Enhanced Glutamatergic and Decreased Gabaergic Synaptic Appositions to GnRH Neurons on Proestrus in the Rat: Modulatory Effect of Aging

Background

Previous work by our lab and others has implicated glutamate as a major excitatory signal to gonadotropin hormone releasing hormone (GnRH) neurons, with gamma amino butyric acid (GABA) serving as a potential major inhibitory signal. However, it is unknown whether GABAergic and/or glutamatergic synaptic appositions to GnRH neurons changes on the day of the proestrous LH surge or is affected by aging.

Methodology/Principal Findings

To examine this question, synaptic terminal appositions on GnRH neurons for VGAT (vesicular GABA transporter) and VGLUT2 (vesicular glutamate transporter-2), markers of GABAergic and glutamatergic synaptic terminals, respectively, was examined by immunohistochemistry and confocal microscopic analysis in young and middle-aged diestrous and proestrous rats. The results show that in young proestrous rats at the time of LH surge, we observed reciprocal changes in the VGAT and VGLUT2 positive terminals apposing GnRH neurons, where VGAT terminal appositions were decreased and VGLUT2 terminal appositions were significantly increased, as compared to young diestrus control animals. Interestingly, in middle-aged cycling animals this divergent modulation of VGAT and VGLUT2 terminal apposition was greatly impaired, as no significant differences were observed between VGAT and VGLUT2 terminals apposing GnRH neurons at proestrous. However, the density of VGAT and VGLUT2 terminals apposing GnRH neurons were both significantly increased in the middle-aged animals.

Conclusions/Significance

In conclusion, there is an increase in glutamatergic and decrease in GABAergic synaptic terminal appositions on GnRH neurons on proestrus in young animals, which may serve to facilitate activation of GnRH neurons. In contrast, middle-aged diestrous and proestrous animals show a significant increase in both VGAT and VGLUT synaptic terminal appositions on GnRH neurons as compared to young animals, and the cycle-related change in these appositions between diestrus and proestrus that is observed in young animals is lost.     

Absence of synapsin I and II is accompanied by decreases in vesicular transport of specific neurotransmitters

Studies of synapsin-deficient mice have shown decreases in the number of synaptic vesicles but knowledge about the consequences of this decrease, and which classes of vesicles are being affected, has been lacking. In this study, glutamatergic, GABAergic and dopaminergic transport has been analysed in animals where the genes encoding synapsin I and II were inactivated. The levels of the vesicular glutamate transporter (VGLUT) 1, VGLUT2 and the vesicular GABA transporter (VGAT) were decreased by approximately 40% in adult forebrain from mice devoid of synapsin I and II, while vesicular monoamine transporter (VMAT) 2 and VGLUT3 were present in unchanged amounts compared with wild-type mice. Functional studies on synaptic vesicles showed that the vesicular uptake of glutamate and GABA was decreased by 41 and 23%, respectively, while uptake of dopamine was unaffected by the lack of synapsin I and II. Double-labelling studies showed that VGLUT1 and VGLUT2 colocalized fully with synapsin I and/or II in the hippocampus and neostriatum, respectively. VGAT showed partial colocalization, while VGLUT3 and VMAT2 did not colocalize with either synapsin I or II in the brain areas studied. In conclusion, distinct vesicular transporters show a variable degree of colocalization with synapsin proteins and, hence, distinct sensitivities to inactivation of the genes encoding synapsin I and II.     

中枢神经系统谷氨酸转运体的研究进展

在中枢神经系统,谷氨酸转运体在谷氨酸一谷氨酰胺循环中发挥着重要作用。谷氨酸转运体有高亲和力转运体,即兴奋性氨基酸转运体（excitatory amino acid transporters,EAATs）和低亲和力转运体,即囊泡谷氨酸转运体（vesicular glutamate transporters,VGLUTs）两种类型。其中,VGLUTs的功能是特异地将突触囊泡外的谷氨酸转运进入突触囊泡内,它包括三个成员,分别是VGLUT1、VGLUT2和VGLUT3。一方面,VGLUT1和VGLUT2标记了所有的谷氨酸能神经元,是谷氦酸能神经元和它们轴突末端高度特异的标志;另一方面,VGLUT1标志着皮质一皮质投射,而VGLUT2则标志着丘脑一皮层投射,VGLUT3则位于抑制性突触末端。     

