Frequency-dependent Facilitation Research Articles

A fast synaptic potential mediated by NMDA and non-NMDA receptors.

A fast synaptic potential mediated by NMDA and non-NMDA receptors. J. Neurophysiol. 78: 2693-2706, 1997. Excitatory synaptic transmission in the CNS often is mediated by two kinetically distinct glutamate receptor subtypes that frequently are colocalized, the N-methyl--aspartate (NMDA) and non-NMDA receptors. Their synaptic currents are typically very slow and very fast, respectively. We examined the pharmacological and physiological properties of chemical excitatory transmission at the mixed electrical and chemical synapses between auditory afferents and the goldfish Mauthner cell, in vivo. Previous physiological data have suggested the involvement of glutamate receptors in this fast excitatory postsynaptic potential (EPSP), the chemical component of which decays with a time constant of <2 ms. We demonstrate here that the pharmacological and voltage-dependent characteristics of the synaptic currents are consistent with glutamatergic transmission and that both NMDA and non-NMDA receptors are involved. The two components surprisingly exhibit quite similar kinetics even at resting potential, with the NMDA response being only slightly slower. Due to its fast kinetics and characteristic voltage dependence, NMDA receptor-mediated transmission at these first-order synapses contributes significantly to paired pulse and frequency-dependent facilitation of successive fast EPSPs during high-frequency repetitive firing, a presynaptic impulse pattern that induces activity-dependent homosynaptic changes in both electrical and chemical transmission. Thus NMDA receptor kinetics in this intact preparation are suited to its functional requirements, namely speed of information transmission and the ability to trigger changes in synaptic efficacy.

Activity-dependent properties of synaptic transmission at two classes of connections made by rat neocortical pyramidal axons in vitro.

1. To compare the dynamics of synaptic transmission at different types of connection, dual intracellular recordings were made from pairs of neurones in slices of adult rat neocortex. Excitatory postsynaptic potentials (EPSPs) were elicited by single spikes, spike pairs and brief spike trains in presynaptic pyramidal cells and responses recorded in postsynaptic pyramidal cells and in interneurones. 2. Pyramid-pyramid EPSPs were strongly voltage dependent and this resulted in a range of paired pulse effects. At thirty-two of sixty-nine pyramid-pyramid connections, the 2nd EPSP was the same shape as the 1st, indicating minimal interaction between active synapses. In these thirty-two connections, paired pulse depression (PPD) was apparent (2nd EPSP integral 46 +/- 21% of the 1st, at 5-20 ms), which recovered within 60-70 ms. 3. In eleven additional pyramid-pyramid pairs, the 2nd EPSP was also the same shape as the 1st, but paired pulse facilitation (PPF, 149 +/- 32%) decaying within 50-60 ms was apparent. Even these connections displayed frequency-dependent depression, however, as 3rd EPSPs were smaller than 1st EPSPs at intervals < 100 ms. 4. At twenty-five pyramid-pyramid connections, 2nd EPSPs were broader than 1st EPSPs and in sixteen of these, voltage- and NMDA receptor-dependent enhancement was large enough to obscure the underlying PPD. PPD was revealed by postsynaptic hyperpolarization (4 pairs), N-methyl-D-aspartate (NMDA) receptor blockade (3 paris), or if Mg2+ was removed (in the one case studied). If synapse location allowed significant depolarization of one active site by another, voltage-dependent enhancement could produce supralinear EPSP summation and overcome PPD. Third EPSPs were, however, consistently smaller than 1st EPSPs. 5. In striking contrast, profound frequency-dependent facilitation, independent of voltage or NMDA receptors was seen at fifteen connections involving two classes of postsynaptic interneurones. 6. At these pyramid-interneurone connections, facilitation of the 2nd EPSP (655 +/- 380% at 5-20 ms) decayed rapidly, within 50-60 ms. Third and fourth EPSPs showed additional facilitation which decayed more slowly, within 90 ms and 2 s, respectively. Facilitation due to five to six spike trains was still apparent at 3 s. Therefore, once initiated by a brief high frequency spike train, facilitation was maintained at lower frequencies.

