Sex Differences in the Effects of Chronic Stress and Oxycodone Associative Learning on Glial Markers in Rat Hippocampus

Ma’ayan Hirschkorn; Annabelle Kim; Garrett Sommer; Sydney Warwick; Lennox Gregoire; Yan Zhou; Joshua Kogan; Jason D Gray; Teresa A Milner

doi:10.1016/j.neuroscience.2026.06.002

. Author manuscript; available in PMC: 2026 Jul 1.

Published in final edited form as: Neuroscience. 2026 Jun 9;610:147–162. doi: 10.1016/j.neuroscience.2026.06.002

Sex Differences in the Effects of Chronic Stress and Oxycodone Associative Learning on Glial Markers in Rat Hippocampus

Ma’ayan Hirschkorn ¹, Annabelle Kim ¹, Garrett Sommer ¹, Sydney Warwick ¹, Lennox Gregoire ¹, Yan Zhou ², Joshua Kogan ³, Jason D Gray ³, Teresa A Milner ^1,^*

PMCID: PMC13316708 NIHMSID: NIHMS2188436 PMID: 42263838

Abstract

Prior studies revealed significant sex differences in hippocampal opioid neuronal networks especially after chronic immobilization stress (CIS) that have important implications for opioid associative learning. Besides neurons, glia play important roles in hippocampal associated learning and can be affected by sex and environmental conditions. Here, we examined the relationship of sex and CIS on microglia (ionized calcium binding adapter molecule, Iba1) and astrocyte (glial fibrillary acidic protein, GFAP) densities in rat hippocampus following conditioned place preference to saline (Sal) or oxycodone (Oxy). Although baseline glial densities were few, Sal females compared to Sal males had lower CA3b Iba1 and CA1. Following Oxy CPP, only females had increased Iba1 densities in CA3b and CA2/3a and GFAP densities in CA1, but both sexes had increased GFAP densities in CA3b. Moreover, compared to Sal counterparts, GFAP densities were lower in dentate gyrus (DG) in Oxy females and in CA1 in Oxy males. CIS females and males compared to unstressed counterparts had decreased CA3b Iba1 and GFAP densities and DG GFAP densities, however, these decreases were more extensive in males. Oxy CIS females, which acquired Oxy CPP, primarily had increased Iba1 and GFAP densities in CA1 and CA3 and decreased GFAP densities in DG. In contrast, Oxy CIS males, which did not acquire Oxy CPP, primarily exhibited increases in GFAP densities in the DG. These studies, together with our prior studies, reveal significant sex differences in the hippocampal glial densities, especially after CIS, which have important implications for Oxy associated learning processes.

Keywords: Microglia, astrocytes, Iba1, GFAP, conditioned place preference

Graphical abstract

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INTRODUCTION

Genetics, environment (e.g., stress) and multi-drug use are all involved in the opioid addiction processes in humans (Sinha, 2007; Strang et al., 2020), complicating the isolation of the influence sex on opioid addiction. The rate of drug overdose involving synthetic opioids and heroin has risen drastically in both sexes, but at a faster rate in women (Scholl et al., 2018; Strang et al., 2020; VanHouten et al., 2019), highlighting the need to further clarify the role sex has on opioid addiction. Women have fluctuating sensitivity to morphine during the menstrual cycle (Ribeiro-Dasilva et al., 2011), suggesting that the ovarian hormones are involved in the addictive processes. Additionally, rodent studies have shown that many sex-related hormones play a part in opioid addiction (Becker & Chartoff, 2019; Becker et al., 2017). For example, heroin self-administration behaviors in rats are affected by differing estrogen levels throughout the estrous cycle (Lacy et al., 2016).

Motivational incentives and associative memory drive the transition from drug use to drug abuse (Koob & Volkow, 2010). These processes can involve the hippocampus directly or indirectly through connections to the mesolimbic reward system (Luo et al., 2011; Vorel et al., 2001). These circuits are the main target of abused drugs such as mu opioid receptor (MOR) agonists, namely the prescription medication oxycodone (Oxy) (Koob & Volkow, 2016). Opioid signaling in the CA3 subregion of the rodent hippocampus can impact both spatial and contextual learning (Kesner & Warthen, 2010; Meilandt et al., 2004). Furthermore, the concept that hormonal states can affect the opioid associative learning processes is supported by our finding that a form of opioid-mediated long-term potentiation (LTP) is present in mossy fiber-CA3 pyramidal cell synapses in female rats at high estrogen states, but not low estrogen states or in male rats (Harte-Hargrove et al., 2015).

Our anatomical studies in rats, particularly those using immuno-electron microscopy, have provided unique insights into sex differences in the distribution of opioid peptides and receptors in specific hippocampal circuits and how these distributions are altered following stress and Oxy-associative learning processes (reviewed in (Chalangal et al., 2021)). We demonstrated that opioid peptides and receptors are distributed within hippocampal circuits in female rats with elevated estrogen states in a way that would enhance sensitivity to both endogenous and exogenous opioids (Ryan et al., 2018). In both female and male rats, Oxy conditioned place preference (CPP) results in a redistribution of opioid receptors in hippocampal circuits in a manner facilitating opioid-associative learning processes. However, the number of circuits changed after Oxy CPP is greater in females. Oxy CPP also results in parallel sex differences in changes in plasticity, stress and kinase markers in the hippocampus (Randesi et al., 2019).

Our prior studies demonstrate that chronic immobilization stress (CIS) primes the opioid system in female rats, but not male rats, in a fashion that would promote opioid-associative learning processes (Reich et al., 2019b). In contrast, CIS has limited effects on the opioid system in male rats and thus reduces its capacity to support opioid-mediated learning processes (Reich et al., 2019b). These sex-specific changes in opioid-receptor trafficking are accompanied by a down-regulation of opioid, stress, plasticity, and kinase/signaling genes in the hippocampus of CIS males (Randesi et al., 2018). Moreover, CIS in males results in elevated glutamatergic GluN1 receptors on the plasmalemmal of CA3 pyramidal cell dendrites (Dolgetta et al., 2022) which may contribute to an increased sensitivity of these neurons to glutamate. Such changes likely contribute to the attenuation of Oxy-CPP in male rats following CIS (Reich et al., 2019b).

Besides neurons, glia may play an important role in opioid associated learning processes. Microglia, the resident macrophages in the brain, are intimately involved in maintaining cognitive heath (Cornell et al., 2022). Microglia are important for maintaining homeostasis in the brain by pruning synapses in the regular cognitive development processes (Hatch et al., 2024). Following chronic stress, activation of microglia can lead to perpetual inflammatory states which have been shown to damage neurons and lead to cognitive decline (Cornell et al., 2022). Microglia, as demonstrated by protein ionized calcium binding adapter molecule (Iba1) (Imai et al., 1996; Sasaki et al., 2001), are activated in models of cognitive and neurodegenerative disorders (Platholi et al., 2023; Prinz et al., 2021).

Like microglia, astrocytes also have been implicated in the emergence of cognitive disorders. Astrocytes play critical roles in blood-brain-barrier formation, brain metabolic, ion, and water homeostasis, neurotransmitter recycling, synapse formation, as well as neuroimmune signaling (Matias et al., 2019). In the context of insult, pathogen infection, or neurological disease, astrocytes undergo functionally complex reactive responses (Chiu et al., 2014; Giovannoni & Quintana, 2020) that are associated with an increase in glial fibrillary acidic protein (GFAP) gene and protein expression (Crespo-Castrillo et al., 2020; Sofroniew, 2020).

