GABAC Antagonist Research Articles

Striatal Neurodegeneration that Mimics Huntington's Disease Modifies GABA-induced Currents.

Huntington’s Disease (HD) is a degenerative disease which produces cognitive and motor disturbances. Treatment with GABAergic agonists improves the behavior and activity of mitochondrial complexes in rodents treated with 3-nitropropionic acid to mimic HD symptomatology. Apparently, GABA receptors activity may protect striatal medium spiny neurons (MSNs) from excitotoxic damage. This study evaluates whether mitochondrial inhibition with 3-NP that mimics the early stages of HD, modifies the kinetics and pharmacology of GABA receptors in patch clamp recorded dissociated MSNs cells. The results show that MSNs from mice treated with 3-NP exhibited differences in GABA-induced dose-response currents and pharmacological responses that suggests the presence of GABAC receptors in MSNs. Furthermore, there was a reduction in the effect of the GABAC antagonist that demonstrates a lessening of this GABA receptor subtype activity as a result of mitochondria inhibition.

γ-Aminobutyric Acid(C) (GABAC) Selective Antagonists Derived from the Bioisosteric Modification of 4-Aminocyclopent-1-enecarboxylic Acid: Amides and Hydroxamates

Series of compounds were generated via the bioisosteric replacement of the carboxylate of 4-ACPCA (2) with hydroxamate or amide groups. All compounds from this study exhibited increased selectivity for GABAC, the most potent being 4-ACPHA (10a, IC50 = 13 μM) and 4-ACPAM (11a, IC50 = 10 μM). This provides evidence that a zwitterionic structure is not essential for GABAC antagonists, rather the emphasis lies in appropriate heteroatoms to participate in hydrogen bonding.

Design, Synthesis, and Pharmacological Evaluation of Fluorescent and Biotinylated Antagonists of ρ1 GABAC Receptors.

The ρ1 GABAC receptor is a ligand-gated chloride ion channel that shows promise as a therapeutic target for myopia, sleep disorders, memory and learning facilitation, and anxiety-related disorders. As such, there is a need for molecular probes to understand the role GABAC receptors play in physiological and pathological processes. To date, no labeled (either radioactive or fluorescent) GABAC selective ligand has been developed that can act as a marker for GABAC receptor visualization and localization studies. Herein, we report a series of fluorescent ligands containing different-sized linkers and fluorophores based around (S)-4-ACPBPA [(4-aminocyclopenten-1-yl)-butylphosphinic acid], a selective GABAC antagonist. One of these conjugates, (S)-4-ACPBPA-C5-BODIPY (13), displayed moderate potency (IC50 = 58.61 μM) and selectivity (>100 times) for ρ1 over α1β2γ2L GABAA receptors. These conjugates are novel lead agents for the development of more potent and selective fluorescent probes for studying the localization and function of GABAC receptors in living cells.

GABA-Induced Relaxation of Porcine Retinal Arterioles in Vitro Depends on Inhibition from the Perivascular Retina and Is Mediated by GABACReceptors

To study the dependence of γ-aminobutyric acid (GABA)-induced relaxation of retinal arterioles on the glutamate agonist N-methyl-D-aspartate (NMDA), adenosine triphosphate (ATP), prostaglandin E2 (PGE2), and adenosine, and to characterize the type and location of GABA-receptor(s) mediating GABA-induced relaxation of retinal arterioles. Porcine retinal arterioles were mounted in a wire myograph, and the effects of agonists and antagonists to NMDA, ATP, PGE2, and adenosine on GABA-induced relaxation were studied. Additionally, experiments were conducted to study relaxation induced by agonists to specific GABA receptors, and GABA-induced relaxation in the presence of specific GABA antagonists. Finally, immunohistochemistry was performed to identify the location of GABA(C) receptors in the porcine retina. GABA induced vasorelaxation during blocking of the glutamate NMDA receptor, PGE2 receptor, and ATP synthesis degradation, but not during blocking of the adenosine receptor or by agonists to any of these compounds. The vasorelaxing effect of GABA could be elicited by a specific GABA(C) agonist, but not by specific GABA(A) or GABA(B) agonists, and could be blocked by a specific GABA(C) antagonist, but not by specific GABA(A) or GABA(B) antagonists. GABA(C) receptor subunits could be identified in the ganglion cell layer, and at the border between the outer plexiform and inner nuclear layers. GABA-induced relaxation of porcine retinal vessels is mediated by the GABA(C) receptor in the perivascular retinal tissue, and depends on blocking of the glutamate NMDA receptor, prostaglandin E2 receptor, or ATP degradation.

