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. Author manuscript; available in PMC: 2024 Feb 1.
Published in final edited form as: Horm Behav. 2022 Dec 15;148:105296. doi: 10.1016/j.yhbeh.2022.105296

The medial preoptic area and acute cocaine’s stimulant effects in rats: Potential influences of estradiol and biological sex

Julia R Martz a,b, Adriana Vasquez a, Ross Gillette c, Andrea C Gore a,c,d,e, Juan M Dominguez a,b,c,e,*
PMCID: PMC9892259  NIHMSID: NIHMS1858761  PMID: 36528006

Abstract

The medial preoptic area (mPOA) in the hypothalamus is an important integrator of neuroendocrine signaling and a key regulator of both natural and drug-induced reward. Although the mPOA modulates sex differences in other behaviors, whether it also modulates sex differences in cocaine response remains unclear. To help us better understand the mPOA’s role in sex differences associated with cocaine response, we examined cocaine-induced changes in locomotion and neural activity in the mPOA of male and female rats. In addition, neural activity in the striatum, a brain area known to be involved in cocaine response, was examined for comparison purposes. Fos, the protein product of the immediate early gene c-fos, was used as the marker of neural activity. Locomotion chambers were used to measure behavior, radioimmunoassays and vaginal lavages were used to determine hormonal status, and immunohistochemical assays were used to quantify Fos. To account for the effects of gonadal hormones, rats were left gonadally intact and categorized as either ‘low-estradiol’ or ‘high-estradiol’ based on their hormonal status on test day. Results indicate that high-estradiol females experienced greater cocaine-induced mPOA Fos-immunoreactivity (Fos-ir) and displayed greater cocaine-induced locomotion than low estradiol females. Conversely, high-estradiol males experienced less cocaine-induced mPOA Fos-ir and displayed less cocaine-induced locomotion than low-estradiol males. Cocaine-induced Fos-ir in the mPOA also correlated with cocaine-induced Fos-ir in areas of the striatum already associated with cocaine response. These findings further support the mPOA’s role in the endocrine-mediated response to cocaine. It also identifies the mPOA as a contributor to sex differences in cocaine response and potential differences in vulnerability to developing cocaine use disorders.

Keywords: Cocaine, Estradiol, Estrous cycle, Fos, Locomotion, Preoptic area, Sex differences, Striatum

1. Introduction

The medial preoptic area (mPOA) in the anterior hypothalamus modulates both natural and drug-induced reward (Numan, 1974; Hull & Dominguez, 2007; Tobiansky et al., 2013). Preoptic regulation of naturally rewarding behaviors, including parental (Rosenblatt, Hazelwood, & Poole, 1996; Champagne et al., 2003) and copulatory behaviors (Powers & Valenstein, 1972; Hull et al., 2007), depends on biological sex. For example, lesions of the mPOA inhibit sexual behavior in male rats (Larsson & Heimer, 1964; Arendash & Gorski, 1983; Paredes et al., 1993) but can either facilitate or disrupt sexual behaviors in females (facilitate: Law & Meagher, 1958; Powers & Valenstein, 1972; disrupt: Whitney, 1986; Yang & Clements, 2000). Although the mPOA provides sexually dimorphic control of naturally rewarding behaviors, whether it also modulates cocaine response in a sexually dimorphic manner remains unclear. The findings reported here elucidate, at least in part, sex differences in the mPOA’s modulation of cocaine response, as indicated by changes in locomotion.

Biological sex and the accompanying sex steroid hormone environment influence response to psychomotor stimulants. Cocaine, for example, increases locomotor activity in both male and female rodents via dopamine neurotransmission in the mesolimbic system (Kelly & Iverson, 1976). However, female rats develop increased locomotor responses more rapidly and to a greater extent than males (Beatty, 1979; Walker et al., 2001, Wissman et al., 2011). This sex difference is partly driven by sex steroid hormones, such as fluctuations in levels of 17β-estradiol (estradiol), the main circulating form of estrogen. When estradiol is elevated, either by exogenous treatment or naturally occurring peaks in the estrous cycle, female rats display greater cocaine-induced locomotion (Quinones-Jenab et al., 2000; Cummings et al., 2014; Quinones-Jenab et al., 1999; Souza et al., 2014). Electrophysiological data suggest that basal dopamine-neuron activity in the ventral tegmental area (VTA) is significantly greater in females compared to males, but only when females are in the proestrus/estrus phase of their estrous cycle, when estradiol levels have peaked. Calipari and colleagues (2017), for example, found augmented VTA dopamine activity during proestrus/estrus was associated with increased phosphorylation of the dopamine transporter at threonine 53, which enhances cocaine’s ability to inhibit dopamine uptake. However, few studies have investigated the role of structures outside of the traditional mesolimbic system in sexually dimorphic responses to cocaine.

The mPOA sends projections to the VTA (Simerly & Swanson, 1988; Zahm et al., 2011; Tobiansky et al., 2013) and contains a high concentration of sex steroid hormone receptors (Simerly et al., 1990). In female rats, estrogen receptors are present in the majority of mPOA efferents to the VTA, whose terminals are in close apposition to dopamine neurons projecting to the nucleus accumbens (NAc; Tobiansky et al., 2016). As evidence of the functionality of these connections, estradiol microinjected into the mPOA of female rats increases conditioned-place preference (CPP) for cocaine, as compared to females that did not receive an estradiol microinjection (Robison, Martz & Dominguez, 2018). In addition, when the mPOA is lesioned, female rats display increased CPP for cocaine, along with increased cocaine-induced neural activity and dopamine release in the NAc (Tobianksy et al., 2013; Tobiansky et al., 2016). The mPOA is also involved in the regulation of locomotion, as demonstrated by lesions in this area, which increase cocaine-induced locomotion in male rats (Will, Martz, & Dominguez, 2016). Taken together, these studies indicate that the mPOA influences the mesolimbic system in both male and female rats.

However, little is known about the effect of cocaine in the mPOA itself. Mattson and colleagues (2005) used the immediate early gene c-fos’s protein product, Fos, as a marker of neural activation in postpartum dams that were exposed to either cocaine- or pup-associated cues in a CPP paradigm. On test day, dams that spent more time in the cocaine-associated chamber experienced greater Fos-immunoreactivity (Fos-ir) in the mPOA compared to controls, but experienced less Fos-ir than dams that preferred the pup-associated chamber. In a study of male rats, McHenry and colleagues (2016) found that expression of deltaFosB (ΔFosB), another member of the Fos family of transcription factors, was not affected by repeated doses of cocaine. Together, these studies begin to elucidate the effects of cocaine in the mPOA. However, to our knowledge, sex comparisons of this effect have not previously been performed.

