Pharmacological properties of l-glutamate receptors associated with the crayfish hindgut.

Pharmacological agents were used to characterize glutamate receptors associated with crayfish hindgut. l-Glutamate reliably increased tonus in isolated hindguts of Procambarus clarkii and suppressed spontaneous hindgut contractions at concentrations of 10 micromol l(-1) or higher. Quisqualate and ibotenate mimicked the effects of L-glutamate. Experiments with strips and rings of hindgut tissue indicate that glutamate acts on both circular and longitudinal muscles. Hindgut contractions were not affected by (+/-)-alpha-amino-3-hydroxy-5-methyl-isoxazole-4-propionoic acid, N-methyl- d-aspartate, domoate or the metabotropic glutamate receptor agonist, (1S,3R)-1-amino-1-carboxycyclopentane-3-acetic acid. Picrotoxin, at 50 micromol l(-1), did not alter the ibotenate-induced reduction in contraction frequency, suggesting that this effect is not produced by inhibitory glutamate receptors. The glutamate-induced increase in tonus was antagonized by Joro spider toxin, JSTX-3. Thus, glutamate receptors associated with crayfish hindgut muscles are of the quisqualate type but are also sensitive to ibotenate. Elevating extracellular potassium concentration mimicked all of the effects of glutamate, suggesting that excessive depolarization may contribute to the suppression of contractions at high agonist concentrations.

Differential effects of neuropeptides on circular and longitudinal muscles of the crayfish hindgut.

Proctolin (Arg-Tyr-Leu-Pro-Thr-OH) and crayfish peptide "DF(2)" (Asp-Arg-Asn-Phe-Leu-Arg-Phe-NH(2)) enhance spontaneous contractions of isolated crayfish hindguts. Both peptides increase the frequency and amplitude of spontaneous, rapid contractions. Proctolin induces a slow contraction, which gives the appearance of an increase in overall tonus. DF(2) has no such effect. To determine whether the peptides affect both longitudinal and circular muscles, hindguts were cut into longitudinal strips and into rings, and contractions were recorded from each. The longitudinal strips generated only rapid contractions, and both peptides increased the frequency and amplitude of such contractions without significantly altering tonus. Rapid contractions were observed in only 1 of 14 preparations of rings. Proctolin induced slow contractions in the rings, and DF(2) had no such effect. The results indicate that neuropeptides have different effects on circular and longitudinal muscles of hindgut.

Effects of a hydroxy-cinnamoyl conjugate of spermidine on arthropod neuromuscular junctions.

N1-coumaroyl spermidine is structurally similar to acylpolyamines found in spider and wasp venoms, which are known to block arthropod glutamate receptors. N1-coumaroyl spermidine reduced the amplitude of excitatory postsynaptic potentials recorded in crayfish muscle. This effect was dose dependent, with an IC50 value of 70 micromol l(-1). N1-coumaroyl spermidine reversibly reduced the amplitude of potentials elicited by iontophoretic application of L-glutamate, indicating a direct effect on postsynaptic glutamate receptors. Neither 1 mmol l(-1) spermidine nor 1 mmol l(-1) coumaric acid altered excitatory postsynaptic potential amplitude, indicating that blockage requires the conjugated phenolic polyamine. N1-coumaroyl spermine, a slightly longer phenolic polyamine, reduced excitatory postsynaptic potential amplitude with approximately the same potency as N1-coumaroyl spermidine. Thus, potency of blockage does not appear to be affected in this experimental preparation by small changes in length of the polyamine. N1-coumaroyl spermidine also reduced excitatory postsynaptic potentials in muscles of the insect Drosophila. The ability of N1-coumaroyl spermidine to attenuate synaptic transmission at insect neuromuscular synapses lends support to the notion that plant-derived phenolic polyamines might serve as natural insecticides.

Protein kinase C is required for long-lasting synaptic enhancement by the neuropeptide DRNFLRFamide in crayfish.

The FMRFamide-related neuropeptide AspArgAsnPheLeuArgPhe-NH2 (DRNFLRFamide, DF2) induces a long-lasting enhancement of synaptic transmission at neuromuscular junctions on the crayfish deep abdominal extensor muscles. Here we investigated the function of protein kinase C (PKC) in this effect because PKC has been implied in the control of long-term synaptic modulation in other systems. The general kinase antagonist staurosporine reduced both the initial increase in excitatory postsynaptic potential (EPSP) amplitude and the duration of synaptic enhancement. Unlike staurosporine, the selective PKC inhibitors, chelerythrine and bisindolylmaleimide, augmented the initial EPSP increase. However, like staurosporine, they also reduced the duration of synaptic enhancement. The PKC activator, phorbol-12-myristate 13-acetate, induced a long-lasting synaptic enhancement that was blocked by chelerythrine. These results show that synaptic enhancement by DF2 is mediated by different intracellular signaling systems that act in temporal sequence. The initial increase in EPSP amplitudes is negatively regulated by PKC and involves another, staurosporine-sensitive, kinase; whereas, the maintenance of synaptic enhancement requires PKC.

