D1‐like dopamine receptors regulate GABAA receptor function to modulate hippocampal neural progenitor cell proliferation

The proliferation and differentiation of neural progenitor (NP) cells can be regulated by neurotransmitters including GABA and dopamine. The present study aimed to examine how these two neurotransmitter systems interact to affect post‐natal hippocampal NP cell proliferation in vitro. Mouse hippocampal NP cells express functional GABA_A receptors, which upon activation led to an increase in intracellular calcium levels via the opening of L‐type calcium channels. Activation of these GABA_A receptors also caused a significant decrease in proliferation; an effect that required the entry of calcium through L‐type calcium channels. Furthermore, while activation of D₁‐like dopamine receptors had no effect on proliferation, it abrogated the suppressive effects of GABA_A receptor activation on proliferation. The effects of D₁‐like dopamine receptors are associated with a decrease in the ability of GABA_A receptors to increase intracellular calcium levels, and a reduction in the surface expression of GABA_A receptors. In this way, D₁‐like dopamine receptor activation can increase the proliferation of NP cells by preventing GABA_A receptor‐mediated inhibition of proliferation. These results suggest that, in conditions where NP cell proliferation is under the tonic suppression of GABA, agonists which act through D₁‐like dopamine receptors may increase the proliferation of neural progenitors.     

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.     

Intracellular blockade of GABAA receptors in the rat hippocampal neurons

The intracellular blockade of GABA_A-receptor-mediated currents is a useful approach to suppress the GABAergic conductance in a single cell and to isolate the glutamatergic component of network-driven activities. Previously an approach has been described allowing intracellular blockade of GABA_A receptors by means of intracellular dialysis of a neuron with the pipette-filling solution, in which fluoride ions that hardly pass through the GABA_A receptor channels substitute for Cl^? and in which Mg²⁺ and ATP are omitted to induce rundown of the GABA_A receptors during whole-cell patch-clamp recordings. However, the kinetics of suppression of GABAergic conductance and the effect on the currents mediated by glutamate receptors remain unknown. Here, using whole-cell recordings with fluoride-based, Mg²⁺- and ATP-free solution on CA3 hippocampal neurons of neonatal rats, we show that after 1 h of such dialysis, both spontaneous and evoked GABA_A-receptor-mediated synaptic currents and responses induced by the GABA_A receptor agonist isoguvacine were completely suppressed. Inward GABAergic postsynaptic currents were suppressed prior to outward currents. Synaptic responses mediated by AMPA receptors were not affected by the dialysis, whereas the NMDA-receptor-mediated postsynaptic currents were reduced by approximately 20%. Dialysis with fluoride-based Mg²⁺, ATP-free solution either fully blocked giant depolarizing potentials (GDPs) in CA3 pyramidal cells (n = 2) or reduced the charge crossing the membrane during GDPs and shifted the GDP reversal potential to more positive values (n = 5). The dialysis-resistant component of GDPs was mediated by glutamate receptors, since: (i) it reversed around 0 mV; (ii) it demonstrated a negative slope conductance at negative membrane voltages, which is characteristic of NMDA receptor-mediated responses; (iii) kinetics of the individual events composing the dialysis-resistant component of GDPs at negative voltages were very similar to those of AMPA receptor-mediated synaptic currents. Thus, this procedure can be useful to isolate the glutamate receptor-mediated component of neuronal network-driven activities.     

Synaptic localization of α5 GABA (A) receptors via gephyrin interaction regulates dendritic outgrowth and spine maturation

