Diversity matters: combinatorial information coding by GABAA receptor subunits during spatial learning and its allosteric modulation

In the hippocampus, GABA inhibition tunes network oscillations and shapes synchronous activity during spatial learning and memory coding. Once released from the presynapse, GABA primarily binds to ionotropic GABA_A receptors (GABA_ARs), which are heteropentamers combinatorially assembled from nineteen known subunits to induce Cl^- currents postsynaptically. Dissecting GABA_AR subtype specificities in neurobiology is daunting because of differences in their developmental dynamics, regional distribution and subcellular compartmentalization. Here, we review recent data to show that the combination of single-cell mRNA-seq and neuroanatomy can reveal unprecedented cell-type and network-specificity of GABA_AR subunits and limit the permutation in subunit configurations, thus rationalizing GABA_AR physiology and pharmacology. By comparing RNA-seq data on principal cells and interneurons we discuss a tight match between GABA_AR subunit allocation, diversity in the origins of GABA inputs and operational rules at synaptic and extrasynaptic sites. We propose that coincident analysis of all GABA_AR subunits, particularly in relation to specific behaviors, could overcome existing pitfalls of the genetic and pharmacological manipulation of single subunits. By using α1 and α5 GABA_AR subunits, we single out hippocampal spatial learning as a process in which, despite the many studies available to date, neither consensus nor causality exists with regards to GABA_AR subtype requirements, curtailing a unifying concept on postsynaptic coding of GABA signals. Finally, we address the modulation of GABA_AR subunits by dopamine and endocannabinoids through receptor heteromerization, cross-modulation of signal transduction and allostery. In sum, data in this review infer that multiparametric computation gains momentum to improve knowledge on GABA_ARs function in cognition and neuropsychiatric illnesses.     

Extrasynaptic GABA(A) receptors and tonic inhibition in rat auditory thalamus

Background

Neural inhibition plays an important role in auditory processing and attentional gating. Extrasynaptic GABA_A receptors (GABA_AR), containing α₄and δ GABA_AR subunits, are thought to be activated by GABA spillover outside of the synapse following release resulting in a tonic inhibitory Cl⁻ current which could account for up to 90% of total inhibition in visual and somatosensory thalamus. However, the presence of this unique type of inhibition has not been identified in auditory thalamus.

Methodology/Principal Findings

The present study used gaboxadol, a partially selective potent agonist for δ-subunit containing GABA_A receptor constructs to elucidate the presence of extrasynaptic GABA_ARs using both a quantitative receptor binding assay and patch-clamp electrophysiology in thalamic brain slices. Intense [³H]gaboxadol binding was found to be localized to the MGB while whole cell recordings from MGB neurons in the presence of gaboxadol demonstrated the expression of δ-subunit containing GABA_ARs capable of mediating a tonic inhibitory Cl⁻ current.

Conclusions/Significance

Potent tonic inhibitory GABA_AR responses mediated by extrasynaptic receptors may be important in understanding how acoustic information is processed by auditory thalamic neurons as it ascends to auditory cortex. In addition to affecting cellular behavior and possibly neurotransmission, functional extrasynaptic δ-subunit containing GABA_ARs may represent a novel pharmacological target for the treatment of auditory pathologies including temporal processing disorders or tinnitus.     

Yellow Fluorescent Protein-Based Assay to Measure GABAA Channel Activation and Allosteric Modulation in CHO-K1 Cells

