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     

Binding sites for [3H]GABA, [3H]flunitrazepam and [35S]TBPS in insect CNS

The CNS of the cockroach Periplaneta americana contains saturable, specific binding sites for [³H]GABA, [³H]flunitrazepam and [³⁵S]TBPS. The [³H]GABA binding site exhibits a pharmacological profile distinct from that reported for mammalian GABA_A and GABA_B receptors. The most potent inhibitors of [³H]GABA binding were GABA and muscimol, whereas isoguvacine, thiomuscimol and 3-aminopropane sulphonic acid were less effective. Bicuculline methiodide and baclofen were ineffective. Binding of [³⁵S]TBPS was partially inhibited by 1.0 × 10⁻⁶ M GABA, whilst binding of [³H]flunitrazepam was enhanced by 1.0 × 10⁻⁷ M GABA. The pharmacological profile of the [³H]flunitrazepam binding site showed some similarities with the peripheral benzodiazepine binding sites of vertebrates, with Ro-5-4864 being a far more effective inhibitor of binding than clonazepam. Thus a class of GABA receptors with pharmacological properties distinct from mammalian GABA receptor subtypes is present in insect CNS.     

Substrate Specificity of [3H] Muscimol Binding to a Particulate Fraction of a Neuron-enriched Culture of Embryonic Rat Brain

Abstract: The effects of some GABA analogues and some drugs on the binding of [³H]muscimol (3.08 nM) to thoroughly washed subcellular particles prepared from a neuron-enriched culture of embryonic rat brain were examined using Na+-free Tris-citrate medium and a centrifugation method. Competition for [³H]muscimol binding sites by excess(10^?5 M) unlabelled GABA provided estimates of “specific” binding. In accord with in vivo neuropharmacological studies on GABA receptors and with in vitro studies on cerebral membrane preparations, [³H]muscimol binding was potently inhibited by muscimol itself (IC₅₀, 2.5 nM), GABA (1C₅₀, 43 nM), isoguvacine (IC₅₀, 61 nM), and 3-aminopropanesulphonic acid (IC₅₀, 160 nM), and less potently inhibited by the GABA antagonist bicuculline methobromide (IC₅₀, 800 nM). δ- Aminovaleric acid (IC₅₀, 2.6 μM), the glycinelp-alanine antagonist strychnine (IC₅₀, 6.6 μM), and the predominantly glial GABA uptake inhibitors β-alanine (IC₅₀, 23 μM) and p-proline (IC₅₀, 66 μM) also inhibited [³H]muscimol binding. Other inhibitors of Na+-dependent GABA uptake, (±)-nipecotic acid, L- 2,4-diaminobutyric acid, and guvacine, as well as picrotoxinin, were relatively inactive as inhibitors of [³H]muscimol binding (IC₅₀≥ 1 mM). In addition to revealing that GABA receptors are present on neuronal membranes before the formation of most synapses, this binding of [³H]muscimol that occurs to neuronal, but not to glial, membranes might be useful as a “neuronal marker” and for the further characterization and isolation of GABA receptors.     

GABA Induces Functionally Active Low-Affinity GABA Receptors on Cultured Cerebellar Granule Cells

The effect of γ-aminobutyric acid (GABA) and its agonists muscimol and 4,5,6,7-tetrahydroisoxazolo[5-4-c]pyridin-3-ol (THIP) on the development of GABA receptors on cerebellar granule cells was studied by cultivation of the cells in media containing these substances. It was found that the presence of 50 μM GABA in the culture media led to the induction of low-affinity GABA receptors (K_D 546 ± 117 nM) in addition to the high-affinity receptors (K_D 7 ± 0.5 nM) which were present regardless of the presence of GABA in the culture media. The functional activity of the GABA receptors was tested by investigating the ability of GABA to modulate evoked glutamate release from the cells. It was found that GABA could inhibit evoked glutamate release (ED₅₀ 10 ± 3 (μM) only when the cells had been cultured in the presence of 50 νM GABA, 50 μM muscimol, or 150 μM THIP, i.e., under conditions where low-affinity GABA receptors were present on the cells. This inhibitory effect of GABA could be blocked by 120 μM bicuculline and mimicked by 50 μM muscimol or 150 μM THIP whereas 150 μM (-)-baclofen had no effect. It is concluded that GABA acting extracellularly induces formation of low-affinity receptors on cerebellar granule cells and that these receptors are necessary for mediating an inhibitory effect of GABA on evoked glutamate release. The pharmacological properties of these GABA receptors indicate that they belong to the so-called GABA_A receptors.     