GABA_A receptor subunit composition is a critical determinant of receptor localization and physiology, with synaptic receptors generating phasic inhibition and extrasynaptic receptors producing tonic inhibition. Extrasynaptically localized α5 GABA_A receptors are largely responsible for tonic inhibition in hippocampal neurons. However, we show here that inhibitory synapses also contain a constant level of α5 GABA_A receptors throughout neuronal development, as measured by its colocalization with gephyrin, the inhibitory postsynaptic scaffolding protein. Immunoprecipitation of the α5 subunit from both cultured neurons and adult rat brain coimmunoprecipitated gephyrin, confirming this interaction in vivo. Furthermore, the α5 subunit can interact with gephyrin independent of other synaptically localized alpha subunits, as shown by immunoprecipitation experiments in HEK cells. By replacing the α5 predicted gephyrin binding domain (Residues 370–385) with either the high affinity gephyrin binding domain of the α2 subunit or homologous residues from the extrasynaptic α4 subunit that does not interact with gephyrin, α5 GABA_A receptor localization shifted into or out of the synapse, respectively. These shifts in the ratio of synaptic/extrasynaptic α5 localization disrupted dendritic outgrowth and spine maturation. In contrast to the predominant view of α5 GABA_A receptors being extrasynaptic and modulating tonic inhibition, we identify an intimate association of the α5 subunit with gephyrin, resulting in constant synaptic levels of α5 GABA_AR throughout circuit formation that regulates neuronal development. © 2015 Wiley Periodicals, Inc. Develop Neurobiol 75: 1241–1251, 2015     

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.     

Neurexins and neuroligins: new partners for GABAA receptors at synapses

Gamma-aminobutyric acid (GABA) is the major inhibitory neurotransmitter in the brain. As one of several types of endogenous receptors, GABA_A receptors have been shown to be essential in most, if not all, aspects of brain functioning, including neural development and information processing. Mutations in genes encoding GABA_A receptors and alterations in the function of GABA_A receptors are associated with many neurologic diseases, and GABA_A receptors have been clinically targeted by many drugs, such as benzodiazepines and general anesthetics. Extensive studies have revealed a number of intracellular chaperons/interactions for GABA_A receptors, providing a protein-protein network in regulating the trafficking and location of GABA_A receptors in the brain. Recently, neurexins and neuroligins, two families of transmembrane proteins present at neurological synapses, are implicated as new partners to GABA_A receptors. These works shed new light on the synaptic regulation of GABA_A receptor activity. Here, we summarized the proteins that were implicated in the function of GABA_A receptors, including neurexins and neuroligins.     

Propofol suppresses synaptic responsiveness of somatosensory relay neurons to excitatory input by potentiating GABAA receptor chloride channels

Propofol is a widely used intravenous general anesthetic. Propofol-induced unconsciousness in humans is associated with inhibition of thalamic activity evoked by somatosensory stimuli. However, the cellular mechanisms underlying the effects of propofol in thalamic circuits are largely unknown. We investigated the influence of propofol on synaptic responsiveness of thalamocortical relay neurons in the ventrobasal complex (VB) to excitatory input in mouse brain slices, using both current- and voltage-clamp recording techniques. Excitatory responses including EPSP temporal summation and action potential firing were evoked in VB neurons by electrical stimulation of corticothalamic fibers or pharmacological activation of glutamate receptors. Propofol (0.6 – 3 μM) suppressed temporal summation and spike firing in a concentration-dependent manner. The thalamocortical suppression was accompanied by a marked decrease in both EPSP amplitude and input resistance, indicating that a shunting mechanism was involved. The propofol-mediated thalamocortical suppression could be blocked by a GABA_A receptor antagonist or chloride channel blocker, suggesting that postsynaptic GABA_A receptors in VB neurons were involved in the shunting inhibition. GABA_A receptor-mediated inhibitory postsynaptic currents (IPSCs) were evoked in VB neurons by electrical stimulation of the reticular thalamic nucleus. Propofol markedly increased amplitude, decay time, and charge transfer of GABA_A IPSCs. The results demonstrated that shunting inhibition of thalamic somatosensory relay neurons by propofol at clinically relevant concentrations is primarily mediated through the potentiation of the GABA_A receptor chloride channel-mediated conductance, and such inhibition may contribute to the impaired thalamic responses to sensory stimuli seen during propofol-induced anesthesia.     

