Metabotropic GABA Receptors Research Articles

GABA modulates Drosophila circadian clock neurons via GABAB receptors and decreases in calcium

Circadian clocks play vital roles in the control of daily rhythms in physiology and behavior of animals. In Drosophila, analysis of the molecular and behavioral rhythm has shown that the master clock neurons are entrained by sensory inputs and are synchronized with other clock neurons. However, little is known about the neuronal circuits of the Drosophila circadian system and the neurotransmitters that act on the clock neurons. Here, we provide evidence for a new neuronal input pathway to the master clock neurons, s-LN(v)s, in Drosophila that utilizes GABA as a slow inhibitory neurotransmitter. We monitored intracellular calcium levels in dissociated larval s-LN(v)s with the calcium-sensitive dye Fura-2. GABA decreased intracellular calcium in the s-LN(v)s and blocked spontaneous oscillations in calcium levels. The duration of this response was dose-dependent between 1 nM and 100 microM. The response to GABA was blocked by a metabotropic GABA(B) receptor (GABA(B)-R) antagonist, CGP54626, but not by an ionotropic receptor antagonist, picrotoxin. The GABA(B)-R agonist, 3-APMPA, produced a response similar to GABA. An antiserum against one of the Drosophila GABA(B)-Rs (GABA(B)-R2) labeled the dendritic regions of the s-LN(v)s in both adults and larvae, as well as the dissociated s-LN(v)s. We found that some GABAergic processes terminate at the dendrites of the LN(v)s, as revealed by GABA immunostaining and a GABA-specific GAL4 line (GAD1-gal4). Our results suggest that the s-LN(v)s receive slow inhibitory GABAergic inputs that decrease intracellular calcium of these clock neurons and block their calcium cycling. This response is mediated by postsynaptic GABA(B) receptors.

Expression of GABAB Receptors in Magnocellular Neurosecretory Cells of Male, Virgin Female and Lactating Rats

GABA is one of the key neurotransmitters that regulate the firing activity of neurones in the supraoptic (SON) and paraventricular (PVN) nuclei. In the present study, we used immunohistochemical techniques to study the distribution and subcellular localisation of metabotropic GABA(B) receptors in magnocellular neurones in the SON and PVN. Robust GABA(B) receptor immunoreactivity (GABA(B)R; both subunit 1 and subunit 2 of the heterodimer), was observed in the SON and PVN. At the light microcope level, GABA(B)R immonoreactivity displayed a clustered pattern localised both intracytoplasmically and at the plasma membrane. Densitometry analysis indicated that GABA(B)R immunoreactivity was significantly more intense in vasopressin cells than in oxytocin cells, both in male, virgin female and lactating rats, and was denser in males than in virgin females. Light and electron microscope studies indicated that cytoplasmic GABA(B)R was localised in various organelles, including the Golgi, early endosomes and lysosomes, suggesting the cycling of the receptor within the endocytic and trafficking pathways. Some smaller clusters at the level of the cell plasma membrane were apposed to glutamic acid decarboxylase 67 immunoreactive boutons, and appeared to be colocalised with gephyrin, a constituent protein of the postsynaptic density at inhibitory synapses. The presence of GABA(B)R immunoreactivity at synaptic and extrasynaptic sites was supported by electron microscopy. These results provide anatomical evidence for the expression of postsynaptic GABA(B) receptors in magnocellular neurosecretory cells.

Determination of the minimal functional ligand-binding domain of the GABAB1b receptor.

In the mammalian central nervous system, slow inhibitory neurotransmission is largely mediated by metabotropic GABA(B) receptors (where GABA stands for gamma-aminobutyric acid), which belong to the G-protein-coupled receptor gene family. Functional GABA(B) receptors are assembled from two subunits GABA(B1) (GABA(B) receptor subtype 1) and GABA(B2). For the GABA(B1) subunit, which binds the neurotransmitter GABA, two variants GABA(B1a) (GABA(B) receptor subtype 1 variant a) and GABA(B1b) have been identified. They differ at the very N-terminus of their large glycosylated ECD (extracellular domain). To simplify the structural characterization, we designed truncated GABA(B1) receptors to identify the minimal functional domain which still binds a competitive radioligand and leads to a functional, GABA-responding receptor when co-expressed with GABA(B2). We show that it is necessary to include all the portion of the ECD encoded by exon 6 to exon 14. Furthermore, we studied mutant GABA(B1b) receptors, in which single or all potential N-glycosylation sites are removed. The absence of oligosaccharides does not impair receptor function, suggesting that the unglycosylated ECD of GABA(B1) can be used for further functional or structural investigations.

