EJP Amplitude Research Articles

Abstract 538: Prolonged Functional Sympathetic Efferent Denervation Following Ablation of the Renal Nerves in SHR

Renal denervation (RDN) has been shown to be an effective and safe treatment for human resistant hypertension and is considered one of the most promising advances in the field of hypertension. However, the mechanisms mediating the sustained decrease in arterial pressure in response to RDN in humans are unknown. This issue is particularly perplexing, given reports that the depressor effect of RDN in animal models is short lived. The current dogma in the literature is that overtime the efferent sympathetic nerves regrow and so the longevity of the depressor effect of RDN in humans cannot be ascribed to the absence of efferent renal sympathetic nerve activity. We hypothesize that the renal sympathetic nerves regenerate following RDN, and arrive at the target tissues, but the functional response is not fully restored and this contributes to the long-term depressor effect of RDN. Our aim was to examine mean arterial pressure (MAP) and vascular function in spontaneously hypertensive rats (SHR) following RDN. In 10 week old male SHR radiotelemetry probes were implanted to measure MAP. At 12 weeks of age, following 3 days basal MAP recording, sham (n=5) or bilateral RDN (n=5) surgery was performed. MAP was recorded until 24 weeks of age (12 weeks post-RDN). Renal lobar arteries were mounted on a myograph and membrane potential and tension responses to perivascular nerve stimulation assessed. Perivascular nerve stimulation with single pulses at increasing voltage evoked excitatory junction potentials (Ejps) of increasing amplitude in SHR-sham. Ejp amplitude was markedly reduced in arteries from SHR-RDN (P<0.01). Repetitive stimulation (1-8Hz, 5s) evoked slow depolarisation and contraction. At 5Hz 5s, slow depolarisation was 12±2 mV in SHR-shams and 7±2 mV in SHR-RDN (P<0.01), and contractions were smaller than SHR-sham (10 ± 3% vs 22 ± 2% HiK; P<0.01). In conclusion, smaller Ejps suggest that close contacting neuroeffector junctions are not reformed and blunted vasoconstrictor responses indicate neuro-vascular function has only partially returned 12 weeks post-RDN (Fig. 2). Our finding of continued ‘functional’ denervation is compatible with our data that RDN causes a sustained decrease in arterial pressure in SHR.

Sympathetic hyperinnervation in obesity is associated with oxidative stress and inflammatory cell NGF synthesis

Obesity is associated with an increased density of the sympathetic nerve plexus over arteries that control blood pressure. Here we examine the role of oxidative stress and inflammatory cell infiltration to changes in perivascular nerve innervation over small mesenteric arteries from C57BL/6 and NOX2−/− mice fed a normal or high fat diet. Changes in smooth muscle membrane potential were measured in response to perivascular nerve stimulation. Innervation density, nerve growth factor (NGF) and inflammatory cell type were examined by immunohistochemistry. Oxidative stress was measured using dihydroethidium fluorescence. Denser nerve plexuses over arteries from obese C57BL/6 mice were associated with larger excitatory junction potentials (EJPs; P<0.05). Oxidative stress and numbers of inflammatory cells positive for NGF were also increased (P<0.05). By contrast, the perivascular nerve plexus in obese NOX2−/− mice was less dense (P<0.05) and EJP amplitude was decreased (P<0.05). Oxidative stress and numbers of NGF positive inflammatory cells were also decreased (P<0.05). We demonstrate that obesity causes an increase in the density of the sympathetic nerve plexus via NOX2dependent and independent processes. Hyperinnervation occurs due to increased oxidative stress and infiltration of NGF producing inflammatory cells. This study was supported by the NHMRC to REH (466009).

Regulation of Postsynaptic Retrograde Signaling by Presynaptic Exosome Release

Retrograde signals from postsynaptic targets are critical during development and plasticity of synaptic connections. These signals serve to adjust the activity of presynaptic cells according to postsynaptic cell outputs and to maintain synaptic function within a dynamic range. Despite their importance, the mechanisms that trigger the release of retrograde signals and the role of presynaptic cells in this signaling event are unknown. Here we show that a retrograde signal mediated by Synaptotagmin 4 (Syt4) is transmitted to the postsynaptic cell through anterograde delivery of Syt4 via exosomes. Thus, by transferring an essential component of retrograde signaling through exosomes, presynaptic cells enable retrograde signaling.