The γ-aminobutyric acid A (GABA_A) ion channels are important drug targets for treatment of neurological and psychiatric disorders. Finding GABA_A channel subtype selective allosteric modulators could lead to new improved treatments. However, the progress in this area has been obstructed by the challenging task of developing functional assays to support screening efforts and the generation of cells expressing functional GABA_A ion channels with the desired subtype composition. To address these challenges, we developed a yellow fluorescent protein (YFP)-based assay to be able to study allosteric modulation of the GABA_A ion channel using cryopreserved, transiently transfected, assay-ready cells. We show for the first time how the MaxCyte STX electroporation instrument can be used to generate CHO-K1 cells expressing functional GABA_A α2β3γ2 along with a halide sensing YFP-H148Q/I152L (YFP-GABA_A2 cells). As a basis for a cell-based assay capable of detecting allosteric modulators, experiments with antagonist, ion channel blocker and modulators were used to verify GABA_A subunit composition and functionality. We found that the I⁻ concentration used in the YFP assay affected both basal quench of YFP and potency of GABA. For the first time the assay was used to study modulation of GABA with 7 known modulators where statistical analysis showed that the assay can distinguish modulatory pEC₅₀ differences of 0.15. In conclusion, the YFP assay proved to be a robust, reproducible and inexpensive assay. These data provide evidence that the assay is suitable for high throughput screening (HTS) and could be used to discover novel modulators acting on GABA_A ion channels.     

Postnatal maturation of gamma-aminobutyric acidA and B-mediated inhibition in the CA3 hippocampal region of the rat

In the adult central nervous system, GABAergic synaptic inhibition is known to play a crucial role in preventing the spread of excitatory glutamatergic activity. This inhibition is achieved by a membrane hyperpolarization through the activation of postsynaptic γ-aminobutyric acid_A (GABA_A) and GABA_B receptors. In addition, GABA also depress transmitter release acting through presynaptic GABA_B receptors. Despite the wealth of data regarding the role of GABA in regulating the degree of synchronous activity in the adult, little is known about GABA transmission during early stages of development. In the following we report that GABA mediates most of the excitatory drive at early stages of development in the hippocampal CA3 region. Activation of GABA_A receptors induces a depolarization and excitation of immature CA3 pyramidal neurons and increases intracellular Ca²⁺ ([Ca²⁺]_i) during the first postnatal week of life. During the same developmental period, the postsynaptic GABA_B-mediated inhibition is poorly developed. In contrast, the presynaptic GABA_B-mediated inhibition is well developed at birth and plays a crucial role in modulating the postsynaptic activity by depressing transmitter release at early postnatal stages. We have also shown that GABA plays a trophic role in the neuritic outgrowth of cultured hippocampal neurons. © 1995 John Wiley & Sons, Inc.     

The diversity of GABAA receptors

The amino acid γ-aminobutyric-acid (GABA) prevails in the CNS as an inhibitory neurotrans-mitter that mediates most of its effects through fast GABA-gated Cl^?-channels (GABA_AR). Molecular biology uncovered the complex subunit architecture of this receptor channel, in which a pentameric assembly derived from five of at least 17 mammalian subunits, grouped in the six classes α, β, γ, δ, ε, and ρ, permits a vast number of putative receptor isoforms. The subunit composition of a particular receptor determines the specific effects of allosterical modulators of the GABA_ARs like benzodiazepines (BZs), barbiturates, steroids, some convulsants, polyvalent cations, and ethanol. To understand the physiology and diversity of GABA_ARs, the native isoforms have to be identified by their localization in the brain and by their pharmacology. In heterologous expression systems, channels require the presence of α, β, and γ subunits in order to mimic the full repertoire of native receptor responses to drugs, with the BZ pharmacology being determined by the particular α and γ subunit variants. Little is known about the functional properties of the β, δ, and ε subunit classes and only a few receptor subtype-specific substances like loreclezole and furosemide are known that enable the identification of defined receptor subtypes. We will summarize the pharmacology of putative receptor isoforms and emphasize the characteristics of functional channels. Knowledge of the complex pharmacology of GABA_ARs might eventually enable site-directed drug design to further our understanding of GABA-related disorders and of the complex interaction of excitatory and inhibitory mechanisms in neuronal processing.     

