The influence of adolescent nicotine exposure on ethanol intake brain gene expression

Background: Nicotine and alcohol are often co-abused. Adolescence is a vulnerable period for the initiation of both nicotine and alcohol use, which can lead to subsequent neurodevelopmental and behavioral alterations. Aim: The aim was to to determine the effect of nicotine exposure during adolescence on ethanol intake, and the effect of both substances on brain gene expression. Methods: RNA was extracted from a randomly selected subset of mice (16 total; 4 samples from each experimental group). Whole brains (including cerebellum) were dissected. Total RNA was extracted with an RNeasy® Midi Kit (QIAGEN, Valencia, CA). RNA quality was assessed using an Agilent 2100 BioAnalyzer™. An Illumina TruSeq® Stranded mRNA Library Prep Kit (Illumina, San Diego, CA) was used for cDNA library preparation following the manufacturers’ protocol. Libraries were sequenced using an Illumina HiSeq 2500 (Illumina, San Diego, CA). Trimmomatic was used to remove sequencing adapters and low-quality ends. The cleaned dataset was analyzed with the Tuxedo pipeline. Subsequently, readings were mapped to the mouse reference genome (Ensembl GRCm38, mm10) using TopHat2 software (http://tophat.cbcb.umd.edu/). The ‘--library_type’ parameter was set to ‘fr-firststrand’. Default settings were preserved for all other TopHat2 parameters. The resulting alignments files from TopHat2 were used to generate a transcriptome assembly. Gene expression was calculated for each condition using the Cufflinks and Cuffmerge utilities. Cuffdiff2 with default settings was used to identify transcripts that were differentially expressed between each treatment group compared to the water only control group.The significance threshold was set at q < 0.05 (FDR corrected). Finally, a Fisher’s exact test was performed using the GeneOverlap R package to test the significance of DEG overlaps. Gene co-expression networks were identified using the Weighted Gene Co-expression Network Analysis (WGCNA) package. Results: RNA Integrity Number (RIN) was on average 8.23 ± 0.26 for all samples, suggesting high RNA integrity and quality. On average, 43 million, 150 base pair single end reads were generated for each sample and used in the analysis. The resulting alignments files from TopHat2 had an average mapping rate of 87.4%. Nicotine Treatment: Mice were randomly assigned into four groups: Water-Water (WW), Water-Ethanol (WE), Nicotine-Water (NW), or Nicotine-Ethanol (NE). During the first six days of the experiment, mice were exposed to 3 glass drinking bottles filled with water or nicotine for 22 h a day, and a single water bottle for 2 h each day (Fig 1). For the WW and WE groups, all 3 bottles were filled with tap water. For the NW and NE groups, all 3 bottles were filled with 200 µg/ml (−)-nicotine freebase (Sigma–Aldrich, St. Louis, MO) dissolved in tap water. Bottles were placed on the cages at 1500 h and were removed and replaced with the single water bottle at 1300 h the next day. The three bottles were read and nicotine consumption (mg/kg) was calculated for each mouse. The 2 h single bottle was weighed and water consumption (ml) was calculated. Leakage/evaporation was accounted for by tubes on control cages handled using the same protocol, but with no animal present. We subtracted the volume lost in control tubes from individual drinking values. These procedures continued throughout the experiment. However, during the last 4 days (PND 42-45) mice were exposed to ethanol via the drinking-in-the-dark (DID) protocol. Drinking-in-the-dark: During experimental days (7-10) nicotine exposure continued as detailed above (22 h/day), however, at 1300 h, all 3 bottles (nicotine or water) were removed and replaced with a single 10 ml serological pipette fitted with a ball bearing drinking spout containing either ethanol or water. Ethanol was prepared from ethyl alcohol (200 proof; Koptec 200) diluted in tap water to produce a 20% v/v solution. Mice had 2 h access to a single bottle of water or ethanol for three days (PND 42-44). On the final day (PND 45), mice had 4 h access to the ethanol bottle. Leakage/evaporation was accounted for by tubes on control cages as described above. At the end of the 4 h drinking session on the final day, blood samples were collected from the tail vein (10µl) and mice were sacrificed via cervical dislocation, whole brains including the cerebellum were dissected and placed into RNAlater® for subsequent RNA extraction.