Synaptic and extrasynaptic GABA-A and GABA-B receptors in the globus pallidus: An electron microscopic immunogold analysis in monkeys

GABA-A and GABA-B receptors mediate differential effects in the CNS. To better understand the role of these receptors in regulating pallidal functions, we compared their subcellular and subsynaptic localization in the external and internal segments of the globus pallidus (GPe and GPi) in monkeys, using pre- and post-embedding immunocytochemistry with antibodies against GABA-A (α1, β2/3 subunits) and GABA-BR1 receptor subtype. Our results demonstrate that GABA-A and GABA-B receptors display a differential pattern of subcellular and subsynaptic localization in both segments of the globus pallidus. The majority of GABA-BR1 immunolabeling is intracellular, whereas immunoreactivity for GABA-A receptor subunits is mostly bound to the plasma membrane. A significant proportion of both GABA-BR1 and GABA-A receptor immunolabeling is extrasynaptic, but GABA-A receptor subunits also aggregate in the main body of putative GABAergic symmetric synapses established by striatal- and pallidal-like terminals. GABA-BR1 immunoreactivity is expressed presynaptically in putative glutamatergic terminals, while GABA-A α1 and β2/3 receptor subunits are exclusively post-synaptic and often coexist at individual symmetric synapses in both GPe and GPi. In conclusion, our findings corroborate the concept that ionotropic and metabotropic GABA receptors are located to subserve different effects in pallidal neurons. Although the aggregation of GABA-A receptors at symmetric synapses is consistent with their role in fast inhibitory synaptic transmission, the extrasynaptic distribution of both GABA-A and GABA-B receptors provides a substrate for complex modulatory functions that rely predominantly on the spillover of GABA.

Gene expression profiling of the rat superior olivary complex using serial analysis of gene expression

The superior olivary complex (SOC) is an auditory brainstem region that represents a favourable system to study rapid neurotransmission and the maturation of neuronal circuits. Here we performed serial analysis of gene expression (SAGE) on the SOC in 60-day-old Sprague-Dawley rats to identify genes specifically important for its function and to create a transcriptome reference for the subsequent identification of age-related or disease-related changes. Sequencing of 31 035 tags identified 10 473 different transcripts. Fifty-seven per cent of the unique tags with a count greater than four were statistically more highly represented in the SOC than in the hippocampus. Among them were genes encoding proteins involved in energy supply, the glutamate/glutamine shuttle, and myelination. Approximately 80 plasma membrane transporters, receptors, channels, and vesicular transporters were identified, and 25% of them displayed a significantly higher expression level in the SOC than in the hippocampus. Some of the plasma membrane proteins were not previously characterized in the SOC, e.g. the purinergic receptor subunit P2X(6) and the metabotropic GABA receptor Gpr51. Differential gene expression between SOC and hippocampus was confirmed using RNA in situ hybridization or immunohistochemistry. The extensive gene inventory presented here will alleviate the dissection of the molecular mechanisms underlying specific SOC functions and the comparison with other SAGE libraries from brain will ease the identification of promoters to generate region-specific transgenic animals. The analysis will be part of the publicly available database ID-GRAB.

Endocannabinoid signalling selectively targets perisomatic inhibitory inputs to pyramidal neurones in juvenile mouse neocortex.

