GABA Synapses Research Articles

Ultrastructural Relationships Between GABAergic Terminals and Cardiac Vagal Preganglionic Motoneurons and Vagal Afferents in the Cat: A Combined HRP Tracing and Immunogold Labelling Study.

The ultrastructural relationships between gamma-aminobutyric acid-immunoreactive (GABA-ir) neurons and other neurons of the nucleus tractus solitarius (NTS) and motoneurons of the nucleus ambiguus (NA) and dorsal motor vagal nucleus (DMVN), were examined by electron microscopic (EM) immunogold labelling with an anti-GABA antiserum on brain stem sections in which vagal motoneurons and vagal afferent fibres were labelled with horseradish peroxidase (HRP). HRP was applied to the cervical vagus or the cardiac vagal branch of anaesthetized cats. After 24 - 48 h survival, brains were glutaraldehyde-fixed and a stable HRP-tetramethylbenzidine reaction product compatible with EM processing was revealed on 250 microm vibratome sections. Following osmium postfixation, dehydration and resin embedding, GABA-ir was localized on ultrathin sections by an immunogold technique. GABA-ir axon terminals, heavily and specifically labelled with gold particles, were very numerous within NTS, DMVN and NA. All terminals contained small, clear, pleomorphic vesicles and a few also contained larger dense cored vesicles. The density of gold particles over clear vesicles, dense cored vesicles and mitochondria was significantly greater than over the cytoplasm of these terminals. GABA-ir synapses were found on the soma and dendrites of neurons, but rarely on other axon terminals within NTS, where GABA-ir cell bodies and dendrites were also seen. These received synaptic contacts from both GABA-ir terminals and from HRP-labelled vagal afferents. In both the DMVN and NA, similar GABA-ir synapses were present on both the soma and dendrites of HRP-labelled motoneurons. GABA synapses were also present on other cell types in DMVN. These observations provide an anatomical basis for a GABAergic inhibition of neurons forming the central pathways of cardiovascular and other autonomic reflexes.

Effects of Barbiturates on GABA System: Comparison to Alcohol and Benzodiazepines.

Central nervous system depressants, e.g. barbiturates, alcohol and benzodiazepines, have a wide spectrum of activity in humans and animals. Evidence accumulated suggests that some of the pharmacological actions exerted by these agents may be mediated through GABA system by mimicking GABAergic transmission. This proceeding briefly summarizes the evidence presented in our previous review (Yu S and Ho Ik: Alcohol 7: 261, 1990) as to how the GABA system plays a part in the barbiturate actions and the development of tolerance to and physical dependence on barbiturates. The comparisons of the effects of alcohol, barbiturates and benzodiazepines at different steps of GABA synapse are also discussed. Furthermore, the results which have been reported in the literature are inconsistent. This may be due to differences in (a) animal models used; (b) brain regions used; (c) protocols (dose, duration, form and route of administration, etc.) used in treating animals and/or (d) techniques (pharmacological, biochemical, physiological, etc.) used.

Effects of acute barbiturate administration, tolerance and dependence on brain GABA system: Comparison to alcohol and benzodiazepines

Central nervous system depressants, e.g., barbiturates, alcohol and benzodiazepines, have a wide spectrum of activity in humans and animals. Evidence accumulated suggests that some of the pharmacological actions exerted by these agents may be mediated through GABA system by mimicking GABAergic transmission. This review attempts to summarize the evidence available as to how the GABA system plays a part in the barbiturate actions and the development of tolerance to and physical dependence on barbiturates. The comparisons of the effects of alcohol, barbiturates and benzodiazepines at different steps of GABA synapse are also presented. Furthermore, the results which have been reported in the literature are inconsistent. This may be due to differences in: (a) animal models used; (b) brain regions used; (c) protocols (dose, duration, form and route of administration, etc.) used in treating animals and/or (d) techniques (pharmacological, biochemical, physiological, etc.) used.

