Inhibitory transmission in locus coeruleus neurons expressing GABAA receptor epsilon subunit has a number of unique properties.

Fast inhibitory synaptic transmission in the brain relies on ionotropic GABA(A) receptors (GABA(A)R). Eighteen genes code for GABA(A)R subunits, but little is known about the epsilon subunit. Our aim was to identify the synaptic transmission properties displayed by native receptors incorporating epsilon. Immunogold localization detected epsilon at synaptic sites on locus coeruleus (LC) neurons. In situ hybridization revealed prominent signals from epsilon, and mRNAs, some low beta1 and beta3 signals, and no gamma signal. Using in vivo extracellular and in vitro patch-clamp recordings in LC, we established that neuron firing rates, GABA-activated currents, and mIPSC charge were insensitive to the benzodiazepine flunitrazepam (FLU), in agreement with the characteristics of recombinant receptors including an epsilon subunit. Surprisingly, LC provided binding sites for benzodiazepines, and GABA-induced currents were potentiated by diazepam (DZP) in the micromolar range. A number of GABA(A)R ligands significantly potentiated GABA-induced currents, and zinc ions were only active at concentrations above 1 muM, further indicating that receptors were not composed of only alpha and beta subunits, but included an epsilon subunit. In contrast to recombinant receptors including an epsilon subunit, GABA(A)R in LC showed no agonist-independent opening. Finally, we determined that mIPSCs, as well as ensemble currents induced by ultra-fast GABA application, exhibited surprisingly slow rise times. Our work thus defines the signature of native GABA(A)R with a subunit composition including epsilon: differential sensitivity to FLU and DZP and slow rise time of currents. We further propose that alpha(3,) beta(1/3,) and epsilon subunits compose GABA(A)R in LC.

Dopamine receptors set the pattern of activity generated in subthalamic neurons.

Information processing in the brain requires adequate background neuronal activity. As Parkinson's disease progresses, patients typically become akinetic; the death of dopaminergic neurons leads to a dopamine-depleted state, which disrupts information processing related to movement in a brain area called the basal ganglia. Using agonists of dopamine receptors in the D1 and D2 families on rat brain slices, we show that dopamine receptors in these two families govern the firing pattern of neurons in the subthalamic nucleus, a crucial part of the basal ganglia. We propose a conceptual frame, based on specific properties of dopamine receptors, to account for the dominance of different background firing patterns in normal and dopamine-depleted states.

Activation of GABA(A) receptors in subthalamic neurons in vitro: properties of native receptors and inhibition mechanisms.

The subthalamic nucleus (STN) influences the output of the basal ganglia, thereby interfering with motor behavior. The main inputs to the STN are GABAergic. We characterized the GABA(A) receptors expressed in the STN and investigated the response of subthalamic neurons to the activation of GABA(A) receptors. Cell-attached and whole cell recordings were made from rat brain slices using the patch-clamp technique. The newly identified epsilon subunit confers atypical pharmacological properties on recombinant receptors, which are insensitive to barbiturates and benzodiazepines. We tested the hypothesis that native subthalamic GABA(A) receptors contain epsilon proteins. Applications of increasing concentrations of muscimol, a selective GABA(A) agonist, induced Cl(-) and HCO currents with an EC(50) of 5 microM. Currents induced by muscimol were fully blocked by the GABA(A) receptor antagonists, bicuculline and picrotoxin. They were strongly potentiated by the barbiturate, pentobarbital (+190%), and by the benzodiazepines, diazepam (+197%) and flunitrazepam (+199%). Spontaneous inhibitory postsynaptic currents were also significantly enhanced by flunitrazepam. Furthermore, immunohistological experiments with an epsilon subunit-specific antibody showed that the epsilon protein was not expressed within the STN. Native subthalamic GABA(A) receptors did not, therefore, display pharmacological or structural properties consistent with receptors comprising epsilon. Burst firing is a hallmark of Parkinson's disease. Half of the subthalamic neurons have the intrinsic capacity of switching from regular-firing to burst-firing mode when hyperpolarized by current injection. This raises the possibility that activation of GABA(A) receptors might trigger the switch. Statistical analysis of spiking activity established that 90% of intact neurons in vitro were in single-spike firing mode, whereas 10% were in burst-firing mode. Muscimol reversibly stopped recurrent electrical activity in all intact neurons. In neurons held in whole cell configuration, membrane potential hyperpolarized by -10 mV whilst input resistance decreased by 50%, indicating powerful membrane shunting. Muscimol never induced burst firing, even in neurons that exhibited the capacity of switching from regular- to burst-firing mode. These molecular and functional data indicate that native subthalamic GABA(A) receptors do not contain the epsilon protein and activation of GABA(A) receptors induces membrane shunting, which is essential for firing inhibition but prevents switching to burst-firing. They suggest that the STN, like many other parts of the brain, has the physiological and structural features of the widely expressed GABA(A) receptors consisting of alphabetagamma subunits.

