Long term facilitation of tension in crustacean muscle and its modulation by temperature,activity and circulating amines

Summary Prolonged stimulation of the motor axon of the opener and stretcher muscles of the crayfish claw leads to long-term facilitation (LTF) of transmitter release at the neuromuscular junction. This facilitation is correlated with enhancement of tension development. Factors shown to enhance LTF of transmitter release, such as increased frequency of excitation, lower temperature, and exposure to ouabain also enhance tension development (Figs. 1, 2 and 4). Prolonged stimulation delivered in a bursting pattern enhances the development of tension more than an equivalent amount of stimulation delivered in a regular pattern (Fig. 3).Two circulating neurohormones, serotonin and octopamine, were examined for their effect on the development of tension during short and long periods of muscle activation. Serotonin and LTF of transmitter release appear to have an additive effect on the development of tension. The threshold for a detectable serotonin effect is 10^–10 M. The effect of octopamine on the development of tension appears to be enhanced by longer periods of maintained muscle activation. LTF of transmitter release resulting from 5 min of continuous activation at 15 Hz is accompanied by a drop in the threshold of an observable octopamine effect on tension from 10^–9Mto 10^–10 M. It is proposed that octopamine's trophic effects on metabolism in muscle act to sustain muscular performance during maintained activity.Abbreviations LTF long term facilitation - ec Membrane potential threshold for contraction - STF short term facilitation - e.j.p. excitatory junction potential This work was supported by a N.S.E.R.C. grant to H.L.A.     

Presynaptic modulation by octopamine at a single neuromuscular junction in the mealworm (Tenebrio molitor)

The effect of octopamine on the neuromuscular junction of the mealworm (Tenebrio molitor) was examined. Octopamine potentiated excitatory junctional potentials (EJPs) recorded intracellularly and extracellularly from ventral longitudinal muscle fibers. The potentiating action of octopamine was blocked in the presence of the alpha-adrenergic blocking agent, phentolamine, but not in the presence of another alpha-blocker, phenoxybenzamine, or the beta-blockers propranolol and dichloroisoproterenol. The resting membrane potential, membrane input resistance, reversal potential of EJP, glutamate potentials, and spontaneous miniature EJPs were found to be unaffected by octopamine. In contrast, quantal contents estimated by the extracellularly recorded EJP failures were greatly increased by octopamine. These results suggest that octopamine acted on the presynaptic terminals via alpha-adrenoceptor-like receptors (octopamine receptors) at the Tenebrio neuromuscular junctions to enhance the transmitter release associated with the motor nerve impulses.     

Effects of octopamine, dopamine, and serotonin on production of flight motor output by thoracic ganglia of Manduca sexta

Effects of biogenic amines on a centrally generated motor pattern in Manduca sexta were examined by pressure injecting nanomole to micromole amounts of octopamine, dopamine or serotonin into thoracic ganglia. Motor output was recorded extracellularly from a pair of antagonistic flight muscles and their motor neurons. The monoamines were found to alter production of a motor pattern that produces rhythmic wing flapping (10 Hz) and exhibits phase relationships similar to those in the flight pattern of intact moths. In mesothoracic ganglia with sensory nerves intact, octopamine (4 X 10(-9) mol) injected into lateral regions evoked regular firing of a single motor neuron, whereas a higher dose (4 X 10(-8) mol) often elicited the flight motor pattern. In the absence of sensory input, these doses of octopamine had little effect. Low doses (10(-10) mol) greatly enhanced motor responses to electrical stimulation of a wing sensory nerve. Dopamine (2 X 10(-10) mol) injected into the medial region of the mesothoracic ganglion elicited the flight motor pattern in the presence or absence of sensory input. Rhythmic output induced by dopamine (5 X 10(-10) mol) was suppressed by injecting serotonin (5 X 10(-10) mol) into the same region. These findings demonstrate that dopamine, octopamine, and serotonin have different effects on motor output in Manduca and suggest that these amines are involved in initiating, maintaining and terminating flight behavior, respectively. Octopamine may elicit flight production by enhancing the efficacy of sensory transmission thereby increasing excitability or arousal. Dopamine may act on interneurons involved in generating the flight motor pattern.     