Retrograde synaptic signalling has long been recognized as a fundamental feature of neural systems. However, the cellular specificity and functional consequences of fast retrograde communication are not well understood. We have focused our efforts on understanding the role that endocannabinoids play in regulating synaptic inhibition in sensory neocortex. Recent studies have implicated endocannabinoids as the retrograde signalling molecules that underlie depolarization-induced suppression of inhibition, or DSI. This short-term form of presynaptic depression is triggered by postsynaptic depolarization and is likely to play an important role in information processing. In the present study we investigated the cellular and synaptic specificity of endocannabinoid signalling in sensory cortex using whole-cell recordings from layer 2/3 pyramidal neurones (PNs) in acute brain slices. We report that GABAergic interneurones that are depolarized by muscarinic receptor stimulation provided the majority of DSI-susceptible inputs to neocortical PNs. This subclass of interneurones generated large, fast postsynaptic currents in PNs which were transiently suppressed by either postsynaptic depolarization or a brief train of action potentials. Neocortical DSI required activation of the type 1 cannabinoid receptor (CB1R) but not metabotropic glutamate or GABA receptors. Using focal drug application, we found that the DSI-susceptible afferents preferentially synapse on the perisomatic membrane of PNs, and not on the apical dendrites. Together, these results suggest that endocannabinoid-mediated DSI in the cortex can transiently and selectively depress a subclass of PN inputs. Although the physiological implications remain to be explored, this suppression of somatic inhibition may alter the excitability of principal neurones and thereby modulate cortical output.

Up-regulation of NMDAR1 subunit gene expression in cortical neurons via a PKA-dependent pathway.

Transcription mediated by protein kinase A and the cAMP response element binding protein (CREB) has been linked to the establishment of long-term memory and cell survival. However, all of the major targets for activated CREB have yet to be identified. Given the fact that CREB-mediated transcription is intimately involved in cellular processes of learning and memory and that CREB activity can be regulated by synaptic N-methyl-d-aspartate receptors (NMDARs) and metabotropic GABA receptors, we have studied the role of the cAMP-dependent signaling pathway in the regulation of the NMDA receptor subunit 1 (NMDAR1), a subunit required for functional receptor formation. We now report that levels of NMDAR1 subunit protein in primary neocortical cultures are increased 66% in response to forskolin, an activator of adenylyl cyclase. Up-regulation of NMDAR1 is paralleled by a twofold increase in mRNA levels and an 83% increase in NMDAR1 promoter/luciferase reporter activity that is dependent on protein kinase A. Three cAMP regulatory elements (CREs) in the rat NMDAR1 promoter (- 228, - 67, and - 39) bind CREB in vitro and forskolin increases binding to two of the sites (- 228 and - 67). Chromatin immunoprecipitation of neuronal rat genomic DNA reveals that CREB is bound in vivo to the endogenous NMDAR1 gene. Increased presence of the activated Ser133 phosphorylated form is dependent on the length of exposure to forskolin. Taken together with the results of mutational analysis, the findings strongly suggest that transcription of NMDAR1 is regulated by the c-AMP signaling pathway, most likely through the binding of CREB and its activation by signal-dependent phosphorylation.

Neurotensin depolarizes globus pallidus neurons in rats via neurotensin type-1 receptor

The globus pallidus is a major component in the indirect pathway of the basal ganglia. There is evidence that neurotensin receptors exist in this nucleus. To determine the electrophysiological effects of neurotensin on pallidal neurons, whole-cell patch-clamp recordings were performed in the acutely prepared brain slices. Under current-clamp recordings, neurotensin at 1 μM depolarized pallidal neurons. Voltage-clamp recordings also showed an inward current induced by neurotensin. The depolarizing effect of neurotensin could be mimicked by the C-terminal fragment, neurotensin (8–13), but not by the N-terminal fragment, neurotensin (1–8). Both SR 142948A, a non-selective neurotensin receptor type-1 and type-2 antagonist, and SR 48692, a selective type-1 receptor antagonist, blocked the depolarizing effect of neurotensin, and which themselves had no effect on membrane potential. Thus, neurotensin type-1 receptors appear to mediate the effect of neurotensin. The depolarization evoked by neurotensin persisted in the presence of tetrodotoxin, ionotropic and metabotropic glutamate and GABA receptor antagonists, indicating that neurotensin excited the pallidal neurons by activating the receptor expressed on the neurons recorded. Current-voltage relationship revealed that both the suppression of a potassium conductance and the activation of a cationic conductance are involved in the neurotensin-induced depolarization. Based on the action of neurotensin in the globus pallidus we hypothesize that alterations of the striatopallidal neurotensin system contribute to symptoms of basal ganglia motor disorders.

GABAB receptor agonist baclofen has non-specific antinociceptive effect in the model of peripheral neuropathy in rat.

