GABAC Receptors Research Articles

Neurochemicals for the Investigation of GABAC Receptors

GABA(C) receptors are being investigated for their role in many aspects of nervous system function including memory, myopia, pain and sleep. There is evidence for functional GABA(C) receptors in many tissues such as retina, hippocampus, spinal cord, superior colliculus, pituitary and the gut. This review describes a variety of neurochemicals that have been shown to be useful in distinguishing GABA(C) receptors from other receptors for the major inhibitory neurotransmitter GABA. Some selective agonists (including (+)-CAMP and 5-methyl-IAA), competitive antagonists (such as TPMPA, (±)-cis-3-ACPBPA and aza-THIP), positive (allopregnanolone) and negative modulators (epipregnanolone, loreclezole) are described. Neurochemicals that may assist in distinguishing between homomeric ρ1 and ρ2 GABA(C) receptors (2-methyl-TACA and cyclothiazide) are also covered. Given their less widespread distribution, lower abundance and relative structural simplicity compared to GABA(A) and GABA(B) receptors, GABA(C) receptors are attractive drug targets.

Novel Cyclic Phosphinic Acids as GABAC ρ Receptor Antagonists: Design, Synthesis, and Pharmacology.

Understanding the role of GABAC receptors in the central nervous system is limited due to a lack of specific ligands. Novel γ-aminobutyric acid (GABA) analogues based on 3-(aminomethyl)-1-oxo-1-hydroxy-phospholane 17 and 3-(guanido)-1-oxo-1-hydroxy-phospholane 19 were investigated to obtain selective GABAC receptor antagonists. A compound of high potency (19, K B = 10 μM) and selectivity (greater than 100 times at ρ1 GABAC receptors as compared to α1β2γ2L GABAA and GABAB(1b,2) receptors) was obtained. The cyclic phosphinic acids (17 and 19) are novel lead agents for developing into more potent and selective GABAC receptor antagonists with increased lipophilicity for future in vivo studies.

Multiple pathways of inhibition shape bipolar cell responses in the retina

Bipolar cells (BCs) are critical relay neurons in the retina that are organized into parallel signaling pathways. The three main signaling pathways in the mammalian retina are the rod, ON cone, and OFF cone BCs. Rod BCs mediate incrementing dim light signals from rods, and ON cone and OFF cone BCs mediate incrementing and decrementing brighter light signals from cones, respectively. The outputs of BCs are shaped by inhibitory inputs from GABAergic and glycinergic amacrine cells in the inner plexiform layer, mediated by three distinct types of inhibitory receptors: GABA(A), GABA(C), and glycine receptors. The three main BC pathways receive distinct forms of inhibition from these three receptors that shape their light-evoked inhibitory signals. Rod BC inhibition is dominated by slow GABA(C) receptor inhibition, while OFF cone BCs are dominated by glycinergic inhibition. The inhibitory inputs to BCs are also shaped by serial inhibitory connections between GABAergic amacrine cells that limit the spatial profile of BC inhibition. We discuss our recent studies on how inhibitory inputs to BCs are shaped by receptor expression, receptor properties, and neurotransmitter release properties and how these affect the output of BCs.

Fragrant Dioxane Derivatives Identify β1-Subunit-containing GABAA Receptors

Nineteen GABA(A) receptor (GABA(A)R) subunits are known in mammals with only a restricted number of functionally identified native combinations. The physiological role of beta1-subunit-containing GABA(A)Rs is unknown. Here we report the discovery of a new structural class of GABA(A)R positive modulators with unique beta1-subunit selectivity: fragrant dioxane derivatives (FDD). At heterologously expressed alpha1betaxgamma2L (x-for 1,2,3) GABA(A)R FDD were 6 times more potent at beta1- versus beta2- and beta3-containing receptors. Serine at position 265 was essential for the high sensitivity of the beta1-subunit to FDD and the beta1N286W mutation nearly abolished modulation; vice versa the mutation beta3N265S shifted FDD sensitivity toward the beta1-type. In posterior hypothalamic neurons controlling wakefulness GABA-mediated whole-cell responses and GABAergic synaptic currents were highly sensitive to FDD, in contrast to beta1-negative cerebellar Purkinje neurons. Immunostaining for the beta1-subunit and the potency of FDD to modulate GABA responses in cultured hypothalamic neurons was drastically diminished by beta1-siRNA treatment. In conclusion, with the help of FDDs we reveal a functional expression of beta1-containing GABA(A)Rs in the hypothalamus, offering a new tool for studies on the functional diversity of native GABA(A)Rs.

