Hormonal regulation of delta opioid receptor immunoreactivity in interneurons and pyramidal cells in the rat hippocampus

Abstract

Clinical and preclinical studies indicate that women and men differ in relapse vulnerability to drug-seeking behavior during abstinence periods. As relapse is frequently triggered by exposure of the recovered addict to objects previously associated with drug use and the formation of these associations requires memory systems engaged by the hippocampal formation (HF), studies exploring ovarian hormone modulation of hippocampal function are warranted. Previous studies revealed that ovarian steroids alter endogenous opioid peptide levels and trafficking of mu opioid receptors in the HF, suggesting cooperative interaction between opioids and estrogens in modulating hippocampal excitability. However, whether ovarian steroids affect the levels or trafficking of delta opioid receptors (DORs) in the HF is unknown. Here, hippocampal sections of adult male and normal cycling female Sprague-Dawley rats were processed for quantitative immunoperoxidase light microscopy and dual label fluorescence or immunoelectron microscopy using antisera directed against the DOR and neuropeptide Y (NPY). Consistent with previous studies in males, DOR-immunoreactivity (-ir) localized to select interneurons and principal cells in the female HF. In comparison to males, females, regardless of estrous cycle phase, show reduced DOR-ir in the granule cell layer of the dentate gyrus and proestrus (high estrogen) females, in particular, display reduced DOR-ir in the CA1 pyramidal cell layer. Ultrastructural analysis of DOR-labeled profiles in CA1 revealed that while females generally show fewer DORs in the distal apical dendrites of pyramidal cells, proestrus females, in particular, exhibit DOR internalization and trafficking towards the soma. Dual label studies revealed that DORs are found in NPY-labeled interneurons in the hilus, CA3, and CA1. While DOR colocalization frequency in NPY-labeled neuron somata was similar between animals in the hilus, proestrus females had fewer NPY-labeled neurons that co-labeled with DOR in stratum oriens of CA1 and CA3 when compared to males. Ultrastructural analysis of NPY-labeled axon terminals within stratum radiatum of CA1 revealed that NPY-labeled axon terminals contain DORs that are frequently found at or near the plasma membrane. As no differences were noted by sex or estrous cycle phase, DOR activation on NPY-labeled axon terminals would inhibit GABA release probability equally in males and females. Taken together, these findings suggest that ovarian steroids can impact hippocampal function through direct effects on DOR levels and trafficking in principal cells and broad indirect effects through reductions in DOR-ir in NPY-labeled interneurons, particularly in CA1.

Introduction

Despite lower rates of use and abuse, clinical evidence suggests that women may be more susceptible to several aspects of addiction than men. For example, women report shorter drug-free periods and higher levels of craving and dysphoria during drug withdrawal (Elman et al., 2001, Griffin et al., 1989, Kosten et al., 1996). Women are also more likely to experience craving following exposure to drug-related cues (Robbins, Ehrman, Childress, & O’Brien, 1999) and relapse to drug-seeking behavior (Elman et al., 2001, McKay et al., 1996, Rubin et al., 1996). Animal models that mimic different phases of the addiction process have been useful in determining whether a biological basis exists for observed sex differences in abuse vulnerability, as these studies control for sociocultural and other factors that may occlude sex differences in clinical studies of drug abuse. In studies of drug relapse or reinstatement, female rats show more extinction responding on the drug-associated lever after drug removal and greater reinstatement after a priming injection than males. Additionally, reinstatement occurs after a lower priming dose in females than in males (Comer et al., 1996, Klein et al., 1997, Lynch and Carroll, 2000). Recent studies using intact and ovariectomized (OVX) rats replaced with estradiol propose that ovarian hormones, particularly estrogen, contribute to the aforementioned sex differences during reinstatement (Fuchs et al., 2005, Kippin et al., 2005, Larson et al., 2005). Taken together, clinical and preclinical studies indicate that women and men differ in relapse vulnerability during abstinence periods. Relapse is frequently triggered by exposure of the recovered addict to objects previously associated with drug use (White, 1996). As the formation of these associations requires the episodic and declarative memory systems engaged by the hippocampal formation (HF) (Hyman and Malenka, 2001, Nestler, 2002, Nestler, 2001, Holden, 2001), studies exploring ovarian hormone modulation of hippocampal function are warranted.

