Sex Differences in Neuroplasticity- and Stress-Related Gene Expression and Protein Levels in the Rat Hippocampus Following Oxycodone Conditioned Place Preference

Highlights

• Hippocampal gene expression and protein levels vary depending on sex after oxycodone (Oxy) conditioned place preference (CPP)

• After Oxy CPP, Npy mRNA expression decreases but a subset of NPY-labeled cells increases in the dentate gyrus (DG) in females

• In CA3, plasticity and stress markers differentially increase in Oxy CPP females (ARC, Crhr2) and males (Arc, CRHR1)

• After Oxy CPP, signaling kinases (Akt1, pAKT, pMAPK) change in opposite directions in females and males in CA1 and DG

• Differences in signaling, kinase, and stress markers suggest sex-specific hippocampal pathways for opioid-associated learning

Abstract

Prescription opioid abuse is a serious public health issue. Recently, we showed that female and male Sprague–Dawley rats acquire conditioned place preference (CPP) to the mu opioid receptor agonist oxycodone. Anatomical analysis of the hippocampus from these rats unveiled sex differences in the opioid system in a way that would support excitation and opiate associative learning processes especially in females. In this study, we examined the expression and protein densities of opioid, plasticity, stress and related kinase and signaling molecules in the hippocampus of female and male rats following oxycodone CPP. Oxycodone CPP females have: a) increases in ARC (activity regulated cytoskeletal-associated protein)-immunoreactivity (ir) in CA3 pyramidal cells; b) decreases in Npy (neuropeptide Y) gene expression in the medial hippocampus but higher numbers of NPY-containing hilar interneurons compared to males; c) increases in Crhr2 (corticotropin releasing factor receptor 2) expression in CA2/3; d) increases in Akt1 (AKT serine/threonine kinase 1) expression in medial hippocampus; and e) decreases in phosphorylated MAPK (mitogen activated protein kinase)-ir in CA1 and dentate gyrus. Oxycodone CPP males have: a) increases in Bdnf (brain derived-neurotrophic factor) expression, which is known to be produced in granule cells, relative to females; b) elevated Mapk1 expression and pMAPK-ir in the dentate hilus which harbors newly generated granule cells; and c) increases in CRHR1-ir in CA3 pyramidal cell soma. These sex-specific changes in plasticity, stress and kinase markers in hippocampal circuitry parallel previously observed sex differences in the opioid system after oxycodone CPP.

Introduction

Prescription opioid abuse has increased dramatically over the past two decades and has become a serious public health issue (CDC, 2015). Notably, drug overdose rates involving synthetic opioids and heroin have dramatically increased in women (VanHouten et al., 2019). The influence of sex in opioid addiction processes has been challenging to elucidate, as it is difficult to separate the role of genes, environment, and multidrug use in humans. However, the finding that women may have altered sensitivity to morphine over the menstrual cycle (Ribeiro-Dasilva et al., 2011) suggests that ovarian hormones could be involved in addictive processes. Indeed, rodent studies have indicated that the hormonal milieu likely plays a part in opioid addiction (Becker et al., 2017). For example, fluctuations in circulating estrogen levels over the estrous cycle in rats have been reported to alter the patterns of heroin self-administration (Lacy et al., 2016).

Neural circuits involved in associative memory formation and the encoding of motivational incentives are critically involved in the pathway to drug addiction for both sexes (Koob and Volkow, 2010). In rodents, opioid signaling in the CA3 region of the hippocampus is implicated in associative learning and spatial memory (Meilandt et al., 2004, Kesner and Warthen, 2010). Additionally, a type of opioid-mediated long-term potentiation (LTP) has been demonstrated in mossy fiber-CA3 synapses from proestrus (high estrogen) female rats, but not diestrus (low estrogen) female or male rats (Harte-Hargrove et al., 2015), suggesting that opioid associative learning processes could be heightened in females in particular hormonal states.

Recently, we have shown that both female and male Sprague–Dawley rats acquire conditioned place preference (CPP) to the mu opioid receptor (MOR) agonist oxycodone (Ryan et al., 2018). Notably, anatomical analysis of the hippocampus from these rats unveiled sex differences in the opioid system in a way that would bolster excitation and opiate associative learning processes to a greater degree in females compared to males (Ryan et al., 2018). In particular, delta opioid receptors (DOR) redistributed within mossy fiber-CA3 synapses in both oxycodone-injected (Oxy) females and males in a manner similar to that demonstrated to be important for opioid-mediated LTP in proestrus females (Harte-Hargrove et al., 2015, Ryan et al., 2018). Additionally, MORs and DORs rearranged in hilar interneurons in Oxy-females in a configuration that could enhance granule cell disinhibition using two different circuits: 1) plasmalemmal associated MORs increased in parvalbumin (PARV)-labeled dendrites (Ryan et al., 2018) which are known to inhibit granule cell soma (Drake et al., 2007); 2) plasmalemmal associated DORs increased on GABAergic interneuron dendrites (Ryan et al., 2018) which are known to contain neuropeptide Y (NPY) and are important for promoting lateral perforant pathway (lpp) LTP (Sperk et al., 2007). However, whether these changes in hippocampal opioid receptor trafficking also are accompanied by changes in gene expression is unknown.

Thus, the goal of this study was to investigate the effect of oxycodone CPP in female and male rats on opioid peptide and receptor gene expression as well as that of synaptic plasticity, stress and related signaling molecules. Genes selected for analysis were the same as those in our recent study in which we demonstrated sex differences in their expression following chronic stress (Randesi et al., 2018). To elucidate these findings, we examined the immunocytochemical distributions of the relevant proteins in the hippocampus from a separate cohort of rats that had been given oxycodone in a CPP paradigm.