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The cycle of chronic cocaine (CC) use and withdrawal results in increased anxiety, depression and disrupted stress-responsiveness. Oxytocin and corticosterone (CORT) interact to mediate hormonal stress responses and can be altered by cocaine use. These neuroendocrine signals play important regulatory roles in a variety of social behaviours, specifically during the postpartum period, and are sensitive to disruption by CC exposure in both clinical settings and preclinical models. To determine whether CC exposure during pregnancy affected behavioural and hormonal stress response in the early postpartum period in a rodent model, Sprague-Dawley rats were administered cocaine daily (30 mg/kg) throughout gestation (days 1–20). Open field test (OFT) and forced swim test (FST) behaviours were measured on postpartum day 5. Plasma CORT concentrations were measured prior to and following testing throughout the test day, while plasma and brain oxytocin concentrations were measured post-testing only. Results indicated increased CORT response following the OFT in CC-treated dams (p≤ 0.05). CC-treated dams also exhibited altered FST behaviour (p≤ 0.05), suggesting abnormal stress responsiveness. Peripheral, but not central, oxytocin levels were increased by cocaine treatment (p≤ 0.05). Peripheral oxytocin and CORT increased following the FST regardless of treatment condition (p≤ 0.05). Changes in stress-responsiveness, both behaviourally and hormonally may underlie some deficits in maternal behaviour, thus a clearer understanding of CC’s effect on the stress response system may potentially lead to treatment interventions which could be relevant to clinical populations. Additionally, these results indicate that CC treatment can have long-lasting effects on peripheral oxytocin regulation in rats, similar to changes observed in persistent social behaviour and stress-response deficits in clinical populations.
Cocaine use, anxiety and dysregulated stress response are highly correlated in clinical populations (1,2), specifically in postpartum women (2,3). Cocaine acutely activates the hypothalamic-pituitary-adrenal (HPA) axis stress response (4), an effect that is intensified by the female sex hormones which are high during pregnancy. Chronic cocaine treatment can significantly raise the elevated basal corticosterone (CORT) concentrations throughout pregnancy in rats (5). Additionally, HPA reactivity is heightened during acute withdrawal, which can occur within hours of the last cocaine exposure, and dysregulation persists during protracted abstinence (4,6). Such drug-induced increases in HPA reactivity are partially responsible for increased anxiety and altered behavioural stress-responsiveness in clinical populations and preclinical models(7,8).
Postpartum mood disorders, including increased anxiety and depression, are not well understood and can negatively impact maternal-infant interactions (9–12). Maintaining stress hormones within a strict range is required for optimal maternal behaviour, as perturbation in either direction can disrupt these behaviours (13–15). Oxytocin, which is well known for its role in the central nervous system in promoting maternal behaviour, has also been described as an ‘anti-stress’ signal (16–21). High brain oxytocin concentrations, similar to concentrations observed during lactation, lower plasma CORT concentrations and mediate some of the HPA hypo-responsiveness observed in the postpartum period (16–21). In addition to its central actions, peripheral oxytocin signaling modulates CORT concentrations; but these effects are dependent on length and concentration of circulating oxytocin exposure (22–24), and may be differently regulated during the postpartum period. Recent evidence suggests that in addition to central actions, peripheral oxytocin signaling is associated with, and may play an important role in response to social interactions in both humans and rodents (25–29), suggesting that peripheral oxytocin affects a variety important behaviors and may serve as a translational biomarker for at-risk women during the postpartum period.
Recent mothers with a history of drug abuse have lower plasma concentrations of oxytocin and cortisol, indicating that neuroendocrine factors in the postpartum period are affected by cocaine use and that stress response may play an important role in the disrupted maternal behaviour observed in these women (16,30–32). Drug-induced deficits in maternal behaviour may be caused by changes in central or peripheral concentrations of oxytocin or HPA hormones; however, it is difficult to determine whether these changes are caused by cocaine or by other confounding factors such as low socioeconomic status, social support systems, or co-abused drugs.
