Neuromuscular transmission in an insect visceral muscle

The electrical properties of the muscles of locust oviduct have been examined using intracellular recordings. The muscle cells are both dye and electrically coupled. They possess a wide array of spontaneous electrical activity ranging from slow oscillations of membrane potential to action potentials. In addition to possessing spontaneous electrical activity, certain regions of the oviduct are under motor control. The amplitude of evoked excitatory junction potentials (EJPs) increased step wise revealing innervation from a maximum of three motor units. These EJPs underwent summation and facilitation, and reached a critical threshold at which point the membrane revealed an active response. Bath applied glutamate, aspartate, proctolin, and octopamine were tested for their ability to alter resting potential and EJPs. L-glutamate (1.6 X 10(-5) M and above) produced a dose-dependent depolarization of membrane potential accompanied by a reduction in amplitude of EJPs. Although L-aspartate resulted in similar effects, the concentrations required were higher than those for glutamate. Proctolin (6.3 X 10(-11) M-6.0 X 10(-9) M) resulted in a dose-dependent depolarization but had little or no effect on amplitude of EJPs. Application of D, L-octopamine (3.2 X 10(-5) M-1.7 X 10(-4) M) induced a small hyperpolarization and a reduction in amplitude of EJP. It is suggested that contractions of locust oviduct appear to be regulated by a combination of a classical neurotransmitter such as glutamate, along with the neuromodulators octopamine and proctolin.     

The pharmacology of insect visceral muscle

• 1.1. At least five sites-of-action must be considered in most visceral nerve-muscle preparations.
• 2.2. L-Glutamate is perhaps the excitatory neuromuscular transmitter in some organs of the viscera but not others. Proctolin is probably a neuromuscular transmitter in some organs of the viscera defined criteria for rapid chemical transmission.
• 3.3. Although 5-HT and certain catecholamines are present in insects and have a pronounced effect on visceral muscle no functional roles has been established for them.
• 4.4. Various peptides released from neuroendocrine organs probably regulate visceral muscle function. Moreover such hormonal actions seem to be mediated by cyclic AMP in some cases.
• 5.5. Recent developments in methods for detecting peptides and prospective transmitters have increased the possibility of rapid progress in insect nerve-muscle pharmacology.

     

The action of iontophoretically applied L-glutamate on an insect visceral muscle

1) lontophoretic application of L-glutamate was employed to study the distribution of glutamate receptors in the superior longitudinal (SL) muscles of the locust (Locusta migratoria) hindgut, in which spontaneous activity was inhibited using normal saline containing 5 mM MgCl_2. 2) Junctional glutamate potentials with a rise time of 50–100 ms (peak) and a decay time of 250–400 ms were recorded at localized sites using ejection pulses in the range 5–10 nC. Most active sites were found in interfiber clefts and were spaced at about 250–300 μm intervals. 3) Desensitization of glutamate receptors occurred using ejection frequencies > 0.2 Hz. Desensitization could be irreversibly blocked using the lectin concanavalin A. 4) Depolarizing (D-) and biphasic depolarizing/hyperpofarizing (DH -) extrajunctional glutamate potentials were observed using ejection pulses > 15 nC. 5) δ-Philanthotoxin (δ-PTX) at concentrations > 0.3 Uml^?1 inhibited junctional glutamate potentials in a dose-dependent manner, 50% inhibition was achieved using 0.45 Uml^?1 δ-PTX. 6) Subthreshold concentrations of proctolin (up to 5 × 10^?10M) had no visible effect on glutamate potentials, suggesting that proctolin possibly does not act by modulating glutamate activity. 7) It is proposed that glutamate plays a transmitter role in SL muscles, while the role of proctolin is still unclear.     

Single glutamate gated channels in insect visceral muscle

Single glutamate-gated ion channels with a conductance of 135 pS are demonstrated in tonic muscle fibres of the locust hindgut. Channel kinetics closely resemble those of glutamatergic channels in locust skeletal muscles. Glutamate concentrations increasing within the range from 5 X 10(-5) to 1 X 10(-3) M result in an increase of the frequency of channel opening and a decrease in channel closed times. Delta-philanthotoxin, a toxin isolated from the venom of the digger wasp Philanthus triangulum, inhibits channel activity by blocking open channels and increasing channel closed times.     