Synaptic and vesicular co-localization of the glutamate transporters VGLUT1 and VGLUT2 in the mouse hippocampus

Vesicular glutamate transporters (VGLUTs) are essential to glutamatergic synapses and determine the glutamatergic phenotype of neurones. The three known VGLUT isoforms display nearly identical uptake characteristics, but the associated expression domains in the adult rodent brain are largely segregated. Indeed, indirect evidence obtained in young VGLUT1-deficient mice indicated that in cells that co-express VGLUT1 and VGLUT2, the transporters may be targeted to different synaptic vesicles, which may populate different types of synapses formed by the same neurone. Direct evidence for a systematic segregation of VGLUT1 and VGLUT2 to distinct synapses and vesicles is lacking, and the mechanisms that may convey this segregation are not known. We show here that VGLUT1 and VGLUT2 are co-localized in many layers of the young hippocampus. Strikingly, VGLUT2 co-localizes with VGLUT1 in the mossy fibers at early stages. Furthermore, we show that a fraction of VGLUT1 and VGLUT2 is carried by the same vesicles at these stages. Hence, hippocampal neurones co-expressing VGLUT1 and VGLUT2 do not appear to sort them to separate vesicle pools. As the number of transporter molecules per vesicle affects quantal size, the developmental window where VGLUT1 and VGLUT2 are co-expressed may allow for greater plasticity in the control of quantal release.     

Effects of subthalamic nucleus lesions and stimulation upon corticostriatal afferents in the 6-hydroxydopamine-lesioned rat

Abnormalities of striatal glutamate neurotransmission may play a role in the pathophysiology of Parkinson's disease and may respond to neurosurgical interventions, specifically stimulation or lesioning of the subthalamic nucleus (STN). The major glutamatergic afferent pathways to the striatum are from the cortex and thalamus, and are thus likely to be sources of striatal neuronally-released glutamate. Corticostriatal terminals can be distinguished within the striatum at the electron microscopic level as their synaptic vesicles contain the vesicular glutamate transporter, VGLUT1. The majority of terminals which are immunolabeled for glutamate but are not VGLUT1 positive are likely to be thalamostriatal afferents. We compared the effects of short term, high frequency, STN stimulation and lesioning in 6-hydroxydopamine (6OHDA)-lesioned rats upon striatal terminals immunolabeled for both presynaptic glutamate and VGLUT1. 6OHDA lesions resulted in a small but significant increase in the proportions of VGLUT1-labeled terminals making synapses on dendritic shafts rather than spines. STN stimulation for one hour, but not STN lesions, increased the proportion of synapses upon spines. The density of presynaptic glutamate immuno-gold labeling was unchanged in both VGLUT1-labeled and -unlabeled terminals in 6OHDA-lesioned rats compared to controls. Rats with 6OHDA lesions+STN stimulation showed a decrease in nerve terminal glutamate immuno-gold labeling in both VGLUT1-labeled and -unlabeled terminals. STN lesions resulted in a significant decrease in the density of presynaptic immuno-gold-labeled glutamate only in VGLUT1-labeled terminals. STN interventions may achieve at least part of their therapeutic effect in PD by normalizing the location of corticostriatal glutamatergic terminals and by altering striatal glutamatergic neurotransmission.     

Activity-dependent regulation of vesicular glutamate and GABA transporters: a means to scale quantal size

The functional balance of glutamatergic and GABAergic signaling in neuronal cortical circuits is under homeostatic control. That is, prolonged alterations of global network activity leads to opposite changes in quantal amplitude at glutamatergic and GABAergic synapses. Such scaling of excitatory and inhibitory transmission within cortical circuits serves to restore and maintain a constant spontaneous firing rate of pyramidal neurons. Our recent work shows that this includes alterations in the levels of expression of vesicular glutamate (VGLUT1 and VGLUT2) and GABA (VIAAT) transporters. Other vesicle markers, such as synaptophysin or synapsin, are not regulated in this way. Endogenous regulation at the level of mRNA and synaptic protein controls the number of transporters per vesicle and hence, the level of vesicle filling with transmitter. Bidirectional and opposite activity-dependent regulation of VGLUT1 and VIAAT expression would serve to adjust the balance of glutamate and GABA release and therefore the level of postsynaptic receptor saturation. In some excitatory neurons and synapses, co-expression of VGLUT1 and VGLUT2 occurs. Bidirectional and opposite changes in the levels of two excitatory vesicular transporters would enable individual neocortical neurons to scale up or scale down the level of vesicular glutamate storage, and thus, the amount available for release at individual synapses. Regulated vesicular transmitter storage and release via selective changes in the level of expression of vesicular glutamate and GABA transporters indicates that homeostatic plasticity of synaptic strength at cortical synapses includes presynaptic elements.     

