GABAergic Nerve Terminals Research Articles

Enhanced Opioid Efficacy in Opioid Dependence Is Caused by an Altered Signal Transduction Pathway

Chronic morphine administration induces adaptations in neurons resulting in opioid tolerance and dependence. Functional studies have implicated a role for the periaqueductal gray area (PAG) in the expression of many signs of opioid withdrawal, but the cellular mechanisms are not fully understood. This study describes an increased efficacy, rather than tolerance, of opioid agonists at mu-receptors on GABAergic (but not glutamatergic) nerve terminals in PAG after chronic morphine treatment. Opioid withdrawal enhanced the amplitudes of electrically evoked inhibitory synaptic currents mediated by GABAA receptors and increased the frequency of spontaneous miniature GABAergic synaptic currents. These effects were not blocked by 4-aminopyridine or dendrotoxin, although both Kv channel blockers abolish acute opioid presynaptic inhibition of GABA release in PAG. Instead, the withdrawal-induced increases were blocked by protein kinase A inhibitors and occluded by metabolically stable cAMP analogs, which do not prevent acute opioid actions. These findings indicate that opioid dependence induces efficacious coupling of mu-receptors to presynaptic inhibition in GABAergic nerve terminals via adenylyl cyclase- and protein kinase A-dependent processes in PAG. The potential role of these adaptations in expression of withdrawal behavior was supported by inhibition of enhanced GABAergic synaptic transmission by the alpha2 adrenoceptor agonist clonidine. These findings provide a cellular mechanism that is consistent with other studies demonstrating attenuated opioid withdrawal behavior after injections of protein kinase A inhibitors into PAG and suggest a general mechanism whereby opioid withdrawal may enhance synaptic neurotransmission.

Pre-embedding staining for GAD67 versus postembedding staining for GABA as markers for central GABAergic terminals.

Pre-embedding immunocytochemistry for the active form of glutamate decarboxylase (GAD67) and postembedding staining for gamma-aminobutyric acid (GABA) were compared as markers for central GABAergic terminals in the phrenic motor nucleus, in which phrenic motor neurons had been retrogradely labeled with cholera toxin B-horseradish peroxidase. Nerve terminals with or without GAD67 immunoreactivity were identified in one ultrathin section. GABA was localized with immunogold in an adjacent section after etching and bleaching. GABA labeling density was assessed over 519 GAD67-positive and GAD67-negative nerve terminals in the phrenic motor nucleus. Frequency histograms showed that statistically higher densities of gold particles occurred over most GAD67-positive terminals. However, some GAD67-negative terminals also showed high densities of gold particles, and some GAD67-positive terminals showed low densities. Preabsorption of the anti-GABA antibody with a GABA-protein conjugate, but not with other amino acid-protein conjugates, significantly reduced gold labeling over both GAD67-positive and GAD67-negative terminals. These results show that the presence of GAD67 immunoreactivity correlates strongly with high densities of immunogold labeling for GABA in nerve terminals in the phrenic motor nucleus. Preabsorption controls indicate that authentic GABA was localized in the postembedding labeling procedure. Only a small proportion of intensely GABA-immunoreactive terminals lack GAD67, suggesting that both GAD67 and GABA are reliable markers of GABAergic nerve terminals.

Involvement of protein kinase C in gamma-aminobutyric acid release from Xenopus oocytes injected with rat brain mRNA.

Involvement of protein kinase C (PKC) in the release of gamma-aminobutyric acid (GABA) was examined in Xenopus laevis oocytes injected with mRNA from rat cerebellum, as compared with findings in slices of rat cerebellum. The mRNA-injected oocytes preloaded with [3H]GABA showed spontaneous release of [3H]GABA, approximately 0.5% of GABA content per 1 min. Stimulation with either Ca2+ ionophore (A23187) or a high K+ concentration increased the release of [3H]GABA from slices of rat deep cerebellar nucleus and mRNA-injected oocytes but not from noninjected and water-injected oocytes. 12-O-Tetradecanoylphorbol 13-acetate (10-300 nM) but not 4alpha-phorbol 12,13-didecanoate (300 nM) potentiated the A23187-stimulated release of [3H]GABA from slices and from mRNA-injected oocytes, in a concentration-dependent manner. Thus, machinery associated with release processes of GABA can be expressed in oocytes by injecting rat cerebellar mRNA, and PKC participates in GABA release from the functionally expressed GABAergic nerve terminals.