Preclinical rodent studies that control drug dose and regimen as well as gestational and postpartum environments, allow for more precise determination of the effects of cocaine on postpartum behaviours. Various cocaine treatment regimens (acute, intermittent, or chronic) disrupt mother-infant interaction dynamics while simultaneously decreasing oxytocin concentrations in the medial preoptic area (MPOA), hippocampus and ventral tegmental area (VTA) in the early postpartum period (33–35). Presently it is unknown if plasma concentrations are similarly altered in cocaine-treated rats as they have been shown to be in humans (31), or if cocaine treatment affects central or circulating oxytocin in the postpartum rat at critical points during stress response. Cocaine-induced disruption of the interactions between oxytocin and CORT in the postpartum period may lead to differential levels of anxiety or depressive-like behaviour which could in part underlie differences in maternal behaviour.
Although there is an established bidirectional relationship between substance abuse and stress-related symptomatology (4,36,37), little is known about how drug use may alter behavioral or hormonal stress response during the postpartum period. Given 1) that cocaine has been shown to disrupt behavioural and HPA activity 2) that cocaine can disrupt oxytocin signalling and maternal behaviour in the postpartum and 3) the importance of the interaction between CORT and oxytocin signalling (both central and peripheral) in stress, we proposed several studies to investigate the relationships between these factors in the postpartum period. We designed studies to determine if CC-treated dams differ from untreated (UN) dams in: 1) measures of anxiety or stress-responsive behaviour in the postpartum period; 2) HPA axis function; 3) plasma and brain oxytocin concentrations following stress. We hypothesized that CC-treated dams would exhibit higher anxiety and greater behavioral and HPA stress-responsiveness and lower plasma and brain region oxytocin concentrations than UN dams.
All methods used standard procedures advocated by the UNC Division of Laboratory Animal Medicine and approved by UNC-Chapel Hill Institutional Animal Care and Use Committee. Sprague-Dawley nulliparous female rats (~200 grams, Charles River, Raleigh, NC) were kept on a 12:12 reverse light cycle (8:00 AM dark) for at least one week and then mated with a single male until conception was noted by the presence of a vaginal plug or sperm in a vaginal smear, and this was defined as gestational day (GD) zero. Seven days following conception (GD7) rats were moved to a colony room and individually housed on a regular 12:12 light cycle (7:00 PM dark). This procedure typically results in 95% of female rats delivering during the afternoon hours (38). Rats were randomly assigned to either treatment or control groups of 9–15 each, as they become pregnant. Gestational weight gain was measured daily for all groups. Water and chow was available ad libitum. Postpartum day (PPD) one was defined as within 18 hours of completed delivery. Following parturition, gestational length, litter weight, number of pups per litter, and sex ratio were recorded. Dams reared a culled litter of 10 of their own biological pups (as close to five male/five female as possible). After testing, dams and their litters were returned to the colony room until the next test session or immediately euthanized.
Dams received 30 mg/kg/day of cocaine HCl (dose calculated as free base, 2 ml total volume, Sigma, St. Louis, MO) in a saline solution. Half of the total cocaine dose (15 mg/kg) was injected twice daily, subcutaneously, at approximately 9:00 AM and 4:00 PM throughout gestation (GD 1–20) and not thereafter. This is the lowest dose which consistent significant effects on postpartum behaviours have been found (34,39). The absorption rate of subcutaneous injections is relatively analogous to “snorting” cocaine commonly reported by humans (40) and this dose (15mg/kg in rat) is roughly equivalent to 1 gram of cocaine in a 160 pound woman. The use of a 27 gauge needle and rotating injection sites significantly reduced the number and severity of cutaneous lesions frequently reported with CC injections in rat models. A topical antibacterial ointment (Polymycin-Bacitracin-Neomycin, Glaxo-Wellcome, Raleigh, NC) was applied on any skin lesions as they were discovered. Subcutaneous administration of cocaine should not cause significant behavioural or biochemical stress when carefully monitored (41).
Dams received no drug treatment during gestation or during the postpartum period, but were weighed daily to control for effects of handling. Saline-treated dams were not used in these studies since previous work has shown that their maternal behaviour did not differ from UN dams (42).