Characteristics of Transmitter Secretion from Individual Sympathetic Varicosities

Electrophysiological recording techniques have been developed to study action potential propagation and neurotransmitter release, at the level of the varicosity, in postganglionic sympathetic nerve terminals. Several techniques have been employed, including differentiation of the rising phases of intracellularly recorded excitatory junction potentials (discrete events) and focal extracellular recording of the nerve terminal action potential and associated excitatory junction currents (NTIs, EJCs). This chapter focuses on the majority of this chapter on results obtained using this preparation. Researchers have shown that transmitter release from individual varicosities occurs intermittently following low-frequency nerve stimulation. The probability of transmitter release increases with stimulation frequency, that is, frequency-dependent facilitation. The mechanisms underlying frequency-dependent facilitation remain fairly poorly understood, but it is clear that calcium plays a pivotal role. One surprising finding is that residual release is inhibited by ryanodine in a use- and time-dependent manner, suggesting that this pharmacologically distinct voltage-dependent calcium channel is located close to, or is in some way coupled to, ryanodine receptors (RyRs). Ryanodine receptors are involved in excitation-contraction coupling in cardiac and skeletal muscle but are not thought to play any role in action potential-evoked transmitter release. The critical determinant of neurotransmitter release is an increase in the intraneuronal calcium concentration following invasion of the nerve terminal by the action potential. This transient increase in the intraneuronal calcium level is thought to arise solely by calcium entering through clusters of voltage-dependent calcium channels located close to the sites of neurotransmitter release, that is, active zones.

Paired pulse facilitation of GABAergic IPSCs in ventral horn neurons in neonatal rat spinal cord

Whole-cell patch-clamp recording of GABAergic inhibitory postsynaptic currents (IPSCs) were made in ventral horn neurons of neonatal rat lumbar spinal cord in slice. In contrast to the hippocampus where paired pulse depression is reported to be observed for GABAergic IPSCs, double pulse stimulation of GABAergic inputs resulted in enhancement in the amplitude of the second IPSC in the spinal ventral horn. The facilitation ratio was decreased during enhanced synaptic transmission by increasing Ca 2+ concentration in the external recording solution. Baclofen and adenosine, which are reported to depress synaptic transmission by presynaptic mechanisms, depressed IPSCs and increased the facilitation ratio. A postsynaptic manipulation such as application of bicuculline or changing the driving force did not affect the facilitation ratio. These results suggest that paired pulse facilitation of GABAergic IPSCs observed in neonatal rat spinal ventral horn appears to be based upon a mechanism similar to that underlying frequency-dependent facilitation of excitatory synaptic transmission, and is sensitive to presynaptic changes in synaptic strength.

ω-conotoxin GVIA-resistant neurotransmitter release in postganglionic sympathetic nerve terminals

Intracellular recording techniques were used to study neurotransmitter release in the guinea-pig isolated vas deferens. Low concentrations of the irreversible and selective N-type calcium channel blocker ω-conotoxin GVIA have previously been shown to block excitatory junction potentials evoked by low frequencies (⩽ 1 Hz) of nerve stimulation. Here we report a component of action potential-evoked release which is insensitive to high concentrations of ω-conotoxin GVIA. We have termed this component “residual release” and show (i) it is positively frequency-dependent, (ii) its magnitude is dependent on both the train length and interval between trains, (iii) “residual release” can be modulated through prejunctional α 2-adrenoceptors and (iv) “residual release” is insensitive to many calcium entry blockers but abolished by ω-grammotoxin SIA and cadmium ions. Although noradrenaline is released by nerve action potentials, residual excitatory junction potentials were abolished by α,β-methylene-ATP and therefore resulted entirely from the actions of neuronally released ATP acting through postjunctional P 2x-purinoceptors. The results suggest that calcium entry through a novel, pharmacologically uncharacterized voltage-dependent calcium channel is responsible for “residual release” in sympathetic nerve terminals. It seems that in response to single or short trains of nerve action potentials, N-type calcium channels dominate the release process. However, at higher frequencies other voltage-dependent calcium channels are recruited and these may have an important role to play in triggering the mechanisms underlying frequency-dependent facilitation.