Positive and Negative Allosteric Modulation of an α1β3γ2 γ-Aminobutyric Acid Type A (GABAA) Receptor by Binding to a Site in the Transmembrane Domain at the γ+-β? Interface

In the process of developing safer general anesthetics, isomers of anesthetic ethers and barbiturates have been discovered that act as convulsants and inhibitors of γ-aminobutyric acid type A receptors (GABA_ARs) rather than potentiators. It is unknown whether these convulsants act as negative allosteric modulators by binding to the intersubunit anesthetic-binding sites in the GABA_AR transmembrane domain (Chiara, D. C., Jayakar, S. S., Zhou, X., Zhang, X., Savechenkov, P. Y., Bruzik, K. S., Miller, K. W., and Cohen, J. B. (2013) J. Biol. Chem. 288, 19343–19357) or to known convulsant sites in the ion channel or extracellular domains. Here, we show that S-1-methyl-5-propyl-5-(m-trifluoromethyl-diazirynylphenyl) barbituric acid (S-mTFD-MPPB), a photoreactive analog of the convulsant barbiturate S-MPPB, inhibits α1β3γ2 but potentiates α1β3 GABA_AR responses. In the α1β3γ2 GABA_AR, S-mTFD-MPPB binds in the transmembrane domain with high affinity to the γ⁺-β⁻ subunit interface site with negative energetic coupling to GABA binding in the extracellular domain at the β⁺-α⁻ subunit interfaces. GABA inhibits S-[³H]mTFD-MPPB photolabeling of γ2Ser-280 (γM2–15′) in this site. In contrast, within the same site GABA enhances photolabeling of β3Met-227 in βM1 by an anesthetic barbiturate, R-[³H]methyl-5-allyl-5-(m-trifluoromethyl-diazirynylphenyl)barbituric acid (mTFD-MPAB), which differs from S-mTFD-MPPB in structure only by chirality and two hydrogens (propyl versus allyl). S-mTFD-MPPB and R-mTFD-MPAB are predicted to bind in different orientations at the γ⁺-β⁻ site, based upon the distance in GABA_AR homology models between γ2Ser-280 and β3Met-227. These results provide an explanation for S-mTFD-MPPB inhibition of α1β3γ2 GABA_AR function and provide a first demonstration that an intersubunit-binding site in the GABA_AR transmembrane domain binds negative and positive allosteric modulators.     

Extrasynaptic α6 Subunit-Containing GABAA Receptors Modulate Excitability in Turtle Spinal Motoneurons

Motoneurons are furnished with a vast repertoire of ionotropic and metabotropic receptors as well as ion channels responsible for maintaining the resting membrane potential and involved in the regulation of the mechanisms underlying its membrane excitability and firing properties. Among them, the GABA_A receptors, which respond to GABA binding by allowing the flow of Cl⁻ ions across the membrane, mediate two distinct forms of inhibition in the mature nervous system, phasic and tonic, upon activation of synaptic or extrasynaptic receptors, respectively. In a previous work we showed that furosemide facilitates the monosynaptic reflex without affecting the dorsal root potential. Our data also revealed a tonic inhibition mediated by GABA_A receptors activated in motoneurons by ambient GABA. These data suggested that the high affinity GABA_A extrasynaptic receptors may have an important role in motor control, though the molecular nature of these receptors was not determined. By combining electrophysiological, immunofluorescence and molecular biology techniques with pharmacological tools here we show that GABA_A receptors containing the α₆ subunit are expressed in adult turtle spinal motoneurons and can function as extrasynaptic receptors responsible for tonic inhibition. These results expand our understanding of the role of GABA_A receptors in motoneuron tonic inhibition.     