Female rats were delivered to the animal colony in cohorts (n=10–60) and allowed to habituate for 5–10 days. Cohorts included many females that were designated for other studies ongoing in the lab, and females from each cohort were randomly assigned to a drug treatment or test type (see below). Following 2 days of travel and test room habituation, all members of a cohort underwent Open Field Testing (OFT) within 2 days of each other. All females were placed in the OFT apparatus 2–5 days prior to mating to obtain a ‘baseline’ anxiety measure to account for the natural variation that can occur within large groups of animals (20). On PPDs 1, 3 and 5, dams were brought to a room and pups were removed for weighing and ultrasonic vocalization (USV) recordings in another room while the dam remained in the home cage for approximately 30 minutes, after which pups were returned to the home cage. Ultrasonic recording equipment included Med Associates model ANL-932-1 ultrasound detectors, sampling at a rate of approximately 30 samples per second, that were connected to transducers, and then to a laptop computer. Med Associates USV software began acquisition of USVs at the session start and terminated one minute later. Number, duration and average frequency of USVs were measured.
On the morning of PPD 5, two hours after pups were returned, CC-treated and UN dams were randomly divided into 3 test groups or ‘types’. Type 1 dams (UN n=7, CC n=6) were tested for all behaviours and hormonal concentrations (CORT and oxytocin). Tail blood was collected for CORT measurement (10:00 AM–12:00 PM) and dams were returned to their home cage in the test room with pups present for 2 hours. The dams were then placed in the OFT chamber for 10 minutes, after which tail blood was again drawn for CORT measurement (12:00 PM–3:00 PM), and the dam placed back in her home cage with pups for a second 2-hour rest period. Dams were then placed in the forced swim test (FST) tank for a 10-minute test after which they were euthanized (2:00 PM–6:00 PM).
Type 2 control dams (UN n=8, CC n=7) were tested for behaviour (OFT and FST) on a similar time schedule, but tail blood was not collected as tail blood collection procedures have been shown to increase CORT concentrations and high CORT concentrations can affect anxiety-like behavior like the OFT (43,44).
Type 3 control dams (UN n=6, CC n=6) were used to measure how plasma CORT concentrations changed throughout the day without having performed the behavioural tasks as CORT exhibits a diurnal rhythm of release that is maintained in lactating rats (45); however, this pattern of release may have been affected by CC treatment (46). All dams were euthanized and trunk blood was collected for CORT and oxytocin measurements. Dam brains were collected and their anterior hypothalamus and amygdala dissected out for oxytocin analysis (see Brain Collection Methods).
The OFT chamber (61 cm × 64 cm × 38 cm high) had dark opaque flooring and walls. Female rats were brought to the testing room and allowed to habituate for at least 30 minutes prior to the test, then removed from the home cage and placed in the OFT chamber for 10 minutes and allowed to explore freely, while the experimenters left the room. Following the test, they were returned to the home cage and any urination or defecation was noted. On postpartum testing days, pups remained in the home caged during habituation and testing, and any nursing behaviour was noted prior to the test. The OFT chamber was cleaned with a non-toxic spray (Greenworks All-purpose Cleaner, Chlorox® Oakland, CA) after each test. A video recorder (either a Panasonic VHS (AG188U) or JVC recorder with low-light sensitivity) placed directly over the test chamber started recording prior to testing and continued until the session ended for later analysis.
The OFT chamber was divided into Wall and Centre compartments. The Wall was defined as the area between the outer edges of the chamber up to 10 cm into the chamber. The Centre was defined as the rest of the chamber (54 cm × 51 cm), approximately 70% of the total space in the chamber. Wall compartment dimensions were chosen based on pilot work determining that this space was typical for female rats to use prior to changing direction along one side of the chamber. Behavioural coding was performed using Noldus Ethovision Version 3.0 software (Noldus Information Technology, Inc. Wageningen, The Netherlands). The rat’s location was tracked and frequency of entries, duration, total distance travelled and velocity of movement in each chamber (Wall, Centre) was recorded. Ethovision recorded data in one-minute bins that were then summed (duration, frequency and distance) or averaged (velocity) across the 10-minute session.
Dams were allowed to rest with pups in the test room prior to the FST. The FST tank (41 cm high; 11 cm radius) was filled with water (average temperature 22–25° C) to a depth so that the rats could not reach the bottom and could not escape the tank. At time of testing, any nursing behaviour was noted and dams were removed from the home cage and placed into the tank for 10 minutes. The dams were then removed from the tank, towel dried and returned to the home cage.