P-872 - Expression of the functional GABAergic nerve terminals and GABAB recepter in Xenopus oocytes

P-872 - Expression of the functional GABAergic nerve terminals and GABAB recepter in Xenopus oocytes

Clonidine differentially modulates the release of endogenous gaba in various rat brain areas

The effects of clonidine on the release of endogenous γ-aminobutyric acid (GABA) have been studied in superfused synaptosomes prepared from rat hypothalamus, nucleus tractus solitarii (NTS) and cerebellum. Clonidine enhanced in a concentration-dependent manner the basal release of GABA from hypothalamus and NTS synaptosomes. In contrast, the imidazoline did not affect the release of the amino acid from the cerebellar preparation. Theα 2-adrenoceptor antagonist yohimbine prevented the releasing effect of clonidine, while theα 1-adrenoceptor antagonist prazosin was ineffective. The results show that the release of GABA from hypothalamus or NTS nerve endings can be enhanced by clonidine through the activation of adrenoceptors of the α 2 subtype. The GABAergic nerve terminals of cerebellum do not seem to possess presynaptic adrenoceptors by which clonidine can regulate the basal outflow of the amino acid. The results suggest that some of the clonidine effects may occur through activation of the GABA system.

A case of adult onset pure pallidal degeneration: II. Analysis of neurotransmitter markers, with special reference to the termination of pallidothalamic tract in human brain

We analyzed neurotransmitter markers in a brain of a very rare case of pathologically confirmed adult-onset pure pallidal degeneration (PPD) as compared with 16 controls. Neurotransmitter concentrations are significantly altered in the globus pallidus (GP), subthalamic nucleus (ST) and the thalamic nuclei. Concentrations of gamma-aminobutyric acid (GABA) in the external segment (GPe) and internal segment (GPi) of GP and ST are decreased to 62, 45 and 55% of the control mean, respectively. Concentrations of glutamic acid are increased in GPi (144%) and ST (134%). Choline acetyltransferase (ChAT) activities are increased in GPe (232%), CPi (218%), ST (161%), and ventroanterior (VA, 210%) and ventrolateral nucleus (VL, 193%) of the thalamus. Noradrenaline (NA) concentrations in GPe and GPi are 56 and 43% of the control mean, respectively. Dopaminergic and serotonergic systems show no remarkable change. The grid microdissection analysis demonstrates a patchy GABA distribution in the thalamus of 3 controls, whereas a small GABA-rich area in the ventro-oral nucleus (VO) according to the atlas of Hopf disappears in adult onset PPD. These results strongly suggest that (1) GP GABAergic neurons are selectively degenerated and striatopallidal GABAergic nerve terminals are hypoactive; (2) ChAT activities in GP, ST, VA and VL are increased; (3) the subthalamopallidal glutamatergic system is not hypoactive; (4) activity of the noradrenergic system in GP is decreased; and that (5) VO in the thalamus specifically receives GABAergic nerve terminals from GP in human brain.

Autoimmune myastenia gravis with thymoma following the spontaneous remission of stiff-man syndrome

A patient who developed generalized autoimmune myasthenia gravis six years after the spontaneous remission of a stiff-man syndrome is described. He also suffered from chronic active hepatitis and had radiological evidence of a thymoma. He did not have diabetes mellitus. Besides anti-nicotinic acetylcholine receptor antibodies, anti-nuclear, anti-DNA, anti-mitochondrial and anti-skeletal muscle antibodies were found in his serum, while islet-cell antibodies were absent. Immunocytochemistry studies failed to demonstrate autoantibodies to GABA-ergic nerve terminals, although an aspecific neuronal immunostaining was observed. The clinical and immunological features of this case support the hypothesis of a dysimmune pathogenesis of SMS, also in cases not associated with autoimmunity to GABA-ergic nerve terminals. Furthermore, a relationship between thymoma and the neurological syndromes discussed could be considered.

