Multiple interneuronal afferents to the giant cells in Aplysia.

1. Several different types of presynaptic neurones to the giant cells of Aplysia have been found in the pleural ganglion. Some of these presynaptic neurones are common to the left giant cell in the pleural ganglion and to the right giant cell in the abdominal ganglion but others make contact only with one. 2. Interneurones of the left giant cell were studied in detail. They can be identified not only physiologically from the type of post-synapitc potential (p.s.p.) which they produce in the left giant cell, but also by their localization in the ganglion. 3. Direct stimulation of these presynaptic neurones produced not only the classical types of post-synaptic potentials known as e.p.s.p. or i.p.s.p. but also a slow e.p.s.p. and more complex post-synaptic potentials consisting of a rapid depolarizing or hyperpolarizing component (e for excitatory; i for inhibitory). According the p.s.p.s. which have been found were classified as being of eight different types: e.p.s.p., slow e.p.s.p., pseudo-slow e.p.s.p., e.i.p.s.p., i.e.p.s.p., i.i.p.s.p., to which is added the biphasic p.s.p. (b.p.s.p.) of electrical origin. 4. The monosynaptic nature of each of these p.s.p.s. was established by four criteria: (a) ability to follow one to one the presynaptic spike, (b) short and constant latency, (c) change of p.s.p. with the presynaptic spike when the duration is prolonged by iontophoretic injection of TEA, (d) sensitivity of the synaptic efficacy to presynaptic polarization. 5. For all p.s.p.s., the hyperpolarization of the interneurone was followed by a decrease in the corresponding amplitude; on the contrary depolarization produced an increase in p.s.p. amplitude. 6. The physiological role of these p.s.p.s. and their possible mechanism are discussed.     

On the transmitter function of 5-hydroxytryptamine at excitatory and inhibitory monosynaptic junctions

1. Two symmetrical giant neurones located in the cerebral ganglion of Aplysia californica contain 4-6 p-mole 5-hydroxytryptamine (5-HT) and are able to synthesize it (Weinreich, McCaman, McCaman & Vaughn, 1973; Eisenstadt, Goldman, Kandel, Koike, Koester & Schwartz, 1973). Stimulation of each of these neurones evokes excitatory and inhibitory potentials in various cells of the ipsilateral buccal ganglion. In nine buccal neurones it evokes excitatory potentials, in other three, ;classical' inhibitory potentials and in one neurone an ;atypical' inhibitory potential.2. The connexion between the giant cerebral neurone and the cells receiving either an excitatory or a ;classical' inhibitory input from it are monosynaptic. TEA injection into the cerebral giant neurone, which prolongs the presynaptic spike, causes a gradual increase of both the excitatory and the inhibitory potentials. On the other hand, high Ca(2+) media, which block polysynaptic pathways, do not suppress these synaptic potentials.3. The iontophoretic application of 5-HT to the buccal neurones receiving excitatory input from the giant cerebral neurones evokes depolarizations showing the pharmacological properties of both A- and A'-responses to 5-HT (see preceding paper). Antagonists which block only the A-receptors (curare, 7-methyltryptamine, LSD 25) block partially the synaptic depolarizing potentials. Bufotenine, which blocks both the A- and A'-receptors, completely blocks the excitatory potentials. Thus, the post-synaptic membrane of these buccal neurones appears to be endowed with both A- and A'-receptors to 5-HT.4. The ;classical' inhibitory potentials elicited in three buccal neurones are hyperpolarizations which reverse at - 80 mV and are due to an increase in K(+)-conductance. The iontophoretic application of 5-HT to these post-synaptic neurones evokes hyperpolarizing B-responses which are also generated by an increase in K(+)-conductance. Antagonists which block the B-responses (bufotenine, methoxygramine) also block the inhibitory potentials.5. The ;atypical' inhibitory potential evoked in one buccal neurone consists in an hyperpolarization which increases in amplitude with cell hyperpolarization. Iontophoretic application of 5-HT to this buccal cell evokes an hyperpolarizing beta-response which also increases in amplitude with cell polarization and results from a decrease in both Na(+)- and K(+)- conductances. The monosynaptic character of the ;atypical' inhibitory potential is not yet fully proven.6. It can be concluded that the excitatory and inhibitory synaptic effects evoked in the buccal neurones by the stimulation of the 5-HT-containing-giant cerebral neurones are very likely mediated by 5-HT.     