Thermal acclimation, heat shock and photoperiod: Do these factors interplay in the adaptive responses of crab neuromuscular systems to temperature?

Evidence is reviewed demonstrating that the adaptive responses to temperature made by the walking leg neuromuscular system of crabs (Carcinus maenas) are in response to local temperature and not in response to hierarchical influences by the CNS and hormonal systems.Evidence is presented showing that the laboratory acclimation responses in muscle membrane resting potential (RP) do not predict the responses in seasonal acclimatized crabs, suggesting that additional environmental factors may have a role.No consistent effect of either short or long day photoperiod was found on muscle membrane RP in 8 or 22°C acclimated crabs. However, short day data for RP were more hyperpolarised than those from long day particularly at temperatures above 17°C. In 8°C acclimated crabs short day exposure resulted in consistently higher EJP amplitudes than following long day exposure. In 22°C acclimated crabs day length had a less marked effect, but short day appeared to alter the pattern of response to temperature towards that for 8°C acclimated crabs.Photoperiod was shown to have an effect on muscle tension, particularly in 8°C acclimated crabs. Long day as compared to short day exposure resulted in a higher force generated at temperatures below about 17°C with a marked temperature dependency. In 22°C acclimated crabs the difference between short and long day exposure was less marked but in this case the force generated was consistently greater following short day exposure. Thus photoperiodic effects may play a role in acclimatization.Heat shock (HS) increased CTMax of 8°C acclimated Carcinus from 33.04±0.2 to 34.17±0.52°C. Following heat shock closer muscle RP was more hyperpolarised but the temperature dependency of RP was unaltered from non-heat shocked controls, furthermore HS increased the temperature at which RP failed from 25.21±0.71 to 34.17±0.52°C. The effect of HS on EJP amplitude was complex, at low temperatures it lowered EJP amplitude but at higher temperatures the amplitude was increased. HS also significantly raised the temperature at which EJP failed from 23.39±0.73 to 27.43±1.16°C.Force generated by the closer muscle was dependent on acclimation temperature. Short day photoperiod increased force generation particularly in cold acclimated crabs. HS reduced force generation over the temperature range measured.

Prejunctional and postjunctional actions of heptanol and 18β-glycyrretinic acid in the rodent vas deferens

Heptanol and 18β-glycyrrhetinic acid (18βGA) block gap junctions, but have other actions on transmitter release that have not been characterised. This study investigates the prejunctional and postjunctional effects of these compounds in guinea pig and mouse vas deferens using intracellular electrophysiological recording and confocal Ca2+ imaging of sympathetic nerve terminals. In mice, heptanol (2 mM) reversibly decreased the amplitude of purinergic excitatory junction potentials (EJPs; 52±5%, P<0.05) while having little effect on spontaneous excitatory junction potentials (sEJPs). Heptanol (2 mM) reversibly abolished the nerve terminal Ca2+ transient in 52% of terminals. 18βGA (10 μM) decreased the mean EJP amplitude, and increased input resistance in both mouse (137±17%, P<0.05) and guinea pig (354±50%, P<0.001) vas deferens indicating gap junction blockade. Further, 18βGA increased the sEJP frequency significantly in guinea pigs (by 71±25%, P<0.05) and in 5 out of 6 tissues in mice (19±3%, P<0.05). Moreover, 18βGA depolarised cells from both mice (11±1%, P<0.01) and guinea pigs (8±1%, P<0.005). Therefore, we conclude that heptanol (2 mM) decreases neurotransmitter release (given the decrease in EJP amplitude) by abolishing the nerve terminal action potential in a proportion of nerve terminals. 18βGA (10 μM) effectively blocks the gap junctions, but the increase in sEJP frequency suggests an additional prejunctional effect, which might involve the induction of spontaneous nerve terminal action potentials.

Electrophysiological methods for recording synaptic potentials from the NMJ of Drosophila larvae.

