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.     

Interaction between octopamine and proctolin on the oviducts of Locusta migratoria

The biogenic amine octopamine and the pentapeptide proctolin are two important neuroactive chemicals that control contraction of the oviducts of the African locust Locusta migratoria. The physiological responses and signal transduction pathways used by octopamine and proctolin have been well characterized in the locust oviducts and this therefore provides the opportunity to examine the interaction between these two pathways. Octopamine, via the intracellular messenger adenosine 3',5'-cyclic monophosphate (cyclic AMP), inhibits contraction of the oviducts, while proctolin, via the phosphoinositol pathway, stimulates contraction. We have examined the physiological response of the oviducts to combinations of octopamine and proctolin and also looked at how combinations of these affect one of the main intracellular mediators of the octopamine response, namely cyclic AMP. It was found that application of octopamine to the oviducts led to a dose-dependent reduction in tonus of the muscle and also a decrease in the amplitude and frequency of spontaneous phasic contractions. Octopamine-induced relaxation was enhanced in the presence of the phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine (IBMX). Octopamine was also able to inhibit proctolin-induced contractions of the oviducts in a dose-dependent manner. A 10(-9) M proctolin-induced contraction was inhibited by 83% in the presence of 10(-5) M octopamine, and was completely inhibited in the presence of 10(-5) M octopamine plus 5x10(-4) M IBMX. Octopamine led to a dose-dependent increase in cyclic AMP content as measured by radioimmunoassay. In the presence of 10(-9) M proctolin, this octopamine-induced increase in cyclic AMP was reduced by as much as 60%. Proctolin also caused a dose-dependent decrease in the cyclic AMP elevation produced by 5x10(-6) M octopamine. These results indicate that octopamine and proctolin can antagonize each other's physiological response when added in combination, and that proctolin is able to modulate the response of the oviducts to octopamine by influencing cyclic AMP levels.     

Effects of octopamine on miniature excitatory junction potentials from developing and adult moth muscle

Intracellular recordings of excitatory junction potentials (EJPs) and miniature EJPs (MEJPs) were made from the dorsal longitudinal muscle of Manduca sexta to determine the sites of action of octopamine. MEJPs increased in amplitude and frequency as the moth developed during the 3 days before eclosion. DL-Octopamine (5 X 10(-6) M) increased the amplitude of excitatory junction potentials in both immature moths (one day before eclosion) and adults. Octopamine (10(-5) M) also increased the amplitude and frequency of MEJPs from immature animals (one and two days before eclosion) but had the opposite effect on adults and pharate adults ready to eclose. Treatment with octopamine (10(-5) M) resulted in a decrease in input resistance and a hyperpolarization in both immature and adult muscle fibers. The results suggest that octopamine acts both presynaptically and postsynaptically but that the increase in the amplitude of the evoked response is due primarily to influences on presynaptic processes.     

Glutamate potential : differences from the excitatory junctional potential revealed by diltiazem and concanavalin A in crayfish neuromuscular junction.

1. The effect of diltiazem and concanavalin A (Con A) on the crayfish neuromuscular junction was investigated in order to compare the action of L-glutamate with that of the excitatory transmitter. 2. When diltiazem (0.3 nM) was added to the perfusion fluid, the iontophoretic glutamate potential was reduced to about half, whereas the amplitude of excitatory junctional potentials (EJPs) increased by about two times. 3. Dose-response curves of L-glutamate suggested that diltiazem acted in a non-competitive manner. The decrease in amplitude of the glutamate potential caused by diltiazem was not due to the acceleration of desensitization of the glutamate receptor. 4. The increase in amplitude of EJPs caused by diltiazem was due to the increase in membrane resistance. The quantal content and size of extracellular EJPs were not affected by diltiazem. 5. In normal saline, bath application of glutamate decreased the amplitude of both glutamate potentials and EJPs because of desensitization of the glutamate receptor. The decrease in amplitude of the glutamate potential was completely prevented by previous application of Con A (10(-6) M). On the other hand, Con A had no influence on the decrease in amplitude of EJPs. 6. Some possible explanations of these pharmacological differences between glutamate potentials and EJPs revealed by diltiazem and Con A are considered.     