β-Alanine as a small molecule neurotransmitter

This review discusses the role of β-alanine as a neurotransmitter. β-Alanine is structurally intermediate between α-amino acid (glycine, glutamate) and γ-amino acid (GABA) neurotransmitters. In general, β-alanine satisfies a number of the prerequisite classical criteria for being a neurotransmitter: β-alanine occurs naturally in the CNS, is released by electrical stimulation through a Ca 2+ dependent process, has binding sites, and inhibits neuronal excitability. β-Alanine has 5 recognized receptor sites: glycine co-agonist site on the NMDA complex (strychnine-insensitive); glycine receptor site (strychnine sensitive); GABA-A receptor; GABA-C receptor; and blockade of GAT protein-mediated glial GABA uptake. Although β-alanine binding has been identified throughout the hippocampus, limbic structures, and neocortex, unique β-alaninergic neurons with no GABAergic properties remain unidentified, and it is impossible to discriminate between β-alaninergic and GABAergic properties in the CNS. Nevertheless, a variety of data suggest that β-alanine should be considered as a small molecule neurotransmitter and should join the ranks of the other amino acid neurotransmitters. These realizations open the door for a more comprehensive evaluation of β-alanine's neurochemistry and for its exploitation as a platform for drug design.

Ionotropic GABA receptors: modelling and design of selective ligands

Ionotropic GABAA and GABAC receptors play an important role in the operation of CNS and serve as targets for many neuroactive drugs. Using the homology modelling and molecular dynamics, the 3D models of the receptors were built and some aspects of ligand-target interactions were elucidated [1,2]. To better understand the structural factors controlling the activity and selectivity of the ligands, a series of QSAR models [3] were derived based on the Molecular Field Topology Analysis (MFTA) [4], CoMFA and Topomer CoMFA approaches. They were compared with each other as well as with the molecular modelling results. Finally, a number of potential selective ligand structures were identified by means of the virtual screening [5] from the available chemicals databases and the generated structure libraries.

Effect of ZnCl2 and Chelation of Zinc Ions by N,N-Diethyldithiocarbamate (DEDTC) on the ERG b-Wave Amplitude from the Isolated Superfused Vertebrate Retina

Purpose: NiCl2 (15 µM) enhances the ERG b-wave amplitude of vertebrate retina, up to 1.5-fold by blocking E/R-type voltage-gated Ca2+ channels, which is mediated by blocking the release of GABA onto ionotropic GABA-A and GABA-C receptors. In vivo, it is likely that zinc, rather than nickel ions, may be involved in the modulation of retinal signalling. Therefore, we tested the effect of both, ZnCl2 (10 to 500 µM) and DEDTC (100 to 500 µM), which chelates zinc ions for the capacity to influence the ERG b-wave amplitude.Methods: Transretinal potentials from the isolated bovine retina were recorded as electroretinograms and Ca2+ inward currents by patch-clamp recordings of stably Cav2.3 transfected HEK-293 cells, yielding an IC50 value of 5.3 µM for ZnCl2.Results: ZnCl2 (10–15 µM) increased the b-wave amplitude by 1.52-fold ± 0.12 (n = 6 retinas), which was partially reversible upon washout. The same 1.5-fold stimulation of the b-wave amplitude was reported recently for 15 µM NiCl2. The superfusion of isolated retinas by DEDTC (100 µM) caused a transient decrease of the ERG b-wave amplitude (0.75-fold ± 0.06; n = 4), suggesting that the co-secretion of Zn2+ ions may occur under scotopic conditions.Conclusion: The stimulatory effect of ZnCl2 on the ERG b-wave amplitude resembles the stimulatory effect of NiCl2 and may be mediated rather by the NiCl2-sensitive, Cav2.3 E-/R-type voltage-gated Ca2+ channels than by NiCl2-sensitive T-type channels.

