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.     

Effects of γ-acetylenic GABA and γ-vinyl GABA on electrically-induced spinal cord convulsions and on spinal cord GABA concentration

The effects of γ-acetylenic GABA and γ-vinyl GABA on electrically-induced spinal cord convulsions were compared to the effects of these same drugs on spinal cord GABA concentration. The data show that the effects of these two compounds on seizure activity do not correlate either positively or negatively with changes in GABA concentration. Although both drugs produced marked increases in the amount of GABA in the spinal cord, their effects on spinal cord convulsions were qualitatively different and failed to correlate temporally with alterations in GABA concentration.     

Effects of GABA natriuresis induced centrally by cholinergic stimulation

Sodium and potassium excretion and urine output have been studied in rats following water loading and intracerebroventricular (i.c.v.) injection of isotonic saline (NaCl-0.15M), gamma-amino butyric acid (GABA), picrotoxin, carbachol, GABA plus picrotoxin, GABA plus carbachol and GABA plus atropine. GABA injection decreased sodium and potassium excretion. Picrotoxin or carbachol injection elicited natriuresis and kaliuresis. GABA injection decreased the effects of the carbachol and atropine injection decreased the effects of the GABA on sodium and potassium excretion. These results suggest an interaction between gabaergic and cholinergic pathways in the control of sodium and potassium excretion.     

Gaba sensitivity of the somata and dendrites of pyramidal cells in isolated slices of rat hippocampus

During experiments on isolated slices slices of rat hippocampus the inhibitory action of -aminobutyric acid (GABA) was investigated on the excitation of field CA, pyramidal neurons, together with the effects of bicuculline, penicillin and thiopentone on this process. It was found that GABA effectively and reversibly reduced the amplitude of the antrodomic population spike in the area of both the somata and the dendrites of these cells. The sensitivity of apical dendrites to GABA exceeded that of the somata by one order, increasing in a proximal-distal direction. The somata of pyramidal neurons were marked by pronounced desensitization to GABA. Bicuculline and penicillin acted as GABA antagonists at all the levels of CA, pyramidal cells investigated. Bicuculline blocked the effects of GABA on somata and dendrites in almost equal measure. The antagonistic effects of penicillin were 10 times greater in the pyramidal layer than in the dendritic region. Thiopentone reinforced the inhibitory effects of GABA. The potentiating effects of thiopentone were exerted most strongly on the dendrites. It is postulated that the membrane of field CA, neurons contain two types of bicuculline-sensitive GABA receptors, differing in their location (mainly on the cell body or dendrite), their pharmacology, and degree of desensitization to GABA.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 17, No. 6, pp. 737–746, November–December, 1985.     

Mutual interactions in the transport of taurine,hypotaurine, and GABA in brain slices

The mutual interactions and the effects of GABA on the saturable transport components of taurine and hypotaurine were investigated with mouse brain slices. The low-affinity taurine transport was competitively inhibited by both hypotaurine and GABA. Hypotaurine did not alter the kinetic parameters of high-affinity taurine uptake, whereas there occurred some stimulation with GABA, possibly by heteroexchange. Taurine had no significant effects on high-affinity hypotaurine uptake, whereas the low-affinity component was reduced by both taurine and GABA, GABA strongly interfered with the high-affinity hypotaurine uptake, being the preferred substrate in simultaneous uptake experiments. The results confirm that taurine, hypotaurine, and GABA are transported into brain slices by only one two-component system with affinities highest for GABA and lowest for taurine.     

Bull sperm acrosome reaction induced by gamma-aminobutyric acid (GABA) is mediated by GABAergic receptors type A