Molecular cloning and functional characterization of human vesicular glutamate transporter 3

Glutamate is the major excitatory neurotransmitter in the mammalian CNS. It is loaded into synaptic vesicles by a proton gradient-dependent uptake system and is released by exocytosis upon stimulation. Recently, two mammalian isoforms of a vesicular glutamate transporter, VGLUT1 and VGLUT2, have been identified, the expression of which enables quantal release of glutamate from glutamatergic neurons. Here, we report a novel isoform of a human vesicular glutamate transporter (hVGLUT3). The predicted amino acid sequence of hVGLUT3 shows 72% identity to both hVGLUT1 and hVGLUT2. hVGLUT3 functions as a vesicular glutamate transporter with similar properties to the other isoforms when it is heterologously expressed in a neuroendocrine cell line. Although mammalian VGLUT1 and VGLUT2 exhibit a complementary expression pattern covering all glutamatergic pathways in the CNS, expression of hVGLUT3 overlaps with them in some brain areas, suggesting molecular diversity that may account for physiological heterogeneity in glutamatergic synapses.     

Heterogeneity of axon terminals expressing VGLUT1 in the cerebral neocortex

Using immunocytochemical techniques and confocal microscopy we have studied the localization of the vesicular glutamate transporters (VGLUTs) 1 and 2 in the mammalian cerebral cortex. The cardinal observations gathered to date can be summarized as follows: 1) Many VGLUT1-positive puncta coexpressing synaptophysin-1 outline pyramidal cell somata and proximal dendrites; of these, a sizeable fraction coexpress VGAT, the vesicular transporter for GABA; 2) VGLUT2-positive puncta are also present in layers II-III and some of them coexpress VGLUTI. These findings suggest that in the cerebral cortex of adult rats axon terminals expressing VGLUT1 are heterogeneous.     

Normal biogenesis and cycling of empty synaptic vesicles in dopamine neurons of vesicular monoamine transporter 2 knockout mice

The neuronal isoform of vesicular monoamine transporter, VMAT2, is responsible for packaging dopamine and other monoamines into synaptic vesicles and thereby plays an essential role in dopamine neurotransmission. Dopamine neurons in mice lacking VMAT2 are unable to store or release dopamine from their synaptic vesicles. To determine how VMAT2-mediated filling influences synaptic vesicle morphology and function, we examined dopamine terminals from VMAT2 knockout mice. In contrast to the abnormalities reported in glutamatergic terminals of mice lacking VGLUT1, the corresponding vesicular transporter for glutamate, we found that the ultrastructure of dopamine terminals and synaptic vesicles in VMAT2 knockout mice were indistinguishable from wild type. Using the activity-dependent dyes FM1-43 and FM2-10, we also found that synaptic vesicles in dopamine neurons lacking VMAT2 undergo endocytosis and exocytosis with kinetics identical to those seen in wild-type neurons. Together, these results demonstrate that dopamine synaptic vesicle biogenesis and cycling are independent of vesicle filling with transmitter. By demonstrating that such empty synaptic vesicles can cycle at the nerve terminal, our study suggests that physiological changes in VMAT2 levels or trafficking at the synapse may regulate dopamine release by altering the ratio of fillable-to-empty synaptic vesicles, as both continue to cycle in response to neural activity.     

