High-hormone female rats exposed to crack cocaine exhibit behavioral alterations accompanied by changes in brain serotonin immunoreactivityAbstract
Crack cocaine induces marked behavioral and neurobiological alterations, including neuropsychiatric symptoms, with sex-dependent differences. This study examined the effects of crack cocaine in female rats in different phases of their estrous cycle. Animals were exposed to crack cocaine (100 mg) for five consecutive days and subsequently either euthanized for plasma cocaine and metabolite quantification or tested in the elevated T-maze (ETM, for avoidance and escape behaviors, related to generalized anxiety and panic disorder, respectively) and in an arena for locomotor activity evaluation (lines crossed). Given the role of serotonin (5-HT) in reward and anxiety/panic regulation, ΔFosB immunoreactivity was analyzed in serotonergic neurons of the dorsal raphe (DR). Plasma cocaine and metabolite concentrations did not differ across estrous cycle phases. Females in metestrus and diestrus showed no behavioral or immunohistochemical alterations following crack exposure. In contrast, proestrus females exhibited reduced avoidance, increased escape latencies, and enhanced locomotor activity, while estrus females showed decreased avoidance and increased locomotion. These effects were accompanied by reduced ΔFosB immunoreactivity in the lateral wings. Double immunoreactivity was increased in the region and in the ventral DR, but decreased in the dorsal DR. These findings reveal that DR neurons modulate crack–induced behavioral alterations in female rats.
Key words
Crack cocaine; Female rats; Reward; Anxiety/panic; Elevated T-maze; Serotonin
INTRODUCTION
Among illicit substances, cocaine is one of the most widely consumed psychostimulants, with over 18 million users worldwide (UNODC 2024). Crack cocaine is a highly toxic derivative of cocaine, produced by combining sodium bicarbonate with cocaine base paste (Falck et al. 2008). The lower cost of crack cocaine compared to powdered cocaine has facilitated its widespread use. In many developing countries, crack use disorder poses a serious public health challenge (Moreira et al. 2015, Butler et al. 2017). Numerous studies have highlighted the long-term cognitive and emotional impairments associated with chronic crack use (Conway et al. 2006, Zubaran et al. 2013, Rosário et al. 2018, Duopah et al. 2024). Commonly reported psychiatric symptoms include anxiety and panic attacks (Conway et al. 2006, Zubaran et al. 2013, Rosário et al. 2018, Duopah et al. 2024). In fact, both anxiety and panic disorders are highly prevalent among individuals with substance use disorders (Compton et al. 2007, Smith & Book 2010). However, few investigations have explored the underlying mechanisms responsible for these neuropsychiatric effects.
It has previously been pointed out that crack cocaine use differs according to gender, with women presenting a faster progression to crack use (Rebelatto et al. 2022). Fluctuations in hormone levels across the menstrual cycle (Motta-Mena & Puts 2017) appear to influence cocaine use in women (Moran-Santa Maria et al. 2014). Although there is evidence reporting no differences regarding the use of smoked cocaine in the follicular (high estrogen) and luteal phases, women in the follicular phase consistently describe cocaine as more pleasurable (Evans et al. 2002). Also, cravings tend to be stronger during the follicular phase (Sinha et al. 2007, Moran-Santa Maria et al. 2014, Reed et al. 2018).
Rodents exhibit a comparable pattern of hormonal variation across their estrous cycle, which lasts approximately 4–5 days and includes the proestrus, estrus, metestrus, and diestrus phases (Sato et al. 2016). A previous review of the literature performed by our research group (Rosário et al. 2022) showed that cocaine self-administration also varies across these phases, with female rats usually exhibiting increased consumption, seeking behavior, craving, relapse, locomotion, among other behavioral alterations during peaks of estrogen. These observations are related to the direct effects played by ovarian hormones (in particularly estradiol), in dopamine, but also in serotonin (5-HT) neurons (Rosário et al. 2022). However, findings are not entirely consistent (Rosário et al. 2022). Thus, the precise relationship between estrous phase and cocaine-related behaviors remains to be fully clarified.
In previous studies from our laboratory, we showed the effects of crack cocaine administered intraperitoneally (IP) (Rosário et al. 2021) or through passive inhalation (Rosário et al. 2026) in male Wistar rats. Results demonstrated that the subchronic IP administration of crack cocaine in male Wistar rats produced either a panicolytic-like effect at lower doses or a panicogenic-like effect at higher doses. The panicolytic-like effects were accompanied by a decrease in ΔFosB immunoreactivity in the dorsomedial, dorsolateral and lateral periaqueductal gray, in the dorsal raphe (DR) dorsal region (DRD) and in the lateral wings/ventrolateral periaqueductal gray (lwDR) (Rosário et al. 2021). Panicogenic-like results generated opposite effects, that is, increases in ΔFosB immunoreactivity in the dorsomedial, dorsolateral and lateral aspects of the periaqueductal gray, in the DRD and in the lwDR (Rosário et al. 2021).
Inhaled crack cocaine, however, induced only panicogenic-like effects (Rosário et al. 2026). These effects were also followed by increases in ΔFosB immunoreactivity in the lwDR and DRD. For this study, double immunoreactivity (ΔFosB/tryptophan hydroxylase, TrpOH, the rate limiting enzyme for 5-HT synthesis) was also analyzed. Results showed that double immunoreactivity was increased in the DRD. lwDR 5-HT neurons, however, were less activated.
The present study investigated, to our knowledge for the first time, behavioral and neurobiological effects of inhaled crack cocaine in female rats across different phases of the estrous cycle. Based on previous evidence demonstrating sex-dependent effects of cocaine and the modulatory influence of ovarian hormones on cocaine-related behaviors (Rosário et al. 2022), we hypothesized that repeated crack cocaine inhalation would induce anxiety- and panic-related behavioral alterations and that these effects would vary depending on the estrous cycle phase.