Retinal Synaptic Pathways Underlying the Response of the Rabbit Local Edge Detector

We studied the circuitry that underlies the behavior of the local edge detector (LED) retinal ganglion cell in rabbit by measuring the spatial and temporal properties of excitatory and inhibitory currents under whole cell voltage clamp. Previous work showed that LED excitation is suppressed by activity in the surround. However, the contributions of outer and inner retina to this characteristic and the neurotransmitters used are currently unknown. Blockage of retinal inhibitory pathways (GABA(A), GABA(C), and glycine) eliminated edge selectivity. Inverting gratings in the surround with 50-microm stripe sizes did not stimulate horizontal cells, but suppressed on and off excitation by roughly 60%, indicating inhibition of bipolar terminals (feedback inhibition). On pharmacologic blockage, we showed that feedback inhibition used both GABA(A) and GABA(C) receptors, but not glycine. Glycinergic inhibition suppressed GABAergic feedback inhibition in the center, enabling larger excitatory currents in response to luminance changes. Excitation, feedback inhibition, and direct (feedforward) inhibition responded to luminance-neutral flipping gratings of 20- to 50-microm widths, showing they are driven by independent subunits within their receptive fields, which confers sensitivity to borders between areas of texture and nontexture. Feedforward inhibition was glycinergic, its rise time was faster than decay time, and did not function to delay spiking at the onset of a stimulus. Both the on and off phases could be triggered by luminance shifts as short in duration as 33 ms and could be triggered during scenes that already produced a high baseline level of feedforward inhibition. Our results show how LED circuitry can use subreceptive field sensitivity to detect visual edges via the interaction between excitation and feedback inhibition and also respond to rapid luminance shifts within a rapidly changing scene by producing feedforward inhibition.

GABAa and GABAc receptor-mediated modulation of responses to color stimuli: electroretinographic study in the turtle Emys orbicularis

GABAergic transmission is involved in color coding in the retina. The specific contribution of different GABA receptors to spectral sensitivity of the retinal responses is not well characterized. We studied GABAa and GABAc receptor-mediated effects on the intensity-response functions of the electroretinographic ON (b-wave) and OFF (d-wave) responses to color stimuli. For this purpose, we compared the effects of GABAa receptor blockade by bicuculline with the effects of GABAa + GABAc receptor blockade by picrotoxin. The blockade of both GABAa and GABAc receptors caused an amplitude increase of the electroretinographic responses, but the effects of the two blockades depended in a specific manner on stimulus intensity and wavelength. The effects of GABAa receptor blockade showed distinct color ON/OFF asymmetry. The absolute and relative sensitivities of the ON responses to blue stimuli and OFF responses to red stimuli were increased to the greatest degree while the sensitivity of the ON responses to red stimuli and OFF responses to blue stimuli was least increased. In contrast, color ON/OFF asymmetry was not typical of the effects of GABAc receptor blockade. The most prominent GABAc effect was the sensitivity increase of the ON and OFF responses to blue stimuli and, to some lesser extent, to green stimuli. The results of this study indicate a specific role of GABAa and GABAc receptor-mediated influences in processing of chromatic information in the distal retina.

Advances and hold-ups in the study of structure, function and regulation of Cys-loop ligand-gated ion channels and receptors

The Cys-loop receptor channels, including the nicotinic acetylcholine (nAChR), serotonin 5-HT3, GABAA, GABAC and glycine receptor channels, are widely expressed in the nervous system (both central and peripheral) where they regulate excitability and neurotransmitter release, as well as at the endplate where they induce neuromuscular contraction. Furthermore dysfunctions in these receptors are thought to be involved in a variety of diseases and disorders, including (but not limited to) Alzheimer's disease, Parkinson's disease, epilepsy, schizophrenia, myasthenic syndromes, depression and substance abuse. Presently the Cys-loop receptors serve as molecular targets for a variety of clinically important drugs, such as muscle relaxants, tranquillizers, anticonvulsants and anti-emetics. Thus, these receptors are presently critical targets for the development of therapeutics to treat a variety of neurological diseases and disorders.