Maximal GABA and muscimol binding to high-affinity sites differ in physiological and in non-physiological buffers

The sensitivity of [³H]GABA and [³H]muscimol high-affinity binding sites to physiological (Krebs-Ringer's bicarbonate) and non-physiological (Tris-citrate) buffers was examined using synaptosomal membranes from bovine retinas. The maximum number of sites (B_max) for [³H]GABA was present when the tissue was assayed in KRB. With only one exception, this effect was independent of the washing conditions used or a small change in pH. In contrast, [³H]muscimol binding sites were maximally present when the tissue was washed in Tris, regardless of the assaying conditions or the small change in pH. Neither [³H]GABA nor [³H]muscimol was displaced by ( - )baclofen. The apparent dissociation constants (K_d) of the ligands did not change under any of the conditions tested. These findings demonstrate a fundamental difference between GABA and muscimol binding sites.     

The stimulation of GABAB receptors increases serotonin release in the rat suprachiasmatic area

The action of γ-aminobutyric acid (GABA) and related compounds on the spontaneous release of newly synthesized [³H]5-hydroxytryptamine ([³H]5-HT) was studied in the suprachiasmatic area (SCA) using a superfusion system. GABA (10 μM) increased [³H]5-HT release from SCA by up to 190%. Bicuculline or picrotoxin (10 μM) failed to inhibit the stimulatory effect of GABA. Muscimol (10 μM), a GABA_A agonist, was ineffective, however β-p-chlorophenyl GABA, R(−)baclofen, enhanced over 200% the release of the indoleamine; this latter effect was stereospecific. RS baclofen was twice less potent than the R(−)isomer in increasing the [³H]5-HT release. S(+)baclofen failed to affect the release of the indoleamine, whereas it attenuated the effect of its enantiomer. The effect of R(−)baclofen was Ca²⁺ dependent and was abolished by tetrodotoxin (TTX).Taken together these results suggest that in the SCA, [³H]5-HT release is facilitated by the stimulation of GABA_B receptors. The possible localization of these receptors is discussed in the light of morphological data recently reported by Bosler et al. (1985) and results obtained after TTX application.     

GABA receptors on the cell-body membrane of an identified insect motor neuron

The pharmacology of a gamma-aminobutyric acid (GABA) receptor on the cell body of an identified motor neuron of the cockroach (Periplaneta americana) was investigated by current-clamp and voltage-clamp methods. Iontophoretic application of GABA increased membrane conductance to chloride ions, and prolonged application resulted in desensitization. Hill coefficients, determined from dose-response data, indicated that binding of at least two GABA molecules was required to activate the chloride channel. Differences between vertebrate GABAA receptors and insect neuronal GABA receptors were detected. For the GABA receptor of motor neuron Df, the following rank order of potency was observed: isoguvacine greater than muscimol greater than or equal to GABA greater than 3-aminopropanesulphonic acid. The GABAB receptor agonist baclofen was inactive. Of the potent vertebrate GABA receptor antagonists (bicuculline, pitrazepin, RU5135 and picrotoxin), only picrotoxin (10(-7) M) produced a potent, reversible block of the response to GABA of motor neuron Df. Both picrotoxinin and picrotin also blocked GABA-induced currents. Bicuculline hydrochloride (10(-4) M) and bicuculline methiodide (10(-4) M) were both ineffective when applied at resting membrane potential (-65 mV), although at hyperpolarized levels partial block of GABA-induced current was sometimes observed. Pitrazepin (10(-4) M) caused a partial, voltage-independent block of GABA-induced current. The steroid derivative RU5135 was inactive at 10(-5) M. In contrast to the potent competitive blockade of vertebrate GABAA receptors by bicuculline, pitrazepin and RU5135, none of the weak antagonism caused by these drugs on the insect GABA receptor was competitive. Flunitrazepam (10(-6) M) potentiated GABA responses, providing evidence for a benzodiazepine site on an insect GABA-receptor-chloride-channel complex.     