According to the ethoexperimental analysis of defensive behaviors in rodents (Blanchard et al. 2001), anxiety is primarily associated with behavioral inhibition and risk assessment, responses typically elicited by potential threats. In the elevated T-maze (ETM), inhibitory avoidance is therefore considered an anxiety-related behavior. In contrast, panic-related responses are characterized by rapid escape or flight reactions in the presence of imminent danger (Blanchard et al. 2001), as modeled by escape from the open arms of the ETM. Supporting this distinction, anxiolytic drugs such as benzodiazepines and buspirone selectively impair inhibitory avoidance, whereas chronic antidepressant treatment reduces escape behavior (Viana et al. 1994, Teixeira et al. 2000, Poltronieri et al. 2003). These pharmacological findings support the interpretation that inhibitory avoidance reflects generalized anxiety-like responses, whereas one-way escape is associated with panic-like behavior (Viana et al. 1994, Teixeira et al. 2000, Poltronieri et al. 2003).
Considering our previous findings (Rosário et al. 2021, 2022, 2026), we further hypothesized that crack cocaine-induced behavioral alterations would differ across estrous cycle phases and be accompanied by differential activation of serotonergic neurons within DR subregions, reflected by changes in ΔFosB and TrpOH immunoreactivity.
MATERIALS AND METHODS
Subjects
Ninety-two female Wistar rats (Universidade Federal de São Paulo, CEDEME, Brazil), 8-9 weeks old and weighing approximately 250 g at the beginning of the experiments, were used in this study. The animals were housed in groups of four within polypropylene cages topped with stainless steel grid lids, under a controlled 12-hour light–dark cycle (lights on from 7:00 a.m. to 7:00 p.m.) and room temperature maintained at 22 ± 1 °C. Throughout the experimental period, food and water were available ad libitum.
Prior to the start of the experiments, the animals underwent a 7-day habituation period in the laboratory. Following habituation, estrous cycle assessment was performed, and the rats were allocated into groups according to their cycle phase (metestrus, diestrus, proestrus, or estrus).
The experimental protocol was approved by the Ethics Committee on the Use of Animals of the Federal University of São Paulo (CEUA–UNIFESP) under protocol number 9406061020. All procedures followed the guidelines of the Brazilian Society for Neuroscience and Behavior for the Care and Use of Laboratory Animals, in accordance with international standards, and every effort was made to minimize animal discomfort and suffering.
Estrous cycle evaluation
The vaginal smears of the rats were collected every day, for a period of approximately 12 days, always at the same time, between 9:00 - 10:00 a.m., and the experimental protocol was only initiated after the occurrence of two complete and regular estrous cycles. Vaginal smears were collected with a pipette filled with 10 μL of distilled water. The tip was inserted shallowly into the rat’s vagina, and the fluid was rinsed. The vaginal fluid was placed on a glass slide and observed under a light microscope with 10x and 40x objective lenses. A different tip and slide were used for each rat.
Nucleated cells keratinized (or cornified) epithelial cells, or leukocytes were observed. The proportion between them was used to determine the phases of the estrous cycle (Figure 1). The animals were divided into groups according to phase of the estrous cycle and sex, as follows: females in, metestrus, diestrus, proestrus or estrus.
Photomicrographs illustrating the different phases of the animal’s estrous cycle. a) Metestrus phase – mixed cell types; b) Diestrus phase - predominance of leukocytes; c) Proestrus phase - predominance of nucleated cells; d) Estrus phase - predominance of cornified cells.
Chemical analysis
The drug was provided as a courtesy by the Criminal Department of Limeira City, São Paulo State, Brazil, exclusively for research purposes. All experiments were conducted using material from the same lot, obtained through law enforcement seizure.
In brief, a 100 mg aliquot of crack cocaine was analyzed by Liquid Chromatography–Tandem Mass Spectrometry (LC-MS/MS) for cocaine quantification. Analyses were performed using a High-Performance Liquid Chromatography (HPLC) system, Agilent 1260 (Agilent Technologies, USA), coupled to a 3200 QTRAP hybrid triple quadrupole/linear ion trap (LIT) mass spectrometer (Sciex, Canada). Certified cocaine reference standards (FE07271503, Cerilliant, USA) were used for calibration. Injections of 10 μL were processed through an Agilent Eclipse XDB-C18 column (4.6 × 50 mm, 1.8 μm) maintained at 25 °C. The elution flow rate was 0.7 mL·min⁻¹.
For positive mode detection, the mobile phase consisted of 0.1% formic acid (LC-MS grade, Sigma-Aldrich, USA) in water (solvent A) and acetonitrile (LC-MS grade, JT Baker, Brazil) (solvent B). A linear gradient was applied at 0.7 mL·min⁻¹, starting with 95% solvent A and 5% solvent B, decreasing solvent A to 5% over 5 minutes, followed by a 1-minute hold. The system was then returned to the initial conditions for 2 minutes. Detection and quantification were carried out using Electrospray Ionization (ESI) in Multiple Reaction Monitoring (MRM) mode, selecting one precursor ion and two product ions for compound identification and confirmation. Data acquisition and processing were performed with Analyst® 1.5.2 software (Sciex, Canada).
The LC-MS/MS analysis determined that the crack cocaine aliquot contained 64.2% cocaine, confirming it as the primary constituent of the sample.
Exposure to crack cocaine
The apparatus consisted of a smoke-generating pump connected, at one end, to a tube in which the substance was burned, and, at the other end, to an acrylic chamber containing the animals. Airflow was maintained at 150 mL/min by a fan, a rate standardized in preliminary pilot studies (B.A. Rosário, unpublished data) to ensure complete combustion of crack cocaine. All experiments were conducted in a controlled environment equipped with a fume hood filter, exhaust fans, and the required personal protective equipment (Figure 2).
Exposure to crack cocaine - The animal is placed in an acrylic box, connected to an automatic system, with air exhaust regulation for the entry and exit of crack and air. The equipment remains inside an exhaust hood, with the presence of filters for the correct release of drug residues into the external environment of the room.
