Analysis of the galanin-induced decrease in membrane excitability in mudpuppy parasympathetic neurons.

Previously, we showed that the neuropeptide galanin hyperpolarizes and decreases membrane excitability of mudpuppy parasympathetic neurons [Konopka L. M., McKeon T. W. and Parsons R. L. (1989) J. Physiol. 410, 107-122]. We also demonstrated that membrane excitability remains depressed when the agonist-induced potential change is negated electrotonically. We hypothesized that galanin inhibits the membrane conductances associated with spike generation. However, we cannot rule out the possibility that the decreased excitability is due to a galanin-induced increase in membrane potassium conductance which reduces the effectiveness of subsequent depolarizing stimuli. Therefore, in the present study we tested, with the galanin-induced hyperpolarization negated, whether the galanin-induced increased membrane potassium conductance was responsible for the decreased excitability. The results showed that the galanin-induced decreased excitability was not dependent on the peak amplitude of the galanin-induced hyperpolarization. Furthermore, the decreased excitability occurred in cells in which there was no measurable galanin-induced hyperpolarization. Moreover, in most cells the galanin-induced decrease in input resistance, measured at the peak of the hyperpolarization (3-25 mV), was less than 15% and when the hyperpolarization was negated electronically, the decrease was even less (approximately 2%). These results indicated that when the hyperpolarization was negated, the galanin-induced increase in potassium conductance was not responsible for the decreased excitability. In preparations pretreated with 5 mM tetraethylammonium, galanin decreased excitability which indicated that a galanin-induced decrease in the calcium-dependent potassium current was not necessary for the decreased excitability. Galanin also decreased excitability in preparations exposed to either 1-3 microM tetrodotoxin or 100-200 microM cadmium. Following galanin application, the threshold for initiation of tetrodotoxin-insensitive spikes was shifted to more positive membrane potentials. Galanin also decreased the amplitude and hyperpolarizing afterpotential of barium spikes in the absence of any agonist-induced hyperpolarization. These observations confirmed that galanin decreased the voltage-dependent calcium conductance. In the present study, we showed that when the hyperpolarization was negated, galanin decreased excitability by shifting the threshold for spike generation regardless of whether voltage-dependent sodium or calcium currents were primarily responsible for the depolarizing component of the action potential.     

Reduction in acetylcholine sensitivity of axotomized ciliary ganglion cells.

1. Isolated cell clusters from ciliary ganglia of 3- to 4-day-old chickens were used to examine the electrical characteristics and sensitivity to iontophoretically applied acetylcholine (ACh) of normal cells and cells that had been axotomized on the day of hatching. 2. Resting potentials, input resistances and capacitances were the same in axotomized cells as in normal cells. These averaged about 70 mV, 165 Momega and 35 pF respectively. 3. Sensitivity to iontophoretically applied ACh was lower in axotomized cells than in normal cells by a factor of about 8. The rise times of the ACh potentials were the same in the two groups; indicating that the reduced sensitivity was not due to a diffusion barrier. 4. The slopes of the dose-reponse curves, plotted on a double-logarithmic scale, suggested that the co-operative action of two ACh molecules was involved in activating a post-synaptic conductance channel. This relation was unaltered by axotomy. 5. The estimated reversal potential for the action of ACh was unchanged after axotomy. 6. Cells in isolated clusters were similar to those in intact ganlia with respect to threshold depolarization, amplitude and time course of action potentials and ability to generate repetitive action potentials. There were no differences in these characteristics between normal and axotomized cells. 7. Cells in the isolated clusters had input resistances which were larger by a factor of 2-3 and capacitances that were smaller by a factor of about 2 than those of cells in intact ganglia. It is suggested that these differences were due to loss of initial segments of axons from the isolated cells. 8. Normal cells in the isolated clusters displayed spontaneous miniature synaptic potentials, indicating that synaptic integrity was maintained during the isolation procedure. As in intact ganglia, no spontaneous activity was observed in axotomized cells.     