Frequenin—A novel calcium-binding protein that modulates synaptic efficacy in the drosophila nervous system

The T(X, Y)V7 rearrangement in Drosophila has originally been recognized as a Shaker-like mutant because of its behavioral and electrophysiological phenotype. The gene whose expression is altered by the V7 rearrangement has been characterized. It encodes a novel Ca 2+-binding protein named frequenin, which is related to recoverin and visinin. In vitro, the frequenin protein functions like recoverin as a Ca 2+-sensitive guanylyl cyclase activator. Anti-frequenin antibodies stain the central and peripheral nervous system in Drosophila embryos and in larval and adult tissue sections. Frequenin appears to be particularly enriched in synapses, such as the motor nerve endings at neuromuscular junctions. Neuromuscular junctions of transgenic flies, which overexpress frequenin upon heat shock, exhibit an extraordinarily enhanced, frequency-dependent facilitation of neurotransmitter release, with properties identical to those observed in V7 junctions. We propose that frequenin represents a new element for the Ca 2+-dependent modulation of synaptic efficacy.

Presynaptic calcium and serotonin-mediated enhancement of transmitter release at crayfish neuromuscular junction

Fura-2 microfluorometric measurements of calcium concentrations in excitor motor nerve terminals of the first walking leg of crayfish Procambrus clarkii were made during application of serotonin (5-HT) to examine whether changes in residual calcium concentration or calcium influx are responsible for the 5-HT-induced increase in transmitter release at this synapse. Calcium concentrations and EPSPs were monitored simultaneously during 0.5-Hz and moderate-frequency (3-16 Hz) stimulation before, during, and after a 5-min application of 5-HT at concentrations between 5 and 100 microM. We did not observe a significant increase in resting calcium concentration [Ca2+]i in presynaptic terminals during or after application of 5-HT despite simultaneously recorded excitatory junction potential (EJP) enhancement. Injection of the calcium buffer EGTA to concentrations sufficient to reduce significantly the buildup of frequency-dependent facilitation during trains of action potentials did not reduce 5-HT-mediated EJP enhancement. We therefore conclude that 5-HT does not release calcium from intracellular stores to an extent sufficient to overcome calcium buffering and removal systems. Spontaneous release of transmitter quanta was increased by 5-HT to the same extent in normal and increased-osmolarity saline in the absence of measured changes in [Ca2+]i. Thus, changes in residual calcium cannot explain 5-HT-induced synaptic enhancement. Increasing the action-potential-mediated calcium influx by broadening the spike or by increasing extracellular calcium increased evoked transmitter release and increased the accumulation of calcium during trains of action potentials. However, though 5-HT produced a comparable synaptic enhancement, we found no evidence for increased calcium accumulation during trains of action potentials, suggesting that net calcium influx, buffering, and removal are not affected by 5-HT. Therefore, we conclude that 5-HT's action does not result from effects on calcium metabolism and suggest that it acts on the neurosecretory apparatus to increase the effectiveness of calcium for stimulating release. Furthermore, based on its relatively constant effects on facilitated and unfacilitated action-potential-evoked release and spontaneous transmitter release, we conclude that 5-HT actions are independent of the concentration of calcium that is driving release.

Action potential broadening and frequency-dependent facilitation of calcium signals in pituitary nerve terminals.