Taurine release modified by GABAergic agents in hippocampal slices from adult and developing mice

Summary. In order to characterize the possible regulation of taurine release by GABAergic terminals, the effects of several agonists and antagonists of GABA receptors on the basal and K⁺-stimulated release of [³H]taurine were investigated in hippocampal slices from adult (3-month-old) and developing (7-day-old) mice using a superfusion system. Taurine release was concentration-dependently potentiated by GABA, which effect was reduced by phaclofen, saclofen and (1,2,5,6-tetrahydropyridin-4-yl)methylphosphinic acid (TPMPA) at both ages, suggesting regulation by both GABA_B and GABA_C receptors. The involvement of GABA_A receptors could not be excluded since the antagonist bicuculline was able to affect both basal and K⁺-evoked taurine release. Furthermore, several GABA_B receptor effectors were able to inhibit K⁺-stimulated taurine release in the adults, while the GABA_C receptor agonists trans-4-aminocrotonic acid (TACA) and cis-4-aminocrotonic acid (CACA) potentiated this release. The potentiation of taurine release by agents acting on the three types of GABA receptors in both adult and developing hippocampus further indicates the involvement of transporters operating in an outward direction. This inference is corroborated by the moderate but significant inhibition of taurine uptake by the same compounds. Received June 28, 1999, Accepted August 31, 1999     

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     

Lack of coupling between GABA release and GABA synthesis in the rat brain via GABAB autoreceptors

Summary. GABA is synthesized within GABA terminals through a highly compartmentalized process in which glial-derived glutamine is a major precursor and its release is modulated by GABA_B autoreceptors. The aim of this work was to ascertain whether or not GABA synthesis and release are coupled in the rat brain through a GABA_B autoreceptor-mediated modulation. It was found that (−)baclofen (30 μM) reduces the K⁺ stimulated release of [³H]GABA in synaptosomes and prisms (10 μM) from cerebral cortex, while at the same concentrations (−)baclofen failed to modify the synthesis of [³H]GABA from [³H]glutamine in cortical and hypothalamic slices, prisms and in cortical synaptosomes. In this latter preparation, identical results were observed when (−)baclofen was added to Krebs-Tris media, containing 5 or 15 mM K⁺ concentration. In agreement with these latter results, glutamic acid decarboxylase (GAD) activity from cortical and hypothalamic prisms was not affected by 1–100 μM (−)baclofen. Similar results on GABA synthesis were also observed when 1–100 μM 3-aminopropil(methyl)-phosphinic acid or GABA was used instead of (−)baclofen to stimulate GABA_B autoreceptors. [³H]GABA release, [³H]GABA synthesis from [³H]glutamine and GAD activity were also insensitive to the action of the GABA_B antagonist CGP 52432 (10–100 μM). Likewise, muscimol (0.3–100 μM) did not affect GABA synthesis. Our results indicate that unlike GABA release, GABA synthesis is not modulated by GABA_B autoreceptors. Received August 31, 1999 Accepted September 20, 1999     

GABAergic Modulation of Striatal Cholinergic Interneurons: An In Vivo Microdialysis Study

