Male and female Sprague-Dawley rats were bred at Rutgers University in the Departmet of Psychology. Twenty-eight days after birth, animals were weaned and housed in goups of 2–3 males and 2–4 females in standard plastic shoebox style cages (44.5 cm long by 21.59 cm wide by 23.32 cm high). Female in the maternal study were housed alone. Animals were given access to food and water ad libitum and maintained on a 12:12 hr light-dark cycle; the light cycle began at 7am and ended at 7pm. All handling and experimental manipulations were carried out in the light portion of the diurnal cycle. Experiments were conducted with full compliance with the rules and regulation specified by the PHC Policy on Human Care and Use of Laboratory Animals and the Guide for the Care and Use of Laboratory Animals. The Rutgers University Animal Care and Facilities Committee approved all procedures.

Experiment 1: What behaviors are expressed during SCAR?

SCAR exposures began when the pubescent female was post-natal day (PND) 35, whereas male breeders varied in age from approximately 120–160 days old. The females in this age range weighed between 120–220-g, whereas the males weighed between 400–700-g. During the experimental manipulation, one pubescent female rat (n = 10) was placed in a novel cage with an adult sexually-experienced male rat for 30-min. The behaviors during the pairing were compared to behaviors during a similar pairing between a pubescent female rat (n = 10) and an adult female rat. All the conditions were the same, regardless of the individual pairings. The exposures occurred each day for eight consecutive days. The pubescent female was exposed to one of two adults that were alternated each day. All interactions were video recorded and behaviors were hand scored by two independent experimenters.

Very few sexual intromissions occurred and therefore, data are not presented here. We counted and analyzed three behaviors as follows: 1) anogenital trackings, 2) pins and 3) escapes. During an anogenital tracking event, the male tracked while presumably sniffing the anogenital region of the female as she ran around the cage. When the snout of the male was touching or nearly touching the anogenital region of the female for a continuous amount of time (>1-sec), we considered this one tracking behavior. During a pin, the adult male would effectively restrain the female, usually by sitting on top of her or turning her over on her back and using his paws to hold her down. During an escape behavior, the female sat up on her rear paws and reached for the top of the cage, as if trying to escape. These three behaviors were counted across the 30-min encounter in 10-min intervals. As noted, these behaviors were compared to the same behaviors expressed by a pubescent female when paired with an adult female (female/female).

Results Experiment 1

During the first SCAR exposure, numbers of anogenital trackings expressed by the adult male (adult male/pubescent female; SCAR) were significantly greater when compared to similar behaviors expressed by an adult female rat paired with a pubescent female (female/female) group (t (18) =6.07; p < 0.001; Fig. 1A). Numbers of escape behaviors expressed by the pubescent female were also greater in number during the interaction with the adult male than the adult female (t (18) = 6.94; p < 0.001; Fig. 1B). Numbers of pins were greater in number when the pubescent female was interacting with an adult male than when interacting with an adult female (t (18) = 5.77, p < 0.001; Fig. 1C). These same behaviors were analyzed during the 8th consecutive day of conspecific exposures. As during the first exposure, numbers of anogenital trackings were elevated (t (18) = 10.51; p < 0.001; Fig. 1D), as were escape behaviors (t (18) = 6.09; p < 0.001; Fig. 1E) and numbers of pins (t (18) = 5.57; p < 0.001; Fig. 1F). Numbers of these behaviors did not change between the first and eighth exposures (p > 0.05). These results suggest that the recorded behaviors did not habituate with continued social interactions between the two conspecifics.

Figure 1 Behavioral measures of SCAR exposures. (A) During the first SCAR exposure, the number of anogenital sniffs was significantly greater in the SCAR (adult male/pubescent female) group than in females paired with another female (female/female). (B) During the first exposure, the female made a greater number of escape behaviors when paired with an adult male than when paired with an adult female. (C) The adult male also pinned the pubescent female down more times than did the adult female.(D–F) These behavioral results were similar during the eighth exposure. The SCAR group received more anogenital sniffs, emitted more escape behaviors and pins when compared to similar behaviors expressed when a pubescent was paired with an adult female. Full size image

Experiment 2: Does the SCAR exposure increase corticosterone?