Quantal analysis of synaptic potentials in neurons of the central nervous system.

This review concentrates on the results of quantal analysis at central synapses, particularly where the morphology and the electrophysiology of the same synaptic connection has been investigated. Results from quantal analysis at peripheral synapses are drawn on to help resolve some issues. The major topics covered in this review are the properties of the quantal synaptic potential and the junctional mechanisms involved in its generation

The gabaergic hypothesis of depression

1. GABAergic mechanisms have been generally ignored in the study of mood disorders and antidepressant drug (AD) action. Recently data have accumulated indicating that GABAergic mechanisms may be involved in both of these. 2. Mood disorders: GABA levels are reported to be low in the CSF and plasma of depressed patients and are related to mood changes. GABA B receptors are decreased in the frontal cortex in two rodent behavioral models of depression and GABA release is reported diminished in the hippocampus. GABAergic drugs (progabide, fengabine) reverse the behavioral deficits in the rodent models and exert clear therapeutic effects in depressed patients. 3. AD action: In behavioral models imipramine upregulates GABA B receptors only in those animals which respond behaviorally to the AD. In naive rats repeated administration of varied ADs upregulates GABA B receptors in the frontal cortex whereas non-ADs (including amphetamine) do not. Bicuculline inhibits the action of imipramine in the learned helplessness model. GABA A receptor stimulation enhances noradrenaline release in the ventral NA pathway. 4. Conclusions: GABAergic mechanisms likely play a role in the modulation of mood and increasing GABAergic tone exerts an antidepressant effect. Actions at GABA synapses appear to be a fundamental facet of ADs, perhaps together with ß-adrenoceptor mediated events.

GABA synapses, depression, and antidepressant drugs.

In the quest for the pathological basis of depression and an understanding of the mechanisms of the clinical action of antidepressant drugs (ADs), multiple hypotheses have been proposed, invoking many central synaptic mechanisms. Of these, the monoaminergic hypotheses have been the most persistent, and propose to link both the etiology of depression and the mechanism of action of ADs. However, during the intense study which these hypotheses have received, several anomalies and paradoxes have come to light which indicate that these monoaminergic theories are insufficient per se to account for the pathogenesis of affective disorders or to provide a common mechanism of AD action.KeywordsFrontal CortexAntidepressant DrugGaba LevelParadoxical SleepLearn Helplessness ModelThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Some factors that influence the decrement in the response to GABA during its continuous iontophoretic application to hippocampal neurons

The response decrement that occurs during continuous iontophoretic application of GABA to hippocampal neurons was characterized by intracellular methods in the rat hippocampal slice. Using several paradigms that compared the responses to GABA with those to poorly transported analogues, we then identified a large component of this decrement that appeared to be independent of GABA uptake and metabolism, and that is probably independent of intracellular chloride accumulation as well. This decrement, which both developed and recovered with half times that average between 3 and 5 s, is too brief to directly account for long-term plasticity of the GABA synapse. However, its time course is appropriate to participation in the development of cellular responses to brief flurries of GABA-mediated inhibitory postsynaptic potentials that may occur normally, or that may occur abnormally during a seizure or artificial tetany.

Synaptic and non-synaptic actions of benzodiazepines on the crayfish sensory neuron

On the stretch-induced discharge activity of the isolated crayfish sensory neuron flurazepam (⩽ 3 × 10 −4 M) and Ro 15-1788 (⩽ 10 −3 M) produced reversible concentration-dependent excitation, but oxazepam only produced depression (⩽ 5 × 10 −4 M). Similar divergent effects on the membrane properties were observed. Oxazepam increased the threshold to firing without changing resting potential, membrane resistance or the GABA-mediated IPSP. In contrast flurazepam and Ro 15-1788 produced a concentration-dependent decrease in threshold. Flurazepam did not alter membrane resistance or resting potential but facilitated GABA transmission. Ro 15-1788 had the opposite effect on the GABA synapse, and also depolarized the resting potential but did not alter membrane resistance. The change in spike threshold appeared to be an important component in producing discharge excitation or depression. These results not only demonstrate the capability of the sensory neuron to discriminate between structures of benzodiazepines, but also that these agents can produce divergent effects on synaptic and non-synaptic properties of a single neuron.