Molecular and electrophysiological evidence for a GABAc receptor in thyrotropin-secreting cells.

In the pituitary, GABA regulates the release of several hormones via different receptors. GABA(C) receptors are heterooligomers that differ from GABA(A) receptors in that they contain p-subunits and are insensitive to bicuculline. However, molecular and functional evidence for the presence of GABA(C) receptors outside the retina has yet to be established. The present work was performed on guinea pig and rat pituitaries. Both Northern blot and RT-PCR analysis showed that, although rho1- and rho2-subunits were expressed at similar levels in the rat retina, rho1 messenger RNA (mRNA) was enriched, relative to rho2 mRNA in the rat pituitary. Northern blot experiments also showed that, in the pituitary, rho1 and rho2 mRNAs are shorter in size than those expressed in the retina. The use of a subunit-specific antibody revealed colocalization of rho1-subunit and anti-TSH labeling on rat pituitary sections. TSH guinea pig pituitary cells were also labeled with a rho-subunit antiserum. Moreover, whole-cell patch clamp on single guinea pig TSH cells showed that GABA induced a bicuculline-insensitive Cl- current. In contrast to the Cl- current generated by GABA(C) receptors in the retina, the bicuculline-insensitive Cl- currents in TSH cells quickly desensitized. These results suggest that a novel GABA(C) receptor may regulate TSH secretion and that the structure and/or biochemical regulation of this pituitary receptor is different from that found in the retina.

Intracellular calcium concentration and hormone secretion are controlled differently by TRH in rat neonatal lactotrophs and somatotrophs.

We studied the effects of TRH on the cytosolic free calcium concentration ([Ca2+]i) of female rat pituitary prolactin-secreting (lactotroph) and GH-secreting (somatotroph) cells in the early postnatal period, i.e. at postnatal days 5 and 10. [Ca2+]i of single identified lactotrophs and somatotrophs was recorded by dual-emission microspectrofluorimetry using the intracellular fluorescent calcium probe indo 1. An application of TRH (100 nM, 10 s) induced a marked [Ca2+]i increase in 65% of neonatal lactotrophs and 34% of neonatal somatotrophs while the remaining cells were unaffected. Most of the responsive cells, both lactotrophs and somatotrophs, exhibited a similar biphasic Ca2+ response, made up of an initial rapid large increase in [Ca2+]i followed by sustained [Ca2+]i fluctuations. In both cell types, removal of Ca2+ from the extracellular medium or addition of the Ca2+ channel blocker, cadmium chloride (500 microM), inhibited the second phase whereas the first phase persisted. Furthermore, in both cell types, protein kinase C (PKC) depletion by incubation in phorbol myristate acetate (1 microM) for 24 h abolished the second phase but did not inhibit the first phase. Conversely, when cells were pretreated with the Ca(2+)-ATPase inhibitor, thapsigargin (100 nM), all TRH-induced [Ca2+]i changes in both cell types disappeared. TRH therefore induces a biphasic increase in [Ca2+]i involving intra- and extracellular Ca2+ in neonatal lactotrophs and somatotrophs as it does in adult lactotrophs. The first phase is presumably due to mobilization of Ca2+ from intracellular stores whereas the second phase presumably results from a PKC-sensitive influx of Ca2+. TRH action on membrane potential was then investigated using the patch-clamp technique in the whole-cell mode. TRH-induced changes in membrane potential consisted of an initial hyperpolarization followed by depolarization and action potential firing. We also investigated TRH action on prolactin and GH secretion by neonatal pituitary cells using RIA. Surprisingly, static assays of prolactin and GH revealed only stimulation of prolactin release by TRH but no effect on GH secretion, although, as expected, GH-releasing factor was a potent agonist of GH secretion. Our results suggest that TRH regulates neonatal lactotrophs and somatotrophs differently, in that the [Ca2+]i changes do not correlate with stimulation of exocytosis in the latter cell type.

