Neural changes after operant conditioning of the aerial respiratory behavior in Lymnaea stagnalis.
In this study, we demonstrate neural changes that occurred during operant conditioning of the aerial respiratory behavior of Lymnaea stagnalis. Aerial respiration in Lymnaea occurs at the water interface and is achieved by opening and closing movements of its respiratory orifice, the pneumostome. This behavior is controlled by a central pattern generator (CPG), the neurons of which, as well as the motoneurons innervating the pneumostome, have previously been identified and their synaptic connections well characterized. The respiratory behavior was operantly conditioned by applying a mechanical stimulus to the open pneumostome whenever the animal attempted to breathe. This negative reinforcement to the open pneumostome resulted in its immediate closure and a significant reduction in the overall respiratory activity. Electrophysiological recordings from the isolated CNSs after operant conditioning showed that the spontaneous patterned respiratory activity of the CPG neurons was significantly reduced. This included reduced spontaneous activity of the CPG interneuron involved in pneumostome opening (input 3 interneuron) and a reduced frequency of spontaneous tonic activity of the CPG interneuron [right pedal dorsal 1 (RPeD1)]. The ability to trigger the patterned respiratory activity by electrical stimulation of RPeD1 was also significantly reduced after operant conditioning. This study therefore demonstrates significant changes within a CPG that are associated with changes in a rhythmic homeostatic behavior after operant conditioning. (+info)
Electrophysiological and behavioral analysis of lip touch as a component of the food stimulus in the snail Lymnaea.
Electrophysiological and video recording methods were used to investigate the function of lip touch in feeding ingestion behavior of the pond snail Lymnaea stagnalis. Although this stimulus was used successfully as a conditioning stimulus (CS) in appetitive learning experiments, the detailed role of lip touch as a component of the sensory stimulus provided by food in unconditioned feeding behavior was never ascertained. Synaptic responses to lip touch in identified feeding motoneurons, central pattern generator interneurons, and modulatory interneurons were recorded by intracellular electrodes in a semi-intact preparation. We showed that touch evoked a complex but characteristic sequence of synaptic inputs on each neuron type. Touch never simply activated feeding cycles but provided different types of synaptic input, determined by the feeding phase in which the neuron was normally active in the rhythmic feeding cycle. The tactile stimulus evoked mainly inhibitory synaptic inputs in protraction-phase neurons and excitation in rasp-phase neurons. Swallow-phase neurons were also excited after some delay, suggesting that touch first reinforces the rasp then swallow phase. Video analysis of freely feeding animals demonstrated that during normal ingestion of a solid food flake the food is drawn across the lips throughout the rasp phase and swallow phase and therefore provides a tactile stimulus during both these retraction phases of the feeding cycle. The tactile component of the food stimulus is strongest during the rasp phase when the lips are actively pressed onto the substrate that is being moved across them by the radula. By using a semi-intact preparation we demonstrated that application of touch to the lips during the rasp phase of a sucrose-driven fictive feeding rhythm increases both the regularity and frequency of rasp-phase motoneuron firing compared with sucrose applied alone. (+info)
Dopamine activates two different receptors to produce variability in sign at an identified synapse.