Drug-induced changes in GABA content of nerve endings in 11 rat brain regions. Correlation to pharmacological effects

For determination of in vivo drug effects on nerve terminal GABA, a procedure was used which allowed the simultaneous measurement of GABA in nerve endings (synaptosomes) from 11 regions of one rat brain. Synaptosomal fractions were prepared by conventional subcellular fractionation procedures and characterized by electron microscopy. Postmortem increases of GABA during removal and dissection of brain tissue, homogenization and fractionation procedures could be sufficiently minimized by rapid processing of the tissue at low temperatures and inclusion of 3-mercaptopropionic acid (1 mM) in the homogenizing medium. In vivo experiments with the GABA-elevating drugs aminooxyacetic acid (30 mg/kg i.p.) and valproic acid (200 mg/kg i.p.) showed that both drugs caused differential effects on synaptosomal GABA levels in different brain regions. A comparison of these biochemical effects with anticonvulsant effects of both drugs in different seizure models supports the concept that GABA synapses in midbrain areas are critically involved in the control of seizure propagation.

Inhibitory mechanism in zebrafish optic tectum: Visual response properties of tectal cells altered by picrotoxin and bicuculline

In previous work we described 4 tyoes of visu al response among tectal cells of the zebrafish 16. Cells of one class,type I, have no spontaneous activity, but respond phasically at ON and OFF. Their responses to moving edges, to stimuli that grow in size, and to stimuli equal in size and shape to the whole receptive field (RF) suggest that these cells may receive inhibitory input from near neigbor cells of the same type in the tectum, as well as excitatory input from retinal fibers 17. In order to further investigate this hypothesis we have studied the effects of drugs on physiological properties of type I cells recorded in the stratum periventriculare layer of the zebrafish tectum. Small (10–50 nl) injections of drugs were made in the tectum while recording 100–500 μm away with extracellular microelectrodes. Both picrotoxin and bicuculline produce the following effects: (1) onset of spontaneous bursting multiunit activity. This noise can be recorded at all depths within the tectum; (2) abolition of the second postsynaptic wave of the optic nerve shock field potential and the current source responsible for it, which occurs in the upper tectal layers at 8 ms latency. This probably represents the secondary activation of inhibitory synapses in those layers; (3) alteration of visual response properties of individual type I tectal cells. The duration of response to small flashing spots and to stimuli that grow in size both increase significantly. Responses to moving edges, which normally occur mostly as the edge is crossing the RF border, become extended to encompass the entire RF. Finally, the cells show reduced negative spatial summation following drug injection. All of these effects are fully reversible with time after injection as the drugs wash out. Control injections (of teleost Ringer's solution, 100 mM HCl, 165 mM NaCl, and strychnine 2 mM or 5 mM) do not elicit any of these effects. The results reported here are consistent with the hypothesis that tectal type I cells receive a delayed inhibitory input, probably via GABA synapses, which determines major properties of the visual resonse.

Interaction of stereoisomers of barbiturates with [ 3H]α-dihydropicrotoxinin binding sites

Racemic depressant barbiturates inhibit the binding of gamma-aminobutyric acid antagonist [ 3H]α-dihydropicrotoxinin (DHP) to rat brain membranes with IC 50 values ranging from 5 to 50 μM. The (−) isomers of pentobarbital and secobarbital were three to four-fold more potent than their (+) isomers in inhibiting [ 3H]DHP binding. In contrast, the (+) isomer of hexobarbital was a better inhibitor than (−) hexobarbital. The stereoisomers of 1-methyl-5-phenyl-5-propyl barbituric acid (MPPB), which show opposite pharmacological activity, inhibited [ 3H]DHP binding in a biphasic manner with a plateau at 4–100 nM MPPB. These results suggest heterogeneity of DHP binding sites. The possibility that depressant and convulsant barbiturates may act at the level of DHP site at the GABA synapse is discussed.

