Inhibition of GABA release by presynaptic ionotropic GABA receptors in hippocampal CA3

Vesicular transmitter release can be regulated by transmitter-gated ion channels at presynaptic axon terminals. The central inhibitory transmitter GABA acts on such presynaptic ionotropic receptors in various cells, including inhibitory interneurons. Here we report that GABA-mediated postsynaptic inhibitory currents in CA3 pyramidal cells of rat hippocampal slices are suppressed by agonists of GABAA receptors. The effect is present for both stimulus-induced and miniature IPSCs, indicating a reduction in the probability of vesicular release by presynaptic, action-potential-independent mechanisms. We conclude that the release of GABA from hippocampal CA3 interneurons is regulated by a negative feedback via presynaptic ionotropic GABA autoreceptors.     

Some pharmacological differences between hippocampal excitatory and inhibitory synapses in transmitter release: an in vitro study.

The effects of adenosine, carbachol, and baclofen on synaptic transmission between neurons in cultured rat hippocampal explants were studied using the tight-seal whole cell clamp technique. In the culture, stimulations of neurites cause postsynaptic currents (PSCs) in nearby neurons under voltage-clamp condition. In the presence of 20 microM bicuculline, most PSCs were considered as glutamatergic excitatory postsynaptic currents (EPSCs), because they were blocked by glutamate antagonist, kynurenate at 1 mM. In the presence of 1 mM kynurenate, PSCs seemed to be inhibitory postsynaptic currents mediated by gamma-aminobutyric acid (GABA), because they were blocked by GABA antagonist, bicuculline at 20 microM. Adenosine at 100 microM and carbachol at 10 microM suppressed these EPSCs to about 35% of control. However, adenosine and carbachol at the same concentration did not suppress the IPSCs. Baclofen at 10 microM suppressed both EPSCs and IPSCs significantly (EPSCs: to about 40% of control, IPSCs: to about 30% of control). In contrast, membrane currents elicited by ionophoretically applied glutamate and GABA were not suppressed by 100 microM adenosine, 10 microM carbachol, and 10 microM baclofen. From these results, it is suggested that the pharmacological sensitivities of transmitter release from presynaptic terminals are different between glutamatergic excitatory synapses and GABAergic inhibitory synapses in hippocampal cultures.     

Synaptic signaling between neurons and glia

Rapid signaling between vertebrate neurons occurs primarily at synapses, intercellular junctions where quantal release of neurotransmitter triggers rapid changes in membrane conductance through activation of ionotropic receptors. Glial cells express many of these same ionotropic receptors, yet little is known about how receptors in glial cells become activated in situ. Because synapses were thought to be the sole provenance of neurons, it has been assumed that these receptors must be activated following diffusion of transmitter out of the synaptic cleft, or through nonsynaptic mechanisms such as transporter reversal. Two recent reports show that a ubiquitous class of progenitors that express the proteoglycan NG2 (NG2 cells) engage in rapid signaling with glutamatergic and gamma-aminobutyric acid (GABA)ergic neurons through direct neuron-glia synapses. Quantal release of transmitter from neurons at these sites triggers rapid activation of aminomethylisoxazole propionic acid (AMPA) or GABA(A) receptors in NG2 cells. These currents exhibit properties consistent with direct rather than spillover-mediated transmission, and electron micrographic analyses indicate that nerve terminals containing clusters of synaptic vesicles form discrete junctions with NG2 cell processes. Although activation of AMPA or GABA(A) receptors depolarize NG2 cells, these receptors are more likely to serve as routes for ion flux rather than as current sources for depolarization, because the amplitudes of the synaptic transients are small and the resting membrane potential of NG2 cells is highly negative. The ability of both glutamate and GABA to influence the morphology, physiology, and development of NG2 cells in vitro suggests that this rapid form of signaling may play important roles in adapting the behavior of these cells to the needs of surrounding neurons in vivo.     