Decreased intracellular GABA levels contribute to spinal cord stimulation-induced analgesia in rats suffering from painful peripheral neuropathy: the role of KCC2 and GABA(A) receptor-mediated inhibition

Elevated spinal extracellular γ-aminobutyric acid (GABA) levels have been described during spinal cord stimulation (SCS)-induced analgesia in experimental chronic peripheral neuropathy. Interestingly, these increased GABA levels strongly exceeded the time frame of SCS-induced analgesia. In line with the former, pharmacologically-enhanced extracellular GABA levels by GABA_B receptor agonists in combination with SCS in non-responders to SCS solely could convert these non-responders into responders. However, similar treatment with GABA_A receptor agonists and SCS is known to be less efficient. Since K⁺ Cl⁻ cotransporter 2 (KCC2) functionality strongly determines proper GABA_A receptor-mediated inhibition, both decreased numbers of GABA_A receptors as well as reduced KCC2 protein expression might play a pivotal role in this loss of GABA_A receptor-mediated inhibition in non-responders. Here, we explored the mechanisms underlying both changes in extracellular GABA levels and impaired GABA_A receptor-mediated inhibition after 30 min of SCS in rats suffering from partial sciatic nerve ligation (PSNL). Immediately after cessation of SCS, a decreased spinal intracellular dorsal horn GABA-immunoreactivity was observed in responders when compared to non-responders or sham SCS rats. One hour later however, GABA-immunoreactivity was already increased to similar levels as those observed in non-responder or sham SCS rats. These changes did not coincide with alterations in the number of GABA-immunoreactive cells. C-Fos/GABA double-fluorescence clearly confirmed a SCS-induced activation of GABA-immunoreactive cells in responders immediately after SCS. Differences in spinal dorsal horn GABA_A receptor-immunoreactivity and KCC2 protein levels were absent between all SCS groups. However, KCC2 protein levels were significantly decreased compared to sham PSNL animals. In conclusion, reduced intracellular GABA levels are only present during the time frame of SCS in responders and strongly point to a SCS-mediated on/off GABAergic release mechanism. Furthermore, a KCC2-dependent impaired GABA_A receptor-mediated inhibition seems to be present both in responders and non-responders to SCS due to similar KCC2 and GABA_A receptor levels.     

GABA—The quintessential neurotransmitter: Electroneutrality,fidelity, specificity,and a model for the ligand binding site of GABAA receptors

Alone of the known neurotransmitters, GABA is an electroneutral zwitterion (pI=7.3) at physiological pH. This confers the highest probability of successfully traversing densely packed synaptic gaps without interacting electrostatically with charged entities enroute, making GABA a high fidelity neurotransmitter. Inhibitory tone in the nervous system is coordinately coupled with physiological activity by means of the GABA system, acidification increasing GABA formation and its Cl^– channel-opening efficacy, while decreasing its removal by transport and metabolic degradation. The above, together with diminution upon acidification of the postsynaptic efficacy of glutamate on excitatory NMDA receptors constitutes a sensitively responsive mechanism by which protons control levels of neural activity, locally and globally. A model made of the GABA binding site of GABA_A receptors based on H-bond and hydrophobic interactions makes it seem unlikely that any other substance known to occur in nerve tissue would give rise to a high noise level at GABA_A receptors.Special issue dedicated to Dr. Claude Baxter.     

Modulation of Ionotropic GABA Receptors by 6-Methoxyflavanone and 6-Methoxyflavone

We evaluated the effects of 6-methoxyflavanone and 6-methoxyflavone on wild-type α1/α2β2γ2L GABA_A and ρ1 GABA_C receptors and on mutant ρ1I307S, ρ1W328 M, ρ1I307S/W328 M GABA_C receptors expressed in Xenopus oocytes using two-electrode voltage clamp and radioligand binding. 6-Methoxyflavanone and 6-methoxyflavone act as a flumazenil-insensitive positive allosteric modulator of GABA responses at human recombinant α1β2γ2L and α2β2γ2L GABA_A receptors. However, unlike 6-methoxyflavone, 6-methoxyflavanone was relatively inactive at α1β2 GABA_A receptors. 6-Methoxyflavanone inhibited [³H]-flunitrazepam binding to rat brain membranes. Both flavonoids were found to be inactive as modulators at ρ1, ρ1I307S and ρ1W328 M GABA receptors but acted as positive allosteric modulators of GABA at the benzodiazepine sensitive ρ1I307S/W328 M GABA receptors. This double mutant retains ρ1 properties of being insensitive to bicuculline and antagonised by TPMPA and THIP. Additionally, 6-methoxyflavanone was also a partial agonist at ρ1W328 M GABA receptors. The relative inactivity of 6-methoxyflavanone at α1β2 GABA_A receptors and it’s partial agonist action at ρ1W328 M GABA receptors suggest that it exhibits a unique profile not matched by other flavonoids.     

