Ethics Statement

All procedures were approved by the University of Calgary Animal Care Committee (protocol numbers: M08110, M10032, M10033, M06105) and conformed to the guidelines outlined by the Canadian Council on Animal Care.

Animals and Parental Environment Conditions

We used 8-week old female and male mice throughout this study. C57BL6 breeding pairs were obtained from Charles River (Laval). Animals were handled in accordance with the animal care policies of the University of Calgary. Animals were maintained on a 12hour light/dark cycle with food and water ad libidum. All mice were provided with nestlets and Plexiglas igloo-style houses as part of the standard enrichment at the University of Calgary animal facility. Female and male mice were mated, where mating was detected by the presence of a plug. Pregnant females were placed into different conditions so that pups would experience different parental conditions until weaning (21 days of age). It has been suggested that mice are particularly sensitive to external disturbances, which increases the likelihood of individual mouse pup and whole litter mortality [27]. Given the high pup mortality rate reported for C57BL6 mice [28], pups born in the different parental conditions were left undisturbed to prevent mortality. As a result, pups were not culled nor were the sex ratios of the litter equalized, as this would involve physical displacement of pups and the nest, leading to disturbance of the cage environment, which has been shown to influence pup mortality [29], [30].

Maternal only condition: Impregnated females were separated into individual cages. After 18 days, females gave birth to a litter of 6–8 pups and raised the pups on their own.

Maternal-virgin condition: Impregnated females were placed into individual cages with a virgin age-matched female and would remain together for duration of pregnancy (18 days). After 18 days, the pregnant females would give birth to a litter of 6–8 pups. The pair would remain together with the pups until weaning.

Maternal-paternal condition: Pairs of male and female mice were placed in individual cages. Mating was detected by the presence of a plug and males remained with their partners for the duration of pregnancy and with the pups until weaning.

After the animals were weaned from their specific rearing environments (at postnatal day 21), they were socially housed with littermates. A total of 269 animals were used in this study. The actual numbers of animals used per analyses are detailed in the figure legends.

Animal Behavioural Tasks

A behavioural test battery, ordered from least to most stressful, was conducted [31], [32]. While the advantages of using a behavioural test battery are many, there is the possibility of mice exhibiting training effects, where one test influences another [31]. As such, we performed a series of tasks that were in similar order outlined in McIlwain et al [31], keeping in mind that some tests may be more sensitive to previous testing experience. Open field was first administered, followed by light-dark, elevated plus maze, Y-Maze, ladder rung, social preference, pre-pulse inhibition, Morris water maze, passive avoidance, and cue/contextual fear conditioning. The duration between conducting the first five tasks was 24 hours. After the social preference task, animals were left undisturbed for 1 week followed by the administration of pre-pulse inhibition. A one-week inter-test duration was employed between the pre-pulse inhibition, Morris Water Maze, passive avoidance and cue/contextual fear conditioning tasks. Naive mice from second generation breeding (i.e. offspring from MO, MV and MP parents) were specifically tested on behavioural assays that first generation biparentally raised mice demonstrated significant differences in. Performance on behavioural tasks, such as cue/contextual fear conditioning, have been demonstrated not to be dependent on previous training [31].

Some studies using rats have demonstrated that performance on spatial memory tasks are independent of estrus cycle phase [33], [34], whereas others have described impaired spatial memory during the proestrus and estrus phase [35], [36], where estrogen is at its peak. In mice, the administration of estradiol has conflicting results on whether it improves or impairs spatial memory [37], [38]. Moreover, a study by Frick et al demonstrated that mice use very different strategies than rats in the Morris water maze [39]. Given that all mice are grouped housed from weaning until behavioural testing, it is possible that these grouped housed mice experience anoestrus [40]. Further Lagace et al demonstrate that hippocampal neurogenesis is not influenced by the estrous cycle in C57BL6 mice and suggest that their data supports the use of female mice without determining estrous cycle stage [41]. As such, female mice underwent behavioural testing without taking into account individual estrous cycle phase.