Animals were exposed daily, for five consecutive days, to crack cocaine (100 mg), following the same experimental protocol adopted in previous studies (Lipaus et al. 2019, Rosário et al. 2022, 2026) (Figure 3). Each rat was first placed individually in the acrylic chamber for 1 minute to acclimate. The pipe was then heated with a torch for an additional 2 minutes. Once the appropriate temperature was reached, the system was activated, and crack cocaine in its “stone” form was placed on the pipe and burned for 2 minutes to ensure complete combustion. After exposure, animals remained in the acrylic chamber for 10 minutes—5 minutes fully enclosed, followed by 5 minutes partially opened to allow gradual ventilation.
The procedure was adopted for both the plasma quantification of cocaine and metabolites (Group 1) and for the behavioral and immunohistochemical experiments (Group 2). After seven days of habituation to the laboratory, the animals were evaluated and separated according to the phase of the estrous cycle. The cycle was measured for an average of 12 days, until two complete regular cycles were observed. After this evaluation, the animals were repeatedly exposed to crack for 5 consecutive days. On the fifth day, they were euthanized by decapitation, and trunk blood was collected for subsequent determination of cocaine and metabolites in the plasma (Group 1). The animals in Group 2 also had their estrous cycle monitored and were repeatedly exposed to crack for 5 consecutive days, being pre-exposed to the open arms of the elevated T-maze (ETM) on the fourth day and tested in the ETM and in the arena on the fifth day. Immediately after the behavioral tests, the animals were euthanized and their brains prepared for immunohistochemistry.
Control animals were also subdivided according to their estrous cycle phase and were placed in an identical apparatus under the same conditions, but without smoke exposure.
Group 1 - Quantification of cocaine and metabolites
Since previous evidence suggests that plasma cocaine and metabolite concentrations may differ across estrous cycle phases due to fluctuating ovarian hormones like estrogen and progesterone (Festa et al. 2004), on the fifth day, immediately after crack cocaine exposure, female animals in different phases of their estrous cycle were euthanized by decapitation, and trunk blood was collected. Plasma samples were separated for the quantification of cocaine and its metabolites—benzoylecgonine (BEC), anhydroecgonine methyl ester (AEME), and ecgonine methyl ester (EME) (Sigma Aldrich/Cerilliant, Germany). AEME, the primary byproduct of crack cocaine pyrolysis, serves as a specific biomarker for crack cocaine use (Scheidweiler et al. 2003).
Sample preparation for LC-MS/MS analysis was performed using the protein precipitation technique. Briefly, 50 μL of plasma were transferred to a 1.5 mL Eppendorf tube, followed by the addition of 200 μL acetonitrile containing the internal standards (IS) cocaine-d₃ and BEC-d₈ (Absolute Standards, USA), both at 50 ng/mL. The mixture was vortexed for 1 minute and centrifuged at 14,000 rpm for 4 minutes. A 75 μL aliquot of the supernatant was collected, diluted with 75 μL ultrapure water, and injected into the LC-MS/MS system.
Chromatographic separation was carried out using an HPLC system (Agilent 1260, Agilent Technologies, USA) coupled to a 3200 QTRAP® hybrid triple quadrupole/linear ion trap (QqLIT) mass spectrometer (Sciex, Canada). A 10 μL injection volume was introduced via an autoinjector into an Agilent Eclipse XDB-C18 column (75 × 3.0 mm, 3.5 μm) with a pre-column (C18, 4.0 × 3.0 mm, Phenomenex, USA), maintained at 25 °C. The flow rate was 0.7 mL·min⁻¹.
The mobile phase consisted of 0.1% formic acid (LC-MS grade, Sigma-Aldrich, USA) in water (solvent A) and acetonitrile with 0.1% formic acid (LC-MS grade, JT Baker, Brazil) (solvent B). A linear gradient was applied at 0.6 mL·min⁻¹, starting with 90% solvent A and 10% solvent B, decreasing solvent A to 10% over 3 minutes, followed by a 2-minute hold. The initial conditions were then restored and maintained for 2 minutes. The total chromatographic run time was 8 minutes, plus 0.5 minutes for conditioning.
Ionization was performed using ESI in positive mode under the following conditions: probe voltage 5.2 kV, temperature 550 °C, nebulizer gas (GS1) 50 psi, heater gas (GS2) 55 psi, and curtain gas 20 psi (with interface heater on). Detection was performed in MRM mode, selecting optimal precursor > product ion transitions for quantification and confirmation: AEME: 182 > 118 and 182 > 122 (RT = 0.73 min) EME: 200 > 182 and 200 > 82 (RT = 0.67 min) BEC: 290 > 168 and 290 > 105 (RT = 2.96 min) Cocaine: 304 > 182 and 304 > 105 (RT = 3.14 min) BEC-d₈ (IS): 298 > 171 and 298 > 105 (RT = 2.96 min) Cocaine-d₃ (IS): 307 > 182 and 307 > 85 (RT = 3.14 min). The well time for each MRM transition was 40 ms, with medium collision-induced dissociation (CID) gas. Quantification was performed by integrating peak areas, using calibration curves prepared by spiking blank rat plasma with known drug concentrations. Calibration curves ranged from 10–400 ng/mL for AEME and EME, and 5–200 ng/mL for BEC and cocaine. The ratio of drug peak area to internal standard peak area was plotted against concentration. Data acquisition and processing were performed using Analyst® 1.5.2 software (Sciex, Canada).
Group 2 – Behavioral analysis
Apparatus
The ETM was constructed from wood and consisted of three arms of equal dimensions (50 × 12 cm). One closed arm, with 40 cm high walls, was positioned perpendicular to two opposing open arms. The entire apparatus was elevated 50 cm above the floor. The open arms were bordered by a 1 cm high transparent acrylic barrier to prevent the animals from falling.
The arena consisted of a circular platform (900 mm in diameter) enclosed by 45 cm high transparent acrylic walls, with a milky acrylic base (1000 × 1000 mm) divided into 12 equal squares.
Illumination at the center of both the ETM and the arena was maintained at 60 Lux. After each experimental session, the apparatuses were cleaned with a 70% ethanol solution to eliminate olfactory cues.