At baseline, there are few sex differences in the densities of Iba1 and GFAP in the rodent hippocampus (Marongiu et al., 2025; Milner et al., 2022), but under certain hormonal, genetic and environmental stress conditions, differences emerge. Proestrus (high estrogen levels) female rats have elevated GFAP densities in the CA1, CA3 and dentate gyrus (DG) compared to diestrus (low estrogen) female rats and male rats (Arias et al., 2009). Following slow pressor angiotensin administration, which increases blood pressure in male mice, but not female mice, GFAP densities increase in the DG of males whereas Iba1 densities increase in the DG and CA2/3a region in females (Milner et al., 2022). In the 5xFAD Alzheimer’s mouse model, GFAP densities in the subiculum are lower in males compared to normal cycling females (Marongiu et al., 2025). Moreover, disruption of fluctuating estrogen levels in a mouse perimenopausal model of cerebral amyloid angiopathy seen in Alzheimer’s disease elevates of GFAP and Iba1 densities in the dorsal subiculum and CA1 (Platholi et al., 2023). Importantly, the distribution of both Iba1 and GFAP substantially overlap the hippocampal opioid system (Chalangal et al., 2021; Milner et al., 2022; Platholi et al., 2023). However, whether there are sex differences in glial markers in the hippocampus following Oxy CPP alone or in combination with CIS is unknown.

This study aims to better understand the influence of sex and CIS on glial expression in the rat hippocampus following CPP to saline (Sal) or Oxy by examining the densities of Iba1 and GFAP in the hippocampi of unstressed and CIS female and male rats following Oxy CPP.

EXPERIMENTAL PROCEDURES

ANIMALS

This study used tissues from two cohorts of adult male and female Sprague Dawley rats (N = 48; RGD Cat# 734476, RRID:RGD_734476) that were collected in our prior studies (Reich et al., 2019b; Ryan et al., 2018). All animal procedures were approved by the Weill Cornell Medicine and Rockefeller University Institutional Animal Care and Use Committees and were in accordance with the National Research Council Guidelines for the Care and Use of Laboratory animals. When received, the rats were ~2.5 months old and the males weighed between 275–325 gm and the females weighed between 225–250 gm. Rats were single housed in R20 rat cages (10.5 in × 19 in × 18 in; Ancare Bellmore NY) with a 12-hour light/dark cycle (lights on 0600–1800) and ad libitum access to food and water. Cohort 1. Naïve females and males that were subjected to either Oxycodone (Oxy) CPP or Saline (Sal) CPP (N=6/group)(Ryan et al., 2018). Cohort 2. CIS females and males that were subjected to either Oxy CPP or Sal CPP (N=6/group) (Reich et al., 2019b). A schematic of the experimental procedures is shown in Figure 1.

Fig. 1. — **a. Cohort 1.** Following arrival, adult rats (N=24) were acclimated and then handled. The CPP behavior followed a 14-day sequence: (**Preconditioning**; **PRE**): On day 1 rats were given free access to the entire CPP apparatus (30 min). **Conditioning:** On days 2 – 9, rats had four 2-day training sessions. Rats were divided into 4 groups (Sal female, Oxy female, Sal male, Oxy male; N = 4/grp). On the first day of each session, Oxy rats were administered oxycodone (3mg/kg, i.p.) and restricted to one compartment of the apparatus (30 min). On the second day of each session, the Oxy rats were administered saline and restricted to the other compartment (30 min). Sal rats were injected with saline before each conditioning session. **CPP test:** On day 14, the doors separating the compartments were removed. The rats were not injected and were put in the central neutral gray compartment and their behavior was monitored for 30 min. Rats were immediately euthanized after the CPP test. **b. Cohort 2.** The experimental design was identical that described in a, except that rats were subjected to CIS after acclimation and handling and before the CPP behavior.

Animal handling and estrous cycle determination:

Handling can cause changes in stress and corticosterone levels (Collins et al., 2016; Deutsch-Feldman et al., 2015). To reduce handling stress, rats were acclimatized to the animal facilities for a week and carefully handled for 3–5 minutes per day for 5 days before beginning experiments. All rats in each cohort were handled by the same experimenters for CPP behavior (cohorts 1 and 2) and CIS protocol (cohort 2).

To reduce confounding variables, behaviors and perfusions were performed on each cohort of rats at the same time of day. Behavior was performed between 9:00 a.m. and 1:00 p.m. for all rats (Reich et al., 2019b; Ryan et al., 2018). Most female rats were in the estrus (elevated estrogen) phase of the estrous cycle (Reich et al., 2019b; Ryan et al., 2018) as evaluated by vaginal smear cytology (Turner & Bagnara, 1971) on the day of perfusion, after the rats were anesthetized and before aortic perfusion.

CIS:

Rats were subjected to CIS for 10 successive days (Mazid et al., 2016; Reich et al., 2019b). For this, rats were placed in plastic cone shaped polyethylene bags with their noses sticking out of a hole at its apex and a Kotex mini-pad underneath them to absorb urine. The bags then were sealed with tape, and the rats were left alone for 30 min. The rats started CPP training two days after the last CIS session.

Oxy CPP:

The CPP apparatus (product # MED-CPP-013, MED Associates Inc) was made of unique compartments (white, black, and a central neutral gray) and could be divided by removable doors. The CPP protocol was conducted over a 14-day period: 1) Day 1 (preconditioning): Rats were given free access to the entire CPP apparatus for 30 minutes.; 2) Days 2 – 9 (conditioning): During conditioning, Oxy was injected in the non-preferred side as determined in the preconditioning session (Reich et al., 2019b; Ryan et al., 2018) Rats had 4 training sessions. On the first day of each session, the rats were administered oxycodone (3mg/kg, i.p.) and restricted to one compartment of the apparatus (e.g., white) for 30 min. On the second day of each session, the rats were administered saline and restricted to the other compartment (e.g., black) for 30 min. Control rats were injected with saline before each conditioning session. Rats were returned to their home cages until the CPP test. 3) Day 14 (CPP test): The doors separating the compartments were removed. The rats were not injected. They were put in the central neutral gray compartment, and their behavior was monitored for 30 min. Rats were euthanized immediately after the CPP test (Reich et al., 2019b; Ryan et al., 2018).

The time that each rat spent in the Oxy-paired compartment was calculated by dividing the time spent in the Oxy section by the total time spent in the CPP apparatus. Preference score was calculated by subtracting the time spent in the Oxy CPP chamber before conditioning and after conditioning. Previous studies have shown that naïve male and female rats (Cohort 1) acquired oxycodone CPP; however, female rats spent more percent time on the Oxy paired side than the male rats (Ryan et al., 2018). Moreover, Oxy males, but not Oxy females, had a significant decrease in locomotion during the first three training sessions (Ryan et al., 2018). Following CIS (Cohort 2), females, but not males, had a significant increase in the percent change in preference score for Oxy-associated chamber (Reich et al., 2019b). There were no significant differences in locomotion in CIS females or males in any of the four training sessions, regardless of treatment (Reich et al., 2019b).

IMMUNOCYTOCHEMISTRY EXPERIMENTS

Antibodies:

A rabbit polyclonal antibody to GFAP (Abcam ab7269, lot GR20948–21, RRID: AB_305808) raised against a full-length protein corresponding to human GFAP was used. On Western blot, this antibody recognizes a band of 55 kDa and a 48-kDA band corresponding to GFAP (manufacture’s datasheet). A rabbit polyclonal antibody raised to a synthetic peptide corresponding to the C terminus of Iba1 (Fujifilm Wako Pure Chemical Corporation SAR6502, 019–19741) was used. The antibody is reactive with human, mouse, and rat Iba1 and recognizes a 17-kDa band protein on Western blot (manufacture’s datasheet). These antibodies have been used in our prior studies (Milner et al., 2022; Platholi et al., 2023)

Immunocytochemical procedures:

Section preparation and immunocytochemistry procedures are described in detail previously (Milner et al., 2011).

