Presynaptic Inhibition in Cuneate blocked by GABA Antagonists

BICUCULLINE has been shown to have an action essentially similar to Picrotoxin in antagonizing both synaptically evoked postsynaptic inhibition and the depressant action of γ-amino-butyric acid (GABA) on cuneate neurones¹. This supports the hypothesis that GABA is the postsynaptic inhibitory transmitter in the cuneate². However, evidence³ indicates that GABA has a dual action in the cuneate, not only depressing the excitability of postsynaptic neurones, but also increasing the excitability of primary afferent terminals in a manner which might be expected of a presynaptic inhibitory transmitter. The experiments reported here show that the alkaloids bicuculline and picrotoxin block presynaptic inhibition and that this action is consistent with them exerting a GABA-antagonist action at primary afferent terminals.     

Gamma-aminobutyric acid, bicuculline, and post-synaptic binding sites

The binding of γ-aminobutyric acid (GABA) to synaptosomal fractions of the rat cerebellar cortex has been examined at 0–4°C in the presence and absence of bicuculline, chlorpromazine, and/or Na⁺. A GABA-binding component has been demonstrated in the synaptosomal fraction which is competitively inhibited by bicuculline. In addition, this binding component persists in the absence of Na⁺ and in the presence of chlorpromazine. It seems likely that this binding component is the post-synaptic binding site or “receptor” of GABA.     

GABA evoked ACh release from isolated guinea pig ileum

To identify the target cells of GABAergic neurons located in the myenteric plexus, the action of γ-aminobutyric acid (GABA) on the release of acetylcholine (ACh) and on the contractions was studied using the isolated guinea pig ileum. GABA evoked a release of ³H-ACh from the contracting ileum, under conditions of loading with ³H-choline. As both the GABA-evoked release of ³H-ACh and the contractions were inhibited by bicuculline, tetrodotoxin and furosemide, but not by hexamethonium, this release seems to be evoked through GABA receptors which are bicuculline sensitive and associated with the Cl- ion channel.     

Actions of avermectin B1a on the γ-aminobutyric AcidA receptor and chloride channels in rat brain

The interaction of avermectin B_1a (AVM) with the γ-aminobutyric acid (GABA) receptor of rat brain was studied using radioactive ligand binding and tracer ion flux assays. Avermectin potentiated the binding of [³H]flunitrazepam and inhibited the binding of both [³H]muscimol and [³⁵S]t-butylbicyclo-phosphorothionate to the GABA_A receptor. Inhibition of muscimol binding by AVM suggested competitive displacement. Two kinds of ³⁶chloride (Cl) flux were studied. The ³⁶Cl efflux from preloaded microsacs was potentiated by AVM and was highly inhibited by the Cl-channel blocker 4,4′-diisothiocyano-2,2′-stilbenedisulfonic acid (DIDS). However, it was not potentiated by GABA nor was it sensitive to the convulsants picrotoxin or bicuculline. On the other hand, ³⁶Cl-influx measurement in a different microsac preparation of rat brain was very sensitive to GABA and other GABA-ergic drugs. Avermectin induced ³⁶Cl influx into these microsacs in a dose–dependent manner, but to only 35% of the maximal influx induced by GABA. The AVM-induced ³⁶Cl influx was totally blocked by bicuculline. It is suggested that AVM opens the GABA_A-receptor Cl channel by binding to the GABA recognition site and acting as a partial receptor agonist, and also opens a voltage–dependent Cl channel which is totally insensitive to GABA but is very sensitive to DIDS.     

Soluble benzodiazepine receptors: Gabaergic regulation

Benzodiazepine receptor binding has been measured in soluble brain extracts with ³H-flunitrazepam as a ligand. Binding to soluble receptors is enhanced by GABAergic agonists with potencies and maximal augmentation essentially the same as on membrane bound benzodiazepine receptors. The GABA induced increase of binding to soluble receptors is reversed by the GABA antagonist bicuculline.     

