Riluzole

Spontaneous tail coiling behavior was evaluated in 20 hpf embryos, after testing each for the lack of a touch-evoked coiling response by means of gentle sensory stimulation. Five embryos at a time were dechorionated and transferred to fish water in a 3.5 mm round petri dish with five niches. Tail coiling was detected at RT during a 5-minute videorecording (time resolution 45 frames/sec) obtained using a digital camera mounted on a stereomicroscope (Leica Microsystems) and for each embryo, a record was made of the frequency of spontaneous tail coiling, the percentage of multiple or complex coilings (i.e. coilings consisting of two or more repeated bends of the trunk before returning to the resting position) and the relative percentages of multiple/complex coilings consisting of contralateral left-right bends of the entire body or ispilateral bends. The effect of riluzole (Sigma-Aldrich) or vehicle (DMSO) on spontaneous tail coilings was tested by gently removing the fish water in order to keep the embryos in the same position, and then adding fish water containing 5 μM riluzole or 0.2% DMSO for five minutes before recording another 5-minute video and making the same analyses.

For these studies, one group of 5XFAD mice (n = 8) was given ad libitum access to Riluzole solution, i.e., 13 mg/kg per mouse per day, a dose that has previously been reported to improve cognition and reduce tau pathology in P301L tau AD mice^{5 (link),17} , from 1 to 6 months of age (total treatment duration = 5 months). A control group of 5XFAD mice (n = 7) and the wild-type (WT) mice (n = 10) had ad libitum access to tap water. For immunohistochemical studies, a separate batch of 5XFAD mice was treated with Riluzole (n = 5) or tap water as control (n = 5). We chose sample sizes based on past literature utilizing these methodologies^{4 (link),7 (link),18 (link)–20 (link)} with the intention to minimize the number of animals to be used for the present study.
Riluzole (R116; Sigma-Aldrich, St Louis, MO, USA) was dissolved in room temperature tap water at a stock concentration of 0.12 mg/ml and stirred for ~6 h, covered by foil to prevent light exposure. The stock solution was diluted into the mouse drinking water based on (1) average animal weight and, (2) water consumption over the previous 24 h period and throughout the study, which was measured by weighing the water bottles. We prepared fresh Riluzole solution after every 2^nd to 3^rd day during the entire treatment period.

Ebselen, riluzole and edaravone were obtained from a commercial supplier (#E3520, #R116, and #443300 respectively Sigma Aldrich). Details of the synthesis and characterisation of MR6-8-2 and MR6-26*2 have been described in our previous report^{25 (link)}.