar slices incubated with physiological K+ (3 mmol/L) and depolarizing K+ (40 mmol/L) concentrations, and during depolarizing conditions with the addition of glutamine and the glutaminase inhibitor 6-diazo-5-oxo-l-norleucine. During K+-induced depolarization glutamate labeling was redistributed from parallel-fiber terminals to glial cells. The nerve terminal content of glutamate was sustained when the slices were supplied with glutamine, which also reduced the accumulation of glutamate in glia. In spite of glutamine supplementation, the depolarized slices treated with 6-diazo-5-oxo-l-norleucine showed depletion of glutamate from parallel-fiber terminals and accumulation in glial cells. We conclude that cerebellar parallel-fiber terminals contain a glutaminase activity enabling them to synthesize glutamate from glutamine. Our results confirm that this is also true for the mossy fiber terminals. In addition, we show that, like for glutamate, the levels of aspartate in parallel-fiber terminals and GABA in Golgi fiber terminals can be maintained during depolarization if glutamine is present. This process is dependent on the activity of a glutaminase, as it can be inhibited by 6-diazo-5-oxo-l-norleucine, suggesting that the glutaminase reaction is important for glutamine to act as a precursor also for aspartate and GABA. The low levels of the kidney type of glutaminase that previously has been shown to be present in the parallel and Golgi fiber terminals could be sufficient to produce the transmitter amino acids. Alternatively, the amino acids could be produced from the liver type of glutaminase, which is not yet localized on the cellular level, or from an unknown glutminase.     

Nicotine-induced and depolarisation-induced glutamate release from hippocampus mossy fibre synaptosomes: two distinct mechanisms

Hippocampus mossy fibre terminals activate CA3 pyramidal neurons via two distinct mechanisms, both quantal and glutamatergic: (i) rapid excitatory transmission in response to afferent action potentials and (ii) delayed and prolonged release following nicotinic receptor activation. These processes were analysed here using rat hippocampus mossy fibres synaptosomes. The relationships between synaptosome depolarisation and glutamate release were established in response to high-KCl and gramicidin challenges. Half-maximal release corresponded to a 52 mV depolarisation step. KCl-induced release was accompanied by transient dissipation of the proton gradient across synaptic vesicle membrane. Nicotine elicited a substantial glutamate release from mossy fibre synaptosomes (EC₅₀ 3.14 μM; V _max 12.01 ± 2.1 nmol glutamate/mg protein; Hill's coefficient 0.99). However, nicotine-induced glutamate release was not accompanied by any change in the membrane potential or in the vesicular proton gradient. The effects of acetylcholine (200 μM) were similar to those of nicotine (25 μM). Nicotinic α7 receptors were evidenced by immuno-cytochemistry on the mossy fibre synaptosome plasma membrane. Therefore, the same terminals can release glutamate in response to two distinct stimuli: (i) rapid neurotransmission involving depolarisation-induced activation of voltage-gated Ca²⁺ channels and (ii) a slower nicotinic activation which does not involve depolarisation or dissipation of the vesicular proton gradient.     

Glutamate-like immunoreactivity revealed in rat olfactory bulb, hippocampus and cerebellum by monoclonal antibody and sensitive staining method

Although there is good evidence favoring L-glutamate as a major excitatory amino acid transmitter, relatively little is known about the distribution of nerve terminals using this substance. A method visualizing glutamate-like immunoreactivity at the light microscopic level by means of a monoclonal antibody, mAb 2D7, is described. --The antigen used for immunization was a glutaraldehyde-linked glutamate-BSA conjugate, and hybridomas were differentially screened by ELISA for production of antibodies recognizing glutamate- but not aspartate-BSA. The crossreactivity of 'anti-glutamate' mAb 2D7 as estimated in absorption tests was low even with conjugates closely related to glutamate-BSA.--Semithin sections from rapidly perfusion-fixed, plastic-embedded rat brain tissues were etched and stained by a combination of the peroxidase-antiperoxidase method and silver enhancement of the diaminobenzidine reaction product. Only this amongst several other immunohistochemical methods tried produced labeling patterns which showed terminal-like elements in brain regions such as olfactory bulb, hippocampus and cerebellum, and which were mostly consistent with already available information on systems using glutamate as neurotransmitter. Particularly striking was the staining of elements reminiscent of mossy fiber terminals in hippocampus and cerebellum as well as of cerebellar parallel fiber terminals.     