Permanence of postsynaptic specializations in the frog sympathetic ganglion cells after denervation

Summary The junctional complex of the axosomatic synapses in the frog sympathetic ganglion is formed by active zones and attachment plates. 16% of the active zones present a dense band or subsynaptic formation on the postsynaptic side. Seven days after the preganglionic fibers have been cut, most of the axon terminals in the ganglion undergo degeneration. The junctional complex is broken by glial cytoplasm, which separates the axon terminals from the neuronal perikaryon. Two distinct morphological types of axonal degeneration are found at this stage: 1. dark and shrunken boutons with a honeycomb appearance, and 2. clear and swollen boutons. These two morphological varieties are interpreted as different aspects of the same degenerative process of the spiral apparatus. Ten days after transection of the preganglionic fibers, most of the degenerating axon terminals have disappeared, and only dark bodies, remnants of such endings, are seen in the glial cytoplasm. Twelve days after the experimental lesion, almost all the axon terminals have disappeared. In all three stages of survival the subsynaptic structures, postsynaptic differentiation and subsynaptic formation, remain unchanged. It is suggested that these structures are not intimately related to the functional integrity of the axon terminals.To the memory of Prof. Fernando de Castro     

Proton-gated sodium current in parasympathetic ganglion cells of frog heart

1. The proton-gated current was investigated in whole-cell configuration of the neurons isolated from bullfrog heart parasympathetic ganglia with the use of the "concentration-clamp" technique, which combines intracellular perfusion and extremely rapid exchange of external solution within 1-2 ms, under a single-electrode voltage-clamp condition. 2. In all isolated neurons, a "step" decrease in extracellular pH (pHo) induced a transient inward current that was followed by a complete inactivation within 1 s. 3. The proton-gated current increased in a sigmoidal fashion as pHo decreased. In the external solution containing 2 mM Ca2+, the threshold of current activation was at pHo 7.4, and the maximum response appeared at pHo 6.5-5.5. The dissociation constant (Kd) and Hill coefficient were 7.1 and 3.2, respectively. 4. The proton-gated current was reduced by decreasing the extracellular Na+ concentration. In the absence of K+, the current produced by reduced extracellular pH reversed at the Na+ equilibrium potential (ENa), indicating that the current was carried by Na+. 5. Kinetics of both the activation and inactivation phases of proton-induced current were single exponential. The time constants of activation (tau a) and inactivation (tau i) had no potential dependence but decreased slightly by decreasing pHo. 6. In the inactivation curve of the proton-induced current obtained by decreasing pHo from various conditioning pHos to 6.5, the half-maximum inactivation occurred at pHo 7.75. 7. The proton-gated current was suppressed as the extracellular Ca2+ concentration [( Ca2+]o) increased from 0.1 to 10 mM, and the half inhibition occurred at greater than 10 mM [Ca2+]o.(ABSTRACT TRUNCATED AT 250 WORDS)     

An electrophysiological study of chemical and electrical synapses on neurones in the parasympathetic cardiac ganglion of the mudpuppy, Necturus maculosus: evidence for intrinsic ganglionic innervation.