Primary afferent responses of a crustacean mechanoreceptor are modulated by proctolin, octopamine, and serotonin

Modulation of sensory responses recorded intracellularly in primary sensory afferents of a crustacean proprioceptor is described. The neuropeptide proctolin enhances the sensory response, whereas the bioamines octopamine and serotonin depress it. The lobster oval organ of the second maxilla, a simple stretch receptor lacking centrifugal control, provides a useful model for studies on nonsynaptic modulation at peripheral sensory loci. Its three large afferents, X, Y, and Z, were prepared for intracellular recording and tested under five experimental conditions: (1) when fully rested, (2) when adapted to maintained stretch and firing tonically, (3) when showing reduced responses after habituation to repetitive stimulation, (4) not stretched but depolarized with current injections, (5) after TTX blockade. The results, taken together, indicate that conductances contributing to the overall amplitude of the receptor potential are major targets for modulators. Thus proctolin increased receptor potential amplitudes with consequent augmentation of spiking, whereas serotonin and octopamine depressed the receptor potentials, often to subthreshold levels with loss of spiking. Octopamine was a less potent agent than serotonin and failed to act upon fibers under TTX blockade. Fibers Y and Z consistently showed sensitivity to the modulators tested. The largest fiber, X, typically was resistant to proctolin, octopamine, and serotonin. Threshold concentrations of 10(-10)-10(-11) M determined in vitro are well below the circulating levels for serotonin and octopamine found in vivo. Proctolin, however, is usually not detectable in the hemolymph, and it is suggested that a significant site of proctolin release may be the oval organ itself.     

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)     

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.     

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.     

Crayfish motor nerve terminal's response to serotonin examined by intracellular microelectrode

Measurements of resting potential and action potential in presynaptic branches of the excitatory motor axon to the crayfish opener muscle were made with intracellular microelectrodes during application of serotonin (10(-9)-10(-3) M). A 5-min exposure to 10(-6) M serotonin produced enhancement of excitatory junction potentials (EJPs) lasting about 1 h. The membrane potential of the presynaptic terminal was depolarized by about 5 mV; the depolarization subsided within 1/2 h. Concomitant reduction in amplitude of the presynaptic action potential, not accompanied by spike broadening, was observed. The presynaptic depolarization, and the enhancement of EJPs, were dependent on the presence of extracellular sodium but not extracellular calcium. A possible mechanism for serotonin's effect involves initial entry of sodium into the nerve terminal, with consequent increased availability of intracellular calcium. The subsequent long-lasting phase of EJP enhancement may result from an additional effect on the metabolism of the nerve terminal.     

Evidence for a possible neurotransmitter/neuromodulator role of tyramine on the locust oviducts

Visualization of the tyraminergic innervation of the oviducts was demonstrated by immunohistochemistry, and the presence of tyramine was confirmed using high-performance liquid chromatography coupled to electrochemical detection. Oviducts incubated in high-potassium saline released tyramine in a calcium-dependent manner. Stimulation of the oviducal nerves also resulted in tyramine release, suggesting that tyramine might function as a neurotransmitter/neuromodulator at the locust oviducts. Tyramine decreased the basal tension, and also attenuated proctolin-induced contractions in a dose-dependent manner over a range of doses between 10(-7) and 10(-4) M. Low concentrations of tyramine attenuated forskolin-stimulated cyclic AMP levels in a dose-dependent manner. This effect was not blocked by yohimbine. High concentrations of tyramine increased basal cyclic AMP levels of locust oviducts in a dose-dependent manner; however, the increases in cyclic AMP were only evident at the highest concentrations tested, 5 x 10(-5) and 10(-4) M tyramine. The tyramine-induced increase in cyclic AMP shared a similar pharmacological profile with the octopamine-induced increase in cyclic AMP. Tyramine increased the amplitude of excitatory junction potentials at low concentrations while hyperpolarizing the membrane potential by 2-5 mV. A further increase in the amplitude of the excitatory junction potentials and the occurrence of an active response was seen upon washing tyramine from the preparation. These results suggest that tyramine can activate at least three different endogenous receptors on the locust oviducts a putative tyramine receptor at low concentrations, a different tyramine receptor to inhibit muscle contraction, and an octopamine receptor at high concentrations.     

