Swelling and cAMP on hyperpolarization-activated Cl- conductance in rat Leydig cells.

We have used the whole cell patch-clamp technique to characterize changes in membrane conductance induced by osmotic swelling in mature rat Leydig cells dialyzed with ATP (control cells) or adenosine 3',5'-cyclic monophosphate (cAMP) plus ATP (cAMP cells). A spontaneous current activation occurs in both groups in isosmotic conditions (300/295 mosM in/out). This development is entirely counteracted in control cells and partly inhibited in cAMP cells by exposing them to a hyperosmotic (350 mosM) bath solution, and these currents increase again in a hyposmotic (205 mosM) bath solution. These currents are sensitive to 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid, a Cl- channel blocker. Taken together, the results indicate that, in the control cells (ATP alone) as well as in the presence of intracellular cAMP, osmotic swelling activates the background hyperpolarization-activated Cl- conductance, osmotic swelling and cAMP appearing to act synergistically.

Responses of ipsilateral retino-tectal (type I1) units of the frog (Rana esculenta) to moving configurational bars.

In frogs, retinal information projecting to the ipsilateral optic tectum uses a complex, at least bi-synaptic, route. Ipsilateral visual units recorded at the tectal level correspond to isthmic axon terminals. For a better approach of their visual function, these units have been stimulated with moving (V = 7.6 degrees/sec) configurational stimuli proved earlier to be able to elicit classical behavioural sequences in amphibians. In the presence of W("worm-like")-stimuli of increasing length (2 degrees < L < 20 degrees), the discharge rate of type I1 units remains rather constant. In response to A("antiworm-like")-stimuli, the discharge rate first increases up to L = 5-6 degrees and then decreases continuously. The ability of these units to discriminate bars of equal dimension but of different configuration was defined using the "contrast-like" formula originally proposed by Ewert et al. (1978). The relationship between the discrimination factor D(W, A) and the length of the stimuli is similar in shape to that found in class R3 ganglion cells. Results suggest thus that the classical functional homology between type I1 ipsilateral units and class R2 retinal neurons is inadequate.

Stimulation, data acquisition, spikes detection and time/rate analysis with a graphical programming system: an application to vision studies.

A Macintosh-based system performs stimulus control and data acquisition, and an off-line analysis, in experiments on visually driven neurons in frog. The stimulus is a target moved on a modified XY recorder. The computer is equipped with a multifunction input/output board to perform stimulus control and data acquisition. The graphical programming system LabVIEW 2 was used to develop the 'Vision 93' package made of 4 main 'virtual instruments' (VIs). By means of DOCS-Exp, the user controls the experiment via screen displays which look like front panels of electronic instruments. DOCS-Preproc performs a user-controlled spike detection and computes mean impulse rate values. DOCS-SAS displays the instantaneous frequency, mean impulse rate, spike counts or interspike time intervals as staircase histograms and/or spline smoothed curves. Finally, DOCS-x-Functions computes and graphs quantitative stimulus/response relationships in terms of velocity, diameter, orientation and configuration of the target. The functionalities of these main VIs are presented and original software components are detailed. System operation is illustrated by using the successive VIs to process a sample signal record.

Involvement of a pertussis toxin-sensitive G-protein in excitation-contraction coupling of intact and cut-end voltage-clamped skeletal muscle fibres.

In voltage-clamped frog muscle fibres 10 ng/ml PTX induced a decrease (approximately 35%) of tension when applied externally. Internal application in cut-end fibres significantly depressed tension after 20 min. This effect increased with time to reach 65% after 60 min. PTX shifted the voltage-dependent inactivation curve of tension by 30 mV towards hyperpolarizations and this was counteracted by raising external calcium concentration. The toxin induced a parallel decrease in tension and voltage-sensitive charge movement (49 +/- 9% and 52 +/- 6% respectively; n = 6). This was not counteracted by prior impregnation with forskolin. Internally applied GTP gamma S (500 microM) induced a simultaneous increase in tension (57 +/- 5%) and charge amount displaced (40 +/- 7%). By contrast, GDP beta S decreased tension and charge movement by 35 +/- 5% and 36 +/- 6% respectively.

The depressing effect of tetracaine and ryanodine on the slow outward current correlated with that of contraction in voltage-clamped frog muscle fibres.