Hormone release from nerve terminals in the neurohypophysis is a sensitive function of action potential frequency. We have investigated the cellular mechanisms responsible for this frequency-dependent facilitation by combining patch clamp and fluorimetric Ca2+ measurements in single neurosecretory terminals in thin slices of the rat posterior pituitary. In these terminals both action potential-induced changes in the intracellular Ca2+ concentration ([Ca2+]i) and action potential duration were enhanced by high-frequency stimuli, all with a frequency dependence similar to that of hormone release. Furthermore, brief voltage clamp pulses inactivated a K+ current with a very similar frequency dependence. These results support a model for frequency-dependent facilitation in which the inactivation of a K+ current broadens action potentials, leading to an enhancement of [Ca2+]i signals. Further experiments tested for a causal relationship between action potential broadening and facilitation of [Ca2+]i changes. First, increasing the duration of depolarization, either by broadening action potentials with the K(+)-channel blocker tetraethylammonium or by applying longer depolarizing voltage clamp steps, increased [Ca2+]i changes. Second, eliminating frequency-dependent changes in duration, by voltage clamping the terminal with constant duration pulses, substantially reduced the frequency-dependent enhancement of [Ca2+]i changes. These results indicate that action potential broadening contributes to frequency-dependent facilitation of [Ca2+]i changes. However, the small residual frequency dependence of [Ca2+]i changes seen with constant duration stimulation suggests that a second process, distinct from action potential broadening, also contributes to facilitation. These two frequency-dependent mechanisms may also contribute to activity-dependent plasticity in synaptic terminals.

Oxytocin release evoked by electrical stimulation of the medial forebrain in the rat: Analysis of stimulus parameters and supraoptic neuronal activity

The role of the medial forebrain area (vertical limb of the diagonal band, medial septum and medial nucleus accumbens) in the control of oxytocin secretion in lactating rats was investigated. Electrical stimulation of the medial forebrain evoked a reproducible rise in intramammary pressure, equivalent to that caused by i.v. injection of 1 mU oxytocin. No presser effect accompanied this response. Radio-immunoassay of plasma samples showed that stimulation caused a significant rise in the concentration of circulating oxytocin. The effects of changing the parameters of stimulation to the medial forebrain were compared with those evoked by stimulation of the neural stalk. The optimal frequency for stimulation of the forebrain was found to be four-fold lower (10–20 Hz) than that for stimulation of the neural stalk (50 Hz). During continuous prolonged stimulation of the forebrain (20 Hz; 2 min) only a single transient response was obtained, whereas a protracted response was obtained as a result of prolonged stimulation of the stalk. Recordings were made from antidromically identified neurosecretory cells in the supraoptic nucleus. Electrophysiological responses to electrical stimulation of the medial forebrain were characterized by two main features. (1) Single-pulse stimulation produced only a small excitation (one or two action potentials), while high-frequency trains produced a profound facilitation of this response, with each pulse evoking short-duration ‘bursting’ behaviour in the supraoptic neurons. (2) During long trains of stimulation this frequency-dependent facilitation declined and could only be renewed after a period of rest. The study indicates that the medial forebrain provides a major excitatory afferent input to the oxytocin-secreting neurons of the supraoptic nucleus, and that the pulse-like secretion of oxytocin following medial forebrain stimulation results from the complex facilitatory and adaptive characteristics of this input.

Dendrotoxin and 4-aminopyridine potentiate neurohypophysial hormone secretion during low frequency electrical stimulation

The K + channel blockers dendrotoxin (10 −9 M) and 4-aminopyridine (10 −6 M) produce similar patterns of augmentation of electrically evoked vasopressin and oxytocin secretion from isolated rat neurointermediate lobes. Both channel blockers produced a greater than two-fold increase in vasopressin release. Oxytocin release, however, was comparably increased only when endogenous opioid inhibition was abolished y the presence of naloxone. The potentiation of hormone release was dependent on the frequency of the applied stimulus. A large effect was seen with 1000 pulses at 4 Hz but not when the same number of pulses was delivered at 12 Hz. These findings suggest that a specific type of K +-channel, sensitive to dendrotoxin and low micromolar 4-aminopyridine, may play a significant role in the frequency-dependent facilitation of secretion from neurohypophysical nerve endings.