Hippocampal output is regulated through a series of principal cell and inhibitory interneuron connections that are susceptible to modulation by endogenous opioids and exogenous opiates. Ovarian steroid hormones have been shown to influence levels of the endogenous hippocampal opioid peptides, enkephalin and dynorphin, which directly modulate hippocampal excitability (Roman et al., 2006, Drake et al., 2007, Torres-Reveron et al., 2008, Torres-Reveron et al., 2009a, Williams et al., 2010). For example, leu-enkephalin levels are increased in sub-regions of the dentate gyrus (DG) and CA3 of young adult females when estrogen levels are relatively high (Torres-Reveron et al., 2008). Dynorphin levels are similarly increased in the DG and select CA3 lamina 24 h following estrogen exposure (Torres-Reveron et al., 2009a). In addition, ovarian steroid hormones modulate mu opioid receptor (MOR) trafficking in hippocampal interneurons. Specifically, elevated levels of estrogens, either in proestrus females or after 72 h of pulsatile estradiol replacement in OVX females, increased the availability of MORs on the plasma membrane of subpopulations of GABAergic basket cells in the hilar region of the DG (Torres-Reveron et al., 2009b). Such altered trafficking of MORs could potentially alter the disinhibitory effects of endogenous or exogenous opiates, given that MOR activation in the DG produces excitation by inhibiting GABAergic transmission (Bramham and Sarvey, 1996, Drake et al., 2007, Morris and Johnston, 1995, Xie and Lewis, 1995a). Activation of the delta opioid receptor (DOR) at the circuit level also affects excitatory transmission and the induction of synaptic plasticity in principal cells of the DG (Bausch and Chavkin, 1997, Bramham and Sarvey, 1996, Piguet and North, 1993) and CA1 (Bao et al., 2007) through both direct effects on principal cells and indirect effects via inhibition of interneurons. In addition, recent reports suggest that opioid peptides acting primarily on DORs play an important role in mediating cue- and context-induced drug-seeking behavior (Marinelli et al., 2009). Thus, ovarian steroid modulation of DOR immunoreactivity (-ir) levels and trafficking in hippocampal neurons merits further investigation.

Prior studies in male rats demonstrated that the highest DOR mRNA levels were found in GABAergic neurons in the principal cell layers, stratum oriens, and dentate hilus (Stumm, Zhou, Schulz, & Hollt, 2004). DOR-ir, moreover, was commonly found in somatostatin (SOM)/neuropeptide Y (NPY) containing GABAergic interneurons in hippocampal lamina (Commons and Milner, 1996, Commons and Milner, 1997). Moreover, in CA1 specifically, DORs were found also on pyramidal cell dendrites and somata and could therefore directly modulate pyramidal cell activity (Commons & Milner, 1997). The present study sought to extend these observations, where applicable, to the female hippocampus. Thus, single and dual immunolabeling approaches were used to assess ovarian hormone influences on DOR-ir and distribution within hippocampal principal cells and interneurons. Quantitative densitometric immunocytochemistry and immunoelectron microscopy were employed to measure altered DOR-ir and trafficking in principal cells of normal cycling proestrus (high estrogen) and diestrus (low estrogen) female rats in comparison to male rats. Dual label immunofluorescence and immunoelectron microscopy was used to examine DOR localization to NPY-labeled interneurons and axon terminals in select hippocampal lamina of males and normal cycling females. The current study focused on findings in the dorsal hippocampus, where estrogen-induced morphological changes have been consistently reported (Cooke & Woolley, 2005).