Tonic inhibition in spinal ventral horn interneurons mediated by α5 subunit-containing GABA(A) receptors

GABA_A receptors mediate synaptic and tonic inhibition in many neurons of the central nervous system. These receptors can be constructed from a range of different subunits deriving from seven identified families. Among these subunits, α₅ has been shown to mediate GABAergic tonic inhibitory currents in neurons from supraspinal nuclei. Likewise, immunohistochemical and in situ hybridization studies have shown the presence of the α₅ subunit in spinal cord neurons, though almost nothing is known about its function. In the present report, using slices of the adult turtle spinal cord as a model system we have recorded a tonic inhibitory current in ventral horn interneurons (VHIs) and determined the functional contribution of the α₅ subunit-containing GABA_A receptors to this current. Patch clamp studies show that the GABAergic tonic inhibitory current in VHIs is not affected by the application of antagonists of the α_4/6 subunit-containing GABA_A receptors, but is sensitive to L-655708, an antagonist of the GABA_A receptors containing α₅ subunits. Last, by using RT-PCR and immunohistochemistry we confirmed the expression of the α₅ subunit in the turtle spinal cord. Together, these results suggest that GABA_A receptors containing the α₅ subunit mediate the tonic inhibitory currents observed in VHIs.     

Homeostatic Competition between Phasic and Tonic Inhibition

The GABA_A receptors are the major inhibitory receptors in the brain and are localized at both synaptic and extrasynaptic membranes. Synaptic GABA_A receptors mediate phasic inhibition, whereas extrasynaptic GABA_A receptors mediate tonic inhibition. Both phasic and tonic inhibitions regulate neuronal activity, but whether they regulate each other is not very clear. Here, we investigated the functional interaction between synaptic and extrasynaptic GABA_A receptors through various molecular manipulations. Overexpression of extrasynaptic α6β3δ-GABA_A receptors in mouse hippocampal pyramidal neurons significantly increased tonic currents. Surprisingly, the increase of tonic inhibition was accompanied by a dramatic reduction of the phasic inhibition, suggesting a possible homeostatic regulation of the total inhibition. Overexpressing the α6 subunit alone induced an up-regulation of δ subunit expression and suppressed phasic inhibition similar to overexpressing the α6β3δ subunits. Interestingly, blocking all GABA_A receptors after overexpressing α6β3δ receptors could not restore the synaptic GABAergic transmission, suggesting that receptor activation is not required for the homeostatic interplay. Furthermore, insertion of a gephyrin-binding-site (GBS) into the α6 and δ subunits recruited α6_GBSβ3δ_GBS receptors to postsynaptic sites but failed to rescue synaptic GABAergic transmission. Thus, it is not the positional effect of extrasynaptic α6β3δ receptors that causes the down-regulation of phasic inhibition. Overexpressing α5β3γ2 subunits similarly reduced synaptic GABAergic transmission. We propose a working model that both synaptic and extrasynaptic GABA_A receptors may compete for limited receptor slots on the plasma membrane to maintain a homeostatic range of the total inhibition.     

Differential Modulation of GABAA Receptors Underlies Postsynaptic Depolarization- and Purinoceptor-Mediated Enhancement of Cerebellar Inhibitory Transmission: A Non-Stationary Fluctuation Analysis Study

Cerebellar GABAergic inhibitory transmission between interneurons and Purkinje cells (PCs) undergoes a long-lasting enhancement following different stimulations, such as brief depolarization or activation of purinergic receptors of postsynaptic PCs. The underlying mechanisms, however, are not completely understood. Using a peak-scaled non-stationary fluctuation analysis, we therefore aimed at characterizing changes in the electrophysiological properties of GABA_A receptors in PCs of rat cerebellar cortex during depolarization-induced “rebound potentiation (RP)” and purinoceptor-mediated long-term potentiation (PM-LTP), because both RP and PM-LTP likely depend on postsynaptic mechanisms. Stimulation-evoked inhibitory postsynaptic currents (eIPSCs) were recorded from PCs in neonatal rat cerebellar slices. Our analysis showed that postsynaptic membrane depolarization induced RP of eIPSCs in association with significant increase in the number of synaptic GABA_A receptors without changing the channel conductance. By contrast, bath application of ATP induced PM-LTP of eIPSCs with a significant increase of the channel conductance of GABA_A receptors without affecting the receptor number. Pretreatment with protein kinase A (PKA) inhibitors, H-89 and cAMPS-Rp, completely abolished the PM-LTP. The CaMKII inhibitor KN-62 reported to abolish RP did not alter PM-LTP. These results suggest that the signaling mechanism underlying PM-LTP could involve ATP-induced phosphorylation of synaptic GABA_A receptors, thereby resulting in upregulation of the channel conductance by stimulating adenylyl cyclase-PKA signaling cascade, possibly via activation of P2Y₁₁ purinoceptor. Thus, our findings reveal that postsynaptic GABA_A receptors at the interneuron-PC inhibitory synapses are under the control of two distinct forms of long-term potentiation linked with different second messenger cascades.     