Multiple inhibitory g-protein-coupled receptors resist acute desensitization in the presynaptic but not postsynaptic compartments of neurons

Acute desensitization is a common property of G(i/o)-coupled receptors. Recent data, however, suggest that, unlike μ-opioid receptors (MORs) located somatodendritically in neurons or expressed in heterologous systems, MORs in the presynaptic compartment of neurons are resistant to acute desensitization. It is not yet clear whether this differential desensitization is a shared property of many G(i/o)-coupled receptors nor whether receptors located presynaptically and postsynaptically in a single cell type display differential desensitization. Here, whole-cell recordings were made from proopiomelanocortin (POMC) neurons in mouse brain slices. Agonists for μ-opioid, nociceptin, and GABA(B) receptors induced postsynaptic currents that desensitized within minutes, whereas inhibition of presynaptic transmitter release mediated by these receptors was maintained throughout agonist exposure. Expression of channelrhodopsin2 in POMC neurons allowed for light-evoked transmitter release from POMC neuron terminals, which was detected by recording postsynaptic currents in downstream neurons. Light-evoked currents were inhibited throughout the application of all agonists tested. Thus, the same receptors that desensitize when expressed in the postsynaptic compartment of POMC neurons resist desensitization when located in the presynaptic compartment. Pharmacologic knockdown of MORs revealed that depletion of receptor reserve does not account for presynaptic resistance to desensitization. In ～25% of recordings with GABA(B) agonist application, presynaptic GABA(B) receptors desensitized, suggesting that resistance to desensitization is not due to an intrinsic property of the terminals themselves. Together, the results indicate that a variety of presynaptic receptors can continue to function after their postsynaptic counterparts desensitize and suggest that a compartment-specific modification may confer resistance to desensitization.     

Developmental profile and mechanisms of GABA-induced calcium signaling in hippocampal astrocytes

GABA (gamma-aminobutyric acid) is a transmitter with dual action. Whereas it excites neurons during the first week of postnatal development, it represents the major inhibitory transmitter in the mature brain. GABA also activates astrocytes by binding to ionotropic (GABA(A)) and metabotropic (GABA(B)) receptors. This results in glial calcium transients which can induce the release of gliotransmitters, rendering GABA an important mediator of neuron-glia interaction. Using whole-cell patch-clamp and ratiometric calcium imaging in hippocampal slices from rats at postnatal days 3-34, we have analyzed the developmental profile as well as the cellular mechanisms of calcium signals induced by GABA(A) and GABA(B) receptor activation in astrocytes. We found that GABA-evoked glial calcium transients are mediated by both GABA(A) and GABA(B) receptors. Throughout development, GABA(A)-receptor activation resulted in immediate calcium transients in the vast majority of astrocytes, most likely by influx of calcium through voltage-gated calcium channels. GABA(B) receptor activation, in contrast, resulted in delayed calcium transients, which were blocked following depletion of intracellular calcium stores and during persistent activation of heterotrimeric G-proteins. GABA(B) receptor-mediated calcium signals exhibited a clear developmental profile with less than 10% of astrocytes responding at P3 or P32-34, and about 60% of cells between P11 and P15. Our data thus indicate that GABA(B) receptor-mediated calcium transients are due to calcium release from intracellular stores following G-protein activation. Moreover, GABA(B) receptor-mediated calcium signaling in astrocytes preferentially occurs at a period during postnatal development when hippocampal networks are established.     

Differences in ratios of GABA,glycine and glutamate immunoreactivities in nerve terminals on rat hindlimb motoneurons: a possible source of post-synaptic variability

Previous pharmacological and physiological data on GABA and glycine receptor-dependent components of miniature inhibitory post-synaptic currents show that the electrophysiological characteristics of synaptic transmission from inhibitory synapses on spinal motoneurons are highly variable. Although post-synaptic factors are thought to be the major underlying cause of this variability, quantitative immunohistochemical data suggest that the transmitter content of afferents also vary from terminal to terminal. To examine whether ratios of amino acid staining densities vary similar to those of components of post-synaptic currents mediated by the corresponding receptors, we quantified immunogold labeling for GABA, glycine and the major excitatory transmitter, glutamate, in nerve terminals contacting the dendrites of motoneurons retrogradely labeled from the rat hindlimb muscle, biceps femoris. Nearly all terminals (94%) were immunoreactive for at least one amino acid and 64% of these contained two or three amino acids. All possible combinations of GABA, glycine and glutamate labeling were found. Over 70% of the terminals contained glycine, of which 60% also labeled for GABA. Of these GABA/glycine boutons, 40% also had glutamate. Half of all terminals contained GABA, but terminals immunoreactive for GABA alone were extremely rare. Immunoreactivity for glutamate occurred in 48% of all terminals and nearly 60% of these also contained glycine. Labeling densities for GABA, glycine and glutamate varied over a wide range from terminal to terminal. We hypothesize that this diversity in amino acid content may be a major underlying cause of variability in GABA- and glycine receptor-mediated components of miniature inhibitory post-synaptic currents in motoneurons.     

