gamma-Aminobutyric acid (GABA) signaling components in Drosophila: immunocytochemical localization of GABA(B) receptors in relation to the GABA(A) receptor subunit RDL and a vesicular GABA transporter

gamma-Aminobutyric acid (GABA) is a major inhibitory neurotransmitter in insects and is widely distributed in the central nervous system (CNS). GABA acts on ion channel receptors (GABA(A)R) for fast inhibitory transmission and on G-protein-coupled ones (GABA(B)R) for slow and modulatory action. We used immunocytochemistry to map GABA(B)R sites in the Drosophila CNS and compared the distribution with that of the GABA(A)R subunit RDL. To identify GABAergic synapses, we raised an antiserum to the vesicular GABA transporter (vGAT). For general GABA distribution, we utilized an antiserum to glutamic acid decarboxylase (GAD1) and a gad1-GAL4 to drive green fluorescent protein. GABA(B)R-immunoreactive (IR) punctates were seen in specific patterns in all major neuropils of the brain. Most abundant labeling was seen in the mushroom body calyces, ellipsoid body, optic lobe neuropils, and antennal lobes. The RDL distribution is very similar to that of GABA(B)R-IR punctates. However, the mushroom body lobes displayed RDL-IR but not GABA(B)R-IR material, and there were subtle differences in other areas. The vGAT antiserum labeled punctates in the same areas as the GABA(B)R and appeared to display presynaptic sites of GABAergic neurons. Various GAL4 drivers were used to analyze the relation between GABA(B)R distribution and identified neurons in adults and larvae. Our findings suggest that slow GABA transmission is very widespread in the Drosophila CNS and that fast RDL-mediated transmission generally occurs at the same sites.     

The gamma 2 subunit of GABA(A) receptors is required for maintenance of receptors at mature synapses

The gamma2 subunit of GABA(A) receptor chloride channels is required for normal channel function and for postsynaptic clustering of these receptors during synaptogenesis. In addition, GABA(A) receptor function is thought to contribute to normal postnatal maturation of neurons. Loss of postsynaptic GABA(A) receptors in gamma2-deficient neurons might therefore reflect a deficit in maturation of neurons due to the reduced channel function. Here, we have used the Cre-loxP strategy to examine the clustering function of the gamma2 subunit at mature synapses. Deletion of the gamma2 subunit in the third postnatal week resulted in loss of benzodiazepine-binding sites and parallel loss of punctate immunoreactivity for postsynaptic GABA(A) receptors and gephyrin. Thus, the gamma2 subunit contributes to postsynaptic localization of GABA(A) receptors and gephyrin by a mechanism that is operant in mature neurons and not limited to immature neurons, most likely through interaction with proteins involved in trafficking of synaptic GABA(A) receptors.     

Influence of subunit configuration on the interaction of picrotoxin-site ligands with recombinant GABA(A) receptors

We have assessed the interaction of picrotoxin and a putative picrotoxin-site ligand [4-dimethyl-3-t-butylcarboxyl-4,5-dihydro (1, 5-a) quinoxaline] (U-93631) with varying configurations of recombinant GABA(A) receptors, using the whole-cell patch clamp technique. In alpha2beta2gamma2 GABA(A) receptors, coapplication of picrotoxin with GABA had minimal effects on initial GABA-activated Cl(-) current amplitude, and subsequently enhanced decay of GABA-activated Cl(-) currents. The half-maximal inhibitory concentration (IC(50)) for picrotoxin in alpha2beta2gamma2 receptors was 10.3+/-1.6 microM. The alpha subunit isoform did not affect picrotoxin-induced inhibition, as IC(50) values for alpha3beta2gamma2 (5.1+/-0.7 microM) and alpha6beta2gamma2 receptors (7.2+/-0.4 microM) were comparable to those obtained in alpha2beta2gamma2 receptors. Interestingly, in receptors lacking an alpha subunit (beta2gamma2 configuration), picrotoxin had a markedly lower IC(50) (0.5+/-0.05 microM) compared to alpha-containing receptors. The inhibitory profile was generally similar for the presumed picrotoxin-site ligand U-93631, i.e., IC(50) values were comparable in all alphabetagamma-containing receptors, but the IC(50) in beta2gamma2 receptors was greater than 10-fold lower. In addition, a modest but significant initial stimulation of GABA-activated current by U-93631 was observed in alpha2beta2gamma2 and beta2gamma2 receptors. A mutation in the second transmembrane domain, shown previously to abolish picrotoxin sensitivity, also greatly attenuated sensitivity to U-93631. Moreover, incubation of receptors with excess U-93631 hindered picrotoxin's ability to gain access to its binding site; both results indicate that U-93631 interacts at the picrotoxin site of the receptor. Our results indicate the presence of an alpha subunit hinders the ability of picrotoxin to block the GABA(A) receptor, and thus provides additional insight into the site of action of picrotoxin. In addition, we have shown that domains important for the actions of picrotoxin also affect U-93631. Thus, this compound should prove to be a useful ligand for analysis of the convulsant site of this receptor.     