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.     

Modulation of Tonic GABA Currents by Anion Channel and Connexin Hemichannel Antagonists

Anion channels and connexin hemichannels are permeable to amino acid neurotransmitters. It is hypothesized that these conductive pathways release GABA, thereby influencing ambient GABA levels and tonic GABAergic inhibition. To investigate this, we measured the effects of anion channel/hemichannel antagonists on tonic GABA currents of rat hippocampal neurons. In contrast to predictions, blockade of anion channels and hemichannels with NPPB potentiated tonic GABA currents of neurons in culture and acute hippocampal slices. In contrast, the anion channel/hemichannel antagonist carbenoxolone (CBX) inhibited tonic currents. These findings could result from alterations of ambient GABA concentration or direct effects on GABA_A receptors. To test for effects on GABA_A receptors, we measured currents evoked by exogenous GABA. Coapplication of NPPB with GABA potentiated GABA-evoked currents. CBX dose-dependently inhibited GABA-evoked currents. These results are consistent with direct effects of NPPB and CBX on GABA_A receptors. GABA release from hippocampal cell cultures was directly measured using HPLC. Inhibition of anion channels with NPPB or CBX did not affect GABA release from cultured hippocampal neurons. NPPB reduced GABA release from pure astrocytic cultures by 21%, but the total GABA release from astrocytes was small compared to that of mixed cultures. These data indicate that drugs commonly used to antagonize anion channels and connexin hemichannels may affect tonic currents via direct effects on GABA_A receptors and have negligible effects on ambient GABA concentrations. Interpretation of experiments using NPPB or CBX should include consideration of their effects on tonic GABA currents.     

Calcium accumulation in neurites and cell bodies of rat cerebellar granule cells in culture: effects on GABAA receptor function

Summary. Accumulation of calcium in rat cerebellar granule cells in culture was studied by two photon laser scanning microscopy. Depolarizations by high extracellular potassium induced short-lived increases in calcium in both cell bodies and neurites. However, although the increase in neurites subsided completely after the initial peak, in cell bodies there was a persistent plateau until the high potassium stimulus was removed. On the contrary, the calcium signal due to NMDA receptors activation was persistent in both cell bodies and neurites and remained until the agonist was present.The nature of these calcium signals provides an interpretation key for the effects of NMDA receptors activation on GABA_A receptors. In particular, the persistent calcium increase in neurites may explain the decrease in GABA activated chloride currents which are related to activation of dendritic/synaptic GABA_A receptors.     

Chloride Accumulators NKCC1 and AE2 in Mouse GnRH Neurons: Implications for GABAA Mediated Excitation