In this video, we describe the electrophysiological methods for recording synaptic transmission at the neuromuscular junction (NMJ) of Drosophila larva. The larval neuromuscular system is a model synapse for the study of synaptic physiology and neurotransmission, and is a valuable research tool that has defined genetics and is accessible to experimental manipulation. Larvae can be dissected to expose the body wall musculature, central nervous system, and peripheral nerves. The muscles of Drosophila and their innervation pattern are well characterized and muscles are easy to access for intracellular recording. Individual muscles can be identified by their location and orientation within the 8 abdominal segments, each with 30 muscles arranged in a pattern that is repeated in segments A2 - A7. Dissected drosophila larvae are thin and individual muscles and bundles of motor neuron axons can be visualized by transillumination1. Transgenic constructs can be used to label target cells for visual identification or for manipulating gene products in specific tissues. In larvae, excitatory junction potentials (EJP’s) are generated in response to vesicular release of glutamate from the motoneurons at the synapse. In dissected larvae, the EJP can be recorded in the muscle with an intracellular electrode. Action potentials can be artificially evoked in motor neurons that have been cut posterior to the ventral ganglion, drawn into a glass pipette by gentle suction and stimulated with an electrode. These motor neurons have distinct firing thresholds when stimulated, and when they fire simultaneously, they generate a response in the muscle. Signals transmitted across the NMJ synapse can be recorded in the muscles that the motor neurons innervate. The EJP’s and minature excitatory junction potentials (mEJP’s) are seen as changes in membrane potential. Electrophysiological responses are recorded at room temperature in modified minimal hemolymph-like solution2 (HL3) that contains 5 mM Mg2+ and 1.5 mM Ca2+. Changes in the amplitude of evoked EJP’s can indicate differences in synaptic function and structure. Digitized recordings are analyzed for EJP amplitude, mEJP frequency and amplitude, and quantal content.

A NEW ELECTROPHYSIOLOGICAL TOOL TO INVESTIGATE THE SPATIAL NEURONAL PROJECTIONS WITHIN THE MYENTERIC ASCENDING REFLEX OF THE MOUSE COLON

1. The intestinal peristaltic reflex is regulated by local microcircuits that, upon activation, result in an oral contraction and anal relaxation of the circular muscle. This contractile response is associated with typical electrophysiological changes in membrane potential resulting from excitatory and inhibitory myenteric pathways. 2. The aim of the present study was to investigate the influence of local electrical stimulation (ES; single pulses, 15 V, 0.3 msec duration) on the ascending gastrointestinal electrophysiological potentials of the mouse colon using a novel 12-channel stimulation electrode in a newly designed model of the ascending myenteric pathways with simultaneous intracellular recording. 3. Local myenteric reflex responses in the proximal colon were initiated by ES (12 bipolar stimulation electrodes (SE) 0.7 mm apart from each other) and excitatory and inhibitory junction potentials (EJP and IJP, respectively) were recorded from circular smooth muscle cells with intracellular recording techniques. In vivo colonic propulsion was determined by measuring the time to expulsion of a 3 mm glass bead inserted 2.5 cm into the distal colon of mouse. 4. Under basal conditions, circular smooth muscle cells displayed a stable membrane potential (-56.7 +/- 6.9 mV; n = 13). Electrical stimulation elicited a tetrodotoxin (3 micromol/L)-sensitive, neuronal-induced EJP (cholinergic; atropine (1 micromol/L) sensitive) and a biphasic IJP. Both the EJP and IJP showed characteristic responses dependent on the distance between stimulation and recording sites. The EJP could be recorded over long distances, resulting in a maximal EJP amplitude at a distance of 10 mm distance (represented by stimulation electrodes (SE) number 6/7) and a maximal projection distance of 18-20 mm. Both components of IJP were maximal during direct stimulation (at SE1; stimulation at the recording site) and gradually decreased to SE6/7 (10 mm). At distances greater than 10 mm apart, ES did not produce IJP. The ganglionic blocker hexamethonium (10-100 micromol/L) concentration dependently abolished all inhibitory junction potentials at distances greater than 10 mm and significantly reduced the amplitude of EJP for the first 10 mm. Colonic propulsion was decreased by hexamethonium (40 mg/kg) and atropine (0.7 mg/kg). 5. Neuronal circuits of the ascending myenteric reflex functionally project distances ranging up to 18-20 mm. Our newly designed setup allows simultaneous electrophysiological investigations of neuronal microcircuitry within the myenteric plexus over short and long distances and enables conclusions to be drawn regarding neuroneuronal and neuromuscular transmission.

