Responses to magnetic stimuli recorded in peripheral nerves in the marine nudibranch mollusk Tritonia diomedea.

Prior behavioral and neurophysiological studies provide evidence that the nudibranch mollusk Tritonia orients to the earth's magnetic field. Earlier studies of electrophysiological responses in certain neurons of the brain to changing ambient magnetic fields suggest that although certain identified brain cells fire impulses when the ambient field is changed, these neuron somata and their central dentritic and axonal processes are themselves not primary magnetic receptors. Here, using semi-intact animal preparations from which the brain was removed, we recorded from peripheral nerve trunks. Using techniques to count spikes in individual nerves and separately also to identify, then count individual axonal spikes in extracellular records, we found both excitatory and inhibitory axonal responses elicited by changes in the direction of ambient earth strength magnetic fields. We found responses in nerves from many locations throughout the body and in axons innervating the body wall and rhinophores. Our results indicate that primary receptors for geomagnetism in Tritonia are not focally concentrated in any particular organ, but appear to be widely dispersed in the peripheral body tissues.

1-Phenoxy-2-propanol is a useful anaesthetic for gastropods used in neurophysiology.

Anaesthesia is often used in neurophysiological, surgical, and neuroanatomical protocols. Several anaesthetics, including magnesium chloride, volatiles (halothane, etc.), and barbiturates, have been used in gastropod neurobiology. 1-Phenoxy-2-propanol (PP) is another anaesthetic option that has not yet been used extensively. We provide an analysis of the neural, muscular and behavioural effects of PP in gastropods. PP eliminates action potentials and reduces muscular contraction force in Hermissenda crassicornis, and eliminates behavioural activity in Tritonia diomedea. Our results show these effects are reversible, with complete action potential recovery, at least partial muscular recovery, and full behavioural recovery. Survival after surgery in T. diomedea was longer with PP than without anaesthetic, and PP also reduced contraction during tissue fixation in Lymnaea stagnalis. Moreover, PP can be bath applied, has low toxicity, and is biodegradable. Thus, PP is an effective anaesthetic in three species of gastropods, and useful in neurophysiological dissection, surgical, and fixation protocols.

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.

Field behavior of the nudibranch mollusc Tritonia diomedea.

The nudibranch mollusc Tritonia diomedea has been a useful model system for studies of how the brain controls behavior. However, no broad study of T. diomedea field behavior exists--an important deficit since laboratory behaviors may differ from what occurs in nature. Here we report analysis of time-lapse video of the slugs in their natural habitat to describe behaviors and their relationships to sensory cues. We found that movements relative to conspecifics, prey, and predators correlated with direction of water flow. These observations lead to three new navigational hypotheses: regardless of the actual heading to the target, T. diomedea crawls (1) upstream toward potential mates, (2) upstream toward food, and (3) downstream away from predators. We also describe both the behavior and its sensory context for feeding, escape swims, mating, and egg-laying, among other behaviors. Field behaviors were similar to published descriptions of laboratory behavior. However, the field observations add contextual detail, including preceding and subsequent behaviors and interactions with suites of habitat features not present in the laboratory. For example, the escape swim, previously studied as an isolated behavior in response to a single stimulus, appears to be affected by multiple sensory modalities and coordinated with several other behaviors. Our work will provide a basis for future neuroethological experimentation and also is the first step in the study of navigation in T. diomedea.

Orientation and navigation relative to water flow, prey, conspecifics, and predators by the nudibranch mollusc Tritonia diomedea.

Progress in understanding sensory and locomotory systems in Tritonia diomedea has created the potential for the neuroethological study of animal navigation in this species. Our goal is to describe the navigational behaviors to guide further work on how the nervous system integrates information from multiple senses to produce oriented locomotion. Observation of T. diomedea in its habitat has suggested that it uses water flow to navigate relative to prey, predators, and conspecifics. We test these hypotheses in the field by comparing slug orientation in time-lapse videos to flow direction in circumstances with and without prey, predators, or conspecifics upstream. T. diomedea oriented upstream both while crawling and after turning. This trend was strongest before feeding or mating; after feeding or mating, the slugs did not orient significantly to flow. Slugs turned downstream away from an upstream predator but did not react in control situations without an upstream predator. These data support the hypothesis that T. diomedea uses a combination of odors (or some other cue transported downstream) and water flow to navigate relative to prey, predators, and conspecifics. Understanding the context-dependent choice between upstream and downstream crawling in T. diomedea provides an opportunity for further work on the sensory integration underlying navigation behavior.

