Sr141716a

Allyl isothiocyanate (AITC; Sigma-Aldrich, Inc., St. Louis, MO, USA) was mixed in mineral oil at a concentration of 10% and injected into the periosteal space in a volume of 10 µl. Microinjection of 10% AITC onto the dura has previously been shown to mimic migraine-like pain in rodents (Edelmayer et al., 2012 (link); Kandasamy et al., 2017b (link)). Δ⁹-tetrahydrocannabinol (Sigma-Aldrich, Inc., St. Louis, MO, USA) was dissolved in vehicle (1:1:18; ethanol:cremophor:saline) and injected intraperitoneally at doses of 0.1, 0.32, and 1.0 mg/kg in a volume of 1 ml/kg. The CB₁ receptor antagonist SR141716A (1.0 mg/kg) and the CB₂ receptor antagonist SR144528 (3.2 mg/kg) (Tocris Bioscience, Minneapolis, MN, USA) were dissolved in the same vehicle as THC and injected intraperitoneally in a volume of 1 ml/kg. These drugs are highly selective for their target receptor (Rinaldi-Carmona et al., 1995 (link); 1998 (link)) and the doses used are known to block the antinociceptive effects of systemically administered THC in female rats(Craft et al., 2012 (link)).

Neurons were treated with HIV-1 Tat_1–86 (10–500 nM; ImmunoDiagnostics; clade 267 B), PF3845 (10–100 nM, Dr. Benjamin Cravatt), CB₁R antagonist SR141716A (SR1, 50 nM, Tocris, Ellisville, MO) and the CB₂R antagonist AM630 (50 nM, Tocris, Ellisville, MO). Concentrations of PF3845 were chosen based on preliminary experiments (data not shown) and previous studies that assessed the activities of these drugs in vitro (Ahn et al., 2009 (link); Niphakis et al., 2013 (link)). Tat concentrations in the 10–500 nM range were selected as these concentrations recapitulate the cellular deficits found in individuals with HIV-1 mediated pathology (Kruman et al., 1998 (link); El-Hage et al., 2008 (link); Perry et al., 2010 (link); El-Hage et al., 2011 (link)). For all experiments PF3845 was added 30 min prior to experiment start. Tat was added for calcium imaging 1 min after the experiment started and for neuronal survival and dendrite morphology assessments at the beginning of experimental studies. In order to determine the contribution of CB₁R and/or CB₂R activity to observed neuroprotective effects, cultures were incubated with SR1 or AM630 30 min prior to PF3845 treatment and were present throughout the duration of the experiment. Antagonist drug concentrations were chosen to maximally block treatments based on preliminary explorative assessments conducted prior to the main experiments.