Activators of protein kinase C potentiate electrically stimulated hormone secretion from the rat's isolated neurohypophysis.

Two activators of protein kinase C, phorbol 12,13-dibutyrate (PDBu) and 1-oleoyl-2-acetylglycerol (OAG), augment electrically stimulated vasopressin and oxytocin secretion from the nerve terminals of the isolated rat neurointermediate lobe. The increased hormone release produced by PDBu is specific to the beta-phorbol conformation, and is dependent upon electrical stimulation in the presence of calcium. Furthermore, the potentiation of release was evident during low frequency stimulation (4 Hz) but not when the same number of pulses were applied at 20 Hz. This occlusion of the phorbol ester's effect by high-frequency stimulation suggests that activation of protein kinase C may play a role in the normal process of frequency-dependent facilitation of secretion in the neurohypophysial system.

Electrical activity at the sympathetic neuroeffector junction in the guinea-pig vas deferens.

1. The relationship between the nerve terminal action potential and transmitter release from sympathetic postganglionic nerve terminals has been studied in vitro by focal extracellular recording. 2. In the absence of stimulation, 'spontaneous excitatory junction currents' (SEJCs) were recorded with amplitudes up to 500 microV, durations of 50-80 ms and frequencies of occurrence of 0.3-0.05 Hz; SEJCs of unusually long time course were also observed. The SEJCs were not recorded in tissues pre-treated with 6-hydroxydopamine to destroy sympathetic nerves, were unaffected by tetrodotoxin (TTX), the competitive alpha-adrenoceptor antagonists, prazosin and phentolamine, the irreversible alpha-adrenoceptor antagonist benextramine but were blocked by alpha,beta-methylene ATP which desensitizes P2-purinoceptors. 3. During trains of supramaximal stimuli at 0.1-4 Hz stimulus locked 'excitatory junction currents' (EJCs) were evoked intermittently from the population of varicosities located under the suction electrode with a probability of occurrence of 0.005-0.8. Although EJCs occurred intermittently, they were always preceded by an associated, non-intermittent, nerve impulse (delay less than or equal to 3 ms). 4. The EJCs reflect transmitter release from nerves because they were abolished by TTX, removal of calcium from the bathing medium, exposure to alpha-beta-methylene ATP and exhibited frequency-dependent facilitation. 5. Amplitude distributions of SEJCs and EJCs recorded in the same attachment were similar and skewed towards low-amplitude events. Individual SEJCs and EJCs could be found which were identical in amplitude and time course. 6. Locally applied TTX blocked impulse propagation and transmitter release in the terminal region; electrotonic invasion of the terminals from the point of block did not activate the transmitter release process. 7. These studies indicate that (1) intermittence of transmitter release is caused by a low probability of release in the invaded varicosity and is not caused by conduction failure in the terminal regions, (2) only a single quantum is normally secreted when the release mechanism of a varicosity is activated by the nerve impulse and (3) active invasion of the terminals is necessary for transmitter release to occur.

Effects of tetraethylammonium ions on frequency-dependent vasopressin release from the rat neurohypophysis.

