Rhanor Gillette

Circadian rhythm of firing rate recorded from single cells in the rat suprachiasmatic brain slice

Abstract Brain slices, containing the suprachiasmatic nuclei, of rats were removed at various times of the fay and the firing rates of single cells were recorded. The firing rates were found to maintain a circadian rhythm in temporal accord with the light/dark cycle of the donor animal, and were highest during the lights on phase.

Interfering with Nitric Oxide Measurements 4,5-DIAMINOFLUORESCEIN REACTS WITH DEHYDROASCORBIC ACID AND ASCORBIC ACID

4,5-Diaminofluorescein (DAF-2) is widely used for detection and imaging of NO based on its sensitivity, noncytotoxicity, and specificity. In the presence of oxygen, NO and NO-related reactive nitrogen species nitrosate 4,5-diaminofluorescein to yield the highly fluorescent DAF-2 triazole (DAF-2T). However, as reported here, the DAF-2 reaction to form a fluorescent product is not specific to NO because it reacts with dehydroascorbic acid (DHA) and ascorbic acid (AA) to generate new compounds that have fluorescence emission profiles similar to that of DAF-2T. When DHA is present, the formation of DAF-2T is attenuated because the DHA competes for DAF-2, whereas AA decreases the nitrosation of DAF-2 to a larger extent, possibly because of additional reducing activity that affects the amount of available N2O3 from the NO. The reaction products of DAF-2 with DHA and AA have been characterized using capillary electrophoresis with laser-induced fluorescence detection and electrospray mass spectrometry. The reactions of DAF-2 with DHA and AA are particularly significant because DHA and AA often colocalize with nitric-oxide synthase in the central nervous, cardiovascular, and immune systems, indicating the importance of understanding this chemistry.

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.

Nitric oxide synthase activity in the molluscan CNS.

Abstract: 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, Cadlina, Armina, Coriphella, and Doriopsilla sp.) and cephalopods (Sepia and Rossia sp.). l‐Arginine/l‐citrulline conversion was β‐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 Ca2+‐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 animals opportunistic predatory lifestyle.

Functional optical coherence tomography for detecting neural activity through scattering changes

We have demonstrated functional optical coherence tomography (fOCT) for neural imaging by detecting scattering changes during the propagation of action potentials through neural tissue. OCT images of nerve fibers from the abdominal ganglion of the sea slug Aplysia californica were taken before, during, and after electrical stimulation. Images acquired during stimulation showed localized reversible increases in scattering compared with those acquired before stimulation. Motion-mode OCT images of nerve fibers showed transient scattering changes from spontaneous action potentials. These results demonstrate that OCT is sensitive to the optical changes in electrically active nerve fibers.

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 β‐NADPH and NBT; α‐NADPH could not substitute for β‐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.

Evolution and Function in Serotonergic Systems

Serotonergic systems of invertebrate and vertebrate central nervous systems (CNS) are functionally similar in multiple characters. Serotonin (5-HT) neurons dispersed throughout the CNS of lophotrochozoan invertebrates (molluscs and leeches) are analogous to vertebrate 5-HT neurons concentrated in the raphe nuclei of mid- and hindbrain: they innervate specific central pattern generators and other circuits of the CNS, receive feedback from them, and support general behavioral arousal. In both groups 5-HT regulates excitatory gain of CNS circuitry and uses similarly diverse 5-HT receptors. Marked contrast, however, exists for roles of 5-HT in regulation of appetite. Where invertebrate 5-HT neurons promote an appetitive state, this role is supplanted in the vertebrates by a peptidergic network centered around orexins/hypocretins, to which the role of 5-HT in arousal is subordinate. In the vertebrates, 5-HT has appetite-suppressant properties. This is paralleled by differing complexities of mechanisms that bring about satiety. Lophotrozoans appear to rely on simple stretching of the gut, with no obvious feedback from true nutrient stores. In contrast, vertebrate appetite is regulated by hypothalamic sensitivity to hormonal signals reporting separately on the status of fat cells and digestive activity, and to blood glucose, in addition to gut stretch. The simple satiety mechanism of a mollusc can be used in value-based foraging decisions that integrate hunger state, taste, and experience (Gillette and others 2000). For vertebrates, where appetite and arousal are regulated by signals from long-lived nutrient stores, decisions can be based on resource need going far beyond simple gut content, enabling value estimation and risk assessment in the longer-term. Thus, connection of nutrient storage depots to CNS circuitry mediating appetite may supply critical substrate for evolving complexity in brain and behavior. This hypothesis may be tested in expanded comparative studies of 5-HT and peptidergic functions in appetite and arousal.

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

Abstract: 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‐µm 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 µM 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 × 10−3M. Hemolymph NO3− in Pleurobranchaea was appreciably higher than values measured for the freshwater pulmonate Lymnaea stagnalis (3.2 ± 0.2 × 10−5M) and for another opisthobranch, Aplysia californica (3.6 ± 0.7 × 10−4M). Capillary electrophoresis methods provide utility and convenience for monitoring NO2−/NO3− levels in single cells and small amounts of tissue.

Directional Avoidance Turns Encoded by Single Interneurons and Sustained by Multifunctional Serotonergic Cells

Avoidance turns in the sea slug Pleurobranchaea are responses to noxious stimuli and replace orienting turns to food stimuli after avoidance conditioning or satiation. Avoidance turns proved to be centrally patterned behaviors, the fictive expression of which could be elicited in reduced preparations and the isolated CNS. Activity in one of a bilateral interneuron pair, the A4 cells, was necessary and sufficient to drive the avoidance turn toward the contralateral side. Single A4 cells appeared to encode both turn direction and angle, in contrast to directional behaviors of other animals in which displacement angle is usually encoded by multiple units. The As1–4 cells, bilateral serotonergic cell clusters, excited the prolonged A4 burst during the turn through electrical and chemical coupling. However, during the escape swim, As1–4 became integral elements of the swim motor network, and A4 activity was entrained to the swim rhythm by alternating excitatory–inhibitory inputs, with only weak spiking. This provides a likely mechanism for the previously observed suppression of the avoidance turn by escape swimming. These observations add significant new aspects to the multiplying known functions of As1–4 and their homologs in other molluscs and point to a pivotal role of these neurons in the organization of gastropod behavior. Simple functional models predict (1) the essential actions of inhibitor neurons in the directionality of the turning network motor output and (2) an integrating role for As1–4 in the behavioral switch between turning avoidance and swimming escape, on the basis of their response to increasing stimulus intensity.

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. J. Comp. Neurol. 382:176‐188, 1997.