The aim of this study was to perform a sex comparison of the relationship between cocaine-induced mPOA neural activity and cocaine-induced locomotion in male and female rats. Fos was used as a marker of neural activation that occurred in response to an acute dose of cocaine. The striatum, an area of the brain already known to be involved in cocaine response (Neisewander et al., 1995; Everitt & Robbins, 2013), was examined for purposes of comparison.To investigate the influence of endogenous gonadal hormones, males and females were gonadally intact. The use of intact rats allowed us to investigate cocaine-induced locomotion and mPOA neural activation in males and in females across the different stages of their estrous cycle. We hypothesized that female rats in proestrus/estrus would experience greater locomotion and mPOA neural activation in response to cocaine compared to females in the other phases of the estrous cycle or males.

2. Materials and Methods

2.1. Animals

Thirty-five female and 19 male Sprague-Dawley rats (Envigo, Indianapolis, IN), aged 9–10 weeks, were used in this experiment. Upon arrival, rats were separated by sex and housed in pairs in a temperature-controlled room on a 12:12 reverse light/dark cycle (lights off at 1000). Food and water was available ad libitum. All experimental procedures were approved by the Animal Care and Use Committee (IACUC) at the University of Texas at Austin and conducted in accordance with the NIH Guide for the Care and Use of Laboratory Animals, 8th edition. Rats were randomly assigned to groups that received either saline or cocaine administration.

2.2. Estrous Cycle Monitoring

Stages of the estrous cycle were monitored in female rats by analyses of cell types in vaginal lavage samples. Females received vaginal lavage each morning for at least 10 consecutive days prior to locomotor testing. Samples were obtained by flushing the vaginal cavity with 25μl 0.9% sterile saline. Using a procedure adapted from Goldman and colleagues (2007), each sample was deposited onto a glass slide and immediately examined under light microscopy to determine cell types present. Samples with clusters of round, nucleated epithelial cells are indicative of females in proestrus, the stage of the cycle when estradiol and progesterone levels peak. Proestrus is followed by estrus, marked by cornified or keratinized cells, the stage of the cycle when females are sexually receptive. Metestrus begins when leukocytes appear alongside the cornified cells, followed by leukocytes and round epithelial cells that are characteristic of diestrus. Metestrus and diestrus are the stages of the cycle when estradiol and progesterone levels are low relative to the peaks that occur during proestrus (Butcher et al., 1973; Smith et al., 1975). Females were assigned to groups based on estrous cycle stage on day of sacrifice: females that were in estrus or proestrus (E/P, n = 17) and females that were in diestrus or metestrus (D/M, n = 18).

2.3. Locomotor Testing

Locomotion was measured during the dark phase of the light cycle in a 70 cm by 21cm apparatus (San Diego Instruments, Inc., San Diego, CA) equipped with a 16 × 4 photocell array. Locomotion testing occurred 17–21 days after arrival to account for cycle stage in the females. On test day, rats were injected with either cocaine (10 mg/kg i.p.) or saline, depending on predetermined group designations, and immediately placed in the testing apparatus for 30 minutes. Rats were not habituated to the chamber prior to this period. This cocaine dose was chosen because it is a moderate dose on the upper ascending limb of the dose response curve that has previously been shown to produce sex differences in locomotor response in intact rats (Walker et al., 2001). In addition, our laboratory has used this dose in prior studies of both males and females (Will et al., 2014; Robison et al., 2018). Rats were tested 1–5 hours after the start of the dark cycle in a room with ambient-white noise. Horizontal locomotion was measured by consecutive beam breaks of the photocell array that were summed and binned into 5 minute segments. Locomotion was computed starting 5-minutes after drug injection, which was meant to minimize the effects of novelty. Four subjects were tested during a software malfunction, but their sessions had been video recorded and an experimenter blind to treatment groups scored the videos according to the same 16 × 4 grid used by the software. Two of the hand scored subjects came from the male cocaine group, one came from the female cocaine group, and one came from the female saline group. The scores done by hand were not significantly different from the automated scores in each group.

2.4. Tissue Preparation

Rats were euthanized by injection of 1.5 ml/kg Euthasol (Animal Health International, Loveland, CO) 90-minutes after the injection of cocaine or saline. Once under deep anesthesia (as defined by lack of response to a toe pinch), the animal was perfused via transcardial perfusion using 50 ml phosphate butter solution (PB; pH 7.35) followed by 300 ml of 4% paraformaldehyde in PB. Brains were removed after perfusion and post-fixed for 1 hour in 4% paraformaldehyde before being transferred to a solution of 30% sucrose in PB, and then refreshing this solution 24-hours later. Brains were coronally sectioned using a frozen microtome at a thickness of 35 μm. The sections were stored in a cryoprotectant solution (30% ethylene glycol and 30% sucrose dissolved in PB with 200 μg/l sodium azide) at −20° C until use.

