**3.1.2 Riluzole in mice**

Riluzole is approved for the treatment of amyotrophic lateral sclerosis and is reported to affect neuronal excitability through a number of different ion-channels (e.g. voltageactivated Na+, glutamate receptors and SK channels). It activates SK channels at concentrations ≥3μM (Cao et al. 2002, Grunnet et al. 2001) and at 30μM (direct injection) it increases the number of TH+ SNc cells in normal mice (Aumann et al. 2011). We therefore tested its ability to increase the number of TH+ SNc in the PD model. Note that riluzole protects SNc neurons from MPTP and 6-OHDA in animal models of PD (Barneoud et al. 1996, Bezard et al. 1998) but does not provide benefit in PD patients, either early (Jankovic & Hunter 2002) or late (Braz et al. 2004) in disease, or in Parkinson plus disorders (Bensimon et al. 2009). To our knowledge riluzole has not been considered as a neuro-restorative agent in PD, such as we are proposing here (i.e. replenishing SNc DA cells by DA phenotype recruitment). We also note that the doses of riluzole used so far in clinical trials for PD have all been relatively low (around 3μM) rather than the 30μM, which we have shown increases the number of SNc TH+ cells in normal mice (Aumann et al. 2011). Therefore, any restorative benefit of riluzole through DA phenotype recruitment may have been missed.

The number of SNc TH+ cells in normal adult mice increased by ~500 following a 2 week infusion of 100μM 1-EBIO (or 30μM riluzole) into midbrain (Aumann et al. 2008, Aumann et al. 2011). To examine whether midbrain 1-EBIO infusion can also increase SNc TH+ cells in a PD model, we repeated this in adult mice with prior depletion of TH+ SNc cells by 6-OHDA. Eight-week old mice received a single injection of 6-OHDA directly into the left SNc. Two weeks later [when TH+ SNc cell loss is maximal (Aumann et al. 2008)] an infusion cannula was implanted into the left SNc, to allow continuous delivery of vehicle, 100μM 1-EBIO or 200μM 1-EBIO by osmotic pump for a further 2 weeks. The number of TH+ cells in the lesioned then treated SNc [expressed relative to the number of TH+ cells in the contralateral (internal control) SNc] is shown in figure 1. Data from individual mice are represented by the square symbols and the mean of each treatment group by the horizontal line. Note: (1) the relative tightness of the number of TH+ SNc cells in the vehicle-treated group, highlighting the consistency of the effect of our 6-OHDA injections across different mice; (2) the much greater variability in the drug-treated groups, indicating 1-EBIO has a restorative effect in some mice, even at 100μM; and (3) the increasing restorative effect with increasing dose of 1-EBIO. Unfortunately, although there is a net (average) increase in the number of TH+ SNc cells from ~50% (vehicle) to ~80% (200μM 1-EBIO) of the normal number of cells, this difference is not significant (p=0.068, one-way ANOVA) due to high variability in the 1-EBIO-treated groups. We also examined the density of immunoreactivity for the DA transporter (DAT) in the striatum in a cohort of vehicle-treated and 200μM 1-EBIO-treated mice to examine whether 1-EBIO improved striatal DA innervation following 6-OHDA depletion. Although there was a trend for DAT density to be higher in the 1-EBIO-treated group, and specifically in the dorsal striatum, not the ventral striatum, this difference was not statistically significant (data

Riluzole is approved for the treatment of amyotrophic lateral sclerosis and is reported to affect neuronal excitability through a number of different ion-channels (e.g. voltageactivated Na+, glutamate receptors and SK channels). It activates SK channels at concentrations ≥3μM (Cao et al. 2002, Grunnet et al. 2001) and at 30μM (direct injection) it increases the number of TH+ SNc cells in normal mice (Aumann et al. 2011). We therefore tested its ability to increase the number of TH+ SNc in the PD model. Note that riluzole protects SNc neurons from MPTP and 6-OHDA in animal models of PD (Barneoud et al. 1996, Bezard et al. 1998) but does not provide benefit in PD patients, either early (Jankovic & Hunter 2002) or late (Braz et al. 2004) in disease, or in Parkinson plus disorders (Bensimon et al. 2009). To our knowledge riluzole has not been considered as a neuro-restorative agent in PD, such as we are proposing here (i.e. replenishing SNc DA cells by DA phenotype recruitment). We also note that the doses of riluzole used so far in clinical trials for PD have all been relatively low (around 3μM) rather than the 30μM, which we have shown increases the number of SNc TH+ cells in normal mice (Aumann et al. 2011). Therefore, any restorative benefit of riluzole through DA phenotype recruitment may have been missed.