Abstract: Striatal cholinergic interneurons have been shown to receive input from Striatal γ-aminobutyric acid (GABA)-containing cell elements. GABA is known to act on two different types of receptors, the GABA_A and the GABA₆ receptor. Using in vivo microdialysis, we have studied the effect of intrastriatal application of the GABA_A-selective compounds muscimol and bicuculline and the GA- BA_B-selective compounds baclofen and 2-hydroxysaclofen, agonists and antagonists, respectively, at GABA receptors, on the output of Striatal acetylcholine (ACh). Intrastriatal infusion of 1 and 10 μmol/L concentrations of the GABA_A antagonist bicuculline resulted in a significant increase in Striatal ACh output, whereas infusion of 1 and 10 /μmol/L concentrations of the GABA_A agonist muscimol significantly decreased the output of Striatal ACh. Both compounds were ineffective in changing the output of Striatal ACh at lower concentrations. Infusion of concentrations up to 100 μmol/L of the GABA_B-selective antagonist 2-hydroxy-saclofen failed to affect Striatal ACh output, whereas infusion of 10 and 100 μmol/L baclofen, but not 0.1 and 1 μmol/L baclofen, significantly decreased the output of Striatal ACh. Thus, agonist-stimulation of GABAA and GABA_B receptors decreases the output of striatal ACh in a dose-dependent fashion, whereas the GABAergic system appears to inhibit tonically the output of striatal ACh via GABA_A receptors, but not via GABA_B receptors. We hypothesize that although GABA_A mediated regulation of striatal ACh occurs via GABA receptors on the cholinergic neuron, the GABA_B mediated effects may be explained by presynaptic inhibition of the glutamatergic input of the striatal cholinergic neuron.     

Interacting Presynaptic k-Opioid and GABAA Receptors Modulate Dopamine Release from Rat Striatal Synaptosomes

Abstract— The presynaptic regulation of stimulated dopa-mine release from superfused rat striatal synaptosomes by opioids and γ-aminobutyric acid (GABA) was studied. It was found that in addition to dopamine D₂ autoreceptors, calcium-dependent K⁺-stimulated [³H]dopamine release was inhibited through activation of a homogeneous population of k -opioid receptors in view of the potent inhibitory effect of the k -selective agonist U69.593 (EC₅₀ 0.2 nM) and its antagonism by norbinaltorphimine. Neither μ-nor δ-selective receptor agonists affected release of [³H]-dopamine. In addition, GABA potently inhibited the evoked [³H]dopamine release (EC₅₀ 0.4 nM) through activation of GABA_A receptors in view of the GABA-mimicking effect of muscimol, the sensitivity of its inhibitory effect to picro-toxin and bicuculline, and the absence of an effect of the GABA_B receptor agonist baclofen. In the presence of a maximally effective concentration of GABA, U69,593 did not induce an additional release-inhibitory effect, indicating that these receptors and the presynaptic D₂ receptor are colocalized on the striatal dopaminergic nerve terminals. The excitatory amino acid agonists N-methyl-d -aspartate and kainate, as well as the cholinergic agonist carbachol, stimulated [³H]dopamine release, which was subject to k -opioid receptor-mediated inhibition. In conclusion, striatal dopamine release is under regulatory control of multiple excitatory and inhibitory neurotransmitter by activation of colocalized presynaptic receptors for excitatory amino acids, acetylcholine, dopamine, dynorphins, and GABA within the dopaminergic nerve terminals. Together, these receptors locally control ongoing dopamine neurotransmission.     

GABA is toxic for mouse striatal neurones through a transporter-mediated process

In the present study, GABA was shown to induce a necrotic neuronal death in cultured striatal neurones from mouse embryos. This effect did not depend on the activation of GABA(A), GABA(B) or GABA(C) receptors as it was neither antagonized by bicuculline, saclofen or picrotoxin, respectively, nor reproduced by the GABA receptor agonists, muscimol and baclofen. Excluding the participation of glutamate, GABA neurotoxicity persisted in the presence of either the antagonists of ionotropic and metabotropic glutamate receptors or glutamate pyruvate transaminase, which induces an immediate catabolism of glutamate. A GABA transport-associated process is involved in GABA neurotoxicity as nipecotic acid and NO 711, two inhibitors of the high-affinity neuronal GABA transporters (GAT-1, in particular), completely prevented the neurotoxic effect of GABA. The activation of a subset of G proteins is also implicated in the GABA transport-mediated neuronal death as GABA neurotoxicity was completely suppressed when striatal neurones were pre-treated with pertussis toxin. Further demonstrating the specificity of this neurotoxic process, GABA-induced neurotoxicity was not observed in cortical neurones which, in contrast to striatal neurones, are largely represented by glutamatergic neurones. In conclusion, our study suggests that glutamate is not the sole neurotransmitter that can be responsible for brain damage and that GABA neurotoxicity involves both GABA transport and G protein transduction pathways.     