Effects of dopamine on the superior cervical ganglion of the rabbit

1. The effects of dopamine on isolated rabbit superior cervical ganglion were investigated with intracellular recording techniques.2. Dopamine (10(-5)-10(-3)M) depressed the amplitude of the excitatory post-synaptic potential (e.p.s.p.) and blocked impulse transmission.3. Dopamine (10(-4)M) induced a slight (2-5 mV) post-synaptic hyperpolarization without altering membrane conductance.4. The post-synaptic membrane sensitivity to acetylcholine (ACh) applied iontophoretically was not affected by dopamine.5. Dopamine decreased the frequency of miniature excitatory post-synaptic potentials (m.e.p.s.p.s) in a high K(+) solution, with no change in the amplitude of m.e.p.s.p.s.6. Dopamine reduced the quantal content of the e.p.s.p. in a low Ca(2+) and high Mg(2+) solution, but had no effect on the quantal size.7. The ganglionic blocking effect of dopamine was antagonized by phenoxybenzamine, but not by propranolol.8. The results show that the ganglionic depressant effect of dopamine is exerted primarily through an alpha-adrenoceptive site at the presynaptic nerve terminal.     

A direct synaptic connexion between the left and right giant cells in Aplysia

1. In Aplysia fasciata, intrasomatic stimulation of the giant cell (RGC) of the right upper quadrant of the abdominal ganglia is followed after a constant delay by the appearance of a synaptic potential recorded in the giant cell (LGC) of the left pleural ganglion.2. The synaptic potential recorded in the LGC soma has a biphasic form (hence biphasic post-synaptic potential or BPSP) consisting of a fast depolarizing phase of about 200-800 muV amplitude and 0.15-0.25 sec duration, followed by a slow hyperpolarizing phase of about 200-800 muV amplitude and 1-3 sec duration.3. During repetitive stimulation summation results in an over-all hyperpolarization at low frequencies (less than 5/sec) and an over-all depolarization for higher frequencies. Very high frequencies (25/sec) of stimulation of the RGC may elicit a spike in the LGC.4. Stimulation with two shocks showed increasing effects with shorter intervals on both the depolarizing and hyperpolarizing phase. These effects were progressive and there was no falling out of one of the two phases as might be expected if the BPSP was a composite of an inhibitory post-synaptic potential (IPSP) and an excitatory post-synaptic potential (EPSP).5. The effects of artificially imposed polarization of the LGC through a second micro-electrode suggest that the BPSP results from a chemical transmission mechanism for both its depolarizing and hyperpolarizing phases but electrical transmission cannot be excluded.6. Curare has no effect on the BPSP and thus excludes a cholinergic transmission mechanism. Chloride ions injected into the LGC soma do not appear to modify the BPSP and hence it is concluded that the hyperpolarizing phase is different from IPSPs of the same cell.7. No synaptic potential is recorded in the RGC following stimulation of the LGC, except in a single preparation in which the RGC soma was situated in the right pleural ganglion. In this case the synaptic potential recorded in both giant cells following stimulation of the other, was biphasic in form.8. It is concluded that the BPSP is a unitary monosynaptic potential which is a characteristic feature of the organization of these two giant cells.     

Synaptic actions of peripheral nerve impulses upon Deiters neurones via the climbing fibre afferents

1. The cerebellar integration of sensory inputs to Deiters neurones was studied in cats under Nembutal anaesthesia.2. Stimulation of peripheral nerves produced in the Deiters neurones a sequence of an initial excitatory post-synaptic potential (e.p.s.p.) and a later inhibitory post-synaptic potential (i.p.s.p.), or a relatively small e.p.s.p.3. The Deiters neurones were classified as forelimb (FL)- or hind limb (HL)-type cells according to the location of the most effective peripheral nerve. In the FL cells stimulation of the forelimb nerves produced the e.p.s.p.-i.p.s.p. sequence (dominant response), while stimulation of the hind limb nerves was ineffective or produced the small e.p.s.p. (non-dominant response). In contrast, in the HL cells the non-dominant response was evoked from the forelimb nerves, and the dominant response from the hind limb nerves.4. The stimulus intensity-response relation indicates that Group I and II muscle afferents and low and high threshold cutaneous afferents contribute to the dominant and non-dominant responses.5. Antidromic identification of these Deiters neurones revealed that 90% of the HL cells and 85% of the FL cells project to the lumbo-sacral and cervico-thoracic segments of the spinal cord, respectively, while 10% of the HL cells and 15% of the FL cells innervate the cervico-thoracic and lumbo-sacral segments, respectively.6. The mean latency of the e.p.s.p. evoked from the forelimb nerves was 14 msec in the FL cells and 13 msec in the HL cells, and the latency of the e.p.s.p. evoked from the hind limb nerves was 17 msec in the FL cells and 18 msec in the HL cells. The later i.p.s.p. regularly followed the onset of the e.p.s.p. with a delay of 3-5 msec.7. The dominant and non-dominant responses in both types of cells exhibited the following three characteristic features: (i) a strong depression after conditioning stimulation of the inferior olive, (ii) an increase of the inferior olivary excitability during the responses, and (iii) a striking frequency depression with stimulation at relatively low frequency (5-10/sec).8. Consequently it was concluded that all of the responses were produced through the climbing fibres originating from the inferior olive, the i.p.s.p.s due to inhibition from Purkyne cells activated by the climbing fibres and the e.p.s.p.s due to excitation from the collaterals of the climbing fibres.     