Section preparation:

Euthanasia occurred between 9am and 1pm over a period of 4 days. The rats were anesthetized with ketamine (100 mg/kg) and xylazine (10 mg/kg) I.P and perfused through the ascending aorta sequentially with: 1) 10–15 mL 0.9% saline and 2% heparin; 2) 50 mL 3.75% acrolein and 2% paraformaldehyde (PFA) in 0.1M phosphate buffer (PB; pH 7.4); 3) 200 mL 2% PFA in PB. Coronal sections (40μm thick) through hippocampus were cut using a vibratome (VT1000X Leica Microsystems, Buffalo Grove, IL) and were stored in cryoprotectant solution (30% sucrose, 30% ethylene glycol in PB) at −20°C until use. For each experiment (Iba1 or GFAP), two dorsal hippocampal sections (−3.5 to −4.2 mm from Bregma (Swanson, 1992)) from each group from Cohorts 1 and 2 (N=6/experimental condition) were coded with hole-punches in the cortex and placed into a single crucible (12 crucibles in total). Crucibles containing tissues from both cohorts 1 and 2 were processed simultaneously throughout the immunocytochemical procedures to ensure identical labeling conditions between groups (Milner et al., 2011). This also enabled comparisons in immunolabeling densities to be made within and between cohorts 1 and 2.

Light microscopic immunocytochemistry:

To neutralize reactive aldehydes, sections were incubated in 1% sodium borohydride in PB for 30 min (Milner et al., 2011) then rinsed ~10 times in PB until bubbles were no longer present. Sections were transferred to 0.1 M Tris saline (TS; pH 7.6) followed by an incubation in 0.5% BSA in TS for 30 min to reduce nonspecific labeling. Sections then were incubated in primary antibodies Iba1 (1:4000) or GFAP (1:6000) diluted in 0.1% BSA and 0.025% Triton-X in TS for 1 day at room temperature and 3 days at 4°C. Next, sections were rinsed in TS and incubated in goat anti-rabbit IgG conjugated to biotin (Jackson Immunoresearch Inc., 111-065-144, RRID: AB_2337965) in 0.1% BSA and TS for 30 min. Sections were subsequently rinsed in TS and incubated with avidin biotin complex (ABC) diluted to half of the manufacture’s recommended dilution (Vectastain Elite kit, Vector laboratories) for 30 min. After rinsing in TS, the bound peroxidase was visualized by reaction in 3,3’ diaminobenzidine (Sigma Aldrich) and 0.003% hydrogen peroxide in TS for 7 minutes (Iba1) and 3 minutes (GFAP). All primary and secondary antibody as well as ABC incubations were carried out at 145 rpm, and all rinses were conducted at 90 rpm on a rotator shaker. Sections were mounted from 0.05 M PB onto gelatin-coated glass slides, dehydrated through ascending series of alcohol through xylene and coverslipped with DPX (Sigma Aldrich).

Analysis and figure preparation:

Densitometric quantification for Iba1- and GFAP-labeling in the dorsal hippocampal layers were accomplished using previously described methods (Pierce et al., 2014; Williams & Milner, 2011; Williams et al., 2011). To ensure unbiased data quantification, the analysis was performed by investigators blinded to experimental conditions. For both Iba1 and GFAP experiments comparisons were made: 1) between rats in Cohort 1; 2) between rats in Cohort 2; and 3) between Sal-injected rats in Cohorts 1 and 2 in each sex.

Sections were photographed with a Nikon Eclipse 80i microscope using a Micropublisher 5.0 digital camera (Q-imagine) and IPLab software (Scanalytics IPlab, RRID:SCR_002775). ImageJ64 (ImageJ, RRID:SCR_003070) was used to measure the average pixel density within the region of interest (ROI) in defined hippocampal subregions. ROIs within four subregions of the dorsal hippocampus were selected: 1) CA1: stratum oriens (SO), stratum radiatum (SR), and stratum lacunosum-moleculare (SLM); 2) CA2/3a: SO, stratum pyramidale (SP), and the near and distal SR (nSR and dSR, respectively); 3) CA3b: SO, SP, stratum lucidum (SLu) and SR; 4) DG: granular cell layer (GCL), supra granular hilus (SGL), central hilus (Cen), and the infragranular hilus (IGL). To control for variations between illuminations of each image, and to account for background labeling, pixel density of a small region lacking labeling (e.g., corpus callosum) was subtracted from ROI measurements. Previous studies (Pierce et al., 2014) have shown a strong linear correlation between pixel density and transmittance, confirming measurement accuracy.

Data are expressed as means +/− SEM. Statistical analyses were conducted using GraphPad Prism 10 software (GraphPad Prism, RRID:SCR_002798), and significance was set to an alpha <0.05. Differences between four groups were compared by using a two-way analysis of variance (ANOVA) followed by Tukey post-hoc tests. Differences between two groups were determined using a Welch’s t-test.

Images were imported into Microsoft PowerPoint 16, where final adjustments to brightness, sharpness, contrast and color balance were achieved. Adjustments were made to the entire image and did not alter the appearance of the initial raw image. Graphs were generated using Prism 10 software.

RESULTS

Iba1 RESULTS

COHORT 1. Sal females and males had few baseline differences in Iba1 densities, however, Iba1 densities increased in select hippocampal subregions in Oxy females

The hippocampal densities of microglia identified by Iba1-labeling were compared between Cohort 1 (Fig. 1a) US (unstressed) females and males that were injected either with Sal or Oxy and subjected to CPP. Our prior studies for Cohort 1showed that both female and male rats in Cohort 1 acquired Oxy CPP (Ryan et al., 2018). Consistent with other studies (Milner et al., 2022; Platholi et al., 2023; Radenovic et al., 2020), Iba1-labeled cells were found scattered throughout all lamina in the CA1, CA3 and DG (Fig. 2a–e). However, fewer Iba1-labeled cells were found in the CA3 pyramidal and DG granule cell layers (Fig. 2a–e). Representative micrographs of Iba1-labeling in regions showing significant differences from each of the four groups are shown in Figure 2f–i (CA2/3a) and k–m (CA3b).

Fig. 2. — a. Low magnification photomicrograph of Iba1 labeling the dorsal hippocampus. Enlarged boxed regions indicate areas of the CA1(b), CA2/3a **(c)**, CA3b **(d)** and dentate gyrus (DG; e) that were sampled. Abbreviations: CH, central hilus; GCL, granule cell layer; IGL, infragranular layer of DG; SP, pyramidal cell layer; SLM, stratum lacunosum-moleculare; SGL, supragranular layer of DG; SO, stratum oriens; SLu, stratum lucidum; SR, stratum radiatum; dSR, distal SR; nSR, near SR. **f-i.** Representative micrographs showing Iba1 labeling in the **CA2/3a** of US Sal female **(f)**, Oxy female **(g)**, Sal male **(h)** and Oxy male **(i)** rats. **j. CA2/3a**: Oxy females compared to Sal females show higher Iba1 densities in the nSR and dSR. **k-n.** Representative micrographs showing Iba1 labeling in the **CA3b** of Sal female **(k)**, Oxy male **(l)**, Sal female **(m)** and Oxy female **(n)** rats. **o. CA3b**: Oxy females compared to Sal females show greater Iba1 densities in SP. Sal males compared to Sal females show higher Iba1 densities in SO and SP. Data are expressed as mean +/− SEM, N = 6 rats/grp. * p < 0.05; ** p < 0.01. Scale Bars: a, 500 microns; **b-e**, 200 microns; **f-i & k-n**, 100 microns

There was a significant interaction of sex and treatment (Sal vs Oxy) on Iba1 labeling in CA3b SP (F(1,20) = 5.886, p = 0.025). There also was a main effect of sex on Iba1 labeling in CA3b SO (F(1,20) = 4.705, p = 0.04), and a main effect of treatment was found in CA2/3a dSR (F(1, 19) = 4.926, p = 0.039). Post hoc showed no significant differences between the density of Iba1-labeling in any subregion of CA2/3a in Sal females compared to Sal males (Fig 2j). However, Sal female rats compared to Sal male rats had less Iba1-density in SO (p = 0.0162) and SP (p = 0.045) of CA3b (Fig. 2o). Following Oxy CPP, Oxy females compared to Sal females showed greater Iba1-labeling densities in nSR (p = 0.009) and dSR (p = 0.012) of CA2/CA3a (Fig. 2j) and in SP (p = 0.01) of CA3b (Fig. 2o). No significant differences in Iba1 densities were seen between Oxy and Sal males in any CA3a or CA3b subregion (Fig. 2j,o).