Electrophysiological studies of fetal mouse olfactory bulb explants during development of synaptic functions in culture

Explants of 18- or 19-day fetal mouse olfactory bulb have been maintained in culture for periods up to 5 weeks. Compound action potentials can be evoked in the bulb explants by 1 day in vitro, and by 3–4 days, synaptically mediated slow wave discharges can be demonstrated in bicuculline (10^?5 M). The capability of the bulb explants to generate these slow-wave discharges has also been revealed by the introduction of picrotoxin (10^?5 M), d-tubocurarine (10^?4 M) and chloride-free medium, but not of strychnine (up to 3 × 10^?5 M). The data indicate early functional development of inhibitory, as well as excitatory, synaptic systems. In addition, the selective and reversible depression of these slow wave potentials by GABA (1–5 × 10^?4 M), but not by glycine (up to 3 × 10^?3 M), indicates a GABA-ergic component in the inhibitory network. Single unit extracellular recordings have been obtained from the presumptive mitral cells which, in culture, are spontaneously active even as early as 1–2 days after explantation. Correlative Bodian silver-impregnations demonstrate the presence of neurons in these explants which resemble typical mitral cells. Studies of mitral cells using paired stimuli suggest the development in vitro of an inhibitory system analogous to that known to suppress the excitability of their in situ counterparts following orthodromic or antidromic activation. These data, as well as the pharmacological sensitivities of the mitral cells in culture to GABA (5 × 10^?4 M) and bicuculline (10^?5 M), indicate that granule-to-mitral synapses may develop characteristic functions in olfactory bulb explants.     

Molecular Conformation of GABA

CURTIS et al.^1,2 have described evidence suggesting that the alkaloid bicuculline is a specific antagonist to the action of γ-aminobutyric acid (GABA), a possible inhibitory transmitter in the mammalian brain^3,4. By considering structural models, they suggested³ as a stereochemical basis for this antagonism a geometrical configuration in which the N and Ol=C1—O2 of GABA is isosteric with the N and C2—Cl—Ol in bicuculline (Fig. 1a and b). An alternative configuration of GABA, which would give a greater degree of stereochemical similarity to bicuculline, is depicted in Fig. 1c. In both molecules the N is now seen to be isosterically related to Ol=Cl—O2 and located at an angle of about 40° to the plane of the O=Cl—O group viewed towards C2 (Fig. 2). In contrast to Curtis's suggestion, there is now not only geometrical congruence but also identical matching of atomic species.     

Effect of inhibitory amino acid antagonists on postsynaptic potentials of motoneurons of the frog Rana ridibunda

On preparations of the isolated spinal cord of the frog Rana ridibunda at intracellular recording from lumbar motoneurons, it is shown that response to the 10 mM GABA application decreased selectively by 40.7 ± 23.7% (n = 6) as a result of the spinal cord treatment with bicuculline (100–150 μM), while response to the Gly application decreased selectively by 50.7 ± 17.8% (n = 10) after the spinal cord treatment with strychnine (5–10 μM). Both strychnine and bicuculline produced potentiation of EPSP by amplitude and duration as well as paroxysmal depolarizational shifts (PDS). Strychnine produced more effectively the potentiation, while bicuculline—PDS. The inhibitory Gly effect decreased significantly the DR and RF EPSP (a decrease of amplitude and duration) as a result of the spinal cord treatment with strychnine (5–10 μM), but not with bicuculline. The inhibitory GABA effect on the DR and RF EPSP decreased as a result of the spinal cord treatment with bicuculline only in a half of the studied motoneurons and to the lesser degree than the inhibitory Gly effect on the same EPSP at the strychnine treatment. Based of the obtained data, it is suggested that the inhibitory effects on the excitatory inputs of the motoneuron in the frog are expressed weaker than in mammals and are related predominantly to the GABA and Gly effects on receptors of interneurons. It is suggested that GABA specifically acts mostly on GABA_A receptors, whereas Gly—on Gly receptors, although there is some part of cross-inhibition. Original Russian Text ? G. G. Kurchavyi, N. I. Kalinina, and N. P. Vesselkin, 2006, published in Zhurnal Evolyutsionnoi Biokhimii i Fiziologii, 2006, Vol. 42, No. 5, pp. 463–471. The present work is a continuation of the work [1].     

The binding of 3H-Isoguvacine to mouse brain synaptic membranes

³H-Isoguvacine, a gamma aminobutyric acid (GABA) agonist, has been shown to bind to a mouse forebrain synaptic membrane preparation. The specific binding is displaceable by GABA, muscimol and bicuculline but not by picrotoxin or diaminobutyric acid. Kinetic data suggest two binding affinities. Highest levels of binding are observed in the cerebellum, cortex and hippocampus. It is suggested that isoguvacine binds to GABA binding sites and therefore represents a new ligand for measuring GABA receptor binding.     

