GABAergic Nerve Endings Research Articles

Histochemical Characterization of the Vestibular Y-Group in Monkey

The Y-group plays an important role in the generation of upward smooth pursuit eye movements and contributes to the adaptive properties of the vertical vestibulo-ocular reflex. Malfunction of this circuitry may cause eye movement disorders, such as downbeat nystagmus. To characterize the neuron populations in the Y-group, we performed immunostainings for cellular proteins related to firing characteristics and transmitters (calretinin, GABA-related proteins and ion channels) in brainstem sections of macaque monkeys that had received tracer injections into the oculomotor nucleus. Two histochemically different populations of premotor neurons were identified: The calretinin-positive population represents the excitatory projection to contralateral upgaze motoneurons, whereas the GABAergic population represents the inhibitory projection to ipsilateral downgaze motoneurons. Both populations receive a strong supply by GABAergic nerve endings most likely originating from floccular Purkinje cells. All premotor neurons express nonphosphorylated neurofilaments and are ensheathed by strong perineuronal nets. In addition, they contain the voltage-gated potassium channels Kv1.1 and Kv3.1b which suggests biophysical similarities to high-activity premotor neurons of vestibular and oculomotor systems. The premotor neurons of Y-group form a homogenous population with histochemical characteristics compatible with fast-firing projection neurons that can also undergo plasticity and contribute to motor learning as found for the adaptation of the vestibulo-ocular reflex in response to visual-vestibular mismatch stimulation. The histochemical characterization of premotor neurons in the Y-group allows the identification of the homologue cell groups in human, including their transmitter inputs and will serve as basis for correlated anatomical-neuropathological studies of clinical cases with downbeat nystagmus.

GABA is a mediator of brain AT1 and AT2 receptor-mediated blood pressure responses.

The nucleus tractus solitarius (NTS), paraventricular nucleus (PVN), and rostral ventrolateral medulla (RVLM) are the most targeted regions of central blood pressure control studies. Glutamate and gamma-aminobutyric acid (GABA) interact within these brain regions to modulate blood pressure. The brain renin-angiotensin system also participates in central blood pressure control. Angiotensin II increases blood pressure through the stimulation of angiotensin II type 1 (AT1) receptors within the PVN and RVLM and attenuates baroreceptor sensitivity, resulting in elevated blood pressure within the NTS. Angiotensin II type 2 (AT2) receptors in cardiovascular control centers in the brain also appear to be involved in blood pressure control and counteract AT1 receptor-mediated effects. The current review is focused on the interaction of GABA with AT1 and AT2 receptors in the control of blood pressure within the RVLM, PVN and NTS. Within the NTS, GABA is released from local GABAergic interneurons that are stimulated by local AT1 receptors and mediates a hypertensive response. In contrast, the local increase in GABA levels observed after AT2 receptor stimulation within the RVLM, likely from GABAergic nerve endings originating in the caudal ventrolateral medulla, is important in the mediation of the hypotensive response. Preliminary results suggest that the hypertensive response to AT1 receptor stimulation within the RVLM is associated with a reduction in GABA release. The current experimental evidence therefore indicates that GABA is an important mediator of brainstem responses to AT1 and AT2 receptor stimulation and that increased GABA release may play a role in hypertensive and hypotensive responses, depending on the site of action.

Delineation of the Role of Astroglial GABA Transporters in Seizure Control.

Studies of GABA transport in neurons and astrocytes have provided evidence that termination of GABA as neurotransmitter is brought about primarily by active transport into the presynaptic, GABAergic nerve endings. There is, however, a considerable transport capacity in the astrocytes surrounding the synaptic terminals, a transport which may limit the availability of transmitter GABA leading to a higher probability of seizure activity governed by the balance of excitatory and inhibitory neurotransmission. Based on this it was hypothesized that selective inhibition of astrocytic GABA transport might prevent such seizure activity. A series of GABA analogs of restricted conformation were synthesized and in a number of collaborative investigations between Prof. Steve White at the University of Utah and medicinal chemists and pharmacologists at the School of Pharmacy and the University of Copenhagen, Denmark, GABA analogs with exactly this pharmacological property were identified. The most important analogs identified were N-methyl-exo-THPO (N-methyl-3-hydroxy-4-amino-4,5,6,7-tetrahydro-1,2-benzisoxazole) and its lipophilic analog EF-1502 ((RS)-4-[N-[1,1-bis(3-methyl-2-thienyl)but-1-en-4-yl]-N-methylamino]-4,5,6,7-tetrahydrobenzo[d]isoxazol-3-ol) both of which turned out to be potent anticonvulsants in animal models of epilepsy.