Procedure
On the morning of the fourth day of treatment, prior to drug exposure, female animals in different phases of their estrous cycle were pre-exposed to one of the open arms of the ETM (Figure 3b). A wooden barrier placed at the junction between the maze’s central area, and the proximal end of the selected open arm isolated it from the remaining apparatus. This forced pre-exposure procedure has been shown to enhance escape behavior by reducing novelty-induced reactions (Teixeira et al. 2000).
On the following day, 30 minutes after treatment administration (fifth day of crack cocaine or vehicle exposure), animals were tested in the ETM. For inhibitory avoidance assessment, each rat was placed at the distal end of the closed arm, and the latency to exit the arm with all four paws toward the open space was recorded (baseline latency). Since the animal does not know that at the end of the closed arm there is an open space, this first measurement is an indicative of locomotor activity. After baseline measurements, the animal was again placed at the distal end of the closed arm, and the latency to exit the arm with all four paws was recorded for two consecutive times (avoidance 1 and avoidance 2) with 30-second intertrial intervals. Avoidance 1 and 2 are measurements of anxiety-related behavior. Subsequently, each animal was placed at the distal end of the same open arm previously used for pre-exposure, and the latency to exit with all four paws was measured (escape 1). This procedure was repeated twice more to obtain escape 2 and escape 3 measurements, again with 30-second intertrial intervals. Escape measurements are panic-related behaviors.
Thirty seconds after completing the ETM protocol, animals were placed in the center of the arena and allowed to freely explore for 5 minutes, during which the number of lines crossed was recorded, thus indicating locomotor activity.
Tissue collection and preparation
Following behavioral testing, female animals from Group 2 were anesthetized with a ketamine (80 mg/kg; Syntec, Brazil)/xylazine solution (12 mg/kg; Syntec, Brazil), fentanyl (0.5 mg/kg, Cristália, Brazil), and acepromazine maleate (1 mg/kg, Syntec, Brazil) and perfused with approximately 100 mL of 0.9% saline for ~1 minute, followed by 500–700 mL of 4% formaldehyde (from paraformaldehyde heated to 60–65 °C) in water at 4 °C, pH 9.5, for ~25 minutes. Brains were post-fixed for 1 hour in the same fixative at 4 °C and stored in phosphate-buffered saline (PBS) containing 20% sucrose at 4 °C. Coronal frozen sections (30 μm thick) were cut in regularly spaced series (5×1-in-5), collected in ethylene glycol-based cryoprotectant, and stored at −20°C.
ΔFosB/tryptophan hydroxylase (ΔFosB/TrpOH-ir) immunoreactivity
To identify neurons immunoreactive for TrpOH (TrpOH-ir), a rabbit polyclonal anti-TrpOH antibody (1:500, AB217024, lot 1023146-1, Abcam, United Kingdom) was used. Immunohistochemistry followed a standard avidin–biotin peroxidase protocol (Hsu & Raine 1981) with Vectastain Elite reagents (Vector Laboratories, USA). The sections were placed in a solution of biotinylated goat anti-rabbit secondary antibody (biotin-conjugated immunoglobulin G) (Vector) at a 1:1000 dilution in KPBS + 0.3% Triton X-100. Sections were pretreated with hydrogen peroxide (0.3%; Sigma, USA) to quench endogenous peroxidase activity before incubation with the primary antibody. The reaction with diaminobenzidine (DAB; 0.05%, Sigma, USA) was developed for 40 seconds without nickel ammonium sulfate, producing a brown cytoplasmic stain.
In the same sections, after TrpOH-ir visualization, neurons immunoreactive for ΔFosB were identified using a rabbit polyclonal antibody (1:500, AV32519, lot QC2248, Sigma-Aldrich, USA). ΔFosB is a marker of chronic neuronal activation (Arnold et al. 2020). The same protocol for the secondary antibody was followed. The avidin–biotin peroxidase protocol was applied, with pretreatment using hydrogen peroxide. The DAB reaction (0.05%) was performed for 40 seconds and amplified with nickel ammonium sulfate, resulting in a black nuclear stain. Neurons showing both brown cytoplasmic (TrpOH) and black nuclear (ΔFosB) labeling were considered double-stained.
Analysis and quantification
Sections were mounted on gelatin-coated slides with DPX mounting medium. Immunostaining was visualized under an Olympus DX50 microscope, and representative photomicrographs were obtained using a digital camera connected to Image Pro Plus 6.0 software (Media Cybernetics, USA). Each brain region was located and analyzed within a standardized frame size. Two sections were selected per animal from each Bregma. Graphs illustrate the average number from these two sections.
Quantification was performed by an observer blinded to treatment conditions, using 200× magnification. Cells were classified as ΔFosB-positive if their nuclei contained dark, punctate gray-black labeling, and as TrpOH-positive if their cytoplasm was brown. Counts were performed manually, with Image Pro Plus software used only to delimit and standardize the analyzed area. Regions were defined relative to the cerebral aqueduct according to the Paxinos & Watson (1982) rat brain atlas.
The DR subdivisions analyzed included the DRD, the lwDR, and the ventral region (DRV) at mid-rostral (−7.20 to −7.80 mm from bregma) and mid-caudal (−7.92 to −8.28 mm) levels, as well as the caudal (DRC) and interfascicular (DRI) regions at caudal intervals (−8.52 to −9.00 mm).
Statistical analysis
All data was first assessed for normality and homogeneity of variance using the Shapiro–Wilk and Levene’s tests, respectively. One-way ANOVA was used to evaluate plasma concentrations of cocaine and its metabolites. ETM results were analyzed using two-way ANOVA, with treatment as the independent factor and trials (avoidance or escape latencies) as the repeated measure. When significant main effects of treatment or treatment × trials interactions were detected, between-group comparisons were performed using the unpaired Student’s t-test. Open field and immunohistochemical data were also analyzed using the unpaired Student’s t-test. To improve the clarity and conciseness of data presentation, only statistically significant t-test results are reported in the Results section. A p-value ≤ 0.05 was considered statistically significant.
