P. Feltz

Coexistence of GABAA and GABAB receptors on Aδ and C primary afferents

1 Intracellular recordings from adult rat dorsal root ganglion neurones were performed in vitro and the coexistence of two γ‐aminobutyric acid (GABA) receptors on the membrane of identified Aδ and C primary afferents was demonstrated. 2 Transient applications of GABA (10−6 − 10−2m) evoked dose‐dependent depolarizations and increased membrane conductance. The responses were mimicked by muscimol, isoguvacine, THIP and 3 amino propane sulphonic acid (3 APS); they were blocked by bicuculline and picrotoxin. Pentobarbitone induced an increase of GABA‐induced depolarizations. 3 Perfusion of tetraethylammonium (TEA, 7.5 mm) and intracellular injection of Cs+ ions unmasked the Ca2+ component of action potentials, which appeared as long‐lasting plateau depolarizations. Such action potentials were shortened in the presence of methoxyverapamil (D600, 5 × 10−6‐ 10−5m) and in a medium without Ca+ ions. 4 Prolonged (5–10 min) perfusion of GABA (10−9‐10−5m) shortened the Ca2+ component of action potentials. This effect was mimicked by baclofen (10−7‐5 × 10−6m) and muscimol (5 × 10−7‐10−5m) and was not affected by bicuculline perfusion (5 × 10−6‐10−5m). Isoguvacine (2.5 × 10−5m) did not affect action potential duration. 5 It is concluded that two GABA receptors coexist on the membrane of slow conducting primary afferents: the bicuculline‐sensitive GABAA receptor mediates depolarizations and the bicuculline‐insensitive GABAB receptor shortens the calcium component of action potentials.

Lipopolyamine‐Mediated Transfection Allows Gene Expression Studies in Primary Neuronal Cells

Abstract: A simple and efficient transfection technique based on lipopolyamine‐coated DNA that can be used for gene transfer in cerebellar granular neurons is described. Gene transfer is achieved by exposure of cells to a DNA/lipid complex obtained by simple mixing of lipopolyamine and plasmid DNA. This procedure may represent a general tool of physiological investigations in primary cells. We show that the promoters of the introduced chimera genes are regulated by their respective trans‐acting factors and may be modulated via membrane receptors and second messengers. This procedure has no noticeable toxic effects, nor does it seem to interfere with complex physiological behavior like neuronal differentiation.

Modulation of GABAA receptor-mediated IPSCs by neuroactive steroids in a rat hypothalamo-hypophyseal coculture model.

1. We have used the whole‐cell configuration of the patch‐clamp technique to investigate the effects of neuroactive steroids on GABAA receptor‐mediated synaptic transmission between rat hypothalamic neurones and pituitary intermediate lobe (IL) cells grown in coculture. In order to discriminate between possible pre‐ and postsynaptic sites of action, the effects of neurosteroids on GABAA receptor‐mediated synaptic currents (IPSCs) were compared with those of GABAA currents (IGABA) triggered by local application of 50 or 500 microM GABA, which yielded approximately half‐maximal and maximal responses, respectively. 2. In primary cultures of rat pituitary IL cells, allopregnanolone (5 alpha‐pregnan‐3 alpha‐ol‐20‐one) reversibly potentiated IGABA in a dose‐dependent manner with a threshold between 0.1 and 1 nM. At a concentration of 10 nM, allopregnanolone increased the response evoked by 50 microM GABA by +21.4 +/‐ 5.1% (n = 8), but had no effect on IGABA induced by 500 microM GABA. The beta‐isomer of allopregnanolone, epipregnanolone (5 beta‐pregnan‐3 beta‐ol‐20‐one, 10 nM), had no effect on IGABA at any concentration of GABA tested. 3. At concentrations lower than 10 microM, pregnenolone sulphate (5‐pregnen‐3 alpha‐ol‐20‐one sulphate) did not significantly inhibit IGABA. However, at 10 microM, a systematic reduction of IGABA evoked by 50 and 500 microM GABA was observed, with mean values of ‐80 and ‐60%, respectively. This blocking effect was reversible and accompanied by a marked acceleration of decay of GABAA currents during the application of GABA. 4. In isolated pairs of synaptically connected hypothalamic neurones and IL cells, allopregnanolone (10 nM) augmented the mean amplitude of spontaneous IPSCs (sIPSCs) and electrically evoked IPSCs (eeIPSCs) by about 40% and increased the mean frequency of sIPSCs. Allopregnanolone (10 nM) also markedly increased the frequency of miniature IPSCs (mIPSCs) recorded in the presence of TTX (0.5 microM), but without modifying their mean amplitude. Epipregnanolone had no effect on the amplitude or frequency of sIPSCs. Neither epipregnanolone nor allopregnanolone modified the time to peak and decay time constants of GABAergic IPSCs. 5. Pentobarbitone (50 microM), a positive allosteric modulator of GABAA receptors, did not affect the amplitude of sIPSCs or eeIPSCs, but significantly increased the decay time constants of both types of IPSCs. Pentobarbitone had no effect on the frequency of sIPSCs. 6. Pregnenolone sulphate (10 microM) completely and reversibly blocked sIPSCs and eeIPSCs. Progressive block of IPSCs was correlated with a gradual decrease of the mean decay time constant. 7. Our results suggest that, under physiological conditions, allopregnanolone might be a potent modulator of GABAergic synaptic transmission, acting at both pre‐ and postsynaptic sites. The involvement of pregnenolone sulphate as a modulator of GABAergic IPSCs under physiological conditions is, however, more questionable. The mechanisms of action of both types of neurosteroids are discussed.