Baclofen, which is a specific agonist of the metabotropic GABA(B) receptor, is used in clinical practice for the treatment of spasticity of skeletal muscles. It also exerts an analgesic effect, but this effect is still not clear and especially controversial in neuropathic pain. In this work, we studied the antinociceptive effects of baclofen in a model of chronic peripheral neuropathic pain - loose ligation of the sciatic nerve (chronic constriction injury, CCI). As controls we used sham-operated animals. The changes of thermal pain threshold were measured using the plantar test 15-25 days after the operation. The obtained results suggest that baclofen increases pain threshold in both groups. The antinociceptive effect of baclofen was dose-dependent and the maximum response without motor deficits was observed at a dose of 15 mg/kg s.c. In the rats with CCI, significant differences between affected (ipsilateral) and contralateral hind paw were present. This difference was dose-dependent, the highest value (6.2+/-1.37 s) was found at the dose of 20 mg/kg. Based on our results and previous findings it could be summarized that baclofen has antinociceptive action, which is attenuated in the model of chronic neuropathic pain probably due to the degeneration of GABA interneurons after chronic constriction injury.

Distribution of metabotropic GABA receptor subunits GABAB1a/b and GABAB2 in the rat hippocampus during prenatal and postnatal development.

Metabotropic gamma-aminobutyric acid receptors (GABAB) play modulatory roles in central synaptic transmission and are involved in controlling neuronal migration during development. We used immunohistochemical methods to elucidate the expression pattern as well as the cellular and the precise subcellular localization of the GABA(B1a/b) and GABAB2 subunits in the rat hippocampus during prenatal and postnatal development. At the light microscopic level, both GABA(B1a/b) and GABAB2 were expressed in the hippocampal primordium from embryonic day E14. During postnatal development, immunoreactivity for GABA(B1a/b) and GABAB2 was distributed mainly in pyramidal cells, with discrete GABA(B1a/b)-immunopositive cell bodies of interneurons present throughout the hippocampus. Using double immunofluorescence, we demonstrated that during the second week of postnatal development, GABA(B1a/b) but not GABAB2 was expressed in glial cells throughout the hippocampal formation. At the electron microscopic level, GABA(B1a/b) and GABAB2 showed a similar distribution pattern during postnatal development. Thus, at all ages the two receptor subunits were located postsynaptically in dendritic spines and shafts at extrasynaptic and perisynaptic sites in both pyramidal and nonpyramidal cells. We further demonstrated that the two subunits were localized presynaptically along the extrasynaptic plasma membrane of axon terminals and along the presynaptic active zone in both asymmetrical and, to a lesser extent, symmetrical synapses. These results suggest that GABAB receptors are widely expressed in the hippocampus throughout development and that GABA(B1a/b) and GABAB2 form both pre- and postsynaptic receptors.

The subcellular localization of GABA(B) receptor subunits in the rat substantia nigra.

The inhibitory effects of GABA within the substantia nigra (SN) are mediated in part by metabotropic GABA(B) receptors. To better understand the mechanisms underlying these effects, we have examined the subcellular localization of the GABA(B) receptor subunits, GABA(B1) and GABA(B2), in SN neurons and afferents using pre-embedding immunocytochemistry combined with anterograde or retrograde labelling. In both the SN pars compacta (SNc) and pars reticulata (SNr), GABA(B1) and GABA(B2) showed overlapping, but distinct, patterns of immunolabelling. GABA(B1) was more strongly expressed by putative dopaminergic neurons in the SNc than by SNr projection neurons, whereas GABA(B2) was mainly expressed in the neuropil of both regions. Immunogold labelling for GABA(B1) and GABA(B2) was localized in presynaptic and postsynaptic elements throughout the SN. The majority of labelling was intracellular or was associated with extrasynaptic sites on the plasma membrane. In addition, labelling for both subunits was found on the presynaptic and postsynaptic membranes at symmetric, putative GABAergic synapses, including those formed by anterogradely labelled striatonigral and pallidonigral terminals. Labelling was also observed on the presynaptic membrane and at the edge of the postsynaptic density at asymmetric, putative excitatory synapses. Double immunolabelling, using the vesicular glutamate transporter 2, revealed the glutamatergic nature of many of the immunogold-labelled asymmetric synapses. The widespread distribution of GABA(B) subunits in the SNc and SNr suggests that GABA(B)-mediated effects in these regions are likely to be more complex than previously described, involving presynaptic autoreceptors and heteroreceptors, and postsynaptic receptors on different populations of SN neurons.

