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R Gillette

Publications and source records attributed to R Gillette.

At least 19 recordsLinked to original sources
PubMed1996

NADPH-diaphorase localization in the CNS and peripheral tissues of the predatory sea-slug Pleurobranchaea californica.

The distribution of putative nitric oxide synthase (NOS)-containing cells in the opisthobranch mollusc Pleurobranchaea californica was studied histochemically via NADPH-diaphorase (NADPH-d) reduction of Nitro Blue Tetrazolium (NTB). Whole mounts and cryostat sections were prepared from the central nervous system and peripheral organs, including the buccal muscles, esophagus, salivary glands, foot, mantle, and gills. NADPH-d-positive neurons were localized predominantly to the buccal and pedal ganglia as well as to distinct areas of the cerebropleural and visceral ganglia. A variety of identified neurons were positive for NADPH-diaphorase in various central ganglia, including the metacerebral cells of the cerebropleural ganglion, putative locomotor neurons of the pedal ganglia, and buccal motoneurons. Specific staining was observed only in somata of central neurons, whereas neuropil areas remained unstained. However, NADPH-d-reactive axons were dense in buccal ganglion nerves, whereas peripheral nerves and connectives of other ganglia had few or no NADPH-d positive terminals. In the periphery, NADPH-d activity was detected only in a few neurons of the rhinophore and tentacle ganglia. NADPH-d staining was marked in the salivary glands and gills, but there was no or very little staining in the esophagus, buccal mass, and foot. Histochemical stain production required the presence of both beta-NADPH and NBT; alpha-NADPH could not substitute for beta-NADPH. The inhibitor of NOS, 2,6-dichlorophenol-indophenol, at 10(-3) M, totally abolished NADPH-d-positive staining. The apparent high activity of central NADPH-d contrasts with much lower activity in the ganglia of the related gastropod Tritonia. These data suggest a role for nitric oxide as a signal molecule in the central nervous system of Pleurobranchaea.

Animals↗
PubMed1996

Nitric oxide synthase activity in the molluscan CNS.

Putative nitric oxide synthase (NOS) activity was assayed in molluscan CNS through histochemical localization of NADPH-diaphorase and through measurement of L-arginine/L-citrulline conversion. Several hundreds of NADPH-dependent diaphorase-positive neurons stained consistently darkly in the nervous system of the predatory opisthobranch Pleurobranchaea californica, whereas stained neurons were relatively sparse and/or light in the other opisthobranchs (Philine, Aplysia, Tritonia, Flabellina, Cadina, Armina, Coriphella, and Doriopsilla sp.) and cephalopods (Sepia and Rossia sp.). L-Arginine/L-citrulline conversion was beta-NADPH dependent, insensitive to removal of Ca2+, inhibited by the calmodulin blocker trifluoperazine, and inhibited by the competitive NOS inhibitor N-nitro-L-arginine methyl ester (L-NAME) but not D-NAME. Inhibitors of arginase [L-valine and (+)-S-2-amino-5-iodoacetamidopentanoic acid)] did not affect L-citrulline production in the CNS. NOS activity was largely associated with the particulate fraction and appeared to be a novel, constitutive Ca(2+)-independent isoform. Enzymatic conversion of L-arginine/L-citrulline in Pleurobranchaea and Aplysia CNS was 4.0 and 9.8%, respectively, of that of rat cerebellum, L-Citrulline formation in gill and muscle of Pleurobranchaea was not significant. The localization of relatively high NOS activity in neuron somata in the CNS of Pleurobranchaea is markedly different from the other opisthobranchs, all of which are grazers. Potentially, this is related to the animal's opportunistic predatory lifestyle.

Animals↗
PubMed1995

Cyclic AMP-gated sodium current in neurons of the pedal ganglion of Pleurobranchaea californica is activated by serotonin.