Evidence that GABAA Receptor Subunit mRNA Expression During Development Is Regulated by GABAA Receptor Stimulation

Abstract: The expression of six mRNA species (α2, α3, α5, β2, β3, and γ2) encoding for GABAA receptor subunits was followed in cultured early postnatal cortical neurons by in situ hybridization histochemistry. In untreated control cultures it was found that these subunit mRNA expression profiles closely follow those seen during development in vivo. α3, α5, and β3 subunit expression declined, α2 expression increased, whereas β2 and γ2 subunit mRNA expression remained relatively constant. To test the hypothesis that GABAA receptor stimulation regulates these expression profiles, we tested the effect of a GABAA receptor positive modulator, allopregnanolone, and a GABAA receptor noncompetitive antagonist, tert‐butylbicyclophosphorothionate (TBPS). It was found that allopregnanolone augmented the rate at which the α3, α5, or β3 subunit mRNA expression declined and prevented the increase in α2 subunit mRNA expression. As well, allopregnanolone down‐regulated β2 subunit mRNA expression. TBPS, on the other hand, up‐regulated α3, α5, β2, and β3 subunit mRNA expression. It also down‐regulated the expression of α2 subunit mRNA. Both allopregnanolone and TBPS had no effect on γ2 subunit mRNA expression. These results imply that the developmental switchover of GABA receptor subunit mRNA expression is regulated by GABAA receptor activity.

Localization of Oxytocin Binding Sites in the Thoracic and Upper Lumbar Spinal Cord of the Adult and Postnatal Rat: A Histoautoradiographic Study

Oxytocin binding sites were detected by autoradiography on films and emulsion‐coated sections in the spinal cord of adult and postnatal rats from C8 to L2, using a highly selective 125l‐labelled oxytocin antagonist. Oxytocin binding sites were detected on all transverse sections in the dorsal horn, where labelling was scattered over laminae I and II. The autonomic areas, i.e. the intermediolateral cell column, the central grey (lamina X) and the nucleus intercalatus were labelled. Binding in the intermediolateral cell column was most frequently observed on sections from T9 to T11 in adult and T7 to T8 in postnatal rats. In this location, oxytocin binding sites were highly concentrated on cell bodies of putative sympathetic preganglionic neurons; however, not all of these cells were labelled. Diffuse labelling occurred on the dorsal part of the central grey, mainly between T8 and L2. Isolated labelled cells belonging to the nucleus intercalatus were scattered between the central canal and the intermediolateral cell column. In addition, oxytocin binding sites were found on some motoneurons of the lateral group of T12‐T13, but only in postnatal rats. The distribution of oxytocin binding sites in the rat spinal cord coincides with that of the oxytocin innervation and strongly suggests a modulatory role of this peptide in sensory and autonomic functions.

Spontaneous and GABA-evoked chloride channels on pituitary intermediate lobe cells and their internal Ca requirements.

On porcine intermediate lobe (IL) endocrine cells, spontaneously opening chloride channels have been studied and compared to GABA-A activated chloride channels. Elementary currents were recorded mainly from outside-out patches excised from IL cells maintained in culture for 1–4 weeks. Spontaneous inward currents were observed in Cs-loaded cells after replacing Na in the extracellular medium by the impermeant ion choline. This activity, at an internal calcium concentration of 10−8 M corresponded to a channel for chloride ions with a main conductance level of 26 pS, and substates around 11 pS. The sequence of permeabilities to halides was I>Br>Cl. These conductance characteristics were common to the GABA-operated channels which also showed a main conductance substate of 23–31 pS. The open time of the 26 pS level mostly encountered in spontaneous activity, was distributed along two modes: one, the most frequent, around 1 ms, and the other around 4 ms. This latter mode was the predominant one observed during GABA and isoguvacine applications but in addition a bursting activity of 19 ms duration was also seen. Specific GABA-A receptor antagonists (bicuculline and SR 42641, 1 μM) blocked activity evoked by GABA (1–10 μM), but did not affect spontaneous events. These spontaneous Cl events were only observed in a restricted range of internal Ca concentrations, i.e. between 1 nM and 0.1 μM, and were practically abolished at Cai 1 μM. The GABA-induced activity of Cl channels was also Ca-sensitive, being reduced when Cai reached 1 μM.