1. The cardiac ganglion of the mudpuppy is situated on a thin sheet of tissue. Two nerve cell types can be distinguished readily in the living preparation - principal cells and smaller interneurones which synapse with the principal cells. The purpose of this study was to investigate synaptic transmission and the functional organization of neuronal connections of ganglion cells with intracellular micro-electrodes. 2. Stimulation of the preganglionic, vagus, nerves evoked a large excitatory response in principal cells. About three quarters of these neurones were innervated by a single vagal axon. The remaining cells received two or more preganglionic nerve fibres. 3. The quantum content of vagal excitatory post-synaptic potentials (e.p.s.p.s) was measured. Normally, the e.p.s.p. was suprathreshold and consisted of about twenty-two quanta, whereas only about nine quanta were required to reach threshold and initiate an action potential. 4. Intracellular stimulation of principal cells evoked e.p.s.p.s in neighbouring principal cells. The responses were blocked by cholinergic antagonists. These potentials were caused by excitation of principal cell axon collateral synapses. 5. Principal cells also formed electrical junctions with each other. These electrical junctions were very weak. Although they transmitted slow potential changes, only a small response was recorded in one cell when an electrically coupled neighbouring cell fired an impulse. The resistance of the electrical junction between principal cells was calculated to be about 5-8 X 10(8) omega. 6. Stable penetrations of interneurones were only rarely achieved, making it difficult to study their functional relationship to principal cells. Action potentials were recorded from interneurones in a few instances. 7. These data demonstrate that parasympathetic ganglion cells in the heart of the mudpuppy receive innervation from more than one source involving both chemical and electrical synapses, and that some of the synapses are intrinsic to the ganglion.     

The contrast sensitivity of retinal ganglion cells of the cat

1. Spatial summation within cat retinal receptive fields was studied by recording from optic-tract fibres the responses of ganglion cells to grating patterns whose luminance perpendicular to the bars varied sinusoidally about the mean level. 2. Summation over the receptive fields of some cells (X-cells) was found to be approximately linear, while for other cells (Y-cells) summation was very non-linear. 3. The mean discharge frequency of Y-cells (unlike that of X-cells) was greatly increased when grating patterns drifted across their receptive fields. 4. In twenty-one X-cells the relation between the contrast and spatial frequency of drifting sinusoidal gratings which evoked the same small response was measured. In every case it was found that the reciprocal of this relation, the contrast sensitivity function, could be satisfactorily described by the difference of two Gaussian functions. 5. This finding supports the hypothesis that the sensitivities of the antagonistic centre and surround summating regions of ganglion cell receptive fields fall off as Gaussian functions of the distance from the field centre. 6. The way in which the sensitivity of an X-cell for a contrast-edge pattern varied with the distance of the edge from the receptive field centre was determined and found to be consistent with the cell's measured contrast sensitivity function. 7. Reducing the retinal illumination produced changes in the contrast sensitivity function of an X-cell which suggested that the diameters of the summating regions of the receptive field increased while the surround region became relatively ineffective.     

The muscarinic effects of acetylcholine on the action potential of bullfrog sympathetic ganglion cells.

The direct effects of acetylcholine (ACh) on Na+- or Ca2+-dependent action potentials of curarized sympathetic ganglion cells in bullfrogs were investigated under a condition where membrane depolarization caused by the muscarinic action of ACh was nullified by means of a hyperpolarizing current. ACh decreased the after-hyperpolarization of Na+-action potentials in Ringer's solution, and increased the after-depolarization of Ca2+-action potentials in the isotonic Ca2+ solution. In both solutions, the maximum rates of rise of the spikes were decreased and the slope membrane resistance at the original resting level was increased. The effects of ACh were abolished by atropine. On the other hand, ACh showed no significant effects on action potentials of bullfrog spinal ganglion cells which possessed no synapses. These results suggest that the ion conductance channels for generation of action potentials of sympathetic ganglion cells are under the direct control of transmitters, such as ACh.     

The effect of iontophoretically applied acetylcholine upon the cat's retinal ganglion cells

