Proposal

The effects of dopamine depletion on searching, crawling and
biting behaviors in the leech.

The cephalic (brain) projection neurons in leeches, Hirudo medicinalis, influence behavioral decisions, such as whether or not to swim or feed. These projection neurons help in our understanding of decision-making processes in other animals as well. Dopamine (DA) is one of the important regulators in animal behavior, and the DA system is dye coupled to the cephalic projection neuron, Tr2, in leeches. Tr2 affects swimming behaviors; its activation stops swimming. The motor rhythm for swimming can be inhibited by DA, but DA does not inhibit crawling. In this way, DA is related to swimming and crawling behaviors. If DA affects some behaviors, it would follow that it has an effect on others, such as searching and feeding.

The purpose of my summer research will be to evaluate if searching, biting, and crawling behaviors in intact leeches are disrupted when DA is depleted. This is one part of a two-part experiment to see if DA changes bite duration, searching, and crawling behaviors. DA content in the leech central nervous system will be selectively depleted using the dopamine synthesis inhibitor a methyl-p-tyrosine (AMT). Other methods of DA depletionóreserpine and the neurotoxin 6-OHDAóhave been shown to reduce serotonin (5-HT) in addition to DA, thus they are less selective. High concentrations of AMT appear to deplete octopamine (OA), but OA does not influence biting in normal leeches; thus an intermediate concentration will be used to target DA. With these facts in mind, immunocytochemistry will be used to stain for the presence of DA, OA, and 5-HT in collected cords of AMT treated leeches. Additional detection methods HPLC and electrochemical detection techniques will be used, if time permits, to test for the presence or absence of DA, OA, and 5-HT in collaboration with Dr. Robert Huber, at Bowling Green State University.

To test biting and crawling behaviors (duration and frequency), experimental and control leeches will be observed for 15 minutes on a piece of Parafilm wax. Frequency of search behaviors, head turns and lifts, will also be analyzed. All tests will be video taped for further analysis, and conducted blind. DA-depleted leeches will be compared to control leeches, as well as the DA-treated leeches from the other part of this experiment.

Summary

The effects of dopamine on locomotion in the medicinal leech.

It would be difficult to overstate how important locomotion is for most animals. The amine, dopamine (DA), is a universal modulator of locomotion, however, very little is understood about DA’s specific role in locomotor rhythm generation. For example, how does DA regulate movement, and how does it interact with motor circuits? The nervous system and the various forms of locomotion in the medicinal leech have been well studied, making it a good model system for addressing these types of questions. In addition, all of the aminergic (i.e., DA and serotonin) neurons have been identified within the leech. The leech central nervous system (CNS) consists of a head brain, a tail brain, and 21 ganglia in between (i.e., the nerve cord). A recent study has shown that when DA is applied to the entire nerve cord, or even just one ganglion, the neural correlate of crawling can be observed (Puhl & Mesce, 2008).

Spontaneous behavior of leeches in both high and low water conditions were observed after they were placed in circular plastic containers with diameters of 30 cm. The low water condition, with barely enough water to cover the bottom of the container, promoted crawling, while high water, 5 cm deep, promoted swimming. Six leeches in 6 separate containers were simultaneously video recorded. The video recording was later analyzed for crawl cycles and time spent swimming.
Two types of treatments were used for behavioral observation: DA treatment, and DA depletion. DA was delivered to the CNS by placing the leech into a DA bath. Although the rate of delivery or the final dose of DA absorbed into the CNS was unkown, this method was a success due to the leech’s negligible blood brain barrier. After treatment with DA there was not any change in behavior in the low water condition, but in the high water condition, crawling increased and swimming decreased. This result is consistent with the hypothesis that intermediate levels of DA activate crawling and suppress swimming.

The other treatment was with injections of 15-25 mg of a-methyl-p-tyrosine (AMT), a DA-synthesis inhibitor. AMT was shown to deplete DA throughout the leech with antibody and glyoxcilic acid staining. 7-15 days post AMT-treatment, leeches would swim significantly more compared to pre-treatment observations (N=4, p=0.03 paired Student’s t-test).
The final aspect of this summer’s research consisted of taking electrophysiological recordings from leech ganglia. A single segmental ganglion was bathed in a solution of DA while recording the extracellular activity of the dorsal posterior (DP) nerve and intracellularly from the mechanosensitive neurons. If fictive crawling was observed from the DP nerve and one of the mechanosensitive cells was stimulated, the crawl pattern would be reset.

In conclusion we found that the behavior of leeches treated with DA supports our central hypothesis that DA biases behavior towards crawling and away from swimming: after treatment with DA, leeches will crawl more and swim less in high water. When DA is depleted in leeches by AMT they will swim significantly more than controls. It was also shown that mechanosensory neurons can influence the crawl rhythm, helping us to understand the circuitry regulating crawling behavior.

Poster (saved as a pdf)