RESULTS
Plasma concentration of cocaine and metabolites
HPLC analysis revealed the following mean ± SEM plasma concentrations (ng/mL) after inhalation exposure 100 mg of crack: metestrus - cocaine 11.8 ± 4.2, AEME 9.4 ± 2.3, BEC 15.3 ± 7.5; diestrus - cocaine 8.5 ± 1.8, AEME 14.7 ± 2.1, BEC 9.6 ± 5.2; proestrus - cocaine 13.4 ± 1.7, AEME 9.4 ± 1.0, BEC 27.4 ± 9.6; estrus - cocaine 14.0 ± 3.3, AEME 7.7 ± 0.5, BEC 12.2 ± 4.6.
One-way ANOVA did not find significant differences for cocaine and metabolites in females exposed to crack in different phases of their estrous cycle: cocaine (F(3,8) = 0.68; p = 0.589); BEC (F(3,8) = 1.24; 0.356); AEME (F(3,8) = 2.31; p = 0.193).
Behavioral results
Metestrus females
Figure 4a shows that crack exposure did not significantly alter avoidance responses in metestrus females. Two-way ANOVA showed significant effects of trials (F (2,36) = 7.88; p = 0.001), but not of treatment (F (1,18) = 0.373; p = 0.549) or of treatment x trials interaction (F (2,36) = 0.296; p = 0.746).
a-b) Avoidance (upper panel a) and escape (lower panel b) latencies (mean ± standard error) in the elevated T-maze, performed by control animals and animals exposed to crack cocaine (5 days) during the metestrus phase of the estrous cycle; c) Total number of crossings in an open field arena. N = 10 per treatment group. Dots represent individual animals. The number of dots may be fewer than the number of animals due to overlapping. *P < 0.05, compared to control animals in the same test (ANOVA followed by the unpaired Student’s t-test).
Figure 4b shows that crack exposure also did not significantly alter escape measurements in metestrus females. Two-way ANOVA showed a significant effect of trials (F (2,36) = 18.41; p < 0.001), but not of treatment (F (1,18) = 0.126; p = 0.727) or of treatment x trials interaction (F (2,36) = 0.177; p = 0.838).
Figure 4c shows the effect of the drug on locomotor activity. Unpaired Student’s t-test did not show significant differences between treatments (p > 0.05).
Diestrus females
Figure 5a shows that crack exposure did not significantly alter avoidance responses in diestrus females. Two-way ANOVA showed significant effects of trials (F (2,36) = 78.71; p < 0.001) and of treatment x trials interaction (F (2,36) = 3.26; p = 0.05), but not of treatment (F (1,18) = 1.23 p = 0.282). Unpaired Student’s t-test did not show significant differences between treatments, in any one of the trials (p > 0.05).
a-b) Avoidance (upper panel a) and escape (lower panel b) latencies (mean ± standard error) in the elevated T-maze, performed by control animals and animals exposed to crack cocaine (5 days) during the diestrus phase of the estrous cycle; c) Total number of crossings in an open field arena. N = 10 per treatment group. Dots represent individual animals. The number of dots may be fewer than the number of animals due to overlapping. *P < 0.05, compared to control animals in the same test (ANOVA followed by the unpaired Student’s t-test).
Figure 5b shows that crack exposure also did not significantly alter escape measurements in diestrus females. Two-way ANOVA showed a significant effect of trials (F (2,36) = 49.96; p < 0.001), but not of treatment (F (1,18) = 0.851; p = 0.369) or of treatment x trials interaction (F (2,36) = 2.09; p = 0.138).
Figure 5c shows the effect of the drug on the number of crossings in an arena. Unpaired Student’s t-test did not show significant differences between treatments (p > 0.05).
Proestrus females
Figure 6a shows that crack exposure significantly altered avoidance responses in proestrus females. Two-way ANOVA showed a significant effect of trials (F(2,36) = 9.56; p < 0.001), treatment (F(1,18) = 5.54; p = 0.03) and treatment x trials interaction (F(2,36) = 4.56; p = 0.017). Unpaired Student’s t-test showed that proestrus crack 100 mg females presented a significant decrease in avoidance 2 latencies when compared to proestrus controls (T(9.11) = 2.96; p = 0.016).
a-b) Avoidance (upper panel a) and escape (lower panel b) latencies (mean ± standard error) in the elevated T-maze, performed by control animals and animals exposed to crack cocaine (5 days) during the proestrus phase of the estrous cycle; c) Total number of crossings in an open field arena. N = 10 per treatment group. Dots represent individual animals. The number of dots may be fewer than the number of animals due to overlapping. *P < 0.05, compared to control animals in the same test (ANOVA followed by the unpaired Student’s t-test).
Figure 6b shows that crack exposure significantly altered escape responses in proestrus females. Two-way ANOVA showed a significant effect of trials (F(2,36) = 42.32; p < 0.001) and treatment (F(1,18) = 10.70; p = 0.004), but not of treatment x trials interaction (F(2,36) = 1.92; p = 0.161). Unpaired Student’s t-test showed that exposure to crack significantly increased escape 1 (T(18) = -2.39; p = 0.028) and 2 latencies (T(18) = -3.88; p = 0.001) in proestrus crack 100 mg when compared to proestrus controls.
Figure 5c shows the effect of the drug on the number of crossings in an arena. Unpaired Student’s t-test showed that proestrus crack 100 mg females presented an increase in the number of crossings when compared to proestrus controls (T(18) = -5.25; p < 0.001).
Estrus females
Figure 7a shows that crack exposure significantly altered avoidance responses in estrus females. Two-way ANOVA showed a significant effect of trials (F(2,36) = 9.21; p = 0.001) and treatment (F(1,18) = 10.10; p = 0.05), but not of treatment x trials interaction (F(2,36) = 1.70; p = 0.198). Unpaired Student’s t-test showed that crack exposure significantly decreased baseline (T(18) = 5.08; p < 0.001) and avoidance 1 (T(18) = 4.73; < 0.001) latencies in estrus crack 100 mg females when compared to estrus controls.
a-b) Avoidance (upper panel a) and escape (lower panel b) latencies (mean ± standard error) in the elevated T-maze, performed by control animals and animals exposed to crack cocaine (5 days) during the estrus phase of the estrous cycle; c) Total number of crossings in an open field arena. N = 10 per treatment group. Dots represent individual animals. The number of dots may be fewer than the number of animals due to overlapping. *P < 0.05, compared to control animals in the same test (ANOVA followed by the unpaired Student’s t-test).