Redefining chemical anatomy of glutamatergic neurotransmission

With the recent identification of the two isoforms of vesicular glutamate transporters VGLUT1 and VGLUT2 and of the presumed neuronal glutamine transporter SAT1 novel tools have been made available to unequivocally define the anatomy of glutamatergic pathways on the cellular and synaptic level. Using highly specific antisera and cRNA probes two distinct glutamatergic pathways expressing either VGLUT1 or VGLUT2 could be detected throughout the central nervous system. Areas where VGLUT1 predominated included the cerebral and cerebellar cortex and the hippocampus. VGLUT2 was mainly expressed in the thalamus, hypothalamus and brain stem. VGLUT1 and VGLUT2 synapses exhibited distinct region- and pathway-specific relationships with each other and with other classical transmitter and peptidergic systems. The glutamine transporter SAT1 was expressed in CNS neurons and in ependymal cells. Neuronal SAT1 expression comprised virtually all glutamatergic neurons but also specific subsets of cholinergic, GABAergic and aminergic neurons in the CNS. In addition to widespread expression of VGLUT1 and VGLUT2 in the CNS, peripheral tissues such as sensory neurons and pancreatic islet cells differentially expressed VGLUT isoforms and SAT1.
Our results suggest pathway-specific functional duality in the regulation of vesicular glutamate release at excitatory synapses and provide evidence for glutamine transport and metabolism in excitatory glutamatergic and diverse nonglutamatergic neurons as well.     

Localization of the glycine transporter GLYT1 in glutamatergic synaptic vesicles

We have previously shown the presence of the glycine transporter GLYT1 in glutamatergic terminals of the rat brain. In this study we present immunohistochemical and biochemical evidence indicating that GLYT1 is expressed not only at the plasma membrane of glutamatergic neurons, but also at synaptic vesicles. Confocal microscopy, immunoblots analysis of a highly purified synaptic vesicle fraction and immunoisolation of synaptic vesicles with anti-synaptophysin antibodies strongly suggested the presence of GLYT1 in synaptic vesicles. Moreover, direct observation with the electron microscope of purified vesicles immunoreacted with anti-GLYT1 and colloidal gold demonstrated that about 40% of the small vesicles of the purified vesicle fraction contained GLYT1. Double labeling for GLYT1 and synaptophysin of this vesicular fraction revealed that more of ninety percent of them were synaptic vesicles. Moreover, a significant part of the GLYT1 containing vesicles (86%) also contained the vesicular glutamate transporter vGLUT1, suggesting a functional role of GLYT1 in a subpopulation of glutamatergic vesicles.     

Electron microscopic examination of the orexin immunoreactivity in the dorsal raphe nucleus

The ultrastructure and the synaptic relationships of the orexin-A-like immunoreactive fibers in the dorsal raphe nucleus were examined with an immunoelectron microscopic method. At the electron microscopic level, most of the immunoreactive fibers, a varicosity appearance at the light microscopic level, were found as axon terminals. The large dense-cored vesicles contained in the immunoreactive axon terminals were the most intensely immunostained organellae. These axon terminals were often found to make synapses. While the axo-dendritic synapses were usually asymmetric in appearance, the axo-somatic synapses were symmetric. Orexin-A-like immunoreactive processes with no synaptic vesicles were also found. These processes often received asymmetric synapses. With less frequency, the synapses were found between the orexin-like immunoreactive processes. The results suggest that the orexin peptides are stored in the large dense-cored vesicles; the orexin-containing fibers may have influences on the physiological activities of the dorsal raphe nucleus through direct synaptic relationships.     

A single vesicular glutamate transporter is sufficient to fill a synaptic vesicle

Quantal size is the postsynaptic response to the release of a single synaptic vesicle and is determined in part by the amount of transmitter within that vesicle. At glutamatergic synapses, the vesicular glutamate transporter (VGLUT) fills vesicles with glutamate. While elevated VGLUT expression increases quantal size, the minimum number of transporters required to fill a vesicle is unknown. In Drosophila DVGLUT mutants, reduced transporter levels lead to a dose-dependent reduction in the frequency of spontaneous quantal release with no change in quantal size. Quantal frequency is not limited by vesicle number or impaired exocytosis. This suggests that a single functional unit of transporter is both necessary and sufficient to fill a vesicle to completion and that vesicles without DVGLUT are empty. Consistent with the presence of empty vesicles, at dvglut mutant synapses synaptic vesicles are smaller, suggesting that vesicle filling and/or transporter level is an important determinant of vesicle size.     