Structure of Excitatory Synapses and GABAA Receptor Localization at Inhibitory Synapses Are Regulated by Neuroplastin-65

Formation, maintenance, and activity of excitatory and inhibitory synapses are essential for neuronal network function. Cell adhesion molecules (CAMs) are crucially involved in these processes. The CAM neuroplastin-65 (Np65) highly expressed during periods of synapse formation and stabilization is present at the pre- and postsynaptic membranes. Np65 can translocate into synapses in response to electrical stimulation and it interacts with subtypes of GABA_A receptors in inhibitory synapses. Here, we report that in the murine hippocampus and in hippocampal primary culture, neurons of the CA1 region and the dentate gyrus (DG) express high Np65 levels, whereas expression in CA3 neurons is lower. In neuroplastin-deficient (Np^−/−) mice the number of excitatory synapses in CA1 and DG, but not CA3 regions is reduced. Notably this picture is mirrored in mature Np^−/− hippocampal cultures or in mature CA1 and DG wild-type (Np^+/+) neurons treated with a function-blocking recombinant Np65-Fc extracellular fragment. Although the number of GABAergic synapses was unchanged in Np^−/− neurons or in mature Np65-Fc-treated Np^+/+ neurons, the ratio of excitatory to inhibitory synapses was significantly lower in Np^−/− cultures. Furthermore, GABA_A receptor composition was altered at inhibitory synapses in Np^−/− neurons as the α1 to α2 GABA_A receptor subunit ratio was increased. Changes of excitatory and inhibitory synaptic function in Np^−/− neurons were confirmed evaluating the presynaptic release function and using patch clamp recording. These data demonstrate that Np65 is an important regulator of the number and function of synapses in the hippocampus.     

GABAergic signaling in primary lens epithelial and lentoid cells and its involvement in intracellular Ca2+ modulation

Primary lens epithelial cell (LEC) cultures derived from newborn (P0) and one-month-old (P30) mouse lenses were used to study GABA (gamma-aminobutyric acid) signaling expression and its effect on the intracellular Ca²⁺ ([Ca²⁺]_i) level. We have found that these cultures express specific cellular markers for lens epithelial and fiber cells, all components of the functional GABA signaling pathway and GABA, thus recapitulating the developmental program of the ocular lens. Activation of both GABA-A and GABA-B receptors (GABA_AR and GABA_BR) with the specific agonists muscimol and baclofen, respectively induces [Ca²⁺]_i transients that could be blocked by the specific antagonists bicuculline and CGP55845 and were dependent on extracellular Ca²⁺. Bicuculline did not change the GABA-evoked Ca²⁺ responses in Ca²-containing buffers, but suppressed them significantly in Ca²⁺-free buffers suggesting the two receptors couple to convergent Ca²⁺ mobilization mechanisms with different extracellular Ca²⁺ sensitivity. Prolonged activation of GABA_BR induced wave propagation of the Ca²⁺ signal and persistent oscillations. The number of cells reacting to GABA or GABA + bicuculline in P30 mouse LEC cultures expressing predominantly the synaptic type GABA_AR did not differ significantly from the number of reacting cells in P0 mouse LEC cultures. The GABA-induced Ca²⁺ transients in P30 (but not P0) mouse LEC could be entirely suppressed by co-application of bicuculline and CGP55845. The GABA-mediated Ca²⁺ signaling may be involved in a variety of Ca²⁺-dependent cellular processes during lens growth and epithelial cell differentiation.     