The specificity of high affinity binding of avermectin B 1a to mammalian brain

Avermectin B 1a, an anthelmintic with pharmacological properties of paralyzing nematodes and causing an increase in the release of GABA from rat brain synaptosomes, was found to bind to dog brain synaptosomes with an apparent- K D of 1–2 nM. This high affinity binding was saturable and the density of binding sites was estimated to be 1.54 pmol/mg protein. Results from binding competition among various analogs of the drug suggested that the binding is stereospecific and the affinities to the binding sites correlated well with the anthelmintic activities. The binding sites are unevenly distributed in the dog brain with the highest density found in cerebellum, less in midbrain, hypothalamus and cerebral cortex, and little or no binding in pons and medulla oblongata. Similar specific binding of avermectin B 1a to synaptosomes of adult rat brains was also observed. The binding sites apparently emerged during the first three weeks of post-natal development of the rat brain. Although all the evidence supports the hypothesis that the binding sites may be associated with GABA synapses, the lack of competition between GABA and the drug in their bindings suggests that the drug receptors are not associated with GABA post-synaptic receptors.

Picrotoxinin binding sites at the GABA synapse: A target for drug action

α-Dihydropicrotoxinin (DHP), a GABA synaptic antagonist, binds to rat brain and crayfish membranes with an apparent K D of 1–2 μ.M. Based on several lines of evidence, it appears that DHP binds to GABA-receptor-ionophore complex at a site distinct from the GABA receptor sites. A variety of centrally acting depressant and convulsant drugs which affect GABAergic transmission inhibit DHP and not GABA binding. The data suggest that DHP-sensitive site at the GABA receptor ionophores may be involved not only in drug action but in possible regulation of the chloride ionophores.

On the gaba transport in fractions of astrocytes and nerve-terminals

Fractions enriched in astrocytes and nerve-terminals have been isolated from cerebral tissues. The GABA metabolism, the uptake and release of 3H-GABA and its ionic requirements have been studied. The relative activities of γ-aminobutyric acid-α-ketoglutarate transaminase (GABA-T) and glutamic acid decarboxylase (GAD) suggest GABA formation in the nerve terminal and breakdown in the astrocyte. The transport system for 3H-GABA present in the nerve and glia is in our hands undistinguishable, both cells utilizing the membrane potential in combination with the Na +-gradient as the driving force. The low GABA levels in the astrocyte, together with its high membrane-potential, make this cell very suitable to serve a transmitter sink at the GABA synapse. Since 3H-GABA transport in astrocytes seems to be reversible, and since the transport systems in glia and neurons show no characteristic differences, the release of 3H-GABA from synaptosomes has been studied. It is suggested that at least 60–70% of the potassium-evoked release of 3H-GABA in the presence of Ca 2+ is non-vesicular, i.e. does not depend on exogenous Ca 2+.