Long-term GABAA receptor activation increases [Ca2+]i in single lactotrophs.

Prolactin (PRL) release by pituitary lactotrophs is inhibited by gamma-aminobutyric acid (GABA). We have investigated the effect of long-lasting activation of GABAA receptors on membrane potential and cytosolic free calcium concentration ([Ca2+]i) in single identified lactotrophs. Membrane potential was recorded using the perforated patch-clamp technique and [Ca2+]i using indo 1 as a fluorescent Ca2+ probe. When cells were bathed in muscimol (10 microM) for 30 min, [Ca2+]i unexpectedly increased in 53% of the lactotrophs. This was due to a Ca2+ influx, since it was inhibited by Ca(2+)-free extracellular medium or by Ca2+ channel blockers such as the dihydropyridine PN 200-110. In cells incubated in muscimol, wash of muscimol from the cell membrane potential and reduced [Ca2+]i to the levels found in control cells. This effect was mimicked by picrotoxin, a GABA-operated Cl- channel blocker, thus supporting the involvement of a muscimol-induced Cl- flux. Conversely, under similar experimental conditions, static assays of PRL release revealed an inhibition of release by muscimol, unaffected by the dihydropyridine PN 200-110. Our observations suggest that GABAA, receptors may not regulate the process of exocytosis within the Ca(2+)- regulated steps.

Recording of a large-conductance chloride channel in normal rat lactotrophs.

Membrane current fluctuations resembling channel openings and closings were observed in the whole cell configuration of the patch-clamp technique in normal rat lactotrophs in primary culture. Using high-gain head stage in whole cell configuration, we characterized the nature and pharmacological properties of the ionic channel underlying these fluctuations. This channel, found in small numbers (< 10 per cell), was specific for Cl- because its reversal potential varied with Cl- gradients, according to the Nernst equation, and its unitary amplitude was linearly related to membrane potential from -100 to 0 mV. Slope conductance was close to 100 pS. Analyzing open times, we demonstrated its Ca2+ and potential dependence. Four sublevels were observed. We suggest that this channel, belonging to the background Cl- channel group, takes part in the regulation of intracellular Cl- concentration of normal rat lactotrophs.

Short applications of gamma-aminobutyric acid increase intracellular calcium concentrations in single identified rat lactotrophs.

We have investigated the direct effect of GABA receptor agonists on the cytosolic free calcium concentration ([Ca2+]i) and the membrane potential of rat lactotrophs in primary culture. [Ca2+]i was recorded in single identified lactotrophs by dual emission microspectrofluorimetry using indo-1 as intracellular fluorescent calcium probe. Whole cell and perforated patch-clamp were performed. A short application of GABA (10(-5) M, 10 s) induced a marked transient [Ca2+]i increase in 66% of lactotrophs, which could be readily mimicked by muscimol (10(-5) M). By contrast, neither L-homocarnosine (10(-3) M) nor baclofen (10(-5) M), a GABAB agonist, had any effect on [Ca2+]i. The GABA-induced [Ca2+]i increase was antagonized by picrotoxin (10(-5) M), bicuculline methiodide (10(-5) M) and strychnine (10(-4) M), demonstrating GABAA receptor specificity. Furthermore, clonazepan (1.5 x 10(-4) M) could potentiate the GABA effect on [Ca2+]i. The [Ca2+]i increase disappeared in the absence of Ca2+ in the extracellular medium or in the presence of Ca2+ channel blockers (cadmium, PN 200-110). GABA and muscimol depolarized the membrane potential with a concomitant fall in cell input resistance, thus suggesting, as in other cell types, the opening of receptor-operated chloride channels. When Ca2+ entry was prevented by the use of cadmium (500 x 10(-6) M), GABA still elicited membrane depolarization but did not raise [Ca2+]i. Our results suggest that a short application of GABA leads to Ca2+ entry through voltage-gated Ca2+ channels in single lactotrophs. This Ca2+ influx is due to depolarization of the prolactin cell.