Two independent scorers blind to rat drug treatment or type scored videotapes and their observations were assessed for reliability within 10% for frequency and latency and within 20% for duration of behaviours of interest. A computer program previously described (35,39) recorded and calculated the frequency, duration, latency and sequence of behaviours displayed by the rat dams as the viewer scored the session. The behaviours of interest were Dive: dam put entire head under water and swam to the bottom of the tank; Climb: dam pressed rapidly alternating forepaws against the wall above the water line, with a vertical body and ventrum against the wall; Swimming was defined by the number of limbs moving; thus there were Swim–2 legs, Swim–3 legs, and Swim-4 legs designations; and Immobile: animal had no more than one leg moving. Tail movements were consistent across behaviours and thus not considered in coding. No Swim-3 behaviour was noted in any test.
Rats were euthanised by rapid decapitation. This method was employed so that the neuropeptide levels could be captured with dams experiencing as little stress as possible while avoiding alteration of neuropeptide levels by anaesthesia. Neuropeptide levels change rapidly in rodents and any behavioural stress can cause rapid release and lead to false reading of oxytocin levels.
Whole brains were removed from the skull, flash-frozen on dry ice, and stored at −80° C until dissection. Brains were incompletely thawed to allow hand dissection using anatomical landmarks in the standard Rat Brain Atlas (47). The amygdala was collected from −2.12 to −3.14 bregma, ventral to the rhinal fissure, and lateral to the corpus callosum. The anterior hypothalamus was collected from −.80 to −2.12 bregma ventral to the anterior commissure and third ventricle and medial to the lateral ventricles. The amount of time any region was allowed out of −80° C conditions was less than 10 minutes; therefore, none of the tissue was completely thawed at any point before the time of assay.
Trunk blood was collected into vials containing 500 KIU/ml of aprotonin (Sigma-Aldrich, St. Louis, MO) and 0.0634M ethylenediaminetetraacetic acid (EDTA; Sigma-Aldrich, St. Louis, MO). Vials were immediately centrifuged at 4°C at 10,000g for 10–15 minutes. Plasma was collected, immediately frozen, and stored at −80°C until further testing. Two UN/Type1 FST blood samples were lost to experimenter error and were not part of the analysis.
Rat dam CORT sample collection was accomplished via a tail clip. Briefly, the dam was wrapped in a cloth towel to provide soft restraint. The collection site was swabbed with betadine (povidone iodine) and alcohol, nicked with a surgical blade, and the resulting blood collected into a centrifuge tube containing 0.0634M EDTA. After collection, direct pressure with sterile gauze and elevation were applied to the site until bleeding stopped. Additionally, Kwik-Stop® Styptic powder with benzocaine was applied to the site to hasten clotting. Once bleeding had been controlled, the dam was released from towel restraint and allowed to recover in her home cage. The entire procedure typically lasted less than three minutes.
A strata-X 33µm polymeric reversed phase solid phase extraction sorbent was equilibrated in a 96-well plate containing 60 mg sorbent per well (Phenomenex, Torrance CA) by adding 1 ml MeOH followed by 1 ml of water. 800 µL of plasma was acidified with 0.4 ml of 1.5% trifluoroacetic acid (TFA) and centrifuged at 6,000g for 20 minutes at 4°C. This supernatant was loaded onto the pre-treated strata-X plate. Wells were washed with 1.5 ml of 0.1% TFA, and then the peptide eluted with 1 ml of 80% acetonitrile. The eluant was collected in a polystyrene tube, evaporated to dryness under a nitrogen (N2) stream, and the residue reconstituted in 200 µL of assay buffer. Extraction efficiency was determined by spiking one positive control with a known amount of hormone and extracting with the other samples.