GABAC receptors in the lateral amygdala: a possible novel target for the treatment of fear and anxiety disorders?

Activation of GABAARs in the lateral nucleus of the amygdala (LA), a key site of plasticity underlying fear learning, impairs fear learning. The role of GABACRs in the LA and other brain areas is poorly understood. GABACRs could be an important novel target for pharmacological treatments of anxiety-related disorders since, unlike GABAARs, GABACRs do not desensitize. To detect functional GABACRs in the LA we performed whole cell patch clamp recordings in vitro. We found that GABAARs and GABABRs blockade lead to a reduction of evoked inhibition and an increase increment of excitation, but activation of GABACRs caused elevations of evoked excitation, while blocking GABACRs reduced evoked excitation. Based on this evidence we tested whether GABACRs in LA contribute to fear learning in vivo. It is established that activation of GABAARs leads to blockage of fear learning. Application of GABAC drugs had a very different effect; fear learning was enhanced by activating and attenuated by blocking GABACRs in the LA. Our results suggest that GABAC and GABAARs play opposing roles in modulation of associative plasticity in LA neurons of rats. This novel role of GABACRs furthers our understanding of GABA receptors in fear memory acquisition and storage and suggests a possible novel target for the treatment of fear and anxiety disorders.

Role of Neurotransmitter Receptors in Mediating Light-Evoked Responses in Retinal Interplexiform Cells

Interplexiform (IP) cells are a long-neglected group of retinal neurons the function of which is yet to be determined. Anatomical study indicates that IP cells are located in the inner nuclear layer, juxtaposed with the third-order neurons. However, the synaptic transmission of IP cells in the inner retina is poorly understood. Using whole cell patch-clamp and pharmacological techniques, we extensively studied synaptic receptors in IP cells. The IP cells in amphibian retinal slices were identified by electrical and morphological properties with voltage-clamp recording and Lucifer yellow dialysis. We find that light-evoked excitatory postsynaptic currents (L-EPSCs) are mediated by AMPA and N-methyl-d-aspartate receptors in IP cells. Although both receptors contributed to the amplitude and kinetics of L-EPSCs, AMPA receptor desensitization substantially shaped L-EPSCs in the neurons, similar to those found in the third-order neurons. The light-evoked inhibitory postsynaptic currents (L-IPSCs) in IP cells were primarily mediated by strychnine-sensitive glycine receptors with a small component of GABA(C) receptors. GABA(C) receptor rho2 subunits were detected in IP cells with single-cell RT-PCR assays. Expression of GABA(C) receptors is one of the special features for IP cells, distinct from most of the third-order neurons that depend on GABA(A) and glycine receptors to relay the inhibitory signals. However, GABA(A) receptors in IP cells acted like nonsynaptic receptors that were activated by exogenous GABA application. Furthermore, L-IPSCs in IP cells were inhibited by the serial inhibitions between amacrine cells in the inner retina. In addition, application of neurotransmitters on the axon terminals of IP cells had no significant current generated in the cells, indicating that the synaptic inputs of IP cells are mainly from the inner retina. This study demonstrates the important role that light signals are encoded by both experiment of inhibitory receptors in IP cells.

Antagonists of ionotropic γ‐aminobutyric acid receptors impair the NiCl2‐mediated stimulation of the electroretinogram b‐wave amplitude from the isolated superfused vertebrate retina

NiCl(2) (15 microM) stimulates the electroretinogram (ERG) b-wave amplitude of vertebrate retina up to 1.5-fold through its blocking of E/R-type voltage-gated Ca(2+) channels. Assuming that such an increase is mediated by blocking the release of the inhibitory neurotransmitter gamma-aminobutyric acid (GABA) via ionotropic GABA receptors, we tested the effect of both GABA itself and GABA-receptor antagonists such as (-)bicuculline (1.51-fold increase) and (1,2,5,6-tetrahydropyridin-4-yl)methylphosphinic acid (TPMPA; 1.46-fold increase) on the b-wave amplitude. Recording of the transretinal potentials from the isolated bovine retina. GABA (100 microM) reduced the b-wave amplitude only when NiCl(2) (15 microM) was applied first. Each antagonist applied on its own stimulated the b-wave amplitude only partially: subsequent NiCl(2) superfusion caused a small but additional increase, leading to a 1.69- and a 1.88-fold total increase of the amplitude by Ni(2+) plus (-)bicuculline or Ni(2+) plus TPMPA, respectively. Only the application of both antagonists in combination, before superfusing low NiCl(2) (15 microM), completely prevented subsequent stimulation by NiCl(2) with a similar 1.90-fold total increase of b-wave amplitude. Those retina segments that did not respond to NiCl(2) could not be stimulated by (-)bicuculline and vice versa. The stimulatory effect of NiCl(2) on the ERG b-wave amplitude is mainly, but not only, mediated by a NiCl(2)-sensitive, Ca(v)2.3-triggered GABA release acting through ionotropic GABA-A and GABA-C receptors.