Modulatory effects of amino acids on neuromuscular transmission on the crayfish fast flexor muscle

The modulatory effects of amino acids on neuromuscular transmission from an identified giant motor neuron (MoG) and abdominal fast flexor muscles of the crayfish were examined using electrophysiological and pharmacological techniques. The distribution of amino acids in the cell body and axon of a single MoG was revealed using high-performance liquid chromatography with electrochemical detection. Eight different amino acids—aspartate, glutamate, glutamine, arginine, glycine, taurine, alanaine and γ-aminobutyric acid—were simultaneously detected in either the cell body or the axon of MoG. Aspartate, glutamate, alanine and arginine were present at relatively high concentration. Local pressure ejection of glutamate caused a depolarization of the abdominal fast flexor muscle fibers. On the other hand, aspartate, alanine and arginine had no clear effects on the same muscle fibers. Aspartate and arginine, however, had modulatory effects on neuromuscular transmission. Alanine had no significant effect on the neuromuscular transmission. Aspartate at a concentration of 200 μM decreased the amplitude of EJPs in the fast flexor muscle mediated by stimulation of both the MoG and non-giant fast flexor motor neurons. Arginine at a concentration of 200 μM reduced the EJP amplitude of the muscle fibers in response to MoG stimulation but enhanced the EJP amplitude of the same muscle fibers by stimulation of non-giant fast flexor motor neurons. Although rather high concentration (1 mM) were required, aspartate increased and arginine decreased the depolarization of the muscle fibers induced by local ejection of glutamate. The opposite effect of arginine on the fast flexor muscles in response to the stimulation of different motor neurons suggested its modulatory role in the different effects of these motor neurons (depression and facilitation) in the fast flexor muscles. J. Exp. Zool. 283:531–540, 1999. © 1999 Wiley-Liss, Inc.

Modulatory effects of amino acids on neuromuscular transmission on the crayfish fast flexor muscle

The modulatory effects of amino acids on neuromuscular transmission from an identified giant motor neuron (MoG) and abdominal fast flexor muscles of the crayfish were exam- ined using electrophysiological and pharmacological techniques. The distribution of amino acids in the cell body and axon of a single MoG was revealed using high-performance liquid chromatogra- phy with electrochemical detection. Eight different amino acids—aspartate, glutamate, glutamine, arginine, glycine, taurine, alanaine and γ-aminobutyric acid—were simultaneously detected in ei- ther the cell body or the axon of MoG. Aspartate, glutamate, alanine and arginine were present at relatively high concentration. Local pressure ejection of glutamate caused a depolarization of the abdominal fast flexor muscle fibers. On the other hand, aspartate, alanine and arginine had no clear effects on the same muscle fibers. Aspartate and arginine, however, had modulatory effects on neuromuscular transmission. Alanine had no significant effect on the neuromuscular transmission. Aspartate at a concentration of 200 μM decreased the amplitude of EJPs in the fast flexor muscle mediated by stimulation of both the MoG and non-giant fast flexor motor neurons. Arginine at a concentration of 200 μM reduced the EJP amplitude of the muscle fibers in response to MoG stimulation but enhanced the EJP amplitude of the same muscle fibers by stimulation of non-giant fast flexor motor neurons. Although rather high concentration (1 mM) were required, aspartate increased and arginine de- creased the depolarization of the muscle fibers induced by local ejection of glutamate. The oppo- site effect of arginine on the fast flexor muscles in response to the stimulation of different motor neurons suggested its modulatory role in the different effects of these motor neurons (depression and facilitation) in the fast flexor muscles. J. Exp. Zool. 283:531n540, 1999. © 1999 Wiley-Liss, Inc.