Some effects of proctolin on the cardiac ganglion of the Maine Lobster, Homarus americanus (Milne Edwards)

The neuropeptide proctolin has excitatory effects on the isolated lobster cardiac ganglion. Selective application to the anterior cell body region produces a dose-dependent (10(-8)--10(-5) M) prolonged depolarization of large anterior cells as well as marked increases in burst frequency and/or duration. In ganglia which have been silenced with tetrodotoxin, proctolin application to anterior cells elicits long-lasting depolarizing responses which are accompanied by a 10-30% increase of the apparent membrane input resistance. Higher proctolin concentrations produce high-frequency trains of driver potentials. It is proposed that a proctolin like peptide may serve a neurohumoral role in the lobster cardiac ganglion and that the anterior motor neurons exhibit endogenous rhythmicity in its presence.     

The association of proctolin with a ventral abdominal muscle of Locusta migratoria

The association of proctolin with the external ventral protractor muscle of the VIIth abdominal segment (M234) of Locusta migratoria was investigated using immunohistochemistry and RP-HPLC in conjunction with the sensitive locust oviduct bioassay. Immunohistochemistry of whole-mount tissues revealed two proctolin-like immunoreactive axons in N2B_2b1 (the nerve branch which innervates M234) as well as immunoreactive processes and varicosities on the surface of M234. Immunogold staining of M234 demonstrated that the proctolin-like immunoreactivity was present in electron-dense granules in its motor terminals. A material indistinguishable from proctolin and with proctolin-like bioactivity co-eluted with authentic proctolin on two different RP-HPLC systems. Bath application of proctolin at concentrations greater than 10^-11 M resulted in a dose-dependent increase in the neurally-evoked fast twitch amplitude and duration of M234. Concentrations greater than 10^-9 M resulted in a dose-dependent increase in basal tonus of M234. These results indicate that proctolin, or a peptide very similar to proctolin, is present in the motor innervation of M234 and acts as a cotransmitter and/or neuromodulator at this typical fast skeletal muscle.Abbreviations M234 external ventral protractor muscle of the Vllth abdominal segment - RP-HPLC reverse-phase high pressure liquid chromatography - TFA trifluoroacetic acid     

Proctolin's role in neurally evoked contractions of the locust oviducts

The effects of proctolin (RYLPT) on neurally evoked contractions of locust oviduct muscle were studied to examine the role of proctolin as a cotransmitter. Increasing the number of stimuli in a burst (from one to 30 stimuli) resulted in an increase in amplitude of contraction of locust oviduct muscle. Proctolin was capable of increasing the amplitude of neurally evoked contractions at lower-stimulus regimes (one- and two-stimulus bursts) but did not do so at higher-stimulus regimes (five- and 10-stimulus bursts). The effects of proctolin were dose dependent within the one- and two-stimulus regimes, with thresholds at 10⁻⁹ M and maxima at 2.5 × 10⁻⁸ M. Addition of proctolin increased the basal tonus and size of a postcontraction relaxation of the oviduct muscle in a dose-dependent manner during all stimulus regimes. However, the effect of proctolin on basal tonus and the postcontraction relaxation was much less at the higher stimulus regimes. Previously, several proctolin analogues have been tested for their ability to antagonize proctolin-induced contractions of the oviduct muscle. Since proctolin is proposed to be a cotransmitter at this neuromuscular junction, one of these analogues, cycloproctolin, was used to antagonize proctolin's effects on neurally evoked contractions. In the presence of the antagonist, the maximum amplitude induced by application of proctolin was decreased by 22.7%, while the proctolin-induced increase in basal tonus was decreased by 45.8%. Finally, the maximum increase in the size of the postcontraction relaxation caused by proctolin was lowered by 32.0%. The results of the present study show that exogenously applied proctolin is an excitant of the oviduct muscle at lower, rather than higher, stimulus regimes, and this latter inaction may be due to the corelease of endogenous proctolin during increased neural stimulation. © 1997 John Wiley & Sons, Inc. J Neurobiol 33: 139–150, 1997     