Presynaptic source of quantal size variability at GABAergic synapses in rat hippocampal neurons in culture

The variability of quantal size depends on both presynaptic (profile of the neurotransmitter concentration in the cleft) and postsynaptic (number and gating properties of postsynaptic receptors) factors. Here we have examined the possibility that at nonsaturated synapses in cultured hippocampal neurons, changes in both the transmitter concentration peak and its clearance from the synaptic cleft may influence the variability of spontaneous miniature synaptic GABAergic currents (mIPSCs). We found that, in contrast to the slow-off GABAA receptor antagonist bicuculline, fast-off competitive antagonists such as SR-95103 and TPMPA differentially blocked small and large mIPSCs. In the presence of flurazepam, a drug believed to increase the affinity of GABA for GABAAR, small mIPSCs were enhanced more efficiently than large events. Moreover, the addition of dextran, which increases the viscosity of the extracellular fluid, preferentially increased small mIPSCs with respect to large ones. These observations suggest that changes in the concentration peak and the speed of GABA clearance in the cleft may be an important source of synaptic variability. The study of the correlation between peak amplitude and kinetics of mIPSCs allowed determination of the relative contribution of transmitter peak concentration vs. time of GABA clearance. Small synaptic responses were associated with fast onset and decay kinetics while large amplitude currents were associated with slow kinetics, indicating a crucial role for GABA synaptic clearance in variability of mIPSCs. By using model simulations we were able to estimate the range of variability of both the concentration and the speed of clearance of the GABA transient in the synaptic cleft.     

Chloride ions in the pore of glycine and GABA channels shape the time course and voltage dependence of agonist currents

In the vertebrate CNS, fast synaptic inhibition is mediated by GABA and glycine receptors. We recently reported that the time course of these synaptic currents is slower when intracellular chloride is high. Here we extend these findings to measure the effects of both extracellular and intracellular chloride on the deactivation of glycine and GABA currents at both negative and positive holding potentials. Currents were elicited by fast agonist application to outside-out patches from HEK-293 cells expressing rat glycine or GABA receptors. The slowing effect of high extracellular chloride on current decay was detectable only in low intracellular chloride (4 mm). Our main finding is that glycine and GABA receptors "sense" chloride concentrations because of interactions between the M2 pore-lining domain and the permeating ions. This hypothesis is supported by the observation that the sensitivity of channel gating to intracellular chloride is abolished if the channel is engineered to become cation selective or if positive charges in the external pore vestibule are eliminated by mutagenesis. The appropriate interaction between permeating ions and channel pore is also necessary to maintain the channel voltage sensitivity of gating, which prolongs current decay at depolarized potentials. Voltage dependence is abolished by the same mutations that suppress the effect of intracellular chloride and also by replacing chloride with another permeant ion, thiocyanate. These observations suggest that permeant chloride affects gating by a foot-in-the-door effect, binding to a channel site with asymmetrical access from the intracellular and extracellular sides of the membrane.     

Colocalization of glycine-like and GABA-like immunoreactivities in Golgi cell terminals in the rat cerebellum: a postembedding light and electron microscopic study