Nicotinic and muscarinic cholinergic receptors coexist on GABAergic nerve endings in the mouse striatum and interact in modulating GABA release

Muscarinic cholinergic receptors (mAChRs) and nicotinic cholinergic receptors (nAChRs) regulating GABA release from striatal nerve endings were studied by monitoring release of previously accumulated [ 3H]GABA or endogenous GABA from superfused mouse striatal synaptosomes. Oxotremorine inhibited the release of [ 3H]GABA elicited by depolarization with 4-aminopyridine (4-AP), an effect antagonized by atropine. Agonists at nAChRs, including the α 4β 2 ∗ subunit-selective RJR2403, provoked the release of [ 3H]GABA as well as of the endogenous transmitter; these effects also were prevented by oxotremorine and pilocarpine suggesting coexpression of functional mAChRs and α 4β 2 ∗ nAChRs on GABAergic nerve endings. The inhibitory effects of oxotremorine on the release of [ 3H]GABA evoked by 4-AP or by RJR2403 were: (i) prevented by the M 2/M 4 mAChR antagonist himbacine; (ii) insensitive to the M2 antagonist AFDX116; (iii) blocked by the selective M 4 mAChR antagonists MT3, thus indicating the involvement of receptors of the M 4 subtype. In conclusion, in the corpus striatum, acetylcholine released from cholinergic interneurons can activate α 4β 2 ∗ nAChRs mediating release of GABA; this evoked release can be negatively modulated by M 4 mAChRs coexpressed on the same GABAergic terminals.

Quantitative immunofluorescence data suggest a permanently enhanced GAD 67/GAD 65 ratio in nerve endings in rat cerebral cortex damaged by early postnatal hypoxia-ischemia: a comparison between two computer-assisted procedures for quantification of confocal laser scanning microscopic immunofluorescence images

The aim of the present study was 2-fold: (1) to determine the ratio between the amount of GAD 67 and GAD 65 (two isoforms of the GABA synthetizing enzyme glutamic acid decarboxylase) in nerve endings in the mature rat cerebral cortex damaged by hypoxia-ischemia during early postnatal life; and (2) to compare two different computer-assisted procedures developed for quantitative analysis of immunofluorescence images obtained with a confocal laser scanning microscope (CLSM). One procedure was based on a program present in the standard Leica CLSM software packet for full-field analysis, the other on a specially written program for object-oriented analysis run on a Kontron IBAS-KAT image analysis system. To this end, rat pups were unilaterally exposed to hypoxic-ischemic conditions and, after a survival period of 6.5 months, sacrificed by perfusion fixation. After dissection of the brain and vibratome sectioning, three animals with substantial damage on one cortical side were selected. Sections of these animals were double-stained with primary antibodies against GAD 67 and GAD 65 and fluorophore-conjugated secondary antibodies and subsequently sampled with a CLSM. Analysis of the CLSM images with both computer-assisted procedures showed for all three animals a clear tendency to higher GAD 67/GAD 65 ratios in cortical GABAergic nerve endings on the hypoxia-damaged side than in matched areas on the contralateral side. This outcome led to the following conclusions. (1) The correspondence between the outcome of both analysis procedures indicates that both procedures are valid for quantification of immunofluorescence images of nerve endings obtained with a CLSM. (2) The outcome lends further support to our view that hypoxic-ischemic encephalopathy, sustained during early postnatal life, may result in an unstable cortical network generating abnormal synchronizations and oscillations which can be amplified and propagated as true epileptic discharges. In such a network both excitatory and inhibitory processes are tonically enhanced, the latter probably as a homeostatic reaction tending to keep abnormal excitation within physiological limits.