Two excitatory motoneurons differ in quantal content of their junctional potentials in abdominal muscle fibers of the cricket, Gryllus bimaculatus

In abdominal muscles 202 and 203 of the cricket, Gryllus bimaculatus, large and small excitatory junctional potentials (l- and s-EJPs) with similar durations can be recorded from the same muscle fibers. At the normal extracellular calcium ion concentration ([Ca 2+] o) of 5 mM, the amplitudes of l-EJPs in both muscles were larger than the threshold membrane potential for muscle action potentials, which is about −40 mV. Below 0.75 mM [Ca 2+] o, the amplitudes became much smaller and were below the firing level for the action potentials. At 0.5 mM, they fluctuated and decreased to 10.3 and 1.9 mV in muscles 202 and 203, respectively, and at 0.25 mM frequent failures occurred. The amplitudes of s-EJPs at 5 mM [Ca 2+] o were 13.3 and 5.1 mV in muscles 202 and 203, respectively, and the fluctuating amplitudes were far below the threshold for muscle action potentials. Below 0.75 mM, s-EJPs were rarely observed. The relation between log(EJP amplitude) and log([Ca 2+] o) was linear within a certain range of [Ca 2+] o and the slopes of the lines for l-EJPs were about twice as steep as those for s-EJPs in both muscles. In muscle 202, the amplitude distribution of l-EJPs obtained at 0.25 mM and that of s-EJPs at 0.75 mM both showed peaks at once and twice the voltage at the first peak, which were coincident with the voltages at the peaks of amplitude distributions of miniature EJPs recorded simultaneously. The reversal potentials for l- and s-EJPs in muscle 202 were +1.02 and +0.22 mV, respectively. In muscle 202, the decreases in amplitude of both EJPs by l-glutamate were similar and concentration-dependent. The results suggest that the difference in amplitude between l- and s-EJPs is attributable mainly to the difference in quantal contents.

Augmentation and suppression of the excitatory and inhibitory neuromuscular transmission induced by the ionophores, X-537A and A23187, in the red muscle of carp, Cyprinus carpio.

The effects of the ionophores, X-537A and A23187, on excitatory junction potentials (ejp), inhibitory junction potentials (ijp), diphasic junction potentials (diphasic jp) composed of ejp and ijp, and miniature excitatory junction potentials (mejp) were examined in the red muscles of carp, Cyprinus carpio. When 25 microM X-537A and 5 microM A23187 were applied, the amplitude of ejp and ijp increased transiently, then decreased gradually, and finally disappeared. The duration of ejp and ijp was little affected by the ionophores. The ionophores induced a transient increase in the frequency of mejp, which then decreased gradually after the maximum increase was attained, and finally fell below the control level. The ionophores had little effect on the resting membrane potential, membrane resistance of the muscle fiber, and amplitude of the compound action potential recorded from nerve bundles innervating this muscle. The ionophores caused ACh-evoked potentials to decrease gradually and finally disappear. These results suggest that the increase in the amplitude of ejp and ijp and in the frequency of mejp might be mainly due to the augmentation of ACh release from nerve terminals caused by an elevation of the intracellular Ca2+ concentration. On the other hand, the decrease and the abolition in the amplitude of ejp and ijp and in the frequency of mejp might be mainly due to the desensitization of the postsynaptic membrane.

The Modulatory Effects of Serotonin, Neuropeptide F1 and Proctolin on the Receptor Muscles of the Lobster Abdominal Stretch Receptor and Their Exoskeletal Muscle Homologues

ABSTRACT The muscle receptor organ (MRO) of the lobster is a complex proprioceptive system lying in parallel with the axial extensor musculature. Two peripherally located sensory neurones extend stretch-sensitive dendrites into individual receptor muscle strands one tonic (RM1) and one phasic (RM2). Previous studies have shown that the sensitivity of the sensory neurones to passive stretch could be enhanced by serotonin and proctolin. Here we show that the receptor muscles and their exoskeletal muscle homologues are also responsive to serotonin, proctolin and, in addition, to neuropeptide F1 (TNRNFLRF-NH2). Two measures of motor performance were enhanced by all three neurohormones: EJP amplitude and nerve-evoked tension development. Serotonin was the most effective modulator of both tonic and phasic muscles. F1 had powerful effects on the phasic extensor muscle. A low incidence of tonic muscle fibres with synapses responding to the neurohormones suggests that there are distinct populations of synapses: those sensitive to specific modulators and others that are insensitive. These findings, taken together with the enhancing effects of modulation on the primary sensory afferents, suggest that circulating neurohormones may act at multiple loci in the MRO system in a concerted and hormone-specific manner to alter the flow of proprioceptive feedback.