Impact of single-site axonal GABAergic synaptic events on cerebellar interneuron activity

Axonal ionotropic receptors are present in a variety of neuronal types, and their function has largely been associated with the modulation of axonal activity and synaptic release. It is usually assumed that activation of axonal GABA_ARs comes from spillover, but in cerebellar molecular layer interneurons (MLIs) the GABA source is different: in these cells, GABA release activates presynaptic GABA_A autoreceptors (autoRs) together with postsynaptic targets, producing an autoR-mediated synaptic event. The frequency of presynaptic, autoR-mediated miniature currents is twice that of their somatodendritic counterparts, suggesting that autoR-mediated responses have an important effect on interneuron activity. Here, we used local Ca²⁺ photolysis in MLI axons of juvenile rats to evoke GABA release from individual varicosities to study the activation of axonal autoRs in single release sites. Our data show that single-site autoR conductances are similar to postsynaptic dendritic conductances. In conditions of high [Cl⁻]_i, autoR-mediated conductances range from 1 to 5 nS; this corresponds to ∼30–150 GABA_A channels per presynaptic varicosity, a value close to the number of channels in postsynaptic densities. Voltage responses produced by the activation of autoRs in single varicosities are amplified by a Na_v-dependent mechanism and propagate along the axon with a length constant of 91 µm. Immunolabeling determination of synapse location shows that on average, one third of the synapses produce autoR-mediated signals that are large enough to reach the axon initial segment. Finally, we show that single-site activation of presynaptic GABA_A autoRs leads to an increase in MLI excitability and thus conveys a strong feedback signal that contributes to spiking activity.     

Brain GABAA receptors studied with subunit-specific antibodies

Brain GABA_A/benzodiazepine receptors are highly heterogeneous. This heterogeneity is largely derived from the existence of many pentameric combinations of at least 16 different subunits that are differentially expressed in various brain regions and cell types. This molecular heterogeneity leads to binding differences for various ligands, such as GABA agonists and antagonists, benzodiazepine agonists, antagonists, and inverse agonists, steroids, barbiturates, ethanol, and Cl⁻ channel blockers. Different subunit composition also leads to heterogeneity in the properties of the Cl⁻ channel (such as conductance and open time); the allosteric interactions among subunits; and signal transduction efficacy between ligand binding and Cl⁻ channel opening. The study of recombinant receptors expressed in heterologous systems has been very useful for understanding the functional roles of the different GABA_A receptor subunits and the relationships between subunit composition, ligand binding, and Cl⁻ channel properties. Nevertheless, little is known about the complete subunit composition of the native GABA_A receptors expressed in various brain regions and cell types. Several laboratories, including ours, are using subunit-specific antibodies for dissecting the heterogeneity and subunit composition of native (not reconstituted) brain GABA_A receptors and for revealing the cellular and subcellular distribution of these subunits in the nervous system. These studies are also aimed at understanding the ligand-binding, transduction mechanisms, and channel properties of the various brain GABA_A receptors in relation to synaptic mechanisms and brain function. These studies could be relevant for the discovery and design of new drugs that are selective for some GABA_A receptors and that have fewer side effects.     

Crayfish sensory terminals and motor neurones exhibit two distinct types of GABA receptors

