Russell C. Wyeth

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

SUMMARY 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.

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.

Towards MEMS Probes for Intracellular Recording

Simultaneous, multi-site recording from the brain of freely behaving animals will allow neuroscientists to correlate neuronal activity with external stimulation and behavior. This information is critical for understanding the complex interactions of brain cells. Recent interest in microelectromechanical systems (MEMS) and in particular in bio-MEMS research has led to miniaturization of microelectrodes for extracellular neuronal recording. MEMS technology offers a unique opportunity to build compact, integrated sensors well suited for multi-site recording from freely behaving animals. These devices have the combined capabilities of silicon-integrated circuit processing and thin-film microelectrode sensing. MEMS probes for intracellular recording may offer significantly improved signal quality. Here we discuss the basic concepts that underlie the construction of intracellular MEMS probes. We first review the basics of neuronal signaling and recording, and the principles of microelectrode technology and techniques. Progress in MEMS technology for neuronal recording is then discussed. Finally, we describe MEMS probes for intracellular recording, viz., fabrication of micro-machined silicon needles capable of penetrating cell membranes. Using these needles, we recorded localized extracellular signals from the hawk moth Manduca sexta and obtained first recordings with silicon-based micro-probes from the inside of neurons, using an isolated brain of the sea slug Tritonia diomedea.

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.

Should Animals Navigating Over Short Distances Switch to a Magnetic Compass Sense

Magnetoreception can play a substantial role in long distance navigation by animals. I hypothesize that locomotion guided by a magnetic compass sense could also play a role in short distance navigation. Animals identify mates, prey, or other short distance navigational goals using different sensory modalities (olfaction, vision, audition, etc.) to detect sensory cues associated with those goals. In conditions where these cues become unreliable for navigation (due to flow changes, obstructions, noise interference, etc.), switching to a magnetic compass sense to guide locomotion toward the navigational goals could be beneficial. Using simulations based on known locomotory and flow parameters, I show this strategy has strong theoretical benefits for the nudibranch mollusk Tritonia diomedea navigating toward odor sources in variable flow. A number of other animals may garner similar benefits, particularly slow-moving species in environments with rapidly changing cues relevant for navigation. Faster animals might also benefit from switching to a magnetic compass sense, provided the initial cues used for navigation (acoustic signals, odors, etc.) are intermittent or change rapidly enough that the entire navigation behavior cannot be guided by a continuously detectable cue. Examination of the relative durations of navigational tasks, the persistence of navigational cues, and the stability of both navigators and navigational targets will identify candidates with the appropriate combination of unreliable initial cues and relatively immobile navigational goals for which this hypothetical behavior could be beneficial. Magnetic manipulations can then test whether a switch to a magnetic compass sense occurs. This hypothesis thus provides an alternative when considering the behavioral significance of a magnetic compass sense in animals.

Videograms: A Method for Repeatable Unbiased Quantitative Behavioral Analysis Without Scoring or Tracking

We present a method that complements both scoring by observers and automated tracking methods for quantifying behaviors. Based on standard motion enhancement, our algorithm converts a behavioral video recording into a single image (‘videogram’) that maps the spatial distribution of activity in the video sequence. This videogram can be used as a visual summary of activity and also as a direct, repeatable, and unbiased measure of animal activity. We describe the algorithm, and then use videograms to show acquisition of odorant-dependent place-conditioning in zebrafish trained in groups. We also demonstrate its potential for determining depth preferences and swimming speeds. This method generates activity measurements suitable for continuous variable statistics, and can be considered as an analysis alternative to behavioral tracking (over which it can have several advantages) for experiments not requiring exact trajectories.

Adaptation of Underwater Video for Near-Substratum Current Measurement

We describe a new method to measure current near the substratum. A variety of current meters are available to record flow data relevant to benthic organisms in their natural habitat. Several factors, including accuracy and precision, sample volume, sample rate, deployment constraints, and cost make them more or less appropriate to different applications. Our method, based on tracking particles in videos from a camera mounted near the substratum, provides an inexpensive option for measuring flow. We validate this “current camera” against a conventional electromagnetic meter, and then use it successfully to estimate flows experienced by the nudibranch Tritonia diomedea in its natural habitat. As we implement it, this current meter samples a large volume and provides a measure of bulk flow in the habitat. However, the method is adaptable to videos acquired from other cameras at a variety of size scales and locations. We therefore conclude that our method is an option that should be considered by biologists interested in measuring flow. In a recent field study (1), we used current direction to partially explain navigation behaviors of the nudibranch mollusc Tritonia diomedea Bergh. To avoid interference with the behaviors and our ability to observe them, we chose to measure flow some distance away from the slugs. As a consequence of this constraint, we needed integrated current headings (over time or space) to overcome the heterogeneous nature of flow in this habitat and thus allow us to estimate flow in nearby regions. Furthermore, we needed measurements made near the substratum, to correspond with flow experienced by the benthic slugs. Several current meter options are available. These include acoustic doppler velocimeters (ADVs), acoustic doppler current profilers (ADCPs), and electromagnetic meters. However, none of

Silicon micro-needles with flexible interconnections

A flexible polyimide-based interconnect scheme was developed to realize isolated needle-like microelectrodes. A simple fabrication approach allows the integration of micromachined silicon needles with a larger silicon base designed to carry elements such as amplifiers, battery or memory. The interconnecting scheme uses two polyimide layers to sandwich a metallic layer. The metal layer forms the electrical connection between the silicon base and the micro-electrodes, while the polyimide layers provide flexible insulation. The current design allows convenient handling of the device during implantation and minimal mechanical load on the implanted region. The device can conform to the surface of neural tissue and allows convenient interfacing with rugged and dynamic tissues. Prototype devices were tested for usability and animal-compatibility. The devices were implanted in sea slugs (Tritonia diomedea) and extracellular signals were acquired. Tritonia diomedea show full recovery from surgery and implantation, and survive up to a minimum of fourteen days with the ability to perform normal behaviors.

A Simple and Effective Method to Condition Olfactory Behaviors in Groups of Zebrafish

We describe a simple assay for studying and conditioning olfactory behaviors of adult zebrafish. The apparatus consists of a circular flow-through tank into which odorants can be administered in a controlled fashion. Odorants (conditioned stimuli; CS) are repeatedly paired with food flakes (unconditioned stimuli; UCS) that are provided inside a tethered floating feeding ring. In response to conditioning, zebrafish develop an odorant-dependent place preference and restrict appetitive swimming behavior to the vicinity of the feeding ring. This robust assay can also be conducted with groups of zebrafish and thus provides a potentially important tool for large behavioral screens.