Permanent increase of the GAD 67/synaptophysin ratio in rat cerebral cortex nerve endings as a result of hypoxic ischemic encephalopathy sustained in early postnatal life: a confocal laser scanning microscopic study

The aim of this study was to investigate whether perinatal hypoxia-ischemia preferentially destroys GABAergic nerve endings in rat cerebral cortex tissue which, in its turn, could then account for the reported higher risk of developing epilepsy later in life. To that end rat pups, with an age of 12–13 days postnatally, were unilaterally to hypoxic-ischemic conditions. After a survival period of to 2 to 6 months, the animals were sacrificed by perfusion fixation and their brains were used for cutting transversal vibratome and frozen sections. These sections were double-stained with primary antibodies against one of the two GABA synthesizing enzymes, glutamic acid decarboxylase with a mol. wt. of 66,600 (GAD 67) and one of the intrinsic membrane proteins synaptic vesicles, synaptophysin, followed by fluorophore-conjugated second antibodies. By using the confocal laser scanning microscope, we determined the ratio between the amount of GAD 67/synaptophysin immunofluorescence in nerve endings per unit volume of tissue in the hypoxia-damaged neocortex. It turned out that this ratio, contrary to expectations, was significantly higher in the hypoxia-damaged cortical areas than in matched areas on the contralateral side. It appeared, moreover, that this effect was directly proportional to the severity of the incurred damage. The conclusion was drawn that these observations do not support the hypothesis that perinatal hypoxia-ischemia ultimately leads to a preferential loss of GABAergic nerve endings in the damaged neocortex and, as such, to a shortage of inhibition.

Evaluation of the mechanisms by which gamma-amino-butyric acid in association with phosphatidylserine exerts an antiepileptic effect in the rat.

The i.p. injection in rats of GABA (740 mg/Kg) after sonication with an equal amount of phosphatidylserine (PS) has an antiepileptic effect. The injection of plain GABA has no such an effect. Blood, brain and synaptosomal accumulation of exogenous labeled GABA under the two circumstances are evaluated. In the case of GABA/PS injection there is a higher passage of the exogenous labeled neurotransmitter into the blood and brain nerve endings (synaptosomes). A higher synaptosomal accumulation of the exogenous labeled neurotransmitter is found even when GABA and PS are injected separately. Since these accumulation increases occur at a time when there is the antiepileptic effect, they seem relevant to it. Our interpretation of the chain of the events resulting in the antiepileptic action is that the phospholipid facilitates from the beginning the first passage of the exogenous neurotransmitter form the peritoneum to the blood. Then a higher passage to the brain tissue and eventually to the GABA-ergic nerve endings ensues. The brisker accumulation of the exogenous neurotransmitter in the nerve endings could be at the basis of a more efficient GABA-ergic inhibitory control in the brain.

Accumulation of labeled gamma-aminobutyric acid into rat brain and brain synaptosomes after i.p. injection.

The accumulation of labeled GABA into brain and brain nerve endings was studied in the adult rat after i.p. injection of large doses of neurotransmitter (740 mg/Kg). In the first 5-30 minutes after the injection the exogenous neurotransmitter reaches a stable plasma level of around 5 mM. The accumulation of radioactive GABA into the brain presents a latency of a few minutes from the time of the injection. Thereafter, the accumulation of the neurotransmitter is almost linear with time. Once in the brain tissue labeled GABA is in part broken down. The exogenous neurotransmitter is taken up in GABA-ergic nerve endings with a steep increase between 20 and 30 minutes after the injection. From a quantitative point of view, the data show that the brain accumulation of labeled GABA at 30 minutes post injection is minimal in the respect of the steady state average concentration of the endogenous neurotransmitter (0.014%). However, the amount of radioactive GABA which accumulates in the nerve endings, at the same post injection time, is around 7% of the endogenous neurotransmitter in that compartment. The data thus show a selective enrichment of exogenous systemic GABA in a physiologically important compartment of the brain.