The effect of gamma-aminobutyric acid (GABA) on the bull sperm acrosome reaction was evaluated, and the interaction of progesterone, a physiologic inducer of the acrosome reaction, with the GABA receptor was explored. The acrosome reaction was stimulated by GABA in a dose-dependent manner. This effect was inhibited completely by bicuculline, a GABA A receptor antagonist, but GABA B and C receptor antagonists had no effect. Accordingly, muscimol, a GABA A receptor agonist, stimulated the acrosome reaction to the same extent as GABA, whereas baclofen (GABA B receptor agonist) and CACA (GABA C receptor agonist), had no effect. Preincubation with progesterone followed by the addition of GABA resulted in a significant increase in the percentage of acrosome reacted spermatozoa compared with progesterone or GABA alone. Taking into account that this increase was less than a simple addition of effects, it might be suggested that GABA and progesterone act through the same receptor and/or use the same mechanism of action. To test this hypothesis, the abilities of GABA and progesterone to induce acrosome reaction were tested in the presence of bicuculline, which suppressed both stimulatory effects. Given that the GABA A receptor is linked to the Cl(-) channel, we tested whether picrotoxin, a blocker of this channel, could modulate the effects of progesterone or GABA. Cl(-) channel blocker picrotoxin dramatically reduced the GABA and progesterone-initiated AR. In conclusion: GABA and progesterone stimulate the acrosome reaction in bull spermatozoa acting through a classical GABA A receptor. The mechanism of action requires the functional integrity of the Ca(2+) Cl(-) channel.     

Acute and long-lasting effects of peripheral injection of caerulein and CCK-8 on the central GABAergic system in mice

Acute and long-lasting effects of peripheral injection of caerulein (CLN) and cholecystokinin octapeptide (CCK-8) on the gamma-aminobutylic acid (GABA) content and the GABA accumulation by aminooxyacetic acid (AOAA) in the discrete brain regions of mice were examined. The content and accumulation of GABA in the striatum, hypothalamus, and frontal cortex was measured with high performance liquid chromatography with electrochemical detection (HPLC-ECD). The GABA content slightly decreased in the striatum 60 min after CLN and CCK-8 were administered, whereas it slightly increased in the hypothalamus and frontal cortex. Moreover, with CLN and CCK-8, the GABA accumulation after AOAA treatment decreased in the striatum and hypothalamus 30 min after injection. Meanwhile, when administering CLN, the GABA content as well as the GABA accumulation after AOAA treatment increased in the striatum and frontal cortex 1 day after injection, and continued to increase the second and third day in the striatum. These results showed that peripheral injection of CLN and CCK-8 had effects on the central GABAergic system with local specific actions, and also the long-lasting and time-dependent biphasic effects of CLN.     

GABA regulates the buccal motor output of Helisoma by two pharmacologically distinct actions

GABA was tested for its effects on patterned motor activity (PMA) underlying feeding. Using buccal motoneuron B19 to monitor PMA through intracellular recordings, GABA was found to exert effects at two levels. First, GABA stimulated rhythmic patterned activity resembling fictive feeding, which is under the control of the buccal CPG. In addition, GABA produced a direct inhibition of neuron B19. Both effects were observed when the buccal ganglia were studied in isolation from the rest of the central nervous system, suggesting local interactions with GABA receptors of buccal neurons. Furthermore, these two actions of GABA were found to be pharmacologically distinguishable. The direct hyperpolarization of neuron B19 was mimicked by muscimol, but not baclofen, and involved an increased chloride conductance, which was blocked by picrotoxin.Baclofen duplicated CPG activation by GABA. Picrotoxin had no effect on GABA- or baclofen-induced PMA.These results demonstrate that the Helisoma buccal ganglia have two GABA receptor types which resemble, pharmacologically, mammalian GABA_A and GABA_B receptors, and that GABA plays a key role in feeding patterned motor activity in Helisoma.Abbreviations CPG central pattern generator - GABA gammaamino butyric acid - HPLC high performance liquid chromatography - IPSP inhibitory postsynaptic potential - PMA patterned motor activity - SLRT supralateral radular tensor muscle     

Effect of ammonia on GABA uptake and release in cultured astrocytes

While the pathogenesis of hepatic encephalopathy (HE) is unclear, there is evidence of enhanced GABAergic neurotransmission in this condition. Ammonia is believed to play a major pathogenetic role in HE. To determine whether ammonia might contribute to abnormalities in GABAergic neurotransmission, its effects on GABA uptake and release were studied in cultured astrocytes, cells that appear to be targets of ammonia neurotoxicity. Acutely, ammonium chloride (5 mM) inhibited GABA uptake by 30%, and by 50-60% after 4-day treatment. GABA uptake inhibition was associated with a predominant decrease in Vmax; the Km was also decreased. Ammonia also enhanced GABA release after 4-day treatment, although such release was initially inhibited. These effects of ammonia (inhibition of GABA uptake and enhanced GABA release) may elevate extracellular levels of GABA and contribute to a dysfunction of GABAergic neurotransmission in HE and other hyperammonemic states.     