Consecutive sections of rat cerebella were incubated with antisera raised against glycine or γ-aminobutyric acid (GABA) conjugated to protein by glutaraldehyde. The sections were subsequently processed according to the peroxidase-antiperoxidase technique (semithin sections) or treated with secondary antibody coupled to colloidal gold particles (ultrathin sections). Corroborating previous light microscopic observations based on pre-embedding immunocytochemistry²⁰, a major proportion (about 70%) of the Golgi cell bodies showed immunoreactivity for both glycine and GABA. Analyses of semithin sections further suggested that the two immunoreactivities were colocalized in the same glomeruli and even in the same Golgi cell terminals. This was confirmed by electron microscopy. Quantification of the immunogold labelling for glycine (which is assumed to play metabolic roles in addition to its presumed role as a transmitter) showed that the net gold particle density was an order of magnitude higher over Golgi cell terminals than over the other constituents of the cerebellar glomeruli (mossy fibre terminals and granule cell dendrites). The total particle density over the latter was only slightly higher than the background level (over empty resin), suggesting that the concentration of ‘metabolic’ glycine is generally low compared to the concentration of glycine in Golgi cells. The stellate and basket cell terminals (which similarly to the Golgi cell are thought to release GABA as transmitter) were immunoreactive for GABA, but (with very few exceptions) virtually unlabelled for glycine, suggesting that our results were not confounded by any crossreactivity of the glycine antiserum with fixed GABA. Direct evidence that the sera reacted selectively with fixed glycine or GABA under the conditions used was obtained by incubating the tissue sections together with test sections containing a series of different amino acid-glutaraldehyde-brain macromolecule conjugates. Adsorption tests with soluble amino acid-glutaraldehyde complexes similarly suggested that the double-labelling of the Golgi terminals indeed reflected a colocalization of glycine and GABA. The results show that two ‘classical’ transmitters, both being inhibitory and acting on Cl⁻ channels, may coexist in the same nerve terminals.     

Unaltered control of extracellular GABA-concentration through GAT-1 in the hippocampus of rats after pilocarpine-induced status epilepticus

The uptake of the inhibitory transmitter GABA (gamma-aminobutyric acid) limits the efficacy of synaptic and tonic inhibition in brain tissue. It has been reported that GABA-uptake is down-regulated in temporal lobe epilepsy. This down-regulation may increase the inhibitory action of GABA but may also limit the anticonvulsant activity of GABA-uptake blockers. We have directly compared the function of GABA-uptake in hippocampal slices from normal and chronically epileptic rats. We raised the global extracellular concentration of GABA by bath-application of the agonist in the absence and presence of the GABA-uptake blocker tiagabine. GABA-induced currents were measured in dentate granule cells and CA1 pyramidal neurons in hippocampal slices. The potentiation of currents by tiagabine was taken as a measure for the efficacy of GABA-uptake in the hippocampal tissue. There was no difference between cells from control- or pilocarpine-treated animals in the response to GABA or in the conductance increase following application of tiagabine. Our data show that in the chronic phase of the pilocarpine-model GABA-uptake maintains its ability to control the extracellular background concentration of GABA.     

Interaction between allopregnanolone and pregnenolone sulfate in modulating GABA-mediated synaptic currents in neurons from the rat medial preoptic nucleus

The two neurosteroids 3alpha-hydroxy-5alpha-pregnane-20-one (allopregnanolone; AlloP) and pregnenolone sulfate (PregS) affect neuronal GABA(A) receptors differently. While AlloP mainly potentiates the currents through GABA(A) receptors, PregS reduces such currents. The present study aimed at clarifying the interaction of AlloP and PregS at GABA(A) receptors in neurons from the medial preoptic nucleus of male rat. AlloP has previously been shown to dramatically prolong GABA-mediated spontaneous inhibitory postsynaptic currents (sIPSCs) in these neurons. Here, by recording sIPSCs under voltage-clamp conditions with the perforated-patch technique, it was shown that PregS by itself did not significantly affect the amplitude or time course of such currents. However, PregS, in a concentration-dependent manner, reduced the AlloP-evoked prolongation of sIPSC decay when the two neurosteroids were applied together. In contrast to sIPSC amplitude and time course, sIPSC frequency was significantly reduced by 10 microM PregS alone. Further, although 1.0 microM AlloP alone induced a clear increase in sIPSC frequency, the frequency was not significantly different from control when 1.0 microM AlloP was applied in combination with 10 microM PregS. In addition to the effects on sIPSC parameters, PregS reduced the baseline current evoked by 1.0 microM AlloP in the absence of GABA application or synaptic activity. PregS by itself did not significantly affect the baseline current. The main effects of AlloP and PregS on the sIPSC time course were mimicked by a simplified model with AlloP assumed to reduce the rate of GABA unbinding from the receptor and PregS assumed to increase the rate of desensitization.     

