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R Gillette

Publications and source records attributed to R Gillette.

At least 19 recordsLinked to original sources
PubMed1999

Central pattern generator for escape swimming in the notaspid sea slug Pleurobranchaea californica.

Escape swimming in the notaspid opisthobranch Pleurobranchaea is an episode of alternating dorsal and ventral body flexions that overrides all other behaviors. We have explored the structure of the central pattern generator (CPG) in the cerebropleural ganglion as part of a study of neural network interactions underlying decision making in normal behavior. The CPG comprises at least eight bilaterally paired interneurons, each of which contributes and is phase-locked to the swim rhythm. Dorsal flexion is mediated by hemiganglion ensembles of four serotonin-immunoreactive neurons, the As1, As2, As3, and As4, and an electrically coupled pair, the A1 and A10 cells. When stimulated, A10 commands fictive swimming in the isolated CNS and actual swimming behavior in whole animals. As1-4 provide prolonged, neuromodulatory excitation enhancing dorsal flexion bursts and swim cycle number. Ventral flexion is mediated by the A3 cell and a ventral swim interneuron, IVS, the soma of which is yet unlocated. Initiation of a swim episode begins with persistent firing in A10, followed by recruitment of As1-4 and A1 into dorsal flexion. Recurrent excitation within the As1-4 ensemble and with A1/A10 may reinforce coactivity. Synchrony among swim interneuron partners and bilateral coordination is promoted by electrical coupling among the A1/A10 and As4 pairs, and among unilateral As2-4, and reciprocal chemical excitation between contralateral As1-4 groups. The switch from dorsal to ventral flexion coincides with delayed recruitment of A3, which is coupled electrically to A1, and with recurrent inhibition from A3/IVS to A1/A10. The alternating phase relation may be reinforced by reciprocal inhibition between As1-4 and IVS. Pleurobranchaea's swim resembles that of the nudibranch Tritonia; we find that the CPGs are similar in many details, suggesting that the behavior and network are primitive characters derived from a common pleurobranchid ancestor.

Action Potentials↗
PubMed1999

Single-cell analyses of nitrergic neurons in simple nervous systems.

Understanding the role of the gaseous messenger nitric oxide (NO) in the nervous system is complicated by the heterogeneity of its nerve cells; analyses carried out at the single cell level are therefore important, if not critical. Some invertebrate preparations, most especially those from the gastropod molluscs, provide large, hardy and identified neurons that are useful both for the development of analytical methodologies and for cellular analyses of NO metabolism and its actions. Recent modifications of capillary electrophoresis (CE) allow the use of a small fraction of an individual neuron to perform direct, quantitative and simultaneous assays of the major metabolites of the NO-citrulline cycle and associated biochemical pathways. These chemical species include the products of NO oxidation (NO2-/NO3-), l-arginine, l-citrulline, l-ornithine, l-argininosuccinate, as well as selected NO synthase inhibitors and cofactors such as NADPH, biopterin, FMN and FAD. Diverse cotransmitters can also be identified in the same nitrergic neuron. The sensitivity of CE methods is in the femtomole to attomole range, depending on the species analysed and on the specific detector used. CE analysis can be combined with prior in vivo electrophysiological and pharmacological manipulations and measurements to yield multiple physiological and biochemical values from single cells. The methodologies and instrumentation developed and tested using the convenient molluscan cell model can be adapted to the smaller and more delicate neurons of other invertebrates and chordates.

Animals↗
PubMed1998

Non-enzymatic production of nitric oxide (NO) from NO synthase inhibitors.

The gaseous signal molecule, nitric oxide (NO*), is generated enzymatically by NO synthase (NOS) from L-arginine. Overproduction of NO contributes to cell and tissue damage as sequelae of infection and stroke. Strategies to suppress NO synthesis rely heavily on guanidino-substituted L-arginine analogs (L-NAME, L-NA, L-NMMA, L-NIO) as competitive inhibitors of NOS, which are often used in high doses to compete with millimolar concentrations of intracellular arginine. We show that these analogs are also a source for non-enzymatically produced NO. Enzyme-independent NO release occurs in the presence of NADPH, glutathione, L-cysteine, dithiothreitol and ascorbate. This non-enzymatic synthesis of NO can produce potentially toxic, micromolar concentrations of NO and can oppose the effects of NOS inhibition. NO production driven by NOS inhibitors was demonstrated ex vivo in the central nervous and peripheral tissues of gastropod molluscs Aplysia and Pleurobranchaea using electron paramagnetic resonance and spin-trapping techniques. These results have important implications for therapeutic regulation of NO homeostasis.

Animals↗
PubMed1998

Serotonin immunoreactivity in the central nervous system of the marine molluscs Pleurobranchaea californica and Tritonia diomedea.