Oxytocin concentrations from extracted plasma were measured using an assay kit and protocol from Assay Designs, Inc (Ann Arbor, MI). The endogenous oxytocin hormone competed with oxytocin linked to alkaline phosphatase for the oxytocin antibody binding sites. After the overnight incubation at 4°C, the excess reagents were washed away and the bound oxytocin phosphatase was incubated with substrate and after 1 hour this enzyme reaction, which generates a yellow color, was stopped. The optical density (OD) was read on a Sunrise plate reader (Tecan, Research Triangle Park, NC) at 405nm. The intensity of the color is inversely proportional to the concentration of oxytocin in the sample. The hormone content (pg/ml) was determined by plotting the OD of each sample against a standard curve. The sensitivity of the assay is 11.6 pg/mL, with a standard range of 15–1000 pg/mL. The intra- and inter- assay variation is 4.8% and 8% respectively. Assay Designs reports cross-reactivity for similar neuropeptides found in mammalian sera at less than 0.001 %.
Sample CORT measurements were measured using the Corticosterone 125I Radioimmunoassay Kit (MP Biomedicals, Orangburg, NY). Samples were brought to room temperature and steroid diluents, 125I-CORT, and anti-CORT were incubated for 2 hours. Precipitant solution was added, vortexed, and centrifuged at 1000g for 15 minutes. The radioactivity in the pellet was measured using a LKB CliniGamma counter, which calculates the nanogram content of CORT in each sample from the standard curve. The intra-assay and inter-assay coefficients of variance were 4.4% and 6.5%, respectively.
For brain regions, tissue was processed as previously described (33). Briefly, tissue was homogenized in buffer and centrifuged. Oxytocin immunoreactive content was assayed in the supernatant according to a protocol from Bachem/Peninsula Labs (Belmont, CA). Samples and standards (1.0 – 128.0 pg) were incubated in duplicate with anti-oxytocin serum. This was followed by incubation with 125I-oxytocin after which time normal rabbit serum and goat anti-rabbit IgG serum were added. The 125I-oxytocin bound to the antibody complex was separated by centrifugation. The radioactivity in the pellet was measured using a LKB CliniGamma counter (PerkinElmer Wizard 1470-005), which calculates the picogram content of oxytocin in each sample from the standard curve.
One-way analysis of variance (ANOVAs) tests were used to evaluate differences amongst cohorts in baseline OFT behaviour. ANOVAs (Treatment × Type) were used to test the comparisons of baseline and postpartum OFT tests. Postpartum behaviour was compared to the baseline behaviours for each animal to investigate differences across time, and 2-way ANOVAs assessed whether CC treatment or Behavioral Testing Type affected this difference. Two-way ANOVAs (Type × Treatment) were used to assess differences in FST behaviour and to determine differences between oxytocin plasma or brain concentrations. Given the non-normality of the CORT data, Kruskal-Wallis tests were used. Linear regression analyses were performed to assess relationships between plasma oxytocin-behaviour, plasma oxytocin-plasma CORT, plasma oxytocin-regional brain oxytocin, and between region-oxytocin concentrations.
All gestational and pup growth data are presented in Table 1. Although gestational length in days was the same across treatment groups, it was observed that CC-treated dams were more likely to start labor earlier (during the dark cycle) compared to UN dams (χ2 (1) = 24.838, p ≤ 0.001). CC-treated dams gained less weight than UN dams during pregnancy (t= −4.146, p≤0.001) and thus weighed significantly less at the end of gestation (t = −3.937, p≤ 0.05). However, CC-treated dams gained more weight during the first postpartum week compared to UN dams (t = 2.827, p≤0.001). There was a small, but significant, effect on PND 1, such that CC-exposed pups weighed less than UN pups (t =−3.076, p≤ 0.001). There were no differences in sex ratio, litter weight, culled litter weight, or weight gain during their first neonatal week. Ultrasonic vocalizations (USVs) were recorded from litters of pups on postnatal days 1, 3, and 5. No differences were observed in the number, duration or frequency of litter USVs (data not shown).
Analyses indicated no significant differences between cohorts on a number of behavioural variables including centre duration, centre frequency, centre distance travelled or velocity. See Table 2 for individual cohort comparisons. Gestational drug treatment and type were randomly assigned, and a 2-way ANOVA revealed there were no significant differences in the baseline centre duration or locomotor activity between treatment or behavioural test types (see Figure 1 A,B).