Presynaptic effects of biogenic amines modulating synaptic transmission between identified sensory neurons and giant interneurons in the first instar cockroach

Intracellular recording was used to investigate the modulatory effects of serotonin and octopamine on the identified synapses between filiform hair sensory afferents and giant interneurons in the first instar cockroach, Periplaneta americana. Serotonin at 10(-4) mol l(-1) to 10(-3) mol l(-1) reduced the amplitude of the lateral axon-to-ipsilateral giant interneuron 3 excitatory postsynaptic potentials. and octopamine at 10(-4) mol l(-1) increased their amplitude. Similar effects were seen on excitatory postsynaptic potentials in dorsal giant interneuron 6. Several lines of evidence suggest that both substances modulate the amplitude of excitatory postsynaptic potentials by acting presynaptically, rather than on the postsynaptic neuron. The fitting of simple binomial distributions to the postsynaptic potential amplitude histograms suggested that, for both serotonin and octopamine, the number of synaptic release sites was being modulated. Secondly, the amplitudes of miniature excitatory postsynaptic potentials recorded in the presence of tetrodotoxin were unaffected by either modulator. Finally, recordings from contralateral giant interneuron 3, which has two identifiable populations of synaptic inputs, showed that each modulator had a more pronounced effect on excitatory postsynaptic potentials evoked by the lateral axon than on those evoked by the medial axon. Immunocytochemistry confirmed that neuropilar processes containing serotonin are present in close proximity to these synapses.     

Neuromuscular modulation in Limulus by both octopamine and proctolin

Both octopamine and proctolin potentiate nerve-evoked skeletal muscle contractions in the horseshoe crab, Limulus. The threshold concentration for octopamine was 10^?9 to 10^?8M, while for proctolin it was 3 × 10^?9M. Norepinephrine and dopamine produced effects similar to octopamine but at higher thresholds; tyramine and serotonin were ineffective. Octopamine caused significant increases in amplitudes of excitatory postsynaptic potentials (epsps) of muscle fibers, but had little effect on muscle fiber input resistance or membrane potential. Also, octopamine did not affect depolarization of muscle fibers and subsequent contraction due to the direct action of exogenously applied glutamate. These results suggest that octopamine potentiates nerve-evoked contractions primarily by facilitating release of neuromuscular transmitter. At concentrations above 10^?7M, however, octopamine sometimes caused muscle spikes in response to motoneuron stimulation, a finding that suggests that octopamine may also have some postsynaptic action. Proctolin potentiated the muscle contractions evoked by glutamate but had little effect on glutamate-evoked muscle fiber depolarization, muscle fiber input resistance, or membrane potential. Thus, proctolin appears to act directly on skeletal muscle to enhance contractility. The proctolin-induced potentiations of contraction were sometimes accompanied by modest increases in epsp amplitude, so that unlike lobster skeletal and Limulus cardiac neuromuscular preparations, proctolin may have a secondary direct synaptic effect. Both octopamine and proctolin have been found in Limulus cardiac ganglion. This potential access to the hemolymph and the relatively low threshold concentrations needed for physiological action suggest that octopamine and proctolin could function as hormonal modulators of neuromuscular function in Limulus.     