The central nervous systems of the marine molluscs Pleurobranchaea californica (Opisthobranchia: Notaspidea) and Tritonia diomedea (Opisthobranchia: Nudibranchia) were examined for serotonin-immunoreactive (5-HT-IR) neurons and processes. Bilaterally paired clusters of 5-HT-IR neuron somata were distributed similarly in ganglia of the two species. In the cerebropleural ganglion complex, these were the metacerebral giant neurons (both species), a dorsal anterior cluster (Pleurobranchaea only), a dorsal medial cluster including identified neurons of the escape swimming network (both species), and a dorsal lateral cluster in the cerebropleural ganglion (Pleurobranchaea only). A ventral anterior cluster (both species) adjoined the metacerebral giant somata at the anterior ganglion edge. Pedal ganglia had the greatest number of 5-HT-IR somata, the majority located near the roots of the pedal commissure in both species. Most 5-HT-IR neurons were on the dorsal surface of the pedal ganglia in Pleurobranchaea and were ventral in Tritonia. Neither the buccal ganglion of both species nor the visceral ganglion of Pleurobranchaea had 5-HT-IR somata. Afew asymmetrical 5-HT-IR somata were found in cerebropleural and pedal ganglia in both species, always on the left side. The clustering of 5-HT-IR neurons, their diverse axon pathways, and the known physiologic properties of their identified members are consistent with a loosely organized arousal system of serotonergic neurons whose components can be generally or differentially active in expression of diverse behaviors.

Animals↗
PubMed1998

Capillary electrophoresis analysis of nitric oxide synthase related metabolites in single identified neurons.

Intracellular concentrations of L-citrulline (Cit) and its metabolites are related to nitric oxide synthase (NOS) activity, an enzyme producing the intercellular messenger NO in animal tissues including the nervous system. A capillary electrophoresis system using laser-induced fluorescence detection is described, and methods are developed to monitor the levels of L-arginine (Arg), Cit, and related molecules in identified neurons of the marine slugs, Pleurobranchaea californica and Aplysia californica. The limits of detection for Arg, Cit, L-arginino-succinate, L-ornithine, and L-arginine phosphate range from 50 amol to 17 fmol (5 nM to 17 microM in the neurons under study); these detection limits are significantly lower than actual intracellular levels of the metabolites, allowing the direct assay of single cells. The levels of NOS metabolites in individual neurons varied form 6 (Arg) and 4 mM (Cit) in putative NOS-containing neurons down to < 1 microM (undetectable) levels in many putative NOS-negative cells. The Arg/Cit ratio is independent of cell volume, correlates with NADPH-diaphorase staining, and appears to be a characteristic parameter for the presence of NOS activity in identified neurons.

Animals↗
PubMed1998

Single neuron analysis by capillary electrophoresis with fluorescence spectroscopy.

A technique to identify and quantitate simultaneously more than 30 compounds in individual neurons is described. The method uses nanoliter volume sampling, capillary electrophoresis separation, and wavelength-resolved native fluorescence detection. Limits of detection (LODs) range from the low attomole to the femtomole range, with 5-hydroxytryptamine (or serotonin [5-HT]) LODs being approximately 20 attomoles. Although the cellular sample matrix is chemically complex, the combination of electrophoretic migration time and fluorescence spectral information allows positive identification of aromatic monoamines, aromatic amino acids and peptides containing them, flavins, adenosine- and guanosine-nucleotide analogs, and other fluorescent compounds. Individual identified neurons from Aplysia californica and Pleurobranchaea californica are used to demonstrate the applicability and figures of merit of this technique.

Animals↗
PubMed1997

Serotonin-immunoreactivity in peripheral tissues of the opisthobranch molluscs Pleurobranchaea californica and Tritonia diomedea.

The distribution of serotonin (5-HT)-immunoreactive elements in peripheral organs of the sea-slugs Pleurobranchaea californica and Tritonia diomedea was studied in cryostat sections. For Pleurobranchaea, 5-HT-immunoreactive (5-HT-IR) neuron cell bodies were found only in the central nervous system (CNS); 5-HT-IR cell bodies were not observed in foot, tentacles, rhinophores, oral veil, mouth, buccal mass, esophagus, gills, salivary glands, skin, reproductive system, and acidic glands, nor in peripheral tentacle and rhinophore ganglia. However, 5-HT-IR neuronal processes were widely distributed in these structures and the patterns of 5-HT-IR elements were characteristic for each particular peripheral tissue. 5-HT-IR elements were most dense in the sole of the foot and the reproductive system, followed by rhinophores, tentacles, oral veil, mouth, buccal mass, and esophagus. The sensory epithelium of rhinophores, tentacles, and mouth showed a highly structured glomerular organization of 5-HT-IR fibers, suggesting a role for 5-HT in sensory signaling. A much lower density of 5-HT-IR innervation was observed in gills, skin, salivary, and acidic glands. 5-HT-IR was observed in neuropil of tentacle and rhinophore ganglia with many transverse 5-HT-IR axons running to peripheral sensory areas. The distribution of 5-HT-IR elements in Tritonia was similar to that of Pleurobranchaea. A significant suggestion of the data is that central serotonergic neurons may modulate afferent pathways from sensory epithelia at the periphery.