Dopamine activates two different receptors to produce variability in sign at an identified synapse

Chemical synaptic transmission was investigated at a central synapse between identified neurons in the freshwater snail, Lymnaea stagnalis. The presynaptic neuron was the dopaminergic cell, Right Pedal Dorsal one (RPeD1). The postsynaptic neuron was Visceral Dorsal four (VD4). These neurons are components of the respiratory central pattern generator. The synapse from RPeD1 to VD4 showed variability of sign, i.e., it was either inhibitory (monophasic and hyperpolarizing), biphasic (depolarizing followed by hyperpolarizing phases), or undetectable. Both the inhibitory and biphasic synapse were eliminated by low Ca2+/high Mg2+ saline and maintained in high Ca2+/high Mg2+ saline, indicating that these two types of connections were chemical and monosynaptic. The latency of the inhibitory postsynaptic potential (IPSP) in high Ca2+/high Mg2+ saline was approximately 43 ms, whereas the biphasic postsynaptic potential (BPSP) had approximately 12-ms latency in either normal or high Ca2+/high Mg2+ saline. For a given preparation, when dopamine was pressured applied to the soma of VD4, it always elicited the same response as the synaptic input from RPeD1. Thus, for a VD4 neuron receiving an IPSP from RPeD1, pressure application of dopamine to the soma of VD4 produced an inhibitory response similar to the IPSP. The reversal potentials of the IPSP and the inhibitory dopamine response were both approximately -90 mV. For a VD4 neuron with a biphasic input from RPeD1, pressure-applied dopamine produced a biphasic response similar to the BPSP. The reversal potentials of the depolarizing phase of the BPSP and the biphasic dopamine response were both approximately -44 mV, whereas the reversal potentials for the hyperpolarizing phases were both approximately -90 mV. The hyperpolarizing but not the depolarizing phase of the BPSP and the biphasic dopamine response was blocked by the D-2 dopaminergic antagonist (+/-) sulpiride. Previously, our laboratory demonstrated that both IPSP and the inhibitory dopamine response are blocked by (+/-) sulpiride. Conversely, the depolarizing phase of both the BPSP and the biphasic dopamine response was blocked by the Cl- channel antagonist picrotoxin. Finally, both phases of the BPSP and the biphasic dopamine response were desensitized by continuous bath application of dopamine. These results indicate that the biphasic RPeD1 --> VD4 synapse is dopaminergic. Collectively, these data suggest that the variability in sign (inhibitory vs. biphasic) at the RPeD1 --> VD4 synapse is due to activation of two different dopamine receptors on the postsynaptic neuron VD4. This demonstrates that two populations of receptors can produce two different forms of transmission, i.e., the inhibitory and biphasic forms of the single RPeD1 --> VD4 synapse.     

Synaptic excitation and inhibition resulting from direct action of acetylcholine on two types of chemoreceptors on individual amphibian parasympathetic neurones.