Differential regulation of GABA(A) and GABA(B) receptors during vestibular compensation

We investigated changes in intrinsic excitability and GABA receptor efficacy in rat medial vestibular nucleus (MVN) neurons following 48 h and 7-10 days of behavioral recovery after unilateral labyrinthectomy (UL) in the rat. The mean in vitro discharge rate of rostral ipsilesional MVN cells at both time points was significantly higher than normal, indicating that the intrinsic excitability of the deafferented cells undergoes a sustained up-regulation during vestibular compensation. In slices from animals that had compensated for 7-10 days after UL, the responsiveness of rostral ipsilesional MVN cells to the GABA(A) agonist muscimol was not different from normal, while the responsiveness to the GABA(B) agonist baclofen was significantly down-regulated. This is in contrast to the situation soon after UL, where the efficacy of both GABA(A) and GABA(B) receptors is markedly down-regulated. The recovery of fast GABA(A) mediated neurotransmission by 7-10 days post-UL presumably enables ipsilesional cells to again respond to vestibular stimulation, through commissural inhibitory modulation from the intact side. The permanent loss of excitatory input from the lesioned side may be, in effect, counteracted by the long-term down-regulation of slow GABA(B) receptors in the de-afferented neurons.     

GABA(A) and GABA(B) receptors on neocortical neurons are differentially distributed

The distribution of functional neurotransmitter receptors on the surface of neurons is highly relevant for synaptic transmission and signal processing. To map functional GABA(A) and GABA(B) receptors on the somadendritic membrane of rat neocortical layer V pyramidal neurons we used patch-clamp recording in combination with infrared-guided laser stimulation to release gamma-aminobutyric acid (GABA) photolytically. The data strongly suggest that relatively more GABA(A) receptors are located at the apical dendrite and relatively more GABA(B) receptors near the soma. Such a specific distribution of GABA(A) and GABA(B) receptors may serve to compensate for differences in electrotonic voltage attenuation between GABA(A) and GABA(B) receptor-mediated inhibitory postsynaptic potentials (IPSPs).     

Involvement of GABA(A) and GABA(B) receptors in afterdischarge generation in rat hippocampal slices

It has been hypothesized that a disruption of gamma-aminobutyric acid (GABA) receptor-mediated processes may be involved in the pathophysiology of focal epilepsy. This disinhibition hypothesis has been postulated from the results of in vitro experiments of the interictal activity of focal epilepsy. Less is known, however, about how disinhibition may be involved in the production of the ictal activity. We therefore examined the pharmacological effects of selective agonists and antagonists of GABA(A) and GABA(B) receptors on ictal-like afterdischarges (ADs) induced following repetitive high-frequency electrical stimulation in the CA1 region of rat hippocampal slices. The GABA(A) receptor antagonist bicuculline (5 microM) fully blocked AD generation, as did the GABA(A) receptor agonist muscimol (2 microM), which is thought to produce a tonic inhibition during application. However, the benzodiazepine receptor agonist diazepam (5 microM), which enhances the inhibitory postsynaptic potential induced by synaptically released GABA, increased the number of spikes in the AD to 148.3% of the control value. On the other hand, the GABA(B) receptor antagonist phaclofen (1 mM) increased the number of spikes in the AD to 234.7% of the control value, while the GABA(B) receptor agonist baclofen (5 microM) reduced it to 46.9%. We therefore conclude that synaptic, but not tonic, activation of GABA(A) receptors appears to be necessary for ictal-like AD generation, while GABA(B) receptor activation plays a protective role. We therefore propose a modification to the simple disinhibition hypothesis.     