1. Isolated rat neurohypophyses were fixed by their stalks to a platinum wire electrode and superfused with oxygenated Krebs-HEPES solution. Vasopressin release into the medium was determined by radioimmunoassay. Vasopressin secretion was increased by electrical stimulation at different frequencies (3-30 Hz) and different train lengths (75-900 pulses). The effects of tetraethylammonium (TEA) ions and of enhanced calcium were tested. 2. Electrical stimulation at 7.5 or 15 Hz evoked a markedly larger release of vasopressin than stimulation at 3 Hz. During continuous stimulation at 7.5 and 15 Hz the evoked vasopressin release per pulse declined rapidly, but with similar time constants for both frequencies indicating that the fatigue of the release process was strongly time dependent. The kinetic analysis showed also that the initial release per pulse was identical for 7.5 and 15 Hz stimulation. Nevertheless, with increasing duration, stimulation at 7.5 Hz became less efficient (in terms of release per total stimulus) than stimulation at 15 Hz and this was due to the time-dependent fatigue. 3. TEA (10 mM) increased the release of vasopressin evoked by 3 Hz stimulation much more than that evoked by 15 Hz stimulation resulting in an equieffective activation of release by both stimuli. On the other hand, elevation of the extracellular calcium from 1.2 to 3 mM did not alter the different efficiency of stimuli of 3 and 15 Hz. In the presence of TEA the time-dependent fatigue of the release during continuous stimulation was prevented, but an additional, slower component of the fatigue became apparent which was release or impulse dependent. 4. As prolongation of the action potential by TEA facilitates preferentially the hormone release evoked by low (ineffective) frequencies, it is suggested that a frequency-dependent broadening of action potentials which reportedly occurs on neurosecretory neurones may play an important role in the frequency-dependent facilitation of hormone release from the rat neurohypophysis.

Characteristic features of transmitter release from sympathetic nerve terminals.

Recently, using intracellular recording techniques, it has been shown that transmitter release from individual varicosities in sympathetic nerves occurs intermittently, and that only a single quantum of transmitter is secreted when the release process of a varicosity is activated by the nerve action potential. Here we discuss results obtained using a novel method of extracellular recording which allows simultaneous measurement of both the nerve action potential and transmitter release from postganglionic sympathetic nerve terminals. We have confirmed that release is intermittent, but the importance of this new approach is that the relationship between the nerve terminal action potential and transmitter release can be studied unambiguously for the first time. Thus, we are able to show unequivocally that: (1) intermittence is caused by a low probability of transmitter release in the invaded varicosity; (2) frequency-dependent facilitation of release is determined by the rate of arrival of the action potential and not by detectable changes in its configuration; (3) the current underlying the exciitatory junction potential (EJP) is brief compared to the total duration of the EJP, and (4) clonidine may inhibit transmitter release by modifying the nerve terminal action potential. These results provide important new insights into the fundamental mechanisms involved in the physiological and pharmacological control of transmitter release from sympathetic nerves.

Action potentials and frequency-dependent secretion in the mouse neurohypophysis.

The frequency-dependence of secretion of arginine vasopressin (AVP) from the mouse neural lobe in vitro was studied and found to be comparable to that reported for the rat neural lobe in vitro. For a stimulus train of 600 pulses, the secretion of AVP per pulse (i.e., facilitation) increased to a maximum at 20 Hz. Compound intracellular action potentials were recorded from the mouse neural lobe using optical recording methods and potentiometric dyes. These extrinsic optical signals reflect the true time courses of transmembrane potential changes (e.g., action potentials), and the action potentials recorded from mouse neural lobes had a duration of 5 ms; at half-maximum peak height. Optical recordings during repetitive stimulation showed that significant spike broadening occurred in each subsequent spike at 10 and 16 Hz stimulation. These data are consistent with a spike broadening hypothesis of frequency-dependent facilitation in the neural lobe. However, 4-aminopyridine, a drug which causes spike broadening in neural tissues by blocking potassium channels, did not produce an increase in secretion of AVP per stimulus from the mouse neural lobe.

Frequency dependent intermittency and ionic basis of impulse conduction in postganglionic sympathetic fibres of guinea-pig vas deferens