Effects of gamma-aminobutyric acid on secretagogue-induced exocrine secretion of isolated, perfused rat pancreas

Because GABA and its related enzymes have been determined in beta-cells of pancreas islets, effects of GABA on pancreatic exocrine secretion were investigated in the isolated, perfused rat pancreas. GABA, given intra-arterially at concentrations of 3, 10, 30, and 100 microM, did not exert any influence on spontaneous or secretin (12 pM)-induced pancreatic exocrine secretion. However, GABA further elevated CCK (10 pM)-, gastrin-releasing peptide (100 pM)-, or electrical field stimulation-induced pancreatic secretions of fluid and amylase dose dependently. The GABA (30 microM)-enhanced CCK-induced pancreatic secretions were completely blocked by bicuculline (10 microM), a GABA(A) receptor antagonist, but were not affected by saclofen (10 microM), a GABA(B) receptor antagonist. The enhancing effects of GABA (30 microM) on CCK-induced pancreatic secretions were not changed by tetrodotoxin (1 microM) but were partially reduced by cyclo-(7-aminoheptanonyl-Phe-D-Trp-Lys-Thr[BZL]) (10 nM), a somatostatin antagonist. In conclusion, GABA enhances pancreatic exocrine secretion induced by secretagogues, which predominantly induce enzyme secretion, via GABA(A) receptors in the rat pancreas. The enhancing effect of GABA is partially mediated by inhibition of islet somatostatin release.     

Identification and functional characterization of a dual GABA/taurine transporter in the bullfrog retinal pigment epithelium

Intracellular microelectrodes, fluorescence imaging, and radiotracer flux techniques were used to investigate the physiological response of the retinal pigment epithelium (RPE) to the major retinal inhibitory neurotransmitter, gamma-aminobutyric acid (GABA). GABA is released tonically in the dark by amphibian horizontal cells, but is not taken up by the nearby Muller cells. Addition of GABA to the apical bath produced voltage responses in the bullfrog RPE that were not blocked nor mimicked by any of the major GABA-receptor antagonists or agonists. Nipecotic acid, a substrate for GABA transport, inhibited the voltage effects of GABA. GABA and nipecotic acid also inhibited the voltage effects of taurine, suggesting that the previously characterized beta- alanine sensitive taurine carrier also takes up GABA. The voltage responses of GABA, taurine, nipecotic acid, and beta-alanine all showed first-order saturable kinetics with the following Km's: GABA (Km = 160 microM), beta-alanine (Km = 250 microM), nipecotic acid (Km = 420 microM), and taurine (Km = 850 microM). This low affinity GABA transporter is dependent on external Na, partially dependent on external Cl, and is stimulated in low [K]o, which approximates subretinal space [K]o during light onset. Apical GABA also produced a significant conductance increase at the basolateral membrane. These GABA-induced conductance changes were blocked by basal Ba2+, suggesting that GABA decreased basolateral membrane K conductance. In addition, the apical membrane Na/K ATPase was stimulated in the presence of GABA. A model for the interaction between the GABA transporter, the Na/K ATPase, and the basolateral membrane K conductance accounts for the electrical effects of GABA. Net apical-to-basal flux of [3H]-GABA was also observed in radioactive flux experiments. The present study shows that a high capacity GABA uptake mechanism with unique pharmacological properties is located at the RPE apical membrane and could play an important role in the removal of GABA from the subretinal space (SRS). This transporter could also coordinate the activities of GABA and taurine in the SRS after transitions between light and dark.     