Serotonin precursor (5-hydroxytryptophan) has a profound effect on the post-copulatory time-fixed sexually refractory stage in the male cricket, <Emphasis Type="Italic">Gryllus bimaculatus</Emphasis> DeGeer

This study addresses a potentially general basis of measuring time in a biological timer. Here, we examined the effects of biogenic amines on the time-fixed post-copulatory sexually refractory stage (ca. 1 h) which is defined as the time interval between spermatophore protrusion and the onset of a calling or a mating response in the reproductive cycle of the male cricket. For subcuticular injection of amines (0.15 ml, 10(-2) mol l(-1)), the interval of the refractory stage was shortened by octopamine, serotonin, 5-hydoxytryptophan and N-acetyl-serotonin but was unchanged by tryptophan, melatonin or 5-hydroxyindol-3-acetic acid. The effect of 5-hydoxytryptophan was most potent (maximum shortening, 38%) and long lasting (ca. 4.5 h) while other amines effected only the injected cycle. Injection of 5-hydoxytryptophan (180 nl, 10(-2) mol l(-1)) into the terminal abdominal ganglion also decreased the interval to a similar extent. Simultaneous injection of 5-hydoxytryptophan with the inhibitor of the serotonin synthesis enzyme reduced the 5-hydoxytryptophan effect suggesting that this effect results from synthesis of serotonin from 5-hydoxytryptophan. The protein synthesis inhibitor cycloheximide had no effect on the interval. These results suggest that the reproductive timer is regulated by serotonergic neurons in the terminal abdominal ganglion without protein synthesis during the interval of the time-fixed sexually refractory stage.     

Octopamine enhances neuromuscular transmission in developing and adult moths, Manduca sexta

The effect of octopamine on neuromuscular transmission was examined in developing and adult Manduca sexta. Intracellular recordings were made from the dorsal longitudinal muscle (DLM), superfused with solutions containing DL-octopamine or other amines. In untreated adult moths and pharate adults nearly ready to enclose (stage Day 19), stimulation of the motor nerve evokes a large excitatory junction potential (EJP), an active membrane response, and a twitch. In adults and Day 19 animals DL-octopamine (10(-7) to 10(-4)M) has no effect on the amplitude and rise-time of the electrical response in normal saline, but 10(-6) to 10(-4) M DL-octopamine increases the amplitude of the excitatory junction potential recorded in saline containing one-third the normal calcium concentration. Immature (Day 16) muscle, which normally produces only small EJPs following stimulation of its motor nerve, responds to 10(-6) to 10(-4) M DL-octopamine by an increase in the EJP above threshold for an active membrane response and a contraction. When the muscle has developed sufficiently to spike and contract in response to nerve stimulation in the absence of exogenous octopamine (Days 17 and 18), application of DL-octopamine increases the maximum rate at which the muscle contracts in response to each stimulus in a train (designated the maximum following frequency, MFF). The threshold dose for an effect on the MFF of Day 18 immature moths is less than 10(-10) M. At this stage 10(-8) M DL-octopamine increases the MFF four-fold. The effect on the MFF is dose-dependent over the range 10(-10) M to 10(-6) M. The biogenic amines DL-epinephrine, DL-norepinephrine, tyramine, DL-phenylethanolamine, 2-phenylethylamine, and dopamine, applied at concentrations of 10(-8) or 10(-4) M, do not change the MFF. Both DL-synephrine (10(-8) M) and serotonin (10(-7) M) mimic the action of 10(-10) M DL-octopamine on the MFF. The action of DL-octopamine (10(-7) M) is blocked by phentolamine (10(-4)M) but not by propranolol (10(-4)M). The octopamine content of hemolymph was determined with a radioenzymtic assay. The concentration of octopamine in the hemolymph increases 3.6-fold, from 5 X 10(-8) M on Day 18 (duration of adult development is 19 days) to 1.85 X 10(-7) M one day following eclosion.(ABSTRACT TRUNCATED AT 400 WORDS)     

Dual excitatory innervation through the different ganglionic roots in the uropod adductor muscle of a crayfish,Procambarus clarkii

Dual excitatory innervation of the crayfish (Procambarus clarkii) uropod adductor muscle, adductor exopodite (ADX), through different roots of the sixth abdominal ganglion, was confirmed histologically and electrophysiologically. ADX contraction induced by the excitor in the third root (R3 excitor), showed more rapid fatigue than that by the excitor in the second root (R2 excitor), possibly owing to a more rapid depletion of the transmitter substance in the process of neuromuscular transmission. The size and position of the cell bodies of the two excitors were not necessarily similar, whereas the dendritic fields appeared to be almost overlapping.     