Some characteristic features of the functional innervation of guinea-pig vas deferens have been determined. Both ganglionic transmission from the hypogastric nerves and impulse propagation in proximal regions (main branch bundles within about 15 mm from the prostatic end of the organ) of the majority of single postganglionic sympathetic fibres of vas deferens nerve, had a high safety factor. Failure at these levels cannot account for the intermittent pattern of electrically-evoked secretion of transmitter from the individual varicosity of the terminals of vas deferens nerves, observed under identical experimental conditions. The shape of the extracellular single fibre action potential recorded by small calibre suction electrodes remained constant in proximal regions of vas deferens nerve, when the frequency of stimulation was varied between 0.5 and 8 Hz. Therefore, frequency-dependent facilitation of transmitter secretion in this tissue cannot be explained by frequency-dependent growth in the amplitude of nerve action potentials, as earlier assumed. However, when recordings were made in distal regions of vas deferens nerve (in small axon bundles, close to their points of insertion into the substance of the epididymal end of the organ), on two occasions fibres were found in which the safety factor for impulse conduction was low and frequency-dependent. The possibility is discussed that this feature, which was an exception in these non-terminal regions of vas deferens nerve, may be shared by the majority of fibres as they proceed distally towards the terminals. Clearly, if this is the case, intermittent failure of transmitter secretion from the individual varicosity may be due, at least in part, to intermittent failure of conduction of the nerve impulse to the terminals. Some useful qualitative information on the ionic basis of the extracellular nerve action potential, that might underlie a proximo-distal decline in the safety factor for impulse conduction in these nerves, was obtained by determining the effects on the shape of the signal, caused by varying the ionic composition of the medium (sodium, calcium), and by local addition of agents with known actions on sodium (tetrodotoxin), potassium (tetra-ethyl ammonium, 4-aminopyridine, rubidium, barium) and calcium channels (cobalt, manganese, lanthanum, nickel, D-600). By these criteria, the action potential that was shown to be a “normal” sodium-potassium spike, in proximal regions of vas deferens nerve, was found to have a different “pharmacological profile”, in distal regions of the nerve, in a manner suggesting that here nerve impulse conduction had become somehow “calcium-dependent”. Although calcium ions alone could not sustain the nerve action potential in the absence of extracellular sodium, variation in the calcium concentration of the medium altered its shape. Furthermore, impulse conduction in distal regions of vas deferens nerve was not only blocked by tetrodotoxin, but also by “calcium channel blockers” of the alkaline earth metal ion class (cobalt, manganese, nickel, lanthanum), but not by the organic “calcium blocker” D-600. In these regions 4-aminopyridine had unique effects, often causing a delayed and prolonged positive-going potential, suggesting promotion of influx of cation, possibly through “calcium channels”. The results suggest that changes in the ionic basis of the nerve impulse as the fibres approach the secretory terminals introduce an element of “plasticity of conduction”, that may be the basis of pharmacological and physiological control of transmitter secretion by regulation of the invasion of terminals by the nerve action potential.

Multiple actions of cocaine on neuromuscular transmission and smooth muscle cells of the guinea-pig mesenteric artery.