Locating GABA in GABA receptor binding sites

The Cys-loop family of ligand-gated ion channels contains both vertebrate and invertebrate members that are activated by GABA (gamma-aminobutyric acid). Many of the residues that are critical for ligand binding have been identified in vertebrate GABA(A) and GABA(C) receptors, and specific interactions between GABA and some of these residues have been determined. In the present paper, I show how a cation-pi interaction for one of the binding site residues has allowed the production of models of GABA docked into the binding site, and these orientations are supported by mutagenesis and functional data. Surprisingly, however, the residue that forms the cation-pi interaction is not conserved, suggesting that GABA occupies subtly different locations even in such closely related receptors.

Contribution of GABAC receptors to inhibition in the rodent accessory optic system

The medial terminal nucleus (MTN) of the mammalian accessory optic system controls vertical compensatory eye movements. It consists of two neuronal populations which respond best either to upward or to downward visual image shifts. The two cell classes are located spatially separate in the dorsal or in the ventral subdivision of the MTN, respectively. Pronounced GABAergic pathways have been described to exist between neurons in the two MTN subdivisions indicating that inhibitory interactions play a significant role for the generation of MTN cell response properties. Yet, the types of GABA receptors which mediate these inhibitory interactions are unknown. Functionally, it is of particular interest to know whether GABA(C) receptors, as in other subcortical visual centers, participate in inhibitory mechanisms in MTN neurons. We therefore performed whole-cell patch clamp recordings from MTN neurons in acute mouse midbrain slices. We monitored excitatory and inhibitory postsynaptic responses to afferent stimulation and applied specific GABA receptor agonists and antagonists to identify the GABA receptor types present in MTN neurons. We found that more than 80% of the neurons in both MTN subdivisions express functional GABA(C) receptors that can be activated by specific receptor agonists. A blockade of GABA(C) receptors, on the other hand, either reduced or enhanced postsynaptic inhibition, indicating that both postsynaptic and presynaptic functions are served by this receptor type. This, together with earlier results, suggests that GABA(C) receptors play a general role for the control of neuronal excitability in subcortical visual pathways.

Novel GABAa ligands, inspired by nature

The major inhibitory neurotransmitter γ-amino butyric acid (GABA) exerts its inhibitory effect by binding to three different classes of receptors: GABAA-, GABAB- and GABAC receptors. GABAA- and GABAC are both ligand gated chloride ion channels, while GABAB are G-protein coupled receptors. GABAA receptors are important therapeutic targets for anxiety disorders, cognitive disorders, epilepsies, mood disorders, schizophrenia and sleep disorders. Due to the commercial interest, many compound classes, such as benzodiazepines, steroids, and barbiturates, are known to allosterically modify the effect of GABA by binding to distinct sites of the GABAA receptors.