Relative enrichment of the lighter 59 kDa form of glutamic acid decarboxylase in nerve endings: An immunoblotting study in pituitary neurointermediate lobe

Previous studies established that glutamate decarboxylase (GAD) is present in the brain of higher vertebrates in two forms composed of the homodimeric association of two subunits 59 and 63 kDa, respectively, that were found to be structurally related. We have performed quantitative comparative immunoblotting of whole brain and pituitary neurointermediate lobe (NIL) extracts. While GAD in the brain is present in cell bodies and nerve endings, it is known to be contained in the NIL exclusively in nerve endings that belong to a relatively long γ-aminobutyric acid (GABA)ergic pathway of central origin. The relative immunolabelling ratio of the 59 kDa to the 63 kDa subunit was at least 10-fold higher in the NIL when compared to whole brain extracts. Accordingly we suggest that the GAD composed of the 59 kDa subunits, which appears to be greatly enriched in GABAergic nerve endings, might represent the form of GAD on which short term activity regulation is exerted in relation to neuronal activity. It might result from the post-translational processing of the GAD formed from the 63 kDa subunits during the axonal transport.

Muscarinic receptors mediate direct inhibition of GABA release from rat striatal nerve terminals

The effects of acetylcholine (ACh) on the depolarization-evoked release of [ 3H]γ-aminobutyric acid ([ 3H]GABA) have been investigated using synaptosomes prepared from rat corpus striatum and depolarized by superfusion with 9 mM KCl. Acetylcholine inhibited the [ 3H]GABA overflow in a concentration-dependent manner. The maximal effect was about 50%. The IC 50 value (concentration producing half-maximal effect) amounted to 1 μM, in the absence of acetylcholinesterase inhibitors. The effect of ACh on the K +-evoked [ 3H]GABA release was counteracted by the muscarinic receptor antagonist atropine, but not by the nicotinic receptor antagonist mecamylamine or by the selective M 1 antagonist pirenzepine. The data show that muscarinic receptors with low affinity for pirenzepine are localized on GABAergic nerve endings in rat corpus striatum where they may directly inhibit the release of GABA.

Two different types of dynorphin-A-immunoreactive terminals in rat substantia nigra

The opioid peptide dynorphin A (1-17) is the third transmitter identified in the striatonigral projection, the other two being gamma-aminobutyric acid (GABA) and substance P. The ultrastructural features of the dynorphinergic terminals in substantia nigra/pars reticulata were studied using pre-embedding immunocytochemistry with the classical peroxidase-antiperoxidase-diaminobenzidine-method; these features were compared with GABAergic boutons visualized with an immunogold method. Two distinct types of dynorphin-A-immunoreactive boutons could be identified: (1) type A (81%) possessing characteristics similar to the GABAergic nerve endings in this region, i.e., large pleomorphic vesicles and symmetric synaptic contacts; (2) type B (19%) displaying asymmetric synaptic zones and small, mostly round vesicles. These results are in agreement with physiological studies suggesting a dual action of dynorphin A in substantia nigra.

GABA terminal autoreceptors in the pars compacta and in the pars reticulata of the rat substantia nigra are GABA B

The depolarization-evoked release of γ-aminobutyric acid (GABA) and its modulation mediated by autoreceptors were studied in superfused synaptosomes prepared from the pars compacta and from the pars reticulata of the rat substantia nigra. The release of [ 3H]GABA evoked by 9 mM KCl was almost totally calcium-dependent in both nigral subregions. In the presence of SK&F 89976A (N-(4,4-diphenyl-3-butenyl)nipecotic acid), a GABA uptake inhibitor added to minimize carrier-mediated homoexchange, GABA (0.3–10 μM) inhibited, in a concentration-dependent way, the K +-evoked overflow of [ 3H]GABA from both pars compacta and pars reticulata synaptosomes. Similarly to GABA, (−)-baclofen (0.3–10 μM) reduced the [ 3H]GABA overflow, being roughly equipotent to GABA in both nigral subregions. The (+) enantiomer of baclofen was ineffective. The overflow of [ 3H]GABA was not consistently affected by muscimol in either the pars compacta or the pars reticulata. The effects of GABA were bicuculline- and picrotoxin-insensitive. However, the inhibition by GABA of the [ 3H]GABA overflow was antagonized by phaclofen. It is concluded that (a) GABA autoreceptors are sited on GABAergic nerve endings in both the pars compacta and pars reticulata of the rat substantia nigra; (b) these autoreceptors belong to the GABA B type.