2.5. Immunohistochemistry

Rats were euthanized 90-minutes after cocaine injection, to capture c-fos activity, which encodes the nuclear phosphoprotein Fos. Tissue sections were double labeled for Fos and a neuronal marker using nickel-labeled 3–3’diaminobenzidine (DAB) and unlabeled DAB, respectively. Free-floating sections of brain tissue containing the mPOA (one representative section from each of plates 18–22 in Swanson (2004)) and the striatum (one section containing the posterior NAc core, the posterior NAc shell, and the posterior central caudate-putamen from plate 13 in Swanson, 2004) were placed on a rotator at room temperature. The tissue was rinsed with PB four times for 5 minutes (20 minutes total) prior to and in between all incubations. Endogenous peroxidase activity was blocked by incubating the tissue in 1% H2O2 for 10 minutes. The tissue was then incubated in a blocking solution containing 2.0% normal goat serum and 0.4% Triton-X in PB for 1 hour before being transferred to a primary rabbit anti-Fos antibody solution (sc-52, Santa Cruz Biotechnology, Inc., Dallas, TX; 1:8000 in blocking solution) for overnight incubation. The tissue was then incubated in biotinylated goat anti-rabbit secondary antibody (Vector Laboratories, Burlingame, CA; 1:500 in blocking solution) for 1-hour. The signal was amplified by incubation with avidin-biotin complex (ABC elite, Vector Laboratories, Burlingame, CA; 1:1000) in PB for 1-hour. The amplified signal was visualized by incubation with 0.02% DAB (Sigma-Aldrich, St. Louis, MO), 2% Ni2SO4 (Thermo Fisher Scientific, Waltham, MA), and 0.01% H2O2 for 10-minutes. The chromogen reaction was terminated by 8 washes in PB. The tissue was then incubated overnight in primary mouse anti-NeuN antibody (MAB377, Millipore, Sigma, Burlington, MA) in blocking solution. The amplification steps were the same as the previous day, but with the use of biotinylated goat anti-mouse secondary antibody and unlabeled DAB. Immunocytochemical controls included omission of primary antibodies for NeuN or Fos. After the final set of washes, stained tissues were mounted to microscope slides, dehydrated, and coverslipped with DPX mounting medium (VWR International, Radnor, PA).

2.6. Cell Counting

Photomicrographs of the stained tissue were taken with a Zeiss AX10 microscope at 20x magnification. NeuN- and Fos-ir cells were quantified in sections in the striatum and across the mPOA by individuals blind to animal identification and group. Unilateral counts were made in a 337 × 450 μm rectangular area for each section. Unilateral counts were used so that researchers could use the single most representative area, as determined by the presence of landmarks (e.g. third ventricle, optic chiasm, and anterior commissure for the mPOA) and absence of tissue defects (e.g. tears). All cell counts were performed in ImageJ (NIH). To account for possible sex differences in the number of total neurons, all cell counts were analyzed as percentages of the total number of neurons ([Fos/NeuN]x100). Therefore, all measures of Fos-ir in this paper are relative to the total NeuN count. Additionally, only Fos-cells colocalized with NeuN were counted in order to exclude non-neuronal Fos expression. The average of the counts from plates 18–21 was classified as the rostrocentral mPOA, and the count from plate 22 was classified as the caudal mPOA. All striatum counts were taken from plate 13.

2.7. Estradiol Radioimmunoassay

The experiment was initially designed to consider only the hormonal status of the female rats, as measured by estrous cycle stage. However, after the experiment was run, blood samples that were collected immediately prior to perfusions were used to investigate serum estradiol levels in the male rats. In males, serum estradiol was measured by radioimmunoassay (RIA) according to the manufacturer’s protocol (Beckman Coulter, Webster, TX, #DSL-4800). All samples were run in duplicate and no values were undetectable.The assay had a range of 2.2 – 750 pg/ul, the sensitivity of the assay was 2.2 pg/ul, and the intra-assay coefficient of variance was 5.27%. The measured values of known control samples were > 97% accurate; Control 1, actual = 23 pg/ul, measured = 23.51 pg/ul, and Control 2, actual 269 pg/ul, measured = 273.52 pg/ul.

2.8. Hormonal Status

To compare the influence of sex steroid hormones in males and females, we created a categorical variable defined as either ‘low-estradiol’ or ‘high-estradiol’. For males, estradiol levels were measured from blood samples taken at sacrifice and categorized as ‘low-estradiol’ or ‘high-estradiol’ based on a median split (range: 5.30 – 13.15 pg/ml; median: 10.10 pg/ml; Fig 1). For females, we relied on estrous cycle stage rather than levels of circulating estradiol. Female rats experience a four-day estrous cycle in which levels of circulating estradiol fluctuate considerably each day. However, because estradiol can stay bound to nuclear receptors for 18–24 hours, decreased levels of circulating estradiol may not necessarily indicate that the hormone is no longer active (Becker et al., 2005). For example, females are sexually receptive during the estrus phase of the cycle, when estradiol levels are lowest. This receptivity is due to the peak of estradiol that occurred the day before, during the proestrus phase of the cycle. Because of this, we chose to rely on lavage samples and categorized E/P females as ‘high-estradiol’ and D/M females as ‘low-estradiol’.

Fig. 1.

Fig. 1

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Distribution of plasma estradiol levels (pg/ml) in male rats. The dashed line represents the median value. Male rats located above the median were classified as ‘high-estradiol’ and males located below the median were classified as ‘low-estradiol.’ Each point represents data from an individual.

2.9. Statistical Analysis

All data were analyzed using R (version 4.0.4) and R studio (version 1.4.1103). All assumptions for parametric tests were met. The Bonferroni Outlier Test was used to determine significant outliers, and the number of outliers removed was limited to one per group. Bartlett’s test was used to check for homogeneity of variance, while the Shapiro-Wilk test in combination with a quantile-quantile (Q-Q) plot was used to check for normality. Total locomotion was analyzed with a 2×2 factorial ANOVA to discriminate between the effects of sex and drug, and a 3×2 factorial ANOVA to analyze the effects of group (males, D/M females, and E/P females) and drug. Linear mixed effects modeling fit by restricted maximum likelihood (REML) was used to assess the influence of time, sex, and drug, and then time, group, and drug in explaining variations in locomotion across the five time points. The linear mixed effects models were generated using the lme function in the R package nlme (v3.1–152; Pinheiro et al., 2021), with drug, sex and time set as fixed factors in the first model, drug, group, and time set as fixed factors in the second model, and individual set as a random factor in both models. Final model selections were decided using Akaike Information Criterion (AIC). For cell count data, a 2×2 factorial ANOVA was used to discriminate between drug and sex effects, and then a 3×2 factorial ANOVA for group and sex effects. Pearson’s r correlation coefficient tests were used in the analysis of all correlations between two continuous variables (i.e. the relationship between Fos-ir and locomotion) independently for each group. Linear regression was used to measure the relationship between hormonal status (categorical variable) and Fos-ir in the mPOA, and then again to meaure the relationship between Fos-ir in the striatum and Fos-ir in the mPOA. Slopes comparisons were made using the emtrends function in the R package emmeans (v1.8.1–1; Lenth et al., 2022). Multiple regression was used to determine predicted locomotion based on mPOA Fos-ir, sex, drug, and hormonal status. Model selection for multiple regression was again decided using AIC. Post hoc comparisons were made using Tukey’s HSD test with statistical significance set at p < .05. Cohen’s d (d) was calculated to measure effect size for all post hoc comparisons. Partial eta squared (η2p) was calculated to measure effect size for all ANOVAs and linear mixed effect models. Adjusted R squared (R2) values were reported for Pearson’s r, regression, and multiple regression analyses.