1. Synaptic transmission was studied in visually identified parasympathetic ganglion cells that modulate the heart beat of the mudpuppy Necturus maculosus).2. The brief pulse of acetylcholine (ACh) released from terminals of the vagus nerve after each impulse can produce two distinct post-synaptic responses in individual principal cells of the ganglion: (i) within a milli-second of release, ACh generates a rapid and strong excitatory post-synaptic potential (e.p.s.p.) that normally initiates a post-synaptic impulse; (ii) this excitation is usually followed by a slow hyperpolarizing inhibitory post-synaptic potential (i.p.s.p.) that lasts for several seconds. The magnitude and time course of the i.p.s.p. depends on the frequency and number of vagal stimuli. When the hydrolysis of ACh is inhibited by prostigmine, a train of nerve stimuli may be followed by an i.p.s.p. lasting half a minute or longer.3. The rapid e.p.s.p. and slow i.p.s.p. result from the direct action of ACh on two different types of chemoreceptors in the post-synaptic membrane of the principal cell. The e.p.s.p. can be preferentially blocked by the nicotinic antagonist dihydro-beta-erythroidine (5 x 10(-7)M), while the i.p.s.p. is selectively blocked by the muscarinic antagonist atropine (5 x 10(-9)M).4. Potentials resembling nerve-evoked e.p.s.p.s and i.p.s.p.s can be produced by iontophoretic release of ACh from micropipettes onto the post-synaptic membrane. Application of the muscarinic agonist bethanechol generates exclusively inhibitory responses.5. The reversal potential for the i.p.s.p. is about -105 mV, which is approximately the equilibrium potential for potassium (E(K)). When the external K(+) concentration is altered, the reversal potential for inhibition is shifted to the new value of E(K) as expected from the Nernst equation. Changes in the external Na(+) and Cl(-) concentrations have no appreciable effect on the reversal potential. Thus, the i.p.s.p. is the result of a conductance increase for K(+).6. The conductance change producing the i.p.s.p. is voltage sensitive. When the membrane potential is shifted from -40 to -60 mV, the i.p.s.p becomes larger and longer. Beyond -60 mV the inhibitory response decreases in proportion to the driving force on K(+) without any further change in time course.7. The inhibitory response produced by an iontophoretically applied pulse of bethanechol has a delayed onset of about 150 msec at 24 degrees C. The early portion of this response, including the delay, is proportional to t(3), where t is time. The proportionality factor (the apparent rate constant) decreases elevenfold when the temperature is lowered by 10 degrees C. This suggests that a multi-step process is involved in the activation of the conductance increase that leads to the inhibitory response. Inhibitory responses with similar kinetics were produced in heart muscles of the mudpuppy upon application of ACh.     

Relaxing effects of catecholamines on mammalian heart

1. The iontophoretic application of acetylcholine (ACh) on to identified neurones in the buccal ganglion of the mollusc Navanax produced a biphasic or monophasic membrane potential change which was a function of the current intensity and site of ACh application.2. Low iontophoretic currents, 200 msec in duration, applied to the somatic surface facing the neuropile, caused a monophasic potential change of 6-10 sec duration, which had a reversal potential of about - 50 mV, varied with changes in the [Cl](o) of the bathing medium, and was not blocked by the cholinolytics tested.3. ACh applied more distal to the soma, in the neuropile, produced a 1-3 sec monophasic response whose reversal potential was more positive than - 30 mV, varied in amplitude with changes in the [Na](o) of the medium, and was blocked by cholinolytics such as tubocurarine, hexamethonium and atropine.4. With larger iontophoretic currents a biphasic response could be obtained, depolarization followed by hyperpolarization, which represented a superposition of the above monophasic potentials.5. The cholinomimetics propionylcholine and butyrylcholine caused a biphasic response like that to ACh. Carbamylcholine and tetramethylammonium also produced a biphasic response but with a more prominent Cl component than that to ACh. Acetyl-beta-methylcholine, oxytremorine and pilocarpine only produced a response comparable to the chloride phase of the ACh response.6. Anticholinesterases prolonged both phases of the ACh response.7. It was concluded that each of the identified neurones possess two types of cholinoceptive sites, which are pharmacologically distinct, produced different changes in membrane permeability and are distributed differently over the axo-somatic membrane complex.     