12-Lipoxygenase Products Attenuate the Glutamate Release and Ca2+ Accumulation Evoked by Depolarization of Hippocampal Mossy Fiber Nerve Endings

Presynaptic correlates of evoked neurotransmitter release include a rise in cytosolic free calcium level and the calcium-dependent liberation of unesterified arachidonic acid. It has been proposed that lipoxygenase metabolites produced from arachidonic acid may constitute an endogenous feedback system for the modulation of neurotransmitter release. The results of the present study are in agreement with this hypothesis. It was demonstrated that membrane depolarization evoked the release of endogenous glutamate from hippocampal mossy fiber synaptosomes, as well as the accumulation of intraterminal free calcium. The presence of 12-lipoxygenase products attenuated both the induced release of glutamate and the increase in calcium content, whereas 5- or 15-lipoxygenase metabolites were ineffective. A role for lipoxygenase products in the negative modulation of mossy fiber secretion processes was further indicated by the observations that low concentrations of the lipoxygenase inhibitor nordihydroguaiaretic acid (0.1-10 microM) potentiated the glutamate release and calcium accumulation induced by membrane depolarization. Therefore, we suggest that 12-lipoxygenase metabolites provide a presynaptic inhibitory signal that limits neurotransmitter release from hippocampal mossy fiber terminals.     

Release of D,L-threo-beta-hydroxyaspartate as a false transmitter from excitatory amino acid-releasing nerve terminals

This study examined whether preaccumulated D,L-threo-beta-hydroxyaspartate (tHA), a competitive substrate for the high-affinity excitatory amino acid (EAA) transporter, is released as a false transmitter from EAA-releasing nerve terminals. Potassium-stimulation (50 mM for 1 min) evoked significant release of the endogenous EAAs (aspartate and glutamate) from superfused neocortical minislices. Endogenous EAA release was largely calcium-dependent and was inhibited by tetanus toxin, a neurotoxin which specifically blocks vesicular exocytosis. In parallel experiments, minislices were pre-incubated with 500 microM tHA. Potassium (50 mM) evoked significant release of tHA and this release was also calcium-dependent and reduced by tetanus toxin. Pre-accumulation of tHA did not affect the release of endogenous glutamate whereas the release of endogenous aspartate was significantly attenuated. These data suggest that tHA selectively accumulates in a vesicular aspartate pool and is released upon depolarization as a false transmitter from EAA nerve terminals.     

Zinc transporter 3 immunohistochemical tracing of sprouting mossy fibres

Zinc transporter 3 (ZNT3) has been shown to transport zinc ions from the cytosol into presynaptic vesicles in the mammalian brain. Several studies have stated that the zinc ion containing synaptic vesicles of zinc-enriched neurons (ZEN) are loaded with ZNT3 proteins in their membranes. This fact makes it possible to trace sprouting mossy fibres in the temporal lobe epileptic hippocampus. In the present study, we examined the expression and distribution patterns of ZNT3 protein and chelatable zinc ions in the mouse hippocampus after pilocarpine treatment. Our results demonstrate that both ZNT3 immunostaining and autometallography reveal identical patterns of sprouting mossy fibres in the inner molecular layer in the mouse hippocampus. Using ZNT3 immuno-electron microscopic analysis we confirmed the presence of ectopic mossy fibre terminals in the inner molecular layer and found additionally by immuno-blotting a significant increase of ZNT3 in the pilocarpine-treated mouse hippocampi compared to age-matched controls. The increase of ZNT3 after pilocarpine treatment was time-dependent. The results support the notion that ZNT3 immunohistochemistry provides an excellent tool for tracing sprouting of ZEN terminals. The progressive increase of ZNT3 immunostaining in the temporal lobe epileptic hippocampus may relate to the increased levels of vesicular zinc ions during seizure.     