**3. Results** 

not shown).

**3.1.2 Riluzole in mice** 

**3.1.1 1-EBIO in mice** 

**3.1 Midbrain infusions of SK channel agonists** 

Fig. 1. Effect of the SK channel agonist 1-EBIO on the number of TH+ SNc cells in 6-OHDAlesioned 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). Two weeks later a cannula was implanted into the left SNc, through which 1-EBIO or vehicle was infused (from an attached minipump) continuously for a further 2 weeks. The number of TH+ cells in the left and right SNc of each mouse was estimated using unbiased stereology by an observer who was unaware of the treatment received. The data for each mouse (filled squares) are expressed as the ratio of the number of TH+ SNc cells on the lesioned and treated (left) side relative to the internal control (right side). The mean of each treatment group is represented by a horizontal line. Lesion and vehicle-treated mice (n=15, black symbols) have a ~50% reduction, on average, in the number of TH+ SNc cells. Treatment with 100μM 1-EBIO (n=9, pale red symbols) appears to have had a beneficial effect (see text) in some mice, but there was no change on average. Treatment with 200μM 1-EBIO (n=10, red symbols) appears to have had an even stronger beneficial effect in some mice, resulting in an average increase in SNc TH+ cells to ~80% of normal. Note, however, that this increase was not statistically significant (p=0.068, one-way ANOVA).

We tested the neuro-restorative effects of orally administered riluzole on motor symptoms and TH+ SNc cell number in a mouse PD model. Three weeks after 6-OHDA SNc lesion, mice were administered riluzole in their drinking water for 2 weeks. Both 3μM and 30μM riluzole doses were tested. Note these were the projected daily systemic concentrations based on the average daily volume of drinking water consumed and body-weight of each mouse. Every 3 days following the lesion and throughout the treatment period, the rotational behavior of the mice in response to amphetamine (5mg/kg i.p.) was measured (figure 2A & A'). Following 6-OHDA lesion and before treatment began (figure 2A), the relative number of ipsiversive rotations (rotations toward the side of the lesion) increased progressively to reach a maximum by 12 days [p=0.035 (time), two-way ANOVA]. Presumably this reflects gradual depletion of DA in the left striatum caused by progressive degeneration of DA neurons in the left SNc. The relative number of contraversive rotations

Activity-Dependent Regulation of the Dopamine

during the cylinder test (data not shown).

**3.2 Striatal infusions of quinpirole** 

OHDA-lesioned rats.

similar in 1-EBIO- and vehicle-infused rats (figure 3B).

shown).

**3.1.3 1-EBIO in rats** 

Phenotype in the Adult Substantia Nigra: Prospects for Treating Parkinson's Disease 285

Following riluzole treatment, we estimated the total number of TH+ SNc cells and measured the density of DAT immunoreactivity in the dorsal striatum in these mice. No differences were found across treatment groups in the number of TH+ SNc cells (figure 2B; p=0.952, one-way ANOVA) or the density of DAT immunoreactivity in the dorsal striatum (data not

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

At the experiment end-point (15 weeks after lesion), the number of SNc TH+ cells was

While infusion of SK channel agonists (1-EBIO or riluzole) into the normal adult mouse SNc recruited ~500 more TH+ SNc cells (Aumann et al. 2008, Aumann et al. 2011), infusion of the D2 DA receptor agonist quinpirole into the normal adult mouse striatum was far more potent in this respect; recruiting ~3000 new TH+ SNc cells (Aumann et al. 2011). We therefore examined the behavioral and cellular effects of striatal quinpirole infusion in 6-

Four different behaviors were measured before and after unilateral 6-OHDA lesion of the SNc and during treatment (100nM quinpirole or vehicle). These are plotted in figure 4A-D. While there was no obvious effect of lesion or treatment on locomotor and rearing behavior (figure 4A-B), an asymmetry in left/right forelimb use (cylinder test and corridor test) and possibly also left/right side attention (corridor test) was evident following lesion (figure 4C-D). The lesion was made in the right SNc in this experiment, resulting in left forelimb

(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 motor dysfunction in this experiment.

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 these data are represented.

Following riluzole treatment, we estimated the total number of TH+ SNc cells and measured the density of DAT immunoreactivity in the dorsal striatum in these mice. No differences were found across treatment groups in the number of TH+ SNc cells (figure 2B; p=0.952, one-way ANOVA) or the density of DAT immunoreactivity in the dorsal striatum (data not shown).