Cholinergic, adrenergic, and purinergic neuromuscular transmission.

A general model of the autonomic neuromuscular junction is proposed which emphasizes muscle effector bundles with gap junctions (or 'nexuses') forming the low resistance pathways allowing electrotonic coupling between neighboring cells, and extensive terminal varicose nerve fibers with 'en passage' release of transmitter. Some variations in autonomic neuromuscular geometry are discussed. Junctional clefts vary from 15nm in densely-innervated tissues such as vas deferens and iris to 2,000 nm in some large elastic arteries. Postjunctional specializations take the form of subsynaptic cysternae (in vas deferens and iris) and aggregations of plasmalemmal vesicles (in circular intestinal muscle). Current views of the synthesis, storage, release, and inactivation of transmitter during cholinergic, adrenergic, and purinergic transmission are summarized.     

Presynaptic inhibition and the participation of GABAB receptors at neuromuscular junctions of the crab Eriphia spinifrons

Presynaptic inhibition exerted by the common inhibitor on the closer and opener muscles and by the specific inhibitor on the opener muscle was investigated in the crab Eriphia spinifrons. In the closer muscle, activation of GABA_B receptors by baclofen reduced the mean quantal content of excitatory junctional currents by about 25%. Blocking GABA_B receptors with CGP 55845 diminished presynaptic inhibition at a similar percentage. GABA_B receptor-mediated presynaptic inhibition is linked to G proteins. Application of pertussis toxin eliminated about 25% of the inhibition exerted by the common inhibitory neuron. GABA_B receptors participate in presynaptic inhibition at release boutons of the slow and the fast closer excitor at a similar percentage. In the opener muscle, presynaptic inhibition of transmitter release from the same endings of the opener excitor was about 15% stronger with the specific inhibitor than with the common inhibitor. About 10% of the presynaptic inhibition produced by either one of the two inhibitors could be abolished by blocking GABA_B receptors. The amplitudes of the excitatory junctional currents in the opener were reduced in the presence of baclofen by about 25%, suggesting that synaptic terminals of the opener excitor are endowed with a similar percentage of GABA_B receptors as terminals of the slow and the fast closer excitors. Baclofen had no effect on postsynaptic inhibition, indicating that GABA_B receptors are not involved in postsynaptic neuromuscular inhibition. Accepted: 8 January 2000     

Structural and functional characteristics of the adenylyl cyclase signaling system regulated by biogenic amines and peptide hormones in the muscle of a worm Lumbricus terrestris

It has been shown for the first time that biogenic amines (catecholamines and tryptophane derivatives) stimulate dose-dependently activity of adenylyl cyclase (AC) and GTP-binding of G-proteins in muscle of the cutaneous-muscle bag of the earthworm Lumbricus terrestris. By efficiency of their stimulating action on the AC activity, biogenic amines can be arranged in the following sequence: octopamine > tyramine > tryptamine = serotonin > dopamine > isoproterenol = adrenalin. The sequence of efficiency of their action on GTP-binding is somewhat different: serotonin > tryptamine > octopamine > dopamine = tyramine > adrenaline > isoproterenol. Sensitivity of AC and G-proteins in the worm muscle to biogenic amines is similar with that in smooth muscle of the molluse Anodonta cygnea (invertebrates), but differs markedly by this parameter from the rat myocardium (vertebrates). It has also been revealed that AC in the worm muscle is regulated by peptide hormones relaxin and somatostatin whose action is comparable with that in the mollusk muscle, but much weaker that the action of these hormones on the rat myocardium AC activity. Use of C-terminal peptides of alpha-subunits of G-proteins of the stimulatory (385-394 Galpha(s)) and inhibitory (346-355 Galpha(i2)) types that disrupt selectively the hormonal signal transduction realized via G(s)- and G(i)-proteins, respectively, allowed establishing that the AC-stimulating effects of relaxin, octopamine, tyramine, and dopamine in the worm muscle are realized via the receptors coupled functionally with G(s)-protein; the AC-inhibiting effect of somatostatin is realized via the receptor coupled with G(i)-protein, whereas serotonin and tryptamine activate both types of G-proteins.     

