**3.1.1 1-EBIO in mice**

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 not shown).