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.     

Experimental studies upon a bundle of tonic fibres in the locust extensor tibialis muscle

The bundle of tonic fibres situated at the proximal end of the locust metathoracic extensor tibialis muscle is innervated by the dorsal unpaired median neurone (DUMETi) as well as by the slow excitatory (SETi)) and common inhibitor (CI) neurones. It is not innervated by the fast excitatory neurone (FETi).These fibres contract spontaneously and rhythmically. The myogenic rhythm can be modified by neural stimulation.Spontaneous slow depolarizing potentials resembling the pacemaker potentials of insect cardiac muscle were demonstrated in these fibres.The actions of glutamate on the tonic muscle fibres are not compatible with its being a specific excitatory transmitter. Glutamate can stimulate weak contractions of the muscle, but this action is inhibited when chloride ions are removed from the saline.10^?6 M Octapamine hyperpolarizes the tonic fibre membrane. Octopamine, GABA and glutamate all inhibit the myogenic contractions and reduce the force of the neurally evoked contractions.The tonic muscle is very responsive to proctolin. At 5 × 10^?11 M proctolin enhances the force and increases the frequency of myogenic contractions. At 10^?9 M it depolarizes the muscle membrane potential, and at that and higher concentrations it causes the muscle to contract. At 2 × 10^?7 M proctolin induces contractures which resemble those evoked by sustained high-frequency neural stimulation. Iontophoretic experiments show that proctolin receptors occur at localized sites on the tonic fibre membrane.     

Some pharmacological properties of neuromuscular transmission in the oviduct of the locust,Locusta migratoria

The effects of various pharmacological agents on neurally evoked contractions of the visceral muscles of the oviduct of Locusta migratoria have been examined. The pentapeptide, proctolin, at low concentrations (10^?11 M?10^?10 M), induced an increase in the amplitude of neurally evoked contractions and basal tonus, and induced the appearance and increased the frequency of myogenic contractions. Glutamate, at 10^?4 M, produced a small transient contraction which in some preparations was accompanied by a reduction in amplitude of neurally evoked contractions. Octopamine, at 10^?6 M, reduced the amplitude of neurally evoked contractions and also resulted in a relaxation of the muscles. The octopaminergic effects were inhibited by the α-aminergic antagonist phentolamine. Neurally evoked contractions were unaffected by dopamine, 5-HT or the acetylcholine receptor antagonists atropine and hexamethonium. Acetylcholine increased the amplitude of neurally evoked contractions, but only at the high concentration of 10^?3 M. The possible role of proctolin and glutamate as excitatory neuro-transmitters and the inhibitory action of octopamine is discussed.     

Effect of stretching on the action potentials of the human and rabbit ventricular myocardium

The effect of stretching from L0 to Lmax on the electrical activity was studied on human myocardial preparations from patients with heart disease and on strips of rabbit ventricular myocardium. Muscular deformation was shown to decrease the amplitude and velocity of depolarization in slow action potentials. The action potentials (AP) possessing a fast depolarization phase were not sensitive to physiological stretching. Antiarrhythmic drugs--ethmozin (2 X 10(-5) M) and ethacizin (2 X 10(-6) M)--caused a decrease in the rate of AP depolarization, thus increasing AP sensitivity to deformation. It is suggested that stretching under the action of ethmozin and ethacizin reduced cardiomyocyte excitability due to suppression of slow Ca-current.     