Phosphorylation-independent desensitization of GABA(B) receptor by GRK4.

Agonist-promoted desensitization of the heterodimeric metabotropic GABA(B) receptor was investigated. Whereas no desensitization was observed in HEK293 cells heterologously expressing the receptor, GABA and the synthetic agonist baclofen induced a robust desensitization in cerebellar granule cells endogenously expressing the receptor. Taking advantage of this cell-specific desensitization phenotype, we identified GRK4 as the kinase involved in the neuronal desensitization. Transfection of small interference RNA directed against GRK4 significantly reduced GRK4 levels in cerebellar granule cells and strongly inhibited the agonist-promoted desensitization. Reciprocally, transfection of GRK4 in HEK293 cells restored agonist-promoted desensitization, confirming that this kinase is sufficient to support desensitization. Surprisingly, this desensitization occurred in the absence of ligand-induced receptor phosphorylation and could be promoted by GRK4 mutants deleted of their kinase domain. Taken together, these results suggest that GRK4 plays a central role in the agonist-promoted desensitization of GABA(B) receptor and that it does so through an atypical mechanism that challenges the generally accepted model linking the kinase activity of GRKs to their role in receptor desensitization.

Dynamic balance of metabotropic inputs causes dorsal horn neurons to switch functional states.

Sensory relay structures in the spinal cord dorsal horn are now thought to be active processing structures that function before supraspinal sensory integration. Dorsal horn neurons directly receive nociceptive (pain) signals from the periphery, express a high degree of functional plasticity and are involved in long-term sensitization and chronic pain. We show here that deep dorsal horn neurons (DHNs) in Wistar rats can switch their intrinsic firing properties from tonic to plateau or endogenous bursting patterns, depending upon the balance of control by metabotropic glutamate (mGlu) and GABA(B) receptors. We further show that this modulation acts on at least one common target, the inwardly rectifying potassium channel (Kir3). Finally, we found that these firing modes correspond to specific functional states of information transfer in which dorsal horn neurons can faithfully transmit, greatly enhance or block the transfer of nociceptive information.

Ethanol antagonizes kainate receptor-mediated inhibition of evoked GABA(A) inhibitory postsynaptic currents in the rat hippocampal CA1 region.

Many studies have demonstrated that ethanol reduces glutamatergic synaptic transmission primarily by inhibiting the N-methyl-D-aspartate subtype of glutamate receptor. In contrast, the other two subtypes of ionotropic glutamate receptor (alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid and kainate) have generally been shown to be insensitive to intoxicating concentrations of ethanol. However, we have previously identified a population of kainate receptors that mediate slow excitatory postsynaptic currents in the rat hippocampal CA3 pyramidal cell region that is potently inhibited by low concentrations of ethanol. In this study, we examined the effect of ethanol on kainate receptor-mediated inhibition of evoked GABA(A) inhibitory postsynaptic currents (IPSCs) in the rat hippocampal CA1 pyramidal cell region. Under our recording conditions, bath application of 1 microM kainate significantly inhibited GABA(A) IPSCs. This inhibition seemed to be mediated by the activation of somatodendritic kainate receptors on GABAergic interneurons and the subsequent activation of metabotropic GABA(B) receptors, because the kainate inhibition was largely blocked by pretreating slices with a GABA(B) receptor antagonist. Ethanol pretreatment significantly antagonized the inhibitory effect of kainate on GABA(A) IPSCs, at concentrations as low as 20 mM. In contrast, ethanol did not block the direct inhibitory effect of a GABA(B) receptor agonist on GABA(A) IPSCs. The results of this study suggest that modest concentrations of ethanol may antagonize presynaptic, as well as postsynaptic, kainate receptor function in the rat hippocampus.

Expression and distribution of metabotropic GABA receptor subtypes GABABR1 and GABABR2 during rat neocortical development.