Synaptic actions of individual vestibular neurones on cat neck motoneurones

1. Unitary synaptic potentials evoked by the activity of single vestibulocollic neurones were recorded by means of spike-triggered signal averaging in neck extensor motoneurones of decerebrate cats. Properties of the vestibulocollic neurones which produced the potentials were examined.2. Vestibulocollic neurones were first identified as projecting to the C3 grey matter by antidromic microstimulation within the C3 extensor motoneurone pool. The spontaneous or glutamate-driven activity of the vestibulocollic neurones was then used to trigger the averaging computer. In this way ten inhibitory and two excitatory neurones were identified (20% of neurones tested).3. Action potentials in local branches of vestibulocollic neurones were usually recorded in the vicinity of motoneurones. Mean orthodromic conduction time from the foot of the extracellular spike, recorded in the vestibular nuclei, that triggered the averager was 0.72 msec. Mean synaptic delay was 0.4 msec.4. I.p.s.p.s had a mean time to peak of 0.81 msec and were readily reversed by injection of hyperpolarizing current. These data, together with the shape indices of i.p.s.p.s indicate that they are generated proximally on motoneurones.5. All vestibulocollic neurones making synapses with motoneurones were monosynaptically driven by stimulation of the ipsilateral vestibular nerve. Four out of seven tested were inhibited by stimulation of the contralateral vestibular nerve (commissural inhibition).6. Two excitatory neurones were located in Deiters' nucleus or on the Deiters'-descending border. Inhibitory neurones were found relatively medially in the vestibular complex in the medial, descending and Deiters' nuclei.7. Vestibulocollic neurones acting on motoneurones were tested for axon branching to more caudal levels of the spinal cord with electrodes placed at C5-7. Both of the excitatory and two out of nine inhibitory neurones branched.     

Synaptic action on Clarke's column neurones in relation to afferent terminal size

1. Excitatory post-synaptic potentials (e.p.s.p.s) were recorded intracellularly from Clarke's column neurones (DSCT neurones) of the cat in response to adequate stimuli applied to a variety of sensory receptors.2. The amplitude of e.p.s.p.s so produced varied from less than 0.2 mV to more than 2-3 mV. The amplitude distribution of e.p.s.p.s suggested that the mean number of ;quanta' of transmitter released by one impulse varied widely from one fibre to another arising from a given type of sensory receptor.3. The average amplitude of e.p.s.p.s evoked by single afferent impulses was significantly smaller for cutaneous inputs than for muscle or joint inputs. However, synaptic action on DSCT neurones produced by different sensory inputs was equally greater, on the average, than that on spinal motoneurones.4. Both small and large e.p.s.p.s in DSCT neurones failed to increase in amplitude during post-synaptic hyperpolarization applied through the cell body. This failure could not be attributed to possible anomalous rectification in the post-synaptic membrane.5. Small and large e.p.s.p.s were comparable in half-decay time, but there was a positive correlation between amplitude and time-to-peak of e.p.s.p.s. It is suggested that the locations of synapses responsible for small and large e.p.s.p.s are intermingled on the dendrites and that large e.p.s.p.s are associated with a longer duration of transmitter action than small e.p.s.p.s.6. Degenerating terminals of primary afferent fibres on DSCT neurones and motoneurones were examined with the electron microscope after chronic section of the dorsal roots.7. Dendritic degenerating terminals showed no significant difference in size between motoneurones and DSCT neurones. Degenerating ;giant' terminals were found on DSCT neurones, but they were located only on or very close to the cell body.8. It is concluded that the major factor responsible for a large number of ;quanta' of transmitter released at synapses on DSCT neurones is the number of multiple synaptic contacts formed by one afferent fibre rather than the size of individual synapses.     

Large amine-containing neurones in the central ganglia of Lymnaea stagnalis.

A histochemical study was made of amine-containing neurones in the central ganglia of Lymnaea stagnalis. Several large catecholamine-containing neurones were detected, including a group in the right parietal ganglion and the largest cell in the right pedal ganglion. Biochemical analyses of the perikaryon of this giant pedal neurone showed that it contained approx 200 pg of dopamine but no detectable noradrenaline. Histochemical evidence for the presence of 5-hydroxytryptamine in some neurones was also obtained. None of the amine-containing neurones gave a positive histochemical reaction (Alcian Blue, Alcian Yellow) for neurosecretory material. Because the dopamine-containing neurone in the right pedal ganglion is readily located in living preparations, it would appear to be a useful model for studying the cellular role of neuronal dopamine.     