Chemical synaptic transmission was investigated at a central synapse between identified neurons in the freshwater snail, Lymnaea stagnalis. The presynaptic neuron was the dopaminergic cell, Right Pedal Dorsal one (RPeD1). The postsynaptic neuron was Visceral Dorsal four (VD4). These neurons are components of the respiratory central pattern generator. The synapse from RPeD1 to VD4 showed variability of sign, i.e., it was either inhibitory (monophasic and hyperpolarizing), biphasic (depolarizing followed by hyperpolarizing phases), or undetectable. Both the inhibitory and biphasic synapse were eliminated by low Ca2+/high Mg2+ saline and maintained in high Ca2+/high Mg2+ saline, indicating that these two types of connections were chemical and monosynaptic. The latency of the inhibitory postsynaptic potential (IPSP) in high Ca2+/high Mg2+ saline was approximately 43 ms, whereas the biphasic postsynaptic potential (BPSP) had approximately 12-ms latency in either normal or high Ca2+/high Mg2+ saline. For a given preparation, when dopamine was pressured applied to the soma of VD4, it always elicited the same response as the synaptic input from RPeD1. Thus, for a VD4 neuron receiving an IPSP from RPeD1, pressure application of dopamine to the soma of VD4 produced an inhibitory response similar to the IPSP. The reversal potentials of the IPSP and the inhibitory dopamine response were both approximately -90 mV. For a VD4 neuron with a biphasic input from RPeD1, pressure-applied dopamine produced a biphasic response similar to the BPSP. The reversal potentials of the depolarizing phase of the BPSP and the biphasic dopamine response were both approximately -44 mV, whereas the reversal potentials for the hyperpolarizing phases were both approximately -90 mV. The hyperpolarizing but not the depolarizing phase of the BPSP and the biphasic dopamine response was blocked by the D-2 dopaminergic antagonist (+/-) sulpiride. Previously, our laboratory demonstrated that both IPSP and the inhibitory dopamine response are blocked by (+/-) sulpiride. Conversely, the depolarizing phase of both the BPSP and the biphasic dopamine response was blocked by the Cl- channel antagonist picrotoxin. Finally, both phases of the BPSP and the biphasic dopamine response were desensitized by continuous bath application of dopamine. These results indicate that the biphasic RPeD1 --> VD4 synapse is dopaminergic. Collectively, these data suggest that the variability in sign (inhibitory vs. biphasic) at the RPeD1 --> VD4 synapse is due to activation of two different dopamine receptors on the postsynaptic neuron VD4. This demonstrates that two populations of receptors can produce two different forms of transmission, i.e., the inhibitory and biphasic forms of the single RPeD1 --> VD4 synapse. (+info)
A molluscan peptide alpha-amidating enzyme precursor that generates five distinct enzymes.
Mechanisms underlying the specificity and efficiency of enzymes, which modify peptide messengers, especially with the variable requirements of synthesis in the neuronal secretory pathway, are poorly understood. Here, we examine the process of peptide alpha-amidation in individually identifiable Lymnaea neurons that synthesize multiple proproteins, yielding complex mixtures of structurally diverse peptide substrates. The alpha-amidation of these peptide substrates is efficiently controlled by a multifunctional Lymnaea peptidyl glycine alpha-amidating monooxygenase (LPAM), which contains four different copies of the rate-limiting Lymnaea peptidyl glycine alpha-hydroxylating monooxygenase (LPHM) and a single Lymnaea peptidyl alpha-hydroxyglycine alpha-amidating lyase. Endogenously, this zymogen is converted to yield a mixture of monofunctional isoenzymes. In vitro, each LPHM displays a unique combination of substrate affinity and reaction velocity, depending on the penultimate residue of the substrate. This suggests that the different isoenzymes are generated in order to efficiently amidate the many peptide substrates that are present in molluscan neurons. The cellular expression of the LPAM gene is restricted to neurons that synthesize amidated peptides, which underscores the critical importance of regulation of peptide alpha-amidation. (+info)
FMRFamide-activated Ca2+ channels in Lymnaea heart cells are modulated by "SEEPLY," a neuropeptide encoded on the same gene.
The cell-attached, patch-clamp technique was used to investigate the modulatory role of the neuropeptide SEQPDVDDYLRDVVLQSEEPLY ("SEEPLY") on FMRFamide-activated Ca2+ channels in isolated Lymnaea heart ventricular cells. Both SEEPLY and FMRFamide are encoded on the same neuropeptide gene and are coexpressed in a pair of excitatory motor neurons that innervate the heart. FMRFamide applied alone was capable of significantly increasing the P(open) time of a Ca2+ channel in isolated heart muscle cells. However, SEEPLY applied alone did not significantly alter the basal level of Ca2+ channel activity in the same cells. Repeated applications of FMRFamide (15 s every min) resulted in a progressive reduction in the number of Ca2+ channel openings and the overall P(open) time of the channel. The fifth successive 15-s application of FMRFamide failed to cause the Ca2+ channels to open in the majority of cells tested. When FMRFamide and SEEPLY were repeatedly applied together (2-min applications every 4 min) the FMRFamide-activated Ca2+ channels continued to respond after the fifth application of the two peptides. Indeed channel activity was seen to continue after repeated 2-min applications of FMRFamide and SEEPLY for as long as the patch lasted (+info)
Roles of G-protein beta gamma, arachidonic acid, and phosphorylation inconvergent activation of an S-like potassium conductance by dopamine, Ala-Pro-Gly-Trp-NH2, and Phe-Met-Arg-Phe-NH2.
