All nicotine doses are expressed as free base concentrations. Saline or nicotine was injected subcutaneously (s.c., 0.05 ml/10 g body weight) 5 min before the test. Experimental Design Cognitive, Affective, and Locomotor Behaviors All behavioral procedures are described in detail in the Supplementary material online. Two cohorts of mice were tested, one (15 ��4+/+ and 14 customer reviews ��4?/? females, 10 ��4+/+ and 11 ��4?/? males) in a battery of cognitive tasks and the other (9 ��4+/+ and 9 ��4?/? females, 8 ��4+/+ and 11 ��4?/? males) to replicate the cognitive tests and examine locomotor activity and affective-like behaviors. For behavioral testing, a within-subjects design was used so that mice of each genotype were subjected to testing in the following order: Y maze, Barnes maze training, Barnes maze probe test, cue- and context-induced conditioned fear, Barnes maze retention, Barnes maze reversal, conditioned fear retention.

After the completion of the cognitive test battery, the second mouse cohort was tested in additional procedures assessing locomotor activity and affective-like behavior. Again, a within-subjects design was employed such that mice of each genotype were subjected to testing in the following order: light�Cdark transfer test, marble burying test, locomotor activity test, tail suspension test, and forced swim test. Nicotine-Induced Analgesia Two additional cohorts of na?ve ��4+/+ and ��4?/? mice were tested for nicotine-induced analgesia using the tail immersion and hot plate tests (see Supplementary material online for details).

The first cohort contained 21 ��4+/+ and 21 ��4?/? females, and the second cohort contained 12 ��4+/+ and 15 ��4?/? females, as well as 17 ��4+/+ and 28 ��4?/? males. In each cohort, mice were first tested in the tail immersion test. Nicotine (0, 0.5, 1, 2, and 3 mg/kg, s.c., 5 min before test) was administered according to a within-subjects Latin square design during 5 consecutive days. One week later, the same mice were tested in the hot plate test. For each sex and genotype, mice were split into three subgroups, and nicotine (0, 1, and 3 mg/kg, s.c., 5 min before test) was administered according to a between-subjects design. Each mouse was only tested once in the hot plate test. Statistical Analyses Statistical analyses were performed using the Biomedical Computer Programs for Personal Computers Statistical Package (BMDP, Los Angeles, CA), the GraphPad Prism statistical package (GraphPad, San Diego, CA), and Statview v.

5.0 (SAS Institute Inc., Cary, NC). Two-way repeated-measures analysis of variance (ANOVA) was used to analyze the data collected from the cognitive test battery. The between-subject factors were Genotype and Sex, and the within-subject factors were Time Intervals (cued and contextual conditioning) and Trials/Blocks (the Barnes maze Carfilzomib test).