1. We studied the roles of adenosine 3',5'-cyclic monophosphate (cAMP) and cAMP-gated Na+ current (INa,cAMP) in the serotonin (5-HT)-induced excitation of putatively serotonergic "G" neurons of the pedal ganglion of Pleurobranchaea californica. Currents were recorded under voltage clamp during 5-HT application and iontophoretic intracellular cAMP injections. INa,cAMP responses to pulsed injections of cAMP were occluded by 5-HT-induced inward current (I5-HT). Occlusion was qualitatively and quantitatively similar to that observed during steady-state activation of INa,cAMP by tonic iontophoretic injection of cAMP. 2. Those neurons exhibiting occlusion of INa,cAMP during 5-HT application also exhibited depolarization-induced (Ca(2+)-dependent) inactivation of both INa,cAMP and I5-HT. The magnitudes of the inactivation to depolarizing pulses of I5-HT or INa,cAMP were similar. Recoveries from inactivation for I5-HT and INa,cAMP followed similar exponentially decaying time courses. 3. The decay rate of the INa,cAMP response is affected by phosphodiesterase inhibitors and can be taken as a sensitive measure of the rate of cAMP degradation. As background steady-state INa,cAMP was increased by larger tonic cAMP injections, the decay rate of super-imposed INa,cAMP responses to pulsed injections of cAMP was slowed as would be expected from saturation of endogenous phosphodiesterase activity. The decay of INa,cAMP responses to pulsed cAMP injections superimposed on I5-HT were similarly slowed, suggesting that 5-HT action is mediated specifically by cAMP. 4. The decay rate constants for INa,cAMP responses to pulsed injections of cAMP superimposed on I5-HT did not differ from those of INa,cAMP responses superimposed on equivalent, background steady-state INa,cAMP induced by tonic injection of cAMP.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1995

Neuronal elements that mediate escape swimming and suppress feeding behavior in the predatory sea slug Pleurobranchaea.

1. The white, bilaterally paired A1 interneurons of the cerebropleural ganglion of Pleurobranchaea californica fire rhythmic bursts of action potentials during escape swimming behavior. We studied the role of the A1s in swimming behavior and pattern generation in whole animal and isolated CNS preparations. 2. The escape swim is a cyclic sequence of dorsal and ventral flexions of the body. During the swim, A1 bursts precede and accompany the dorsal flexion phase of the cycle. Hyperpolarization of A1 to prevent spike activity interrupts swimming behavior in the whole animal and fictive swimming in the isolated CNS. Stimulated A1 activity was not observed to cause swimming in whole animals, and was only occasionally sufficient to trigger fictive swimming activity in the isolated CNS. 3. In quiescent whole animal preparations, stimulation of a single A1 normally causes a single dorsal flexion followed by body flexion to the side contralateral to the stimulated cell; characteristically, A1 spike activity stimulates feedback inhibition coinciding with the end of dorsal flexion and the onset of contralateral flexion. 4. A1 spike activity suppresses feeding behavior and causes proboscis retraction in whole animal preparations induced to feed. A1 activity also suppresses fictive feeding driven by stimulation of the critical phasic paracerebral neurons (PCps) of the motor network of feeding in the isolated CNS. Concomitantly, A1 spikes cause potent inhibition of the PCp interneurons. 5. The A1s are specifically excited by noxious mechanical and chemical stimuli, but are not affected by feeding stimuli or the occurrence of feeding behavior. 6. We conclude that the A1 neurons are elements of an escape swimming pattern generator, and that they are probably homologous to the similar C2 neurons of the nudibranch Tritonia diomedea. One of their functions outside of generating the swim pattern may be the suppression of feeding behavior in response to noxious stimulation. These observations provide a neural mechanism for the original observations of the dominance of escape swimming behavior over feeding.

Action Potentials↗
PubMed1995

From Polyplacophora to Cephalopoda: comparative analysis of nitric oxide signalling in mollusca.