[Electrophysiological study of the action mechanism of somatoliberin (GH-RH) on hypophyseal GH3 tumor cells]. / Etude électrophysiologique du mécanisme d'action de la somatolibérine (GH-RH) sur des cellules hypophysaires tumorales GH3.

We have investigated the electrical response of patched GH3 cells to Growth-Hormone Releasing-Hormone (GH-RH). GH-RH (100 nM) enhanced firing frequency of action potentials. This is accompanied by membrane depolarization (5-10 mV) and conductance increase. Voltage clamp studies reveal that GH-RH potentiates calcium inward currents and a calcium-dependent chloride current; transient outward current is diminished. These changes in membrane conductance account for the cytosolic free calcium rise shown by Indo-1 fluorescence measurements.

[Prevention of recurrent hemorrhage in patients with cirrhosis. Results of a controlled trial of propranolol versus endoscopic sclerotherapy]. / Prévention des récidives hémorragiques chez des malades atteints de cirrhose. Résultats d'une étude contrôlée comparant propranolol et sclérose endoscopique.

Between July 1984 and March 1986, we conducted a prospective randomized trial comparing propranolol and endoscopic sclerotherapy in the prevention of recurrent variceal hemorrhage in a group of non selected alcoholic cirrhotics. Seventy-six patients with variceal hemorrhage were randomized to receive propranolol (P) (34 patients), or sclerotherapy (S) (42 patients) approximately 12 days after initial bleeding. The 2 groups were similar as concern age, sex, etiology of cirrhosis, severity of liver failure, the number of previous hemorrhages, and the severity of initial hemorrhage. No side effects were observed in the P group; 20 patients (48 percent) in the group S had at least one side effect although minor. After an average follow-up of 36 months, 18 patients in group P (53 percent) and 23 in group S (55 percent) had hemorrhagic recurrence. Rebleeding occurred from other sources than esophageal varices in 5 patients, in the group S only. Five patients in group P and 8 patients in group S died of rebleeding. During the follow-up period, 8 patients in group P (23 percent) and 13 patients in group S (31 percent) died. No significant difference could be demonstrated between the 2 groups as regards the percentages of patients without variceal rebleeding or survival, calculated according to the Kaplan Meier method. In conclusion, in this trial, no significant difference could be demonstrated between propranolol and endoscopic sclerosis in the prevention of recurrent variceal hemorrhage in alcoholic cirrhotic patients.

Electrophysiological response to thyrotropin-releasing hormone of rat lactotrophs in primary culture.

The response of rat pituitary cells to thyrotropin-releasing hormone (TRH) in primary culture was studied in the whole-cell configuration with the patch-clamp technique. Prolactin (PRL)-containing cells were identified in the culture with a peroxidase-antiperoxidase immunocytochemical method. The cells were cultured from the pituitaries of diestrous (D) and lactating (L) female rats. Membrane electrophysiological properties (resting potential and input resistance) of pituitary cells in primary culture varied widely. Under the recording conditions reported here, the mean resting potential of lactotrophs was about -30 mV. There were spontaneous fluctuations in membrane resting potential (10-15 mV) as well as of membrane input resistance, making these parameters difficult to evaluate accurately. Most of the cells exhibited spontaneous firing activity that was shown to be mainly calcium-dependent. There was no difference between L and D cells in resting membrane electrophysiological properties. TRH (10(-7) M) induced a transient hyperpolarization of the membrane similar to that previously described in the GH3 clonal pituitary cell line. Voltage-clamp studies showed that this hyperpolarization resulted from activation of an outward current, the reversal potential of which ranged from -48 to -86.5 mV. Experimental manipulations of the ionic composition of internal and external recording media suggested that both K+ and Cl- were involved. This hyperpolarizing response was observed both in D and L cells, although L cells had larger and faster responses than D cells. This observation may be of physiological significance because lactotrophs have been reported to exist in various subtypes.

[Phorbol myristate acetate promotes entry of calcium in hypophysial cells GH3 B6 via potential dependent calcium channels]. / Le phorbol myristate acétate favorise l'entrée de calcium dans les cellules hypophysaires GH3 B6 par les canaux calciques potentiel-dépendants.

