A common SNP in Chrna5 enhances morphine reward in female mice

Abstract

The single nucleotide polymorphism (SNP) D398N (rs16969968) in CHRNA5, the gene encoding the α5 subunit of the nicotinic acetylcholine receptors (nAChR), has been associated with both nicotine and opiate dependence in human populations. Expression of this SNP on presynaptic VTA dopaminergic (DA) neurons is known to cause a reduction in calcium signaling, leading to alterations in transmitter signaling and altered responses to drugs of abuse. To examine the impact of the Chrna5 SNP on opiate reward and underlying dopaminergic mechanisms, mice harboring two copies of the risk-associated allele (Chrna5 A/A) at a location equivalent to human rs16969968 were generated via CRISPR/cas9 genome editing. We sought to determine whether Chrna5 A/A mice show differences in sensitivity to rewarding properties of morphine using the conditioned place preference paradigm. When mice were tested two weeks after conditioning, female Chrna5 A/A mice showed significantly enhanced preference for the morphine-paired chamber relative to WT females, suggesting that this genotype may enhance opioid reward specifically in females. In contrast, Chrna5 genotype had no effect on locomotor sensitization in male or female mice. Relative to WT females, peak amplitude of ACh-gated currents recorded from VTA DA neurons in Chrna5 A/A females was potentiated 1 day after conditioning with morphine. Increased FOS expression was also observed in Chrna5 A/A mice relative to WT mice following exposure to the morphine CPP chamber. We propose that impaired α5 nAChR subunit function alters DA neuron response following repeated morphine exposures, and that this early cellular response could contribute to enhanced opiate reward two weeks after conditioning.

Keywords: Conditioned place preference; Mice; Morphine; Nicotinic acetylcholine receptor; Reward; Single nucleotide polymorphism.

Fig. 1.. Derivation of the Chrna5^A/A line.

(A) Relevant sequence of the mouse Chrna5 gene. Blue: repair template with left and right homology arms and the G to A change marked as the red box. Purple: guide RNA (gRNA) used to induce double strand break in combination with the Cas9 enzyme. Green and orange: PCR primers. (B) PCR genotyping of a litter of 8 mice obtained from Chrna5A/G to Chrna5A/G mating’s. PCR products were digested with the Taq1 restriction enzyme. The G allele produces two bands of 119 and 50 bp; the A allele one of 169 bp. Mouse numbers 1, 4, 6 and 7 are Chrna5^A/A homozygotes. (C) Sanger sequencing of the PCR product of mouse 7 confirms the presence of the A allele.

Fig. 2.. Chrna5 A/A genotype enhances morphine reward in female mice.

CPP preference score (time spent in morphine-paired chamber minus time spent in saline-paired chamber) in male (A) and female (B) mice (n = 9–13/group). Preference scores were obtained one day before conditioning (Pretest), and one day and two weeks after conditioning (1 Day Post and 2 Weeks Post). **p < 0.01, ***p < 0.001, ****p < 0.0001 relative to Pretest. #p < 0.05 relative to WT. Error bars represent SEM.

Fig. 3.. Chrna5 genotype does not impact morphine-induced locomotor sensitization.

Locomotor activity counts in male (A, C) and female (B, D) mice (n = 9–13/group) on morphine (A, B) and saline (C, D) conditioning days. For panels A and C: ****p < 0.0001 WT relative to Morphine Conditioning Session 1. #p < 0.05, ###p < 0.001, ####p < 0.0001 Chrna5 A/A relative to Morphine Conditioning Session 1. For panels B and D: *p < 0.05, **p < 0.01, ***p < 0.001 WT relative to Saline Conditioning Session 1. #p < 0.05 Chrna5 A/A relative to Saline Conditioning Session 1.Error bars represent SEM.

Fig. 4.. Peak current amplitude recorded from VTA DA neurons is higher in Chrna5 A/A females compared to G/G females 1 day after morphine conditioning.

(A) Typical traces of hyperpolarization-activated potassium currents (Ih) which were activated by hyperpolarizing voltage steps (−10 mV each) from the holding potential (−60 mV), indicative of dopaminergic neurons. (B) Example of a biocytin back-filled neuron from the VTA stained for tyrosine hydroxylase. (C) Representative current traces and (D) peak current amplitude (pA) recorded from VTA dopamine neurons in mice (n = 3–6/group) that were drug-naïve, 1-day post-conditioning with 10 mg/kg morphine, or 2 weeks post-conditioning with 10 mg/kg morphine. Red arrows indicate puff application of ACh. **p < 0.01. Error bars represent SEM.

Fig. 5.. Augmented neuronal activation in Chrna5 A/A mice after morphine conditioning.

(A) Representative images of FOS expression in the nucleus accumbens (NAc) of female WT and Chrna5 A/A mice conditioned with morphine. FOS expression was quantified in the prelimbic cortex (PL) and infralimbic cortex (IL) (B), nucleus accumbens core (NAcC) and shell (NAcSh) (C), and ventral tegmental area (VTA) of morphine-conditioned mice (n = 5–10/group) and data from morphine-treated mice were normalized to genotype- and time point-matched, saline-conditioned controls to compute fold change. Representative images of each region are shown with quantification areas outlined in white. ac, anterior commissure; cc, corpus callosum. Error bars represent SEM.