The distribution of putative nitric oxide synthase (NOS)-containing cells has been analysed using NADPH-d histochemistry in the CNS and peripheral tissues in more than 2D ecologically and systematically different molluscan genera representing 3 main classes of the phylum MOLLUSCA: Polyplacophora (Lepidopleurus, Lepidozona, Katharina), Gastropoda (Littorina, Lymnaea, Aplexa, Physa, Planorbarius, Planorbis, Helisoma, Biomphalaria, Helix, Limax, Cepaea, Bulla, Aplysia, Phyllaplysia, Philine, Pleurobranchea, Tritonia, Armina, Flabellina, Cadlina) and Cephalopoda (Octopus, Sepia, Rossia, Loligo). Several species were used for more detailed immunohistochemical, biochemical, biophysical and physiological studies to further assay of NOS activity and to analyse functional roles of nitric oxide (NO) in these animals. The main conclusions of our comparative analysis and literature survey can be summarised as following: (i) There is strong evidence for the presence of NO-dependent signalling pathways in different molluscan species. (ii) We hypothesise that a general tendency in the evolution of NADPH-d-reactive cells in Mollusca is a migration of nitrergic function from periphery to the CNS. Also, different isoforms of NOS appear to be present in any one species. (iii) One of the main functional targets of NO signalling is the feeding system. However, there are obvious differences between predators (many labelled central motoneurons) and herbivorous species (many labelled peripheral putative sensory cells) as well as between land/freshwater and marine animals. Nevertheless, in all species tested NO-activated feeding-like motor patterns in the buccal ganglia. Additional functional and cellular targets for NO in molluscs are also considered. We briefly review neuromodulatory mechanisms of NO action and we consider molluscs as useful model systems for investigations of the roles of NO.

Animals↗
PubMed1994

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Delivery of Health Care↗
PubMed1993

Co-regulation of cAMP-activated Na+ current by Ca2+ in neurones of the mollusc Pleurobranchaea.

1. The cAMP-gated Na+ current (INa, cAMP) was studied in axotomized neurons of the pedal ganglion of the sea slug Pleurobranchaea. INa, cAMP responses were elicited by iontophoretic injection of cAMP and recorded in voltage clamp. 2. The current-voltage relation for INa, cAMP was flat between -90 and -50 mV, but declined steeply with depolarization from -50 to -30 mV. Depolarizing pulses also suppressed the INa, cAMP response, which recovered slowly over tens of seconds. 3. The inactivating effects of depolarization on the current were abolished both by blockade of Ca2+ current and intracellular injection of Ca2+ chelator. Thus, Ca2+ influx through voltage-dependent Ca2+ channels probably mediates inactivation of INa, cAMP within its normal physiological range of action. 4. Increasing intracellular cAMP levels antagonized the effects of Ca2+ influx on INa, cAMP. The mutual antagonism of the ions suggests that cAMP and Ca2+ act competitively in regulation of the INa, cAMP channel. 5. Measures of fractional inactivation of INa, cAMP provided evidence for the existence of an appreciable basal level of current, and hence cAMP, in the unstimulated neuron. Since INa, cAMP is a direct function of cAMP activity, measures of fractional inactivation permit quantification of cAMP levels in the living neuron. 6. Calcium inactivation of INa, cAMP completes a negative feedback loop that can contribute to endogenous burst activity. Over the burst cycle, depolarization and action potential activity driven by INa, cAMP would lead to Ca2+ influx, consequent inactivation of the inward current, and hyperpolarization. This mechanism of endogenous bursting resembles other in which the burst cycle has been found to be regulated by kinetics of Ca2+ influx and removal. However, INa, cAMP may vary in its Ca2+ sensitivity in different neurons and these variations may affect the functional expression of endogenous oscillatory activity.

Animals↗
PubMed1993

cAMP-activated Na+ current of molluscan neurons is resistant to kinase inhibitors and is gated by cAMP in the isolated patch.

The cAMP-dependent Na+ current (INa,cAMP) modulates excitability in many molluscan neurons. Rapid activation of INa,cAMP by cyclic nucleotide, its ion dependence, and its blockade by divalent cations resemble cyclic nucleotide-activated cation currents in vertebrate photoreceptors and olfactory receptors, where activation has been found to be independent of kinase activity. We tested the phosphorylation dependence of INa,cAMP in neurons of the feeding and locomotory networks of the predatory marine snail Pleurobranchaea. Identified neurons of pedal and buccal ganglia were axotomized for recording the INa,cAMP response to iontophoretic injection of cAMP under two-electrode voltage clamp. Intracellular injections of specific peptide inhibitor of protein kinase A had no blocking effects on activation of INa,cAMP by iontophoretic injection of cAMP. Inward single-channel currents were activated in excised inside-out patches during exposure to cAMP in salines without added ATP. Sodium was the major current carrying ion. Two distinct types of INa,cAMP channel activity were observed, where opening probability and open times differed, but conductance was similar, 36.7 pS. These observations suggest that INa,cAMP activation occurs by direct binding of cAMP to a regulatory site at the channel, rather than by phosphorylation.