A developmental “switch” in chloride transporters occurs in most neurons resulting in GABA_A mediated hyperpolarization in the adult. However, several neuronal cell subtypes maintain primarily depolarizing responses to GABA_A receptor activation. Among this group are gonadotropin-releasing hormone-1 (GnRH) neurons, which control puberty and reproduction. NKCC1 is the primary chloride accumulator in neurons, expressed at high levels early in development and contributes to depolarization after GABA_A receptor activation. In contrast, KCC2 is the primary chloride extruder in neurons, expressed at high levels in the adult and contributes to hyperpolarization after GABA_A receptor activation. Anion exchangers (AEs) are also potential modulators of responses to GABA_A activation since they accumulate chloride and extrude bicarbonate. To evaluate the mechanism(s) underlying GABA_A mediated depolarization, GnRH neurons were analyzed for 1) expression of chloride transporters and AEs in embryonic, pre-pubertal, and adult mice 2) responses to GABA_A receptor activation in NKCC1^-/- mice and 3) function of AEs in these responses. At all ages, GnRH neurons were immunopositive for NKCC1 and AE2 but not KCC2 or AE3. Using explants, calcium imaging and gramicidin perforated patch clamp techniques we found that GnRH neurons from NKCC1^-/- mice retained relatively normal responses to the GABA_A agonist muscimol. However, acute pharmacological inhibition of NKCC1 with bumetanide eliminated the depolarization/calcium response to muscimol in 40% of GnRH neurons from WT mice. In the remaining GnRH neurons, HCO₃ ^- mediated mechanisms accounted for the remaining calcium responses to muscimol. Collectively these data reveal mechanisms responsible for maintaining depolarizing GABA_A mediated transmission in GnRH neurons.     

GABAA Receptor in the Thalamic Specific Relay System Contributes to the Propofol-Induced Somatosensory Cortical Suppression in Rat

Interaction with the gamma-aminobutyric-acid-type-A (GABA_A) receptors is recognized as an important component of the mechanism of propofol, a sedative-hypnotic drug commonly used as anesthetic. However the contribution of GABA_A receptors to the central nervous system suppression is still not well understood, especially in the thalamocortical network. In the present study, we investigated if intracerebral injection of bicuculline (a GABA_A receptor antagonist) into the thalamus ventral posteromedial nucleus (VPM, a thalamus specific relay nuclei that innervated S1 mostly) could reverse propofol-induced cortical suppression, through recording the changes of both spontaneous and somatosensory neural activities in rat’s somatosensory cortex (S1). We found that after injection of bicuculline into VPM, significant increase of neural activities were observed in all bands of local field potentials (total band, 182±6%), while the amplitude of all components in somatosensory evoked potentials were also increased (negative, 121±9% and positive, 124±6%).These data support that the potentiation of GABA_A receptor-mediated synaptic inhibition in a thalamic specific relay system seems to play a crucial role in propofol-induced cortical suppression in the somatosensory cortex of rats.     

Differential Drug Responses on Native GABAA Receptors Revealing Heterogeneity in Extrasynaptic Populations in Cultured Hippocampal Neurons

Hippocampal pyramidal neurons potentially express multiple subtypes of GABA_A receptors at extrasynaptic locations that could therefore respond to different drugs. We activated extrasynaptic GABA_A receptors in cultured rat hippocampal pyramidal neurons and measured single-channel currents in order to compare the actions of two drugs that potentially target different GABA_A receptor subtypes. Despite the possible difference in receptor targets of etomidate and diazepam, the two drugs were similar in their actions on native extrasynaptic GABA_A receptors. Each drug produced three distinct responses that differed significantly in current magnitude, implying heterogeneous GABA_A receptor populations. In the majority of patches, drug application increased both the single-channel conductance (>40 pS) and the open probability of the channels. By contrast, in the minority of patches, drug application caused an increase in open probability only. In the third group high-conductance channels were observed upon GABA activation and drug application increased their open probability only. The currents potentiated by etomidate or diazepam were substantially larger in patches displaying high-conductance GABA channels compared to those displaying only low-conductance channels. Factors contributing to the large magnitude of these currents were the long mean open time of high-conductance channels and the presence of multiple channels in these patches. In conclusion, we suggest that the local density of extrasynaptic GABA_A receptors may influence their single-channel properties and may be an additional regulating factor for tonic inhibition and, importantly, differential drug modulation. This work is dedicated to the memory of Professor P. W. Gage.     