Effect of baclofen on in vitro noradrenaline release from rat hippocampus and cerebellum: an action at an α2-adrenoceptor

The release of [³H]noradrenaline from rat hippocampal synaptosomes by 25 mM K⁺ and 5 μM veratridine, but not by the Ca²⁺ ionophore A23187 was depressed by baclofen. This depression was reversed by 8-Bromo-cAMP. This action of baclofen was stereospecific and mimicked both that of GABA in the presence of bicuculline and that of clonidine. The α₂-adrenoceptor antagonists yohimbine and Wy25309 antagonised the action of clonidine and baclofen but not that of GABA. Specific binding of [³H]clonidine was displaced by Wy25309 and baclofen, but not by GABA. Specific binding of [³H]GABA in the presence of Ca²⁺ was displaced by baclofen but not by Wy25309. It is concluded that baclofen is not a specific agonist at GABA_B receptors in the brain.     

Decreased GABA<Subscript>A</Subscript> Receptor Function in the Brain Stem during Pancreatic Regeneration in Rats

Gamma amino butyric acid is a major inhibitory neurotransmitter in the central nervous system. In the present study we have investigated the alteration of GABA receptors in the brain stem of rats during pancreatic regeneration. Three groups of rats were used for the study: sham operated, 72 h and 7 days partially pancreatectomised. GABA was quantified by [³H]GABA receptor displacement method. GABA receptor kinetic parameters were studied by using the binding of [³H]GABA as ligand to the Triton X-100 treated membranes and displacement with unlabelled GABA. GABA_A receptor activity was studied by using the [³H]bicuculline and displacement with unlabelled bicuculline. GABA content significantly decreased (P < 0.001) in the brain stem during the regeneration of pancreas. The high affinity GABA receptor binding showed a significant decrease in B _max (P < 0.01) and K _d (P < 0.05) in 72 h and 7 days after partial pancreatectomy. [³H]bicuculline binding showed a significant decrease in B _max and K _d (P < 0.001) in 72 h pancreatectomised rats when compared with sham where as B _max and K _d reversed to near sham after 7 days of pancreatectomy. The results suggest that GABA through GABA receptors in brain stem has a regulatory role during active regeneration of pancreas which will have immense clinical significance in the treatment of diabetes.     

[3H]Bicuculline Methochloride Binding to Low-Affinity γ-Aminobutyric Acid Receptor Sites

Abstract: The binding of [³H]bicuculline methochloride (BMC) to mammalian brain membranes was characterized and compared with that of [³H]γ-aminobutyric acid ([³H]GABA). The radiolabeled GABA receptor antagonist showed significant displaceable binding in Tris-citrate buffer that was improved by high concentrations of chloride, iodide, or thiocyanate, reaching >50% displacement in the presence of 0.1 M SCN^?. An apparent single class of binding sites for [³H]BMC (K_D= 30 nM) was observed in 0.1 M SCN^? for fresh or frozen rat cortex or several regions of frozen and thawed bovine brain. The B_max was about 2 pmol bound/mg of crude mitochondrial plus microsomal membranes from unfrozen washed and osmotically shocked rat cortex, similar to that for [³H]GABA. Frozen membranes, however, showed decreased levels of [³H]BMC binding with no decrease or an actual increase in [³H]GABA binding sites. [³H]BMC binding was inhibited by GABA receptor specific ligands, but showed a higher affinity for antagonists and lower affinity for agonists than did [³H]GABA binding. Kinetics experiments with [³H]GABA binding revealed that low- and high-affinity sites showed a similar pharmacological specificity for a series of GABA receptor ligands, but that whereas all agonists had a higher affinity for slowly dissociating high-affinity [³H]GABA sites, bicuculline had a higher affinity for rapidly dissociating low-affinity [³H]GABA sites. This reverse potency between agonists and antagonists during assay of radioactive antagonists or agonists supports the existence of agonist- and antagonist-preferring conformational states or subpopulations of GABA receptors. The differential affinities, as well as opposite effects on agonist and antagonist binding by anions, membrane freezing, and other treatments, suggest that [³H]BMC may relatively selectively label low-affinity GABA receptor agonist sites. This study, using a new commercially available preparation of [³H]bicuculline methochloride, confirms the report of bicuculline methiodide binding by Mohler and Okada (1978), and suggests that this radioactive GABA antagonist will be a valuable probe in analyzing various aspects of GABA receptors.     