The loading of neurotransmitters into synaptic vesicles

Classical (non-peptide) transmitters are stored into secretory vesicles by a secondary active transporter driven by a V-type H(+)-ATPase. Five vesicular neurotransmitter uptake activities have been characterized in vitro and, for three of them, the transporters involved have been identified at the molecular level using cDNA cloning and/or Caenorhabditis elegans genetics. These transporters belong to two protein families, which are both unrelated to the Na(+)-coupled neurotransmitter transporters operating at the plasma membrane. The two isoforms of the mammalian vesicular monoamine transporter, VMAT1 and VMAT2, are related to the vesicular acetylcholine transporter (VACHT), while a novel, unrelated vesicular inhibitory amino acid transporter (VIAAT), also designated vesicular GABA transporter (VGAT), is responsible for the storage of GABA, glycine or, at some synapses, both amino acids into synaptic vesicles. The observed effects of experimentally altered levels of VACHT or VMAT2 on synaptic transmission and behavior, as well as the recent awareness that GABAergic or glutamatergic receptors are not always saturated at central synapses, suggest a potential role of vesicular loading in synaptic plasticity.     

Into great silence without VGLUT3

The vesicular glutamate transporters VGLUT1 and VGLUT2 fill synaptic vesicles with glutamate, an essential prerequisite for glutamatergic transmission in the CNS. In contrast, the third isoform, VGLUT3, is not confined to glutamatergic neurons, and its function has remained enigmatic. In this issue of Neuron, Seal et al. show that mice lacking VGLUT3 are profoundly deaf and exhibit nonconvulsive seizures.     

Interaction between the vesicular glutamate transporter type 1 and endophilin A1, a protein essential for endocytosis

In the nerve terminal, neurotransmitter is actively packaged into synaptic vesicles before its release by Ca2+-dependent exocytosis. The three vesicular glutamate transporters (VGLUT1, -2 and -3) are highly conserved proteins that display similar bioenergetic and pharmacological properties but are expressed in different brain areas. We used the divergent C-terminus of VGLUT1 as a bait in a yeast two-hybrid screen to identify and map the interaction between a proline-rich domain of VGLUT1 and the Src homology domain 3 (SH3) domain of endophilin. We further confirmed this interaction by using different glutathione-S-transferase-endophilin fusion proteins to pull down VGLUT1 from rat brain extracts. The expression profiles of the two genes and proteins were compared on rat brain sections, showing that endophilin is most highly expressed in regions and cells expressing VGLUT1. Double immunofluorescence in the rat cerebellum shows that most VGLUT1-positive terminals co-express endophilin, whereas VGLUT2-expressing terminals are often devoid of endophilin. However, neither VGLUT1 transport activity, endophilin enzymatic activity nor VGLUT1 synaptic targeting were altered by this interaction. Overall, the discovery of endophilin as a partner for VGLUT1 in nerve terminals strongly suggests the existence of functional differences between VGLUT1 and -2 terminals in their abilities to replenish vesicle pools.     

Differential sorting of the vesicular glutamate transporter 1 into a defined vesicular pool is regulated by light signaling involving the clock gene Period2

Synaptic strength depends on the amount of neurotransmitter stored in synaptic vesicles. The vesicular transmitter content has recently been shown to be directly dependent on the expression levels of vesicular neurotransmitter transporters indicating that the transport capacity of synaptic vesicles is a critical determinant for synaptic efficacy. Using synaptic vesicles prepared from whole brain at different times of the day we now show that the amount of vesicular glutamate transporter (VGLUT) 1 undergoes strong diurnal cycling. VGLUT1 protein levels are high before the start of the light period, decline at noon, increase again before start of the dark period, and decline again at midnight. Mice kept in complete darkness showed within a 24-h period only a single peak of VGLUT1 expression in the middle of the rest phase. In contrast, mice lacking the period gene Period 2, a core component of the circadian clock, did not show any light-cycle-dependent changes of VGLUT1 levels. No other of several synaptic vesicle proteins examined underwent circadian cycling. Circadian cycling of VGLUT1 was not seen when analyzing homogenate or synaptosomes, the starting fraction for vesicle preparation. Circadian cycling of VGLUT1 was also not reflected at the mRNA level. We conclude that nerve terminals are endowed with mechanisms that regulate quantal size by changing the copy number of transporters in synaptic vesicles. A reduced amount of VGLUT1 per vesicle is probably achieved by means of selective sorting controlled by clock genes.     