Summary The effect of iontophoretically applied acetylcholine was tested in 278 retinal ganglion cells of the cat.26% of 122 ganglion cells were influenced by acetylcholine. In animals, which were pretreated with intravenously administered physostigmine, the proportion of acetylcholine sensitive cells rose to 87%. Acetylcholine had a differential effect upon retinal reaction types. Off-center neurons were excited, On-center neurons were inhibited. The time course of the acetylcholine effect resembled that observed in the cerebral cortex. In a considerable proportion of units, the effect used to diminish with repeated application of the drug (desensitation).Intravenously applied atropine blocked the excitatory response of Off-center neurons to acetylcholine, but failed to prevent acetylcholine inhibition of On-center neurons. The excitatory response appears thus predominantly mediated by muscarinic receptors. Nicotinic receptors are obviously of little importançe in mediating the response to acetylcholine, since acetylcholine sensitivity remained unchanged after intravenous injection of the curariform agent dihydro--erythroidine. Present histochemical and histological knowledge together with our results suggest, that the response of retinal ganglion cells to light stimulation of their receptive field periphery is possibly transmitted through cholinergic amacrine cells.This work was supported by the Deutsche Forschungsgemeinschaft (S.F.B. Kybernetik).     

Actions of noradrenaline and acetylcholine on sympathetic ganglion cells

1. The responses of the post-synaptic membrane of sympathetic ganglion cells to noradrenaline (NA) and to acetylcholine (ACh) were studied in relation to the slow inhibitory post-synaptic potential (S-IPSP) and slow excitatory one (S-EPSP) respectively.2. NA produced an hyperpolarization of about 4 mV in cells of rabbit superior cervical ganglia.3. The hyperpolarizing response to NA was not accompanied by any detectable change in membrane resistance, and it was depressed by conditioning depolarization.4. NA also depressed all the post-synaptic potentials, presumably by an action on presynaptic function.5. ACh produced a large depolarization in ganglion cells of rabbit and of frog (paravertebral) ganglia, which was accompanied by a large decrease in membrane resistance.6. When ACh was applied during nicotinic blockade, achieved with high concentration of nicotine (frog ganglia) or D-tubocurarine (rabbit ganglia), it still produced a considerable depolarization. This response could be blocked by atropine, and is presumably a muscarinic type of action.7. The muscarinic-ACh response was not accompanied by a decrease in membrane resistance. Instead, the frog ganglion cells exhibited increased resistances of up to more than twice the resting value during both the muscarinic-ACh depolarization and the S-EPSP.8. The muscarinic-ACh depolarization and the S-EPSP were both depressed by conditioning hyperpolarization (in nicotinized frog cells). An initial hyperpolarizing phase now appeared in both of these responses.9. It is concluded that the hyperpolarizing response to NA and the depolarizing response to muscarinic-ACh action are not generated by increases in ionic mobilities in the post-synaptic membrane; and that these two responses are produced by the same electrogenic mechanisms which underlie the S-IPSP and the S-EPSP respectively.     

The effects of hypoxia in the new-born lamb before and after denervation of the carotid chemoreceptors

1. Twenty-seven unanaesthetized new-born lambs, 6 hr-10 days old, responded to two levels of inspired oxygen, 125 and 110 mm Hg (alveolar CO(2) being controlled) with a sustained increase in minute ventilation (V), a small increase in heart rate and a less consistent rise in systemic blood pressure.2. An increase in V was observed when arterial oxygen tension (P(a), (O2)) had fallen by 6-15 mm Hg. There appeared to be no fixed threshold of P(a), (O2) at which ventilation started to increase.3. The increase in ventilation caused by these levels of hypoxia was significantly and directly related to the age of the lamb and to its control alveolar CO(2).4. More severe hypoxia caused a progressive increase in V until P(a), (O2) was about 25 mm Hg when respiration failed. This increase at P(a), (O2) > 25 mm Hg was markedly potentiated when alveolar P(CO2) (P(A), (CO2)) was increased and abolished after bilateral denervation of the carotid chemoreceptors.5. Significant (> 10%) left-to-right shunts were found in ten out of twelve lambs lightly anaesthetized with pentobarbitone sodium, breathing air. Hypoxia diminished the left-to-right pressure gradient largely by its pressor effect on the pulmonary circulation. When inspired O(2) tension (P(I), (O2)) was 70 mm Hg, all seven lambs studied showed a reversal of the pressure gradient and evidence of right-to-left shunts (11-42%) across the ductus arteriosus.6. The implications of these findings have been discussed with reference to previous studies of the new-born response to hypoxia and it is concluded that the peripheral chemoreceptors are fully active at birth and in the new-born period.     