Animals↗
PubMed1997

Nitrite and nitrate levels in individual molluscan neurons: single-cell capillary electrophoresis analysis.

Cell and tissue concentrations of NO2- and NO3- are important indicators of nitric oxide synthase activity and crucial in the regulation of many metabolic functions, as well as in nonenzymatic nitric oxide release. We adapted the capillary electrophoresis technique to quantify NO2- and NO3- levels in single identified buccal neurons and ganglia in the opisthobranch mollusc Pleurobranchaea californica, a model system for the study of the chemistry of neuron function. Neurons were injected into a 75-microm separation capillary and the NO2- and NO3- were separated electrophoretically from other anions and detected by direct ultraviolet absorbance. The limits of detection for NO2- and NO3- were <200 fmol (<4 microM in the neurons under study). The NO2- and NO3- levels in individual neurons varied from 2 mM (NO2-) and 12 mM (NO3-) in neurons histochemically positive for NADPH-diaphorase activity down to undetectable levels in many NADPH-diaphorase-negative cells. These results affirm the correspondence of histochemical NADPH-diaphorase activity and nitric oxide synthase in molluscan neurons. NO2- was not detected in whole ganglion homogenates or in hemolymph, whereas hemolymph NO3- averaged 1.8 +/- 0.2 x 10(-3) M. Hemolymph NO3- in Pleurobranchaea was appreciably higher than values measured for the freshwater pulmonate Lymnaea stagnalis (3.2 +/- 0.2 x 10(-5) M) and for another opisthobranch, Aplysia californica (3.6 +/- 0.7 x 10(-4) M). Capillary electrophoresis methods provide utility and convenience for monitoring NO2-/NO3- levels in single cells and small amounts of tissue.

Animals↗
PubMed1997

Cyclic AMP levels, adenylyl cyclase activity, and their stimulation by serotonin quantified in intact neurons.

In molluscan central neurons that express cAMP-gated Na+ current (INa,cAMP), estimates of the cAMP binding affinity of the channels have suggested that effective native intracellular cAMP concentrations should be much higher than characteristic of most cells. Using neurons of the marine opisthobranch snail Pleurobranchaea californica, we applied theory and conventional voltage clamp techniques to use INa,cAMP to report basal levels of endogenous cAMP and adenylyl cyclase, and their stimulation by serotonin. Measurements were calibrated to iontophoretic cAMP injection currents to enable expression of the data in molar terms. In 30 neurons, serotonin stimulated on average a 23-fold increase in submembrane [cAMP], effected largely by an 18-fold increase in adenylyl cyclase activity. Serotonin stimulation of adenylyl cyclase and [cAMP] was inversely proportional to cells' resting adenylyl cyclase activity. Average cAMP concentration at the membrane rose from 3.6 to 27.6 microM, levels consistent with the expected cAMP dissociation constants of the INa,cAMP channels. These measures confirm the functional character of INa,cAMP in the context of high levels of native cAMP. Methods similar to those employed here might be used to establish critical characters of cyclic nucleotide metabolism in the many cells of invertebrates and vertebrates that are being found to express ion currents gated by direct binding of cyclic nucleotides.

Adenylyl Cyclases↗
PubMed1997

Fast and slow activation kinetics of voltage-gated sodium channels in molluscan neurons.

Whole cell patch-clamp recordings of Na current (I(Na)) were made under identical experimental conditions from isolated neurons from cephalopod (Loligo, Octopus) and gastropod (Aplysia, Pleurobranchaea, Doriopsilla) species to compare properties of activation gating. Voltage dependence of peak Na conductance (gNa) is very similar in all cases, but activation kinetics in the gastropod neurons studied are markedly slower. Kinetic differences are very pronounced only over the voltage range spanned by the gNa-voltage relation. At positive and negative extremes of voltage, activation and deactivation kinetics of I(Na) are practically indistinguishable in all species studied. Voltage-dependent rate constants underlying activation of the slow type of Na channel found in gastropods thus appear to be much more voltage dependent than are the equivalent rates in the universally fast type of channel that predominates in cephalopods. Voltage dependence of inactivation kinetics shows a similar pattern and is representative of activation kinetics for the two types of Na channels. Neurons with fast Na channels can thus make much more rapid adjustments in the number of open Na channels at physiologically relevant voltages than would be possible with only slow Na channels. This capability appears to be an adaptation that is highly evolved in cephalopods, which are well known for their high-speed swimming behaviors. Similarities in slow and fast Na channel subtypes in molluscan and mammalian neurons are discussed.

Action Potentials↗
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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