GABAC receptors are expressed in GABAergic and non-GABAergic neurons of the rat superior colliculus and visual cortex

GABAC receptors are expressed in GABAergic and non-GABAergic neurons of the rat superior colliculus and visual cortex

Activation of the Tonic GABACReceptor Current in Retinal Bipolar Cell Terminals by Nonvesicular GABA Release

Within the second synaptic layer of the retina, bipolar cell (BC) output to ganglion cells is regulated by inhibitory input to BC axon terminals. GABA(A) receptors (GABA(A)Rs) mediate rapid synaptic currents in BC terminals, whereas GABA(C) receptors (GABA(C)Rs) mediate slow evoked currents and a tonic current, which is strongly regulated by GAT-1 GABA transporters. We have used voltage-clamp recordings from BC terminals in goldfish retinal slices to determine the source of GABA for activation of these currents. Inhibition of vesicular release with concanamycin A or tetanus toxin significantly inhibited GABA(A)R inhibitory postsynaptic currents and glutamate-evoked GABA(A)R and GABA(C)R currents but did not reduce the tonic GABA(C)R current, which was also not dependent on extracellular Ca(2+). The tonic current was strongly potentiated by inhibition of GABA transaminase, under both normal and Ca(2+)-free conditions, and was activated by exogenous taurine; however inhibition of taurine transport had little effect. The tonic current was unaffected by GAT-2/3 inhibition and was potentiated by GAT-1 inhibition even in the absence of vesicular release, indicating that it is unlikely to be evoked by reversal of GABA transporters or by ambient GABA. In addition, GABA release does not appear to occur via hemichannels or P2X(7) receptors. BC terminals therefore exhibit two forms of GABA(C)R-mediated inhibition, activated by vesicular and by nonvesicular GABA release, which are likely to have distinct functions in visual signal processing. The tonic GABA(C)R current in BC terminals exhibits similar properties to tonic GABA(A)R and glutamate receptor currents in the brain.

Guanidino Acids Act as ρ1 GABAC Receptor Antagonists

GABA(C) receptors play a role in myopia, memory-related disorders and circadian rhythms signifying a need to develop potent and selective agents for this class of receptors. Guanidino analogs related to glycine, beta-alanine and taurine were evaluated at human rho(1)GABA(C) receptors expressed in Xenopus oocytes using 2-electrode voltage clamp methods. Of the 12 analogs tested, 8 analogs were active as antagonists and the remaining were inactive. (S)-2-guanidinopropionic acid (IC(50) = 2.2 microM) and guanidinoacetic acid (IC(50) = 5.4 microM; K (B) = 7.75 microM [pK (B) = 5.11 +/- 0.06]) were the most potent being competitive antagonists at this receptor. In contrast, the beta-alanine and GABA guanidino analogs showed reduced activity, indicating the distance between the carboxyl carbon and terminal nitrogen of the guanidino group is critical for activity. Substituting the C2-position of guanidinoacetic acid with various alkyl groups reduced activity indicating that steric effects may impact on activity. The results of this study contribute to the structure-activity-relationship profile required in developing novel therapeutic agents.

GABAC Receptor-Mediated Inhibition Is Altered But Not Eliminated in the Superior Colliculus of GABAC ρ1 Knockout Mice

GABA(C) receptors (GABA(C)Rs) are widely expressed in the mammalian subcortical visual system, particularly in the retina and superior colliculus (SC). GABA(C)Rs are composed of specific rho1-3 subunits the expression of which varies among visual structures. Thus rho1 subunits are most abundant in retina, and their loss eliminates GABA(C)R expression and function. In the SC, rho2 subunit expression may be equal to or stronger than rho1 subunit expression; however, results across studies vary considerably. To more directly assess the expression of GABA(C)R subunits, we characterized inhibition in the SC of wild-type (WT) and GABA(C) rho1 Null mice that lack expression of GABA(C) rho1 subunits. We used whole cell patch-clamp recordings and evaluated GABA(C)R-mediated modulation of electrically evoked post synaptic currents using either agonists or antagonists in WT mice. In GABA(C) rho1 Null stratum griseum superficiale (SGS) cells, inhibitory postsynaptic currents were shorter in duration and their excitatory postsynaptic currents (EPSCs) were longer, indicating that a slow GABA(C)R-mediated inhibitory component was reduced in each case. In contrast to retina, GABA(C)R-mediated currents in the SC were altered but not eliminated in GABA(C) rho1 Null mice. In the majority of SC cells in GABA(C) rho1 Null mice, GABA(C)R activation could still be induced to alter EPSC peak amplitudes in putative interneurons and in many projection neurons. These results, compared with previously published data, indicate a fundamental difference between retina and SC in the control of GABA(C)R expression and subunit composition.