GABA receptor modulation of 5-HT neuronal firing: characterization and effect of moderate in vivo variations in glucocorticoid levels

Evidence from electrophysiological studies suggests that 5-HT neuronal firing in the dorsal raphe nucleus (DRN) may be regulated by both GABA(A) and GABA(B) receptors. Here, we addressed the question of whether the activity of individual 5-HT neurons is regulated by both GABA(A) and GABA(B) receptors. In addition, we examined the concentration-response relationships of GABA(A) and GABA(B) receptor activation and determined if GABA receptor regulation of 5-HT neuronal firing is altered by moderate alterations in circulating corticosterone. The activity of 5-HT neurons in the DRN of the rat was examined using in vitro extracellular electrophysiology. The firing of all individual neurons tested was inhibited by both the GABA(A) receptor agonist 4,5,6,7-tetrahydroisoxazolo-[5,4-c]-pyridin-3-ol hydrochloride (THIP) (25 microM) and the GABA(B) receptor agonist baclofen (1 microM). Responses to THIP (5, 10, 25 microM) and baclofen (1, 3, 10 microM) were concentration dependent and attenuated by the GABA(A) and GABA(B) receptor antagonists, bicuculline (50 microM) and phaclofen (200 microM), respectively. To examine the effects of corticosterone on the sensitivity of 5-HT neurons to GABA receptor activation, experiments were conducted on adrenalectomized animals with corticosterone maintained for two weeks at either a low or moderate level within the normal diurnal range. These changes in corticosterone levels had no significant effects on the 5-HT neuronal response to either GABA(A) or GABA(B) receptor activation. The data indicate that the control of 5-HT neuronal activity by GABA is mediated by both GABA(A) and GABA(B) receptors and that this control is insensitive to moderate changes in circulating glucocorticoid levels.     

Effects of GABA on electrical properties of cultured rat pituitary tumor cells: An intracellular recording study

In this study we show that the electrophysiological properties of a clonal line of prolactin secreting (PRL) rat pituitary cells (GH3/B6) are altered by local application of γ-aminobutyric acid (GABA). The effects of GABA on these cells are: 1) decrease in membrane conductance and 2) hyperpolarization of 5 to 10 mV. When the cells were spontaneously active, GABA reduced and usually arrested action potential firing. This effect was completely reversible. No desensitization of GABA effects was observed even after several applications. The reversal potential of the GABA induced responses was found to be near — 40 mV. Pharmacological studies were performed in order to assess the specificity of the response to GABA and to attempt to characterize the ionic mechanism involved. GABAergic receptor antagonists such as picrotoxin and bicuculline prevented the effect of GABA, whereas dopaminergic receptor antagonists, such as haloperidol or chlorpromazine had no effect. Furosemide, known as a blocker of chloride ion transport in a number of systems, competed with GABA. This substance induced a response similar to that observed with GABA and reduced the effect of GABA when administered before GABA. This study demonstrates a direct and specific effect of GABA on the electrical activity of a tumoral line of rat pituitary cells. Since this electrical activity has previously been shown to be calcium dependent and involved in the secretion of PRL by these cells, the data presented here suggest GABA as an inhibitory regulator of PRL secretion directly at the pituitary level.     

The role of amino acid neurotransmitters in the regulation of pituitary gonadotropin release in fish.

Both glutamate and gamma-aminobutyric acid (GABA) are involved in pituitary hormone release in fish. Glutamate serves 2 purposes, both as a neurotransmitter and as a precursor for GABA synthesis. Glutamate can be catabolized to GABA by the actions of 2 distinct but related enzymes, glutamate decarboxylase 65 (GAD65) and GAD67. They derive from 2 different genes that likely arose from an early gene duplication prior to the emergence of teleosts more than 400 million years ago. There is good evidence for the involvement of GABA in luteinizing hormone (LH) release in fish. The mechanism of GABA action to stimulate LH release appears to be a combination of effects on GnRH release, potentiation of gonadotropin hormone-releasing hormone (GnRH) action, and in some cases directly at the LH cell. These actions appear to be dependent on such factors as sex or sex steroid levels, and there may also be species differences. Nevertheless, the stimulatory effects of GABA on LH are present in at least 4 fish species. In contrast, convincing data for the inhibitory effects of GABA on LH release have only been observed in 1 fish species. The sites and mechanisms of action of amino acid neurotransmitters on LH release have yet to be fully characterized. Both 130N-methyl-D-aspartic acid (NMDA) and S-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) type glutamate receptors are likely to have important roles. We suggest that it is a receptor similar to the GABA(A) type which mediates the effects of GABA on LH release in fish, at least partially acting on the GnRH neuron, but likely directly acting at the gonadotroph as well. GABA may also be involved in regulating the release of other pituitary hormones in fish, namely follicle stimulating hormone (FSH = GTH-I), prolactin, and growth hormone. Based on the findings described in this review, a working model for the involvement of glutamate and GABA in the regulation of LH release in teleost fish is proposed.     