10 GH3/B6 cells were patched-clamped using a pipette containing NMG as internal cation, 2 mM ATP and 100 microM leupeptin. Whole-cell calcium or barium currents were recorded prior and after PMA (10(-8) or 10(-7) M). PMA increased the inward calcium current at potential levels close to threshold in 8 cells; 7 cells only exhibited an increase in transitory calcium current at potential levels close to threshold; in one cell, both transitory and conventional calcium currents were increased. 2 cells did not respond to PMA.

The relationship between the transient inward current (TI) and other components of slow inward current in mammalian cardiac muscle.

When rabbit sino-atrial node preparations and isolated guinea-pig ventricular cells are subjected to Na-K pump blockade (either by reducing external K+ by a factor of 10: sinus node; or by the presence of 10(-7) M ouabain: ventricular cells) they develop oscillatory transient inward currents of the kind already recorded in Purkinje fibres and ventricular muscle strands. The time course of these transient currents, generally known as TI's, closely resembles that of the slow component of second inward current (isi,2) previously reported by us as occurring in rabbit sinus node when recorded near its threshold (-40 mV). Moreover, we have found that, under voltage clamp conditions, the 'envelope' of isi currents activated by depolarization from negative membrane potentials matches the outline of the iTI which develops during the initial hyperpolarization. In the sinus node, oscillations of iTI become smaller near O mV but are never flat and there is no clear cut reversal potential, whilst in ventricular cells oscillations and contractions cease at very positive membrane potentials (+35 mV) without the TI current ever becoming net outward. Replacing 75% of the external Na+ with Li+ reduces isi and iTI in the node by about the same proportion strongly suggesting that both are carried by a Na-Ca exchange mechanism. This idea is supported by reproducing the conditions of Na-K pump block in a computer model of the sinus node activity++, when oscillatory currents are generated by variations in activity of the Na-Ca exchange mechanism triggered by fluctuating levels of intracellular calcium. The same model when used to test the hypothesis that isi,2 might be carried by a non-specific ion channel showed that considerable distortion of the action potential would then occur. From the experimental and computed results it is concluded that the majority of isi,2 and iTI currents are both mediated by Na-Ca exchange.

Relationship between the transient inward current and slow inward currents in the sino-atrial node of the rabbit.

In low K+ (0.3 mM) solutions rabbit sinus node preparations show the oscillatory transient inward current, iTI, already recorded in these conditions in Purkinje and ventricular preparations. The time course of iTI closely resembles that of the slow component of the slow inward current (isi) previously reported by us (Brown, Kimura, Noble, Noble & Taupignon, 1984a) in rabbit sinus node, when recorded near its threshold (-40 mV). When the duration of voltage-clamp steps is varied there is a strong correlation between the 'envelope' of isi amplitudes on depolarization and the time course of iTI on hyperpolarization. Although oscillations of iTI become smaller near 0 mV, there is no potential at which the current records are completely flat, suggesting that there is no simple reversal potential. 75% substitution of Na+ by Li+ greatly reduces both iTI and slow isi in about the same proportion. Reducing the activity of the Na-K exchange pump by the amount expected in 0.3 mM-K+ solutions is sufficient to induce oscillatory iTI in a computer model of the sino-atrial node (Noble & Noble, 1984). The model reproduces the current as variations in the Na-Ca exchange current dependent on intracellular Ca2+ concentration ([ Ca]i). The model was also used to test the alternative hypothesis that the slow inward currents might be generated by [Ca]i-activated non-specific cation channels. It is shown that this would distort the shape of the repolarization phase of the action potential. It is concluded that the experiments and computations are consistent with the hypothesis that a large fraction of iTI and the slow component of isi could both be generated by Na-Ca exchange and that only a relatively small fraction might be generated by non-specific channels.

The slow inward current, isi, in the rabbit sino-atrial node investigated by voltage clamp and computer simulation.

