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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.

Microelectrode studies of the evoked activity of neuronal columns in the barrel cortex in rats showed that the GABAC receptor blocker 1,2,5,6-tetrahydropyridin-4-ylmethylphosphonic acid (TPMPA) had an activatory effect on the secondary components of evoked potentials (EP) mainly of the upper afferent modules of columns as compared with the lower efferent layers. This is evidence that GABAC receptors are located on the presynaptic terminals of thalamocortical glutamatergic afferents and ascending apical dendrites of pyramidal cells. Blockade of GABAC influences with TPMPA led to dose-dependent depolarization of afferents with development of presynaptic inhibition and suppression of the primary responses (PR) of EP. The different effects of the GABAC receptor blocker on the secondary components of EP in the upper layers of the cortex are due to the development of afterhyperpolarization of neurons after high-amplitude PR and afterdepolarization after low-amplitude responses, with subsequent activation of different voltage-gated channels and the formation of different gradients in the direct current (DC) potential of the cortex.

The mechanisms of action of antagonists of the γ‐aminobutyric acid C (GABAC) receptor picrotoxin, quercetin and pregnanolone were studied. Ionic currents (chloride), mediated through human homomeric GABAρ1 receptors expressed in Xenopus oocytes, were recorded by two‐electrode voltage clamp. Dose–response (D–R) curves and kinetic measurements of GABAρ1 currents were carried out in the presence or absence of antagonists. Use‐dependent actions were also evaluated. Picrotoxin, quercetin and pregnanolone exerted noncompetitive actions. IC50 values measured at the EC50 for GABA (1 μM) were as follows: picrotoxin 0.6±0.1 μM (Hill coefficient n=1.0±0.2); quercetin 4.4±0.4 μM (n=1.5±0.2); pregnanolone 2.1±0.5 μM (n=0.8±0.1). These antagonists produced changes only in the slope of the linear current–voltage relationships, which was indicative of voltage‐independent effects. The effect of picrotoxin on GABAρ1 currents was use‐dependent, strongly relied on agonist concentration and showed a slow onset and offset. The mechanism was compatible with an allosteric inhibition and receptor activation was a prerequisite for antagonism. The effect of quercetin was use‐independent, showed relatively fast onset and offset, and resulted in a slowed time course of the GABA‐evoked currents. The effect of pregnanolone was use‐independent, presented fast onset and a very slow washout, and did not affect current activation. All the antagonists accelerated the time course of deactivation of the GABAρ1 currents. British Journal of Pharmacology (2004) 141, 717–727. doi:10.1038/sj.bjp.0705657

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 reduction of evoked inhibition and 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.

Lanthanide‐induced modulation of GABAC receptors expressed in Xenopus oocytes was studied. We obtained two‐electrode voltage‐clamp recordings of ionic currents mediated by recombinant homomeric GABAρ1 receptors and performed numerical simulations of kinetic models of the macroscopic ionic currents. GABA‐evoked chloride currents were potentiated by La3+, Lu3+ and Gd3+ in the micromolar range. Lanthanide effects were rapid, reversible and voltage independent. The degree of potentiation was reduced by increasing GABA concentration. Lu3+ also induced receptor desensitization and decreased the deactivation rate of GABAρ1 currents. In the presence of 300 μM Lu3+, dose–response curves for GABA‐evoked currents showed a significant enhancement of the maximum amplitude and an increase of the apparent affinity. The rate of onset of TPMPA and picrotoxin antagonism of GABAρ1 receptors was modulated by Lu3+. These results suggest that the potentiation of the anionic current was the result of a direct lanthanide–receptor interaction at a site capable of allosterically modulating channel properties. Based on kinetic schemes, which included a second open state and a nonconducting desensitized state that closely reproduced the experimental results, two nonexclusive probable models of GABAρ1 channels gating are proposed. British Journal of Pharmacology (2005) 146, 1000–1009. doi:10.1038/sj.bjp.0706411