3. Results

3.1. Locomotion

3.1.1. Acute Cocaine Increases Locomotion in Males and Females

Cocaine increased total locomotion in both male and female rats (Fig 2a). A 2×2 factorial ANOVA revealed a main effect of drug (F1,51 =44.21, p <.001, η2p = .46) and sex (F1,51=6.70, p = .013, η2p = .12) on overall locomotion, with no interaction effect of drug and sex. Post hoc comparisons revealed that both males and females experienced greater locomotion in response to cocaine than saline (p < .001, d = 1.75; p < .001, d = 1.91), and there was no difference in total cocaine-induced locomotion between males and females. Looking at the data across the five time points, a linear mixed effect model fit by REML revealed a main effect of time (F4, 212 = 36.0055, p < .001, η2p = .61), as well as drug (F1,51 = 44.21, p < .001, η2p = .50) and sex (F1, 51 = 7.19, p = .01, η2p = .44), with rats generally expressing less locomotor activity later in the test (Fig 2b). There were no significant two or three way interactions between time, drug, and sex. When analyzed at individual time points, post hoc comparisons indicate that males experienced significantly greater locomotion in response to cocaine compared to saline at the first four time points (p < .05, d = 1.44; p < .001, d = 1.83; p < .05, d = 1.21; p < .05, d = 1.63), but not at the last. Females experienced significantly greater locomotion in response to cocaine compared to saline at the last four time points (p < .01, d = 1.70; p < .001, d = 1.73; p < .05, d = 1.65; p < .01, d = 1.57), but not at the first. Males and females did not experience significant differences in ambulatory activity in response to cocaine at any time point.

Fig. 2.

Fig. 2

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Locomotor activity in an open field after acute saline or cocaine administration. (a) Males and females that received cocaine (10 mg/kg) expressed greater total locomotor activity during the testing period than males (*** p < .001) and females (*** p < .001) that received saline (Mal Coc, n = 10; Fem Coc, n = 18; Mal Sal, n = 9; Fem Sal, n = 17), during a 30-minute locomotion task. Each point represents data from an individual. (b) In a 30-minute locomotor task, binned into 5, 5-minute segments starting at minute 5, locomotion decreased across time for all groups. Cocaine males expressed greater locomotion than saline males during the first four time points (* p < .05, *** p < .001), whereas cocaine females expressed greater locomotion than saline females during the last four time points (* p < .05, ** < .01, *** p < .001). Male and female rats did not display differences in cocaine-induced locomotion at any time point. (c) After females were divided by phase of estrous cycle (E/P = estrus/proestrus and D/M = diestrus/metestrus), males and E/P females that received cocaine expressed greater total locomotion than males and E/P females that received saline (*** p < .001; Mal Coc, n = 10; E/P Fem Coc, n = 10; Mal Sal, n = 9; E/P Fem Sal, n = 7). Each point represents data from an individual. (d) Analysis revealed a main effect of time, drug, and group (E/P females, D/M females, and males), and an interaction between drug and group. Locomotion decreased across time for all groups. Cocaine E/P females expressed greater locomotion than saline E/P females at the second and third time points (* p < .05, ** p < .01), while cocaine D/M females did not differ from saline D/M females (D/M Fem Coc, n = 8; D/M Fem Sal, n = 10) at any time point. Cocaine males expressed greater locomotion than saline males at the first four time points (* p < .05, *** p < .001).

3.1.2. Acute Cocaine-Induced Locomotion in Females is Dependent on Stage of Estrous Cycle

When females were grouped by phase of estrous cycle (D/M and E/P), results showed that cocaine increased total locomotion in males and only E/P females (Fig 2c). A 3×2 factorial ANOVA revealed a main effect of group (D/M females, E/P females, males; F2,50 = 4.81, p < .05, η2p = .16) and drug (F1,50 = 42.60, p < .001, η2p = .46) on total locomotion, as well as an interaction effect of group and drug (F2,48 = 3.30, p < .05, η2p = .12). Post hoc analysis revealed that locomotor activity significantly increased in males and E/P females that received cocaine compared to saline (p < .001, d = 1.75; p < .001, d = 3.22). However, locomotion did not increase in D/M females that received cocaine compared to those that received saline. When examining sex differences, the locomotor response to cocaine in males versus E/P females did not differ significantly, but the response in D/M females was significantly lower than that of males (p < .01, d = 1.67). When analyzing locomotion across the five time points, a linear mixed effect model fit by restricted maximum likelihood revealed a main effect of time (F4,212 = 36.3165, p < .001, η2p = .61), group (F2,48 = 5.47, p < .01, η2p = .45), and drug (F1,48 = 49.90, p < .001, η2p = .50) on locomotion, as well as an interaction between group and drug (F2,48 = 3.52, p < .05, η2p = .44). Locomotion generally decreased across time for all groups (Fig 2d). Post hoc comparisons indicate that D/M females did not express increased locomotion in response to cocaine compared to saline at any time point, while E/P females experienced significantly increased locomotion in response to cocaine compared to saline at the second and third time points (p < .05, d = 2.59; p < .01, d = 2.84), but not at any of the other three time points. Males experienced increased ambulatory activity in response to cocaine compared to saline across the first four time points (p < .05, d = 1.44; p < .001, d = 1.83; p < .05, d = 1.21; p < .05, d = 1.63), but not at the last. Cocaine-induced locomotion in males and E/P females did not differ significantly at any time point. Locomotion was not significantly different between males, E/P females, and D/M females that received saline.