A role for calcium/calmodulin-dependent protein kinase in mediating synaptic modulation by a neuropeptide.

The crayfish neuropeptide DRNFLRFamide (DF2) has previously been shown to increase the amplitude of excitatory post-synaptic potentials (EPSPs) in crayfish muscles by enhancing transmitter release from synaptic terminals [18]. This effect involves at least two types of kinase enzyme: one or more kinases which mediate the initial rise in EPSP amplitude, and protein kinase C (PKC) which prolongs the elevation in EPSP amplitude for several minutes [6,7]. The present investigation was aimed at identifying kinases that participate in the initial response. KN-62, an inhibitor of Ca2+/calmodulin-dependent protein kinase, delayed the rise in EPSP amplitude induced by DF2. The maximal response to the peptide occurred in about 40 min when KN-62 was present and in 15 min when the inhibitor was absent. KN-62 did not significantly alter EPSP amplitude by itself. KN-04, a structural analog of KN-62 which does not block Ca2+/CaM kinase activity, did not alter EPSP amplitude or the response to the neuropeptide. These data strongly suggest that Ca2+/CaM kinase participates in the initial increase in transmitter release.

Temperature dependence of synaptic modulation by a FMRFamide-related neuropeptide in crayfish.

The present study examined the temperature dependence of synaptic transmission and peptidergic modulation of chemical synapses on the phasic abdominal extensor muscles of crayfish. Decreasing the temperature from 25 degrees C to 5 degrees C in saline, decreased the EPSP amplitude by 88% and increased the EPSP half-decay time four-fold. The putative neurohormone DRNFLRFamide (DF2) increased EPSP amplitudes, but was more effective at 7-9 degrees C than at 15-17 degrees C. DF2 might play a hormonal role in counteracting low transmitter release at low temperature.

Isolation of two FMRFamide-related peptides from crayfish pericardial organs.

Pericardial organs of the crayfish, Procambarus clarkii, were removed, and material from them was extracted and fractionated using two sequential reverse-phase HPLC columns. Fractions were analysed using a radioimmunoassay (RIA) specific for the C-terminal peptide sequence -Arg-Phe-NH2. The peak immunoreactive fraction from the second column appeared to contain two peptides with the sequences DRNFLRFamide and NRNFLRFamide, based on microsequencing analysis and on the absolute requirement for the amide in the RIA. These two peptides, respectively, have been given the names NF1 and DF2 due to similarity with previously identified lobster peptides F1 and F2. NF1 and DF2 are cardioexcitatory and augment synaptic transmission at a neuromuscular synapse, and evidence is presented for the calcium-dependent release of these peptides from the pericardial organs.

Long-term adaptation of crayfish neurons depends on the frequency and number of impulses.

Increasing the impulse activity of crustacean neurons for a few days causes long-lasting changes in transmitter release, which are termed 'long-term adaptation' (LTA) in previous studies. Both the amount of transmitter released at the beginning of a stimulus train, and synaptic fatigue during repetitive stimulation, are reduced. The present study examines the dependence of these synaptic changes on the frequency and number of impulses used to elicit LTA. Fatigue resistance develops consistently when crayfish phasic motor neurons are stimulated for 3 days with as few as 9,000 impulses per day, and occurs in response either to low frequency stimulation (0.2 or 0.5 Hz), or to stimulation in short bursts at a moderate average frequency (2.5 Hz). In contrast, the reduction in initial transmitter release does not appear consistently when the frequency and number of impulses are both low (9,000 impulses per day delivered at 0.2 Hz), but does occur at the moderate stimulus frequency (2.5 Hz) and when a larger number of impulses (18,000) are delivered at a low frequency (0.5 Hz). The data suggest that the two changes in synaptic transmission that comprise LTA have different stimulus requirements.

Modulation of Crayfish Hearts by FMRFamide-related Peptides.

The present study examines effects of FMRFamide-related peptides (FaRPs) on crayfish heart. Lobster peptides F1 (TNRNFLRFamide) and F2 (SDRNFLRFamide) increase the rate and amplitude of heart beat in hearts isolated from Procambarus clarkii. Thresholds for these effects were between 10-10 and 10-9 M for F2 and between 10-9 and 10-8 M for F1. FMRFamide and FLRFamide elicited similar cardioexcitatory effects, but at thresholds of approximately 10-7 M. Thus, the aminoterminal extensions "TNRN" and "SDRN" enhance the excitatory actions of FMRFamide and FLRFamide. SchistoFLRFamide (PDVDHVFLRFamide) and leucomyosuppressin (pQDVDHVFLRFamide) markedly decrease the rate of cardiac contractions at 10-9 to 10-8 M and can suppress the cardiac rhythm for one minute or more at 10-7 M. The amino-terminal extensions of these two peptides, therefore, are necessary for inhibition of heart rate. Both of these peptides cause an initial reduction in contraction amplitude, but contractions subsequently increase in the presence of schistoFLRFamide. Thus, crayfish hearts are sensitive to several FMRFamide-related peptides, but the sites and mechanisms of action remain to be determined.