1. The effects of cocaine on the neuromuscular transmission and smooth muscle cells of the guinea-pig mesenteric artery were observed using various experimental procedures.2. Cocaine (10(-7) M) depolarized the membrane and increased the membrane resistance of single smooth muscle cells. Outward current pulses produced neither spikes nor graded responses in Krebs solution, but in the presence of 10(-5) M-cocaine, outward current did produce spikes.3. Perivascular nerve stimulation evoked excitatory junction potentials (e.j.p.s). Repetitive stimulation (0.25-1.0 Hz) produced a frequency-dependent facilitation. Application of cocaine (10(-7) M) reduced the amplitude of the first e.j.p. (e.j.p.(f)) and also after completion of facilitation (e.j.p.(s)). However, the facilitation process was not affected by cocaine (10(-5) M).4. On pre-treatment with phentolamine (3 x 10(-7) M), both e.j.p.(f) and e.j.p.(s) were enhanced, but on pre-treatment with yohimbine (3 x 10(-7) M), e.j.p.(f) was inhibited and e.j.p.(s) was enhanced. Both phentolamine and yohimbine accelerated the facilitation of e.j.p.s in the absence and presence of cocaine (10(-7) M).5. The conduction velocity of nerve excitation measured from the latency of generation of e.j.p.s was slightly lowered by cocaine. The number of nerve fibres or varicosities contributing to the generation of an e.j.p. was not reduced in the presence of cocaine (10(-5) M).6. Mechanical responses could be recorded on perivascular nerve stimulation, and direct muscle stimulation on treatment with tetrodotoxin. Cocaine (10(-7) to 10(-4) M) enhanced the contraction evoked by direct muscle stimulation and inhibited the contraction evoked by perivascular nerve stimulation.7. Cocaine (10(-5) M) enhanced the contraction evoked by 5 x 10(-6) M-noradrenaline (NA) and direct muscle stimulation (5 sec pulse) but no effect was observed on the K-induced contraction (39.2 mM-K). On pre-treatment with guanethidine (10(-6) M) these effects of cocaine were not affected.8. In the presence of cocaine (10(-5) M), the depolarization of the membrane induced by NA was additively increased, and the dose response curve for NA was shifted to the left with no change in the maximum amplitude of contraction.9. When 10(-5) M-cocaine was applied during contractions evoked by alternate perivascular nerve stimulation and exogenously applied NA, the contraction evoked by perivascular nerve stimulation was reduced, while that evoked by NA was enhanced.10. In saponin-treated skinned muscles, the pCa-tension relationship was not affected by application of 10(-4) M-cocaine. The effects of cocaine on the Ca accumulation and release from the store site were estimated. It was found that cocaine (10(-4) M) slightly inhibited the Ca accumulation (0.89 times the control) but did not modify the Ca-release mechanism.11. The overflow of NA, 3,4-dihydroxyphenylglycol (DOPEG) and 3-methoxy-4-hydroxyphenylglycol (MOPEG) were measured in the same tissue before and after application of perivascular nerve stimulation in the presence or absence of cocaine (10(-7) to 10(-5) M). Cocaine induced a concentration-dependent increase in the overflow of NA and a reduction in the amounts of DOPEG and MOPEG.12. We conclude from these studies that cocaine mainly inhibits the sensitivity of the intra-junctional adrenoceptor, but increases the sensitivity of the extrajunctional adrenoceptor distributed on the post-junctional muscle membrane, with increase in the overflow of NA. The enhancement of mechanical response in the presence of cocaine is probably due to an increased sensitivity of the extra-junctional adrenoceptor and changes in the post-junctional muscle membrane, without any marked effect on the prejunctional mechanism.

Central control of swimming in the cubomedusan jellyfishCarybdea rastonii

1. Swimming in the cubomedusaCarybdea rastonii is controlled by a subumbrellar nerve net. Neurons that make up this net, including “giant” neurons, make random synaptic contacts with each other and with the circular subumbrellar swimming muscles (Figs. 1–3). 2. Extracellularly recorded swimming impulses originate in the rhopalia and spread throughout the subumbrellar nerve net, initiating contractions of the subumbrellar musculature (Fig. 4). 3. Intracellular recordings from the subumbrellar giant neurons indicate that all-or-none overshooting action potentials precede each swimming contraction (Fig. 6). Synaptic depolarizations were occasionally recorded alone, and triggering an action potential (Fig. 6C, E). 4. Extracellularly recorded muscle potentials exhibit frequency-dependent facilitation, with normal swimming at about 80% of the maximal contraction of the muscle sheet (Figs. 4 and 5). 5. Intracellular recordings from subumbrellar muscle cells reveal graded depolarizations with each contraction of the muscle sheet. The jagged potentials are initially small and increase in amplitude with the first few contractions in a series (Fig. 7). The increases in muscle cell depolarizations may be related to the facilitation in the size of extracellularly recorded muscle potentials. 6. Pacemakers of the four rhopalia interact by a dominance hierarchy; the rhopalium with the highest firing frequency controls swimming. Dominance shifts have been observed in two-rhopalia preparations (Fig. 8 A), and facilitation of the musculature often occurs independently of rhopalial sequence (Fig. 8B). 7. The swimming system ofCarybdea is comparable to the “giant fiber nerve net (GFNN)” of other scyphomedusae.