The blood draw procedure increased centre time in the OFT in Type 1 dams compared to Type 2 dams regardless of CC treatment (F(1,24)= 6.021, p≤ 0.05, Figure 1C). Qualitatively, CC-treated dams were more likely to decrease activity as the session continued compared to UN dams. There were no differences between CC-treated and UN dams on OFT behaviour or urine and feces responses (Figure 1 C,D). However, when compared to baseline measures (Figure 1E,F), ANOVA tests indicated that the CC-treated group (regardless of Type) had a strong trend for shorter centre duration (F(1,24)= 3.909, p≤ 0.06). UN dams did not significantly differ from their own baseline measures, however, testing Type qualitatively affected the direction of change.
At baseline (morning), CC-treated dams showed a non-significant trend for lower CORT concentrations compared to UN dams (z=1.753, p≤ 0.08). Type 1 UN dams did not exhibit a change in CORT concentrations following the OFT (Figure 2A); however, CC-treated dams showed a significant increase (z= 2.073, p≤ 0.05). Type 3 dams were tested for CORT concentrations at corresponding times of day but without having performed the behavioural tests and there were no differences between treatment groups or within groups between the two time points (Figure 2B).
CC-treated dams (Type 1 and Type 2) were immobile for a shorter duration (F (1,22) = 5.771, p≤ 0.05, see Figure 3A) compared to UN dams. Experimental procedure type also influenced FST behaviour. Type 1 dams were immobile less often (F (1,22) = 9.295, p≤ 0.01) and for a shorter duration (F (1, 22) = 9.672, p≤ 0.01) compared to Type 2 dams, regardless of treatment.
All hormonal data are presented in Figure 3. A two-way ANOVA (Treatment × Type) of plasma oxytocin concentrations revealed FST exposure (Type 1 and Type 2) increased oxytocin concentrations compared to No FST (Type 3) dams (F (1,22) = 7.006, p≤ 0.01, Figure 3B). CC-treated dams had higher oxytocin concentrations than UN dams regardless of Type (F (1,22) = 5.145, p≤ 0.05, Figure 3B). A two-way ANOVA (Treatment × Type) revealed that CORT concentrations were significantly raised in all Type 1 and Type 2 dams by exposure to the FST compared to Type 3/No FST dams (F (2,22) = 26.612, p≤ 0.001) with no effect of treatment (see Figure 3C). Correlations between the FST behaviours and plasma concentrations of oxytocin and CORT revealed that a greater amount of total swim time (Swim-2 plus Swim-4) was positively related to plasma oxytocin concentrations within all dams regardless of treatment (F (1,12) = 6.047, p≤ 0.05, Figure 3D). No correlation was observed between plasma CORT and FST behaviors (see Figure 3E). Linear regression analyses indicated that oxytocin and CORT had no direct relationship with each other at this time point in UN dams (Figure 3F). However, CC-treated dams showed a strong positive relationship between the two hormones following the FST (R2 = 0.76; F (1,6) = 19.552, p≤ 0.005).
There were no differences between Type 1 and Type 2 dams’ oxytocin tissue content, thus their data were pooled for comparisons between animals who experienced the FST and those that did not (Type 3) and correlations with behaviors. Type 3 dams showed a trend for higher hypothalamic oxytocin content compared with Type 1 and Type 2 dams regardless of treatment (F (2,25) = 5.157, p≤ 0.05. Figure 3G). Hypothalamic oxytocin showed a positive relationship with plasma concentrations following the FST (R2= 0.318; F (1,14) = 6.528, p≤ 0.05, Figure 3H). There were no differences in amygdala oxytocin concentrations caused by CC treatment and no significant relationships with behavior were observed (Figure 3G).
We predicted that CC-treated dams would exhibit behaviours indicating higher levels of anxiety and HPA stress-responsiveness than would UN dams and that these differences would correlate with lower plasma and brain oxytocin concentrations. Our hypotheses were partially confirmed as CC treatment resulted in subtle differences in anxiety-like and stress-coping behaviours, as well as important differences in endocrine stress signalling in the early postpartum period.