Effect of baclofen on in vitro noradrenaline release from rat hippocampus and cerebellum: an action at an α2-adrenoceptor

The release of [³H]noradrenaline from rat hippocampal synaptosomes by 25 mM K⁺ and 5 μM veratridine, but not by the Ca²⁺ ionophore A23187 was depressed by baclofen. This depression was reversed by 8-Bromo-cAMP. This action of baclofen was stereospecific and mimicked both that of GABA in the presence of bicuculline and that of clonidine. The α₂-adrenoceptor antagonists yohimbine and Wy25309 antagonised the action of clonidine and baclofen but not that of GABA. Specific binding of [³H]clonidine was displaced by Wy25309 and baclofen, but not by GABA. Specific binding of [³H]GABA in the presence of Ca²⁺ was displaced by baclofen but not by Wy25309. It is concluded that baclofen is not a specific agonist at GABA_B receptors in the brain.     

Glutamic Acid and γ-Aminobutyric Acid Modulate Each Other's Release Through Heterocarriers Sited on the Axon Terminals of Rat Brain

Abstract: The effects of γ-aminobutyric acid (GABA) on the spontaneous release of endogenous glutamic acid (Glu) or aspartic acid (Asp) and the effects of Glu on the release of endogenous GABA or [³H]GABA were studied in superfused rat cerebral cortex synaptosomes. GABA increased the outflow of Glu (EC₅₀17.2 μM) and Asp (EC₅₀ 18.4 μM). GABA was not antagonized by bicuculline or picrotoxin. Neither muscimol nor (-)-baclofen mimicked GABA. The effects of GABA were prevented by GABA uptake inhibitors and were Na⁺ dependent. Glu enhanced the release of [³H]GABA (EC₅₀ 11.5 μM) from cortical synaptosomes. Glu was not mimicked by the glutamate receptor agonists N-methyl-d -aspartic, kainic, or quisqualic acid. The Glu effect was decreased by the Glu uptake inhibitor D-threo-hydroxyaspartic acid (THA) and it was Na⁺ sensitive. Similarly to Glu, D-Asp increased [³H]GABA release (EC₅₀ 9.9 μM), an effect blocked by THA. Glu also increased the release of endogenous GABA from cortex synaptosomes. In this case the effect was in part blocked by the (RS)-α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor antagonist 6-cyano-7-nitroquinoxaiine-2, 3-dione, whereas the 6-cyano-7-nitroquinoxaline- 2, 3-dione-insensitive portion of the effect was prevented by THA. GABA increased the [³H]D-Asp outflow (EC₅₀ 13.7 μM) from hippocampal synaptosomes in a muscimol-, (-)- baclofen-, bicuculline-, and picrotoxin-insensitive manner. The GABA effect was abolished by blocking GABA uptake and was Na⁺ dependent. Glu increased the release of [³H]- GABA from hippocampal synaptosomes (EC₅₀ 7.1 μM) in an N-methyl-d -aspartic acid-, kainic acid-, or quisqualic acid-insensitive way. The effect of Glu was prevented by THA and was Na⁺ dependent. As in the cortex, the effect of Glu was mimicked by D-Asp in a THA-sensitive manner. It is proposed that high-affinity GABA or Glu heterocarriers are sited respectively on glutamatergic or GA- BAergic nerve terminals in rat cerebral cortex and hippocampus. The uptake of GABA may modulate Glu and Asp release, whereas the uptake of Glu may modulate the release of GABA. The existence of these heterocarriers is in keeping with the reported colocalization of GABA and Glu in some cortical and hippocampal neurons. Preliminary data suggest that these mechanisms may also be present in rat cerebellum and spinal cord.     

