Experimental design

The main purpose of this experiment was to evaluate whether continuous EE (24 h/day) could alter ethanol consumption after periods of withdrawal and after a stressful situation. The experimental design is shown in Fig 1. Twenty-one mice were exposed to the drinking-in-the-dark (DID) paradigm for 15 days for the acquisition and stabilization of ethanol consumption. To facilitate the escalation of ethanol consumption, we included three periods of ethanol deprivation (2 days off). Immediately after the last consumption session (day 15), approximately 20 μl blood samples were collected from the submandibular vein using a 5.0 mm lancet [35]. Blood was collected in a heparinized tube and centrifuged at 2,000 x g for 10 min at 4°C to separate plasma and then stored at -20°C until the time of the assay. The plasma was used to quantify blood ethanol concentrations (BECs) using an enzymatic assay and Analox GL6 Multiassay Analyser (Analox Instruments, Lunenburg, MA, USA). Blood ethanol concentrations are expressed as milligrams of ethanol per milliliter of blood (mg/ml). Following the acquisition phase, the animals were randomly distributed into two groups: standard housing conditions (SC; n = 10) and EE for 24 h/day (EE24h; n = 11). After six days of ethanol deprivation, the mice were offered two bottles containing either 20% ethanol or tap water for 2 h only, once a week, for 4 weeks. The mice were tested in the elevated plus maze (EPM) on week 5. On weeks 5 and 6, mice were given 24 h free access to ethanol and the consumption was measured 2 and 24 h from the start of drinking. Next, in order to evaluate if restraint stress alters ethanol consumption, mice were subjected to a 1-h restraint stress session before being given 24-h free access to ethanol at week 6 of the experiment. Ethanol consumption can decrease immediately after exposure to a restraint stress [36], so we also measured the consumption after 2 and 24 h of ethanol drinking.

The short arrows represent weekly exposure to the two-bottles choice paradigm (20% ethanol and water) for 2 h. The long arrows represent re-exposures for additional 22 h. Cross = elevated plus maze test. • = BEC measurement.

This experiment followed similar experimental design (Fig 1), with the exception that mice were exposed to EE for 3 h/day instead of 24 h/day. Twenty animals were subjected to the acquisition phase and then randomly allocated to two groups: control (SC, n = 10) and EE3h (n = 10). In this experimental procedure, the objects were placed in the cages immediately after lights were off (9:00 AM) and then removed after 3 h (12:00 PM). Cages of the control mice were manipulated by the experimenter as a control for the disturbances caused by manipulating the objects in the EE3h cages. Considering that in Experiment 1 we observed no significant differences in ethanol consumption during the first five re-exposures to ethanol, we limited the re-exposures to 4 weeks in Experiment 2. During re-exposures at weeks 3 and 4, intake was measured twice (i.e., at the end of the first 2 h and at 24 h after the onset of consumption). The EPM test was performed on the 3rd week, and the animals were exposed to 1-h restraint stress on the 4th week, immediately before the ethanol consumption test. Considering the differences in ethanol consumption between the EE and SC groups during the 24 h of ethanol access after stress exposure in Experiment 1, we measured plasma corticosterone levels in this experiment. Blood samples were collected after euthanasia, and plasma corticosterone levels were determined in duplicate using a Corticosterone EIA Kit (Cayman Chemical, Ann Arbor, MI, USA) according to the manufacturer’s instructions.

A supplementary experiment to investigate whether stress alters ethanol intake was performed following the same protocol as described in Experiment 2, with the exception that the mice (n = 9) had access only to the ethanol bottle during the 2h re-exposure tests. Water bottles were not offered (S1 Appendix).