Dopamine and the neuropeptides Ala-Pro-Gly-Trp-NH2 (APGWamide or APGWa) and Phe-Met-Arg-Phe-NH2 (FMRFamide or FMRFa) all activate an S-like potassium channel in the light green cells of the mollusc Lymnaea stagnalis, neuroendocrine cells that release insulin-related peptides. We studied the signaling pathways underlying the responses, the role of the G-protein betagamma subunit, and the interference by phosphorylation pathways. All responses are blocked by an inhibitor of arachidonic acid (AA) release, 4-bromophenacylbromide, and by inhibitors of lipoxygenases (nordihydroguaiaretic acid and AA-861) but not by indomethacin, a cyclooxygenase inhibitor. AA and phospholipase A2 (PLA2) induced currents with similar I-V characteristics and potassium selectivity as dopamine, APGWa, and FMRFa. PLA2 occluded the response to FMRFa. We conclude that convergence of the actions of dopamine, APGWa, and FMRFa onto the S-like channel occurs at or upstream of the level of AA and that formation of lipoxygenase metabolites of AA is necessary to activate the channel. Injection of a synthetic peptide, which interferes with G-protein betagamma subunits, inhibited the agonist-induced potassium current. This suggests that betagamma subunits mediate the response, possibly by directly coupling to a phospholipase. Finally, the responses to dopamine, APGWa, and FMRFa were inhibited by activation of PKA and PKC, suggesting that the responses are counteracted by PKA- and PKC-dependent phosphorylation. The PLA2-activated potassium current was inhibited by 8-chlorophenylthio-cAMP but not by 12-O-tetradecanoylphorbol 13-acetate (TPA). However, TPA did inhibit the potassium current induced by irreversible activation of the G-protein using GTP-gamma-S. Thus, it appears that PKA targets a site downstream of AA formation, e.g., the potassium channel, whereas PKC acts at the active G-protein or the phospholipase. (+info)
Measurement of calcium channel inactivation is dependent upon the test pulse potential.
We have developed two methods to measure Ca2+ channel inactivation in Lymnaea neurons-one method, based upon the conventional double-pulse protocol, uses currents during a moderately large depolarizing pulse, and the other uses tail currents after a very strong activating pulse. Both methods avoid contamination by proton currents and are unaffected by rundown of Ca2+ current. The magnitude of inactivation measured differs for the two methods; this difference arises because the measurement of inactivation is inherently dependent upon the test pulse voltage used to monitor the Ca2+ channel conductance. We discuss two models that can generate such test pulse dependence of inactivation measurements-a two-channel model and a two-open-state model. The first model accounts for this by assuming the existence of two types of Ca2+ channels, different proportions of which are activated by the different test pulses. The second model assumes only one Ca2+ channel type, with two closed and open states; in this model, the test pulse dependence is due to the differential activation of channels in the two closed states by the test pulses. Test pulse dependence of inactivation measurements of Ca2+ channels may be a general phenomenon that has been overlooked in previous studies. (+info)
The Northern Bolivian Altiplano: a region highly endemic for human fascioliasis.
The worldwide importance of human infection by Fasciola hepatica has been recognized in recent years. The endemic region between Lake Titicaca and the valley of La Paz, Bolivia, at 3800-4100 m altitude, presents the highest prevalences and intensities recorded. Large geographical studies involving Lymnaea truncatula snails (malacological, physico-chemical, and botanic studies of 59, 28 and 30 water bodies, respectively, inhabited by lymnaeids; environmental mean temperature studies covering a 40-year period), livestock (5491 cattle) and human coprological surveys (2723 subjects, 2521 of whom were school children) were conducted during 1991-97 to establish the boundaries and distributional characteristics of this endemic Northern Altiplano region. The endemic area covers part of the Los Andes, Ingavi, Omasuyos and Murillo provinces of the La Paz Department. The human endemic zone is stable, isolated and apparently fixed in its present outline, the boundaries being marked by geographical, climatic and soil-water chemical characteristics. The parasite distribution is irregular in the endemic area, the transmission foci being patchily distributed and linked to the presence of appropriate water bodies. Prevalences in school children are related to snail population distribution and extent. Altiplanic lymnaeids mainly inhabit permanent water bodies, which enables parasite transmission during the whole year. A confluence of several factors mitigates the negative effects of the high altitude. (+info)