Hippocampal hyperexcitability induced by GABA withdrawal is due to down-regulation of GABA(A) receptors.

The sudden interruption of an intracortical instillation of exogenous gamma-aminobutyric acid (GABA) generates an epileptic focus in mammals. Seizures elicited by GABA withdrawal (GW) last for weeks. A similar withdrawal-induced hyperexcitability is also produced by several GABA(A) receptor agonists. This work reports a quantitative analysis of GW-induced hyperexcitability produced in the hippocampus in vitro. GW produced a left-ward displacement of the input/output (I/O) function, suggesting that the postsynaptic component is predominant to explain the hyperexcitability. A decrease in the inhibitory efficacy of the GABA(A) receptor agonist, muscimol, confirmed that inhibition was impaired. Binding saturation experiments demonstrated a decrease in [(3)H]-muscimol binding after GABA withdrawal showing a close correlation with the development of hyperexcitability. All these modifications coursed without changes in receptor affinity (K(D)) for muscimol or bicuculline as demonstrated by both binding studies and Schild analysis. It is concluded that, in the CA1 region of the hippocampus, it is the number of functional GABA(A) receptors, and not the affinity of the receptor, what is decreased during GW-induced hyperexcitability.     

Delayed upregulation of GABA(A) alpha1 receptor subunit mRNA in somatosensory cortex of mice following learning-dependent plasticity of cortical representations.

Experience-dependent modifications of cortical representational maps are accompanied by changes in several components of GABAergic inhibitory neurotransmission system. We examined with in situ hybridization to 35S-labeled oligoprobe changes of expression of GABA(A) receptor alpha1 subunit mRNA in the barrel cortex of mice after sensory conditioning training. One day and 5 days after the end of short lasting (3 daily sessions) training an increased expression of GABA(A) alpha1 mRNA was observed at the cortical site where the plastic changes were previously found. Learning associated activation of the cerebral cortex increases expression of GABA(A) receptor mRNA after a short post-training delays.     

Diversity of inhibitory neurotransmission through GABA(A) receptors

In the brain, highly connected and heterogeneous GABAergic cells are crucial in controling the activity of neuronal networks. They accomplish this task by communicating through remarkably diverse sets of inhibitory processes, the complexity of which is reflected by the variety of interneuron classification schemes proposed in recent years. It is now becoming clear that the subcellular localization and intrinsic properties of heteropentameric GABA(A) receptors themselves also constitute major sources of diversity in GABA-mediated signaling. This review summarizes some of the factors underlying this diversity, including GABA(A) receptor subunit composition, localization, activation, number and phosphorylation states, variance of GABA concentration in the synaptic cleft, and some of the presynaptic factors regulating GABA release.     

The effects of stiripentol on GABA(A) receptors

The anticonvulsant stiripentol (Diacomittm) has been shown to have a positive impact on control of seizures for many patients with Dravet syndrome. As with most antiepileptic drugs, stiripentol has multiple mechanisms of action. Its direct anticonvulsant activity is likely due to enhancement of inhibitory, γ-aminobutyric acid (GABA)ergic neurotransmission. Stiripentol was shown to increase the activity of both neuronal and recombinant GABA(A) receptors at clinically relevant concentrations. At recombinant receptors, stiripentol was found to act through a unique site in a subunit-dependent manner. Positive modulation by stiripentol was most effective at GABA(A) receptors containing an α3 subunit. The expression of the α3 subunit is developmentally regulated, with highest levels in the immature brain. This subunit selectivity may explain the greater clinical efficacy of stiripentol in childhood-onset epilepsies, including Dravet syndrome.     