Stimulation of TM3 Leydig cell proliferation via GABA<Subscript>A</Subscript> receptors: A new role for testicular GABA

The neurotransmitter gamma-aminobutyric acid (GABA) and subtypes of GABA receptors were recently identified in adult testes. Since adult Leydig cells possess both the GABA biosynthetic enzyme glutamate decarboxylase (GAD), as well as GABA_A and GABA_B receptors, it is possible that GABA may act as auto-/paracrine molecule to regulate Leydig cell function. The present study was aimed to examine effects of GABA, which may include trophic action. This assumption is based on reports pinpointing GABA as regulator of proliferation and differentiation of developing neurons via GABA_A receptors. Assuming such a role for the developing testis, we studied whether GABA synthesis and GABA receptors are already present in the postnatal testis, where fetal Leydig cells and, to a much greater extend, cells of the adult Leydig cell lineage proliferate. Immunohistochemistry, RT-PCR, Western blotting and a radioactive enzymatic GAD assay evidenced that fetal Leydig cells of five-six days old rats possess active GAD protein, and that both fetal Leydig cells and cells of the adult Leydig cell lineage possess GABA_A receptor subunits. TM3 cells, a proliferating mouse Leydig cell line, which we showed to possess GABA_A receptor subunits by RT-PCR, served to study effects of GABA on proliferation. Using a colorimetric proliferation assay and Western Blotting for proliferating cell nuclear antigen (PCNA) we demonstrated that GABA or the GABA_A agonist isoguvacine significantly increased TM3 cell number and PCNA content in TM3 cells. These effects were blocked by the GABA_A antagonist bicuculline, implying a role for GABA_A receptors. In conclusion, GABA increases proliferation of TM3 Leydig cells via GABA_A receptor activation and proliferating Leydig cells in the postnatal rodent testis bear a GABAergic system. Thus testicular GABA may play an as yet unrecognized role in the development of Leydig cells during the differentiation of the testicular interstitial compartment.     

Depolarizing action of GABA on neurons of the central nervous system during early postnatal development

Within the early period of mammalian ontogenic development, activation of GABA_A receptors evokes in the central nervous system depolarization of the cellular membranes, instead of inhibition. In this review, we describe phenomenology and mechanisms underlying the effects related to this peculiarity, in particular a GABA-activated increase in the intracellular calcium concentration and oscillations of the latter in the hippocampal neuronal network. The physiological role of the GABA-mediated depolarizing synaptic activity (as a possible factor influencing formation of a glutamatergic synaptic network) is discussed.     

Calcium channels and GABA receptors in the early embryonic chick retina

The properties of calcium channels were studied at the period of neurogenesis in the early embryonic chick retina. The whole neural retina was isolated from embryonic day 3 (E3) chick and loaded with a Ca²⁺-sensitive fluorescent dye (Fura-2). The retinal cells were depolarized by puff application of high-K⁺ solutions. Increases in intracellular Ca²⁺ concentrations were evoked by the depolarization through calcium channels. The type of calcium channel was identified as l-type by the sensitivity to dihydropyridines. The Ca²⁺ response was completely blocked by 10 μM nifedipine, whereas it was remarkably enhanced by 5 μM Bay K 8644. Then we sought a factor to activate the calcium channel and found that GABA could activate it by membrane depolarization at the E3 chick retina. Puff application of 100 μM GABA raised intracellular Ca²⁺ concentrations, and this Ca²⁺ response to GABA was also sensitive to the two dihydropyridines. Intracellular potential recordings verified clear depolarization by bath-applied 100 μM GABA. The Ca²⁺ response to GABA was mediated by GABA_A receptors, since the GABA response was blocked by 10 μgM bicuculline or 50 μM picrotoxin, and mimicked by muscimol but not by baclofen. Neither glutamate, kainate, nor glycine evoked any Ca²⁺ response. We conclude that l-type calcium channels and GABA_A receptors are already are already expressed before differentiation of retinal cells and synapse formation in the chick retina. A possibility is proposed that GABA might act as a trophic factor by activating l-type calcium channels via GABA_A receptors during the early period of retinal neurogenesis. © 1993 John Wiley & Sons, Inc.