Stimulation-induced changes at crayfish (Procambarus clarkii) neuromuscular terminals

Summary The fine structure of neuromuscular terminals of the single excitor axon was examined in the limb stretcher muscle of the crayfish Procambarus clarkii. A morphometric comparsion of the neuromuscular terminals of the left and right limbs of a control crayfish showed them to be similiar in qualitative as well as quantitative features. The excitor axon to the stretcher muscle of the right side was stimulated, by backfiring its branches in the adjacent opener muscle, at 20 Hz for 4–5 h per day over 4–5 days. The stretcher muscle on the left side was not stimulated and served as a control. Morphometric analysis of stimulated terminals revealed an increase in the number of dense bars and synaptic vesicles compared to their non-stimulated, contralateral counterparts. Since dense bars are regarded as active sites of transmitter release, changes in their number provide a morphological basis for synaptic plasticity.     

Effect of prolonged inactivity on skeletal motor nerve terminals during aestivation in the burrowing frog, <Emphasis Type="Italic">Cyclorana alboguttata</Emphasis>

This study examined the effect of prolonged inactivity, associated with aestivation, on neuromuscular transmission in the green-striped burrowing frog, Cyclorana alboguttata. We compared the structure and function of the neuromuscular junctions on the iliofibularis muscle from active C. alboguttata and from C. alboguttata that had been aestivating for 6 months. Despite the prolonged period of immobility, there was no significant difference in the shape of the terminals (primary, secondary or tertiary branches) or the length of primary terminal branches between aestivators and non-aestivators. Furthermore, there was no significant difference in the membrane potentials of muscle fibres or in miniature end plate potential (EPP) frequency and amplitude. However, there was a significant decrease in evoked transmitter release characterised by a 56% decrease in mean EPP amplitude, and a 29% increase in the failure rate of nerve terminal action potentials to evoke transmitter release. The impact of this suite of neuromuscular characteristics on the locomotor performance of emergent frogs is discussed.     

Insect Neuromuscular Synapses

Miniature end-plate potentials were used in studying severalaspects of the neuromuscular systems in the cockroach femur.The similar sizes and time courses of miniatures associatedwith fast and slow type excitatory axons suggest that they employthe same transmitter. There is other evidence also indicatingthat the essential difference between these two excitatory systemsis in the number of packets of transmitter released per nerveimpulse rather than different transmitter substances. From theshapes of miniatures it was suspected that typical muscle fibersmight have a branching structure. This was confirmed by histologicalexamination, intracellular stimulation, and intracellular dyeinjection. That inhibitory transmission is quantal is indicatedby hyperpolarizing miniatures which occur at random time intervals.Inhibitory transmission can be made to fail and recover in astepwise manner by manipulating the Ca/Mg ratio. In studiesof toxins which affect transmitter release at vertebrate motorend-plates, botulinal toxin was found to be without effect ateither excitatory or inhibitory junctions in cockroach muscle.However, black widow spider venom acted as it does in vertebrates,promoting massive release of transmitters and then permanentinactivation of the junctions.     