Layer-specific immunocytochemical localization of GABA(B)R1a and GABA(B)R1b receptors in the rat piriform cortex

A peculiar, layer-segregated immunoreactive distribution of GABABR1a and GABABR1b receptor antibodies is present in the piriform cortex of adult rats. The GABABR1a antibody selectively marked the neuropile in layer Ia, where afferent olfactory fibres and intrinsic GABAergic (gamma-aminobutyric acid) axons terminate on the distal apical dendrites of pyramidal neurons. The GABABR1b antibody was detected in the soma and the large basal dendrites of layer II and III neurons. The pattern of distribution observed supports the hypothesis that (presynaptic) GABABR1a receptors in the superficial molecular layer modulate neurotransmitter release in a feedforward synaptic circuit, whereas GABABR1b (postsynaptic) receptors mediate feedback inhibitory potentials on principal cells.     

Taurine activates GABA(A) but not GABA(B) receptors in rat hippocampal CA1 area

We investigated if taurine, an endogenous GABA analog, could mimic both hyperpolarizing and depolarizing GABA(A)-mediated responses as well as pre- and postsynaptic GABA(B)-mediated actions in the CA1 region of rat hippocampal slices. Taurine (10 mM) perfusion induced changes in membrane potential and input resistance that are compatible with GABA(A) receptor activation. Local pressure application of taurine and GABA from a double barrel pipette positioned along the dendritic shaft of pyramidal cells revealed that taurine evoked a very small change of membrane potential and resistance compared with the large changes induced by GABA in these parameters. Moreover, in the presence of GABA(A) antagonists, local application of GABA on the dendrites evoked a GABA(B)-mediated hyperpolarization while taurine did not induce any change. Taurine neither mimicked baclofen inhibitory actions on presynaptic release of glutamate and GABA as judging by the lack of taurine effect on paired-pulse facilitation ratio and slow inhibitory postsynaptic potentials, respectively. These results show that taurine mainly activates GABA(A) receptors located on the cell body, indicating therefore that if taurine has any action on the dendrites it will not be mediated by either GABA(A) or GABA(B) receptors activation.     

Pre- and postnatal development of GABA receptors in Macaca monkey visual cortex.

GABA is a putative inhibitory neurotransmitter in adult mammalian visual cortex but also has been implicated as playing a crucial role in cortical information processing during development. In order to understand better the role of GABA during primate visual cortex development, we have examined the time course of GABAA and GABAB receptor ontogenesis in 18 Macaca nemestrina monkeys ranging from fetal day 61 (F61d) to adulthood. The GABA and benzodiazepine binding sites of the GABAA receptor were detected by 3H-muscimol (3H-MS) and 3H-flunitrazepam (3H-FZ), respectively. GABAB receptors were detected by 3H-baclofen (3H-BA). All ligands were visualized by in vitro autoradiography. Quantitative analysis of film density was done to compare laminar changes during pre- and postnatal development. Saturation binding experiments were done for MS and FZ binding sites to determine receptor number (Bmax) and affinity (Kd) at selected pre- and postnatal ages. Both MS and FZ binding sites were present at F61d-72d throughout the cortical plate and marginal zone. FZ binding sites were more dense than MS binding sites over the cortical plate at young ages and were especially dense over the marginal zone. FZ binding sites also were present in lesser amounts over the subplate and intermediate zone, but not over the subventricular zone. By F119d-126d, layer 4 could be distinguished by its higher density for both ligands. The basic adult laminar pattern was established for both MS and BZ binding sites by birth (birth = F165d-170d). After birth, MS density increases dramatically in all layers, but layer 4C remains most dense to adulthood. FZ labeling is heavy in both layers 4 and 3 at birth but after 4 weeks after birth (P4 wk) it declines somewhat in the supragranular layers so that layer 4C now predominates. Labeling in layers 5/6 virtually disappears after birth. BA binding sites were present at F126d, at which time layer 4 was slightly lighter than the remainder of striate cortex; this laminar pattern remained basically the same throughout our series to adulthood. Competitive binding of agonist and antagonists for the GABAA receptor showed that MS binding characteristics were similar at F126d and P8.5 years (yr). MS binding site Bmax was about 8% of adult values at F72d, 24% by F126d, and 56% at F152d. Bmax then rose rapidly after birth to peak at P18wk at 169% of adult values, and then declined to P1yr. A second peak of 143% was found around P3.5yr, with adult values reached by P8.5yr.(ABSTRACT TRUNCATED AT 400 WORDS)     

