Serial-section atlas of the Tritonia pedal ganglion.

The pedal ganglion of the nudibranch gastropod Tritonia diomedea has been the focus of neurophysiological studies for more than 50 yr. These investigations have examined the neural basis of behaviors as diverse as swimming, crawling, reflex withdrawals, orientation to water flow, orientation to the earth's magnetic field, and learning. Despite this sustained research focus, most studies have confined themselves to the layer of neurons that are visible on the ganglion surface, leaving many neurons, which reside in deeper layers, largely unknown and thus unstudied. To facilitate work on such neurons, the present study used serial-section light microscopy to generate a detailed pictorial atlas of the pedal ganglion. One pedal ganglion was sectioned horizontally at 2-µm intervals and another vertically at 5-µm intervals. The resulting images were examined separately or combined into stacks to generate movie tours through the ganglion. These were also used to generate 3D reconstructions of individual neurons and rotating movies of digitally desheathed whole ganglia to reveal all surface neurons. A complete neuron count of the horizontally sectioned ganglion yielded 1,885 neurons. Real and virtual sections from the image stacks were used to reveal the morphology of individual neurons, as well as the major axon bundles traveling within the ganglion to and between its several nerves and connectives. Extensive supplemental data are provided, as well as a link to the Dryad Data Repository site, where the complete sets of high-resolution serial-section images can be downloaded. NEW & NOTEWORTHY Because of the large size and relatively low numbers of their neurons, gastropod mollusks are widely used for investigations of the neural basis of behavior. Most studies, however, focus on the neurons visible on the ganglion surface, leaving the majority, located out of sight below the surface, unexamined. The present light microscopy study generates the first detailed visual atlas of all neurons of the highly studied Tritonia pedal ganglion.

Different functions for homologous serotonergic interneurons and serotonin in species-specific rhythmic behaviours.

Closely related species can exhibit different behaviours despite homologous neural substrates. The nudibranch molluscs Tritonia diomedea and Melibe leonina swim differently, yet their nervous systems contain homologous serotonergic neurons. In Tritonia, the dorsal swim interneurons (DSIs) are members of the swim central pattern generator (CPG) and their neurotransmitter serotonin is both necessary and sufficient to elicit a swim motor pattern. Here it is shown that the DSI homologues in Melibe, the cerebral serotonergic posterior-A neurons (CeSP-As), are extrinsic to the swim CPG, and that neither the CeSP-As nor their neurotransmitter serotonin is necessary for swim motor pattern initiation, which occurred when the CeSP-As were inactive. Furthermore, the serotonin antagonist methysergide blocked the effects of both the serotonin and CeSP-As but did not prevent the production of a swim motor pattern. However, the CeSP-As and serotonin could influence the Melibe swim circuit; depolarization of a cerebral serotonergic posterior-A was sufficient to initiate a swim motor pattern and hyperpolarization of a CeSP-A temporarily halted an ongoing swim motor pattern. Serotonin itself was sufficient to initiate a swim motor pattern or make an ongoing swim motor pattern more regular. Thus, evolution of species-specific behaviour involved alterations in the functions of identified homologous neurons and their neurotransmitter.

A stereo-compound hybrid microscope for combined intracellular and optical recording of invertebrate neural network activity.

Optical recording studies of invertebrate neural networks with voltage-sensitive dyes seldom employ conventional intracellular electrodes. This may in part be due to the traditional reliance on compound microscopes for such work. While such microscopes have high light-gathering power, they do not provide depth of field, making working with sharp electrodes difficult. Here we describe a hybrid microscope design, with switchable compound and stereo objectives, that eases the use of conventional intracellular electrodes in optical recording experiments. We use it, in combination with a voltage-sensitive dye and photodiode array, to identify neurons participating in the swim motor program of the marine mollusk Tritonia. This microscope design should be applicable to optical recording studies in many preparations.

Comparative mapping of serotonin-immunoreactive neurons in the central nervous systems of nudibranch molluscs.