Immunocytochemistry of glutamate at the synaptic level

High concentrations of glutaraldehyde (2-5%) were found optimal for fixation of glutamate. In the absence of glutaraldehyde, (para)formaldehyde does not permanently retain L-[3H]-glutamate or D-[3H]-aspartate previously taken up into brain slices. Rats were fixed by rapid transcardial perfusion with 2.5% glutaraldehyde/1% (para)formaldehyde, and brain samples osmicated, embedded in epoxy resin, sectioned, and exposed to specific antisera to glutamate (conjugated to carrier protein by glutaraldehyde), followed by colloidal gold-labeled second antibody. The gold particle density was higher over putative glutamatergic nerve terminals than over any other tissue elements (two to three times tissue average in cerebellum and hippocampus). Calibration by test conjugates containing known concentrations of fixed glutamate processed in the same fluid drops as the tissue sections indicated that the concentration of fixed glutamate in putative glutamatergic terminals in hippocampus CA1 was c. 20 mmol/liter. The grain density over the parent cell bodies was only slightly higher than the tissue average. (Grain densities over test conjugates of other amino acids, aldehyde-fixed to brain macromolecules, were similar to that over empty resin. Labeling was blocked by glutamate-glutaraldehyde but not by other glutaraldehyde-treated amino acids.) In other experiments, brain slices were incubated in oxygenated artificial cerebrospinal fluid (CSF) and then immersion-fixed and processed as above. Here, the ration of grain densities in putative glutamatergic terminals vs other tissue elements was greater than in perfusion-fixed material. Comparison of intra-terminal areas poor and rich in synaptic vesicles suggested that in this preparation vesicles contained at least three times the glutamate concentration of cytosol. In the glutamatergic synapses of the giant reticulospinal axons in lamprey the ratio was over 30. Prolonged K+ depolarization of hippocampal and cerebellar slices reduced the nerve terminal glutamate immunoreactivity in a Ca2(+)-dependent manner. The results suggest that glutamate is released by exocytosis at excitatory synapses and show that immunocytochemistry can be used to study the cellular processing of small molecules.     

Activation of group II metabotropic glutamate receptors blocks zinc release from hippocampal mossy fibers

Background

The hippocampal CA3 area contains large amounts of vesicular zinc in the mossy fiber terminals which is released during synaptic activity, depending on presynaptic calcium. Another characteristic of these synapses is the presynaptic localization of high concentrations of group II metabotropic glutamate receptors, specifically activated by DCG-IV. Previous work has shown that DCG-IV affects only mossy fiber-evoked responses but not the signals from associational-commissural afferents, blocking mossy fiber synaptic transmission. Since zinc is released from mossy fibers even for single stimuli and it is generally assumed to be co-released with glutamate, the aim of the work was to investigate the effect of DCG-IV on mossy fiber zinc signals.

Results

Studies were performed using the membrane-permeant fluorescent zinc probe TSQ, and indicate that DCG-IV almost completely abolishes mossy fiber zinc changes as it does with synaptic transmission.

Conclusions

Zinc signaling is regulated by the activation of type II metabotropic receptors, as it has been previously shown for glutamate, further supporting the corelease of glutamate and zinc from mossy fibers.     

Kainate receptors and the induction of mossy fibre long-term potentiation

There is intense interest in understanding the molecular mechanisms involved in long-term potentiation (LTP) in the hippocampus. Significant progress in our understanding of LTP has followed from studies of glutamate receptors, of which there are four main subtypes (alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA), N-methyl-D-aspartate (NMDA), mGlu and kainate). This article summarizes the evidence that the kainate subtype of glutamate receptor is an important trigger for the induction of LTP at mossy fibre synapses in the CA3 region of the hippocampus. The pharmacology of the first selective kainate receptor antagonists, in particular the GLU(K5) subunit selective antagonist LY382884, is described. LY382884 selectively blocks the induction of mossy fibre LTP, in response to a variety of different high-frequency stimulation protocols. This antagonist also inhibits the pronounced synaptic facilitation of mossy fibre transmission that occurs during high-frequency stimulation. These effects are attributed to the presence of presynaptic GLU(K5)-subunit-containing kainate receptors at mossy fibre synapses. Differences in kainate receptor-dependent synaptic facilitation of AMPA and NMDA receptor-mediated synaptic transmission are described. These data are discussed in the context of earlier reports that glutamate receptors are not involved in mossy fibre LTP and more recent experiments using kainate receptor knockout mice, that argue for the involvement of GLU(K6) but not GLU(K5) kainate receptor subunits. We conclude that activation of presynaptic GLU(K5)-containing kainate receptors is an important trigger for the induction of mossy fibre LTP in the hippocampus.     