IP3-mediated octopamine-induced synaptic enhancement of crayfish LG neurons

The biogenic amines, octopamine and serotonin, modulate the synaptic activity of the lateral giant interneuron (LG) circuitry of the crayfish escape behavior. Bath application of both octopamine and serotonin enhances the synaptic responses of LG to sensory stimulation. We have shown previously (Araki et al. J Neurophysiol 94:2644-2652, 2005) that a serotonin-induced enhancement of the LG response was mediated by an increase in cAMP levels following activation of adenylate cyclase; however, octopamine acts independently. Here, we clarify how octopamine enhances the LG response during sensory stimulation using physiological and pharmacological analyses. When phospholipase C inhibitor U-73122 was directly injected into the LG before biogenic amine application, it abolished the enhancing effect of octopamine on direct sensory input to the LG, but did not block indirect input via sensory interneurons or the effect of serotonin. Direct injection of IP(3), and its analogue adenophostin A, into the LG increased the synaptic response of the LG to sensory stimulation. Thus, IP(3) mediates octopamine-induced synaptic enhancement of the LG, but serotonin acts independently. These results indicate that both octopamine and serotonin enhance the synaptic responses of the LG to sensory stimulation, but that they activate two different signaling cascades in the LG.     

Hormonal Effects on the in vitro Larval Growth of the Swine Intestinal Roundworm,Ascaris suum

Summary

Several biogenic amines and insect juvenile hormone III were tested in a growth bioassay of the parasitic nematode, Ascaris suum. Compounds (1 to 1000μzmol) were placed in culture with third-stage larvae for 24 hr, larvae were then rinsed several times, and larval cultures were returned to incubators for 6 more days. By this time, larvae had developed to the fourth-stage. The larvae were fixed in hot formalin, and their lengths were measured. Epinephrine and norepinephrine oxidized and were nematocidal under these culture conditions. Histamine and serotonin had no effect on length of the larvae. Octopamine (10–50μmol) exposure resulted in a significant dose-dependent increase in length. When incubated with octopamine (10μzmol) for 7 days, larvae grew more slowly than controls without octopamine (P< 0.05). Juvenile hormone III stimulated a dose-dependent (0 to 10μmol) increase in length after a 24 hr exposure. No synergism was detected between juvenile hormone III and ecdysone when co-incubated with larvae. These results indicate that Ascaris larvae are growth-insensitive to low concentrations of biogenic amines of host origin. Conversely, biological transmitters of invertebrate origin are potent stimulators of larval growth.     

Multimodality in the amplitude distribution of miniature endplate potentials due to the action of spatially discrete areas of transmitter release

Miniature end plate potentials (MEPPs) were simultaneously recorded in frog sartorius muscle by two intracellular microelectrodes. Some isolated groups of points (clouds) were found on the diagram of MEPPs scatter. Several peaks each of which was composed of signals from certain clouds (or from several clouds) on the scatter diagram were found on the histograms of MEPPs amplitude distribution. It is assumed that the clouds on the scatter histogram and the peaks on the histogram of MEPPs amplitudes are formed at the cost of secretion of quantum acetylcholine from spatial separate areas of transmitter release. The data obtained do not correspond with the subquantum hypothesis of transmitter release in neuromuscular junctions.     

Aminergic modulation of the myogenic heart in the branchiopod crustacean Triops longicaudatus

Although crustaceans typically have a neurogenic heart, the primitive crustacean Triops longicaudatus has a myogenic heart with the heartbeat arising from the endogenous rhythmic activity of the myocardium. In the present investigation, the effects of six biogenic amines, epinephrine, norepinephrine, dopamine, octopamine, serotonin and histamine, on the myogenic heart of T. longicaudatus were examined. Epinephrine, norepinephrine, dopamine and octopamine accelerated the heartbeat, increasing both the frequency and amplitude of the action potential of the myocardium in a concentration dependent manner. The ability of epinephrine and norepinephrine to produce the acceleratory effects was more potent than that of dopamine and octopamine; the threshold concentrations of epinephrine and norepinephrine were approximately 10(-10) M and those of dopamine and octopamine approximately 10(-7) M. Serotonin weakly inhibited the heartbeat, decreasing both the frequency and amplitude of the myocardial action potential in a concentration dependent manner with a threshold concentration of approximately 10(-6) M. Histamine exhibited no effect on the heartbeat. The results provide the first evidence for direct effects of amines on the crustacean myocardium and suggest neurohormonal regulation of the myogenic heart in T. longicaudatus.