Anxiolytic-like Effect of Testosterone in Male Rats: GABAC Receptors Are Not Involved.

The effect of testosterone on anxiety-like behaviors has been the subject of some studies. There is evidence that testosterone modulates anxiety via GABA (gama aminobutyric acid) and GABAergic system. The involvement of GABAC receptors in those effects of testosterone on anxiety-like behaviors of the rats was investigated in the present study. A group of rats received subcutaneous injections of testosterone (5, 10 and 20 mg/kg). Two groups of rats received intracerebroventricular injections of either CACA (GABAC agonist, 0.125 μg/rat) or TPMPA (GABAC antagonist, 3 microg/rat) following administration of testosterone (5, 10 and 20 mg/kg). After the injections, the rats were submitted to the elevated plus-maze test of anxiety. The rats received testosterone alone, showed a decreased in anxiety-like behaviors (P< 0.01). Administration of either CACA or TPMPA did not modify animals' behavior compared to the rats received testosterone alone. The results of the present study showed that administration of testosterone induces anxiolytic-like behaviors in the rats and GABAC receptors possibly are not involved in the anxiolytic effect of testosterone.

The Synaptic Mechanism of Direction Selectivity in Distal Processes of Starburst Amacrine Cells

Patch-clamp recordings revealed that distal processes of starburst amacrine cells (SACs) received largely excitatory synaptic input from the receptive field center and nearly purely inhibitory inputs from the surround during both stationary and moving light stimulations. The direct surround inhibition was mediated mainly by reciprocal GABA(A) synapses between opposing SACs, which provided leading and prolonged inhibition during centripetal stimulus motion. Simultaneous Ca(2+) imaging and current-clamp recording during apparent-motion stimulation further demonstrated the contributions of both centrifugal excitation and GABA(A/C)-receptor-mediated centripetal inhibition to the direction-selective Ca(2+) responses in SAC distal processes. Thus, by placing GABA release sites in electrotonically semi-isolated distal processes and endowing these sites with reciprocal GABA(A) synapses, SACs use a radial-symmetric center-surround receptive field structure to build a polar-asymmetric circuitry. This circuitry may integrate at least three levels of interactions--center excitation, surround inhibition, and reciprocal inhibitions that amplify the center--surround antagonism-to generate robust direction selectivity in the distal processes.

Long-Term Plasticity Mediated by mGluR1 at a Retinal Reciprocal Synapse

The flow of information across the retina is controlled by reciprocal synapses between bipolar cell terminals and amacrine cells. However, the synaptic delays and properties of plasticity at these synapses are not known. Here we report that glutamate release from goldfish Mb-type bipolar cell terminals can trigger fast (delay of 2-3 ms) and transient GABA(A) IPSCs and a much slower and more sustained GABA(C) feedback. Synaptically released glutamate activated mGluR1 receptors on amacrine cells and, depending on the strength of presynaptic activity, potentiated subsequent feedback. This poststimulus enhancement of GABAergic feedback lasted for up to 10 min. This form of mGluR1-mediated long-term synaptic plasticity may provide retinal reciprocal synapses with adaptive capabilities.

Glycine receptors and glycinergic synaptic input at the axon terminals of mammalian retinal rod bipolar cells.

We investigated the properties of glycine receptors and glycinergic synaptic inputs at the axon terminals of rod bipolar cells (RBCs) in rats by patch-clamp recording. Glycine currents recorded from isolated axon terminals were larger than those from isolated somata/dendrites; this was confirmed by puffing glycine onto these two regions in retinal slices. The current density at terminal endings was more than one order of magnitude higher than the density at somatic/dendritic regions. Glycine currents from isolated terminals and isolated somata/dendrites showed similar sensitivity to picrotoxinin blockade. Single-channel opening recorded from isolated terminals and somata/dendrites displayed a similar main-state conductance of ~46 pS. Application of glycine effectively suppressed depolarization-evoked increases in intracellular Ca2+ at the terminals. In the presence of GABAA and GABAC antagonists, strychnine-sensitive chloride currents were evoked in RBCs in retinal slices by puffing kainate onto the inner plexiform layer. No such currents were observed if the recorded RBCs did not retain axon terminals or if Ca2+ was replaced by Co2+ in the extracellular solution. The currents displayed discrete miniature-like events, which were partially blocked by tetrodotoxin. Consistent with early studies in the rabbit and mouse, this study demonstrates that glycine receptors are highly concentrated at the axon terminals of rat RBCs. The pharmacological and physiological properties of glycine receptors located in the axon terminal and somatic/dendritic regions, however, appear to be the same. This study provides evidence for the existence of functional glycinergic synaptic input at the axon terminals of RBCs, suggesting that glycine receptors may play a role in modulating bipolar cell synaptic transmission.

