**3.1.3 1-EBIO in rats**

284 Neuroscience – Dealing with Frontiers

(rotations away from the side of the lesion) remained constant throughout this experiment (data not shown). There was no significant difference in rotational behavior amongst the 3 treatment groups in this post-lesion and pre-treatment period [figure 2A, p=0.107 (treatment)], nor was there a treatment by time interaction [figure 2A, p=0.981 (treatment x time)]. Drug (or vehicle) treatment began 18 days following 6-OHDA (figure 2A'). Despite this, the rotational phenotype of the mice in each treatment group remained unchanged throughout the treatment period [figure 2A'; p=0.923 (treatment), p=0.986 (time), p=0.949 (treatment x time), two-way ANOVA]. Thus, riluzole had no effect on 6-OHDA-induced

Fig. 2. Effects of the SK channel agonist riluzole on rotational behavior in response to amphetamine and the number of TH+ SNc cells in 6-OHDA-lesioned adult mice. 6-OHDA was injected into the left SNc of 8-week old male C57Bl/6J mice to reduce the number of SNc TH+ cells to ~50% of normal (see vehicle-treated mice, black symbols in B). (A & A') Every 3rd day for the next 18 days the rotational behavior in response to amphetamine was examined. The relative mean ± SE number of ipsiversive rotations performed over a 25 minute interval, beginning 25 minutes following amphetamine injection, is plotted over time for each treatment group. Note, the software used to count rotations was not as accurate as manual counting or other methods (e.g. rotometers); therefore the number of rotations here should not be compared with data collected using these other methods. However, the software consistently counted rotations at different times; therefore any changes in number of rotations accurately reflect behavioral changes. (A) 6-OHDA resulted in progressive increase in the number of ipsiversive rotations over time (p=0.035, two-way ANOVA). No differences in number of contraversive rotations occurred over this same period (data not shown). (A') Over days 20-33, vehicle (black bars), 3µM riluzole (pale red bars), or 30µM riluzole (red bars) was administered to the mice in their drinking water. During this time there was no change in the number of ipsiversive (or contraversive, data not shown) rotations by treatment or by time (two-way ANOVA). (B) At the end of day 33, there were no differences in the number of TH+ cells in the 6-OHDA-lesioned SNc across the three treatment groups (p=0.952, one-way ANOVA). See figure 1 legend for details about how

motor dysfunction in this experiment.

these data are represented.

We repeated the 200µM 1-EBIO infusion experiment in mice detailed above in unilateral 6- OHDA-lesioned rats, because motor impairments can be more accurately quantified in rats using locomotor cells, the cylinder test, and rotational response to amphetamine. The timecourse of this experiment was longer than the mouse experiment because it takes longer for SNc DA cell degeneration to occur following 6-OHDA, and because we wanted to see whether a treatment period longer than we used in mice (2 weeks) produced any effect.

In summary, there were no effects of 200µM 1-EBIO infusion on motor behavior or number of TH+ SNc cells following 6-OHDA lesion in rats. The rotational behavior in response to amphetamine is plotted against time in figure 3A. This shows that following 6-OHDA, there was a gradual increase in the ratio of ipsiversive:contraversive rotations, as expected, which peaked at 4 weeks. Two weeks later (i.e. 6 weeks following lesion) half the rats were implanted with osmotic pumps infusing 200µM 1-EBIO directly into the lesioned midbrain, and the other half were implanted with osmotic pumps infusing vehicle. Following the onset of drug (or vehicle) treatment, there was a trend (non-significant) for animals receiving 200µM 1-EBIO to show improvement in their rotational response at 9 & 11 weeks (3 & 5 weeks after treatment onset; figure 3A). However, no such trend was apparent at 13 & 15 weeks (7 & 9 weeks after treatment onset; figure 3A). A similar scenario, including a nonsignificant trend for improvement at 9 & 11 weeks, was also observed in motor behavior during the cylinder test (data not shown).

At the experiment end-point (15 weeks after lesion), the number of SNc TH+ cells was similar in 1-EBIO- and vehicle-infused rats (figure 3B).