γ-Aminobutyric acid inhibits the release of α-melanocyte-stimulating hormone from rat hypothalamic slices

The effect of γ-aminobutyric acid (GABA) on release of α-melanocyte-stimulating hormone (α-MSH) from hypothalamic neurons was investigated in vitro using the perifusion technique. Rat hypothalamic slices were continuously superfused with Krebs-Ringer medium and the release of α-MSH in the effluent perifusate was monitored by means of a sensitive and specific radioimmunoassay method. Infusion of 50 mM K + for 15 min induced a transient increase of α-MSH release (5- to 8-fold above the spontaneous level). Infusion of the same dose of K + for 75 min caused a brief discharge of α-MSH during the first 30 min followed by sustained release of the neuropeptide. The effect of GABA was investigated 27 min after the onset of KCl infusion. Application of GABA (5 × 10 −5 M) resulted in a significant and reversible inhibition of K +-induced α-MSH release. The GABA A agonist, muscimol (10 −4M), produced a prolonged inhibition of K + -evoked α-MSH release, while the GABA B agonist, baclofen (10 −4 M), was devoid of effect on hypothalamic α-MSH release. Bicuculline (10 −4 M), a specific GABA A antagonist, had no effect when added alone to the medium but totally reversed the inhibitory effect of GABA on K +-induced α-MSH release. Taken together these data suggest that exogenous GABA exerts an inhibitory control on α-MSH neurons. Our data also show that the effect of GABA on α-MSH release by hypothalamic neurons is mediated through GABA A-type receptors. To investigate further the involvement of GABA neuronal systems in the regulation of α-MSH release, rats were treated with the GABA transaminase inhibitor AOAA and the α-MSH content was assessed in both the intermediate lobe of the pituitary and in the hypothalamus. Three hours after intraperitoneal injection of aminooxyacetic acid hemihydrochloride (AOAA; 30 mg/kg), a significant increase of α-MSH concentration was observed in pars intermedia and hypothalamic extracts. These results, together with the presence of an abundant network of GABAergic nerve terminals in the basal hypothalamus, support the notion that the neurotransmitter GABA may play a physiological role in the regulation of α-MSH release by hypothalamic neurons.

GABA‐immunoreactive neurons and terminals in the lateral cervical nucleus of the cynomolgus monkey

An antiserum against the inhibitory transmitter substance gamma-aminobutyric acid (GABA) was used to investigate the distribution of GABAergic nerve terminals and cell bodies in the lateral cervical nucleus (LCN) of the cynomolgus monkey. Light microscopic immunohistochemistry demonstrated GABA-immunoreactive puncta, suggestive of nerve terminals, scattered throughout the LCN. The terminal-like profiles are often present along the somata of unlabeled neurons, but most are located in the neuropil. GABA-immunoreactive neurons are present in the LCN, but constitute a very small number of the LCN neurons. Electron microscopy showed that the GABA-positive neurons are small with a relatively large nucleus. They are contacted by few somatic boutons. Numerous GABA-immunoreactive terminals containing densely packed round to oval synaptic vesicles were also found. Most GABA-positive terminals make synaptic contact with dendrites, but synapses with cell bodies are also present. Synaptic contacts between labeled and unlabeled terminals were not observed. Some GABA-positive terminals make contact with GABA-positive neurons. The present findings suggest that GABA is a major inhibitory transmitter substance in the LCN of the monkey. However, in comparison with other somatosensory relay nuclei, there are few GABA-immunoreactive neurons in the LCN. This may imply that the GABA-positive neurons branch extensively in the LCN or that an extrinsic source of GABAergic input exists.