3.2. Fos Expression

3.2.1. mPOA Fos Expression is Dependent on Sex and Estrous Cycle Stage in Females

Female rats had higher levels of Fos-ir across the mPOA compared to males (Fig 3b). A 2×2 factorial ANOVA revealed a main effect of sex (F1,46 = 15.00, p < .001, η2p = .25), with increased Fos-ir in females compared to males, but no main effect of drug. This pattern is conserved across the sub regions of the mPOA, with a main effect of sex in the rostrocentral (F1,51 = 13.23, p < .001, η2p = .21) and caudal mPOA (F1,46 = 5.74, p < .05, η2p = .11). Post hoc analysis revealed that females display greater Fos-ir than males, regardless of drug treatment, in the rostrocental (p < .001, d = 1.09) and caudal mPOA (p < .05, d = 0.71). After the females were divided into groups based on estrous cycle stage, 3×2 factorial ANOVAs revealed a main effect of group in the rostrocentral (F2,50 = 14.80, p < .001, η2p = .37) and caudal mPOA (F2,45 = 4.50, p < .05, η2p = .17), but not drug (Fig 3c). Post hoc comparisons between groups showed that E/P females, regardless of drug treatment, express more Fos-ir than D/M females in the rostrocentral mPOA (p < .01, d = 1.17), but not in the caudal mPOA. E/P females express greater Fos-ir than males in the rostrocentral (p < .001, d = 1.85) and caudal mPOA (p < .05, d = 1.05).

Fig. 3.

Fig. 3

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Fos-ir (cells with Fos-immunoreactivity) in the mPOA is influenced by sex and estrous cycle stage in females. (a) Coronal sections from Swanson (2004) depicting areas where cell counts were performed in the mPOA (b) Females expressed greater Fos-ir than males in the rostrocentral (*** p < .001; Mal Coc, n = 10; Fem Coc, n = 18; Mal Sal, n = 9; Fem Sal, n = 17), and caudal mPOA (* p < .05; Mal Coc, n = 10; Fem Coc, n = 16; Mal Sal, n = 8; Fem Sal, n = 15), regardless of drug treatment. (c) After dividing females into groups based on estrous cycle stage, E/P females expressed greater Fos-ir in the rostrocentral mPOA than D/M females (** p < .01), regardless of drug treatment. E/P females also expressed significantly greater Fos-ir than males in the rostrocentral (*** p < .001; Mal Coc, n = 10; E/P Fem Coc, n = 10; Mal Sal, n = 9; E/P Fem Sal, n = 7; D/M Fem Coc, n = 8; D/M Fem Sal, n = 10) and caudal (* p < .05; Mal Coc, n = 10; E/P Fem Coc, n = 9; Mal Sal, n = 9; E/P Fem Sal, n = 6; D/M Fem Coc, n = 7; D/M Fem Sal, n = 9) mPOA. (d) Demonstrative photomicrograph of cells in the mPOA with NeuN and with NeuN colocalized with Fos (scale bar is 40 μm; BV = blood vessel). Each point represents data from an individual.

3.2.2. Striatal Fos Expression is Dependent on Sex, Drug, and Estrous Cycle Stage in Females

Cocaine administration influenced Fos-ir in the caudate, NAc core, and NAc shell (Fig 4). In the caudate, a 2×2 factorial ANOVA revealed a main effect of drug (F1,51 = 57.01, p < .001, η2p = .53), with increased cocaine-induced Fos-ir in both males and females, and no main effect of sex (Fig 4b). Post hoc comparisons revealed that males and females experienced greater locomotion after receiving cocaine, compared to rats that received saline (p < .001, d = 2.66; p < .001, d = 1.89). After the females were grouped based on stage of estrous cycle, a 3×2 factorial ANOVA revealed a group by drug interaction (F2,48 = 3.39, p = .04, η2p = .12; Fig 4c). Post hoc comparisons showed males receiving cocaine expressed greater Fos-ir compared to saline males (p < .001, d = 2.66), and only the cocaine E/P females expressed greater Fos-ir compared to saline E/P females (p < .001, d = 2.23). Cocaine D/M females did not express an increase in Fos-ir compared to saline D/M females. In the NAc shell, a 2×2 factorial ANOVA yielded a main effect of drug (F1,51 = 6.72, p = .01, η2p = .12) and a main effect of sex (F1,51 = 11.1109, p < .01, η2p = .18, Fig 4b). With females divided into groups based on estrous cycle stage, a 3×2 factorial ANOVA showed a main effect of drug (F2,50 = 6.43, p = .01, η2p = .11) and a main effect of group (F2,50 = 5.50, p < .01, η2p = .18; Fig 4c). Post hoc analysis revealed no significant comparisons. In the NAc core, a 2×2 factorial ANOVA revealed a main effect of drug (F1,51 = 7.10, p = .01, η2p = .12; Fig 4b), and no effect of sex. With females divided into groups based on estrous cycle stage, a 3×2 factorial ANOVA again revealed only a main effect of drug (F2,50 = 8.23, p < .01, η2p = .14; Fig 4c). Post hoc comparisons showed that only the cocaine E/P females expressed greater Fos-ir compared to saline E/P females (p < .05, d = 1.49).

Fig. 4.

Fig. 4

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Fos-ir in the striatum of males and females. (a) Coronal section from Swanson (2004) depicting areas where cell counts were performed in the striatum (b) 90-minutes after cocaine administration, males (*** p < .001) and females (*** p < .001) expressed greater caudate Fos-ir than rats that received saline (Mal Coc, n = 10; Fem Coc, n = 18; Mal Sal, n = 9; Fem Sal, n = 17). (c) When females were divided by cycle stage, only E/P females (*** p < .001) and males (*** p < .001) had greater cocaine-induced Fos-ir in the caudate compared to saline animals. E/P females also had greater cocaine-induced Fos-ir in the NAc shell than E/P females that received saline (* p < .05; Mal Coc, n = 10; E/P Fem Coc, n = 10; Mal Sal, n = 9; E/P Fem Sal, n = 7; D/M Fem Coc, n = 8; D/M Fem Sal, n = 10). Each point represents data from an individual.