Development of neuromuscular transmission in a larval tunicate

1. The time sequence of the development of acetylcholinesterase (AChE), acetylcholine (ACh) receptors and functional synapses on the embryonic muscle membrane in a tunicate larva (Halocynthia roretzi) was investigated in vivo.2. The fertilized tunicate egg was incubated in natural sea water at 9 degrees C. Sixty-eight hr after fertilization the free-swimming larva was hatched, which had six striated muscle fibres in the tail. The developmental stage of the embryo was indicated by the developmental hours after fertilization.3. The transmitter at the neuromuscular junction in the hatched larva is ACh. (i) Neuromuscular transmission was completely blocked by D-tubocurarine (1-5 x 10(-5)M). (ii) Eserine (5-10 x 10(-7)M) approximately doubled the time constant of the falling phase of miniature excitatory junctional currents (e.j.c.s). (iii) The reversal potential of the membrane response to iontophoretically applied ACh was -10 mV and similar to that of e.j.c.s. (iv) AChE was present on the muscle membrane surface.4. AChE activity became visible histochemically on the embryonic cell membrane in the presumptive muscle region as early as the late gastrula stage (27 hr after fertilization, 12 hr before the ACh response appeared).5. The response to iontophoretically applied ACh was present at 39 hr after fertilization but could not be evoked at 38 hr.6. Between 39 and 41 hr after fertilization, the ACh responses increased rapidly, then remained relatively unchanged until larval hatching.7. The stage of the initial appearance of the ACh response corresponded to the stage when the Ca current abruptly increased in the muscle membrane.8. The first sign of neuromuscular transmission was appearance of a giant excitatory junctional potential (e.j.p.) with uniform amplitude (about 15-20 mV) and slow time course (time constant of the falling phase of a giant e.j.c. was 23.4 +/- 6.9 msec, mean and S.D., at -60 mV and 11 degrees C).9. Within a few hours, these giant e.j.p.s disappeared and were successively replaced by medium-sized e.j.p.s and then e.j.p.s similar to those seen in hatched larvae (time constant of the falling phase of a miniature e.j.c. was 8.5 +/- 1.8 msec at -60 mV and 11 degrees C).     

The mechanism of excitation by acetylcholine in the cerebral cortex

1. The muscarinic depolarizing action of ACh on cortical neurones is associated with an increase in membrane resistance (mean DeltaV/DeltaR = 3.16 mV/MOmega).2. ACh also promotes repetitive firing by slowing repolarization after spikes.3. The depolarizing effect has a mean reversal level of -86.7 mV (with mean resting potential -56 mV).4. It is concluded that as a muscarinic excitatory agent, ACh probably acts by reducing the resting K(+) conductance of cortical neurones, and also the delayed K(+) current of the action potential.5. These results are discussed in relation to the possible role of ACh in cortical function.     

Synaptic connexions of two symmetrically placed giant serotonin-containing neurones

1. Each giant serotonin cell in Helix pomatia makes synaptic connexions with three non-amine-containing neurones: the anterior, middle and posterior buccal cells.

2. Individual e.p.s.p.s, of 500-600 msec duration, were observed in both left and right middle cells following each evoked giant serotonin cell action potential. They were facilitated with repetitive stimulation of the giant serotonin cells and summed to give rise to an action potential. The membrane resistance of the middle cells was reduced when the giant serotonin cells were stimulated to fire rapidly. Evidence is presented which suggests that the link between each giant serotonin cell and each middle cell is monosynaptic.

3. Iontophoretically applied serotonin produced a depolarizing potential change in the middle cell perikaryon; the response rapidly desensitized on repetitive application.

4. Morphine abolished reversibly the middle cell serotonin potential and antagonized transmission from the giant serotonin cells to the middle cells. Lowering the Na concentration of the medium reversibly diminished the size of the serotonin potential and the giant serotonin cell elicited e.p.s.p.s in the middle cells.

5. Reserpine, which depletes serotonin in the giant serotonin cell, impaired transmission from these cells to the middle cells.

6. The results suggest that serotonin is the synaptic transmitter released from the giant serotonin cells on to the middle cells and that this system is a suitable model for further analysis of the neuronal role of serotonin.

     

Inactivation of the asymmetrical displacement current in giant axons of Loligo forbesi.

1. Asymmetrical displacement currents ('gating currents') have been recorded in intracellularly perfused squid giant axons by averaging the currents associated with depolarizing and hyperpolarizing pulses. The relation between 'gating current' and Na inactivation was studied by investigating the effect of pulse duration and conditioning pulses. 2. Increasing the pulse duration from 0-3-1 msec to 10-20 msec reduced the off-response of the 'gating current' to 50-70% of its normal size; the time constant was 5 msec at +20 mV and 8 degrees C. The decrease of the Na current during a 10-20 msec pulse was stronger and faster; it decayed to 10-26% with a time constant of 1-35 msec. 3. The effect of pulse duration could also be demonstrated by using only depolarizing pulses. The charge displacement at the end of single or averaged depolarizing pulses was smaller for long pulse durations than for short. A long depolarizing pulse was followed by a small long-lasting tail of inward current. 4. A conditioning depolarizing pulse of 10-20 msec duration to a potential of -30 or +10 mV, followed by a short recovery period at -70 mV, decreased the on-response of the 'gating current'. Its size was reduced to 46-71% and 61-94%, respectively, for a recovery interval of 1-75 and 5 msec at 2-3 degrees C. The reduction of the Na current, measured under similar conditions, was more pronounced; the Na current was decreased to less than 50% of its normal value. 5. The observations about the effect of pulse duration and conditioning pulses on the 'gating current' are qualitatively consistent with those of Bezanilla & Armstrong (1974, 1975) and support the view that part of the asymmetrical charge displacement is inactivated during a 10-20 msec depolarization.     