The serotonergic systems in nudibranch molluscs were compared by mapping the locations of serotonin-immunoreactive (5-HT-ir) neurons in 11 species representing all four suborders of the nudibranch clade: Dendronotoidea (Tritonia diomedea, Tochuina tetraquetra, Dendronotus iris, Dendronotus frondosus, and Melibe leonina), Aeolidoidea (Hermissenda crassicornis and Flabellina trophina), Arminoidea (Dirona albolineata, Janolus fuscus, and Armina californica), and Doridoidea (Triopha catalinae). A nomenclature is proposed to standardize reports of cell location in species with differing brain morphologies. Certain patterns of 5-HT immunoreactivity were found to be consistent for all species, such as the presence of 5-HT-ir neurons in the pedal and cerebral ganglia. Also, particular clusters of 5-HT-ir neurons in the anterior and posterior regions of the dorsal surface of the cerebral ganglion were always present. However, there were interspecies differences in the number of 5-HT-ir neurons in each cluster, and some clusters even exhibited strong intraspecies variability that was only weakly correlated with brain size. Phylogenetic analysis suggests that the presence of particular classes of 5-HT-ir neurons exhibits a great deal of homoplasy. The conserved features of the nudibranch serotonergic system presumably represent the shared ancestral structure, whereas the derived characters suggest substantial independent evolutionary changes in the number and presence of serotonergic neurons. Although a number of studies have demonstrated phylogenetic variability of peptidergic systems, this study suggests that serotonergic systems may also exhibit a high degree of homoplasy in some groups of organisms.

Dopamine modulation of Ca(2+) dependent Cl(-) current regulates ciliary beat frequency controlling locomotion in Tritonia diomedea.

The physiological mechanisms controlling ciliary beating remain largely unknown. Evidence exists supporting both hormonal control of ciliary beating and control via direct innervation. In the present study we investigated nervous control of cilia based locomotion in the nudibranch mollusc, Tritonia diomedea. Ciliated pedal epithelial (CPE) cells acting as locomotory effectors may be electrically excitable. To explore this possibility we characterized the cells' electrical properties, and found that CPE cells have large voltage dependent whole cell currents with two components. First, there is a fast activating outward Cl(-) current that is both voltage and Ca(2+) influx dependent (I(Cl(Ca))). I(Cl(Ca)) is sensitive to DIDS and 9-AC, and resembles currents of Ca(2+)-activated Cl(-) channels (CaCC). Ca(2+) dependence also suggests the presence of voltage-gated Ca(2+) channels; however, we were unable to detect these currents. The second current, a voltage dependent proton current (I(H)), activates very slowly and is sensitive to both Zn(2+) and changes in pH. In addition we identify a new cilio-excitatory substance in Tritonia, viz., dopamine. Dopamine, in the 10 mumol l(-1)-1 mmol l(-1) range, significantly increases ciliary beat frequency (CBF). We also found dopamine and Tritonia Pedal Peptide (TPep-NLS) selectively suppress I(Cl(Ca)) in CPE cells, demonstrating a link between CBF excitation and I(Cl(Ca)). It appears that dopamine and TPep-NLS inhibit I(Cl(Ca)) not through changing [Ca(2+)](in), but directly by an unknown mechanism. Coupling of I(Cl(Ca)) and CBF is further supported by our finding that DIDS and zero [Cl(-)](out) both increase CBF, mimicking dopamine and TPep-NLS excitation. These results suggest that dopamine and TPep-NLS act to inhibit I(Cl(Ca)), initiating and prolonging Ca(2+) influx, and activating CBF excitation.

Nervous control of ciliary beating by Cl(-), Ca(2+) and calmodulin in Tritonia diomedea.

In vertebrates, motile cilia line airways, oviducts and ventricles. Invertebrate cilia often control feeding, swimming and crawling, or gliding. Yet control and coordination of ciliary beating remains poorly understood. Evidence from the nudibranch mollusc, Tritonia diomedea, suggests that locomotory ciliated epithelial cells may be under direct electrical control. Here we report that depolarization of ciliated pedal epithelial (CPE) cells increases ciliary beating frequency (CBF), and elicits CBF increases similar to those caused by dopamine and the neuropeptide, TPep-NLS. Further, four CBF stimulants (zero external Cl(-), depolarization, dopamine and TPep-NLS) depend on a common mode of action, viz. Ca(2+) influx, possibly through voltage-gated Ca(2+) channels, and can be blocked by nifedipine. Ca(2+) influx alone, however, does not provide all the internal Ca(2+) necessary for CBF change. Ryanodine receptor (RyR) channel-gated internal stores are also necessary for CBF excitation. Caffeine can stimulate CBF and is sensitive to the presence of the RyR blocker dantrolene. Dantrolene also reduces CBF excitation induced by dopamine and TPep-NLS. Finally, W-7 and calmidazolium both block CBF excitation by caffeine and dopamine, and W-7 is effective at blocking TPep-NLS excitation. The effects of calmidazolium and W-7 suggest a role for Ca(2+)-calmodulin in regulating CBF, either directly or via Ca(2+)-calmodulin dependent kinases or phosphodiesterases. From these results we hypothesize dopamine and TPep-NLS induce depolarization-driven Ca(2+) influx and Ca(2+) release from internal stores that activates Ca(2+)-calmodulin, thereby increasing CBF.