Withdrawal Symptoms

Alcohol administration through the voluntary consumption of gelatin containing alcohol was presented to adolescent rats (PND 30–50) in a gel matrix consisting of distilled water (76.67%), Knox Gelatin (3.33%), polyose (10%), and 190 proof ethanol (10%), whereas the control gelatin contained distilled water in place of ethanol. Preparation was as described (Rowland et al., 2005 (link); Nasrallah et al., 2011 (link); Schindler et al., 2014 (link)). Gels were available 24 h a day with ad libitum access to food and water. Gel intake levels were measured daily and expressed in g/kg of body weight. All rats had access to only control gelatin for the first three days; after which rats were divided into either ethanol or control gelatin groups matched by weight and baseline intake for 20 days of assigned gelatin intake.
While this mode of administration produces enduring effects on cognition (Nasrallah et al., 2009 (link), 2011 (link); Schindler et al., 2014 (link), 2016 (link); Spoelder et al., 2015 (link); Kruse et al., 2017 (link)), it does not achieve blood-alcohol concentrations that model heavy episodic drinking in adolescents. Therefore, we also utilized a second model of AAU that produces higher blood-alcohol concentrations (Crews et al., 2016 (link)). Adolescent intermittent ethanol administration via intragastric (IG), alcohol was presented to adolescent rats (PND 25–55) as a mixture of 190-proof ethanol and distilled water (16 g/kg, 20% ethanol, weight over volume). One cohort of rats received a single daily IG administration of ethanol and the other cohort received a single daily IG administration of distilled water (comparable volumes of water) on a 2-day on/off schedule Animals were then weighed and monitored daily.
For both IG and gelatin methods, after the last day of administration, the animals underwent three to four weeks of withdrawal and were monitored daily for withdrawal symptoms (e.g., seizures, weight loss, lack of grooming, and anxious behavior). It is important to mention that no overt signs of withdrawal were observed. Once the withdrawal period was completed, the rats began a food restriction diet of 90 ± 2% of their bodyweight and were exposed to 45 mg sucrose pellets (Bio-Serv, Frenchtown, NY) in their home cage to reduce neophobia. Additionally, prior to the start of the behavioral tasks, the rats underwent one magazine-training session in a standard operant chamber (Med Associates, St. Albans, VT) where they were given 15 min to consume 10 sucrose pellets in the magazine tray.

This work was performed in compliance with the Declaration of Helsinki and was approved by the Ethics Committee of the Affiliated Hospital, Chengdu University of Traditional Chinese Medicine (NO: 2021KL-094). A WeChat mini-program was used to execute questionnaires for selecting volunteers in Chengdu, China. All participants provided written informed consent before the study. MPASD volunteers and healthy controls were recruited by the MPATS and PSQI. The MPATS contains 16 questions, including withdrawal symptoms, salience, social comfort, and mood changes. The inclusion criteria of MPASD subjects were ≥ 40 (MPATS) and ≥ 7 (PSQI) scores (31 ). To minimize the interference of severe sleep disorders upon MPA, those characterized as “poor” sleepers (7 ≥ PSQI ≥ 15 scores) were recruited from a non-clinical population (32 (link)). The PQSI score was made up of questions including sleep quality, sleep latency, sleep duration, sleep efficiency, sleep disturbance, daytime dysfunction, and medication use. The exclusion criteria included one of the following conditions: (1) Oral diseases, including periodontitis, bleeding gums or tooth decay. (2) The antibiotic regimen within the last 3 months. (3) Upper respiratory tract infection or rhinitis or pharyngitis whinth 1 month. (4) Smokers and alcohol abusers and other types of addicts. Healthy controls were simultaneously recruited into group N. All enrolled subjects completed the 72-h constant routine.