Overall, Sal females compared to Sal males had similar densities of Iba1-labeling in the hippocampus except for subregions of CA3b. Following Oxy CPP, the density of Iba1-labeling increased in females, but not males, in select subregions of CA2/CA3a and CA3b.

COHORT 2. CIS Sal males had elevated Iba1 in CA1 and decreased Iba1 in CA3b, however, Oxy CIS females had elevated Iba1 in CA1

The Iba1-densities in hippocampus in Cohort 2 (Fig. 1b) were compared between CIS females and CIS males that were injected either with Sal or Oxy and subjected to CPP. Our prior studies for Cohort 2 showed that CIS female rats, but not CIS male rats, acquired Oxy CPP (Reich et al., 2019b). Representative micrographs of Iba1-labeling in regions showing significant differences from each of the four groups are shown in Figure 3a–d (CA1) and f–i (CA3b).

ANOVA showed a significant interaction between sex and treatment (Sal vs Oxy) in CA1 SO (F(1,20) = 15.20, p = 0.001), CA1 SR (F(1,20) = 15.36, p = 0.001) and CA3b SP (F(1,20) = 7.879, p = 0.01). Additionally, there was a sex effect seen in the CA1 SP (F 1,20) = 5.22, p = 0.03) and the CA3b SP (F(1,20) = 8.166, p = 0.01). Post hoc showed lower (p = 0.012) Iba1-labeling density in the CA1 SR in CIS Sal females compared to CIS Sal males (Fig. 3e), but significantly higher Iba1 densities in CA3b SP (p = 0.001) and CA3b SLu (p = 0.02) in CIS Sal females when compared to CIS Sal males (Fig. 3j).

Following Oxy CPP, Oxy CIS females compared to CIS Sal females had increased Iba1 labeling in CA1 SO (p = 0.01), CA1 SP (p = 0.02) and CA1 SR (p = 0.001) (Fig. 3e). However, Oxy CIS females compared with CIS Sal females had decreased (p = 0.01) Iba1 labeling densities in CA3b SR (Fig. 3j). Unlike Oxy CIS females, CIS Oxy males had a reduced (p = 0.01) Iba1-labeling density in the CA1 SO when compared to CIS Sal males (Fig. 3e). Moreover, Oxy CIS females compared to Oxy CIS males had a greater Iba1-labeling density in CA1 SO (p = 0.001), CA1 SP (p = 0.007) and CA1 SR (p = 0.01) (Fig. 3e).

Thus, CIS Sal females compared to CIS Sal males had lower densities of Iba1 in SO and SR of CA1 and higher densities of Iba1 in most subregions of CA3b. Following Oxy CPP, CIS females exhibited increases in Iba1 labeling in SO and SR of CA1. In contrast, CIS males that were injected with Oxy, but did not achieve CPP, had a decrease in Iba1density in SO of CA1 compared to the Sal CIS males.

COHORTS 1 & 2 Sal rats. CIS prominently decreased Iba1 densities in the DG of males

To better understand the baseline effects of CIS on microglia in females compared to males, the Iba1 densities were compared between Sal-injected US and CIS female rats and Sal-injected US and CIS male rats (Sal rats Cohorts 1 & 2; Fig 1). Representative micrographs showing Iba1-labeling in regions showing significant differences are shown in Figure 4 a–d (CA1), f-i (CA2/3a), k–n (CA3b), and q-t (DG).

Fig. 4. — **a-d.** Representative micrographs showing Iba1 labeling in the **CA1** of Sal US female **(a)**, Sal CIS female **(b)**, Sal US Male **(c)**, and Sal CIS male **(d)** rats. **e. CA1:** Sal US females compared to Sal CIS females have lower Iba1 labeling densities in CA1 SLM. **f-i.** Representative micrographs showing Iba1 labeling in **CA2/3a** of Sal US female **(f)**, Sal CIS female **(g)**, Sal US male **(h)** and Sal CIS male **(i)** rats. j. **CA2/3a:** Sal US males had lower Iba1 densities than Sal CIS males in SO. **k-n.** Representative micrographs showing Iba1 labeling in **CA3b** of Sal US female **(k)**, Sal CIS female **(l)**, Sal US male **(m)**, and Sal CIS male **(n)** rats. **o,p. CA3b:** Sal US females compared to Sal CIS females had higher Iba1 densities in the SO. Sal US males compared to Sal CIS males had higher Iba1 densities in SO, SP, SLu and SR. **q-t.** Representative micrographs showing Iba1 labeling in the DG of Sal US female **(q)**, Sal CIS female **(r)**, Sal US male **(s)**, and Sal CIS male **(t)** rats. **u. DG:** Sal CIS males compared to Sal US males had lower Iba1 densities in GCL. Data are expressed as mean +/− SEM, N = 5–6 rats/grp. *p < 0.05, **p<0.01, ***p<0.001, ****p<0.0001. Scale Bars: **a-d, f-i, k-n**, & **q-t**, 100 microns

Alterations in Iba1 densities US Sal females compared to CIS Sal females were limited to select lamina in CA1 and CA3b. CIS females showed more Iba1-labeling in the CA1 SLM (t(6.142) = 3.333, p = 0.015) when compared to US females (Fig. 4e). Conversely, CIS females in the CA3b SO (t(9.98) = 2.3, p = 0.044) showed less Iba1-labeling when compared to US females (Fig. 4o).

Alterations in Iba1 densities US Sal males compared to CIS Sal males were more extensive than those seen in females. In CA2/3a, CIS males showed greater Iba1densities in SO (t(7.584) = 3.395, p = 0.01) compared to US males (Fig. 4j). In CA3b, CIS males compared to US males had reduced Iba1 densities in all subregions (SO (t(9.371) = 7.684, p < 0.0001; Welch’s t-test), SP (t(6.748) = 5.039, p = 0.002; Welch’s t-test), SLu (t(7.694 = 6.766, p = 0.0002) and SR (t(5.142) = 4.058, p = 0.009)) (Fig. 4p). Moreover, CIS males compared to US males had decreased Iba1 densities in the GCL of DG (t(9.976) = 2.672, p = 0.023) (Fig. 4u).

GFAP RESULTS

COHORT 1. Following Oxy CPP, GFAP densities in hippocampus changed in opposite directions in females and males

The densities of astrocytes identified by GFAP were compared between US females and males that were injected either with Sal or Oxy and subjected to CPP (Cohort 1; Fig. 1a). Consistent with other studies (Arias et al., 2009; Milner et al., 2022; Platholi et al., 2023), GFAP-labeled cells were found throughout all lamina in the CA1, CA3 and DG but were particularly dense in the CA1 SLM region and the hilus of the DG (Fig. 5a–e). Fewer GFAP-labeled cells were found in the granule cell layer (Fig. 5a & e). Representative micrographs showing the distribution GFAP-labeling in regions showing significant differences from each of the four groups are shown in Figure 5f–i (CA1), k–n (CA3b) and p-s (DG).