To understand the possible contribution of metabotropic gamma-aminobutyric acid receptors (GABABR) in cortical development, we investigated the expression pattern and the cellular and subcellular localization of the GABABR1 and GABABR2 subtypes in the rat neocortex from embryonic day 14 (E14) to adulthood. At the light microscopic level, both GABABR1 and GABABR2 were detected as early as E14. During prenatal development, both subtypes were expressed highly in the cortical plate. Using double immunofluorescence, GABABR1 colocalized with GABABR2 in neurons of the marginal zone and subplate, indicating that these proteins are coexpressed and could be forming functional GABABRs during prenatal development in vivo. In contrast, only GABABR1 but not GABABR2 was detected in the tangentially migratory cells in the lower intermediate zone. During postnatal development, immunoreactivity for GABABR1 and GABABR2 was distributed mainly in pyramidal cells. Discrete GABABR1-immunopositive cell bodies of interneurons were present throughout the neocortex. In addition, GABABR1 but not GABABR2 was found in identified Cajal-Retzius cells in layer I. At the electron microscopic level, immunoreactivity for GABABR1 and GABABR2 was found in dendritic spines and dendritic shafts at extrasynaptic and perisynaptic sites throughout postnatal development. We further demonstrated the presynaptic localization of GABABR1 and GABABR2, as well as the association of the receptors with asymmetrical synaptic junctions. These results indicate potentially important roles for the GABABRs in the regulation of migratory processes during corticogenesis and in the modulation of synaptic transmission during early development of cortical circuitry.

Cholinergic transmission via central synapses in the locust nervous system.

In this study we examine the nature of chemical synaptic transmission between identified filiform hair receptors on the prothoracic segment of a locust and the identified postsynaptic projection interneuron (A4I1). The effects of pressure ejected acetylcholine, and various ligands of acetylcholine receptors on the activity of the postsynaptic neuron A4I1, or on wind-elicited responses in A4I1 are reported. It is suggested that the transmitter of the afferent fibers is acetylcholine, and that fast transmission is mediated by nicotinic acetylcholine-receptors. Both nicotine and carbachol act as agonists, whereas d-tubocurarine and alpha-bungarotoxin act as antagonists. The presence of muscarinic acetylcholine receptors was also evident from the modulatory effects of muscarine, oxotremorine and pilocarpine, which were blocked by bath application of atropine. GABA, and its agonists muscimol and cis-4-amino-crotonic-acid lead to inhibition of A4I1 responses. This inhibition was prevented by the additional application of picrotoxin. This suggests involvement of a ligand-gated GABA receptor which, most likely, increases chloride conductance. Metabotropic GABA-receptors do not seem to be involved, since baclofene, diazepam and bicuculline ejections had no effects. Glutamate also inhibits wind elicited A4I1 responses. Although attempts were made to further characterize the receptor involved, tested substances such as kainic acid, glycine, CNQX or GDEE had no effect.

Negative Cross Talk between Anionic GABAAand Cationic P2X Ionotropic Receptors of Rat Dorsal Root Ganglion Neurons

Using whole-cell patch-clamp recording and intracellular Ca(2+) imaging of rat cultured DRG neurons, we studied the cross talk between GABA(A) and P2X receptors. A rapidly fading current was the main response to ATP, whereas GABA elicited slowly desensitizing inward currents. Coapplication of these agonists produced a total current much smaller than the linear summation of individual responses (68 +/- 5% with 10 microm ATP plus 100 microm GABA). Occlusion was observed regardless of ATP response type. Neurons without functional P2X receptors manifested no effect of ATP on GABA currents (and vice versa). Occlusion was also absent in the presence of the P2X blocker trinitrophenyl-ATP (TNP-ATP) or of the GABA blocker picrotoxin, indicating a lack of involvement by metabotropic ATP or GABA receptors. Less occlusion was obtained when ATP was applied 2 sec after GABA than when GABA was applied after ATP. Changing the polarity of GABA currents by using intracellular SO(4)2- instead of Cl(-) significantly reduced the occlusion of ATP currents by GABA, suggesting an important role for Cl(-) efflux in this phenomenon. Occlusion was enhanced whenever intracellular Ca(2+) ([Ca(2+)](i)) was not buffered, indicating the cross talk-facilitating role of this divalent cation. Ca(2+) imaging showed that ATP (but not GABA) increased [Ca(2+)](i) in voltage-clamped or intact neurons. Our data demonstrated a novel Cl(-) and Ca(2+)-dependent interaction between cationic P2X and anionic GABA(A) receptors of DRG neurons. Such negative cross talk might represent a model for a new mechanism to inhibit afferent excitation to the spinal cord as GABA and ATP are coreleased within the dorsal horn.