The action of the venom of Philanthus triangulum F. on an insect visceral muscle

• 1.1. Low concentrations (0.05−0.38 BU/ml) of a crude venom extract from P. triangulum F. potentiate nerve-evoked contractions of the locust hindgut, possibly due to contamination of the venom preparation with proctolin.
• 2.2. Higher venom concentrations inhibit nerve-evoked contractions to a dose-independent plateau level.
• 3.3. The venom has no effect on responses to bath-applied proctolin, but responses to bath-applied L-glutamate are inhibited.
• 4.4. Spontaneous contractions are unaffected by the venom.
• 5.5. It is concluded that the plateau contractions are the result of excitation by non-glutamatergic transmission, and are possibly the result of proctolin release.

     

The functional role of octopaminergic neurons in insect motor behavior

The role of efferent, octopaminergic dorsal unpaired median (DUM) neurons in insects is examined by recording from them during motor behaviour. This population of neuromodulatory neurons is divided into sub-populations which are specifically activated or inhibited during ongoing motor behavior. These neurons are always activated in parallel to the respective motor circuits, and in addition to their modulatory effects on synaptic transmission may also cause metabolic changes in their target tissues.     

Roles of octopaminergic and dopaminergic neurons in appetitive and aversive memory recall in an insect

Background  

In insect classical conditioning, octopamine (the invertebrate counterpart of noradrenaline) or dopamine has been suggested to mediate reinforcing properties of appetitive or aversive unconditioned stimulus, respectively. However, the roles of octopaminergic and dopaminergic neurons in memory recall have remained unclear.     

Evidence for postsynaptic modulation of muscle contraction by a Drosophila neuropeptide

DPKQDFMRFamide, the most abundant FMRFamide-like peptide in Drosophila melanogaster, has been shown previously to enhance contractions of larval body wall muscles elicited by nerve stimulation and to increase excitatory junction potentials (EJPs). The present work investigated the possibility that this peptide can also stimulate muscle contraction by a direct action on muscle fibers. DPKQDFMRFamide induced slow contractions and increased tonus in body wall muscles of Drosophila larvae from which the central nervous system had been removed. The threshold for this effect was approximately 10(-8)M. The increase in tonus persisted in the presence of 7x10(-3)M glutamate, which desensitized postsynaptic glutamate receptors. Thus, the effect on tonus could not be explained by enhanced release of glutamate from synaptic terminals and, thus, may represent a postsynaptic effect. The effect on tonus was abolished in calcium-free saline and by treatment with L-type calcium channel blockers, nifedipine and nicardipine, but not by T-type blockers, amiloride and flunarizine. The present results provide evidence that this Drosophila peptide can act postsynaptically in addition to its apparent presynaptic effects, and that the postsynaptic effect requires influx through L-type calcium channels.     

Effects of leucomyosuppressin on the excitation-concentration coupling of insect Leucophaea maderae visceral muscle

1. Leucomyosuppressin (LMS) inhibited neurally evoked contractions of the hindgut of the cockroach Leucophaea maderae. The threshold for this inhibition of LMS was in the range of 1 × 10⁻¹⁰ M.2. LMS caused a sharp reduction in both l-glutamate and proctolin induced contractions. Dose-response profiles of the effect of LMS (held constant at 10⁻⁸M) on variable amounts of proctolin showed an inhibitory effect at 10⁻⁹ M proctolin and below, but at 5 × 10⁻⁹ M proctolin and above, LMS caused no inhibition.3. Potassium (158 mM) depolarized hindguts treated with LMS (10⁻⁸ M) showed a marked reduction (76% ± 2.1) in the proctolin (10⁻⁸ M) response.4. When calcium depleted preparations were returned to normal calcium levels (2 mM) in the presence of proctolin (10 ⁻⁸ M) a contraction occurred that was 45% ± 4 of the maximum in normal saline solution. However, LMS (10⁻⁸ M) reduced this response to only 28% ± 2 of the maximum.5. Proctolin (10⁻⁸ M) induced contractions in the presence of the manganous ions (2mM) fell to 63% ± 4 of the maximum but on the addition of LMS (10⁻⁸M), such responses fell to only 16% ± 5 of the maximum.6. These results offer evidence for a non-synaptic site of action for LMS and a perturbation of key calcium dependent events in the excitation-contraction coupling sequence of visceral muscle by this peptide.     