Chloride-mediated junction potentials in circular muscle of the guinea pig ileum

Junction potentials were recorded from circular muscle cells of the guinea pig ileum at various sites oral and anal to a transmural stimulus in the presence of atropine, apamin, and substance P antagonism (desensitization) at 30 degrees C. A short train of pulses produced an inhibitory junction potential (slow IJP), which preceded an excitatory junction potential (slow EJP). The slow IJP was observed up to 56.4 +/- 2.9 mm oral and 65.4 +/- 2.2 mm anal to the stimulus. The slow EJP was observed up to 50.4 +/- 1.9 mm oral and 58.3 +/- 2.1 mm anal to the stimulus. Hexamethonium (400 microM) decreased the amplitudes of the slow IJP and slow EJP at all sites. After hexamethonium, the slow IJP was observed up to 37.3 +/- 2.3 mm oral and 39.8 +/- 2.1 mm anal to the stimulus and the slow EJP was 44.2 +/- 2.5 mm oral and 43.3 +/- 2.6 mm anal to the stimulus. The slow IJP and slow EJP were associated with an increase and decrease in membrane resistance, respectively. Conditioning depolarizations of the circular muscle cells reduced the amplitudes of the slow IJP and slow EJP. Both were abolished at a membrane potential of approximately -25 mV. Conditioning hyperpolarizations increased the amplitude of both the slow IJP and slow EJP. Ionic substitution experiments with low external chloride solution (12.4 mM) resulted in an immediate increase in slow IJP and slow EJP amplitudes, whereas more prolonged perfusion resulted in a significant decrease in slow IJP and slow EJP amplitudes. 4,4'-Diisothiocyanostilbene-2,2'-disulfonic acid (400 microM) resulted in decreases in slow IJP and slow EJP amplitudes. These results suggest that the slow IJP and slow EJP are due to a decrease and increase in membrane chloride conductance, respectively. The noncholinergic neural pathways responsible for the slow IJP and slow EJP extend approximately 40 mm orally and anally along the longitudinal axis of the guinea pig ileum.

Cromakalim and K + channels in canine trachealis

The effects of cromakalim and glibenclamide on membrane properties and responses to acetylcholine of canine trachea were studied in the double sucrose gap to evaluate the presence and function of ATP-sensitive K + channels. Cromakalim produced a concentration-dependent hyperpolarization of muscle membrane potential which at maximum brought the membrane potential near the potassium equilibrium potential. Current clamping by hyperpolarizing current to this equilibrium potential abolished the hyperpolarization but not the membrane resistance decrease to cromakalim. Glibenclamide had no effect on resting membrane properties but reduced or abolished effects of cromakalim. Another K + channel antagonist, tetraethylammonium at 20 mM, also reduced the effects of cromakalim, but 4-aminopyridine (5 mM), Ba 2+ (1 mM, and apamin (10 −6M) had no antagonistic effect. The EJP produced on stimulation of cholinergic nerves sometimes increased just after cromakalim-induced hyperpolarization, but within 5–10 min as membrane resistance dramatically fell it was reduced, as was the depolarization to infused acetylcholine. Initially the reduction in EJP amplitude could be partially overcome by applying hyperpolarizing currents or by applying a second field stimulation; later the EJP was reduced further and was unaffected by these procedures. Even when depolarization to acetylcholine was markedly reduced, the contraction was not. Glibenclamide had no effects alone but antagonized all the effects of cromakalim. These results suggest that ATP-sensitive cromakalim activated K + channels are present in canine trachea but are usually closed during resting conditions under our experimental conditions. When they are opened by cromakalim, they hyperpolarize to near E K, markedly decrease membrane resistance and reduce the depolarization response to acetylcholine, probably by short circuiting the acetylcholine-induced current. There also appear to be ATP-sensitive K + channels in cholinergic nerves which, when activated, reduced acetylcholine output.