The effects of tetracaine (10-50 microM) and ryanodine (0.1-10 microM) were tested on the slow outward K+ current (Iso) and the mechanical tension of isolated frog muscle fibres in a voltage-clamp device (double mannitol-gap) connected to a mechanoelectric transducer. In the concentration range tested, both drugs induced a simultaneous inhibition of tension and current. In all cases the effect on tension was twice that on current. The tetracaine-induced current and tension blocks were fully reversible and dose-dependent. In contrast the ryanodine effects on current and tension were not reversible and did not exhibit a dose dependence except for the delay before the onset of the response, which was shortened when the concentration was raised. Linear regression analysis of the time-dependent and dose-dependent effects of both drugs indicated a strong correlation between the decreases in tension and current. It is concluded that the slow outward current is partly under the control of the Ca2+ release from sarcoplasmic reticulum during contraction.

Stimulation frequency and external ionic composition control the repriming of caffeine-induced contractures in frog skeletal muscle.

The effect of stimulation rate and of external ionic composition on the repriming period of contractures induced by 6 mM caffeine was tested on isolated skeletal muscle fibres of the frog (Rana ridibunda). The repriming period, which was 11.2 +/- 0.1 min (mean +/- SEM, n = 9) on quiescent fibres, was shortened in fibres stimulated at a frequency ranging from 3 to 12 min-1 (optimal rate, 8 min-1; full repriming 5.7 +/- 0.2 min; n = 10). A 10-fold increase in the extracellular calcium concentration shortened the repriming period on both stimulated and quiescent fibres, whereas decreasing external calcium (1/10) delayed it. In a Na+-free solution (Li+ substituted) the repriming period of stimulated fibres was markedly delayed (14 min), whereas quiescent fibres never recover more than 10% of their ability to develop subsequent caffeine contractures. In contrast, with a 35% Na solution, the repriming period was greatly shortened (stimulated, 5.4 +/- 0.2 min, n = 7; quiescent, 6.2 +/- 0.5 min, n = 8). It is concluded that repriming depends on three mechanisms that seem to refill a calcium store and trigger recovery: the slow inward calcium current, a Na+-Ca2+ exchange, and perhaps a passive Ca2 influx.

Electrophysiological study of single Leydig cells freshly isolated from rat testis. I. Technical approach and recordings of the membrane potential in standard solution.

Single Leydig cells were isolated from rat testis by a collagenase digestion procedure and purified through a 21,000 g self generated densities gradient of 35% Percoll. A method including collagen and fibronectin was proposed to attach freshly prepared Leydig cells to the bottom of plastic Petri dishes. Four hours after the isolation of the cells, it was simultaneously possible to determine their membrane potential by a standard electrophysiological technique using intracellular microelectrodes and to judge cellular integrity by direct microscopic observations. In standard Earle's solution, changes of membrane potentials appeared to be biphasic. On 198 impaled cells, 18 +/- 1 S after the impalement was effective, the membrane potential reached a most negative value (MP1) (-37.6 +/- 0.7 mV), followed by a gradual depolarization to a steady state (MP2) (-25.1 +/- 0.6 mV) which remained constant for a few minutes. In standard Earle's solution, the membrane resistance was low or decreasing towards the most negative potential, then it increased towards the steady potential. At this state, the average value of the cell input resistance was 65.9 +/- 6.0 M omega (n = 16). No action potential was observed either in standard Earle's solution or under a depolarizing current state. It was concluded that the electrophysiological characteristics of the Leydig cell are similar to those of fibroblasts and macrophages, three types of cells with the same mesenchymal origin, present in the interstitial tissue of the rat testis. But the resting potential of the Leydig cell is higher and this secreting cell does not elicit hyperpolarizing oscillations at the steady state, under mechanical or electrical stimuli.

Voltage clamp with double sucrose gap technique. External series resistance compensation.

In this paper we deal with the double sucrose-gap voltage clamp technique. To perform a reliable clamp or to analyze the intracellular potential distribution, any external series resistance in the artificial node must be taken into account for it induces an instability in the external potential as soon as a current develops. A circuit was designed to compensate for this error, it has been found effective on an analog model and on experimental uni- or multicellular preparations. The attenuation in series resistance frequently causes ringing in the step response. This behavior was studied theoretically and also simulated with analog models where a selective bridged-T network was found to represent the electrical characteristics of the preparation when associated with the chamber and control electronics. A residual series resistance was found and is considered to be a part of the preparation. Characteristics necessary to obtain best results are proposed, for a preparation to be studied in experiments utilizing the double sucrose gap technique with external series resistance compensation.