Sex- and Genotype-Dependent Nicotine-Induced Behaviors in Adolescent Rats with a Human Polymorphism (rs2304297) in the 3'-UTR of the CHRNA 6 Gene

Abstract

In human adolescents, a single nucleotide polymorphism (SNP), rs2304297, in the 3'-UTR of the nicotinic receptor subunit gene, CHRNA6, has been associated with increased smoking. To study the effects of the human CHRNA6 3'-UTR SNP, our lab generated knock-in rodent lines with either C or G SNP alleles. The objective of this study was to determine if the CHRNA6 3'-UTR SNP is functional in the knock-in rat lines. We hypothesized that the human CHRNA6 3'-UTR SNP knock-in does not impact baseline but enhances nicotine-induced behaviors. For baseline behaviors, rats underwent food self-administration at escalating schedules of reinforcement followed by a locomotor assay and a series of anxiety tests (postnatal day (PN) 25-39). In separate cohorts, adolescent rats underwent 1- or 4-day nicotine pretreatment (2×, 30 μg/kg/0.1 mL, i.v.). After the last nicotine injection (PN 31), animals were assessed behaviorally in an open-field chamber, and brain tissue was collected. We show the human CHRNA6 3'-UTR SNP knock-in does not affect food reinforcement, locomotor activity, or anxiety. Further, 4-day, but not 1-day, nicotine exposure enhances locomotion and anxiolytic behavior in a genotype- and sex-specific manner. These findings demonstrate that the human CHRNA6 3'-UTR SNP is functional in our in vivo model.

Keywords: CHRNA6; adolescent; anxiety-like behavior; locomotor activity; pharmacogenetics.

Figure 1

Generation of Humanized CHRNA6 3′-Untranslated Region (UTR) SNP Rodents. Site-specific double-stranded breaks (DSB) were introduced at the CHRNA6 3′-UTR of the wild-type (WT) Sprague-Dawley (green, 95 nucleotides) via Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated (Cas9) endonuclease methods followed by the insertion of a donor vector through homologous recombination (HR) DNA repair. A human CHRNA6 3′-UTR (gold, 559 nucleotides) containing either the minor or major SNP allele at nucleotide position 123 (SNP location indicated by *C or *G, respectively) was introduced by a donor vector. F2 offspring resulting from confirmed founders and F1 crossbred pups resulted in homozygous major (α6^GG) and homozygous minor (α6^CC) humanized CHRNA6 3′-UTR SNP rodents. Ampicillin (Amp); base pair (bp); AscI and NotI: restriction enzyme recognition sites; plasmid origin of replication (pUC ori).

Figure 2

The human CHRNA6 3′-UTR knock-in does not impact baseline behavior. (A) Experimental paradigm for baseline behaviors. Postnatal Day (PN), Elevated Plus Maze (EPM), and Light/Dark Box Test (LDT). (B) Mean daily 30 min response ± SEM for food self-administration at Fixed Ratio (FR)1, FR2, and FR5 schedules of reinforcement, n = 16/group. **** p < 0.0001; *** p < 0.001; ** p < 0.01; * p < 0.05 Reinforced (Reinf) vs. Non-Reinforced (Non Reinf) responses. (C) Mean breakpoint ± SEM for food self-administration under a progressive ratio (PR) schedule of reinforcement, n = 16/group. (D) Mean total ambulatory counts ± SEM, n = 15–16/group. (E) Mean percentage (%) of time spent in the center of an open field ± SEM, n = 15–16/group. (F) Mean % of time spent in the open arm of an EPM ± SEM, n = 16/group. (G) Mean % of time spent on the light side of a Light/Dark box ± SEM, n = 8/group. * p < 0.05 females vs. males. Circles represent females and squares represent males.

Figure 3

Nicotine-induced locomotion in humanized CHRNA6 3′-UTR SNP rats. Mean total ambulatory counts ± SEM for (A) 1-day nicotine exposure, collapsed by sex n = 13–19/group. (B) 4-day nicotine exposure, * p < 0.05, *** p < 0.001 Nic vs Sal; ^ p < 0.05 α6^CC Nic vs α6^GG Nic n = 7–10/group. Saline (Sal), Nicotine (Nic). Circles represent females, and squares represent males.

Figure 4

Nicotine-induced anxiolytic behavior in humanized CHRNA6 3′-UTR SNP rats. Mean percent (%) time spent in center ± SEM for (A) acute (1-day) nicotine exposure *** p < 0.001 α6^CC Nic vs α6^CC Sal, ^^ p = 0.01 α6^CC Nic vs α6^GG Nic n = 13–18/group. (B) Sub-chronic (4-day) nicotine exposure, * p < 0.05, ** p < 0.01 Nic vs Sal; ^ p < 0.05, ^^ p < 0.01 α6^CC Nic vs α6^GG Nic, n = 7–10/group. Saline (Sal), Nicotine (Nic). Circles represent females, and squares represent males.

Figure 5

Nicotine does not alter α6 nicotinic receptor subunit mRNA expression in humanized CHRNA6 3′-UTR SNP Rats. Mean dpm/mg ± SEM for sub-chronic nicotine exposed male and female rats in the (A) substantia nigra (SN), (B) anterior ventral tegmental area (aVTA), (C) posterior ventral tegmental area (pVTA), and (D) interpeduncular nucleus (IPN), n = 3–5/group. Saline (Sal), Nicotine (Nic). Circles represent females, and squares represent males.