Odours detected by rhinophores mediate orientation to flow in the nudibranch mollusc, Tritonia diomedea.

Tritonia diomedea is a useful neuroethological model system that can contribute to our understanding of the neural control of navigation. Prior work on both sensory and locomotory systems is complemented by recent field experiments, which concluded that these animals primarily use a combination of odours and water flow as guidance cues. We corroborate these field results by showing similar navigation behaviours in a flow tank. Slugs crawled upstream towards both prey and conspecifics, and turned downstream after crawling into a section of the flow tank downstream of a predator. Controls without upstream odour sources crawled apparently randomly. We then tested whether these behaviours depend on odours detected by the rhinophores. Outflow from a header tank was used to generate prey, predator and unscented control odour plumes in the flow tank. Slugs with rhinophores crawled upstream towards a prey odour plume source, turned downstream in a predator odour plume, and showed no reaction to a control plume. Slugs without rhinophores behaved similarly to controls, regardless of odour plume type. Finally, we used extracellular recordings from the rhinophore nerve to demonstrate that isolated rhinophores are chemosensitive. Afferent activity increased significantly more after application of all three odour types than after unscented control applications. Responses were odour specific. We conclude that rhinophores mediate orientation to flow, and suggest that future work should focus on the integration of mechanosensation and chemosensation during navigation in T. diomedea.

Immunological localization of Tritonia peptide in the central and peripheral nervous system of the terrestrial snail Helix aspersa.

We report here evidence that the pedal peptides (Peps) first discovered in mollusks may be neurotransmitters with a general role in control of molluscan somatic and visceral muscles. Using Tritonia peptide (TPep) antiserum we obtained morphological evidence for such a role in Helix aspersa. We localized 1,200-1,400 small and medium-sized (5-40 microm) TPep-IR neurons in the central nervous system of Helix and demonstrated the presence of these neurons in each ganglion. Many TPep-immunoreactive (IR) neurons were motoneurons that sent axons to almost all peripheral nerves. TPep-IR fibers innervated the foot, esophagus, hermaphroditic duct, optic tentacles, salivary gland, heart, and proximal and distal aorta. In peripheral tissues TPep-IR fiber ramifications were mostly associated with muscles and with ciliated epithelia. In addition, TPep-IR fibers were in the neuropil of the ganglia, the commissures, and the connectives, and they formed axosomatic terminals in the central nervous system. TPep-IR neurons were found in the esophagus and hermaphroditic duct and as sensory receptors in the bulb of the optic tentacles. These results from Helix, and those reported elsewhere from other mollusks, suggest a general involvement of TPep-like substances in control of muscle- and ciliary-driven motor activities, including perhaps their antecedent sensory and central axosomatic integrative activity.

Localization of serotonin and its possible role in early embryos of Tritonia diomedea(Mollusca: Nudibranchia).

A classical neurotransmitter serotonin (5-HT) was detected immunochemically using laser scanning microscopy at the early stages of Tritonia diomedea development. At the one- to eight-cell stages, immunolabeling suggested the presence of 5-HT in the cytoplasm close to the animal pole. At the morula and blastula stages, a group of micromeres at the animal pole showed immunoreactivity. At the gastrula stage no immunoreactive cells were detected, but they arose again at the early veliger stage. Antagonists of 5-HT(2) receptors, ritanserin and cyproheptadine, as well as lipophilic derivatives of dopamine blocked cleavage divisions or distorted their normal pattern. These effects were prevented by 5-HT and its highly lipophilic derivates, serotoninamides of polyenoic fatty acids, but not by the hydrophilic (quaternary) analog of 5-HT, 5-HTQ. The results confirm our earlier suggestion that endogenous 5-HT in pre-nervous embryos acts as a regulator of cleavage divisions in nudibranch molluscs.

Immunocytochemical localization of pedal peptide in the central nervous system of the gastropod mollusc Tritonia diomedea.