3.2.3. Cocaine-Induced Fos Expression in the mPOA Predicts Cocaine-Induced Fos Expression in the Striatum

Cocaine-induced Fos-ir in the rostrocentral mPOA predicted cocaine-induced Fos in the caudate and NAc core and shell (Fig 5). Regression analyses revealed that rostrocentral mPOA Fos-ir did not predict Fos-ir in any sub region of the striatum among saline treated rats. However, cocaine-induced Fos-ir in the rostrocentral mPOA predicted cocaine-induced Fos-ir in the caudate of females (b = 1.53, t16 = 2.96, p < .01), and explained a significant portion of the variance in caudate Fos-ir (F1,16 = , p < .01, R2 = .35). Fos-ir in the rostrocentral mPOA did not predict caudate Fos-ir in cocaine males. The slopes of the relationships between mPOA Fos and caudate Fos were significantly different between saline females and cocaine females (t33 = 2.24, p < .05), but not between male treatment groups or between the sexes. Cocaine-induced Fos-ir in the rostrocentral mPOA positively correlated with cocaine-induced Fos-ir in the NAc core in males (b = .80, t8 = 2.34, p < .05), but negatively correlated in females (b = −0.76, t16 = 2.65, p < .05). A significant portion of the variance in NAc core Fos-ir was explained in both groups (F1,8 = 5.47, p < .05, R2 = .41; F1,16 = 7.03, p < .05, R2 = .31). The slopes of the relationships between mPOA Fos and NAc core Fos were significantly different between cocaine males and cocaine females (t26 = 2.46, p < .05), but not between saline males and saline females. There were also no differences in slopes among male or female only treatment groups (i.e. saline females versus cocaine females and saline males versus cocaine males). Cocaine-induced Fos-ir in the rostrocentral mPOA predicted cocaine-induced Fos-ir in the NAc shell in males (b = 0.99, t8 = 2.80, p < .05, R2 = .49) and females (b = 0.47, t16 = 2.38, p < .05), and explained a significant portion of the variance in both groups (F1,8 = 7.83, p < .05, R2 = .49; F1,16 = 5.65, p < .05, R2 = .26). The slopes of the relationships between mPOA Fos and NAc Shell Fos did not differ among male and female treatment groups or between males and females.

Fig. 5.

Fig. 5

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Rostrocentral mPOA Fos-ir predicts striatal Fos-ir in males and females that received cocaine. In the caudate, cocaine-induced rostrocentral mPOA Fos-ir predicted caudate Fos-ir in females (R2 = .35, ** p < .01), but not males. In the NAc core, cocaine-induced Fos-ir positively correlated with rostrocentral mPOA Fos-ir in males (R2 = .41, * p < .05), but negatively correlated in females (R2 = .31, * p < .05). The slopes of the lines were significantly different between cocaine males and cocaine females in the NAc core. (* p < .05). In the NAc shell, cocaine-induced Fos-ir positively correlated with Fos-ir in the rostrocentral mPOA of males (R2 = .49, * p < .05) and females (R2 = .26; * p < .05). Each point represents data from an individual.

3.3. Relationship Between mPOA Fos, Locomotion, and Hormonal Status

3.3.1. Cocaine-Induced mPOA Fos Predicts Cocaine-Induced Locomotion in Males and Females

Cocaine-induced Fos-ir in the rostrocentral, but not the caudal, mPOA predicted cocaine-induced locomotion in males and females (Fig 6). In the rostrocentral mPOA, regression analyses revealed that Fos-ir predicted locomotion in cocaine males (b = 86.57, t8 = 2.36, p < .05) and cocaine females (b = 28.94, t16 = 2.45, p < .05), and explained a significant portion of the variance in locomotion among these groups (F1,8 = 5.57, p < .05, R2 = .41; F1,16 = 5.99, p < .05, R2 = .27). Rostrocentral mPOA Fos-ir did not predict locomotion among male or female rats that received saline. Furthermore, the slopes of the relationships were not significantly different between cocaine females and saline females, cocaine males and saline males, or between males and females in either drug condition. In the caudal mPOA, there was no relationship between locomotion and Fos-ir in males or females, and there were no significant differences in slopes.

Fig. 6.

Fig. 6

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Cocaine-induced Fos-ir in the rostrocentral mPOA predicts cocaine-induced locomotion in males and females. A positive relationship between Fos-ir and locomotion was present in the rostrocentral, but not caudal mPOA, in males (R2 = .41, * p < .05) and females (R2 = .27, * p < .05) that received cocaine. There were no significant correlations present in saline treated animals. There were no significant differences in slopes between males and females or between cocaine and saline among groups. Each point represents data from an individual.

3.3.2. Hormonal Status Predicts mPOA Fos Expression in Males and Females

We found that stage of estrous cycle was associated with mPOA Fos-ir regardless of drug, therefore regression analyses were performed to determine whether hormonal status was predictive of mPOA Fos-ir, in both males and females (Fig 7). Hormonal status is a categorical variable that we defined above as either ‘low-estradiol’ or ‘high-estradiol’. In saline-treated females, hormonal status predicted Fos-ir in the rostrocentral mPOA (b = 7.21, t15 = 3.04, p < .01), and explained a significant portion of the variance in Fos-ir (F1,15 = 9.26, p < .05, R2 = .38). The slope of this relationship in saline-treated females is positive, indicating that high-estradiol status is associated with greater Fos-ir. For cocaine-treated females, there was no significant relationship between hormonal status and Fos-ir in the rostrocentral mPOA. In cocaine-treated males, hormonal status predicted Fos-ir in the rostrocentral mPOA (b = −5.51, t8 = −2.70, p < .05), and explained a significant portion of the variance in Fos-ir (F1,8 = 7.31, p < .05, R2 = .48). The slope of this relationship in cocaine-treated males is negative, indicating that high-estradiol status is associated with less Fos-ir. For saline-treated males, there was no significant relationship between hormonal status and Fos-ir in the rostrocentral mPOA. In addition, the slopes of the lines were significantly different between saline females and saline males (t = 2.50, p < .05), and between cocaine females and cocaine males (t = 2.53, p < .05).

Fig. 7.

Fig. 7

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Hormonal status predicts mPOA Fos-ir differently in males and females. In males, high-estradiol status predicted low cocaine-induced Fos-ir in the rostrocentral mPOA (R2 = .48, * p < .05). In females, high-estradiol status predicted high Fos-ir in the rostrocentral mPOA (R2 = .38, * p < .05), but only in saline-treated rats. The slopes of the lines were significantly different between males and females in both drug conditions (* p < .05). Each point represents data from an individual.