Aza-THIP and related analogues of THIP as GABAC antagonists

The potency of a series of eight compounds structurally related with 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol (THIP), a potent GABAA partial agonist exhibiting GABAC ρ1 antagonist effect (Ki=25 μM), was determined electrophysiologically using homomeric human GABAC ρ1 receptors expressed in Xenopus oocytes. Protolytic properties (pKa values for the acidic bioisosteric groups) and the presence of steric bulk in the molecules appear to be structural parameters of importance for blockade of the GABAC ρ1 receptor. Within this series of moderately potent GABAC antagonists, only 4,5,6,7-tetrahydropyrazolo[5,4-c]pyridin-3-ol (Aza-THIP) does not interact detectably with GABAA receptors, and Aza-THIP has the potential of being a useful tool for molecular and behavioural pharmacological studies.

Modulation of the components of the rat dark-adapted electroretinogram by the three subtypes of GABA receptors

The separate components of the dark-adapted electroretinogram (ERG) are believed to reflect the electric activity of neurones in both the inner and the outer layers of the retina, although their precise origin still remains unclear. The purpose of this study was to examine whether selective blockage or stimulation of the different subtypes of GABA receptors might help further elucidate the cellular origin of the components of the dark-adapted ERG. The rat retina is of interest since the localization and physiology of GABA receptors in that retina have been examined in great detail. GABA agonists and antagonists, known to affect the responses of neurons in the inner plexiform layer, were injected into the vitreous of one eye while ERG responses evoked by flashes of white light were recorded. GABA and the GABAa agonist isoguvacine completely removed the oscillatory potentials (OPs) and reduced the amplitude of the a- and b-waves. TPMPA, a GABAC antagonist, reduced the a- and b-waves but had no significant effect on the OPs. Baclofen, a GABAb agonist, reduced the amplitude of the a- and b-waves, without having any effects on the amplitude of the OPs. The GABAb antagonist CGP35348 increased the amplitudes of the a- and b-wave without having an effect on the amplitudes of the OPs. The GABAb receptor ligands had significant and opposite effect on the latency of the OPs. These results indicate that retinal neurons, presumably a subpopulation of amacrine cells, that have GABAb receptors are not the source of the OPs of the ERG, although they may modulate these wavelets in some manner, while contributing to the generation of the dark-adapted a- and b-waves. OPs are modified by stimulation of GABAa receptors, and the a- and b-waves by stimulation of all GABA receptor subtypes.

Temporal modulation of scotopic visual signals by A17 amacrine cells in mammalian retina in vivo.

We examined function of the feedback pathway from A17 GABAergic amacrine cells to rod bipolar cells (A17 feedback), a critically located inhibitory circuit in the classic rod pathway of the mammalian retina whose role in processing of scotopic visual information is still poorly understood. We show evidence that this A17 feedback has a profound influence on the temporal properties of rod-driven postphotoreceptoral responses (assessed with the scotopic electroretinogram b-wave). Application of a GABA(c) antagonist prolonged preferentially the decay of the scotopic b-wave. The degree of prolongation increased as the light intensity decreased. Application of selective GABA(a) antagonists accelerated the kinetics of the scotopic b-wave. This effect was abolished when the GABA(c) antagonist was coapplied. Selective ablation of A17 cells mimicked the action of the GABA(c) antagonist. In A17 cell-ablated retinas, the GABA(c) antagonist was no longer very effective to slow the decay of the scotopic b-wave. Thus the A17 feedback, activated by light stimulation and mediated mainly by the GABA(c) receptors, makes the scotopic b-wave more transient by accelerating preferentially its decay. The strength of the feedback can be modulated by GABA(a) receptor-mediated inhibition and by light intensity. Our results also suggest that in the mammalian retina the feedback may be a novel mechanism that contributes postphotoreceptorally to the termination of rod signals, especially those elicited by very dim light stimuli.

An improved, versatile synthesis of the GABAC antagonists (1,2,5,6-tetrahydropyridin-4-yl)methylphosphinic acid (TPMPA) and (piperidin-4-yl)methylphosphinic acid (P4MPA)

A simple, versatile and economical synthesis of the highly selective GABAC antagonist (1,2,5,6-tetrahydropyridin-4-yl)methylphosphinic acid (TPMPA) and the saturated analogue P4MPA is described. TPMPA is prepared in high yield in five steps via a palladium catalysed C–P bond forming reaction.