Tritonia pedal ganglion peptides (TPeps) are a trio of pentadecapeptides isolated from the brain of the nudibranch Tritonia diomedea. TPeps have been shown both to increase the beating rate of ciliated cells of Tritonia and to accelerate heart contractions in the mollusc Clione limacina. Here we examine the immunocytochemical distribution of TPeps in the Tritonia central nervous system. We found the brain and buccal ganglia to be rich sources of TPep immunoreactivity. Specific cells in both structures, some of them previously identified, were immunoreactive. Moreover, immunoreactive fibers were seen connecting ganglia and exiting almost all the major nerves. In the brain, we found that the paired, ciliated statocysts apparently receive TPep innervation. In addition, we observed unstained cell bodies in each buccal ganglion with extensive TPep immunoreactive projections surrounding their somata and primary neurites. Similar projections were not observed in the brain. We also compared the TPep immunoreactivity with that of SCP(b) in the buccal ganglia. We observed many neurons and processes that were immunoreactive to both peptides. One neuron that contains both TPep- and SCP(b)-like peptides (B12) has an identified role in the Tritonia feeding network. Together, these findings suggest that TPeps may play an active role in the central nervous system of Tritonia as neurotransmitters modulating orientation, swimming, and feeding.

Computer-assisted visualizations of neural networks: expanding the field of view using seamless confocal montaging.

Microscopic analysis of anatomic relationships within the neural networks of adult and developing tissues often requires sampling large spatial regions of neuronal architecture. To accomplish this, there are two common imaging approaches: (1) image the entire area at once with low spatial resolution; or (2) image small sections at higher magnification/resolution and then join the sections back together by mosaic reconstruction (photomontaging). Low magnification imaging is relatively rapid to perform, resulting in a visualization that encompasses a large field of view with an extended depth of field. However, for fluorescence microscopy, low magnification visualizations are often plagued by poor spatial resolution. High magnification imaging possesses superior spatial resolution, but it produces an image with limited depth of field. When creating a larger field of view, the final image is also fragmented at the boundaries where multiple images are stitched together. Using confocal microscopy as well as features of common image processing programs, we outline a new method to transform individual, spatially contiguous z-series into a montage with a seamless field of view and an extended depth of field. In addition, we show that the manual alignment of images our method requires does not introduce significant errors into the final image. We illustrate our method for visualizing neural networks using tissues from the adult gastropod mollusc, Tritonia diomedea, and the developing zebrafish, Danio rerio.

[The possible functional interaction of serotonin and neuropeptides in embryogenic regulatory processes (experiments on embryos of the mollusk Tritona diomedea)]. / O vozmozhnom funktsional'nom vzaimodeistvii serotonina i neiropeptidov v reguliatornykh protsessakh émbriogeneza (opyty na zarodyshakh molliuska Tritona diomeda).

Ritanserin and inmecarb hydrochloride, antagonists of serotonin, act cytostatically and teratogenically on early embryos of Tritonia diomedea, a nudibranch mollusk. On the basis of a pharmacological analysis and the type of developmental abnormalities observed, this action appears to be due to disturbances in the functional activity of endogenous serotonin and is associated with damage of to the cytoskeleton. The effects of ritanserin and inmecarb are prevented or attenuated by lipophilic serotonin analogs (serotoninamides of polyenoic fatty acids), as well as by polypeptides isolated from neurons Pd5 and Pd6 of the pedal ganglia of the adult Tritonia. In late embryos (stage of veligers), serotonin and to a lesser extent its lipophilic analogs strongly increase embryonic motility. This effect of serotonin is potentiated by some neuropeptides and inhibited by others. These results provide evidence for functional interaction between serotonin and neuropeptides in the control processes of embryogenesis.

Serotonin inhibits ciliary transport in esophagus of the nudibranch mollusk Tritonia diomedea.

Both serotonin and the molluskan pedal neuropeptides (TPEPs) cause increased ciliary beating rate of cells of the foot epithelium of the nudibranch mollusk, Tritonia diomedea. Here we compared responses of the ciliated epithelium of the esophagus with that of the foot, and report fundamental differences. Serotonin reduces the ciliary transport rate of the esophagus. We find also that the serotonin driven inhibition of esophagus is blocked and the excitation of foot epithelium is reduced by the serotonin receptor blocker ketanserin. On the contrary, ergometrine completely blocked the serotonin effect in the esophagus, and does not block the serotonin effect in the foot. Neither the TPEP driven excitation of ciliated cells of the foot nor that of the esophagus is blocked by ketanserin and ergometrine. Clearly, serotonin and TPEP regulation of different ciliated epithelia involve different receptors. Thus, mechanisms of serotonin control of different ciliated epithelia in the same animal are apparently fundamentally different, and unlike responses in all previous reports, 5HT here inhibits a ciliated epihelium.

Differential effects of serotonergic and peptidergic cardioexcitatory neurons on the heart activity in the pteropod mollusc, Clione limacina.