The rat inferior olive as seen with immunostaining for glutamate decarboxylase

Boutons presumed to use gamma-aminobutyric acid as neurotransmitter (GABAergic boutons) were detected by glutamate decarboxylase (GAD) immunocytochemistry in all regions of the rat inferior olive. The remarkably high concentration of these boutons allowed a clear visualization of olivary subnuclei boundaries. Regional variations in GAD immunostaining intensity were observed within the nuclear complex and were graded both visually and photometrically. The regional staining variations, for the most part, followed subnuclei boundaries and olivary zonal compartments that have been delineated by the topography of climbing fibre projections. Some subnuclei were grouped by similar staining intensities. The beta nucleus and a medial region in the ventral fold of the dorsal accessory olive were most intensely immunostained, followed by the subnucleus c of the medial accessory olive. Lower staining intensities were observed in the dorsomedial cell column, the dorsal fold of the dorsal accessory olive and the dorsal cap. The lowest intensities were observed in the subnuclei a and b of the medial accessory olive, the ventrolateral outgrowth, the rostral lamella of the medial accessory olive, the principal olive, and the lateral part of the ventral fold of the dorsal accessory olive. The factors contributing to the variations in immunostaining intensity (bouton size and frequency of occurrence) were investigated. The largest boutons were observed in the beta nucleus. Intermediate sized boutons were observed in the dorsomedial cell column, dorsal cap and the dorsal fold of the dorsal accessory olive. The smallest boutons were present in the remaining regions of the inferior olive, including the principal olive, the rostral lamella of the medial accessory olive, and the ventral fold of the dorsal accessory olive. The medial region of the dorsal accessory olive ventral fold contained a higher density of GABAergic boutons than other regions. GABAergic bouton size and innervation density therefore largely accounted for the variations in GAD immunostaining intensity. This study provides a map of the rat inferior olive based on the distribution of GABAergic nerve terminals, and may serve as a basis for characterizing different GABAergic afferent systems in the inferior olive.

Phosphatidylserine increases in vivo the synaptosomal uptake of exogenous GABA in rats

A sonicated liposome suspension of γ-aminobutyric acid (GABA) and phosphatidylserine (liposome-entrapped GABA), intraperitoneally administered in rats, inhibited EEG epileptic activity induced by penicillin, whereas GABA did not. A significant increase (20.4%) in brain radioactivity accumulation occurred at 5 min after i.p. administration of [ 14C]GABA associated with phosphatidylserine in comparison with the administration of [ 14C]GABA; such an increase persisted after 20 min. However, the accumulation of radioactivity into brain synaptosomes demonstrated a 24.1% increase at 5 min and subsequently showed a 43.3% increase at 20 min after injection of liposome-entrapped GABA. The above findings suggest that phosphatidylserine stimulates exogenous GABA uptake into brain GABAergic nerve terminals.

GABAergic nerve terminals in rat hippocampus possess α2-adrenoceptors regulating GABA release

Noradrenaline enhanced in a concentration-dependent way the basal release of endogenous GABA from superfused rat hippocampus synaptosomes. The α2-adrenoceptor antagonist yohimbine prevented the releasing effect of noradrenaline while the α1-adrenoceptor antagonist prazosin was ineffective. It is concluded that GABAergic nerve terminals in rat hippocampus possess adrenoceptors of the α2-subtype whose activation causes enhancement of GABA release.

Structure and Location of a Gabaa Receptor Complex in the Central Nervous System

GABA-gated chloride channels in the central nervous system contain a regulatory site, the benzodiazepine receptor, through which drugs can modulate the efficiency of GABAergic synaptic transmission and thereby affect the degree of anxiety, muscle tension, vigilance and convulsions. The biochemical analysis of the purified receptor complex with monoclonal antibodies shows a heterooligomeric composition of two glycosylated subunits (alpha, beta). The immunoprecipitated complex contains the binding sites for GABA, benzodiazepines and the convulsant TBPS. The receptor complex was located, immunocytochemically, in synapses of brain regions rich in GABAergic nerve terminals.

Presynaptic muscarinic and alpha-adrenoceptor-mediated regulation of GABA release from myenteric neurones of the guinea-pig small intestine.