Figure 7b shows that crack exposure did not significantly alter escape measurements in estrus females. Two-way ANOVA did not show a significant effect of trials (F(2,36) = 1.07; p = 0.354), treatment (F(1,18) = 0.877; p = 0.362) or treatment x trials interaction (F(2,36) = 0.982; p = 0.385).
Figure 7c shows the effect of the drug on the number of crossings in an arena. Unpaired Student’s t-test showed that crack exposure significantly increased the number of crossings (T(18) = -2.94; p = 0.009) in estrus crack 100 mg when compared to estrus controls.
Immunohistochemical results
ΔFosB immunoreactivity in non-serotonergic neurons
Table I illustrates ΔFosB immunoreactivity (Fos-ir) in non-serotonergic neurons of distinct subregions of the DR for female animals in different phases of their estrous cycle. For metestrus and diestrus females, unpaired Student’s t-test did not show significant differences between treatments in any subnucleus (p > 0.05).
Number (Mean ± SEM) of cells immunoreactive to ∆FosB in different brain regions in female rats in different phases of their estrous cycle.
For proestrus crack 100 mg females, unpaired Student’s t-test showed significant decreases in ΔFosB immunoreactivity in the mid-rostral (T(10) = 5.72; p < 0.001) and mid-caudal lwDR (T(10) = 6.48; p < 0.001) when compared to proestrus controls (see Table I and Figure 8).
Number (mean ± S.E.M.) of ΔFosB immunoreactive neurons counted in the mid-rostral (a), mid-caudal (b) and caudal (c) dorsal raphe subnuclei of rats from each treatment group in the proestrus and estrous phase of the estrous cycle. N = 6 per treatment group. Dots represent individual animals. The number of dots may be fewer than the number of animals due to overlapping. *p < 0.05 with respect to the other groups (unpaired Student’s t-test). DR: dorsal raphe; lwDR: lateral wings of the dorsal raphe; DRD: dorsal region of the dorsal raphe; DRV: dorsal raphe ventral subnucleus; DRC: dorsal raphe caudal subnucleus; DRI: dorsal raphe interfascicular subnucleus.
Similar results were found for estrus females. Significant decreases in ΔFosB immunoreactivity were found both in the mid-rostral (T(10) = 6.21; p < 0.001) and mid-caudal lwDR (T(10) = 6.70; p < 0.001), when compared to estrus controls (see Table I and Figure 8).
TrpOH immunoreactivity
Table II shows TrpOH immunoreactivity (TrpOH-ir). Unpaired Student’s t-test did not show any significant difference between treatments.
Number (Mean ± SEM) of cells immunoreactive to tryptophan hydroxylase (TrpOH) in different brain regions in female rats in different phases of their estrous cycle.
ΔFosB/TrpOH immunoreactivity
Table III illustrates double immunoreactivity in the different subregions of the DR. For metestrus and diestrus females, unpaired Student’s t-test did not show significant differences between treatments in any subnucleus of the region (p > 0.05).
Number (Mean ± SEM) of cells immunoreactive to double immunoreactive (∆FosB + TrpOH) in different brain regions in female rats in different phases of their estrous cycle.
For proestrus females, unpaired Student’s t-test showed significant increases in double immunoreactivity in the mid-rostral (T(10) = -3.91; p = 0.003) and mid-caudal lwDR (T(10) = -5.0; p = 0.001), and in the mid-caudal DRV (T(10) = -4.32; p = 0.002), and significant decreases in double immunoreactivity in the mid-rostral (T(10) = 5.28; p < 0.001) and mid-caudal DRD (T(10) = 7.70; p < 0.001) induced by crack 100 mg in comparison to proestrus controls (see Table III and Figures 8 and 9).
As for estrus females, unpaired Student’s t-test also found significant increases in double immunoreactivity in the mid-rostral (T(10) = -7.75; p < 0.001) and mid-caudal lwDR (T(10) = -3.79; p = 0.004), and in the mid-caudal DRV (T(10) = -2.53; p = 0.031), and significant decreases in double immunoreactivity in the mid-rostral (T(10) = 5.43; p < 0.001) and mid-caudal DRD (T(10) = 5.35; p = 0.002) induced by crack 100 mg when compared to estrus controls (see Table III and Figures 8 and 9).
Number (mean ± S.E.M.) of double-labeled (ΔFosB + tryptophan hydroxylase immunoreactive) neurons counted in the different dorsal raphe subnuclei of rats from each treatment group in the proestrus phase of the estrous cycle. N = 6 per treatment group. Dots represent individual animals. The number of dots may be fewer than the number of animals due to overlapping. *p < 0.05 with respect to the other two groups (unpaired Student’s t-test). lwDR: lateral wings of the dorsal raphe; DRD: dorsal raphe dorsal subnucleus; DRV: dorsal raphe ventral subnucleus; DRC: dorsal raphe caudal subnucleus; DRI: dorsal raphe interfascicular subnucleus.
DISCUSSION
The findings of the present study show that female rats, in different phases of their estrous cycle and exposed to 100 mg of crack cocaine for 5 consecutive days, presented similar plasma levels of cocaine and metabolites. Nevertheless, these animals behaved differently, according to their estrous cycle phase, thus suggesting an interaction between ovarian hormones and crack cocaine. Metestrus and diestrus females (low hormone) did not show any behavioral or immunohistochemical alterations in response to crack cocaine exposure. On the other hand, proestrus and estrus females (high hormone) presented important behavioral and immunohistochemical alterations. Proestrus females exhibited a significant decrease in avoidance latencies, an increase in escape latencies and an increase in locomotor activity, when compared to proestrus controls. Estrus females also showed a decrease in avoidance latencies and an increase in locomotor activity, when compared to estrus controls. These behavioral effects were followed by decreases in ΔFosB immunoreactivity in the lwDR. Double immunoreactivity, however, was also increased both in the lwDR and in the DRV. On the other hand, 5-HT neurons in the DRD were less activated. No other behavioral or immunohistochemical results were found.