Modulation of tension production by octopamine in the metathoracic dorsal longitudinal muscle of the cricket Teleogryllus Oceanicus

ABSTRACT Application of octopamine to the metathoracic dorsal longitudinal muscle (DLM) of the cricket Teleogryllus oceanicus produced dose-dependent increases of twitch amplitude, contraction rate and relaxation rate. The threshold for octopamine effects was between 10−8 and 10−7mol l−1, while maximal effects were seen at approximately 10−5 mol l−1. The octopamine receptors were classified as octopamine2 receptors on the basis of the differential responsiveness of the muscle to the octopamine agonists naphazoline, tolazoline, clonidine and the octopamine antagonists metoclopramide and chlorpromazine. It was not possible to distinguish between octopamine2A or octopamine2B receptors in this preparation. Octopamine had both presynaptic and postsynaptic effects, since it increased both miniature end-plate potential (mEPP) frequency and muscle relaxation rate. Ata calcium concentration of 11 mmol l−1, octopamine did not affect muscle membrane potential, input resistance or EJP amplitude, but the EJP duration at half amplitude (T1/2) was slightly increased. In low-calcium saline (1.8 mmol l−1), octopamine did not affect membrane potential or T1/2, but EJP amplitude was increased. Stimulation of the octopaminergic dorsal unpaired median neuron (DUMDL), which innervates the metathoracic DLM, increased twitch amplitude in about 25% of the preparations. Failure in the other preparations was apparently due to spike conduction failure within the metathoracic ganglion. These results show that octopamine can be an important modulator of metathoracic DLM tension production.

Transmitter release modulated by isoprenaline in the dog isolated mesenteric vein

1. The effects of isoprenaline on the release of transmitter substances from perivascular adrenergic nerves were estimated from the excitatory junction potential (ejp) and outflow of noradrenaline in the dog mesenteric vein. 2. Isoprenaline increased the ejp amplitude and decreased the evoked release of noradrenaline. Yohimbine potentiated the former and converted the latter to an increased outflow. 3. Therefore, stimulation of prejunctional β-adrenoceptor by isoprenaline increases the release of noradrenaline from perivascular adrenergic nerves. 4. Possible involvement of prejunctional α-adrenoceptors in the isoprenaline-induced modulation of transmitter release was also suggested.

The relationship between the number of synaptic vesicles and the amount of transmitter released

The relationship between the number of synaptic vesicles and the amount of transmitter released from identified synapses was investigated in the dorsal longitudinal flight muscle (DLM) of the temperature-sensitive endocytosis mutant of Drosophila melanogaster, shibirets-1(shi). In the shi fly at 29 degrees C, vesicle recyling is blocked, but transmitter release proceeds normally. Thus, by inducing transmitter release at 29 degrees C, shi synapses gradually become depleted of synaptic vesicles. In this way it was possible to regulate the number of vesicles in a synapse. Intracellular recordings were made from individual fibers of the DLM in shi flies after various periods at 29 degrees C while stimulating at 0.5 Hz. The amplitude of the evoked excitatory junction potential (ejp), gradually decreased with longer exposure and was brought to various levels. The fiber was then rapidly fixed for electron microscopy. The number of vesicles per synapse was compared with the amplitude of the ejp at the time of fixation. It was observed that the smaller the ejp amplitudes became, the fewer vesicles were in the synapses. Also, as the ejp amplitude decreased, an increased number of synapses contained no vesicles. It is concluded that synaptic vesicles are directly involved in the release process.

Some membrane properties of the circular muscle of chicken rectum and its non-adrenergic non-cholinergic innervation.