Retrograde suppression of GABAergic currents in a subset of SCN neurons

Many postsynaptic neurons release a retrograde transmitter that modulates presynaptic neurotransmitter release. In the suprachiasmatic nucleus (SCN), retrograde signaling is suggested by the presence of dendritic dense-core vesicles. Whole-cell voltage-clamp recordings were made from rat SCN neurons to determine whether a retrograde messenger could modulate the activity of afferent gamma-aminobutyric acid (GABA)ergic inputs. The frequency and amplitude of spontaneous GABAergic currents was significantly reduced in a subpopulation of SCN neurons (eight out of 13) following a postsynaptic depolarization. Similarly, a postsynaptic depolarization significantly reduced the amplitude of evoked GABAergic currents during both day and night recordings. A postsynaptic depolarizing pulse eliminated paired-pulse inhibition of GABAergic currents consistent with a presynaptic mechanism. Muscimol-activated currents were not altered by postsynaptic depolarization, demonstrating that the activity of GABA(A) receptors was not altered. Depolarization-induced inhibition of the GABAergic currents was not observed when a Ca2+ chelator was included in the microelectrode. Postsynaptic depolarization significantly increased the Ca2+ concentration in both the soma and dendrites. The dendritic Ca2+ levels increased faster, to a higher concentration and decayed faster than in the soma. The depolarization-induced inhibition of the evoked GABAergic current was blocked by the G-protein uncoupling agent N-ethylmaleimide, suggesting that the retrograde messenger acts on a pertussis toxin-sensitive G-protein-coupled receptor. Because the majority of SCN neurons receive GABAergic input from neighboring cells, these results describe a retrograde signaling mechanism by which SCN neurons can modulate GABAergic synaptic input.     

Physiological identification of GABA as the inhibitory transmitter for mammalian cortical neurons in cell culture

(1) Rat cortical neurons grown in dissociated cell culture exhibit IPSPs which appear to be generated by an increase in membrane conductance to chloride.

(2) The neurons are all sensitive to GABA in micromolar concentrations and GABA mimics the inhibitory transmitter.

(3) The neurons are much less sensitive to glycine and insensitive to taurine.

(4) Bicuculline and strychnine both block essentially all IPSPs and at the same concentrations block GABA effects.

(5) It is concluded that GABA is the main, or only, inhibitory transmitter utilized by the cortical neurons in vitro. The relevance of this conclusion to in situ transmitter identification is discussed.

Pharmacological characterization of GABAB-mediated responses in the CA1 region of the rat hippocampal slice.

It is generally accepted that the bicuculline-resistant responses to GABA are mediated through the activation of GABAB receptors that mediate a slow IPSP. However, a number of reported observations are difficult to reconcile with this model. Specifically, GABAB antagonists only partially block bicuculline-resistant GABA responses, and both 4-aminopyridine (4-AP) and carbachol have been reported to block responses to the selective GABAB agonist baclofen, but not GABA itself. Thus, it has been argued that baclofen and GABA increase potassium conductance through separate receptor mechanisms. This suggestion is not easily reconcilable with the postulated physiological role of GABAB receptors in mediating the slow IPSP. We have addressed these discrepancies by using the new GABAB antagonists 2-hydroxy-saclofen (2-OH-SAC) and CGP 35348 in the presence of the GABA uptake inhibitor SKF 89976A. The weak antagonism of 2-OH-SAC against the bicuculline-resistant GABA response was improved when the GABA uptake was inhibited with SKF 89976A, allowing for the application of lower GABA concentrations. Under these circumstances, 2-OH-SAC and CGP 35348 strongly antagonized GABA and baclofen responses, but did not have any effect on outward currents evoked by 5-HT. The slow IPSP evoked in the presence of glutamate antagonists was reversibly inhibited by CGP 35348 (IC50 = 14 microM), without affecting the fast IPSP. Carbachol (0.3-20 microM) had no effect on outward currents evoked by either baclofen or GABA. 4-AP (5 microM to 1 mM), despite causing a large increase in cell excitability, did not change baclofen responses. Higher concentrations of 4-AP (5 mM) induced inward current, and reduced both baclofen and GABA outward currents to a similar extent.(ABSTRACT TRUNCATED AT 250 WORDS)     