Synaptic actions of peripheral nerve impulses upon Deiters neurones via the mossy fibre afferents

1. The cerebellar integration of sensory inputs to Deiters neurones was investigated in decerebrate cats. In some preparations decerebration was combined with transection of the olivocerebellar fibres.2. In the latter preparations peripheral nerve impulses generally produced a response consisting of a sequence of the following post-synaptic potentials: (i) an initial e.p.s.p. (d(1)), (ii) early i.p.s.p. (h(1)), (iii) later i.p.s.p. (h(2)).3. The mean latencies of d(1), h(1) and h(2) were 5.7, 7.3 and 9.8 msec from the forelimb nerves, and 7.5, 9.0 and 13.4 msec from the hind limb nerves, respectively.4. The stimulus intensity-response relation indicates that the Group I muscle afferents as well as the low threshold cutaneous afferents contribute to the response.5. In the preparations with the intact inferior olive there were additional components of the post-synaptic potentials: a later e.p.s.p. (d(2)) and another later i.p.s.p. (h(3)), their mean latencies being 15.3 and 19.7 msec from the forelimb nerves, and 18.0 and 21.3 msec from the hind limb nerves, respectively.6. The d(1) and h(2) components were attributed to the mossy fibre afferents and d(2) and h(3) to the climbing fibres; d(1) and d(2) were due to excitation through the collaterals of the mossy and climbing fibres, and h(2) and h(3) to inhibition from Purkyne cells activated by the mossy and climbing fibres, respectively. h(1) was too early to be produced through the cerebellum, and was probably mediated by inhibitory neurones in the reticular formation.     

The acetylcholine sensitivity of the surface membrane of multiply-innervated parasympathetic ganglion cells in the mudpuppy before and after partial denervation.

1. The surface chemosensitivity to iontophoretically applied acetylcholine (ACh) of single nerve cells in the cardiac ganglion of the mudpuppy was examined. 2. Some synapses on the neurones can be recognized in the living preparation with differential interference contrast optics. Identified synaptic regions of the ganglion cells were more sensitive to ACh than were other areas. The mean sensitivity of synaptic areas was 509 mV/nC, but that of random spots on the cell surface (which were mainly non-synaptic) was only 190 mV/nC. The mean rise time of ACh responses at synapses was 23 msec and at random spots was 36 msec. These data suggest that the density of ACh receptors is highest under the synapses on the post-synaptic membrane. 3. When some, but not all, of the presynaptic terminals on the ganglion cells are destroyed by cutting the vagus nerve, the sensitivity of the entire surface membrane to applied ACh increases. This increase in sensitivity reaches a maximum about 4-6 weeks after the operation. 4. Synaptic transmission at excitatory collateral synapses which remain after vagal degeneration is not altered by this hypersensitivity. 5. Neurones from ganglia which have been isolated and maintained in organ culture also become hypersensitive to applied ACh. this heightened chemosensitivity deveoops much faster in vitro; hypersensitivity in cultured ganglia becomes manifest within 4-5 days, in contrast with 4-6 weeks after vagus degeneration in vivo.     

Transmission from preganglionic fibres in the hypogastric nerve to peripheral ganglia of male guinea-pigs