This study investigated the effects of a number of GABA analogues on rat ρ3 GABAC receptors expressed in Xenopus oocytes using 2‐electrode voltage clamp methods. The potency order of agonists was muscimol (EC50=1.9±0.1 μM) (+)‐trans‐3‐aminocyclopentanecarboxylic acids ((+)‐TACP; EC50=2.7±0.9 μM) trans‐4‐aminocrotonic acid (TACA; EC50=3.8±0.3 μM) GABA (EC50=4.0±0.3 μM) > thiomuscimol (EC50=24.8±2.6 μM) > (±)‐cis‐2‐aminomethylcyclopropane‐carboxylic acid ((±)‐CAMP; EC50=52.6±8.7 μM) > cis‐4‐aminocrotonic acid (CACA; EC50=139.4±5.2 μM). The potency order of antagonists was (±)‐trans‐2‐aminomethylcyclopropanecarboxylic acid ((±)‐TAMP; KB=4.8±1.8 μM) (1,2,5,6‐tetrahydropyridin‐4‐yl)methylphosphinic acid (TPMPA; KB=4.8±0.8 μM) > (piperidin‐4‐yl)methylphosphinic acid (P4MPA; KB=10.2±2.3 μM) 4,5,6,7‐tetrahydroisoxazolo[5,4‐c]pyridin‐3‐ol (THIP; KB=10.2±0.3  μM) imidazole‐4‐acetic acid (I4AA; KB=12.6±2.7 μM) > 3‐aminopropylphosphonic acid (3‐APA; KB=35.8±13.5 μM). trans‐4‐Amino‐2‐methylbut‐2‐enoic acid (2‐MeTACA; 300 μM) had no effect as an agonist or an antagonist indicating that the C2 methyl substituent is sterically interacting with the ligand‐binding site of rat ρ3 GABAC receptors. 2‐MeTACA affects ρ1 and ρ2 but not ρ3 GABAC receptors. In contrast, (±)‐TAMP is a partial agonist at ρ1 and ρ2 GABAC receptors, while at rat ρ3 GABAC receptors it is an antagonist. Thus, 2‐MeTACA and (±)‐TAMP could be important pharmacological tools because they may functionally differentiate between ρ1, ρ2 and ρ3 GABAC receptors in vitro. British Journal of Pharmacology (2002) 135, 883–890; doi:10.1038/sj.bjp.0704432

The synaptic terminals of mammalian rod bipolar cells are the targets of multiple presynaptic inhibitory inputs arriving from glycinergic and GABAergic amacrine cells. To investigate the contribution of these different inhibitory receptor types, we have applied the patch-clamp technique in acutely isolated slices of the adult mouse retina. By using the whole-cell configuration, we measured and analyzed the spontaneous postsynaptic currents (PSCs) in rod bipolar cells. The spontaneous synaptic activity of rod bipolar cells was very low. However, when amacrine cells were depolarized by AMPA or kainate, the PSC frequency in rod bipolar cells increased significantly. These PSCs comprised several types that could be distinguished by pharmacological and kinetic criteria. Strychnine-sensitive, glycinergic PSCs were characterized by a mean peak amplitude of −43.5 pA and a weighted decay time constant (τw) of 10.9 ms. PSCs that persisted in the presence of strychnine, but were completely inhibited by bicuculline, were mediated by GABAARs. They had a mean peak amplitude of −20.0 pA and a significantly faster τw of 5.8 ms. Few PSCs remained in the presence of strychnine and bicuculline, suggesting that they were mediated by GABACRs. These PSCs were characterized by much smaller amplitudes (−6.2 pA) and a significantly slower decay kinetics (τw = 51.0 ms). We conclude that rod bipolar cells express at least three types of functionally different inhibitory receptors, namely GABAARs, GABACRs, and GlyRs that may ultimately regulate the Ca2+ influx into rod bipolar cell terminals, thereby modulating their glutamate release.