Sensitivity of hippocampal interneurons and pyramidal cells to GABA and some of its agonists:in vitro experiments

Effects of GABA and its agonists baclofen and muscimol on the background spike activity of single hippocampal neurons were studied in rat brain slices using an intracellular recording technique. Interneurons localized in thestratum alveus-oriens and pyramidal neurons of thestratum pyramidale showed high sensitivity to GABA (mean ID₅₀=65 µM and 40 µM, ranges 10–140 µM and 3–200 µM), baclofen (ID₅₀=2.6 µM and 3.5 µM, ranges 0.6–20.0 µM and 0.4–30.0 µM), and muscimol (ID₅₀=0.85 µM and 0.21 µM, ranges 0.11–4.0 µM and 0.05–0.45 µM, respectively). Responses of hippocampal neurons to application of GABA or either of its agonists were predominantly inhibitory. A part of interneurons (30%) differed from pyramidal neurons in their irresponsivity or low sensitivity to baclofen applications. GABA- or muscimol-induced inhibition of spike activity in many pyramidal cells was preceded by a short-lasting excitation. Our findings indicate that a part of hippocampal interneurons are very poorly supplied with GABAb receptors. Inhibition of pyramidal cells evoked by activation of GABAa receptors probably develops against the background of accompanying depolarization, which in some cases can result in a provisional excitation of these neurons. The excitatory effects of GABA on the pyramidal cells are mediated by GABAa receptors.Neirofiziologiya/Neurophysiology, Vol. 27, No. 1, pp. 36–44, January–February, 1995.     

[3H]muscimol binding in the rat retina

Crude membrane fractions were prepared from rat retinae and used to study the specific binding of [³H]muscimol, a potent GABA agonist. Specific [³H]muscimol binding was enhanced 2–3 fold by pretreatment of the membranes with 0.025% Triton X-100. Two muscimol binding sites were demonstrated with K_D values of 4.4 and 12.3 nM. GABA, muscimol, and 3-aminopropanesulfonic acid were the most potent inhibitors of specific [³H]muscimol binding with K_I values of 15, 10, and 50 nM, respectively. These data are consistent with binding to the synaptic GABA receptor.     

Effects of External pH Variations on Brain Presynaptic Sodium and Calcium Channels; Repercussion on the Evoked Release of Amino Acid Neurotransmitters

The effects of external pH (pH _out) variations on the Na⁺ and on the Ca²⁺ dependent fractions of the evoked amino acid neurotransmitter release were separately investigated, using GABA as a model transmitter. In [³H]GABA loaded mouse brain synaptosomes, the external acidification (pH _out6.0) markedly decreased the Na⁺ dependent fraction of [³H]GABA release evoked by veratridine (10 M) in the absence of external Ca²⁺, as well as the Ca²⁺ dependent fraction of [³H]GABA release evoked by high (20 mM) K⁺ in the absence of external Na⁺. The depolarization-induced elevation of [Na _i ] (monitored in synaptosomes loaded with the Na⁺ indicator dye, SBFI) and the depolarization-induced elevation of [Ca _i ] (monitored in synaptosomes loaded with the Ca²⁺ indicator dye fura-2) were also markedly decreased at pH _out 6. On the contrary, the external alkalinization (pH _out 8) facilitated all the above responses. A slight increase of the baseline release of the [³H]GABA was observed when pH _out was changed from 7.4 to 8. This effect was only observed in the presence of Ca²⁺. pH _out changes from 7.4 to 6 or to 7 did not modify the baseline release of the transmitter. All the effects of pH _out variations on [³H]GABA release were independent on the presence of HCO-₃. It is concluded that external H⁺ regulate amino acid neurotransmitter release by their actions on presynaptic Na⁺ channels, as well as on presynaptic Ca²⁺ channels.     

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.     

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.