1. Intracellular records were obtained from ganglion cells of the pelvic plexus of male guinea-pigs.2. The input resistance of cells which responded to intracellular stimulation varied from 40 to 150 MOmega. Slope resistance decreased when the membrane was hyperpolarized. Time constants varied from 5 to 200 msec. Resting membrane potentials ranged from 40 to 70 mV.3. Action potentials in response to direct stimulation were followed by a prolonged phase of after-hyperpolarization.4. A second type of cell was also impaled which did not respond to electrical stimulation. These cells had resting membrane potentials in the range 60-70 mV, input resistances of less than 20 MOmega and time constants of less than 3 msec.5. In most ganglion cells, stimulation of the hypogastric nerve evoked action potentials which were often followed by a secondary phase of depolarization indicating continuing transmitter action.6. Orthodromic responses were generally ;all-or-nothing' and could not be graded with changes in stimulus strength. The latency of orthodromic responses indicated that ganglion cells were innervated by both B and C fibres in the hypogastric nerve.7. Orthodromic responses were blocked by tubocurarine, 5 x 10(-5) g/ml., and dihydro-beta-erythroidine, 10(-5) g/ml.8. Spontaneous, excitatory post-synaptic potentials of up to 4.8 mV in amplitude were observed. The frequency of their discharge was greatly increased by repetitive stimulation of the hypogastric nerve.9. The ultrastructure of the pelvic ganglia was studied by electronmicroscopy. Two types of ganglion cell process were observed, fine (0.1 mu) branching tufts thrown up from the soma within the surrounding capsule and longer, thicker (1 mu) extracapsular processes. Synapses were found to occur most frequently between the varicose terminal segments of preganglionic axons and the small intracapsular processes.10. Similarities between the properties of the pelvic ganglia innervated by the hypogastric nerve and those of the parasympathetic division of the autonomic nervous system are discussed.     

A study of synaptic transmission in the absence of nerve impulses

1. The axo-axonic giant synapse in the stellate ganglion of the squid has been used to study synaptic transmission.2. When nerve impulses have been eliminated with tetrodotoxin, synaptic transfer of potential changes can still be obtained by applying brief depolarizing pulses to the presynaptic terminal.3. Suitably matched pulses are as effective as the normal presynaptic spike in evoking post-synaptic potentials. The synaptic delay and the time course of the post-synaptic potential are very similar to that in the normal preparation.4. The synaptic transfer (input/output) characteristic has been studied under different experimental conditions. With brief (1-2 msec) current pulses, post-synaptic response becomes detectable when the presynaptic depolarization exceeds about 30 mV. The post-synaptic potential increases about tenfold with 10 mV increments of presynaptic depolarization.5. Calcium increases, magnesium reduces the slope of the synaptic transfer curve. The influences on this curve of (i) duration of the pulse, (ii) preceding level of membrane potential, (iii) position of recording electrode, (iv) rate of repetitive stimulation are described.6. After loading the synaptic terminal with tetraethylammonium ions, large inside-positive potentials can be produced in the terminal and maintained for many milliseconds.7. By raising the internal potential to a sufficiently high level, synaptic transfer becomes suppressed during the pulse, and the post-synaptic response is delayed until the end of the pulse.8. This observation is in accord with a prediction of the ;calcium hypothesis', viz. that inward movement of a positively charged Ca compound, or of the calcium ion itself, constitutes one of the essential links in the ;electro-secretory' coupling process of the axon terminal.     

Effects of enkephalin and endorphin on the inhibitory junction potentials in the duodenal smooth muscle cells of the guinea-pig

Inhibitory junction potentials (i.j.p.s) evoked by field stimulation were recorded from the smooth muscle cells of the guinea-pig duodenum intracellularly. The membrane potential was -54.3 mV. The parameters of the i.j.p. were as follows: latency, 71 msec; time to peak, 146 msec; amplitude, 15.5 mV; rate of hyperpolarization, 107 mV/sec; and half decay time of the i.j.p., 193 msec. Met-enkephalin (10(-7)-10(-6) M) had no effect on the membrane potential and the i.j.p. The membrane potential was decreased by beta-endorphin (1.7 X 10(-7)-6.8 X 10(-7) M). Increase in the latency and the time to peak and decrease in the amplitude and the rate of hyperpolarization of the i.j.p. were observed for beta-endorphin. "Spontaneous" excitatory junction potentials (e.j.p.s) were generated by beta-endorphin. Naloxone (3.1 X 10(-6)-3.1 X 10(-4) M) hyperpolarized the membrane of the muscle cells. At high concentrations of naloxone (3.1 X 10(-4) and 3.1 X 10(-3) M), inhibition of the i.j.p. was observed. Levallorphan (2.3 X 10(-4) M) prolonged the latency and the time to peak and reduced the amplitude of the i.j.p. The membrane potential was slightly decreased by levallorphan. "Spontaneous" e.j.p.s were generated by levallorphan in a certain population of the cells. It is concluded that Met-enkephalin does not contribute to the non-adrenergic inhibitory transmission and that beta-endorphin acts as a modulator in the control mechanism of the intestinal motility. The effects of naloxone and levallorphan on the i.j.p. are discussed.