Gabapentin

AAV2/5 GfaABC₁D-hMADi-mCherry was
microinjected into the dorsal striatum of R6/2 or NCAR mice at 4 weeks old.
Two weeks after the microinjection, 1 mg/kg clozapine
N-oxide (CNO; Tocris Bioscience, 4936) was administered to
mice by i.p. injection every other day for 3-6 weeks to chronically activate
hM4Di in dorsal striatal astrocytes in vivo. Behavioral
tests, brain slice experiments and immunohistochemistry were performed two
hours after the last CNO administration. In some cases, 100 mg/kg Gabapentin
(Tocris Bioscience, 0806) was administered by i.p. injection to mice 1 hour
before every CNO injection. Previous work shows that the effects of striatal
astrocyte hM4Di DREADD activation in mice are reversible (Nagai et al., 2019 (link)), which is consistent with
prior assessments in other cells (Alexander
et al., 2009). However, the reversibility experiment could not be
performed in R6/2 mice for the present study, because by ~12 weeks
the mice are sick from the disease in other parts of the body (recall we
microinject AAV for hM4Di only into the striatum, whereas the disease
affects other parts of the body as well). Hence, the humane endpoint
considerations of our experiments mandate that the mice be euthanized before
they succumb to disease throughout the body.

AM251, 1-(2,4-Dichlorophenyl)-5-(4-iodophenyl)-4-methyl-N-1-piperidinyl-1H-pyrazole-3-carboxamide, and naloxone were purchased from Sigma-Aldrich (St. Louis, MO, USA). SB-334867, N-(2-Methyl-6-benzoxazolyl)-N’−1,5-naphthyridin- 4-yl urea, and gabapentin were purchased from Tocris Bioscience (Bristol, UK). For i.p. injections, SB-334867 and AM251 were dissolved in 0.9% saline solution containing 10% (w/v) encapsin and 2% (v/v) dimethylsulfoxide (DMSO). Morphine and naloxone were dissolved in 0.9% saline. All drugs were prepared as the working concentrations for i.p. injections.

To induce opioid analgesic tolerance, morphine sulfate (West Ward Pharmaceuticals, Eatontown, NJ) was injected intraperitoneally at a dose of 5 mg/kg (in rats) or 10 mg/kg (in mice) twice a day for 8 consecutive days^{4 (link),7 (link)}. The α2δ-1Tat peptide and scrambled control peptide were synthesized by Bio Basic Inc. (Marham, Ontario, Canada) and validated by using liquid chromatography and mass spectrometry. The α2δ-1Tat peptide and Tat-fused scrambled control peptide were dissolved in saline and injected intrathecally, followed by a 10 μl saline flush 20 min before morphine administration on each testing day. Gabapentin (Tocris Bioscience, Ellisville, MO) was dissolved in saline and injected intraperitoneally before morphine administration on each testing day.