A group of four cardioexcitatory neurons has been identified in the intestinal ganglia of the mollusc Clione limacina. Relatively weak stimulation of the intestinal neurons induced auricle contractions only, while strong stimulation produced initial auricle contractions followed by full-cycle auricle-ventricle contractions. Intestinal cardioexcitatory neurons probably utilized as their transmitter a peptide similar to Tritonia pedal peptide--they showed pedal peptide-like immunoreactivity, and their effects were mimicked by application of the exogenous pedal peptide. The pedal cardioexcitatory neuron was found to produce strong excitatory effects only on the ventricle contractions. Its stimulation induced ventricle contractions in the quiescent heart or significantly accelerated the rate of ventricle contractions in the rhythmically active heart. The pedal cardioexcitatory neuron apparently utilized serotonin as a neurotransmitter, based upon serotonin immunoreactivity, blocking effect of serotonin antagonists mianserin and methysergide, and the observation that exogenous serotonin mimicked its effect. A dense network of pedal peptide-like immunoreactivity was found both in the auricle and ventricle tissue. Serotonin immunoreactivity was densely present in the ventricle, while the auricle contained only a separate serotonin-immunoreactive unbranched axon. Thus, there are two separate groups of central cardioexcitatory neurons with different effects on heart activity, which together might provide a complex cardio-regulatory function in Clione.

Modulation of ciliary beat frequency by neuropeptides from identified molluscan neurons.

Prior work in the nudibranch Tritonia diomedea indicated that certain identifiable pedal ganglion neurons (Pd5 and 6) innervating the foot synthesize three novel peptides (TPeps) that resemble Pedal peptide (Pep) identified in the sea hare Aplysia californica. We report here that when TPeps are applied directly to isolated ciliated patches of Tritonia diomedea foot epithelium, there is an increase in ciliary beating that normally drives locomotion. Exposure to TPeps also increases the ciliary beat frequency of cells isolated from the pedal epithelium, suggesting that the observed ciliomotor effects are direct and not mediated by intervening cells. Antibodies to TPep bind to specific cells of the brain and foot and to ciliated peripheral tissues in Tritonia diomedea and in the pulmonate gastropod Lymnaea stagnalis. We suggest, therefore, that TPeps may regulate the activity of ciliated cells responsible for pedal locomotion and other functions in gastropod molluscs.

Function of identified nerves in orientation to water flow in Tritonia diomedea.

We determined which sensory and motor nerves mediate orientation to flow in the marine slug Tritonia diomedea, and tested the hypothesis that the slug orients to water flow by comparing the intensities of water flow stimulation on each side of its body. Lesion experiments revealed which nerves carried information necessary for flow orientation. The lateral branches of Cerebral Nerve #2 were the only cerebral nerves necessary for flow orientation. Cutting all cerebral nerves except the lateral branches of Cerebral Nerve #2 did not eliminate flow orientation. Thus, the lateral branches of Cerebral Nerve #2 were both necessary and sufficient (among the cerebral nerves) for flow orientation. Denervation of one side of the head by cutting Cerebral Nerves #1-4 on one side did not eliminate normal flow orientation. We have revised our model of how Tritonia diomedea orients to flow to allow for this unilateral determination of flow direction. Unilaterally cutting Pedal Nerve #3, which contains many pedal motor axons, reduced turning toward that side, but did not affect final orientation to flow. The ability to detect flow direction was not compromised by the inability to initially turn towards flow.

Purification and sequencing of neuropeptides from identified neurons in the marine mollusc, Tritonia.

Neuropeptides were characterized in two similar identified neurons termed pedal 5 and 6 (Pd5 and Pd6). Both neurons appear white, a characteristic of peptidergic neurons, and send peripheral axons down several nerves that innervate the foot and control locomotion. Gel electrophoresis of neurons incubated with labeled amino acids indicated that individually dissected Pd5 and Pd6 neurons synthesized peptide precursors of the same size and processed them in parallel. Using HPLC, several absorbance peaks that had retention times typical of peptides were identified that were specific to extracts of Pd5 and Pd6. Three peptides were purified from extracts of many pooled Pd5 and Pd6 neurons. The complete sequences of two 15-amino acid peptides were obtained and the sequence of a third 15-amino acid peptide was inferred from the partial sequence of an apparent processing intermediate. Each of the three peptides show sequence homology to Aplysia pedal peptide (Pep). HPLC of neurons incubated with labeled amino acids demonstrated that Pd5 and Pd6 each synthesized all three sequenced peptides.