Motor neurones of the crayfish walking system display inhibitory responses evoked either by γ-amino butyric acid (GABA) or glutamate, possibly involving the same receptor (Pearlstein et al. 1994). In order to test if this sensibility to both GABA and glutamate was a specific property of crayfish GABA receptors, pharmacological characteristics of GABA-evoked responses in both sensory terminals from CB chordotonal organ and motor neurones of the walking system have been compared. Both receptors are GABA-gated Cl⁻ channels activated by specific GABA_A (muscimol, isoguvacine), GABA_B (3-aminopropyl phosphinic acid), and GABA_C (cis-4-amino crotonic acid) agonists, and blocked by competitive (β-guanidino propionic acid) and non-competitive (picrotoxin) antagonists. They were insensitive to specific GABA_A (bicuculline, SR-95531) and GABA_B (phaclofen) antagonists. Furthermore, in both cases, nipecotic acid and the modulatory drug diazepam had no effect. However, our results demonstrate that GABA receptors of sensory terminals are different from those of motor neurones. GABA-induced desensitisation only occurred in sensory terminals. Moreover, glutamate was shown to activate GABA-gated Cl⁻ channels in motor neurones, but not in sensory terminals. Therefore, GABA is likely to be the endogenous neurotransmitter of presynaptic inhibition in sensory terminals, whereas inhibition between antagonistic motor neurones would be achieved by glutamate. Accepted: 10 July 1996     

GABA<Subscript>A</Subscript> Receptor and Glycine Receptor Activation by Paracrine/Autocrine Release of Endogenous Agonists: More Than a Simple Communication Pathway

It is a common and widely accepted assumption that glycine and GABA are the main inhibitory transmitters in the central nervous system (CNS). But, in the past 20 years, several studies have clearly demonstrated that these amino acids can also be excitatory in the immature central nervous system. In addition, it is now established that both GABA receptors (GABARs) and glycine receptors (GlyRs) can be located extrasynaptically and can be activated by paracrine release of endogenous agonists, such as GABA, glycine, and taurine. Recently, non-synaptic release of GABA, glycine, and taurine gained further attention with increasing evidence suggesting a developmental role of these neurotransmitters in neuronal network formation before and during synaptogenesis. This review summarizes recent knowledge about the non-synaptic activation of GABA_ARs and GlyRs, both in developing and adult CNS. We first present studies that reveal the functional specialization of both non-synaptic GABA_ARs and GlyRs and we discuss the neuronal versus non-neuronal origin of the paracrine release of GABA_AR and GlyR agonists. We then discuss the proposed non-synaptic release mechanisms and/or pathways for GABA, glycine, and taurine. Finally, we summarize recent data about the various roles of non-synaptic GABAergic and glycinergic systems during the development of neuronal networks and in the adult.     

Effect of furosemide on GABA<Subscript>A</Subscript>-induced <Superscript>36</Superscript>Cl transport and Cl--ATPase activity in synaptic membranes of carp brain (<Emphasis Type="Italic">Cyprinus carpio</Emphasis> L.)

We studied the effect of furosemide on GABA_A-induced ³⁶Cl transport and GABA_A-induced Cl^--ATPase activity in synaptic membranes of fish brain. At physiological pH 7.4, GABA (0.1–100 µM) stimulated ³⁶Cl influx in synaptoneurosomes and Cl^--ATPase activity in synaptic membranes. Furosemide (0.1–0.5 mM) removed the activating effect of the mediator on chloride transport and enzyme activity (I₅₀ equaled 0.16 and 0.12 mM, respectively). In the absence of the mediator, picrotoxin (50 µM) activated the basal ³⁶Cl influx in synaptoneurosomes and the basal Mg²⁺-ATPase activity of synaptic membranes. Furosemide (1 mM) removed the activating effect of picrotoxin on both biochemical processes. The obtained data demonstrated similar sensitivities of GABA_A-induced transport of ³⁶Cl in synaptoneurosomes and of GABA_A-induced Cl^--ATPase activity in the synaptic membranes to furosemide and indicated the involvement of the ATPase in GABA_A-induced processes. The soluble ATPase, recovered by sodium deoxycholate solubilization of the membranes, remained sensitive to GABA_A-ergic ligands, which suggested proximity of their binding sites with ATP hydrolysis sites in the protein molecule and their structural coupling.Translated from Izvestiya Akademii Nauk, Seriya Biologicheskaya, No. 1, 2005, pp. 18–22.Original Russian Text Copyright © 2005 by Menzikov, Menzikova.     