Pharmacological blockade of the globus pallidus-induced inhibitory response of subthalamic cells in the rat

The aim of the present study was to investigate, with extracellular recording and microiontophoretic techniques, the possibility that gamma-aminobutyric acid (GABA) is the transmitter substance in the pallido—subthalamic (GP-STN) pathway. Experiments were carried out in 94 rats, anesthetized with ketamine. Among the 236 recorded STN neurons, 85 were inhibited by GP stimulation. This inhibition lasted 10–20 msec(mean duration±S.E.M.13.45 ± 0.5msec). Control stimulations located either in the internal GP or in the striatum never elicited the same type of response as they did in the STN. STN neurons were inhibited by microiontophoretically applied GABA or muscimol. Iontophoretically applied bicuculline or picrotoxin reversibly blocked the GP-evoked inhibition at doses which blocked the GABA inhibitory responses but not those produced by glycine. The results are consistent with the hypothesis that GABA is the transmitter released by the inhibitory pallido—subthalamic pathway, and are in agreement with recent biochemical data. Nevertheless, on a few cells, strychnine blocked the GP-induced inhibition. This result is discussed in relation to the specificity of GABA and glycine antagonists.     

Development of Action Potential-dependent and Independent Spontaneous GABAA Receptor-mediated Currents in Granule Cells of Postnatal Rat Cerebellum

The postnatal development of spontaneous GABAergic transmission between cerebellar Golgi cells and granule cells was investigated with voltage-clamp recording from rat cerebellar slices, in symmetrical Cl^-conditions. Between postnatal days 7 and 14 (P7–14), bicuculline-and TTX (tetrodotoxin)-sensitive spontaneous inhibitory postsynaptic currents (sIPSCs), occurred at high frequency in 56% of granule cells. Between P10 and P14, sIPSCs were superimposed on tonic current of-12 ± 1.8 pA at -70 mV, that was accompanied by noise with variance of 17 ± 3 pA². Both the current and noise were inhibited by bicuculline. TTX blocked the bicuculline-sensitive current and noise by?60%. Between P18 and P25, sIPSCs were less frequent; all cells showed tonic, bicuculline-sensitive currents, but these were partially inhibited by TTX (?35%). Between P40 and P53, slPSCs were rare; tonic, bicuculline-sensitive currents and noise were greater in amplitude, with mean values of-17 pA and 22 pA² at-70 mV, they were present in all cells but they were not inhibited by TTX. Glycine receptor channels that were expressed in immature, but not adult cells, did not mediate spontaneous currents. Our results indicate that spontaneous transmission onto cerebellar granule cells in immature animals consists primarily of action potential-dependent, phasic release of vesicular GABA. This generates GABA_A receptor-mediated slPSCs. The effects of GABA transporter blockers suggest that it also produces the TTX-sensitive current-noise, as GABA spills out of synapses to activate extrasynaptic receptors or receptors in neighbouring synapses. In older animals, action potential-independent release of transmitter is predominant and results in tonic activation of GABA_A receptors. This does not appear to be spontaneous vesicular release of GABA. Neither does it appear to be reversed uptake of GABA, although further work is required to rule out these possibilities.     

Differential regulation of gamma-aminobutyric acid receptor channels by diazepam and phenobarbital

The anticonvulsant activity of diazepam and phenobarbital may be mediated in part by enhancement of inhibition involving gamma-aminobutyric acid (GABA). While both diazepam and phenobarbital increase GABA receptor chloride current, they may have different mechanisms of action, since they bind to different sites on the GABA receptor-chloride channel complex. We used the patch clamp technique to compare the effects of diazepam and phenobarbital on single GABA receptor currents. Outside-out patches were obtained from mouse spinal cord neurons grown in cell culture for 2 to 4 weeks. GABA (2 microM) evoked single channel currents that occurred as single brief openings or in bursts of multiple openings. Diazepam (20 nM) and phenobarbital (500 microM) both increased the GABA receptor current by increasing mean open time without altering channel opening frequency. However, the temporal grouping of openings into bursts suggested that the enhancement occurred via different mechanisms. Diazepam increased the frequency of bursting GABA receptor currents with minimal effect on the duration of bursts. Phenobarbital increased the duration of bursting GABA receptor currents without altering the frequency of bursts. These results suggest that diazepam binds to a site that may enhance single channel burst frequency by increasing the affinity of GABA binding, while phenobarbital may stabilize the bursting open state of the channel by binding to a different modulatory site at or near the chloride channel.     