There was a significant interaction between sex and treatment (Sal vs Oxy) in the CA1 SO (F(1,17) = 11.27, p = 0.004), CA1 SR (F(1,17) = 8.345, p = 0.01), DG IGL (F(1,17) = 12.77, p = 0.002) and DG SGL (F(1,17) = 17.07, p = 0.001). There were main effects of treatment (Sal vs Oxy) in the CA3b SO (F(1,17) = 19.41 p = 0.0004), CA3b SLu (F(1,17) = 18.79 p = 0.0005) and CA3b SR (F(1,17) = 53.43, p < 0.0001).

When comparing Sal females to Sal males, post hoc showed decreased GFAP densities in the CA1 SO (p = 0.006) and CA1 SR (p = 0.039) (Fig. 5j), but an increase in GFAP labeling in the DG SGL (p = 0.007) (Fig. 5t).

In Oxy females compared to Sal females, post-hoc showed increased densities of GFAP-labeling in the CA1 SO (p = 0.028), CA3b SO (p = 0.002) and CA3b SR (p = 0.001) (Fig. 5o). Conversely, the density of GFAP-labeling was decreased in the DG SGL (p = 0.002) and DG IGL (p = 0.004) in Oxy females compared to Sal females (Fig. 5t). In contrast to females, Oxy males compared with Sal males had less GFAP labeling density in CA1 SO (p = 0.032), CA1 SP (p = 0.036), and CA1 SR (p = 0.025) (Fig. 5j). Like females, Oxy males compared with Sal males had increased GFAP densities in SO (p = 0.022), SLu, (p = 0.0006) and SR (p < 0.0001) of CA3b (Fig. 5o). In the DG, Oxy females compared to Oxy males had lower densities of GFAP-labeling in the SGL (p = 0.012) and IGL (p = 0.06) (Fig. 5t).

Overall, Sal females compared to Sal males had lower densities of GFAP-labeling in the CA1 but similar GFAP densities in CA3b and DG. Following Oxy CPP, the density of GFAP-labeling changed in opposing directions females compared to males in subregions of the CA1 and DG. However, Oxy CPP resulted in elevations in the density of GFAP-labeling in both females and males in the CA3b.

COHORT 2. GFAP densities in DG prominently decreased in Oxy CIS females but increased in Oxy CIS males

GFAP densities were compared between CIS females and CIS males that were injected either with Sal or Oxy and subjected to CPP (Cohort 2; Fig. 1b). Representative micrographs showing the distribution GFAP-labeling in regions showing significant differences from each of the four groups are shown in Figure 6a–d (CA1) and f-i (CA2/3a), k–n (CA3b) and p–s (DG).

There was a main effect of sex in the CA1 SO (F(1,20) = 9.283, p = 0.007), CA1 SR (F(1,20) = 15.51, p = 0.001), CA1 SLM (F(1,20) = 73.92, p < 0.0001) and DG SGL (F(1,20) = 9.739, p = 0.005). There was a significant effect of treatment (Sal vs Oxy) in the CA1 SLM (F(1,20) = 6.272, p = 0.02), CA3b SLu (F(1,20) = 5.017, p = 0.037) and CA3b SR (F(1,20) = 4.390, p = 0.049). There also was an interaction of sex and treatment (Sal vs Oxy) in the CA1 SO (F(1,20) = 15.21, p = 0.001), CA1 SR (F(1,20) = 25.69, p < 0.0001), CA1 SLM (F(1,20) = 20.80, p = 0.0002), CA2/CA3a SO (F(1,20) = 6.885, p = 0.016), DG IGL (F(1,20) = 38.86, p < 0.0001) and DG CH (F(1,20) = 64.66, p < 0.0001).

Few differences in the densities of GFAP were seen in Sal CIS females compared to Sal CIS males in CA1, CA2/3a and CA3b (Fig. 6e, j, o). However, post hoc results show a greater (p = 0.01) in GFAP density in Sal CIS females compared to Sal CIS males in the CA1 SLM (Fig. 6e). Moreover, Sal CIS females compared to Sal CIS males had greater densities of GFAP in the SGL (p = 0.002), IGL (p < 0.0001) and CH (p < 0.0001) of the DG (Fig. 6t).

Oxy CIS females compared to Sal CIS females had increased densities of GFAP labeling in CA1 SO (p = 0.015), CA1 SR (p = 0.006), CA1 SLM (p < 0.0001) and in the CA3b SR (p = 0.03) (Fig. 6e, o). In contrast, Oxy CIS females compared to Sal CIS females had decreased densities of GFAP labeling in the DG IGL (p = 0.0012) and DG CH (p < 0.0001) (Fig. 6t). Unlike Oxy CIS females, Oxy CIS males compared to CIS Sal males had decreased GFAP densities in CA1 SO (p = 0.01), CA1 SR (p = 0.001) and CA2/3a SO (p = 0.01) (Fig. 6e, j). Moreover, Oxy CIS males compared to Sal CIS males had increased GFAP densities of DG SGL (p = 0.014), DG IGL (p < 0.0001), and DG CH (p < 0.0001) (Fig. 6t).

When comparing Oxy CIS females to Oxy CIS males, a greater GFAP-labeling densities were seen in CA1 SO (p < 0.0001), CA1 SR (p < 0.0001), CA1 SLM (p < 0.0001) and CA2/3a SO (p = 0.01) (Fig. 6e,j). However, Oxy CIS females compared to Oxy CIS males had less dense GFAP in the IGL (p = 0.0063) and DG CH (p = 0.0001) (Fig. 6t).

Thus, CIS alone decreased the density of GFAP-labeling in select but different hippocampal subregions in both females and males (DG in Sal CIS females; CA1 in Sal CIS males). However, the direction of change of the density of GFAP, especially in the CA1 and DG, was the opposite in Oxy CIS females (which acquired CPP) compared to Oxy CIS males (which did not acquire Oxy CPP).

COHORT 1 & 2 Sal rats. CIS Sal females and males had decreased GFAP densities in DG but they were more prominent in males

To better understand the baseline effects of CIS on hippocampal GFAP, the densities of GFAP-labeling were compared between Sal-injected US and CIS female rats and Sal-injected US and CIS male rats (Sal rats Cohorts 1 & 2; Fig 1). Representative micrographs showing the distribution GFAP-labeling in regions showing significant differences shown in Figure 7a–b, d–e (CA2/3a) and g–h, j–k (DG).

Fig. 7. — **a-b, d-e.** Representative micrographs showing GFAP labeling in the **CA1** of Sal US female **(a)**, Sal CIS female **(b)**, Sal US Male **(d)**, and Sal CIS male **(e)** rats. **c. CA2/3a:** Sal CIS females compared to Sal US females had higher GFAP densities in SP. **f. CA2/3a**: Sal CIS males compared to Sal US males had higher GFAP densities in SO. **g-h, j-k.** Representative micrographs showing GFAP labeling in the **CA2/3a** of Sal US female **(g)**, Sal CIS female **(h)**, Sal US male **(j)**, and Sal CIS male **(k)** rats. i. DG: Sal CIS females and males compared to their unstressed counterparts had lower GFAP densities in the SGL, IGL, and CH Moreover, Sal CIS males compared to Sal US males had decreased GFAP densities in the GCL. Data are expressed as mean +/− SEM, N = 5–6 rats/grp. *p < 0.05, **p<0.01, ***p<0.001, ****p<0.0001. Scale Bars: **a-d** & **g-j**, 100 microns

Following CIS, the directions of the GFAP density changes were similar in Sal females and males and occurred primarily in the DG. CIS Sal females and males showed increased GFAP labeling compared to their US counterparts in SP (female, t(9.0) = 2.373, p = 0.042; Welch’s t-test) and SO (male, t(8.46) = 3.306, p = 0.01; Welch’s t-test) of CA2/3a (Fig. 7c,f). Conversely, GFAP densities decreased in the DG SGL (female, t(8.98) = 4.22, p = 0.002; male, (t(9.113) = 7.964, p < 0.0001) IGL (female, t(8.989) = 2.322, p = 0.045; male, t(9.958) = 9.595, p < 0.0001) and CH (female, t(5.609) = 2.846, p = 0.032; male, t(9.913) = 4.884, p = 0.001) (Fig. 7i,l). CIS males also demonstrated decreased GFAP densities compared to their US counterparts in the DG GCL (t(8.011) = 3.965, p = 0.004) (Fig. 7l).

