Supporting Information
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Supporting Information Hill et al. 10.1073/pnas.0914661107 SI Text (Fig. S1 C and D respectively). Thus, in our hands, over the range of Determination of Doses for Pharmacological Agents Targeting Endo- concentrations studied, the highest doses for AM404 and URB597 cannabinoid Signaling. To obtain dosages suitable for detecting re- did not alter the acute corticosterone response in the rat. Based on peated stress-dependent shifts in CB1 receptor requirements and the results of these pilot studies, the doses used for all subsequent AEA metabolism, we determined concentrations for AM251, tests of repeated restraint were 1 mg/kg for AM251, 0.3 mg/kg for URB597, and AM404 that were subthreshold with respect to the URB597, and 2 mg/kg for AM404. corticosterone response to acute restraint, and a concentration of AM251 that was subthreshold for basal corticosterone secretion Daily Administration of Drugs That Modulate Endocannabinoid (Fig. S1). Under basal conditions, corticosterone concentrations Signaling on Stress Habituation. Daily administration of the CB1 increased after acute injections of 2 mg/kg and 5 mg/kg doses of the receptor antagonist AM251 (1 mg/kg) before stress exposure pre- CB1 receptor antagonist AM251, but not after a 1-mg/kg dose (Fig. vented the habituation of stress-induced corticosterone secretion A S1 ). There was no effect of the 1 mg/kg dose of AM251 on the that was seen in animals that received daily injections of vehicle magnitude of the corticosterone response to a single episode of before stress exposure for 9 consecutive days. Similarly, animals that 30-min restraint (Fig. S1B). There was no significant effect of the anandamide hydrolysis inhibitor URB597 (0.1 and 0.3 mg/kg) or received daily administration of URB597 (0.3 mg/kg) or AM404 the anandamide hydrolysis/uptake inhibitor AM404 (0.5, 1, and (2 mg/kg) demonstrated comparable habituation of stress-induced 2 mg/kg) to alter the corticosterone response to 30-min restraint corticosterone secretion as vehicle treated animals. Fig. S1. Mean (±SEM) corticosterone (CORT; ng/mL) concentrations in circulation in animals receiving single systemic injections of AM251 under basal (A)or acute restraint conditions (B), and in animals receiving URB597 (C) or AM404 (D) under acute restraint. *P < 0.05 vs. vehicle (VEH) injections (n = 5/drug dose). Hill et al. www.pnas.org/cgi/content/short/0914661107 1of2 Fig. S2. Mean (±SEM) corticosterone (CORT; ng/mL) concentrations in circulation on first (Acute) and last (Repeat) day of restraint at 30-min exposure in animals bearing daily systemic injections of vehicle (VEH), CB1 receptor antagonist AM251, anandamide hydrolysis inhibitor URB597, or anandamide uptake/ hydrolysis inhibitor AM404. *P < 0.05 vs. acute counterpart (n = 5/group). Fig. S3. (A) Representative photomicrograph of needle track terminating in basolateral amygdala. As defined by morphological features highlighted by thionin staining (1), structures labeled for reference are as follows: BMA, basomedial amygdala; CEA, central amygdala; and MEA, medial amygdala. (B)Di- agrams of brain sections from all rats included in the analyses, bearing proper needle tracks terminating only in the basolateral amygdala. 1. Swanson LW (2004) Brain Maps: Structure of the Rat Brain (Academic, San Diego), 3rd Ed. Table S1. Mean (±SEM) tissue content levels (nmol/g protein) for 2-arachidonoylglycerol (2- AG) in unstressed animals and at 30 min and 24 h after last bout of repeated restraint exposure (n = 8/group) Region Unstressed Repeat (30 min) Repeat (24 h) Prefrontal cortex 3.0 ± 0.1 2.9 ± 0.3 3.1 ± 0.3 Hippocampus 4.0 ± 0.3 4.1 ± 0.2 4.5 ± 0.4 Hypothalamus 7.9 ± 1.2 6.1 ± 0.5 6.5 ± 0.3 Thalamus 3.5 ± 0.5 3.3 ± 0.5 3.5 ± 0.2 Hill et al. www.pnas.org/cgi/content/short/0914661107 2of2.