The properties of the slow inward current, isi, in the sino-atrial (s.a.) node of the rabbit have been investigated using two microelectrodes to apply voltage clamp to small, spontaneously beating, preparations. Many of the experimental results can be closely simulated using the computer model of s.a. node electrical activity (Noble & Noble 1984) which has been developed from models of Purkinje fibre activity (Noble 1962; DiFrancesco & Noble 1984). Comparison of the computed reconstructions with experimental results provides a test of the validity of the modelling. Experiments using paired depolarizing clamp pulses show that inactivation of isi is calcium-entry dependent although, unlike the inactivation of Ca2+ currents in some other systems, it also shows some voltage-dependence. Re-availability (recovery from inactivation) of isi in s.a. node is much slower than inactivation at the same potential, showing that isi is not controlled by a single first order process. This very slow recovery from inactivation of isi in the s.a. node and the slow time course of its activation and inactivation at voltages near threshold (-40 to -50 mV) can be closely modelled by assuming that there are two components of 'total isi': a fast inward current, iCa,f' representing the 'gated' fraction and a second, slower, inward current component, iNaCa which, we propose, is caused by the sodium-calcium exchange that ensues when the initial Ca2+ -entry triggers the release of stored intracellular Ca2+. When repetitive trains of clamp pulses are given, a 'staircase' of isi magnitude is seen which can be increasing ('positive') or decreasing ('negative') according to the potential level and frequency of the pulse train given. When computer reconstructions of such staircases are made, it is found that the positive staircases (which, in contrast to negative staircases, imply that more complex processes than simple inactivation are present) can be closely simulated by a model which incorporates slower processes (suggested Na-Ca exchange current) in the total isi in addition to the gated current component.(ABSTRACT TRUNCATED AT 400 WORDS)

The ionic currents underlying pacemaker activity in rabbit sino-atrial node: experimental results and computer simulations.

The membrane currents underlying the pacemaker depolarization have been investigated in rabbit s.a. node preparations using the two-microelectrode voltage clamp technique. Many of the experimental results have been simulated using a computer model of s.a. node electrical activity. Changes of three time-dependent membrane currents which could contribute to pacemaker depolarization are found to occur in the relevant potential range: decay of the potassium current, iK, and activation of the inward current, if, and of the slow inward current, isi. The contribution of if activation to the pacemaker depolarization ranges from nil to an appreciable part depending on the preparation; when Cs (1 mM) blocks if, it nevertheless does not prevent pacemaking. In the model, holding the if activation variable at zero slows but does not stop pacemaking; doubling if conductance and shifting its activation curve by 15 mV in the positive direction causes a 15% faster rate of pacemaking. The slow time course of re-availability of isi must be allowed for when determining the isi threshold. A voltage clamp protocol designed to mimic as closely as possible an action potential followed by a pacemaker depolarization gives an estimate of isi threshold at the potential level of the last third of the pacemaker depolarization. This has been confirmed in experiments in which the voltage clamp was switched on at different points in the pacemaker depolarization. In the computer simulation, 'blocking' isi depolarizes the membrane to the zero current level (close to the potential reached at the end of a pacemaker depolarization) and stops the generation of action potentials. The decay of iK contributes to the pacemaker depolarization; with both our own model and that of K. Yanagihara, A. Noma and H. Irisawa, Jap. J. Physiol. 30, 841-857 (1980) 'blocking' iK decay abolishes pacemaker activity. Computations of extracellular K+ concentration changes compared with iK decay in a cylindrical model allow re-assessment of the interpretation of K+ concentration measurements during pacemaking made by J. Maylie, M. Morad and J. Weiss, J. Physiol., Lond. 311, 167-178 (1981).(ABSTRACT TRUNCATED AT 400 WORDS)

The effect of external potassium on the blockade of the inward-going rectification by cesium ions in the frog atrial trabeculae.

The effect of Cs ions on the inward-going rectification of the frog atrial trabeculae was investigated in various external K concentrations using the double sucrose gap technique. In the presence of 10 mM Cs the inward-going rectification disappeared and the steady-state background current was related linearly to the membrane potential. The Cs-sensitive current was obtained by the difference between control and membrane currents measured in the presence of Cs ions. The current rectified in the inward direction and its reversal potential was very close to the K equilibrium potential. At potentials positive to the resting value the effect of Cs was progressively reduced, leaving the outward rectifier component of the background current unaffected. The analysis of the conductances calculated from the current-voltage relationships showed that the Cs effect was insensitive to moderate changes in the external K concentration, indicating that Cs ions strongly reduced K conductance. In the presence of high K concentrations the blocking effect of Cs appeared to be diminished. The lack of Cs action on the outward rectifier component and the results obtained in high K solutions were discussed.