GABA and the behavioral effects of anxiolytic drugs

Much recent research has shown that benzodiazepine binding sites in the central nervous system are associated with GABA receptors. It is therefore possible that the pharmacological and therapeutic effects of benzodiazepines and drugs with similar profiles are mediated through GABAergic mechanisms. In this paper the evidence is considered for a possible involvement of GABA in the behavioral effects of anxiolytic drugs. There are a number of reports that the behavioral actions of anxiolytics can be antagonised by GABA antagonists such as bicuculline or picrotoxin but there are many contradictory findings and these drugs are difficult to use effectively in behavioral studies. In general, GABA agonists do not exert anxiolytic-like behavioral effects after systemic injection but intracerebral administration of muscimol has been shown to produce benzodiazepine-like actions. Although a number of questions remain unanswered, current evidence does not provide strong support for a role for GABA in the behavioral effects of anxiolytic drugs.     

Effects of GABA receptor stimulation on the release of [3H]GABA from slices of rat neostriatum.

Slices of rat neostriatum were incubated in Krebs-Henseleit medium. Modulation of [3H]GABA release by GABA agonists and antagonists was investigated. The GABAA receptor agonists muscimol (0.1 microM) and isoguvacine (5 microM) enhanced the stimulated release of [3H]GABA. The antagonists picrotoxin (1 microM) and bicuculline (50 microM) prevented the effects of the agonists. In the presence of naloxone (1 microM), which blocked the effects of enkephalinergic neurons within the slice preparation, muscimol (1 microM) no longer affected the release of [3H]GABA.     

Sensitivities of Achatina giant neurones to putative amino acid neurotransmitters

1. GABA receptors in Achatina identifiable giant neurones were classified into the muscimol I, muscimol II and baclofen types. Muscimol I and II type GABA receptors were sensitive to GABA and muscimol but insensitive to baclofen, whereas baclofen type receptors were sensitive to GABA and baclofen but insensitive to muscimol. Muscimol I and baclofen types were associated with the inhibition caused by GABA, while muscimol II type with the GABA excitation.2. GABA, muscimol and TACA produced a transient outward current (I_out) with an increase in membrane conductance (g) of an Achatina neurone, TAN, having the muscimol I type GABA receptors. Their relative potency values (RPV) at GABA ed₅₀ (approximately 10⁻⁴ M) were: GABA: muscimol: TACA = 1:0.6:0.3. The GABA effects were potentiated by pentobarbitone, antagonized competitively by pitrazepin and non-competitively by picrotoxin and diazepam, and unaffected by bicuculline. The ionic mechanism of effects of GABA and its two analogues was the increase in membrane Cl⁻ conductance (g_Cl).3. GABA and (±)-baclofen produced a slow I_out with a g increase of another Achatina neurone, RPeNLN, having the baclofen type GABA receptors. The two compounds were almost equipotent (ed₅₀: approximately 3 × 10⁻⁴ M). The ionic mechanism of their effects was the increase in g_k. The two compounds hardly affected the voltage-gated and slowly inactivating calcium current. I_out produced by GABA and (±)-baclofen were reduced by TEA, but unaffected by 4-AP, bicuculline, pitrazepin and picrotoxin.4. β-hydroxy-l-glutamic acid (l-BHGA) showed the marked effects on the Achatina giant neurones; the two neurones were excited by the compound, whereas the three inhibited. D-BHGA, l-Glu, d-Glu and NMDA were less effective than l-BHGA or almost ineffective. Erythro-l-BHGA was more or less effective than threo-l-BHGA according to the neurones tested.5. α-Kainic acid and domoic acid excited the two neurones, which were excited by l-BHGA. l-Quisqualic acid showed the similar effects to l-BHGA, which were mostly much stronger than l-BHGA. Erythro-l-tricholomic acid and dl-ibotenic acid showed the effects similar to l-BHGA selectively on some neurones.6. It was pointed out that the pharmacological features of GABA on the Achatina neurones are simpler than those of l-BHGA, due to the simpler structure of the former compound having less binding sites than the latter.     