Mover is a novel vertebrate-specific presynaptic protein with differential distribution at subsets of CNS synapses

Presynaptic nerve terminals contain scaffolding proteins that orchestrate neurotransmitter release at active zones. Here we describe mover, a yet unknown non-transmembrane protein that is targeted to presynaptic terminals when overexpressed in cultured neurons. Confocal immunomicroscopy revealed that mover colocalizes with presynaptic markers in the calyx of Held. In the hippocampus, mover localizes to mossy fibre terminals, but is absent from inhibitory nerve terminals. By contrast, mover localizes to inhibitory terminals throughout the cerebellar cortex. Our results suggest that mover may act in concert with generally expressed scaffolding proteins in distinct sets of presynaptic terminals.     

Postnatal accumulation of zinc by the rat hippocampus

Timm's staining material has been detected in the rat hippocampus as early as day 1 postnatally. However, staining was diffuse and widespread and light granulation was observed only in the mossy fiber layer. By day 6 postnatally most diffuse staining had disappeared and the characteristic pattern of granulation had intensified in the mossy fiber layer. Pronounced staining of the mossy fibers became apparent from day 6. Electron microscopic autoradiography indicated that⁶⁵Zn injected intraperitoneally into suckling pups became localized largely in the axons and axon terminals of the mossy fiber layer in the CA3 and CA4 regions of the Horn of Ammon. In vitro studies with hippocampal slices have demonstrated that zinc is accumulated by an active transport system, but the kinetic characteristics of this uptake do not appear to alter with age. Zinc located intracellularly in the hippocampus appears to be associated mainly with large molecular weight ligands, with more than 75% of newly acquired zinc being bound to substances having molecular weights greater than 70,000 Daltons.     

The projection of spinocerebellar neurons from the sacrococcygeal region of the spinal cord in the cat. An experimental study using anterograde transport of WGA-HRP and degeneration.

The projection from the sacro-coccygeal region of the spinal cord to the cerebellum was studied by two different techniques in the cat. In five cats wheat germ agglutinin-horseradish peroxidase conjugate (WGA-HRP) was injected caudal to a preceding unilateral cordotomy at the sacral level, aimed at interrupting the spinocerebellar tracts on one side completely, and the distribution of WGA-HRP labeled mossy fibers and mossy fiber terminals was studied in the cerebellum. In three additional cats, degenerating fibers were examined in Fink-Heimer stained sections following unilateral transection of the lateral and ventral funiculi at L7 or S3 level. In the WGA-HRP experiments the labeled mossy fiber terminals were located bilaterally in lobules I-V. Most of them were found in the anterior part of lobule II. In addition, labeled terminals were observed in sublobule VIIIB and in pars copularis of the paramedian lobule, contralateral to the cordotomy. The terminals in the anterior lobe were concentrated in longitudinal zones parallel to the mid sagittal plane. In lobule II, the terminals were most abundant in the superficial, apical parts of the folia. Some presumed terminals were also seen in the cerebellar nuclei. Labeled fibers were found contralateral, but not ipsilateral to the cordotomy in the superior and inferior cerebellar peduncles, as well as in the spinal cord rostral to the cordotomy. The results of the degeneration experiments were the same as those of the WGA-HRP experiments with regard to the detailed projections in the cerebellar cortex. This is strong support against the possibility that WGA-HRP labeled cerebellar mossy fiber terminals, following WGA-HRP injections in the spinal cord, would represent terminals of collaterals of retrogradely labeled neurons. It also lends strong support in favour of WGA-HRP as a reliable anterograde tracer for studying cerebellar cortical projections of spinocerebellar neurons in the cat.     

Glutamate-immunoreactive climbing fibres in the cerebellar cortex of the rat

The climbing fibre system, one of the two main excitatory inputs to the cerebellar cortex, is anatomically and physiologically well characterized, while the nature of its neurotransmitter is still a matter of debate. We wished to determine whether glutamate-immunoreactive profiles with the morphological characteristics of climbing fibres could be found in the rat cerebellar cortex. For this purpose, a monoclonal anti-glutamate antibody has been used in combination with a sensitive postembedding immunoperoxidase method on semithin sections or in combination with a postembedding immunogold method on ultrathin sections. At the light microscopic level, climbing fibres appeared as strongly stained fibrous profiles, chains of interconnected varicosities or heavily labelled dots of various sizes, often in close apposition to principal Purkinje cell dendrites. At the electron microscopic level, certain labelled varicosities or more elongated profiles resembling climbing fibre terminals were in synaptic contact with dendritic spines of Purkinje cells. Quantitative analysis of gold particle densities showed that such elements were about three to four times more heavily labelled than their postsynaptic partners. The results obtained in this study demonstrate that at least a subset of climbing fibres and their terminals contain relatively high levels of glutamate-like immunoreactivity and provide additional evidence for a role of glutamate as transmitter in these cerebellar afferents.     