Picrotoxin binding sites at the gaba synapse: A target for drug action

Picrotoxin binding sites at the gaba synapse: A target for drug action

γ‐AMINOBUTYRIC ACID RECEPTOR BINDING and UPTAKE IN MEMBRANE FRACTIONS OF CRAYFISH MUSCLE

Abstract— The uptake and binding of [3H]GABA and the binding of [3H]muscimol were measured in cell‐free fractions of crayfish muscle. The uptake of GABA was saturable, of high affinity (Km= 0.5μm), and inhibited by low concentrations of compounds believed to block GABA uptake specifically, such as nipecotic acid and 2,4,diaminobutyric acid. The GABA uptake activity was localized to sucrose gradient fractions enriched in sarcolemma as demonstrated by marker enzymes and electron microscopy. The binding of the potent GABAergic agonist muscimol was also localized to the sarcolemma. The binding was saturable, of high affinity (KD= 9 nm), and inhibited by GABA (K1= 125 nm) and by low concentrations of receptor‐specific GABA analogues, such as isoguvacine, imidazole acetic acid, and 3‐aminopropane sulfonic acid. The rank order for inhibition by GABA analogues of [3H]muscimol binding sites correlated very well with activity on GABA synapses in invertebrates, consistent with specific postsynaptic receptor labeling.

Mechanisms of Action of Benzodiazepines

This chapter discusses the mechanisms of action of benzodiazepines. “Mechanisms” is used in the plural because the actions of a psychotropic agent may be expressed on multiple levels. The greatest progress in revealing the mechanisms of action of benzodiazepines has been at the biochemical level. Biochemical and pharmacological evidence strongly implicates the γ-aminobutyric acid (GABA) synapse as a major site of action of the benzodiazepines. An action on the GABA synapse may account for alterations in the effects of other neurotransmitters, such as norepinephrine, serotonin, and acetylcholine. One of the most ubiquitous neurotransmitters in brain tissue, GABA is known to impinge upon aminergic and cholinergic neurons throughout the CNS. Benzodiazepines may have direct effects on the activity of neurochemicals other than GABA. Two preliminary reports suggest that alterations in GABA sensitivity do not account for all of the pharmacological activity of diazepam. Most drugs that influence the CNS are known to alter the disposition of more than one neurotransmitter, and there is no evidence to suggest that the benzodiazepines act differently.

Bicuculline and Picrotoxin Blockade of Positive Dorsal Root Potentials

MOUNTING evidence suggests that GABA functions as a transmitter in the pathways mediating presynaptic inhibition in the spinal cord1–10 and cuneate nucleus11–14. In spite of recent attempts to locate a GABA synapse in these multi-synaptic pathways at the axo-axonic terminal4,13,15, the clarification of GABA's role in this system awaits the elucidation of the complex circuits mediating afferent terminal polarization. This complexity may include such features as tonic activity of interneurones and convergence of phasic inputs from different fibre groups. For example, Mendell16, recording spinal cord dorsal root potentials (DRPs), has observed that stimulation of high threshold afferent fibres from muscle nerve generates a positive going DRP, reflecting primary afferent hyperpolarization. From studies of the interactions between positive and negative DRPs he has postulated that the positive DRP results from a phasic inhibition of tonic activity in the pathway for the negative DRP17. This implies that the axoaxonic synapse is common to the pathways for both the positive and negative DRPs. Thus, if the only GABA synapse in this system is located at the axo-axonic junction, antagonists of GABA should block DRPs of both polarities.

The effect of the GABA antagonists bicuculline and picrotoxin on primary afferent terminal excitability

Summary (1) The effects of the convulsants bicuculline, picrotoxin, and strychnine on the polarization of gastrocnemius Ia primary afferent terminals have been ascertained with the excitability testing technique. Phasic increases in the excitability of these terminals were induced by conditioning stimuli applied to the hamstring nerve. (2) Bicuculline blocked the phasic excitability increases without appreciably altering the tonic state of terminal excitability. (3) Picrotoxin also blocked phasic increases and in addition increased the tonic excitability of the terminal. (4) Convulsant doses of strychnine failed to block phasic excitability increases but in larger doses increased tonic excitability. (5) The capacity of the GABA antagonists bicuculline and picrotoxin to block phasic increases in terminal excitability supports the hypothesis that the presynaptic inhibitory pathway contains a GABA synapse. (6) Although its position remains uncertain, these data can best be explained by locating this synapse at a site removed from the afferent terminal, where it functions to disinhibit tonically active interneurons which depolarize the terminal.