Two of the γ-aminobutyric acid (GABA) receptors, GABAA and GABAC, are ligand-gated chloride channels expressed by neurons in the retina and throughout the central nervous system. The different subunit composition of these two classes of GABA receptor result in very different physiological and pharmacological properties. Although little is known at the molecular level as to the subunit composition of any native GABA receptor, it is thought that GABAC receptors are homomeric assemblies of ρ-subunits. However, we found that the kinetic and pharmacological properties of homomeric receptors formed by each of the ρ-subunits cloned from perch retina did not resemble those of the GABAC receptors on perch bipolar cells. Because both GABAA and GABAC receptors are present on retinal bipolar cells, we attempted to determine whether subunits of these two receptor classes are capable of interacting with each other. We report here that, when coexpressed in Xenopus oocytes, heteromeric (ρ1Bγ2) receptors formed by coassembly of the ρ1B-subunit with the γ2-subunit of the GABAA receptor displayed response properties very similar to those obtained with current recordings from bipolar cells. In addition to being unresponsive to bicuculline and diazepam, the time-constant of deactivation, and the sensitivities to GABA, picrotoxin and zinc closely approximated the values obtained from the native GABAC receptors on bipolar cells. These results provide the first direct evidence of interaction between GABA ρ and GABAA-receptor subunits. It seems highly likely that coassembly of GABAA and ρ-subunits contributes to the molecular organization of GABAC receptors in the retina and perhaps throughout the nervous system.

A preparation of isolated presynaptic terminals of rat retinal rod bipolar cells was developed. Patch-clamp recordings were performed on the isolated terminal to determine the type(s) of voltage-activated Ca2+ channels and the contribution of GABAA and GABAC receptor-mediated currents localized in the terminal region. Both low-voltage-activated (LVA) and high-voltage-activated (HVA) Ca2+ currents, with properties similar to those found in intact cell recordings, were observed in the isolated terminal recordings. Consistent with previous studies, the HVA Ca2+ currents are L-type since the currents were blocked by low micromolar concentrations of nimodipine and potentiated by BayK 8644. Also, both GABAA and GABAC receptor-mediated currents were observed in the isolated terminal. The current density of GABAC receptors in the terminal was more than three times higher than that in the soma. In contrast, the current density of GABAA currents between the terminal and the soma was not significantly different. Assessed by 100 μM GABA, the contributions of GABAA and GABAC receptors to the total GABA-mediated currents at the terminal were comparable. This study directly demonstrates the localization of LVA Ca2+ channels at the axon terminal of mammalian rod bipolar cells, suggesting that LVA Ca2+ channels may play a role in bipolar cell transmitter release. Results of this study also support the notion that both types of ionotropic GABA receptors regulate synaptic transmission in mammalian rod bipolar cells. In addition, this study reports for the first time the feasibility of direct patch-clamp recordings of isolated axon terminals of mammalian retinal bipolar cells. The isolated presynaptic terminal preparation of mammalian retinal bipolar cells could be a valuable system for the study of transmitter release in the central nervous system (CNS).

High levels of endogenous cholecystokinin (CCK) are present in the rat retina (Eskay & Beinfeld, 1982), but the cellular localization and physiological actions of CCK in the rat retina are uncertain. The goals of this study were to characterize the cells containing CCK, identify cell types that interact with CCK cells, and investigate the effects of CCK on rod bipolar cells. Rat retinas were labeled with antibody to gastrin-CCK (gCCK) using standard immunofluorescence techniques. Patch-clamp methods were used to record from dissociated rod bipolar cells from rats and mice. Gastrin-CCK immunoreactive (-IR) axons were evenly distributed throughout the retina in stratum 5 of the inner plexiform layer of the rat retina. However, the gCCK-IR somata were only detected in the ganglion cell layer in the peripheral retina. The gCCK-IR cells contained glutamate decarboxylase, and some of them also contained immunoreactive substance P. Labeled axons contacted PKC-IR rod bipolar cells, and recoverin-IR ON-cone bipolar cells. CCK-octapeptide inhibits GABAC but not GABAA mediated currents in dissociated rod bipolar cells.