Glutamate-like immunoreactivity revealed in rat olfactory bulb,hippocampus and cerebellum by monoclonal antibody and sensitive staining method

Summary Although there is good evidence favoring l-glutamate as a major excitatory amino acid transmitter, relatively little is known about the distribution of nerve terminals using this substance. A method visualizing glutamate-like immunoreactivity at the light microscopic level by means of a monoclonal antibody, mAb 2D7, is described. — The antigen used for immunization was a glutaraldehyde-linked glutamate-BSA conjugate, and hybridomas were differentially screened by ELISA for production of antibodies recognizing glutamate- but not aspartate-BSA. The cross-reactivity of anti-glutamate mAb 2D7 as estimated in absorption tests was low even with conjugates closely related to glutamate-BSA. — Semithin sections from rapidly perfusion-fixed, plastic-embedded rat brain tissues were etched and stained by a combination of the peroxidase-antiperoxidase method and silver enhancement of the diaminobenzidine reaction product. Only this amongst several other immunohistochemical methods tried produced labeling patterns which showed terminal-like elements in brain regions such as olfactory bulb, hippocampus and cerebellum, and which were mostly consistent with already available information on systems using glutamate as neurotransmitter. Particularly striking was the staining of elements reminiscent of mossy fiber terminals in hippocampus and cerebellum as well as of cerebellar parallel fiber terminals.     

Monosynaptic GABAergic signaling from dentate to CA3 with a pharmacological and physiological profile typical of mossy fiber synapses

Mossy fibers are the sole excitatory projection from dentate gyrus granule cells to the hippocampus, where they release glutamate, dynorphin, and zinc. In addition, mossy fiber terminals show intense immunoreactivity for the inhibitory neurotransmitter GABA. Fast inhibitory transmission at mossy fiber synapses, however, has not previously been reported. Here, we show that electrical or chemical stimuli that recruit dentate granule cells elicit monosynaptic GABA(A) receptor-mediated synaptic signals in CA3 pyramidal neurons. These inhibitory signals satisfy the criteria that distinguish mossy fiber-CA3 synapses: high sensitivity to metabotropic glutamate receptor agonists, facilitation during repetitive stimulation, and NMDA receptor-independent long-term potentiation. GABAergic transmission from the dentate gyrus to CA3 has major implications not only for information flow into the hippocampus but also for developmental and pathological processes involving the hippocampus.     

Abundance of Zinc Ions in Synaptic Terminals of mocha Mutant Mice: Zinc Transporter 3 Immunohistochemistry and Zinc Sulphide Autometallography

The mocha mouse is an autosomal recessive pigment mutant on mouse chromosome 10 caused by a deletion in the gene for the delta subunit of the adaptor-like complex AP-3. Based on zinc transporter 3 (ZnT3) immunohistochemistry, zinc TSQ fluorescence and a modified Timm method, previous studies found a lack of histochemically-detectable zinc and a substantial reduction in the ZnT3 immunoreactivity. It has, therefore, been suggested that the mocha mouse could serve as a model for studies of the significance of zinc ions in zinc-enriched (ZEN) neurons. We have chosen the mocha-zinc-model in a study of the significance of ZEN neurons in hypoxia-caused damage in mouse brain. In order to establish that the model was either void of zinc ions or had a significantly decreased level of zinc ions in their ZEN terminals, we repeated the studies that had lead to the above assumption, the only methodology difference being that we used the zinc specific Neo-Timm method instead of the Timm method applied in the original study. We found that, although the ZnS autometallography (AMG) technique revealed a reduction in staining intensity as compared to the littermate controls, there were still plenty of zinc ions in the ZEN terminals, in particular visible in telencephalic structures like neocortex and hippocampus. At ultrastructural levels the zinc ions were found in a pool of vesicles of the ZEN terminals as in the control animals, but additionally zinc ions could be traced in ZEN neuronal somata in the neocortex and hippocampus. The mossy fibres in the hippocampus of mocha mice also bind with TSQ, though less than in the controls. We found ZnS AMG grains in ZEN neuronal somata, which were also immunoreactive for ZnT3. Our study confirmed the decreased ZnT3 immunoreactivity in ZEN terminals of the mocha mouse found in the original study. Based on these findings, we suggest that the mocha mouse may not be an ideal model for studies of the histochemically-detectable zinc ion pool of the central nervous system.     