In a second experiment, we analyzed the effects of SCAR exposure on the concentrations of the stress hormone corticosterone at two time points. First, we compared the amount of corticosterone released within the pubescent female 30-min after the exposure to an adult male versus exposure to an adult female. Pubescent females were exposed to either an adult male breeder (n = 6) or an adult female (n = 5, PND 60–120) for 30-min and following the single exposure, trunk blood was collected 30-min later. Animals were given a lethal dose of pentobarbital intraperitoneal injection and trunk blood was collected. Blood was transferred into heparin tubes (BD Biosciences, Franklin Lakes, NJ), centrifuged at 2500 RPM for 20-min and stored at −20 °C. Corticosterone immunoassay was performed according to manufacturer’s protocol (Corticosterone EIA Kit, Arbor Assays, Ann Arbor, MI). In separate groups, a pubescent female was exposed to an adult male for 30-min (n = 8) or was placed alone in a novel cage for 30-min (n = 7). The concentration of corticosterone in the blood of the pubescent female exposed to the adult male was compared to the amount released in response to a novel context, which is mildly stressful for a rodent. Two hours after the interaction had ceased, females were given a lethal dose of pentobarbital as above and blood was collected for radioimmunoassay of corticosterone concentrations.

Results Experiment 2

The SCAR experience was stressful for the female, as indicated by elevated concentrations of the stress hormone corticosterone, which is released from the adrenal glands during a stressful experience. Concentrations were elevated in the pubescent female 30-min after the first exposure to an adult male when compared to the concentrations that were released when she was placed with an adult female in a novel setting (t (13) = 2.59; p < 0.05; Fig. 2A). In a separate experiment, corticosterone concentrations in pubescent females exposed to the adult male for 30-min were elevated two hours later when compared to concentrations in a pubescent female that was left alone in a novel context for 30-min and returned to the home cage (t (9) = 3.07, p < 0.05; Fig. 2B). These data suggest that social interaction with the opposite sex is more stressful than interaction with the same sex and more stressful than being left alone in a novel context, at least in the pubescent female rodent.

Figure 2 SCAR increases stress hormones and disrupts learning. (A) Corticosterone concentrations were significantly elevated in pubescent females thirty minutes after they were exposed to the adult male when compared to concentrations in pubescent females that were paired with an adult female. (B) Concentrations were elevated two hours later in pubescent females that were paired with an adult male when compared to concentrations in pubescent females that were placed in novel context. (C) Learning the classically conditioned eyeblink response was assessed in females exposed to the adult male. Performance during trace conditioning was reduced in those females (SCAR) when compared to females that were not exposed to an adult male (No SCAR). The dotted line indicates the 60% learning criterion that was established as a measure of successful learning of the conditioned response. Full size image

Experiment 3: Does SCAR disrupt associative learning in the pubescent female?

In a third experiment, we examined the effect of SCAR exposures on learning the classically-conditioned eyeblink response using a trace procedure. Electromyography (EMG) activity from the eyelid was used to assess eyeblink activity through the muscle. Electrodes were implanted around the eyelid to deliver an unconditioned stimulus (US). During surgery, rodents were injected with sodium pentobarbital (35mg/kg), which was supplemented with an isoflurane inhalant. Two pairs of electrodes (insulated stainless steel wire 0.005 in.) were attached to a head stage and implanted through the upper eyelid (orbicularis occuli muscle). Insulation around the wire was removed from a section of each electrode in order to make contact with the muscle. The head stage was positioned using four screws and secured with dental acrylic. After surgery, rats were kept warm and under observation until recovery from anesthesia. Rats were provided Children’s Acetaminophen (conc. 32mg/ml), after surgery at a 112mg/kg dose, administered orally and allowed at least 2 days recovery prior to training.

At PND 35, a female pubescent rat (n = 6) was exposed to an adult sexually-experienced male for 30-min each day or placed alone (n = 6) in the cage for 30-min. After the fifth SCAR exposure, eyeblink electrode surgery was performed, as described above. After two days of recovery, the females were once again exposed to the adult male each day (SCAR) or left alone in a cage without the male (No SCAR). On the eighth day, each female was exposed to the male for 30-min and then removed from the SCAR exposure and transferred to the conditioning room. The electrodes were connected to the recording equipment and they were acclimated to the training apparatus for one hour. The next day, each female was exposed to the adult male, as before and then trained with 200 trials of trace conditioning. This procedure was repeated for four days, for a total of 800 trials of training.