3.3.3. mPOA Fos Expression, Hormonal Status, Sex, and Drug Predict Locomotion

Multiple regression analysis was used to predict locomotion based on rostrocentral mPOA Fos-ir, hormonal status, sex, and drug. The best fitting model (F8,45 = 6.72, p < .001, R2 = .67), as determined by the model with the lowest AIC, has rostrocentral mPOA Fos-ir (b = 22.55, t45 = 2.28, p < .05) and the interaction between sex, drug, and hormonal status (b = −1854.14, t45 = −4.66, p < .001) as significant predictors of locomotion. The interaction indicated that hormonal status predicts cocaine-induced locomotion differently in males and females. High-estradiol status predicts increased cocaine-induced locomotion in females, but decreased cocaine-induced locomotion in males (Fig 8).

Fig. 8.

Fig. 8

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Locomotion is predicted by mPOA Fos-ir, hormonal status, sex, and drug. Multiple regression analysis revealed that Fos-ir in the rostrocentral mPOA and the interaction between sex, drug, and hormonal status best predicted locomotion (R2 = .67, p < .001). In males, high-estradiol status and low mPOA Fos-ir was associated with lower locomotion in rats that received cocaine. In females, high-estradiol status and high mPOA Fos-ir was associated with higher locomotion in rats that received cocaine. Each point represents data from an individual.

4. Discussion

Because the mPOA is implicated in cocaine response, neuroendocrine regulation, and sex differences in various behaviors (Tobiansky et al., 2016; Champagne et al., 2006; Hull & Dominguez, 2007), we sought to investigate the influence of biological sex and hormonal status on cocaine-induced activity in the mPOA. However, results show that the mPOA is not directly activated by acute cocaine administration, as measured by Fos-ir. However, cocaine-induced Fos-ir in the rostrocentral mPOA did correlate with cocaine-induced locomotion in both male and female rats, suggesting that the rostrocentral mPOA influences cocaine response indirectly. This finding is consistent with the pattern of mPOA innervation of the mesolimbic system. Previous work from our laboratory demonstrated that mPOA efferents to the VTA in female rats are primarily located in the rostrocentral, but not caudal, mPOA (Tobiansky et al., 2013). These findings indicate that specifically the rostrocentral portion of the mPOA is involved in the downstream modulation of cocaine response.

Our results also show that cocaine-induced Fos-ir in the rostrocentral mPOA correlated with cocaine-induced activation of the striatum, a region of the brain well known for its involvement in cocaine response (Neisewander et al., 1995; Everitt & Robbins, 2013). In females, cocaine-induced Fos-ir in the rostrocentral mPOA positively correlated with cocaine-induced Fos-ir in the caudate and NAc shell, but negatively correlated with cocaine-induced Fos-ir in the NAc core. In males, cocaine-induced Fos-ir in the rostrocentral mPOA positively correlated with cocaine-induced Fos in the NAc shell and core, but not in the caudate. These correlations are present even though only the caudate, and not the NAc core or shell, was activated by an acute dose of cocaine in both males and females. The cause of these correlations is unknown, but they are indicative of possible mPOA influences on striatal responses to cocaine.

The influence of the mPOA on cocaine response is mediated by endocrine inputs. While much work remains to be done to support or disprove this hypothesis, the abundance of sex steroid hormone receptors present in the mPOA of males and females (Simerly et al., 1990), along with past work showing that estradiol microinjections into the mPOA increase cocaine CPP in female rats (Robison, Martz, & Dominguez, 2018), would point us in that direction. Results presented here further support this idea by showing that estradiol status influenced mPOA Fos-ir in both males and females. Regardless of drug treatment group, mPOA Fos-ir was elevated in females during the P/E phase of the cycle (high-estradiol status), when estradiol levels had peaked, compared to those in D/M phases (low-estradiol status). In males, the effect of endogenous estradiol may have an opposite effect, as evidenced by our findings. We found that cocaine-induced Fos-ir in the mPOA was elevated in males with lower levels of circulating estradiol (low-estradiol status) compared to males with higher levels of estradiol (high-estradiol status). These findings were not surprising, given that previous work has shown that sensitization and cocaine self-administration is enhanced by estradiol administration in ovariectomized females, but not in castrated males (Jackson, Robinson, & Becker, 2006), and that activation of the g-protein coupled estrogen receptor (GPER1) in the striatum increases motivation to self-administer cocaine in females, but not in males (Quigley et al., 2021). The present study did not address the profile of activated neurons in the mPOA, but previous work in female rats has shown that the majority of rostrocentral mPOA neurons that project to the VTA contain sex steroid hormone receptors, specifically estrogen receptor alpha (ERα) and GPER1, but not progesterone receptor (Tobiansky et al., 2016). Our findings further support the hypothesis that the rostrocentral mPOA influences the mesolimbic system through neuroendocrine priming. It is possible, for example, that the mPOA integrates information on hormonal status and relays this information to the VTA, where dopamine neurons respond to reinforcing stimuli. Selective silencing of sex steroid hormone receptors in the mPOA of both sexes could provide insight into neuroendocrine modulation of drug response in this area of the brain.

Several studies have investigated the effects of multiple doses or chronically administered stimulants on locomotion between male and female rats and they show that females generally express greater amphetamine- (Forgie & Stewart, 1993; Milesi-Hallé, 2007) and cocaine-induced locomotion and sensitization after repeated doses of cocaine than do males (van Haaren & Meyer, 1991; Walker et al., 2001; Chin et al, 2002; Wissman et al, 2011; Cummings et al, 2014). Here, after a single dose of 10 mg/kg cocaine, we found that cocaine-naïve male and female rats experienced comparable increases in locomotion. While these findings speak to the effects of acute cocaine administration, it is possible that sex differences might become clearer after administering different doses of cocaine or after chronic administration. In addition, although stereotyped behaviors (e.g. head weaving and bobbing, paw treading, and patterned locomotion) were not measured in this experiment, sex differences in these behaviors could exist. For example, female rats have been shown to respond to lower doses of cocaine (van Haaren & Meyer, 1991) and display stereotyped behaviors at lower doses relative to males (Walker et al., 2001). When the females in our study were examined by estrous cycle phase, only E/P (high-estradiol status) females experienced an increase in cocaine-induced locomotion that was comparable to males, while D/M (low-estradiol status) females did not differ from saline treated rats. The attenuated response to cocaine in D/M females is likely due to low levels of estradiol that occur during diestrus and metestrus in the rat estrous cycle, an effect that others have observed (Quinones-Jenab et al., 1999; Sell et al., 2000; Souza et al., 2014). Like the findings of Quinones-Jenab (1999) and Souza (2014) and their colleagues, we found no differences in locomotion in control rats that received saline, meaning that cycle stage status did not influence baseline levels of locomotion in the absence of drug.