GABA axon terminals in synaptic contact with enkephalin neurons in the hypothalamus of the guinea pig. Demonstration by double immunocytochemistry

By using a method combining pre-embedding immunoperoxidase staining for enkephalin and postembedding immunocolloidal gold labeling for γ-aminobutyric acid (GABA) it has been demonstrated that many GABAergic boutons made synapses on enkephalin-reacting some in the magnocellular dorsal nucleus of the guinea pig hypothalamus. The gold particles revealing the presence of GABA were essentially located over the small clear vesicles and mitochondria present in these GABAergic nerve endings. All the synapses observed were symmetrical. Taking into account the great number of these nerve endings, we conclude for a strong regulatory role of GABA on enkephalin-containing cells of the magnocellular dorsal nucleus.

Interrelationships between ornithine, glutamate and GABA-III. an ornithine aminotransferase activity that is resistant to inactivation by 5-fluoromethylornithine

5-Fluoromethylornithine (5-FMOrn) is a specific inactivator of l-ornithine:2-oxoacid aminotransferase (OAT). However, a certain proportion of the OAT activity in mouse brain, liver and kidney is not inactivated by this compound. In the present work, the occurrence, distribution and subcellular localization of this 5-FMOrn-resistant OAT is reported. It was shown that the 5-FMOrn-resistant brain enzyme is kinetically different from the corresponding liver enzyme, and it also differs from the 5-FMOrn-sensitive OAT. The most conspicuous difference between the 5-FMOrn-resistant OAT of liver and brain is the sensitivity of the latter against excessive concentrations of its substrate 2-oxoglutarate. 5-FMOrn and GABA are reversible inhibitors of the 5-FMOrn-resistant enzyme. Both compounds compete with Orn for the enzymes active site. A number of known inactivators of GABA-T which are at the same time inactivators of OAT, and canaline, a natural inhibitor of OAT, inactivate both the 5-FMOrn-sensitive and the 5-FMOrn-resistant enzyme. Gabaculine is the most potent inhibitor of the 5-FMOrn-resistant enzyme that is presently known. Our results are compatible with the suggestion that the 5-FMOrn-resistant OAT is an isoenzyme. From the fact that this form of OAT prevails in the brain, and its occurrence in the nerve ending fraction of brain homogenates supports the view that 5-FMOrn-resistant OAT may be involved in the intraneuronal generation of neurotransmitter glutamate and/or GABA from Orn as precursor. Further support in favour of this notion are previous findings which suggest feedback inhibition of OAT by GABA in GABAergic nerve endings.

Gaba nerve endings in the rat red nucleus combined detection with serotonin terminals using dual immunocytochemistry

Immunocytochemical methods were used to examine the ultrastructural features and cellular interrelationships of GABA and serotonin afferent fibers to the rat red nucleus. GABAergic nerve endings were identified in two ways, either using a pre-embedding immunoperoxidase procedure with an antibody against glutamate decar☐ylase, the GABA-synthesizing enzyme, or after post-embedding immunogold labelling with an anti-GABA antibody. With the latter approach, it was possible to simultaneously visualize the GABAergic and serotoninergic innervation of the red nucleus (magnocellular part) in electron microscope preparations. This procedure involved GABA labelling of ultrathin sections obtained from specimens previously immunostained for serotonin using the pre-embedding peroxidase-antiperoxidase technique. The doubly stained sections showed gold and peroxidase markers to be present in two distinct populations of axonal varicosities. Unlike the GABAergic nerve endings, which were found to be profusely distributed throughout the nucleus, the serotonin nerve endings were relatively scarce. They contacted dendrites of large-sized neurons usually endowed with several GABA-gold labelled terminals. Not uncommonly, direct appositions between serotonin and GABA-positive terminals were also encountered. These data provide morphological evidence that red nucleus outputs may be dually regulated by GABAergic and serotoninergic afferents, while suggesting that presynaptic GABA/serotonin interactions might also play a significant part in red nucleus fucntions.