Overall, the direction of the changes in the GFAP densities were similar in Sal CIS females and males compared to their Sal US counterparts and most of the changes were seen in the DG. However, compared to Sal CIS females, Sal CIS males had greater reductions of GFAP densities in most DG subregions and included the GCL.

DISCUSSION

The relationship of sex and CIS on microglia and astrocytic densities in the young adult rat hippocampus at baseline and following Oxy CPP was investigated. Baseline sex differences in Iba1 and GFAP densities in Sal US rats were few and limited to select hippocampal subregions. Following Oxy CPP, Iba1 densities increased in CA3 subregions only in US females whereas GFAP densities were altered in both sexes, but the direction of change was regionally and sex dependent. Following Oxy CPP, only females had increased Iba1 densities in CA3b and CA2/3a and GFAP densities in CA1, but both sexes had increased GFAP densities in CA3b. CIS females and males compared to unstressed counterparts had decreased CA3b Iba1 and GFAP densities and DG GFAP densities, however, these decreases were more extensive in males. Oxy CIS females, which acquired Oxy CPP, primarily had increased Iba1 and GFAP densities in CA1 and CA3 and decreased GFAP densities in DG. In contrast, Oxy CIS males, which did not acquire Oxy CPP, primarily exhibited increases in GFAP densities in the DG. These studies reveal significant regional sex differences in the hippocampal glial densities, especially after CIS, which together with our prior studies, have important implications for Oxy associated learning processes (see summary Fig. 8).

Fig. 8. — Comparisons of glial markers Iba1 (orange) and GFAP (blue) before and after Oxy CPP behavior. (black = for both Iba1 and GFAP) Up arrows indicate density increases in Oxy compared to Sal counterparts; down arrows indicate density decreases in Oxy compared to Sal counterparts; = indicates no differences

Methodological Considerations

Hippocampal tissue from Cohorts 1 and 2 (Reich et al., 2019a; Ryan et al., 2018) was processed for immunocytochemistry together so that baseline effects of CIS on Iba1 and GFAP densities could be determined on Sal rats. However, injections in themselves may have affected baseline glial densities in these rats. Increased spine density of CA1 pyramidal cells have been observed in male rats injected with saline compared to unhandled (Horner et al., 1991). Moreover, repeated intraperitoneal Sal injections shift the dose-response curve of corticotrophin releasing factor in the locus coeruleus of male rats (Curtis et al., 1995; Curtis et al., 1999), suggesting that injections alone can impact of stress responses.

Our prior studies have demonstrated that Iba1 and GFAP densitometry is useful for determine differences glial levels in large datasets (Marongiu et al., 2025; Milner et al., 2022; Platholi et al., 2023). However, a limitation of this approach is that it did not quantify morphological changes of the glial cells and thus restricts the assessment the activation of these cells (Reddaway et al., 2023).

Baseline sex differences in glial expression

Few baseline hippocampal densities for Iba1 and GFAP were observed between unstressed Sal female and male rats (Fig. 8). Sal females compared to Sal males had lower Iba1 densities in CA3b SO and SP and lower GFAP densities in CA1 SO and SR. However, Sal females compared to Sal males had higher GFAP densities in DG SGL. Of note, the female rats used in the current study were in mostly in estrus (i.e., elevated estrogen state)(Reich et al., 2019b; Ryan et al., 2018). Thus, it is possible the observed sex differences in glia densities were due in part to estrogen levels. In support, ovariectomy, which removes circulating hormones, leads to increases in Iba1 (Sárvári et al., 2017) and macrophage antigen complex-1, a marker of reactive microglia (Benedusi et al., 2012), in the rat hippocampus. Moreover, erratic fluctuations in estrogen levels seen in mice with accelerated ovarian failure (AOF) compared to normal cycling females have increase GFAP density in CA1 SP (Marongiu et al., 2025).

The differences in the baseline densities of Iba1 and GFAP Sal female and male rats in hippocampal subregions correlate with prior sex differences observed in the anatomy and function of the rat opioid system (reviewed in (Chalangal et al., 2021)). The lower densities of GFAP-labeling CA1 in Sal females compared to Sal males parallel elevations in plasmalemmal DORs in CA1 pyramidal cells in high estrogen females compared to males (Williams et al., 2011). The lower Iba1 densities observed in CA3b in Sal females compared to Sal males parallel elevations of mossy fiber LEnk levels and DORs in MF-CA3 synapses in high estrogen females compared to males (Mazid et al., 2016; Pierce et al., 2014). Moreover, the higher densities of GFAP in DG SGL, where the PARV interneurons reside (Chalangal et al., 2021), in Sal females compared to Sal males are concomitant with elevated plasmalemmal MORs PARV interneurons in high estrogen females (Milner et al., 2013; Torres-Reveron et al., 2009). Notably, the changes in Iba1 and GFAP densities in CA3 and DG occur in regions importantly involved in MOR regulation of mossy fiber transmission and a DOR-dependent LTP at MF-CA3 synapses in high estrogen females (Harte-Hargrove et al., 2015).

Oxy CPP elevated CA3b GFAP densities in both sexes

Following Oxy CPP, the changes in density of Iba1 labeling were limited to select subregions in females whereas the changes in the GFAP densities were more extensive and the direction of change varied with subregion and sex. In general, Oxy CPP changed the densities of Iba1 in CA2/CA3a and CA3b in females so that the resultant levels were equivalent to those seen in both Sal and Oxy CPP males. Likewise, Oxy CPP reduced GFAP densities in CA1 in males and in DG in females so that they were equivalent to groups in the opposite sex. Thus, following OXY CPP sex differences in the glial densities in CA1 and the DG were no longer apparent.

In contrast, Oxy CPP elevated GFAP densities in both sexes in most subregions of CA3b (Fig. 8). Oxy-CPP has been shown to redistribute DORs to MF-CA3 synapses in males and remain elevated in MF-CA3 synapses in females (Ryan et al., 2018). The presence of DORs in MF-CA3 synapses are important for opioid-mediated LTP in high estrogen females (Harte-Hargrove et al., 2015). Oxy CPP associated elevations in GFAP densities in CA3b subregions could highlight the role of astrocytes at MF-CA3 synapses. Astrocytes are known to possess MORs, the activation of which leads to the opening of TREK-1 and TWIK-1 containing K2P channels, facilitating astrocytic glutamate release at MF-CA3 synapses (Nam et al., 2021). MOR activation also leads to a downregulation of GLT1 in astrocytes, resulting in decreased reuptake of glutamate from MF-CA3 synapses (Sanderson et al., 2019).

Oxy CPP elevated Iba1 densities in SR of CA2/3a in females. CA2/3a SR also contains high levels of phosphorylated DORs, which are important for opioid receptor internalization and trafficking(Bellamy et al., 2019; Deng et al., 2001; Doll et al., 2011; Law et al., 2000). Moreover, following Oxy CPP, SO and the PCL of CA2/3a shows increases in Arc (Randesi et al., 2019), a gene that is important for synaptic plasticity (Bramham et al. (2010) and LTP processes (Plath et al. (2006).