Features of the chlorine-dependent effect of acetylcholine and neutral amino acids on neurons of the Achatina snail

Responses of neurons in cerebral ganglia of an African giant snail (Achatina fulica) to acetylcholine (ACh), gamma-aminobutyric acid (GABA) and glycine (Gly) were investigated. Cl-dependent currents induced by these mediators in neurons of one and a half month old snail sibs were inhibited by dibutyryl-cAMP and strychnine. Inhibition of ACh-responses by 10(-8) mol/l GABA was mimicked by applications of dibutyryl-cAMP and isobutylmethylxanthine. GABA- and Gly-responses exhibited complete cross-desensitization, but this effect was absent for ACh and GABA (or Gly). On the basis of pharmacological dependences of GABA- and Gly-responses it is suggested that these amino acids act on the same receptor-channel complex in neurons of young snails. ACh-, GABA- and Gly-induced chloride currents were independent in neurons of four years old snails.     

Block by picrotoxin of a GABAergic chloride channel expressed on crayfish muscle after axotomy

Outside-out patches containing a γ-aminobutyric acid (GABA)-activated chloride channel expressed after axotomy on crayfish deep extensor abdominal muscle were excised. GABA and the blocker picrotoxin (PTX) were applied to the patches using a liquid filament switch to study the effects of picrotoxin on the GABA-elicited currents. Coapplication of GABA and PTX resulted in a reduction of the current amplitude compared with that elicited by the same GABA concentration alone. This reduction of the amplitude was dependent on both the GABA and PTX concentrations. The rise time of the current decreased after coapplication of GABA and PTX. Evaluation of the single channel currents and off-currents in the presence of GABA and PTX showed a dramatic shortening of the burst duration of the channel. The open time distributions were not altered, whereas in the closed time distributions a new closed time was apparent in presence of PTX. Preincubation with PTX prior to the GABA pulse resulted in an increase of the rise time. This effect was dependent on the PTX concentration only. Possible mechanisms are discussed to explain the effects of PTX and are implemented into the existing molecular reaction scheme of the channel.     

Cross-inhibitory interactions between GABAA and P2X channels in myenteric neurones

Inhibitory interactions between GABA(A)[induced by gamma-aminobutyric acid (GABA)] and P2X [activated by adenosine 5'-triphosphate (ATP)] receptors of myenteric neurones from the guinea pig small intestine were characterized using whole-cell recordings. Currents induced by GABA (I(GABA)) or ATP (I(ATP)) were inhibited by picrotoxin or pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid, respectively. Currents induced by GABA + ATP (I(GABA+ATP)) were only as large as the current induced by the most effective transmitter, revealing current occlusion. This occlusion requires maximal activation of at least one of these receptors. Sequential applications of neurotransmitters, and kinetic and pharmacological properties of I(GABA+ATP) indicate that they are carried through both GABA(A) and P2X channels. ATP did not affect I(GABA) in neurones: (i) in which P2X channels were not present; (ii) after inhibiting P2X channels with Ca2+ (iii) in the presence of pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid, a P2X receptor antagonist; (iv) after P2X receptor desensitization or (v) at I(ATP) reversal potential. Similarly, GABA did not affect P2X-mediated currents in neurones: (i) in which GABA(A) channels were not present; (ii) in the presence of picrotoxin, a GABA(A) channel blocker; (iii) after GABA(A) receptor desensitization or (iv) at the I(GABA) reversal potential. Current occlusion occurred as fast as current activation and it was still present in the absence of Ca2+, at 11 degrees C, after adding to the pipette solution a cocktail of protein kinase inhibitors (staurosporine + genistein + K-252a), after substituting the GTP in the pipette with GDP-beta-S and after treating the cells with N-ethylmaleimide. Taken together, all of these results are consistent with a model of cross-inhibition between GABA(A) and P2X.