Possible occurrence of ornithine-omega-aminotransferase in GABAergic neurons.

The specific precursors for neurotransmitter pools of glutamate giving rise to GABA in GABAergic neurons and nerve endings have not been clearly established. Glutamate is the immediate precursor for the production of GABA and it is suggested that ornithine (from arginine) might be serving as one of the precursors of glutamate for the formation of neurotransmitter pool of GABA. Damage to GABAergic neurons in different regions of the brain in anoxia is well known. If arginine and ornithine act as precursors for GABA in GABAergic neurons, a decrease in the activities of arginase and ornithine-omega-transferase (Orn-T) is possible in areas having the lesions involving the GABAergic neurons due to anoxia. Estimation of Orn-T and arginase in different regions of the brain of rats exposed to anoxia revealed such a possibility.

Synaptic vesicles from hog brain—their isolation and the coupling between synthesis and uptake of γ-aminobutyrate by glutamate decarboxylase

Purified synaptic vesicles were isolated from hog cerebral cortex by a rapid procedure consisting of homogenization of cerebral cortex slices in iso-osmotic sucrose, differential centrifugation and sucrose density-gradient centrifugation. The purity of the vesicles was evaluated both biochemically and morphologically. The vesicles contained high amounts of ?-aminobutyrate (GABA) and acetylcholine at specific concentrations of 390 nmol/mg protein and 7.2 nmol/mg protein respectively. Glutamate decarboxylase, the enzyme which catalyses GABA formation, binds to the synaptic vesicles in a calcium-dependent manner. The percentage of glutamate decarboxylase bound to the vesicles increases from about 5% without calcium, reaching a plateau of about 60% at 4 mM Ca(2+). Magnesium in concentrations 0.2-10 mM has no significant effect on glutamate decarboxylase binding. Also in phospholipid vesicles (small unilamellar phosphatidylserine-phosphatidylcholine. 2:1 liposomes) Ca(2+), but not Mg(2+), induced the binding of glutamate decarboxylase, reaching a plateau of 50% at 2 mM Ca(2+). Both in synaptic vesicles and in phospholipid vesicles the calcium-dependent glutamate decarboxylase binding seems to be specific, and not caused by unspecific association of proteins, since the specific binding (bound enzyme activity/mg bound protein) increases 3-fold from 0 to 4 mM Ca(2+). The functional role of this binding was studied in GAD containing vesicles by measuring the relationship between the accumulation of [(3)H]GABA, newly synthetized from [(3)H]glutamate, and the uptake of added [(14)C]GABA. No significant uptake of [(14)C]GABA was found under the experimental conditions used, whereas large amounts of [(3)H]GABA were found within the vesicles. It appears that the [(3)H]GABA accumulation process is functionally linked to [(3)H]GABA synthesis and is mediated by the membrane-bound glutamate decarboxylase. This synthesis-coupled uptake of GABA into synaptic vesicles possibly serves to bring about a plasticity effect in previously stimulated GABAergic nerve endings.