CIS results in prominent sex-specific glial changes in hippocampal opioid circuits

Most changes in glial densities induced by CIS in Sal injected rats were limited to CA3b and the DG. Although CIS resulted in decreases in glial densities in these regions in both sexes, the males had more. In particular, Iba1 densities decreased in all lamina of CA3b in CIS Sal males, but only decreased in SO of CA3b in CIS Sal females. Moreover, Iba1 densities were decreased Iba1 in the DG granular cell layer of males. Although GFAP densities decreases in DG hilar regions of both CIS Sal male and female rats, CIS Sal rats also had decreased GFAP densities in the granule cell layer. Notably, CA3b SR contains the basal dendrites of pyramidal cells which undergo dendritic atrophy in male rats (Galea et al., 1997; Vyas et al., 2002; Watanabe et al., 1992), but not female rats (Conrad et al.; Luine et al., 2007), following chronic stress. Also, CIS in males, but not females, results in a loss of PARV interneurons in the DG subgranular zones (Czeh et al., 2005; Hu et al., 2010; Milner et al., 2013) and in NPY interneurons in the central hilus (Mazid et al., 2016). The negative impact of chronic stress on CA3 and DG neuronal populations in males importantly contributes to the impairment of cognitive performance and reduction of LTP seen in males following chronic stress (reviewed in (McEwen, 1999; McEwen & Milner, 2007)). Recent studies support a key role of microglia in mediating neuroplasticity and highlight potential mechanisms underlying the involvement of microglia in memory deficits in response to stress (Bravo-Jimenez et al., 2025; Flury et al., 2025; Hu et al., 2022).

The observed sex differences in glial densities in the CA3b and DG following CIS could contribute to previously reported sex differences in the hippocampal dentate-CA3 opioid circuits (reviewed in (Chalangal et al., 2021)). Briefly, following CIS, females from all estrous cycle stages have: 1) elevated Lenk-levels in mossy fibers (Pierce et al., 2014) suggesting greater stores of Enk are available for release; 2) continued elevations of DORs in MF-CA3 synapses (Mazid et al., 2016) indicating the mechanisms for promoting LTP (Harte-Hargrove et al., 2015) are still in place; 3) increased cytoplasmic of MORs in hilar PARV interneurons (Milner et al., 2013) indicating greater availability of MOR pools; and 4) increased plasmalemmal DORs on hilar NPY/SOM interneurons (Mazid et al., 2016) known to project to granule cell dendrites where they converge with entorhinal afferents (Milner & Bacon, 1989; Milner & Veznedaroglu, 1992). As DORs inhibit NPY release, activation of DORs on NPY-containing interneurons could promote lateral perforant pathway LTP (Sperk et al., 2007). Conversely, the effects of CIS on the opioid system in males dramatically differs those seen in females. Following CIS, males have: 1) increased OPRM1-expression in DG granule cells (Johnson et al., 2021) which could make them more sensitive to the hyperpolarization effects of MORs mu opioids (Piguet & North, 1993); 2) decreased plasmalemma DORs in NPY/SOM-containing DG interneurons (Mazid et al., 2016); and 3) elevated PDYN expression in DG granule cells (Johnson et al., 2021), which could negatively impact spatial learning processes as well as granule cell-perforant path excitability and LTP (Wagner et al., 1993).

CIS in males, but not females, results in a down regulation of gene expression for corticotrophin-releasing hormone receptor 1 (Crhr1) and another stress gene (Avpr1a) as well as several plasticity genes (Arc, Cdh2, Ntrk2) and kinase/signaling genes (Akt1, and Arrb) in the CA3 region (Randesi et al., 2018). Additionally, Crh gene expression is upregulated in the DG of CIS males (Randesi et al., 2018). CIS in males, but not females, results in the redistribution of CRFR1 from the cytoplasm to the near plasmalemmal compartment of CA3 pyramidal cell dendrites and decrease in total CRFR1 in DG interneurons (McAlinn et al., 2018). CIS in males, but not females, decreases plasmalemmal DOR densities in CA3 pyramidal cell dendrites and hilar interneurons (Mazid et al., 2016). Thus, the observation that CIS males, but not females, also had increased densities of GFAP and decrease densities of Iba1 in CA3 suggest that glia may play a role in the sex differences observed stress responses in the DG-CA3 opioid system following CIS.

Following CIS, sex differences in Oxy CPP behaviors are paralleled by regional differences in glial densities

CIS females, but not CIS males, acquire Oxy CPP (Reich et al., 2019b). Like behavioral responses, divergent and regionally specific changes in hippocampal glial densities were seen in CIS Oxy female and male rats (Fig. 8). In the DG hilus, Oxy CIS females has prominent decreases in GFAP densities whereas Oxy CIS males had increases in GFAP densities. Moreover, Oxy CIS females had decreases in Iba1 densities and increases in GFAP densities in CA3 SR where the apical dendrites of CA3 pyramidal cells reside. Conversely, Oxy CIS males had increases in GFAP densities in the DG hilus and SO CA2/3a. Importantly, Oxy injections not paired with CPP have little effect on the opioid system in the DG and CA3 in either female or male rats (Ashirova et al., 2021). Thus, together with sex specific changes in glial densities following CIS alone described in section above, these findings suggest additional regions where glia could contribute to sex differences in the behavioral differences to Oxy CPP training seen following CIS.

Notable sex differences in glial densities in Oxy CIS rats also were observed in CA1. Oxy CIS females had increases in both Iba1 and GFAP densities in most CA1 lamina. Conversely, Oxy CIS males relative to either Sal CIS males or Oxy CIS females had decreased Iba1 and GFAP densities in several subregions of CA1. These findings are congruent with our prior studies showing sex differences in the opioid and CRF systems in CA1 following CIS. CA1 pyramidal cell dendrites from CIS females have increased cytoplasmic and total DORs suggesting greater DOR pools, whereas those from CIS males have elevated near plasmalemma DORs, suggesting greater sensitivity to opioid ligands (Rubin et al., 2020). Moreover, CIS females have higher levels of CRF receptor (CRFR1) in every cellular compartment of CA1 pyramidal cells dendrites compared to unstressed females or CIS males (McAlinn et al., 2018). As DORs are thought to be neuroprotective (Charron et al., 2008; Feng et al., 2009; Hayashi et al., 2002), this redistribution of DORs and CRFR1 could render CA1 pyramidal cells in females less vulnerable to neuronal damage from CRF following CIS (Maecker et al., 1997). Interestingly, as CRF has been shown to decrease glutamate excitotoxicity (Elliott-Hunt et al., 2002; Hollrigel et al., 1998), this unopposed increased CRFR1 signaling in CA1 dendrites in CIS females could also enhance neuroprotection (Charron et al., 2009). Moreover, the present observation that Iba1, and to a lesser extent GFAP, increases in CA1 in CIS females, but not CIS males, suggests that glia could be involved in this neuroprotection.

As with GFAP in CA3b, increases in GFAP densities in CA1 could demonstrate the role of astrocytes in modulating the excitability of Schaffer collaterals. MOR activation facilitates astrocytic glutamate release via TREK-1 and TWIK-1 containing K2P channels, which increases the excitability of Schaffer collaterals synapsing on CA1 pyramidal cells via mGluR1 receptors (Nam et al., 2021). CA1 pyramidal cell hyperexcitability is a hallmark of neurodegeneration and is prominently observed in AD models (Lei et al., 2012; Phelan et al., 2024; Šišková et al., 2014). Significantly, astrocytes contribute to CA1 pyramidal cell activity (Bohmbach et al., 2022; Courtney et al., 2023; Wang et al., 2021), and reactive astrocytes are associated with decreased inhibitory synaptic currents and CA1 hyperexcitability (Ortinski et al., 2010). Moreover, the activation of astrocytic MOR alone potentiates synaptic plasticity at Schaffer collateral-CA1 synapses, facilitating the memory acquisition associated with CPP (Nam et al., 2019).

