Neuroethology of Melibe leonina swimming behavior

American Zoologist, Sep 2001 by Watson, Winsor H III, Lawrence, Kaddee A, Newcomb, James M

Behavior

Long-term patterns of Melibe swimming activity were determined by analysis of time-lapse videotapes. Experiments took place in a 20-liter aquarium continuously perfused with natural seawater, at 8-12 deg C. Individual Melibe (n = 12) were exposed to a natural light regime of 14L:10D, as well as constant darkness and constant light conditions. During constant darkness the tank was illuminated with a 40-watt ceramic coated red light and constant light was provided by a 30-watt light.

Experiments ran for 40-72 hr and were monitored by a low-light video camera connected to a Panasonic AG-RT600P timelapse VCR. Videotapes were analyzed to determine the number and duration of spontaneous bouts of swimming.

Semi-intact and isolated brain preparations

Experiments with semi-intact preparations were carried out using the same chamber and methods employed by Willows to study swimming in Tritonia (Willows, 1967; Willows et al., 1973). Each Melibe was suspended by hooks from its dorsal integument in a chamber continuously perfused with natural seawater. A small opening was made in the integument just over the brain, and the CNS was immobilized by pinning it to a small wax-covered platform. Typically, this procedure stabilized the brain enough so that intracellular recordings could be obtained for up to an hour while the Melibe exhibited intermittent bouts of swimming.

In other experiments brains were removed from animals by cutting all nerve roots, except for the pedal-pedal connectives. Isolated brains were pinned in a 2-ml recording dish embedded in an aluminum plate. Coolant or ambient seawater was circulated through the aluminum plate to keep the recording chamber at 8-12 deg C.

Intracellular recording

Intracellular recordings were obtained using 20-40 megil microelectrodes filled with 2 M potassium acetate. Certain neurons were filled with lucifer yellow (5% dissolved in 0.15 M LiCl^sub 2^) by passing hyperpolarizing current pulses (5-10 nA, 50% duty cycle). After neurons were filled the ganglia were fixed overnight in 4% paraformaldehyde in filtered seawater, and then dehydrated, cleared and mounted in DPX. Permanent images were obtained with a Nikon Optiphot-2 epifluorescence microscope or a Bio-Rad MRC-600 confocal microscope.

Nitric oxide synthase histology

Putative nitric oxide synthase (NOS) containing neurons were localized using both immunocytochemical and NADPH-diaphorase histochemical staining methods. The immunocytochemical methods have been described in detail in Watson and Willows (1992). Isolated brains were fixed, rinsed in 0.1 M phosphate buffer (PB) and then treated with 0.1% trypsin for 15 min and 4% Triton X-100 in PB for 1 hr. They were then rinsed in PB containing 0.4% Triton X-100 and 0.1% sodium azide (PTA) for 24 hr and incubated overnight in 6% goat serum in PTA (PTA-GS). This was followed by incubation in primary antibodies (anti-universal NOS, Affinity Bioreagents, Golden, CO., 1:100 in PTA-GS) for 48 hr, a 24 hr rinse in PTA, incubation in goat anti-rabbit secondary antibodies conjugated to fluorescein (1:100 in PTA-GS, 24 hr), and then a final rinse in PB. Brains were then dehydrated, cleared in methyl salicylate, mounted in DPX and viewed as described above in the lucifer methods.