A trace conditioning procedure was used, during which the animal is trained to learn the temporal relationship between a white noise conditioned stimulus (CS) and an unconditioned stimulus (US) of periorbital eyelid stimulation. The white noise was delivered at 80 dB for 250 ms, separated by a 500 ms trace interval and ending with stimulation of the eyelid at 0.5 mA for 100 ms. EMG activity was recorded throughout each trial (excluding the US) to assess and analyze percentage of adaptive eyeblink responses (those that occurred during the trace interval). Eyeblinks in response to the CS were assessed as significant changes in the magnitude and duration from the baseline EMG response. An eyeblink was counted if the EMG activity exceeded 10-ms, 0.3-mV and was at least three standard deviations (SD) more than the baseline prestimulus EMG response. Those responses that occurred during the 500-ms trace interval and before the US were considered conditioned responses (CRs). As noted, all rats were provided 200 trials each day for 4 consecutive days. Animals that emitted at least 60% conditioned responding in any one session over the course of four days were considered to have learned the CR.

Results Experiment 3

A repeated measures ANOVA was conducted using performance on eight blocks of 100 trials as the dependent measures. As expected, the main effect of training was highly significant [F (7,70) = 7.89, p < 0.001], indicating that the number of CRs increased over blocks and therefore learning occurred. During the first 100 trials, when most of the learning occurs, pubescent females exposed to the adult male emitted fewer CRs than the females that were not exposed to the adult male [F (4,40) = 3.28; p < 0.05]. Females exposed to the adult male (SCAR) also emitted fewer CRs across blocks of 100 trials over the four days of training [F (1,10 = 5.78; p < 0.05; Fig. 2C). These results suggest that both groups learned, but females exposed to the adult male produced fewer well-timed CRs (i.e. during the trace interval). The percentage of CRs was neither increasing on the last day (p = 0.11), suggesting a plateau in learning; yet performances remained different between females exposed to the adult male and those not exposed (p < 0.001). The conditioning data were further analyzed using an arbitrary learning criterion of 60% responding. This criterion is shown as a dotted line in Fig. 2C to indicate 60% conditioned responding. All females in the control group (No SCAR; 6/6) reached a learning criterion of 60% responding with 800 trials, whereas only 50% of females (3/6) in the SCAR group did.

Experiment 4: Does SCAR disrupt maternal sensitization?

Adult virgin females can express maternal behaviors over time in response to newborn pup exposure14,20 through a process known as maternal sensitization. These same behaviors were expressed by females in puberty, as shown in Fig. 3A. To determine whether the SCAR exposures reduce maternal sensitization, each pubescent virgin female rat (n = 8) was exposed to the adult male (SCAR) for 21 consecutive days beginning on PND35. As a control, a group of pubescent females (n = 8) were each placed alone in an empty cage according to the same schedule. On the fifth day of SCAR exposures, PND39, two newborn postnatal pups (PND 1–10) were placed in the pubescent female’s home cage for 24-h. The pups were born from non-experimental dams and therefore returned to their original dams for nutrition and care every 24 hours, spending 24-h with their lactating dams. Newborn pup health was fair; if pups were neglected by their original dam, they were removed from the study. For maternal behavior observations, pups were place at opposite sides of the home cage and maternal behaviors were observed and recorded for the first 10 minutes after placement. The recorded behaviors were 1) licking/grooming of pups, 2) retrieving of either one or two pups and 3) grouping of pups. Once the full complement of maternal behaviors was expressed for two consecutive days, the female was considered to have expressed maternal sensitization.