The peri- and post- partum periods are typically characterised by high basal blood CORT concentrations, and a hypo-responsiveness to stress evidenced by blunted CORT responses to physiological and psychological stressors (16,19,48–50); however, CC-treated dams showed an increase in CORT in response to the OFT, a response that was absent from UN dams, suggesting that this test was more stressful to CC-treated dams. This change was not due to a typically occurring diurnal rhythm, as CC-treated dams that did not undergo OFT testing did not show a similar increase in CORT. Interestingly, CC-treated dams’ CORT concentrations did not change significantly throughout the day, while CORT concentrations in UN dams increase by the third blood draw. This rise in the UN dams’ CORT concentrations may have been in response to the multiple handlings on PPD5, and a lack of response in the CC-treated dams may suggest that the type of stressors that cause a CORT response differs across drug treatments. Additionally, CC-treated dams show differences in baseline plasma oxytocin (Figure 3B), which has also been tied to anxiety and CORT levels (51). There was a trend for CC dams to show a relationship between plasma oxytocin and CORT at rest (p<0.09), suggesting a mechanism for the underlying changes in CORT signaling. However, high levels of peripheral oxytocin can modulate circulating CORT concentrations in the rat dependent on the dose, timing, and endocrine state of the animal (22–24), warranting future studies to better understand the interactions between these endocrine systems and their control of anxiety-like and stress-responsive behaviors in the postpartum period.
Surprisingly, CC dams exhibited significantly less immobility than UN dams. The traditional interpretation of immobility, described as ‘despair’ which decreases upon antidepressant administration (52), does not seem appropriate given the high amount of immobility in PPD5 dams compared to virgin female rats observed both here and in other studies (53). There are a number of physiological reasons why immobility or a ‘reactive’ coping style would be an advantageous strategy for postpartum females including changes in fat content, body density, and differences in metabolic and stress mobilization (54). Interestingly, Type 1 dams were also exhibited more immobility, suggesting that the multiple blood draws may have an important effect on later stress-responsive behaviors. Taking this into consideration, future studies could test depressive-like behaviors in rodents with alternative methods, such as measuring learned helplessness or sucrose consumption; however these tests may suffer from similar confounds when measured during lactation. Measuring intracranial electrical self-stimulation behavior to test anhedonia may also prove useful in understanding depressive-like symptomology in this context. An alternative interpretation of the FST defines greater struggling (i.e. less immobility and greater climbing) as observed in the CC dams, as a ‘proactive’ coping response to the highly stressful FST (20,52). Proactive coping responses are associated with high levels of aggression and CC dams have consistently shown increased maternal aggression (55–57), suggesting a specific test of coping responses would be highly informative. The neurobiology underlying FST behaviour was altered by CC treatment, and although other cognitive effects of CC treatment may play important roles, neuroendocrine mechanisms are likely major contributors to these behavioral differences.
Following the FST, CC-treated dams showed much higher plasma oxytocin concentrations compared to UN dams, perhaps indicating a greater stress response, since oxytocin is known to be released into the blood stream in response to stress (58,59). This difference could be a result of altered release concentrations, timing or degradation mechanisms of oxytocin and deserves further study. In the current study, all dams exposed to the FST (Type 1 and 2) exhibited increased plasma oxytocin concentrations compared to control (No FST/Type 3) animals. Although our measurement of plasma oxytocin can only be temporally associated with the end of the FST, previous studies in male rats show an initial exposure to uncontrollable swimming resulted in massive release of oxytocin into the plasma, amygdala, and SON (measured by microdialysis) (18,60,61). In contrast, our study found lower hypothalamic content following the FST, suggesting perhaps that hypothalamic oxytocin was dendritically released and had diffused away from the hypothalamus by the time of collection. These data must be interpreted with caution, as they were collected after the completion of the test and cannot distinguish between intracellular and extracellular oxytocin concentrations. SON oxytocin content during the stressful experience appears to correspond with the degree of uncontrollability of the behavioural context in male rats (18), indicating that future studies using more anatomically and temporally specific measurement techniques, such as microdialysis, may reveal changes in central oxytocin that explain the greater behavioural stress reactivity in CC-treated dams compared to UN dams. Additionally, in FST-exposed animals a positive correlation was observed between hypothalamic and plasma concentrations (regardless of Type), suggesting a coordination of release during the FST.