GABAA Receptors Modulate Early Spontaneous Excitatory Activity in Differentiating P19 Neurons

Abstract: P19 embryonic carcinoma (EC) stem cells are pluripotent and are efficiently induced to differentiate into neurons and glia with retinoic acid (RA) treatment. Within 5 days, a substantial number of differentiating P19 cells express gene products that are characteristic of a neuronal phenotype. P19 neurons were used as a model to explore the relationship between neuronal “differentiation” in vitro and the acquisition of γ-aminobutyric acid (GABA_A) receptors and functional GABA responses. Pulse-labeling experiments using bromodeoxyuridine indicated that all neurons had become postmitotic within 3–4 days after treatment with RA. This was confirmed by a reduction in the immunocytochemical detection of the undifferentiated stem cell antigen SSEA-1. Subsequently, a transient expression of nestin was observed during the first 5 days in vitro (DIV) after exposure to RA. By 5–10 DIV after RA, a significant number of neurons (～80–90%) expressed immunocytochemically detectable glutamate decarboxylase and GABA coincident with the acquisition of membrane binding sites for tetanus toxin. These phenotypic markers were maintained for >30 DIV after RA. Under current-clamp conditions, random, low-amplitude, spontaneous electrical activity appeared in neurons within the first few days after RA treatment and this was blocked by the specific GABA_A receptor antagonist bicuculline. Thereafter, the appearance and progressive increases in the frequency of spontaneous action potentials in P19 neurons were observed that were similarly attenuated by bicuculline. In neurons > 5 DIV after RA, exogenous application of GABA elicited similar action potentials. The onset of excitatory responses to GABA or muscimol in voltage-clamped neurons appeared immediately after the cessation of neuronal mitosis and before the previously reported acquisition of responses to glutamate. In fura-2 imaging studies, the exogenous application of GABA resulted in neuron-specific increases in intracellular Ca²⁺. Thus, P19 neurons provide an in vitro model for the study of the early acquisition and properties of electrical excitability to GABA and the expression of functional GABA_A receptors.     

ISOLATION OF HYDROPHOBIC PROTEINS BINDING AMINO ACIDS: γ-AMINOBUTYRIC ACID BINDING IN THE RAT CEREBRAL CORTEX

—The binding of [¹⁴C]GABA to nerve-ending membranes isolated from rat cerebral cortex follows a hyperbolic curve saturating at 0·4pmol/μg protein. This binding is about 60% inhibited by chloropromazine, and about 40%, inhibited by bicuculline. A hydrophobic protein fraction binding [¹⁴C]GABA was separated from the total. lipid extract of nerve-ending membranes. The binding follows a hyperbolic curve that saturates at 10·5 pmol of [¹⁴C]GABA/μg of protein, with an apparent K_d= 30 μm . The binding is competitively inhibited by bicuculline with a K_i= 273 μm . These results are compared with those previously obtained on a GABA binding protein from crustacean muscle.     

Studies on the mechanism of modulation of [3H]noradrenaline release from rat hippocampal synaptosomes by GABA and benzodiazepine receptors

Release of [³H]noradrenaline from rat hippocampal synaptosomes was triggered by pulses of 25 mM K⁺, 5 μM veratridine or superfusion with the Ca²⁺ ionophore A23187. GABA with bicuculline or chlordiazepoxide depressed the release of [³H]noradrenaline evoked by depolarisation but not by the Ca²⁺ ionophore. 8 Br-cAMP with [Ca²⁺]₀ 0.3 mM had no effect on spontaneous or K⁺-evoked release of [³H]noradrenaline and completely blocked the effect of chlordiazepoxide and GABA with bicuculline. With [Ca²⁺]₀ 1 mM 8 Br-cAMP enhanced spontaneous and K⁺-evoked release of [³H]noradrenaline, and reversed the depression caused by GABA with bicuculline. GABA alone evoked Ca²⁺-dependent release of [³H]noradrenaline which was sensitive to [Cl^?]₀. The results suggest that the GABA_A-receptor mediated release of [³H]noradrenaline is due to depolarisation resulting from increased Cl^? conductance whereas the depression of depolarisation-dependent release of [³H]noradrenaline by GABA_B or benzodiazepine receptors is mediated by a cAMP-dependent decrease in the voltage-dependent Ca²⁺ conductance.     

Post-synaptic Inhibition in the Cuneate blocked by GABA Antagonist

PICROTOXIN has been shown to antagonize specifically the inhibitory action of λ-aminobutyric acid (GABA) as opposed to glycine (Gly) on cells of the cuneate nucleus, either spontaneously active or excited by peripheral stimulation¹. Bicucul-line also seems to be a specific antagonist of the depressant action of GABA in several regions of the mammalian nervous system², although probably not in others³.     