The effects of cholinomimetic and sympathomimetic drugs on the release of [3H]-gamma-aminobutyric acid ([3H]-GABA) evoked by high K+ from the isolated small intestine of the guinea-pig were investigated, in the presence of tetrodotoxin. Acetylcholine and oxotremorine, at concentrations ranging from 10(-9) to 10(-6) M inhibited the evoked release of [3H]-GABA in a concentration-dependent manner, while nicotine was without effect. Scopolamine and pirenzepine inhibited the effect of oxotremorine, while hexamethonium had no effect. The IC50 values for scopolamine and pirenzepine of the oxotremorine (3 X 10(-8) M)-induced inhibition were 1.02 X 10(-9) M and 9.78 X 10(-10) M, respectively. Noradrenaline, but not isoprenaline inhibited the evoked release of [3H]-GABA. Clonidine (10(-10)-10(-6) M) reduced the evoked release of [3H]-GABA in a concentration-dependent manner, but phenylephrine had no effect. The inhibitory effect of clonidine was antagonized by yohimbine but not by prazosin. These findings provide evidence for the localization of M1-muscarinic and alpha 2-adrenoceptors on GABAergic nerve terminals and their involvement in the presynaptic control of the release of GABA from the guinea-pig small intestine.

A calculation method for evaluating the time course of GABA removal from a synaptic cleft by presynaptic uptake systems

A calculation method for evaluating the time course of γ-aminobutyric acid (GABA) removal from a synaptic cleft by presynaptic uptake is suggested. The evaluation of the actual time required to remove GABA requires the knowledge of: (a) K M's and V max's (mol/min/mg protein) of the synaptosomal uptake systems in a certain brain area; (b) the synaptosomal volume per mg of protein in the synaptosomal preparation used; (c) the mean sphere diameter for synaptic boutons in the brain area considered and the proportion of GABAergic nerve terminals.

Evidence for specific immunolysis of nerve terminals using antisera against choline acetyltransferase, glutamate decarboxylase and tyrosine hydroxylase

Synaptosomes prepared from striatum or cerebral cortex of rat brain were incubated with antibodies raised against three neurotransmitter biosynthetic enzymes, choline acetyltransferase, glutamate decarboxylase and tyrosine hydroxylase in the presence or absence of complement. Immunolysis was first assessed by measuring the release of lactic dehydrogenase or reduction in potassium from synaptosomes, and lysis of neurochemically specific subpopulation of synaptosomes was detected by measuring release of either transmitters, their biosynthetic enzymes or by blockade of sodium-dependent uptake of transmitter or precursor. In both striatum and cortex, antibodies to choline acetyltransferase lysed only cholinergic while those against glutamate decarboxylase only lysed GABAergic nerve terminals. Antibodies against tyrosine hydroxylase lysed only the dopaminergic terminals in striatum but not noradrenergic terminals in cortex. The lysis occurred only in the presence of complement, and was never observed in the absence of complement. The studies indicate that antibodies to the neurotransmitter biosynthetic enzymes recognize antigens in the synaptosomal membrane specific only to neurons harboring the transmitters. The results suggest that the antibody-positive peptides in the membrane and neurotransmitter biosynthetic enzyme share common antigenic sites, probably common peptides.

GABA efflux from synaptosomes: effects of membrane potential, and external GABA and cations.

Presynaptic GABAergic nerve terminals accumulate gamma-aminobutyric acid (GABA) by a sodium-dependent carrier mechanism in which two Na+ are co-transported with one GABA. We have examined the influence of external GABA and cations on GABA efflux from 3H-GABA loaded rat brain synaptosomes, to determine whether or not the carriers can also mediate GABA efflux. We observed that, in Ca-free media (to minimize Ca-dependent, evoked release), external GABA promotes GABA efflux when the medium contains Na+, but inhibits GABA efflux in the absence of Na+. The efflux of GABA into Ca-free media is stimulated by depolarization (either with veratridine or increased external K+). These data, and published data on the internal Na+ dependence of GABA efflux into Ca-free media, indicate that exiting GABA is cotransported with Na+. The stimulatory effect of depolarization is consistent with efflux of Na+ along with the uncharged GABA. The (carrier-mediated) efflux is also stimulated when the carriers cycle inward with Na+ + GABA. The inhibitory effect of GABA in Na+-free media implies that GABA can bind to unloaded carriers and that the carriers loaded only with GABA cycle very slowly, if at all. Our data, and data from the literature, can be fitted to a simple model with sequential binding of solutes: external GABA binds to the carrier first, and only the free or fully-loaded (with 2Na+ + 1GABA) carriers can cycle. Other binding sequences and random binding, do not fit the experimental observations.

