Locomotive Behavioral Choice in the Leech Kathryn McCormick and Peter D. Brodfuehrer. Dept. of Biology, Bryn Mawr College, Bryn Mawr, PA, Introduction In the medicinal leech, Hirudo medicinalis, recent research has suggested that interneurons in the head brain may be multifunctional. Tr1, one such cell, was initially characterized to trigger only one behavior: swimming; but has recently been found to trigger crawling as well (Graybeal (BMC ’04) and McCormick). In this study, we tested whether there is a correlation between the firing frequency of cell Tr1 and the level of activity in the connectives with the type of locomotive behavior triggered. Experimental Methods All experiments were performed using an isolated nerve cord preparation from the head ganglion to the tail ganglion (Fig. 1). Extracellular recordings were made using suction electrodes from the dorsal posterior (DP) nerve, and from the three locations along the ventral nerve cord - between ganglions (3,4), (9,10), and (15-16). The firing frequency of cell Tr1 was manipulated by passing current through an intracellular electrode (Fig. 2). Fig. 2. A) Schematic drawing the soma and porcesses of cell Tr1 in the head ganlgion. B) Confocal picture of Tr1 filled with Lucifer Yellow dye. Tr1 is found symmetrically in both halves of the head. B Results Swimming was identified by short duration bursts of large amplitude spikes in the dorsal posterior (DP) nerve, with a period of approximately 0.5 – 1 s (Fig 3b). Crawling was characterized by bursts of large and small amplitude spikes in the DP, with a period greater than 8 s. Bursts of activity in the medial anterior (MA) nerve also occurred approximately simultaneously with small amplitude DP spikes (Fig 3a). Stimulation frequencies of Tr1 were calculated for each trial that induced either swimming or crawling. Data was separated by preparation and then averages were calculated for both swimming and crawling (Table 1). No significant difference in the firing frequency of cell Tr1 was found between stimulation trials that led to swimming and those that led crawling. Intracellular Electrode in Cell Tr1 Fig. 3. Intracellular stimulation of cell Tr1 can trigger either a) crawling or b) swimming. a b Table 2. Comparison between the amount of connective activity between Tr1 stimulation trials that led to swimming and crawling. The level of connective activity was quantified by measuring the variance of the neuronal signals in the connectives one second before (pre) and one second after (post) Tr1 stimulation. Table 1. Comparison between the firing frequency of cell Tr1 that led to swimming or crawling. Conclusion The firing frequency of cell Tr1 and the amount of activity elicited in the connectives following Tr1 stimulation do not determine whether swimming or crawling will be elicited in the isolated nerve cord. Other factors in the nerve system must control this decision. Fig. 1. Schematic drawing of the isolated ventral nerve cord showing locations of extracellular and intracellular recording electrodes. H = head ganglion, T= tail ganlgion, numbers = segmental ganglia. The amount of activity in the connectives one second before and one second after stimulating Tr1 was calculated by measuring variance. We found that Tr1 stimulation greatly increases the amount of activity in the connectives independent of the behavior elicited, and swimming caused a greater level of activity in the connective than crawling once behavior began. However, there was no difference the amount of activity directly after swimming or crawling was triggered. There was also no difference in the amount of activity in the connectives that preceded Tr1 stimulation.