Evidence for chitin synthesis in an insect cell line

Cells from the continuous MRRL-CH line derived from embryos of the tobacco hornworm synthesized chitin. Digestion of the washed pellet from [¹⁴C]-N-acetylglucosamine-labeled cells by chitinase yielded a water-soluble labeled compound. The lyophilized residue from the supernatant of the chitin digestion was analyzed by gas-liquid chromatography as its trimethylsilyl derivative. The major component cochromatographed with derivitized chitobiose. The presence of chitobiose was confirmed by gas chro-matography-mass spectrometry. The synthesis of chitin by this cell line is inhibited by diflubenzuron.     

Inositol phospholipid hydrolysis may mediate the action of proctolin on insect visceral muscle

The formation of inositol phosphates in response to the neuropeptide proctolin was studied in locust oviducts. Glycerophosphoinositol, inositol 1-phosphate, inositol 1,4-bisphosphate, and inositol 1,4,5-trisphosphate were identified in the locust oviducts using anion-exchange chromatography. Proctolin stimulated the release of inositol 1-phosphate, inositol 1,4-bisphosphate, and inositol 1,4,5-trisphosphate during a 5-min incubation. In the presence of lithium ions the effects of proctolin were enhanced, with elevations of 98%, 42%, and 45% of inositol 1-phosphate, inositol 1,4-bisphosphate, and inositol 1,4,5-trisphosphate, respectively. Physiologically the effects of proctolin upon muscular contraction of locust oviducts were mimicked by the active phorbol ester, phorbol 12-myristate 13-acetate, and by the diacylglycerol analogue, 1-oleoyl-2-acetylglycerol. The inactive phorbol ester, 12-myristate 13-acetate 4-O-methyl ether, was without effect. The effects of the active phorbol ester and the diacylglycerol analogue were calcium-dependent requiring micromolar concentrations of calcium. The results indicate that the locust oviducts possess proctolin receptors that are linked to phosphatidylinositol metabolism and that inositol phospholipid hydrolysis may mediate the physiological action of proctolin.     

The role of proctolin and glutamate in the excitation-contraction coupling of insect visceral muscle

Proctolin (1 X 10(-10) to 1 X 10(-9) M) had a minimal effect on the spontaneous and evoked electrical events of the hindgut of the cockroach Leucophea maderae. Spontaneous action potentials and contractile activity stopped when the hindgut was exposed to 2 mM Mn2+. Eighty per cent of the response of the hindgut to glutamate was blocked by manganese, but only 35% of the response to proctolin. Hindguts were responsive to proctolin in a calcium-free medium, but not to glutamate. Moreover, proctolin appeared to facilitate the reentry of calcium after depleted preparations were returned to normal levels of external calcium. The results offer evidence for two calcium transmembrane channels in insect visceral muscle.     

Evidence for long-distance, chemical gall induction by an insect

Abstract We report that a chemical stimulus from a herbivore, a galling insect, changes plant morphology and physiology to benefit the herbivore. Previous studies could not determine whether insect galls are induced by mechanical or chemical stimuli because feeding and oviposition both occurred at the site of gall formation. We report that the mouthparts of a spruce‐galling insect, Adelges cooleyi, were inserted in stem phloem cells far from induced galls, that tissues between mouthparts and galls appeared normal, and that the ability to initiate galls was inversely correlated with distance from buds (potential gall sites). Thus the effects of chemical stimuli were unambiguously separated from any mechanical influence of probing stylets or ovipositors. Our results strongly suggest that galls were induced by a chemical stimulus transported to buds via vascular tissue and that its efficacy was dose‐dependent.     