4-Aminotetrolic Acid : New Conformational-restricted Analogue of γ-Aminobutyric Acid

RECENT studies strongly support a role for γ-aminobutyric acid (GABA) as an inhibitory transmitter at certain synapses in the mammalian central nervous system. Structure activity correlations of many GABA analogues implicate both the intramolecular distance between the zwitterionic centres and the rotational freedom of the molecule as important factors governing the synaptic activity of these substances¹. The following observations provide pertinent information about the active conformation(s) of GABA recognized by the receptor. (1) Muscimol, an isoxazole isolated from Amanita muscaria, seems to function as a GABA analogue as its inhibitory action on central neurones is comparable with that of GABA both in potency² and with respect to antagonism by bicuculline³. Molecular orbital calculations suggest that GABA and muscimol can assume similar conformations as zwitterions with the charged centres (N⁺ and 0^?) at least 5 and, more likely, 6 Å apart⁴. (2) The selective GABA antagonist bicuculline exhibits some degree of structural similarity with particular conformations of GABA and muscimol³. (3) X-ray crystallography indicates that GABA exists in a partially folded conformation in the solid state^5,6. (4) A model of the GABA receptor proposes that GABA adopts a folded conformation with a distance of less than 4.4 Å between the charged centres⁷. Observations (1) and (2) suggest extended conformations for GABA, while (3) and (4) suggest folded conformations.     

GABAergic neuron-to-astrocyte signaling regulates dendritic branching in coculture

Effects of neurotransmitters on dendritic morphology were analyzed in cocultures of neurons and astrocytes from the neonatal rat olfactory bulb by means of immunocytochemical staining and morphometry. About 70% of the neurons were γ-aminobutyric acid (GABA)-immunoreactive on day 7 of the coculture. Morphometric analysis of neurons having no contact with other neurons revealed that incubation of the coculture with either a sodium channel blocker, tetrodotoxin, or GABA_A receptor antagonists such as bicuculline or picrotoxin resulted in a decreased number of dendritic branch points as compared to neurons in control cultures, while the same treatment did not affect radial dendritic outgrowth or the number of primary dendrites. Application of a GABA_B receptor antagonist, phaclofen, or an AMPA-type glutamate receptor antagonist, 6-cyano-7-nitroquinoxaline-2,3-dione, had no detectable effect on dendritic morphology. Incubation of the coculture with a GABA_A receptor agonist, muscimol, enhanced branching and reversed the inhibitory effect of tetrodotoxin. Branching was also enhanced by increasing extracellular K⁺. The inhibitory effect of tetrodotoxin or bicuculline and the stimulatory effect of muscimol or elevated K⁺ were abolished when neurons were grown on a monolayer of dead astrocytes, indicating that the morphoregulatory action of GABA requires living astrocytes to operate. Astrocytes pretreated with muscimol before the addition of neurons supported branching better than those without pretreatment. These results suggest that various aspects of dendritic growth are regulated by distinct mechanisms, and that neuron-to-astrocyte signaling mediated by GABA promotes dendritic branching. © 1998 John Wiley & Sons, Inc. J Neurobiol 37: 251–264, 1998     

A comparison of GABA and beta-alanine transport and GABA membrane binding in the rat brain

It was found that rat brain nerve endings contain a high affinity and Na^- dependent transport system for [³H]β-alanine ([³H]β-ala). As determined from Michaelis-Menten plots, the [³H]β-ala K_m was 2.8 × 10^-5 M and the V_max was 0.29 nmol/mg protein/5 min. Under similar incubation conditions the [³H]GABA K_m was 3.8 x 10^-6M and the V_max was 6.3 nmol/mg protein/5 min. The [³H]β-ala and [³H]GABA transport systems were further characterized by determining the IC₅₀ values for a number of compounds. The compounds tested were GABA, β-ala, l -2,4-diaminobutyric acid. DL-3-hyd-roxy-GABA, β-guanidopropionic acid, strychnine, γ-guanidobutyric acid, imidazole-4-acetic acid, DL-proline, bicuculline, L-serine, glycine, l -α-ala and taurine. DABA, dl -3-hydroxy-GABA, β-guanidopro-pionic acid and γ-guanidobutyric acid were more potent inhibitors of [³H]GABA than [³H]β-ala transport. Strychnine, imidazole-4-acetic acid, proline and glycine were between 2 and 6 times more potent inhibitors of [³H]β-ala than [³H]GABA transport. β-Ala, bicuculline, serine, α-alanine and taurine were all markedly more potent (12–150 times) inhibitors of [³H]β-ala than [³H]GABA transport. IC₅₀ values were also determined for the above compounds for the sodium-dependent and the sodium-independent binding of [³H]GABA to both fresh and frozen brain membranes. In general, the potency of these compounds to inhibit either sodium-independent or sodium-dependent binding was greater in fresh tissue. It was also observed that the neurophysiologically‘glycine-like’amino acids were more potent inhibitors in the presence of NaCl. No significant correlations were found between [³H]GABA binding under any condition and [³H]GABA or [³H]β-ala transport into nerve endings.