Ionotropic and metabotropic GABA and glutamate receptors in primate basal ganglia

The functions of glutamate and GABA in the CNS are mediated by ionotropic and metabotropic, G protein-coupled, receptors. Both receptor families are widely expressed in basal ganglia structures in primates and nonprimates. The recent development of highly specific antibodies and/or cDNA probes allowed the better characterization of the cellular localization of various GABA and glutamate receptor subtypes in the primate basal ganglia. Furthermore, the use of high resolution immunogold techniques at the electron microscopic level led to major breakthroughs in our understanding of the subsynaptic and subcellular localization of these receptors in primates. In this review, we will provide a detailed account of the current knowledge of the localization of these receptors in the basal ganglia of humans and monkeys.

Cloning and tissue distribution of a novel isoform of the rat GABA(B)R1 receptor subunit.

We have identified a novel splice variant of the metabotropic GABA(B) receptor (R) subunit I, designated GABA(B)R1f, from a rat hippocampus cDNA library screen. GABA(B)R1f shares sequence homology with rat GABA(B)R1a, with the exception of an in-frame deletion of exon 4, resulting in a 21 bp deletion in the coding sequence of the N-terminal extracellular domain. In addition, GABA(B)R1f also contains a 93 bp in-frame insertion in a region of the sequence corresponding to the second extracellular loop and the fifth transmembrane domain, similar to that found in rat GABA(B)R1c. While being ubiquitously (but variably) expressed, reverse-transcription polymerase chain reaction analysis revealed the GABA(B)R1f isoform to be most prevalent in peripheral vs central tissues, suggesting a potential role for this novel isoform in either the mediation of inhibitory transmission in these various tissues, or in as yet defined actions unrelated to central synaptic regulatory mechanisms attributable to GABA(B)R.

Multireceptor GABAergic regulation of synaptic communication in amphibian retina.

The synaptic output of retinal bipolar cells was monitored by recording light-evoked EPSCs in ganglion cells. Application of (RS)-2-amino-3-(3-hydroxy-5-tert-butyl-4-isoxazolyl (ATPA), a selective agonist at kainate receptors, depolarized amacrine cells and reduced the light-evoked excitatory current (L-EPSC) in ganglion cells. ATPA had only a slight effect on the light responses of bipolar cells. Therefore, ATPA suppresses bipolar cell synaptic output to ganglion cells. ATPA reduced the transient L-EPSC, but had comparatively little effect on sustained L-EPSC, of ganglion cells. The transient ON L-EPSC was more suppressed than the transient OFF L-EPSC. Thus, ATPA preferentially suppressed transient output from bipolar cells.GABA receptor antagonists blocked the effect of ATPA. This indicates that ATPA stimulated an endogenous inhibitory feedback pathway that suppressed bipolar cell output.CGP55845 and CGP35348 reduced the ATPA-induced suppression of L-EPSCs in ganglion cells, signifying that part of the feedback pathway is mediated by metabotropic GABA receptors.(1,2,5,6-Tetrahydropyridine-4-yl)-methylphosphinic acid (TPMPA) and picrotoxin, GABAC receptor antagonists, reduced the ATPA effect. Picrotoxin was more effective than ATPA. However, picrotoxin blocked only a part of this GABAC effect, while imidazole-4-acetic acid (I4AA) blocked another segment of the effect. This indicates that two pharmacologically distinct GABAC receptors mediate feedback to bipolar cells. SR95531 produced a very small suppression of the ATPA effect. Thus, GABAA receptors provide a negligible component to this feedback pathway. The experiments indicate that endogenous GABAergic feedback to bipolar cells suppresses their output, and that this feedback is mediated by at least three types of GABA receptor, both metabotropic and ionotropic.In conjunction with previous studies, the results indicate that feedback inhibition is the predominant factor regulating transient signalling in ganglion cells, while feedforward inhibition is the primary regulator of tonic ganglion cell signals.