Relaxation and immunity enhancement effects of gamma-aminobutyric acid (GABA) administration in humans

The effect of orally administrated gamma-aminobutyric acid (GABA) on relaxation and immunity during stress has been investigated in humans. Two studies were conducted. The first evaluated the effect of GABA intake by 13 subjects on their brain waves. Electroencephalograms (EEG) were obtained after 3 tests on each volunteer as follows: intake only water, GABA, or L-theanine. After 60 minutes of administration, GABA significantly increases alpha waves and decreases beta waves compared to water or L-theanine. These findings denote that GABA not only induces relaxation but also reduces anxiety. The second study was conducted to see the role of relaxant and anxiolytic effects of GABA intake on immunity in stressed volunteers. Eight acrophobic subjects were divided into 2 groups (placebo and GABA). All subjects were crossing a suspended bridge as a stressful stimulus. Immunoglobulin A (IgA) levels in their saliva were monitored during bridge crossing. Placebo group showed marked decrease of their IgA levels, while GABA group showed significantly higher levels. In conclusion, GABA could work effectively as a natural relaxant and its effects could be seen within 1 hour of its administration to induce relaxation and diminish anxiety. Moreover, GABA administration could enhance immunity under stress conditions.     

Diazepam Increases γ-Aminobutyric Acid in Human Cerebrospinal Fluid

In 11 neurological patients, levels of the inhibitory neurotransmitter gamma-aminobutyric acid (GABA) were determined in cerebrospinal fluid (CSF) before and 1, 3, 5, and 8 min after intravenous injection of diazepam (2 or 5 mg). GABA levels increased progressively after intravenous injection of 5 but not 2 mg of the benzodiazepine, the differences from preinjection values being significant at 3, 5, and 8 min. Furthermore, when relative CSF GABA alterations determined after injection of diazepam were compared to those determined in sequential CSF aliquots of 10 patients without diazepam injection, mean GABA increases after diazepam were significantly different from controls in all CSF fractions. The data suggest that, in addition to its well-known effects on postsynaptic GABA function, diazepam may exert effects on endogenous GABA concentrations and/or on GABA release in the human CNS as reflected by elevation of GABA levels in human CSF.     

GABA maintains the proliferation of progenitors in the developing chick ciliary marginal zone and non-pigmented ciliary epithelium

GABA is more than the main inhibitory neurotransmitter found in the adult CNS. Several studies have shown that GABA regulates the proliferation of progenitor and stem cells. This work examined the effects of the GABA(A) receptor system on the proliferation of retinal progenitors and non-pigmented ciliary epithelial (NPE) cells. qRT-PCR and whole-cell patch-clamp electrophysiology were used to characterize the GABA(A) receptor system. To quantify the effects on proliferation by GABA(A) receptor agonists and antagonists, incorporation of thymidine analogues was used. The results showed that the NPE cells express functional extrasynaptic GABA(A) receptors with tonic properties and that low concentration of GABA is required for a baseline level of proliferation. Antagonists of the GABA(A) receptors decreased the proliferation of dissociated E12 NPE cells. Bicuculline also had effects on progenitor cell proliferation in intact E8 and E12 developing retina. The NPE cells had low levels of the Cl-transporter KCC2 compared to the mature retina, suggesting a depolarising role for the GABA(A) receptors. Treatment with KCl, which is known to depolarise membranes, prevented some of the decreased proliferation caused by inhibition of the GABA(A) receptors. This supported the depolarising role for the GABA(A) receptors. Inhibition of L-type voltage-gated Ca(2+) channels (VGCCs) reduced the proliferation in the same way as inhibition of the GABA(A) receptors. Inhibition of the channels increased the expression of the cyclin-dependent kinase inhibitor p27(KIP1), along with the reduced proliferation. These results are consistent with that when the membrane potential indirectly regulates cell proliferation with hyperpolarisation of the membrane potential resulting in decreased cell division. The increased expression of p27(KIP1) after inhibition of either the GABA(A) receptors or the L-type VGCCs suggests a link between the GABA(A) receptors, membrane potential, and intracellular Ca(2+) in regulating the cell cycle.