Quantitative evaluation of the properties of a pyridazinyl GABA derivative (SR 95531) as a GABAA competitive antaginist. An electrophysiological approach

We have investigated the effects of an aryl-aminopyridazine derivative of GABA (SR 95531) on dose-response curves of GABA-induced depolarizations from dorsal root ganglion neurones recorded intracellularly. The reversible shift to the right of the dose-response curves in a parallel fashion and the dissociation constant (KB) value of 0.13 +/- 0.02 microM (n = 15) indicate that this compound is a potent competitive GABAA antagonist. The competitive nature of SR 95531-induced antagonism was confirmed by single channel analysis. In excised membrane patches from bovine chromaffin cells (outside out configuration), 0.2-0.5 microM SR 95531 did not alter the mean open time of GABA-activated channels and did not introduce further short closing gaps within bursts. Whole cell recordings from cultured nodose ganglion neurones indicated that SR 95531 (10 microM) did not modify significantly any of the 3 types of calcium currents already reported in sensory neurones. This result might be of importance for further studies of presynaptic GABA actions on transmitter release.

Sumatriptan inhibits the release of CGRP and substance P from the rat spinal cord.

The possible presynaptic action of the anti-migraine drug sumatriptan on primary afferent fibres containing substance P and/or calcitonin gene-related peptide was investigated on superfused rat horizontal spinal cord slices with attached dorsal roots. Electrical stimulation of dorsal roots triggered a significant overflow of both peptides; this could be reduced by sumatriptan in a concentration-dependent manner. As expected from the involvement of 5-HT1B/1.D beta receptors, methiothepin, (-)tertatolol and GR 127,935, but not WAY 100,635, prevented the inhibitors effect of sumatriptan. These data support the idea that the anti-migraine action of sumatriptan may involve, at least in part, a presynaptic inhibitory control of nociceptive (trigeminovascular) substance P- and/or calcitonin gene-related peptide-containing sensory fibres.

GABAA and GABAB receptors on porcine pars intermedia cells in primary culture: Functional role in modulating peptide release

A primary culture of porcine pars intermedia cells with particularly high yields has been developed. The cells, grown in monolayers, secrete the pro-opiomelanocortin-derived peptide alpha-melanocyte-stimulating hormone over several weeks. The patch-clamp technique has been used to demonstrate the presence of gamma-aminobutyrateA (GABAA) receptors on the cells. GABA or the selective GABAA receptor agonist isoguvacine produced a depolarizing increase in chloride conductance that desensitized rapidly. The response was antagonized by bicuculline and by the aminopyridazine derivative of GABA (SR 95103), a novel GABAA receptor antagonist. The effects of specific agonists for each receptor were tested on peptide release from cells maintained in a perfusion system. Isoguvacine (10 microM) potentiated Ba2+-evoked release of alpha-melanocyte-stimulating hormone, whereas (-)-baclofen (50 microM) decreased both basal and stimulated hormone release. This negative effect on peptide secretion was reproduced when GABA (50 microM) was perfused in the presence of bicuculline (10 microM) to block GABAA receptor activation. The possible mechanisms underlying these GABAA and GABAB effects on stimulus-secretion coupling in this neuroendocrine model are discussed.

Dual population of GABAA and GABAB receptors in rat pars intermedia demonstrated by release of αMSH caused by barium ions

1 We have studied the effects of selective GABAA and GABAB agonists on α‐melanophore stimulating hormone (αMSH) release from intact rat neurointermediate lobes (NIL) in vitro. Agonist effects were tested against either basal αMSH output or BaCl2 (5 mM)‐evoked release. 2 GABA (50 μM) produced a biphasic effect on basal release, with an enhancement followed by inhibition of release. The enhancement but not the inhibition was blocked by bicuculline methiodide (100 μM). 3 Baclofen (10 μM), a specific GABAB agonist, reduced the basal and Ba2+‐evoked hormonal release in a stereospecific manner. (−)−Baclofen (5 μM) was active whereas the (+)‐isomer was inactive at the same concentration. 4 Isoguvacine (50 μM) a specific GABAA agonist, potentiated the Ba2+‐evoked release of αMSH. GABA (50 μM) mimicked this effect, and its action was antagonized by bicuculline methiodide (200 μM). 5 The results suggest that both GABAA and GABAB receptors are present on the endocrine cells of the intermediate lobe.