FMRFamide-like immunoreactivity in the crayfish nervous system.

FMRFamide-like immunoreactivity (FLI) was detected in the nervous system of the crayfish Procambarus clarkii using an antiserum that recognizes extended RFamide peptides. Immunocytochemistry revealed FLI in neuronal somata, axons and varicose processes within the central nervous system. In the periphery, plexuses of immunoreactive varicosities were present in the pericardial organs (POs), in thoracic roots and on the hindgut. The hindgut plexus arose from 3-5 axons leaving the sixth abdominal ganglion (A6) via the intestinal nerve. The presence of FLI in these locations was confirmed by radioimmunoassay. In contrast, no FLI was detected in motor axons innervating exoskeletal muscles of the abdomen. The POs contained by far the largest amount of FLI of all tissues examined. The immunoreactive material was partially characterized by extraction and separation on two consecutive reversed-phase high performance liquid chromatography (RP-HPLC) columns. The largest amount of immunoreactivity on the second column co-eluted with a synthetic peptide, SDRNFLRFamide (F2), previously identified as one of two or more FMRFamide-related peptides contained in lobster POs. The immunoreactive fractions and peptide F2 elicited similar effects on isolated crayfish hearts; all increased the rate and amplitude of spontaneous cardiac contractions. As with the immunoreactivity, the highest level of bioactivity was contained in the fraction that co-eluted with F2. The results suggest that FMRFamide-related peptides act as neurohormones in crayfish and are likely to play roles in controlling circulation and defecation.

Pericardial peptides enhance synaptic transmission and tension in phasic extensor muscles of crayfish.

Two identified peptides, which are structurally related to FMRF-NH2 and are known to be associated with lobster pericardial organs, increase nerve-evoked tension and excitatory postsynaptic potentials (EPSPs) recorded from crayfish deep abdominal extensor muscles. At low stimulus frequencies, which produce marked depression of muscle twitches with successive stimuli, the peptides quickly and reversibly restore tension. Increased quantal content of transmitter release, rather than changes in postsynaptic input resistance, accounted for most of the increase in EPSP amplitude. The results support earlier suggestions that these two peptides may act as circulating neurohormones and provide the first evidence for peptidergic modulation of a phasic neuromuscular system in a crustacean.

Long-term adaptation in lobster motor neurons and compensation of transmitter release by synergistic inputs.

Earlier studies with crayfish have shown that chronic increases in neural activity, by electrical stimulation, cause a long-lasting reduction in the amount of transmitter released at low stimulus frequencies or at the beginning of a stimulus train. When such chronic stimulation is applied to phasic extensor motor neurons of the lobster abdomen, a similar change in transmitter release is apparent, as indicated by a decrease in excitatory postsynaptic potential (EPSP) size at 0.1 Hz. However, the EPSPs from unstimulated axons which innervate the same target muscle from a different nerve increase in size. Thus, activity-dependent reduction in transmitter release at one set of synapses appears to be compensated for by increased synaptic efficacy from less active synergistic inputs. The mechanism of such compensation is not known.

Formation, maintenance, and functional uncoupling of connections between identified Helisoma neurons in situ.

Previous work with identified Helisoma neurons has characterized an array of neuroplastic responses to axotomy that include the generation of new neuritic outgrowth, the reinnervation of target organs, and the formation of new electrical synapses. These responses are not random, but rather occur in a precise, predictable manner under a variety of culture conditions. The present investigation demonstrates that specific identified neurons display similar neuroplastic "behavior" within the living animal. In response to in situ nerve crushes, neurons B4 and B5 generate new neuritic outgrowth, neuron B4 functionally reinnervates the salivary glands, and new electrical synapses form between the left and right neurons B5. The in situ paradigm employed in the present experiments made it possible to examine responses to axotomy over longer periods than in earlier studies with organ cultures. New B5R-B5L connections, previously found to be stable over the short term in culture, gradually decreased in strength in situ, and the cells effectively uncoupled by 8 weeks after axotomy. This uncoupling did not depend upon target reinnervation and occurred in the continued presence of neurites in the buccal commissure. It is suggested that the stability of new connections is related to whether the connection previously existed in the unperturbed nervous system. The similarities between the ability of identified neurons to grow and to form synaptic connections in situ and in culture suggests that neurons are endowed with a specific program of regenerative responses that can be expressed reliably in a wide variety of environmental conditions.

Modulatory effects of proctolin on a crab ventilatory muscle.

Proctolin enhances nerve-evoked, phasic contractions of a selected respiratory muscle of the shore crab, Carcinus maenas, but has no effect on muscle tonus. Proctolin also increases the work and power output of this muscle. These effects are functionally appropriate in view of previous reports that proctolin stimulates the ventilatory rhythm. They also suggest that proctolin exerts coordinated modulatory control at the central and peripheral levels of the gill ventilatory system. In contrast, serotonin, dopamine and octopamine have no effect on this muscle.