Neuromodulatory effects of acetylcholine and serotonin on the sensitivity of leech mechanoreceptors.

1. Each segmental ganglion of the leech Hirudo medicinalis contains 6 touch (T) cells, 4 pressure (P) cells and 4 nociceptive (N) cells. The receptive terminals of these cells innervate the skin in discrete areas. These cells are known to have extrasynaptic receptors. 2. We tested the effect of transmitter substances present in leech CNS on the sensitivity of T and P cells to mechanical stimuli. Substances tested included octopamine, FMRFamide, proctolin, substance P, glutamate, GABA, acetylcholine and serotonin. 3. Only acetylcholine and serotonin had consistent effects. Serotonin (1 x 10(-3) M) increased the number of action potentials of T cells elicited by a standard stimulus. Serotonin (1 x 10(-4) M) and acetylcholine (1 x 10(-3) M) increased the number and frequency of action potentials in P cells.     

Proctolin: A possible releasing factor in the corpus cardiacum/corpus allatum of the locust

The corpus cardiacum (CC) and corpus allatum (CA) of the locust, Locusta migratoria, contain intense proctolin-like immunoreactivity (PLI) within processes and varicosities. In contrast, in the cockroach, Diploptera punctata, although a similar staining pattern occurs within the CC, PLI appears absent within the CA. The possible role of proctolin as a releasing factor for adipokinetic hormone (AKH) and juvenile hormone (JH) was investigated in the locust. Proctolin caused a dose-dependent increase in AKH I release (determined by RP-HPLC) from the locust CC over a range of doses with threshold above 10(-8)M and maximal release at about 10(-7)M proctolin. Isolated glandular lobes of the CC released greater amounts of AKH I following treatment with proctolin and in these studies AKH II was also released. Confirmation of AKH I release was obtained by injecting perfusate from incubated CCs into locusts and measuring hemolymph lipid concentration. Perfusate from CC incubated in proctolin contained material with similar biological activity to AKH. Proctolin was also found to significantly increase the synthesis and release of JH from locust CA, with the increase being greatest from CAs that had a relatively low basal rate of JH biosynthesis (<35 pmol h(-1) per CA). In contrast, proctolin did not alter the synthesis and release of JH from the cockroach CA. These results suggest that proctolin may act as a releasing factor for AKHs and JH in the locust but does not act as a releasing factor for JH in the cockroach.     

Effects of octopamine and forskolin on excitatory junction potentials of developing and adult moth muscle

Intracellular recordings were made from the dorsal longitudinal muscle of Manduca sexta to determine the effects of development and octopamine on the excitatory junction potential (EJP) produced in response to electrical stimulation of the motor nerve. Observations were made on pharate moths during the last 3 days before eclosion and on adults. In saline, the highest values for EJP amplitude and maximum rate of rise and for resting membrane potential are reached on the nineteenth day of the pupal period, the day the animal ecloses; adult values are slightly lower. In animals of all ages tested, DL-octopamine (5 X 10(-6) M) increases EJP amplitude and maximum rate of rise. Increases in amplitude are greater in animals at stage day 17 and 18 than in animals at stage day 19 and adult. Octopamine has no effect on EJP rise time (onset to peak) or recovery time (peak of EJP to 70% recovery). Octopamine causes a hyperpolarization of about 6 mV. The results show that developmental changes in synapse properties are paralleled only in part by changes induced by octopamine. Both development and octopamine increase EJP amplitude and maximum rate of rise, and neither alter rise time. EJP recovery time changes with development but not in response to octopamine. Forskolin (10(-4) M) mimics the effects of octopamine on day 17 animals. EJP amplitude and maximum rate of rise are increased by forskolin, and rise time and recovery time are unaffected. Forskolin, like octopamine, causes a 6 mV hyperpolarization of the muscle fiber. These results suggest that octopaminergic modulation at the Manduca sexta dorsal longitudinal neuromuscular junction may be mediated by changes in intracellular levels of cyclic AMP.