Figure 3 SCAR disrupts maternal behavior and sensitization. (A) Pubescent females that were exposed to the adult male during puberty (SCAR) were less likely to learn to express maternal behaviors over the course of 17 days. Only three of these females (3/8) expressed maternal behaviors whereas all of the virgin females that were not exposed to the adult male did (8/8). (B) The numbers of maternal behaviors (licking, retrieving and pup grouping) were tallied each day for a potential total score of 3. Pubescent females exposed to the adult male (SCAR) expressed fewer of these behaviors than did females not exposed to the adult male (No SCAR). Full size image

Results Experiment 4

The following maternal behaviors were analyzed: licking, retrieving and grouping of pups. The numbers of maternal behaviors were tallied each day for a potential total score of 3. Repeated-measures analysis of variance across days of exposure to the pups and the SCAR condition indicated a significant increase in maternal behavior [F (16) = 8.39; p < 0.05; Fig. 3B] and an interaction with the SCAR exposures [F (1,16) = 2.18; p < 0.01]. Significant differences between the group behaviors emerged within seven days of pup exposure (p < 0.05). Most of the SCAR females did not express all three maternal behaviors whereas females not exposed to the male (8/8) expressed maternal behaviors, usually within 5–7 days (Fig. 3A).

Experiment 5. Does SCAR disrupt newly-generated cells in the hippocampus?

First, we assessed the potential impact of SCAR exposures on the number of cells proliferating in the dentate gyrus within the first two hours of a SCAR exposure. Females were injected with one intraperitoneal injection of 5-bromo-2-deoxyuridine (BrdU; 200 mg/kg) immediately before a single 30-min SCAR exposure and sacrificed 2 hours following the BrdU injection (n = 5). Cell numbers were compared to those in a group that were injected with BrdU and sacrificed two hours later (n = 6). Second, we assessed the potential impact of SCAR exposures on the number of cells that were labeled with BrdU after exposure to the adult male over the course of one week. To do this, a group of pubescent females were exposed to the adult male each day for 8 consecutive days beginning at PND35 (n = 7). They were injected with BrdU before the 6th exposure (PND 40) and sacrificed one week after the injection. Another group of females were left alone in their home cages (n = 4), given a BrdU injection on PND 40 and sacrificed one week later. To examine the effects of SCAR on cell survival, a group of animals was injected with BrdU once and sacrificed twenty-one days after the one BrdU injection (No SCAR; n = 7). The number of cells that were labeled with BrdU was compared to the numbers in a group (SCAR; n = 5) that was injected with BrdU and then exposed for 30-min to the adult male each day for 21 days beginning at PND35.

Immunohistochemistry was conducted to analyze the number of BrdU-labeled cells. Animals were deeply anaesthetized with sodium pentobarbital (100 mg/kg; Butler Schein, Indianapolis, IN, USA) and transcardially perfused with 4% paraformaldehyde in 0.1 M phosphate buffer. Brains were extracted and post-fixed in 4% paraformaldehyde at 4 °C for 24–48-h to preserve tissue structure, before being transferred to phosphate buffered saline (PBS). A vibratome was used to cut 40μm coronal sections through the entire rostral-caudal extent of the dentate gyrus in one hemisphere. This is the standard practice in our laboratory, as no hemispheric differences in proliferation have been observed between the left and right dentate gyrus21,22. Every twelfth slice was mounted onto a superfrost glass slide (Fisher Scientific, Suwane, GA, USA) and allowed to air dry. Once dry, the tissue was stained using standard peroxidase methods to visualize the cells that incorporated BrdU as described previously22. Tissue was pretreated with heated 0.1 M citric acid (pH 6.0), rinsed with 0.1 M PBS, incubated in trypsin for 10-min and denatured in 2N HCl for 30-min with PBS rinses in between. Tissue was incubated overnight in primary mouse anti-BrdU (1: 200; Becton-Dickinson, Franklin Lakes, NJ, USA) and 0.5% Tween-20 (Vector Laboratories, Burlingame, CA, USA). The next day, tissue was rinsed and incubated in biotinylated anti-mouse antibody (1: 200, Vector Laboratories) for 60-min and placed in avidin-biotin-horseradish peroxidase (1: 100; Vectastain ABC Kit, Vector Laboratories) for 60-min. Tissue was placed in diaminobenzidine (DAB SigmaFrost tablets, Sigma Aldrich) for four minutes, rinsed, counterstained with 0.1% cresyl violet, dehydrated, cleared and cover slipped with Permount glue (Fisher Scientific).