Our prior studies have noted that Oxy CPP in naïve rats differentially alters the opioid system and associated signaling molecules in the CA1 from female and male rats (reviewed in (Chalangal et al., 2021)). Oxy CPP elevates phosphorylated MOR levels in CA1 in females but not males (Bellamy et al., 2019). As phosphorylation is important for opioid receptor internationalization and trafficking (Deng et al., 2001; Doll et al., 2011; Law et al., 2000), this finding suggests that Oxy CPP differentially activates opioid receptors in females and males in the CA1. Following Oxy CPP, Akt1 (serine/threonine kinase 1), a signal transduction intermediate involved in synaptic protein translation (Akama & McEwen, 2003; Brunet et al., 2001; Chong et al., 2005), increases in samples containing CA1/DG from females and decreases in samples containing CA1/DG from males. Moreover, phosphorylated pAKT levels decrease in the pyramidal cell layer in Oxy CPP males. These findings suggest that oxycodone CPP maintains AKT-mediated signaling events in female rats but reduces them in male rats. Our observation that Oxy CPP in CIS females increases Iba1 and GFAP in CA1 whereas Oxy in the absence of CPP in CIS males decreases Iba1 and GFAP parallels these changes.

Conclusion

The implications of altered Iba1 and GFAP densities on sex differences in hippocampal function following Oxy CPP alone or in addition to CIS can only be speculated at present. The neuroprotective and plasticity-promoting activity of both microglia and astrocytes has been well documented, and it is understood that dysregulation of glial functions contributes to the neuropathology observed in conditions such as brain ischemia, pathogen infection, trauma, and other deleterious states (Li & Barres, 2018; Matias et al., 2019). Given the importance of microglia in synaptic pruning (Kim et al., 2017), synaptic plasticity and remodeling (Madry et al., 2018), and learning and memory (Elmore et al., 2018; Worthen et al., 2020), and considering the negative effects of stress on microglial activity and spatial memory (Hu et al., 2022), it can be understood that microglia may play a crucial role in Oxy associative learning and contribute to the cognitive deficits observed following chronic stress. Given the role of astrocytes in synaptic regulation and their involvement in neurotransmission, particularly via opioid receptor activation in the hippocampus (Nam et al., 2019; Nam et al., 2021; Sanderson et al., 2019), it is understandable that astrocytes are inextricably linked to contextual memory formation following Oxy CPP and/or CIS. Together, the findings of this study demonstrate that altered hippocampal glial densities following Oxy CPP alone and in addition to CIS play a role in associative learning processes. These differences in glia-specific changes represent a potential novel target underlying the sex-specific changes during the formation of addictive behaviors.

HIGHLIGHTS.

There were few baseline sex differences in hippocampal Iba1 and GFAP densities.
Oxycodone CPP increased Iba1 (females) and GFAP (both sexes) densities in CA3.
Chronic immobilization stress (CIS) decreased Iba1densities in CA3 more in males.
CIS females acquired Oxycodone CPP whereas CIS males did not.
Glial densities changed in opposite directions in Oxycodone CIS females and males.

Acknowledgements

We thank Drs. Bruce S. McEwen (died January 2, 2020), Mary Jeanne Kreek (died March 27, 2021) for their guidance and support with the conception of the experiments from which the tissues used in this study were derived. We thank Ms. June Chan and Dr. Diane Lane for technical assistance.

Funding:

This work was supported by National Institutes of Health grants R01 DA08259 and R01 HL136520.

ABBREVIATIONS

ABC: avidin-biotin complex
BSA: bovine serum albumin
CIS: chronic immobilization stress
CPP: conditioned place preference
DAB: diaminobenzidine
DG: dentate gyrus
DOR: delta opioid receptor
GABA: Gamma-amino butyric acid
GFAP: Glial fibrillary acid protein
Iba1: ionized calcium binding adapter molecule
ir: immunoreactivity
LTP: long-term potentiation
MOR: mu opioid receptor
NMDA: N-methyl-D-aspartate
NPY: neuropeptide Y
Oxy: oxycodone
PARV: parvalbumin
PFA: paraformaldehyde
PB: phosphate buffer
PCL: pyramidal cell layer
ROI: region of interest
Sal: saline
SLM: stratum lacunosum-moleculare
SLu: stratum lucidum
SO: stratum oriens
SOM: somatostatin
SR: stratum radiatum
TS: tris-buffered saline
US: unstressed

Footnotes

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Declaration of Interest: none

Data statement:

Data available upon request to the corresponding author.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Data Availability Statement

Data available upon request to the corresponding author.

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PERMALINK

Sex Differences in the Effects of Chronic Stress and Oxycodone Associative Learning on Glial Markers in Rat Hippocampus

Ma’ayan Hirschkorn

Annabelle Kim

Garrett Sommer

Sydney Warwick

Lennox Gregoire

Yan Zhou

Joshua Kogan

Jason D Gray

Teresa A Milner

Abstract

Graphical abstract

INTRODUCTION

EXPERIMENTAL PROCEDURES

ANIMALS

Fig. 1. Schematic of experimental cohorts.

Animal handling and estrous cycle determination:

CIS:

Oxy CPP:

IMMUNOCYTOCHEMISTRY EXPERIMENTS

Antibodies:

Immunocytochemical procedures:

Section preparation:

Light microscopic immunocytochemistry:

Analysis and figure preparation:

RESULTS

Iba1 RESULTS

COHORT 1. Sal females and males had few baseline differences in Iba1 densities, however, Iba1 densities increased in select hippocampal subregions in Oxy females

Fig. 2. Iba1-labeling in the hippocampus from US female and male rats following Oxy CPP.

COHORT 2. CIS Sal males had elevated Iba1 in CA1 and decreased Iba1 in CA3b, however, Oxy CIS females had elevated Iba1 in CA1

Fig. 3. Iba1-labeling in the hippocampus from Oxy CIS female and male rats.

COHORTS 1 & 2 Sal rats. CIS prominently decreased Iba1 densities in the DG of males

Fig. 4. Iba1-labeling in the hippocampus from Sal US female and male rats compared to their CIS counterparts.

GFAP RESULTS

COHORT 1. Following Oxy CPP, GFAP densities in hippocampus changed in opposite directions in females and males

Fig. 5. GFAP-labeling in the hippocampus from US female and male rats following Oxy CPP.

COHORT 2. GFAP densities in DG prominently decreased in Oxy CIS females but increased in Oxy CIS males

Fig. 6. GFAP-labeling in the hippocampus from CIS female and male rats following Oxy CPP.

COHORT 1 & 2 Sal rats. CIS Sal females and males had decreased GFAP densities in DG but they were more prominent in males

Fig. 7. GFAP-labeling in the hippocampus from Sal female and male rats following CIS.

DISCUSSION

Fig. 8. Summary of glial density differences in the rat hippocampus in US and CIS rats following Oxy CPP.

Methodological Considerations

Baseline sex differences in glial expression

Oxy CPP elevated CA3b GFAP densities in both sexes

CIS results in prominent sex-specific glial changes in hippocampal opioid circuits

Following CIS, sex differences in Oxy CPP behaviors are paralleled by regional differences in glial densities

Conclusion

HIGHLIGHTS.

Acknowledgements

Funding:

ABBREVIATIONS

Footnotes

Data statement:

REFERENCES

Associated Data

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

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