Animals↗
PubMed1991

Kinetic analysis of cAMP-activated Na+ current in the molluscan neuron. A diffusion-reaction model.

cAMP-activated Na+ current (INa,cAMP) was studied in voltage-clamped neurons of the seaslug Pleurobranchaea californica. The current response to injected cAMP varied in both time course and amplitude as the tip of an intracellular injection electrode was moved from the periphery to the center of the neuron soma. The latency from injection to peak response was dependent on the amount of cAMP injected unless the electrode was centered within the cell. Decay of the INa,cAMP response was slowed by phosphodiesterase inhibition. These observations suggest that the kinetics of the INa,cAMP response are governed by cAMP diffusion and degradation. Phosphodiesterase inhibition induced a persistent inward current. At lower concentrations of inhibitor, INa,cAMP response amplitude increased as expected for decreased hydrolysis rate of injected cAMP. Higher inhibitor concentrations decreased INa,cAMP response amplitude, suggesting that inhibitor-induced increase in native cAMP increased basal INa,cAMP and thus caused partial saturation of the current. The Hill coefficient estimated from the plot of injected cAMP to INa,cAMP response amplitude was close to 1.0. An equation modeling INa,cAMP incorporated terms for diffusion and degradation. In it, the first-order rate constant of phosphodiesterase activity was taken as the rate constant of the exponential decay of the INa,cAMP response. The stoichiometry of INa,cAMP activation was inferred from the Hill coefficient as 1 cAMP/channel. The equation closely fitted the INa,cAMP response and simulated changes in the waveform of the response induced by phosphodiesterase inhibition. With modifications to accommodate asymmetric INa,cAMP activation, the equation also simulated effects of eccentric electrode position. The simple reaction-diffusion model of the kinetics of INa,cAMP may provide a useful conceptual framework within which to investigate the modulation of INa,cAMP by neuromodulators, intracellular regulatory factors, and pharmacological agents.

1-Methyl-3-isobutylxanthine↗
PubMed1990

pH-sensitive, Ca2+/calmodulin-dependent phosphorylation of unique protein in molluscan nervous system.

Intracellular pH and Ca2+ are prominent co-regulators of neuron excitability that act on ion channels. In looking for a possible mechanism of their action, we tested their combinatorial effect on the phosphorylation state of nervous system proteins. 32PO4 labelling in endogenous phosphorylation reactions of homogenates of nervous tissue of the sea-slug Pleurobranchaea showed steep pH sensitivity in protein migrating at a molecular mass of 108 kDa with pI 6.9-7.0 (pp108). Phosphorylation of pp108 was highest below reaction pH 7.0 and declined steeply as pH rose to 7.4 pp108 phosphorylation was Ca2+/calmodulin-dependent. pp108 constituted a significant part of the total protein (0.15%) and phosphoprotein (8.9%) of the nervous system. The specifically and uniquely combinatorial pH and Ca2+ sensitivity of the phosphorylation of pp108, and its relative abundance, suggest that it could mediate integrated actions of H+ and Ca2+ in the molluscan neuron.

Animals↗
PubMed1988

Enhancement of cyclic AMP-dependent sodium current by the convulsant drug pentylenetetrazol.

The convulsant drug pentylenetetrazol (PTZ) causes paroxysmal depolarizing shifts (PDS) and bursting in molluscan neurons. PDS has been found to be accompanied by increased levels of cyclic AMP (cAMP) and supported by persistent Na+ current. In neurons of the snail Lymnaea stagnalis the blocker of cAMP degradation isobutylmethylxanthine (IBMX) mimicks PTZ action. Na+ dependence of PTZ-induced inward shift in holding current in voltage-clamped cells supports the potential Na+ current origin of PDS. Intracellular cAMP injection elicits a transient Na+ current whose amplitude and duration are enhanced by both PTZ and IBMX. PTZ may cause PDS partly through slowing cAMP degradation, thus enhancing the cAMP-dependent Na+ current. PDS-generated bursts cause partial inactivation of the Na+ current, which may contribute towards burst termination.