Quantitative microscopic analysis was performed blind to the experimental condition by coding each slide. Estimates of the total number of BrdU-positive cells were determined using a modified unbiased stereology protocol23,24. Number of BrdU-positive cells in the dentate gyrus of each slice (granule cell layer and hilus) was counted by hand at 1000X on a Nikon Eclipse 80 i light microscope. Ten slices throughout the rostral caudal extent of the hippocampus were collected on the slides and the number was multiplied by 24 to obtain an estimate of the total number of BrdU-positive cells in the dentate gyrus in both hemispheres.

To assess whether maternal “learning” rescued new neurons from death and/or whether SCAR would prevent their survival, groups of pubescent females that were exposed to the adult male (n = 7) or not (No SCAR; n = 7) in the previous experiment were injected once with BrdU and cell numbers were compared to those in additional groups that were not exposed to pups (SCAR, n = 5; No SCAR, n = 7). As noted, one week later, just as most of the new cells would undergo programmed cell death, maternal sensitization with the offspring began. Females were housed each evening with offspring and their maternal behaviors were recorded and analyzed, as described in Experiment 4. Three weeks after the BrdU injection, four groups of females were given a lethal dose of sodium pentobarbital and brains were prepared for immunohistochemistry and microscopic analyses. Due to the nature of BrdU injections, number of animals in these groups was smaller than numbers from data presented in Experiment 4. In addition, we analyzed potential differences in cell numbers between the dorsal and ventral hippocampus. To accomplish this, BrdU labeled cells in the ventral region were compared to those in the dorsal according to interaural coordinates. The dorsal hippocampus was associated with slices from the rostral hippocampus (interaural 3.70 mm to 6.88 mm), whereas the ventral was associated with slices from the caudal hippocampus (interaural 2.28 mm to 3.70 mm), as described25.

Results Experiment 5

The numbers of BrdU-labeled cells did not differ between females exposed to the adult male and sacrificed 2-hr or 1-week later (p > 0.05; Fig. 4A,B). We did not observe any differences between the dorsal and ventral hippocampi (p > 0.05) on any of these measures (2 hour, 1 week, 3 weeks). Also, exposure to the adult male alone did not significantly affect the number of surviving BrdU-labeled cells (p = 0.94; Fig. 4C and Fig. 5A). However, the number of BrdU-labeled cells increased in females that had been exposed to pups during maternal sensitization (F (1,25) = 10.03; p < 0.005; Fig. 5A). These data suggest that presence of the pups in the cage may be sufficient to increase the survival of newly-generated neurons in the dentate gyrus of the hippocampus. The interaction between pup exposure and SCAR exposure was nearly significant [F (1,22) = 3.66; p = 0.068). Planned comparisons indicated that the females that were not exposed to adult male but were exposed to pups had more BrdU-labeled cells in the dentate gyrus granule cell layer than females that were not exposed to pups or the adult male (p = 0.002). In contrast, the females that were exposed to the adult male and were exposed to pups did not have significantly more BrdU-labeled cells than those that were not exposed to pups (p = 0.41). There was a significant correlation between the number of cells remaining in the hippocampus at 3 weeks and numbers of maternal behaviors expressed in the presence of the pups (r = 0.55; p < 0.05). Females that were less likely to express maternal behavior during sensitization retained fewer of the new cells. Therefore, the potential impact of SCAR on the survival of new cells in the hippocampus is not necessarily mediated by the stress of the SCAR experience itself but because it reduced the learning of maternal behavior, which does appear to increase the survival of the newly generated cells. These data are novel for two reasons: first, they indicate that exposure to the offspring may be sufficient to increase the survival of newly-generated cells in the hippocampus. Second, the data suggest that the SCAR experience reduces the survival of newly-generated cells in the female hippocampus through deficits in learning to become maternal.

Figure 4 SCAR did not reduce proliferation of newly-generated cells in the hippocampus. (A) SCAR exposures did not alter the number of newly-generated (BrdU-labeled) cells two hours later. (B) The number of BrdU-labeled cells increased during the week after the BrdU injection but SCAR exposures did not alter the numbers of cells. (C) Three weeks later, most BrdU-labeled cells were no longer present and therefore, had presumably died. (D,E) Representative photomicrographs of BrdU-labeled cells at 400X and 1000X in the dentate gyrus (granule cell layer) of a pubescent female. Full size image