1. Membrane properties and innervation of the circular muscle of chicken rectum were investigated by recording intracellularly electrotonic potentials evoked by passing current, and excitatory and inhibitory junction potentials (EJPs and IJPs) evoked by electrical stimulation of the extrinsic or intrinsic nerves. 2. The membrane potential was -55 +/- 0.6 mV (n = 95). Action potentials of long duration (1.2-4.0 s) discharged spontaneously, or were generated when the membrane depolarization due to either electrotonic potential or EJP reached the threshold. The drug D600 blocked the generation of action potentials. 3. Electrotonic potentials spread fairly well in the longitudinal direction of the muscle fibres but not in the transverse direction. The longitudinal space constant was 1.7 +/- 0.2 mm (n = 10) and the membrane time constant was 205 +/- 21 ms (n = 10). 4. Field stimulation of intramural nerves evoked an EJP followed by a long-lasting IJP (3-12 s in total duration) in most cells, and an EJP alone or an IJP alone in a small number of cells. The EJP and IJP were preserved in the simultaneous presence of atropine and guanethidine, but abolished with tetrodotoxin. 5. Stimulation of Remak's nerve trunk or its branches produced EJPs which were atropine resistant and guanethidine resistant just like the EJP elicited by intramural nerve stimulation. The extrinsic nerve stimulation was ineffective in eliciting IJPs. 6. The EJP amplitude declined in a linear manner as the distance from the stimulating site of intramural nerves was increased. The decline was much greater along the transverse axis than the longitudinal axis of circular muscle fibres. 7. The reversal potential for the EJP was estimated by extrapolation to be about - 15.3 +/- 0.3 mV (n = 7). 8. Apamin did not inhibit the IJP. During the hyperpolarization of a single IJP or summed IJPs, electrotonic potentials remained unchanged or slightly decreased in amplitude.

Distribution of excitatory junction potential amplitude in cat cerebral arteries: Examination of its quantal nature and modulation by opiates

We used scorpion venom to release small amounts of an excitatory neurotransmitter from adventitial nerves in cat left anterior descending cerebral artery. We used glass microelectrodes to measure and record postsynaptic electrical events of minimal amplitude. These events were similar to postsynaptic spontaneous and electrically evoked excitatory junction potentials (ejp's) seen in skeletal muscle. We performed a frequency analysis of the ejp amplitudes to determine if they fit a unimodal or multimodal distribution. We also investigated the effects of phentolamine, norepinephrine, hydromorphone, and morphine on ejp amplitude and frequency in the artery. Statistical analysis of the ejp frequency and amplitude revealed a multipeaked distribution with decreasing peaks. These results were similar to the distribution reported for acetylcholine release in skeletal muscle. The ejps were inhibited by phentolamine, which suggested that these events were adrenergically mediated. Norepinephrine and the opiates, hydromorphone and morphine, reduced the frequency and amplitude of the ejp's. The vessels also constricted to increasing doses of norepinephrine both under control conditions and in the presence of opiate. These results suggest that norepinephrine blocks the ejp's by a feedback mechanism at the presynaptic membrane and that endorphins and/or enkephalins, also acting at this presynaptic site, may modulate neurotransmission in the cerebral circulation.

Physiology and motor innervation of the supralateral radular retractor muscles of the pulmonate snail,Planorbarius corneus

1. Some electrophysiological properties of a multifunctional molluscan muscle, the supralateral radular retractor (SLR), were examined. The resting membrane potential was 62.3±10.5 mV and was dependent upon permeabilities to K+, Na+ and Cl−. Current-voltage relationships for SLR muscle fibres were linear, and the fibres had input resistances of 22.5±5.6 MΩ. Indirect evidence suggested that they were not electrically coupled. 2. Miniature EJPs occurred spontaneously in the muscle. Action potentials in the identified motoneurones elicited monosynaptic, chemical EJPs in muscle fibres. The majority of fibres were polyneuronally innervated by up to four motor axons. Action potentials did not normally occur in the muscle fibres, and contraction was proportional to the degree of depolarization produced by EJPs. 3. Contractions elicited in the SLRs by the stimulation of motoneurones were bilaterally symmetrical, and were temporally and spatially characteristic of the type of neurone excited. Temporal differences in contraction between motor units were determined mainly by differences in initial EJP amplitude. The relatively small degree of facilitation shown by all motor units had little effect on the speed of contraction. A linear summation model adequately described the growth of facilitation at terminals of one type of motoneurone, but not at those of the other two types of motoneurones examined.