1-Methyl-3-isobutylxanthine↗
PubMed1988

Cyclic AMP-stimulated sodium current in identified feeding neurons of Lymnaea stagnalis.

Iontophoretic injection of cAMP elicits a slow, transient inward current in identified buccal feeding motoneurons and in the giant cerebral interneuron of the snail, Lymnaea stagnalis. The current is voltage independent, and is abolished in the absence of extracellular Na+. Application of the phosphodiesterase inhibitor isobutylmethylxanthine (IBMX) causes a marked increase in both amplitude and duration of cAMP-stimulated inward current. The amplitude of the current is reduced following prolonged application of depolarizing pulses to the cell. However, generation of high-frequency bursts of action potentials lasting up to 20 s has no significant effect on the amplitude of the cAMP-induced current measured subsequently. Bath application of the cAMP analogue 8-chlorophenylthio-cAMP or of IBMX leads to enhanced bursting activity in buccal motoneurons. It is suggested that cAMP sensitivity in feeding motoneurons provides a mechanism for adjusting the cells' responsiveness to rhythmic synaptic inputs during the generation of feeding motor output.

1-Methyl-3-isobutylxanthine↗
PubMed1988

Regulation of cAMP-stimulated ion current by intracellular pH, Ca2+, and calmodulin blockers.

1. Iontophoretic injection of adenosine 3',5'-cyclic monophosphate (cAMP) into identified neurons elicited a slow transient Na+ current whose amplitude and duration were sensitive to altered intracellular pH (pHi), calmodulin blocking drugs, depolarization, and manipulations of internal and external Ca2+. 2. Intracellular acidification between resting pHi to several tenths of a pH unit increased the amplitude of the cAMP-stimulated current and prolonged its duration. 3. Intracellular alkalinization of similar magnitude also increased the amplitude and duration of the current response. The effects of alkalinization were somewhat labile. In cells alkalinized by NH4+-containing salines, washout of NH4+ with normal saline caused acidification and further enhanced the cAMP current response. The immediacy of the increase and the dual acid/basic sensitivity of the response suggest an accommodative process whereby the responsiveness of the cell to cAMP adapts to a maintained pHi. 4. The calmodulin blockers trifluoperazine and N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide increased the amplitude and duration of the current response. Phorbol ester activators of Ca2+/phospholipid-dependent kinase had no effect on the current. 5. Periods of depolarization preceding tests significantly reduced current response amplitude. This effect was dependent on saline Ca2+ and was blocked by Co2+. 6. Intracellular injection of the Ca2+ chelator ethylene glycol-bis(beta-aminoethyl ether)N,N,N',N',-tetraacetic acid also augmented the amplitude and duration of the current response. 7. The above effects are consistent with a possible common site of action on cAMP degradation. This interpretation is consistent with previous evidence for pH-sensitive and Ca2+/calmodulin-dependent cAMP phosphodiesterase activity in Pleurobranchaea nervous tissue.

Animals↗
PubMed1987

Calcium dependence of voltage sensitivity in adenosine 3',5'-cyclic phosphate-stimulated sodium current in Pleurobranchaea.