Sex‐specific alterations in GABA receptor‐mediated responses in laterodorsal tegmentum are associated with prenatal exposure to nicotine

Smoking during pregnancy is associated with deleterious physiological and cognitive effects on the offspring, which are likely due to nicotine‐induced alteration in the development of neurotransmitter systems. Prenatal nicotine exposure (PNE) in rodents is associated with changes in behaviors controlled in part by the pontine laterodorsal tegmentum (LDT), and LDT excitatory signaling is altered in a sex and age‐dependent manner by PNE. As effects on GABAergic LDT signaling are unknown, we used calcium imaging to evaluate GABA_A receptor‐ (GABA_AR as well as GABA_A‐_ρR) and GABA_B receptor (GABA_BR)‐mediated calcium responses in LDT brain slices from female and male PNE mice in two different age groups. Overall, in older PNE females, changes in calcium induced by stimulation of GABA_AR and GABA_BR, including GABA_A‐_ρR were shifted toward calcium rises. In both young and old males, PNE was associated with alterations in calcium mediated by all three receptors; however, the GABA_AR was the most affected. These results show for the first time that PNE is associated with alterations in GABAergic transmission in the LDT in a sex‐ and age‐dependent manner, and these data are the first to show PNE‐associated alterations in functionality of GABA receptors in any nucleus. PNE‐associated alterations in LDT GABAergic transmission within the LDT would be expected to alter output to target regions and could play a role in LDT‐implicated, negative behavioral outcomes following gestational exposure to smoking. Accordingly, our data provide further supportive evidence of the importance of eliminating the consumption of nicotine during pregnancy.     

Optical Imaging of Hippocampal Neurons with a Chloride-Sensitive Dye: Early Effects of In Vitro Ischemia

Abstract: We determined if changes in intraneuronal Cl^? occur early after ischemia in the hippocampal slice. Slices from juvenile rats (14–19 days old) were loaded with the cell-permeant form of 6-methoxy-N-ethylquinolinium chloride (MEQ), a Cl^?-sensitive fluorescent dye. Real-time changes in intracellular chloride concentration ([Cl^?]_i) were measured with UV laser scanning confocal microscopy in multiple neurons within each slice. In vitro ischemia (26–28°C, 10 min) was confirmed by the loss of synaptic transmission (evoked field excitatory postsynaptic potentials) from pyramidal cells in area CA1. After ischemia and reoxygenation (10 min), MEQ fluorescence decreased significantly in CA1 pyramidal cells and interneurons. The decreased fluorescence corresponded to an ischemia-induced increase in [Cl^?]_i of ～10 mM. Pretreatment with the GABA_A-gated Cl^? channel antagonist picrotoxin (100 µM) blocked the ischemia-induced change in [Cl^?]_i. Analysis of the superfusates indicated that ischemia also caused a transient amino acid (GABA, glutamate, and aspartate) release that was maximal at ～10 min, returning to baseline shortly thereafter. Recovery from ischemia was confirmed by the return of synaptic transmission in area CA1, the return toward baseline of the ischemia-induced decrease in MEQ fluorescence, and exclusion of propidium iodide from MEQ fluorescent cells. Furthermore, pyramidal cells did not undergo cell swelling during this early phase of reoxygenation, as indicated by the volume-sensitive dye calcein. Thus, mild ischemia induces the accumulation of [Cl^?]_i secondary to GABA_A receptor activation, in the absence of cellular swelling or death. In contrast, depolarization of the slice with K⁺ (50 mM) decreased MEQ fluorescence significantly but caused cell swelling. Picrotoxin did not prevent the K⁺-induced increase in [Cl^?]_i. It is possible that an increased [Cl^?]_i, following either an ischemic event or an episode of depolarization, would reduce the Cl^? driving force and thereby limit synaptic transmission by GABA. To support this hypothesis, ischemia caused a reduction in the ability of the GABA agonist muscimol to increase [Cl^?]_i after 20-min reoxygenation.