Laminal distribution of gamma-aminobutyric acid (GABA) in the occipital cortex of rats: evidence as a neurotransmitter

Serotonergic pathways from brainstem to spinal cord play an important role in the modulation of pain perception. To establish where that modulation occurs, we examined serotonin-immunoreactive axonal contacts on individually characterized laminae I and II dorsal horn neurons intracellularly filled with horseradish peroxidase. We found serotonin axonal contacts on marginal, stalked, and islet cells. Contacts preferentially occurred on dendritic shafts rather than on spines. Marginal and stalked cells received the heaviest innervation.     

Saturation and self-inhibition of rat hippocampal GABA(A) receptors at high GABA concentrations

Current responses to ultrafast gamma-aminobutyric acid (GABA) applications were recorded from excised patches in rat hippocampal neurons to study the gating properties of GABA(A) receptors at GABA concentrations close to saturating ones and higher. The amplitude of currents saturated at approximately 1 mm, while the onset rate of responses reached saturation at 4-6 mm GABA. At high GABA concentrations (> 10 mm), the amplitude of current responses was reduced in a dose-dependent manner with a half-blocking GABA concentration of approximately 50 mm. The peak reduction at high GABA doses was accompanied by a tendency to increase the steady-state to peak ratio. At concentrations higher than 30 mm, this effect took the form of a rebound current, i.e. during the prolonged GABA applications, the current firstly declined due to desensitization onset and then, instead of decreasing towards a steady-state value, clearly increased. Both the self-inhibition of GABA(A) receptors by high GABA doses and rebound were clearly voltage dependent, being larger at positive holding potentials. The fast desensitization component accelerated with depolarization at all saturating [GABA] tested. The rebound phenomenon indicates that the self-block of GABAA receptors is state dependent, and suggests that the sojourn in the desensitized conformation provides a 'rescue' from the block. We propose that high GABA concentrations inhibit the receptors by direct occlusion of the channel pore having no effect on the receptor gating.     

Differential alcohol modulation of GABA(A) and NMDA receptors

NMDA and GABA(A) receptors are believed to be important CNS targets of alcohol action. In mouse hippocampal neurons, n-alcohols from ethanol to dodecanol enhanced GABA-activated ion current, whereas higher alcohols had no effect. Alcohols below pentanol affected NMDA receptors more potently than GABA(A) receptors. Increasing alcohol carbon chain length produced a greater average change in apparent binding energy and potency for modulation of GABA(A) than of NMDA receptor-channels, with the result that alcohols above pentanol affected GABA(A) receptors more potently than NMDA receptors. The anesthetic potency of n-alcohols in rats more closely reflected NMDA receptor modulatory potency for lower alcohols and GABA(A) receptor modulatory potency for higher alcohols. The results suggest that there may be fundamental differences in the sites through which alcohols affect NMDA and GABA(A) receptor function.     

GABA(A) receptors mediate inhibition of T cell responses

We describe the presence of functional GABA(A) receptors on T cells. GABA inhibited anti-CD3 and antigen-specific T cell proliferation in vitro in a dose-dependent manner that was 1) mimicked by the GABA(A) receptor agonist muscimol (but not the GABA(B) receptor agonist baclofen), 2) blocked by GABA(A) receptor antagonists and a GABA(A) receptor Cl- channel blocker (picrotoxin) and 3) enhanced by pentobarbital. These data suggest that GABA(A) receptors mediate this immune inhibition and that these receptors can be modulated in a similar fashion to their neuronal counterparts. Finally, GABA inhibited DTH responses in vivo. Thus, pharmacological modulation of GABA(A) receptors may provide new approaches to modulate T cell responses in inflammation and autoimmune disease.