Dorsal unpaired median neurons, and ventral bilaterally paired neurons, project to a visceral muscle in an insect

Cobalt backfilling, Lucifer yellow injection and neurophysiological recordings have been used to identify the neurons, in particular dorsal unpaired median neurons, which contribute axons to the oviducal muscles of the locust Locusta migratoria. A total of eight neurons within the VIIth abdominal ganglion have axons passing to the oviducts. Three pairs of bilaterally symmetrical neurons have ventrally located cell bodies. One neuron from each pair projects to the left side of the oviducts and the other the right side of the oviducts. These cells lie ipsilateral to the nerve root through which they exit. The neuropilar branches are intraganglionic and lie mainly in the ipsilateral neuropile, however one of the neurons from each side possesses a giant process, reaching 10 micron in diameter, which passes dorsally to the contralateral side of the ganglion. The other two neurons are dorsal unpaired median neurons, and have large cell bodies which lie at the posterior end of the ganglion. Lucifer yellow injection into these two dorsal unpaired median neurons reveals a single neurite passing anteriorly from the cell body which bifurcates into two bilaterally symmetrical processes which exit to the oviducts through both the left and right sternal roots. Similar to other identified dorsal unpaired median neurons, the cell bodies stain with neutral red and can support overshooting action potentials. The possibility that these two cells contain octopamine is discussed.     

Evidence from insect fibrillar muscle about the elementary contractile process

Bundles of myofibrils prepared from the dorsal longitudinal flight muscles of giant water bugs show oscillatory contractile activity in solutions of low ionic strength containing ATP and 10^-8-10^-7 M Ca²⁺. This is due to delay between changes of length and changes of tension under activating conditions. The peculiarities of insect fibrillar muscle which give rise to this behavior are (1) the high elasticity of relaxed myofibrils, (2) a smaller degree of Ca²⁺ activation of ATPase activity in unstretched myofibrils and extracted actomyosin, and (3) a direct effect of stretch on ATPase activity. It is shown that the cross-bridges of striated muscle are probably formed from the heads of three myosin molecules and that in insect fibrillar muscle the cycles of mechanochemical energy conversion in the cross-bridges can be synchronized by imposed changes of length. This material is more suitable than vertebrate striated muscle for a study of the nature of the elementary contractile process.     

Evidence for peripheral inhibition in an arachnid muscle

Summary Blectrophysiological investigations on the promotor tibiae muscle in the tarantulaDugesiella hentzi showed for the first time the existence of peripheral inhibition in an arachnid. The observed effects of GABA and picrotoxin are similar to those in crustacean and insect muscle.This investigation was supported by grants No. Ra 113/4 to Prof. W. Rathmayer and SFB 138 of the Deutsche Forschungsgemeinschaft.I thank Professors W. Rathmayer and E. Florey and Mr. C. Kleinsteuber for their support during various stages of this work.     

Fine structure of an octopaminergic neuron and its terminals

The large octopaminergic dorsal unpaired median neuron of the locust that innervates the extensor tibiae muscle, DUMETi, was examined electronmicroscopically. Its soma contains many Golgi complexes apparently making dense-core vesicles similar to those found in peripheral branches and terminals. There are also larger stores of the dense material in the soma, especially near the exit of the principal neurite, that are not in vesicular form. Since the neurons can be penetrated and stimulated by microelectrodes, they form favorable subjects for direct studies of the control of neurosecretion. Preterminal fine branches of the neuron were located in proximal outer bundles of muscle fibers into which they had been traced electrophysiologically. They contain numerous large dense-core vesicles arrayed in rows near microtubules. These fine branches have a thick layer of collagenous connective tissue between the axon and the muscle fiber. Final terminals have varicosities containing many vesicles, lying inside the outer layers of the sarcolemmal complex of muscle fibers. They do not form synaptic structures. Terminals of another DUM neuron, one that innervates the dorsal longitudinal flight muscles (DUMDL), were similar in detail to those of DUMETi. DUMETi swelled about 20-fold in cross-sectional area above a ligature, in a 12-hr period, indicating that there is an extensive centrifugal flow of material in it, and sprouted a branch.