Vesicular glutamate transporter 2 is expressed in different nerve fibre populations that selectively contact pulmonary neuroepithelial bodies

Pulmonary neuroepithelial body (NEB) receptors in rats receive at least four different nerve fibre populations. In addition to a spinal sensory innervation that contacts NEBs at their basal side, extensive vagal nodose sensory terminals and separate nitrergic and cholinergic nerve endings protrude between NEB cells. In the present study, antibodies against the vesicular glutamate transporter 2 (VGLUT2), a transmembrane protein responsible for loading glutamate into synaptic vesicles, were used to investigate whether some of the nerve terminals contacting NEBs in rat lungs might use glutamate as a neurotransmitter. VGLUT2 immunoreactivity (IR) was detected in extensive intraepithelial arborising nerve terminals that appeared to contact most of the NEBs. Multiple immunostaining showed VGLUT2 IR in the vagal nodose and spinal sensory nerve terminals contacting NEBs, and in another, most likely sensory, intraepithelial nerve fibre population, the origin and further characteristics of which remain to be elucidated. At least part of the VGLUT2-immunoreactive nerve fibres that contact NEBs were shown to be myelinated. The expression of VGLUT2 indicates that glutamate is stored and released as a neurotransmitter in terminals of several pulmonary (sensory) nerve fibre populations that selectively relate to the complex NEB receptors. The present study strongly suggests an involvement of glutamatergic mechanisms in the peripheral transduction of sensory stimuli from the lungs, via the release of glutamate from nerve terminals, thereby modulating the activity of NEB receptor cells or the excitability of afferent nerves.     

Presynaptic glycine receptors on hippocampal mossy fibers

Presynaptic glycine receptors (GlyRs) have been implicated in the regulation of glutamatergic synaptic transmission. Here, we characterized presynaptic GlyR-mediated currents by patch-clamp recording from mossy fiber boutons (MFBs) in rat hippocampal slices. In MFBs, focal puff-application of glycine-evoked chloride currents that were blocked by the GlyR antagonist strychnine. Their amplitudes declined substantially during postnatal development, from a mean conductance per MFB of ∼600 pS in young to ∼130 pS in adult animals. Single-channel analysis revealed multiple conductance states between ∼20 and ∼120 pS, consistent with expression of both homo- and hetero-oligomeric GlyRs. Accordingly, estimated GlyRs densities varied between 8-17 per young, and 1-3 per adult, MFB. Our results demonstrate that functional presynaptic GlyRs are present on hippocampal mossy fiber terminals and suggest a role of these receptors in the regulation of glutamate release during the development of the mossy fiber - CA3 synapse.     

Glutamate metabolism in the brain of young rats exposed to organic and inorganic lead

The synthesis of glutamate and its conversion to glutamine and GABA were studied using labelled glucose in cerebral cortex, cerebellum and brainstem of rats intoxicated acutely with tetraethyl lead and chronically with lead acetate. To assess the interconversion and the synaptosomal accumulation of these amino acids, the labelling of glutamate, glutamine and GABA were measured in whole tissue and synaptosomes after giving labelled glutamate. The radioactive carbon dioxide production from labelled glutamate by brain slices was measured to evaluate the oxidation of glutamate. The tissue levels of glutamate, glutamine and GABA and the activity of glutamate decarboxylase were also measured in both conditions.In inorganic lead toxicity, even though the glutamate pool size was reduced, the glutamate-glutamine cycling between synaptosomes and astrocytes was increased. The oxidation of glutamate and the glutamate-GABA cycling were reduced. These findings suggest that brain tries to maintain the endogenous glutamate levels by decreasing the oxidation of glutamate and increasing the uptake systems and the cycling through glutamine in inorganic lead toxicity. In organic lead toxicity, the glutamate pool as well as glutamate turnover was reduced markedly resulting in complete distortion of glutamate metabolism.