Autoradiography of GABA in the rat hypothalamic median eminence

Light microscopy autoradiographs of the rat hypothalamic median eminence were prepared after injection of high specific activity tritiated GABA and GABA structural analogs. Following intracardiac injection of labeled GABA with short (15 min) survival time, a dense accumulation of silver grains was observed over the external layer of the median eminence. The silver grains appeared much less numerous and randomly scattered over the internal layer. No conspicuously labeled cells could be detected in the median eminence. A similar pattern of labeling was observed after 10 min in vitro incubation of the median eminence with a low concentration(2.5 × 10 −7M) of labeled GABA. Clusters of silver grains were also visible over the external layer following intraventicular injection of labeled GABA. In this latter case, however, other sites of labeling were revealed over the internal and ependymal layers. The dense labeling over the external layer with tritiated GABA was partially reduced by a simultaneous intracardiac injection of a 50-fold excess of non-radioactive cis-aminocyclohexane car☐ylic acid — a reported preferential substrate for GABA neuronal uptake — but it was not displaced by a 2000-fold excess of non-radioactive β-alanine — a reported specific substrate for GABA glial uptake. Intracardiac injection of triated β-alanine led to a faint and even labeling over the entire median eminence with no preferential accumulation of silver grains over the various layers. Following intraventricular injection of labeled β-alanine the tanycytes and their processes as well as numerous glial cells appeared heavily labeled. These results suggested that there exist cell elements in the external layer of the hypothalamic median eminence which are capable of accumulating exogenous GABA according to its neuronal uptake characteristics. Although the exact nature of these cells is not readily apparent at this stage of our investigations, these findings led us to speculate that there might be a subpopulation of GABAergic nerve endings in the vicinity of the primary plexus capillaries.

Central Actions of Benzodiazepines: General Introduction

After a brief review of the characteristic somatic and psychotropic effects of benzodiazepines evidence is presented which supports a specific facilitatory action of these drugs on GABA ergic synapses within the mammalian central nervous system. Benzodiazepines enhance presynaptic inhibition in the spinal cord and dorsal column nuclei as well as postsynaptic inhibition in dorsal column nuclei, hippocampus, hypothalamus, cerebral cortex, cerebellar cortex, which are all examples of recurrent and collateral inhibition mediated by GABA ergic intrinsic neurones. In addition, the compounds also enhance the inhibitory effect of GABA ergic long projection neurones in the substantia nigra and the lateral vestibular nucleus of Deiters. Several problems remain to be solved, such as the exact site at which benzodiazepines initiate their action (pre-synaptically at GABA ergic nerve endings or postsynaptically at the target cells) and the possible existence of endogenous ligands for the benzodiazepine receptor. Some suspected implications which studies on benzodiazepine binding sites could have for a deeper understanding of the mode of action of these drugs are discussed.

High affinity uptake of GABA in presumed GABA-ergic nerve endings in rat brain

After interruption of the striato-nigral pathway uptake of γ-aminobutyric acid (GABA) in the substantia nigra decreased within 7 days to a constant level 30–40% of the normal. Concomitantly glutamate decar☐ylase (GAD) was reduced to 10%. Hence about two-thirds of the GABA uptake activity in substantia nigra are localised to the alleged GABA-ergic nerve elements originating from corpus striatum. The lesion resistant part of the uptake is probably not localised in cell bodies or large processes, since it was the same in tissue prisms as in whole homogenates and crude nerve ending fractions. It was also not influenced by aminooxyacetic acid, which would argue against a localisation in glia. Whereas GAD was recovered mainly in a ‘heavy’ nerve ending fraction, a large proportion of the GABA uptake was situated in a ‘light’ fraction. After hemisections, GABA uptake was reduced to a similar extent in both fractions. It is suggested that whereas GAD is concentrated in nerve terminals, a significant proportion of the GABA uptake may be localised in preterminal axon branches in the substantia nigra. GABA uptake in the dorsal part of the lateral vesticular nucleus was not reduced by interruption of the Purkinje axons from the cerebellar vermis whereas GAD was reduced by 50%. This indicates that the reuptake mechanism is not concentrated in the Purkinje axon terminals. In the hippocampus neither GABA nor GAD were reduced by lesions of afferent nerve pathways, in accordance with previous results showing that in this region GABA producing neurones are intrinsic. The order of ratios of GABA uptake to particulate GAD activity in different regions was: hippocampus > cerebellar cortex > substantia nigra > dorsal part of lateral vesticular nucleus ≈ nucleus interpositus. The ratio may reflect the degree of specific localisation of the GABA uptake mechanism to the GABA-ergic structures.