In addition to the increased oxytocin following FST, CC-treated dams showed a significant relationship between plasma oxytocin and plasma CORT concentrations that was not observed in UN dams, indicating that in response to stressful stimuli, the regulation of the hypothalamus, specifically the parvocellular cells of the PVN, may be altered in CC-treated dams; however, a significant relationship may have been found in the UN dams as well with a larger sample size. Alternatively, these results could represent different set-points for endocrine responsiveness, given that the Type 3 CC-treated dams show differences compared to UN dams in resting CORT (lower) and oxytocin (higher) concentrations, taken at the same time of day. As CC treatment has a multitude of effects on HPA and PVN signalling, during other reproductive stages (8,62,63), future studies may focus more attention on these cells to better understand changes that may occur in the postpartum period following CC treatment.
An increase in anxiety-like behaviours is observed in postpartum drug-abusing women (31), and although it is commonly associated with prepartum anxiety, it has been observed that anxiety can spontaneously occur postpartum (64). On PPD 5, CC-treated dams exhibited a strong trend towards an increased anxiety-like profile compared to baseline, with the majority of CC dams showing decreased centre duration. Cocaine withdrawal-induced anxiogenesis typically peaks 3–5 days following the last drug administration in non-lactating rats (65), suggesting that CC-treated dams may have been undergoing withdrawal from cocaine treatments that ended on their GD 20 (4–5 days prior to testing). One possible mediator of anxiety would be changes in HPA reactivity (44). Previous work indicated that continued cocaine exposure during lactation results in similar deficits in maternal behaviour compared to dams undergoing withdrawal on PPD1 (66), suggesting that the timing of testing is critical to understanding the underlying mechanisms responsible for deficits in postpartum behaviors and neuroendocrine signalling. CC dams also exhibited oxytocin concentrations that were two times greater than UN dams when taken from rest (see Figure 3B), similar to what is observed in depressed women (67), patients with high state anxiety (68) and social anxiety (69).
Significant differences between CC-treated and UN dams were not observed in the direct comparisons of OFT behaviour data, potentially because the variability was very high within the groups. UN dams also showed high variability in the change from baseline, suggesting that there could be normal variation in the progression to the low anxiety typically observed in the second week of lactating rats (16). It was noted in these studies that centre duration was quite low (see Figure 1C) especially in Type 2 dams; however, this could be a habituation effect as pilot work showed that dams without baseline measurements had higher centre duration (unpublished data). Alternatively, Type 2 dams may be reacting to removal from the nest to a greater extent than Type 1 dams. Additionally, the OFT may not be the most sensitive test of anxiety-like behavior in postpartum rat dams. Future studies using light-dark boxes or elevated plus mazes may reveal differences. Alternatively, the multiple handling sessions during the first postpartum week may have increased anxiety-like behavior across all groups, and future studies could determine whether this was the case. Our procedures introduce a light cycle switch on gestation day 7, which may result in subtle long lasting changes in anxiety-like behavior or hormonal signalling; and since cocaine has been shown to affect circadian endocrine rhythms (70,71), changes in endocrine signalling during gestation may be perpetuated into the postpartum period, affecting CC-treated dams differently than UN dams.
These data indicate that CC treatment during pregnancy alters peripheral endocrine signalling in a behaviourally context-specific fashion. Such disruptions likely interact and modulate the dams’ physiological state and thus impact her behaviour. The finding that the changes are dependent on the environment, suggest a complex tuning of these endocrine systems, not just a simple knock-down of their function. Stress during pregnancy can reduce maternal behaviour in rodents; however, rats that naturally exhibited low amounts of maternal behaviour are not affected by stress, suggesting that optimal maternal care can be reduced but only in certain populations (72). CC effects on hormonal and behavioural stress response differ from those recently reported following chronic stress during pregnancy (73) suggesting that signalling mechanisms outside of the HPA-axis may be involved in mediating the observed effects following CC treatment. Future work directly comparing CC treatment with chronic stress and their combination would be highly informative and perhaps better model the human condition.
We thank the invaluable assistance of Dr. Hsiao Tien, Dr. Matthew McMurray, Dr. Sheryl Moy, Elizabeth Cox, Dave Gardner, Thomas Jarrett, Cara Heaton, Nisel Desai, Marlana Radcliffe, and Ben Thompson for their help in the preparation of this manuscript and data collection as well as their thoughtful discussions with the authors. The authors were supported by Award Number P01DA022446 (JMJ) from the National Institute on Drug Abuse. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institute on Drug Abuse or the National Institutes of Health.