Metabolism and brain uptake of gamma-aminobutyric acid in galactosamine-induced hepatic encephalopathy in rats.

Kinetic studies of [3H]gamma-aminobutyric acid ([3H]GABA) after an intravenous injection were performed in normal rats and in rats with severe degree of hepatic encephalopathy due to fulminant hepatic failure induced by galactosamine. Moreover, plasma and brain GABA levels, and GABA and glutamic acid decarboxylase activity were studied in some brain areas. After intravenous injection, [3H]GABA disappeared very rapidly in the blood of normal rats, with a prompt increase of 3H metabolites. In comatose rats, a delayed disappearance of [3H]GABA was parallelled by a lower amount of metabolites, indirectly indicating a peripheral decrease of GABA-transaminase activity. The amount of [3H]GABA in brain was lightly but constantly lower in comatose rats than in controls, indicating that the change in permeability of the blood-brain barrier in hepatic encephalopathy does not affect the [3H]GABA uptake of the brain. Furthermore, the assay of endogenous GABA in blood, whole brain, and brain areas did not show any significant difference in any of the two groups. The finding that glutamic acid decarboxylase activity in brain was reduced, together with the indirect evidence of a reduction in GABA-transaminase, may account for the steady state of GABA in hepatic encephalopathy. However, the reduction in glutamic acid decarboxylase activity is in favor of a functional derangement at the GABA-ergic nerve terminals in this pathological condition.

Increased glutamate decarboxylase activity in the red nucleus of the adult cat after cerebellar lesions

Glutamate decarboxylase (GAD)activity, a marker for GABAergic structures, was studied in the cat red nucleus. GAD is more concentrated in the rostral than in the caudal third of the structure. GAD levels were measured after chronic unilateral lesions of the cerebellum. Destruction of the dentate area and of the nucleus interpositus induced increases of GAD in the contralateral but not in the ipsilateral red nucleus. Similar changes also occurred in the denervated nucleus ventralis lateralis (VL) and nucleus ventralis anterior (VA) of the thalamus. Results show that loss of the excitatory cerebellar input could lead to changes in inhibitory GABAergic nerve terminals. This increase may be induced transsynaptically within existing neurons or, more likely, additional GAD-containing nerve terminals may be formed by axonal sprouting.

Inhibition by apomorphine of the potassium-evoked release of [3H]-gamma-aminobutyric acid from the rat substantia nigra in vitro.

1 The spontaneous and potassium-evoked release of tritium from the rat substantia nigra prelabelled with [(3)H]-gamma-aminobutyric acid [(3)H]-GABA were assessed in vitro under conditions of superfusion.2 Kainic acid lesions performed in the right caudate nucleus resulted in a 70% reduction in the ability of the homolateral nigral cells to take up and retain [(3)H]-GABA when compared with the unlesioned side. The potassium-evoked release of [(3)H]-GABA remained proportional to the radioactivity retained in the tissue suggesting that the nigral GABA neurones that survived kainic acid treatment were still functional.3 The spontaneous outflow of [(3)H]-GABA was significantly increased by exposure to different concentrations of exogenous GABA (10 to 1000 muM) when amino-oxyacetic acid was present in the incubation medium.4 Apomorphine in concentrations ranging from 1 to 30 muM inhibited the calcium-dependent release of [(3)H]-GABA induced by 1 min exposure to 30 mM K(+). These concentrations of apomorphine did not affect the spontaneous outflow of radioactivity. In vivo administration of haloperidol 0.2 mg/kg antagonized the in vitro inhibition by apomorphine of the K(+)-evoked release of [(3)H]-GABA.5 The results obtained with apomorphine and haloperidol suggest the presence of presynaptic dopamine-like inhibitory receptors in gabaergic nerve terminals.6 Dopamine in concentrations ranging up to 300 muM did not modify either the spontaneous or the K(+)-evoked release of [(3)H]-GABA from the substantia nigra. These concentrations of dopamine effectively displaced [(3)H]-dopamine recently taken up into the substantia nigra.7 Our results do not support the view that dendritic release of dopamine from the substantia nigra might be involved in the physiological modulation of the spontaneous or the stimulation-evoked release of GABA.