In a previous study performed with male animals (Rosário et al. 2026), we did not find any significant effects of a five-day exposure to 100 mg of crack cocaine. A higher dose (250 mg) induced decreases in escape latencies, a panicogenic-like effect. In the present study, metestrus and diestrus females behaved like male animals. On the other hand, high hormone females showed decreases in avoidance latencies and an increase in the number of lines crossed in an arena, thus suggesting hyperlocomotion, even though baseline and escape latencies were not decreased. In fact, proestrus females took a longer time to escape from the open arms of the ETM, suggesting a panicolytic-like effect of crack cocaine exposure. These results indicate that high hormone females are more sensitive than low hormone females and males to the positive, rewarding effects, of crack cocaine exposure.
Although no previous studies have directly examined the effects of crack cocaine exposure in female rats, clinical and preclinical evidence demonstrates sex-dependent differences in response to cocaine. Clinical studies indicate that women initiate cocaine use at younger ages, progress more rapidly to dependence, and exhibit greater vulnerability to its effects (Griffin et al. 1989, McKay et al. 1996, Elman et al. 2001, Becker & Hu 2008, Becker et al. 2012). Menstrual cycle phases also appear to influence psychological and physiological responses to cocaine, although the specific roles of estradiol and progesterone remain unclear (Evans et al. 2002).
Preclinical studies support these observations, showing that female rodents are more sensitive than males to cocaine’s behavioral effects (Becker et al. 1982, Roberts et al. 1989, van Haaren & Meyer 1991, Haney et al. 1994, Lynch & Carroll 1999, 2000, Sell et al. 2000), with these differences linked to variations in ovarian hormone levels (Festa et al. 2004, Larson et al. 2005, Yang et al. 2007, Kerstetter et al. 2013, Souza et al. 2014).
In humans, the menstrual cycle lasts approximately 25–34 days and involves a coordinated sequence of ovarian, uterine, and hormonal changes (Motta-Mena & Puts 2017). The menstrual cycle is divided into three phases: follicular, characterized by the growth of ovarian follicles and a rise in estradiol levels; ovulatory, triggered by a luteinizing hormone surge that leads to rupture of the dominant follicle and release of an oocyte; and luteal, during which the corpora lutea forms and primarily secretes progesterone, with smaller amounts of estradiol (Motta-Mena & Puts 2017). In contrast, rats and mice exhibit an estrous cycle lasting only 4–5 days, comprising four phases: proestrus, estrus, metestrus, and diestrus. During proestrus, ovarian follicles mature rapidly, producing high levels of estradiol until ovulation occurs at the end of this phase. At the onset of estrus, the follicles transform into corpora lutea, which secretes progesterone while estradiol levels decline. The corpora lutea regresses during metestrus and continues to regress through diestrus (Sato et al. 2016). As a result, hormone concentrations are higher during proestrus and estrus and lower during metestrus and diestrus.
Estrogen and progesterone are known to play critical roles in the regulation of serotonergic neurotransmission, including the modulation of 5-HT synthesis, release, reuptake, and receptor signaling (Bethea et al. 1998, Sánchez et al. 2010, Hernández-Hernández et al. 2019). Estrogen has been shown to increase the expression and activity of TrpOH, while also altering the 5-HT transporter (SERT) availability and the expression of several 5-HT receptor subtypes, including 5-HT1A and 5-HT2A receptors (Hernández-Hernández et al. 2019). Progesterone and its neuroactive metabolites can further modulate serotonergic neurotransmission through interactions with both serotonergic and GABAergic signaling pathways (Kolatorova et al. 2022). These hormonal influences are particularly relevant within the DR, the principal source of forebrain 5-HT, where estrogen and progesterone receptors are expressed in serotonergic neurons and associated neural circuits. For these reasons, we aimed to verify how crack cocaine exposure, aside from altering behavior in females in different phases of their estrous cycle, altered ΔFosB immunoreactivity in serotonergic neurons of this raphe nucleus.
Our immunohistochemical results show that the behavioral effects found in the present study were followed by decreases in ΔFosB immunoreactivity in non-serotonergic neurons of the lwDR. Double immunoreactivity, however, was increased in the lwDR and in the DRV. On the other hand, 5-HT neurons in the DRD were less activated. FosB family proteins, particularly ΔFosB, accumulate in response to repeated stimuli and remain stably expressed, reflecting long-lasting—but not acute—changes in neuronal activity (Nestler et al. 2001, Carle et al. 2007, Ulery-Reynolds et al. 2009). Therefore, our immunohistochemical results illustrate the effects of the repeated exposure to crack cocaine on DR neurons activation. These results suggest that, at least in females, the main DR nuclei that modulate crack’s behavioral effects are the DRD, the DRV and the lwDR.
The DR plays a significant role in a wide spectrum of essential neuronal functions ranging from social cognition to emotional learning, primarily through its 5-HT neurons (Ren et al. 2018, Garcia-Garcia & Soiza-Reilly 2019). These neurons project to various brain regions, including the amygdala, the frontal cortex, the dorsal periaqueductal gray, and the ventral tegmental area, influencing behavioral/emotional responses (Paul & Lowry 2013, Ren et al. 2018, Garcia-Garcia & Soiza-Reilly 2019). Evidence also shows that the DR is not a homogenous structure (Paul & Lowry 2013). Different subpopulations of 5-HT neurons have distinct functions and responses to reward and stress, with some promoting anxiety/panic-like behavior and others potentially mediating coping mechanisms (Garcia-Garcia & Soiza-Reilly 2019). Activation of the DR can produce a strong reinforcement signal, often carried out by dual 5-HT-glutamatergic neurons, that project to the ventral tegmental area, enhancing “waiting” or goal-directed behavior (Luo et al. 2015, Feng et al. 2024). The DR is also activated by reward-associated drugs like cocaine and heroin, and this activation modulates locomotor responses, important predictors of vulnerability to drug use and addiction, particularly for psychostimulants like cocaine and amphetamine (Yuen et al. 2022, Li et al. 2025).