Physiological and morphological correlations of horizontal cells in the mudpuppy retina.

1. Horizontal cells (HCs) of the mudpuppy (Necturus maculosus) retina were physiologically characterized with the use of intracellular recordings in a superfused, dark-adapted, retina-eyecup preparation. 2. Physiological analysis included an evaluation of rod versus cone input and a determination of the receptive field size with the use of a displaced slit of light. 3. The morphology of HCs was established through intracellular staining with horseradish peroxidase (HRP) and Lucifer yellow mixed in a single electrode. 4. Three types of horizontal cells were identified, each associated with a distinct morphology. Physiological subtypes included luminosity (L) and chromaticity (C) cells. Morphological diversities included single axon-bearing, multiple axon-bearing and, nonaxon-bearing cells. All C-type HCs lacked axons. 5. Approximately 90% of HCs encountered in this study were L-type cells, which received sign-conserving inputs from both rods and cones. These cell types contained one or more long axons that often stretched greater than 500 microns. This group was morphologically diverse, particularly with respect to variations in the number of axons, but we were unable to correlate this diversity with any unique set of physiological properties. 6. Several C-type HCs were identified (n = 8). These cells depolarized to a low-intensity, short-wavelength (SW) stimulus, whereas they hyperpolarized to high-intensity, long-wavelength stimuli. Morphologically, these cells were axonless (n = 4), with relatively small dendritic fields. 7. A third group of HCs were classified as "short wavelength preferring" HCs (n = 7). These cells responded better to a SW stimulus at all intensity levels. They were thus dissimilar to the common L-type HCs, which showed an apparent rod to cone transition as the stimulus intensity increased, suggestive of a shift from rod to cone preference. Morphologically, these cells were axonless (n = 2), but had broader dendritic fields than the C-type HCs. 8. Our observations indicate that the horizontal cell population of the mudpuppy retina is considerably more complex than previously supposed. The existence of both axon-bearing and axonless HCs, which could be correlated with L- and C-type physiology, implies that HCs may support more than one function in outer retina processing.     

Changes in blood erythrocytes after partial denervation of the kidneys in rabbits

Summary Bilateral partial denervation of the kidneys in rabbits stimulated bone marrow erythropoiesis, slowed erythroblast maturation and caused a rise of the blood reticulocyte count. A brief period of anemia set in during the first three weeks after the operation though the development of polycythaemin was a possible alternative.At the time of anemia the serum erythropoietic activity was lowered.(Presented by Academician V. N. Chernigovskii) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 57, No. 3, pp. 41–44, March, 1964     

The effect of hydrolytic enzymes on the acetylcholine sensitivity of the skeletal muscle cell membrane

Isometric tension developed by rat soleus and extensor digitorum longus (EDL) muscles in response to acetylcholine (Ach) applied in vitro was recorded. Tension of contractures elicited in response to Ach increased after muscles had been incubated with phospholipase C, pepsin, or soluble fractions prepared from muscle homogenate.Using intracellular microelectrodes, resting membrane potential (RMP) and depolarisation in response to Ach added to the bathing medium were recorded in endplate-free regions of the muscle fibres. No significant change in RMP was observed in muscles incubated with soluble muscle fraction or phospholipase C, but depolarisation in response to Ach or carbachol was significantly increased. The time course for the increase in depolarisation and the contracture response to Ach was similar.When all available receptors were blocked with -bungarotoxin prior to incubation so that no response to Ach could be elicited, with subsequent incubation in muscle soluble fraction or phospholipase C, both contractures and depolarisation in response to Ach returned. These results support the hypothesis that receptors, not previously available to interact with Ach or -bungarotoxin were revealed following incubation.