Furthermore, and of relevance to better understanding the effects of estradiol directly in the mPOA, estradiol implanted into the mPOA of female rats enhanced wheel running activity, but not locomotion in an open field test (Fahrbach et al., 1985). This finding suggests that increased estradiol in the mPOA of females does not alone increase open field locomotion, in the absence of cocaine. Our laboratory has also shown that female rats that received estradiol microinjections directly into the mPOA prior to undergoing locomotor testing in an open field did not express increased cocaine-induced locomotion compared to females that received vehicle microinjections of artificial cerebral spinal fluid (aCSF; Robison et al., 2018). However, females that received the estradiol microinjections and cocaine experienced non-statistically significant increases in locomotion compared to rats that received only cocaine. This difference may have been greater if all rats in this study had not received systemic estradiol replacement, regardless of microinjection treatment. Systemic estradiol replacement may have activated cells in the mPOA prior to microinjection treatment, impeding enhanced locomotion following microinjections of estradiol. We hypothesize that estradiol microinjections into the mPOA of naturally cycling females in diestrus, or in females with lower levels of systemic estradiol replacement, comparable to physiological levels during diestrus, would express increased cocaine-induced locomotion compared to aCSF controls. Future experiments will further investigate the effects of estradiol in the mPOA.

To our knowledge, few studies have examined both sex and hormonal differences in the response to an acute dose of cocaine administered to cocaine-naïve male and female rats. In line with our findings, Craft & Stratmann (1996) reported no difference in cocaine-induced locomotion (as measured by beam breaks) between males and naturally cycling female Sprague Dawley rats that were naïve to cocaine before receiving a 10 mg/kg (i.p.) injection of cocaine. In studies of male and female Fischer rats, Chin and colleagues (2002) found no difference in cocaine-induced locomotion (as measured by total ambulations) after rats received an acute i.p injection of 15 mg/kg cocaine on day 1 of the experiment, but Festa and colleagues (2003) found that female Fischer rats experienced greater cocaine-induced locomotion (as measured by total ambulations) than male rats during a three-hour period following a 15 mg/kg injection (i.p) of cocaine. In an experiment with Wistar rats, van Haaren & Meyer (1991) found that naturally cycling female rats experienced greater locomotion (as measured by total distance traveled) than males after receiving a 10 mg/kg (s.c.) injection of cocaine. In this experiment, rats underwent five 40-minute locomotion sessions on consecutive days in which they received a 1 mg/kg dose of cocaine, a 10 mg/kg dose, and three doses of saline on the remaining days, which is to say that rats might not have been entirely naïve to cocaine when they were tested with the 10 mg/kg dose. Walker and colleagues (2001) also found that naturally cycling female Sprague-Dawley rats expressed greater cocaine-induced locomotion in response to 10, 20, and 40 mg/kg (i.p.) injections of cocaine than did males. However, since locomotion was measured with multiple doses (in random order), these rats were not entirely cocaine naïve for some of the locomotion tests. Additionally, Walker and colleagues (2001) and Festa and colleagues (2003) performed testing during the light portion of the day cycle, which could be an additional factor worth considering in interpretation (as noted by Nelson et al., 2021). While sex differences between males and naturally cycling females were examined in all these studies, none reported the effect of estrous cycle stage in females. Our finding that males and E/P female rats display comparable levels of cocaine-induced locomotion, while D/M females express less, is the first to show this and, thus, provides insight into both sex and cycle stage differences that are present at first exposure to cocaine. Furthermore, our correlational analyses of hormonal status and locomotion indicates that males with elevated endogenous estradiol experience lower levels of cocaine-induced locomotion, compared to males with lower levels of endogenous estradiol.

Lastly, locomotion was a straightforward behavioral assay to measure in conjunction with Fos-ir. This assay enabled our laboratory to account for acute effects of cocaine and control for cycle stage and estradiol effects in male and female rats. However, chronic and self-administration models would provide insights that are more translational to humans and the alterations that occur in motivation and reward systems in the brain. These two areas of research are brought together when one considers the possible long term effects of the first exposure to cocaine. For example, does hormonal status at first exposure to cocaine have long-lasting effects on motivation for cocaine or is the impact of this first experience insignificant? Initial experiences of motivated behaviors, such as sexual behavior, have been show to influence long term mating preferences (Pfaus et al., 2013), so it is possible that this is the case for drug taking behaviors as well.

In summary, the present study shows that sex and sex steroid hormone status influence the neural and behavioral response to cocaine at first exposure, and that this influence may be modulated by the mPOA. While acute cocaine administration (10mg/kg) did not increase Fos expression in the mPOA, it is possible that different doses or chronic administration could lead to increased cocaine-induced activity in this area, especially because there is a population of cocaine- and amphetamine- related transcript (cart)-expressing cells in the mPOA (Tsuneoka et al., 2017). Future investigations into the nature of the connections between the mPOA and the mesolimbic system should yield important insights into sex differences in development and maintenance of substance use disorders. Furthermore, while a large body of work has focused on the enhancing effect of estradiol on drug response in females, less work has focused on the effects of estradiol in males. Here, we show that estradiol may affect cocaine response in males in a manner that is opposite to that seen in females. Specifically, the presence of peripheral estradiol is associated with enhanced cocaine responses in females, but attenuated cocaine responses in males. In addition, these findings do not account for potential effects of local estradiol synthesis, in which peripheral testosterone could be converted to estradiol in the brain. The results of this study report a role for the mPOA in the regulation of these neuroendocrine-induced sex differences in neural and behavioral responses to cocaine.

Highlights.

  • Fos in the mPOA correlates with cocaine-induced locomotion in male and female rats.

  • Hormonal status correlates with cocaine-induced locomotion and Fos in the mPOA.

  • Sex and hormonal status influence acute cocaine-induced locomotion.

Acknowledgements

We wish to thank Dr. James Curley and Dr. Novin Ghaffari for consult and expertise in statistical methodology. This work was supported by the National Institutes of Health grant R01-DA032789 and a Ransom Faculty Fellowship, The University of Texas at Austin, to JMD. JRM was funded by training grant T32-AA007471. The authors report no biomedical financial interests or potential conflict of interest.

Footnotes

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