Molecular cloning and functional identification of mouse vesicular glutamate transporter 3 and its expression in subsets of novel excitatory neurons

We have cloned and functionally characterized a third isoform of a vesicular glutamate transporter (VGLUT3) expressed on synaptic vesicles that identifies a distinct glutamatergic system in the brain that is partly and selectively promiscuous with cholinergic and serotoninergic transmission. Transport activity was specific for glutamate, was H(+)-dependent, was stimulated by Cl(-) ion, and was inhibited by Rose Bengal and trypan blue. Northern analysis revealed higher mRNA levels in early postnatal development than in adult brain. Restricted patterns of mRNA expression were observed in presumed interneurons in cortex and hippocampus, and projection systems were observed in the lateral and ventrolateral hypothalamic nuclei, limbic system, and brainstem. Double in situ hybridization histochemistry for vesicular acetylcholine transporter identified VGLUT3 neurons in the striatum as cholinergic interneurons, whereas VGLUT3 mRNA and protein were absent from all other cholinergic cell groups. In the brainstem VGLUT3 mRNA was concentrated in mesopontine raphé nuclei. VGLUT3 immunoreactivity was present throughout the brain in a diffuse system of thick and thin beaded varicose fibers much less abundant than, and strictly separated from, VGLUT1 or VGLUT2 synapses. Co-existence of VGLUT3 in VMAT2-positive and tyrosine hydroxylase -negative varicosities only in the cerebral cortex and hippocampus and in subsets of tryptophan hydroxylase-positive cell bodies and processes in differentiating primary raphé neurons in vitro indicates selective and target-specific expression of the glutamatergic/serotoninergic synaptic phenotype.     

Molecular and functional analysis of a novel neuronal vesicular glutamate transporter.

Glutamate is the major excitatory neurotransmitter in the mammalian central nervous system. Packaging and storage of glutamate into glutamatergic neuronal vesicles requires ATP-dependent vesicular glutamate uptake systems, which utilize the electrochemical proton gradient as a driving force. VGLUT1, the first identified vesicular glutamate transporter, is only expressed in a subset of glutamatergic neurons. We report here the molecular cloning and functional characterization of a novel glutamate transporter, VGLUT2, from mouse brain. VGLUT2 has all major functional characteristics of a synaptic vesicle glutamate transporter, including ATP dependence, chloride stimulation, substrate specificity, and substrate affinity. It has 75 and 79% amino acid identity with human and rat VGLUT1, respectively. However, expression patterns of VGLUT2 in brain are different from that of VGLUT1. In addition, VGLUT2 activity is dependent on both membrane potential and pH gradient of the electrochemical proton gradient, whereas VGLUT1 is primarily dependent on only membrane potential. The presence of VGLUT2 in brain regions lacking VGLUT1 suggests that the two isoforms together play an important role in vesicular glutamate transport in glutamatergic neurons.     

Ultrastructure and morphometric parameters of glutamatergic synapses on nigrothalamic and unidentified neurons of the reticular zone of theSubstantia nigra in cats

Nigrothalamic neurons were identified into thesubstantia nigra by their retrograde labelling with horseradish peroxidase. Axon terminals that contain glutamate (the excitatory transmitter) were revealed immunocytochemically with an immunogold electron microscopic technique. Ultrastructural parameters (the large and small diameters of axon terminals, area of their profiles, coefficient of form of profiles, large and small diameters of synaptic vesicles) were analyzed in all 240 synapses under study. Synaptic contacts localized on both nigrothalamic and unidentified neurons belonged to three morphologically specific groups. Synapses of the groups I and III, according to classification by Rinvik and Grofova, were characterized by a symmetric type of synaptic contact and contained polymorphic synaptic vesicles. Contacts in group-II synapses were asymmetric, and respective terminals contained round vesicles. Among the studied synapses, 65.8% were classified as group-I contacts, 25.0% belonged to group II, and 9.2% belonged to group III. Glutamate-positive axon terminals formed predominantly group-II synapses; such connections constituted 70% of this group's synapses. Sixty percent of glutamate-positive synapses were localized on the distal dendrites and 23% on the proximal dendrites, while 17% of such synapses were distributed on the somata of nigral neurons. Such a pattern of distribution of glutamate-positive synapses was observed on both nigrothalamic and unidentified nigral neurons. About 7% of glutamate-positive synapses were formed by very large axon terminals containing round synaptic vesicles; yet, the contacts of these terminals were of a symmetric type. Twenty percent of group-I synapses, i.e., synapses considered inhibitory connections, were found to manifest a weak immune reaction to glutamate.Neirofiziologiya/Neurophysiology, Vol. 28, No. 6, pp. 285–295, November–December, 1996.