Light-evoked responses of bipolar cells in a mammalian retina.

We recorded light-evoked responses from rod and cone bipolar cells using patch-clamp techniques in a slice preparation of the rat retina. Rod bipolar cells responded to light with a sustained depolarization (ON response) followed at light offset by a slight hyperpolarization. ON and OFF cone bipolar cells were encountered, both with diverse temporal properties. The responses of rod bipolar cells were composed primarily of two components, a nonspecific cation current and a chloride current. The chloride current was reduced greatly in axotomized cells and could be suppressed by coapplication of the GABA(A) antagonist bicuculline and the GABA(C) antagonist (1,2,5,6-tetrahydropyridine-4-yl)methylphosphinic acid. This suggests that it largely reflects feedback from GABAergic amacrine cells. The response latency of intact rod bipolar cells was shorter than that of the axotomized cells, and the sensitivity curve covered more than twice the dynamic range. Application of the GABA receptor antagonists partially mimicked the effects of axotomy. These findings suggest that functional properties of the axon terminal system-notably synaptic feedback from amacrine cells-play an important role in defining the response properties of mammalian bipolar cells.

Inhibitors of histamine methylation in brain promote formation of imidazoleacetic acid, which interacts with GABA receptors.

In brain, the precursor of imidazoleacetic acid (IAA), a GABAA agonist but a GABAC antagonist, is not known. In the periphery, IAA derives from oxidation of histamine. But in brain, histamine is thought to be metabolized solely by histamine methyltransferase (HMT), forming tele-methylhistamine (t-MH) and tele-methylimidazoleacetic acid (t-MIAA). We showed that [3H]-histamine (intracerebroventricularly) could be converted to IAA in brains of rats, a process increased by inhibition of HMT. This demonstrated that brain can oxidize histamine and suggested that endogenous histamine might also be oxidized if HMT activity were reduced. We examined in rat cerebral cortex, effects of the following HMT inhibitors (mg/kg i.p.): metoprine (10), tacrine (10), velnacrine (10, 30), and physostigmine (1,2). Tacrine was a potent inhibitor (Ki approximately 22 nM). To measure histamine in tissue that contained HMT inhibitors, we developed a gas chromatography-mass spectrometry method. After 2 h, all drugs reduced endogenous levels of t-MH and t-MIAA and increased levels of histamine and IAA. Our results show that inhibition of HMT promotes oxidation of histamine in brain, probably by shunting histamine to an alternative metabolic pathway. Formation of IAA provides a novel interaction between histaminergic and GABAergic systems in brain. Accumulation of IAA should be considered when inhibitors of HMT are used to probe brain histamine function.

Receptive field of the retinal bipolar cell: a pharmacological study in the tiger salamander.

1. It is widely believed that signals contributing to the receptive field surrounds of retinal bipolar cells pass from horizontal cells to bipolar cells via GABAergic synapses. To test this notion, we applied gamma-aminobutyric acid (GABA) agonists and antagonists to isolated, perfused retinas of the salamander Ambystoma tigrinum while recording intracellularly from bipolar cells, horizontal cells, and photoreceptors. 2. As we previously reported, administration of the GABA analogue D-aminovaleric acid in concert with picrotoxin did not block horizontal cell responses or the center responses of bipolar cells but blocked the surround responses of both on-center and off-center bipolar cells. 3. Surround responses were not blocked by the GABA, antagonists picrotoxin or bicuculline, the GABAB agonist baclofen or the GABAB antagonist phaclofen, and the GABAC antagonists picrotoxin or cis-4-aminocrotonic acid. Combinations of these drugs were similarly ineffective. 4. GABA itself activated a powerful GABA uptake mechanism in horizontal cells for which nipecotic acid is a competitive agonist. It also activated, both in horizontal cells and bipolar cells, large GABAA conductances that shunted light responses but that could be blocked by picrotoxin or bicuculline. 5. GABA, administered together with picrotoxin to block the shunting effect of GABAA activation, did not eliminate bipolar cell surround responses at concentrations sufficient to saturate the known types of GABA receptors. 6. Surround responses were not blocked by glycine or its antagonist strychnine, or by combinations of drugs designed to eliminate GABAergic and glycinergic pathways simultaneously. 7. Although we cannot fully discount the involvement of a novel GABAergic synapse, the simplest explanation of our findings is that the primary pathway mediating the bipolar cell's surround is neither GABAergic nor glycinergic.