1. Ionophoretic injection of cyclic AMP into a voltage-clamped molluscan neurone caused a transient slow inward current (Isi) whose amplitude was enhanced by depolarization. Na+-replaced salines abolished the current, placing it with cyclic AMP-stimulated Na+ currents of other gastropod species. 2. Isi amplitude was suppressed by extracellular Ca2+. The amplitude increased up to 4-fold at holding potentials of -50 mV in nominally Ca2+-free saline. Ion substitutions showed that Ca2+ suppressed Isi more effectively than Mg2+, Co2+, Cd2+, Mn2+, Ba2+ or Sr2+. 3. Voltage sensitivity of Isi was abolished by low-Ca2+ salines, by the Ca2+ current blocker Co2+ and by substitution of Ba2+ or Sr2+ as Ca2+ channel current carriers. In such salines Isi showed no appreciable change in amplitude at holding potentials between -70 and -25 mV. 4. Intracellular injection of the Ca2+ chelator EGTA both augmented the amplitude of the current and its duration. EGTA injection failed to suppress the Ca2+-dependent voltage sensitivity of Isi. Intracellular injection of concentrated 3-N-(morpholino) propanesulphonic acid (MOPS) pH buffer to inhibit secondary, Ca2+-dependent intracellular acidification also failed to suppress the voltage sensitivity, as did injections of a mixed EGTA and MOPS solution. 5. While the data indicate a requirement for extracellular Ca2+ in conferring voltage sensitivity, they do not support a role for an intracellular action. An extracellular binding site for Ca2+ could mediate the voltage sensitivity, either by local depolarization-dependent changes in extracellular Ca2+ concentration or through direct voltage-sensitive block of the Isi channel.

Action Potentials↗
PubMed1985

pH sensitivity of calmodulin distribution in nervous tissue fractions.

Alkalinization of nervous system extracts of the mollusk, Pleurobranchaea, from pH 7.0 to 8.0 markedly increases the ratio of soluble to total calmodulin. This effect is independent of pH effects on free Ca2+ concentration and is pronounced at micromolar (near intracellular) levels of Ca2+. These data may relate to recent evidence that Ca2+/calmodulin-activated cyclic nucleotide phosphodiesterase mediates the effects of small changes in intracellular pH (0.1-0.2 units) on the electrical activity of neurons. Calmodulin redistribution could reflect altered availability to stimulate phosphodiesterase activity and supports a role for calmodulin in mediating effects of intracellular pH fluxes on cellular activity.

Animals↗
PubMed1984

Functional roles and circuitry in an inhibitory pathway to feeding command neurones in Pleurobranchaea.

The paracerebral neurones (PCNs) of the brain of Pleurobranchaea californica serve a command role in the initiation of feeding behaviour (Gillette, Kovac & Davis, 1978). The PCNs are synaptically excited by food stimuli applied to the oral veil of hungry, naive animals. In food avoidance-conditioned animals, the PCNs are inhibited by a barrage of inhibitory postsynaptic potentials concomitant with the suppression of feeding (Davis & Gillette, 1978). In this paper, an interneuronal pathway is described which causes inhibition of the PCNs and potentially mediates the effects of learning. The inhibitory pathway consists of three serially connected interneurones. One population, designated the Interneurone 1s (Int-1s), monosynaptically inhibits the PCNs. A second population, the Interneurone 2s (Int-2s), excites the Int-1 population. They also excite other neurones of the brain including the metacerebral giant neurones. A third population, the Interneurone 3s (Int-3s), monosynaptically excites the Interneurone 2 population. Dual intracellular recordings and current injection show that ipsilateral members of the Int-2 population are electrically coupled via a nonrectifying connection. Contralateral members of the Int-2 population are excitatorily coupled via a polysynaptic pathway. The Int-1 population is phasically active during the rhythmic motor activity that underlies feeding. In the isolated nervous system Int-1 activity is phase-locked with rhythmic PCN activity; Int-1 activity occurs maximally at the end of a PCN burst, during the retraction phase of the cycle. Int-2 activity also occurs during the retraction phase. During actual feeding in the whole animal preparation, the Int-2s are also phasically active; maximal excitation occurs during buccal mass retraction and maximal inhibition during protraction and the bite. Stimulated activity in a single Int-2 can entirely suppress the rhythmic motor activity of the feeding network evoked by electrical stimulation of the stomatogastric nerve. The suppressant effects of Int-2 activity must be mediated widely within the feeding network because the rhythmic motor output so driven is not dependent on PCN spiking. Application of an appetitive chemosensory stimulus to whole and semi-intact animal preparations initiated feeding and elicited excitation of the Int-1 and Int-2 populations. Noxious chemosensory stimuli, such as a dilute soap solution or ethanol, elicited oral veil withdrawal and inhibition of the Int-2s by multiple inhibitory postsynaptic potentials.(ABSTRACT TRUNCATED AT 400 WORDS)

Action Potentials↗