Exactly how the different subnuclei of the DR participate in the effects of reward-associated drugs is still a matter of debate. However, in the present study increased activation of serotonergic neurons was found in the lwDR and DRV only in estrus and proestrus females exposed to crack cocaine, thus suggesting that sexual hormones interact with 5-HT neurons in these subnuclei of the DR, possibly increasing susceptibility to the reinforcing effects of the drug. The specific contribution of the lateral and ventral regions to crack cocaine behavioral effects may be linked to their roles in reward processing (McDevitt et al. 2014, Chang et al. 2021). It has been previously shown that ventral tegmental area-projecting neurons from the DR are primarily characterized by their neurochemical profile (5-HT neurons that also release glutamate) and general distribution in the dorsal and ventral borders (Qi et al. 2014).
Aside from modulating the reinforcing effects of drugs, DR neurons also modulate anxiety/panic responses. Previous evidence indicates that anxiety-related responses seem to be mainly coordinated by serotonergic neurons in the DRD (Spiacci et al. 2012, 2016, Lopes et al. 2019, Lemes et al. 2023). In fact, results from our research group (Lopes et al. 2019) demonstrated that exposure to an enriched environment produced anxiolytic and panicolytic-like effects in the ETM, accompanied by reduced double immunolabeling in the DRD. Activation of the DRD through local injection of the excitatory amino acid kainic acid also facilitated ETM avoidance and inhibited escape reactions (Spiacci et al. 2012). Similarly, blockade of 5-HT1A receptors in the DRD with the antagonist WAY 100635 produced comparable results (Spiacci et al. 2016). In contrast, administration of the 5-HT1A agonist 8-OH-DPAT in the same subnucleus elicited opposite effects (Spiacci et al. 2016). Our findings support these observations and suggest that the anxiolytic-like effects observed after exposing high hormone animals to 100 mg of crack cocaine are possibly linked to decreased 5-HT activation in this DR subnucleus.
Neurons in the lwDR, on the other hand, are strongly associated with regulating active defensive responses triggered by panic-inducing stimuli (Johnson et al. 2005, Paul & Lowry 2013, Spiacci et al. 2016). Spiacci et al. (2016), using the ETM, demonstrated that increased escape responses are linked to heightened activity of non-serotonergic neurons in the lwDR. Consistent with this observation, Roche et al. (2003) reported that situations evoking active defense behaviors—such as escape reactions—predominantly increase c-Fos expression in the lwDR. Importantly, they identified these neurons as non-serotonergic. If this interpretation holds true, it might explain why exposure to 100 mg of crack cocaine produced panicolytic-like effects in proestrus females, while simultaneously reducing ΔFosB expression in non-serotonergic neurons of this subregion of the DR. Among the non-serotonergic populations within the region are GABAergic, nitrergic, glutamatergic, CRFergic, and dopaminergic neurons (Roche et al. 2003, Fu et al. 2010, Calizo et al. 2011, Huang et al. 2019). Nevertheless, it is important to emphasize that panicolytic-like effects were only found for proestrus females, although the same immunohistochemical findings were found for both estrus and proestrus animals. Thus, future studies are necessary to identify which non-serotonergic neurons of the lwDR are involved with the panicolytic-like effects found for proestrus animals.
This study has several limitations that should be considered when interpreting the findings. First, although GC–MS analysis confirmed the presence of cocaine and did not detect additional active substances, we were unable to fully characterize the composition of the crack rock used. Therefore, the potential contribution of trace contaminants to the observed effects cannot be completely excluded. Nevertheless, the behavioral and neurobiological alterations reported here are largely consistent with findings obtained using purified cocaine in animal models, suggesting that the effects observed are primarily attributable to cocaine exposure and its interaction with ovarian hormones. Second, because behavioral assessments were not performed following a single crack cocaine exposure, the present study cannot determine whether similar effects would occur after acute administration. Third, although the inclusion of ovariectomized animals would have provided a more direct assessment of the contribution of ovarian hormones, our design focused on naturally occurring hormonal fluctuations across the estrous cycle. Future studies incorporating ovariectomy and hormone replacement protocols will be important for establishing causal relationships between ovarian hormones and crack cocaine-induced behavioral and neurobiological alterations.
In addition, future investigations should examine dose-dependent effects in female rats, including higher crack cocaine doses previously shown to induce panicogenic-like responses in males, and should incorporate neurochemical approaches, such as quantification of 5-HT and its metabolites, to further elucidate the mechanisms underlying the serotonergic alterations associated with crack cocaine exposure.
CONCLUSIONS
To conclude, the results of our present study suggest that high hormone female rats exhibit hyperlocomotion and rewarding, anxiolytic-like effects, after the exposure to 100 mg of crack cocaine. No significant effects of crack cocaine were found in low hormone females, thus suggesting that high hormone females are more sensitive to the positive effects of crack cocaine exposure. Proestrus females also showed panicolytic-like effects after the exposure to 100 mg of crack cocaine. Double immunoreactivity was increased in the lwDR and in the DRV, what might explain some of the positive effects of crack cocaine. 5-HT neurons in the DRD were less activated, possibly explaining the anxiolytic-like effects of crack cocaine. On the other hand, decreases in ΔFosB immunoreactivity in the lwDR are possibly associated to the panicolytic-like effects of the drug. These observations suggest that DR neurons modulate, at least in part, the behavioral effects of crack cocaine in high hormone females.
Acknowledgements
We thank Centro de Espectrometria de Massas Aplicada (CEMSA – Universidade de São Paulo) for cocaine and metabolites quantification in plasma. This study was supported by Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP, grant number 2022/11908-2). Bárbara A. Rosário was the recipient of a PhD fellowship grant from Coordenação de Aperfeiçoamento de Pessoal de Ensino Superior (CAPES, grant number 001). Jéssica A. Lemes was the recipient of a post-doctorate fellowship grant from FAPESP (grant number 2023/07571-5). Milena B. Viana was the recipient of a research fellowship grant from Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq, grant number 304930/2023-7).
Data availability
References
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