A surgical procedure was necessary to carry out telemetric measures. Therefore, telemetry was monitored in separate groups of animals that did not undergo the behavioural test battery. Home cage activity was telemetrically monitored in mice as described in Antle et al [42]. Male mice were surgically implanted with an intraperitoneal transmitter. Animals were allowed 7 days to recover from the surgery in individual cages before testing. Locomotor activity was assessed by frequencies emitted by the implanted transmitters. Frequencies were collected by individual receivers placed under each cage for 1 week. Data from the receivers were sent to a separated computer equipped with ER-4000 Energizer/Receivers and running Vital View data collection software (Minimitter, Bend, OR). Locomotor activity was recorded across the entire week of observation and averaged to obtain mean values for 10 minute intervals.

Open field analysis was conducted using the methods outlined in Sgado et al [43]. Briefly, open field behaviour was recorded and quantified using an automated video tracking system. The open field arena was circular (1.55 m diameter, 0.46 m height) constructed of white fiberglass. Mice were permitted to freely explore for 5 minutes while an image tracking system (HVS Image, Hampton, UK) recorded total distance traveled, average walking speed, and percent time spent in the core, middle, and periphery of the arena. The arena was cleaned between trials with 70% ethanol.

The Y-maze task was conducted based on methods described in Saxe et al [44]. Briefly, the Y-maze was constructed out of three Plexiglas arms of equal size joined together in a Y configuration. Each arm was 40 cm long and 10 cm wide with 12 cm high walls. The floor of each arm was white and the walls were clear. The apparatus was placed at ground level and all sessions were video captured for subsequent analysis. Individually, mice were allowed to explore two out of the three arms for a total of 10 minutes. The third arm was block with a piece of Plexiglas that could be slid in and out. Thirty minutes later the mice were individually returned to the maze for 5 minutes with all arms open. The number of entries and time spent in each arm were quantified.

The elevated plus maze was conducted based on methods previously described in Malleret et al [45]. Briefly, the plus maze consisted of four black Plexiglas arms, two open arms (67 cm×7 cm) and two enclosed arms (67 cm×7 cm×17 cm) that forms a cross shape with the open arms opposite of each other. The maze was elevated 55 cm above ground. Animals were placed in the center of the maze and explored for 10 minutes. A camera was mounted directly above the maze to record the behaviour of the animal. The number of entries into the open versus enclosed arms were counted, as well as the time spent in the open versus enclosed arms. An anxiety ratio was calculated for each animal by dividing the amount of time spent in the open arms by the total time in the open plus enclosed arms.

The light-dark task was based on methods described in Saxe et al [44]. Briefly, the light-dark task was conducted in a Plexiglas square arena measuring 44 cm×44 cm with white floors and clear walls. A dark tinted Plexiglas cover was created to fit over half of the arena. An opening at the floor level in the centre of the tinted Plexiglas cover allowed for passage between the light and dark compartments. The light compartment was brightly illuminated with a fluorescent light. Mice would be placed in the dark compartment to begin with and allowed to freely explore both compartments for 5 minutes, while being video recorded. Time spent in each of the compartments was quantified.

The horizontal ladder rung consists of clear Plexiglas walls and removable metal rungs (3 mm diameter) with a minimum distance of 1 cm between each rung. The rung walkway is 1 meter in length, the walls span 1 m and measures 19 cm high from the height of the rungs. The entire ladder is elevated 30 cm above the ground, the width of the 1 meter walkway is 6 cm to prevent the mouse from turning around with ease. During training and testing, the home cage was placed at one end of the rung. Animals were trained over several sessions with rungs all separated by a 1 cm distance until they were able to walk the length of the rung with speed and without hesitation. The difficulty of the task was modified by varying the position of the rungs by removing rungs along a 40 cm portion midway along the length of the walkway. An irregular pattern of spacing the rungs was changed each trial session to trial session to prevent animals from learning the pattern. The distance of the rungs varied from 1 cm to 3 cm for the irregular patterns. Trial sessions consisted of 5 complete walks along the length of the apparatus. Between trials the apparatus was cleaned with 70% ethanol. A video camera recorded paw placement on the rungs. Evaluation of forelimb and hindlimb paw placement was assessed using a modified version presented in Metz and Whishaw [46]. The total number of steps taken within the 40 cm portion was quantified in addition to the number of full slips, which were given a score of 1 and half-slips, which were given a score of 0.5. A full slip would entail the animal’s entire paw and portion of leg falling in the space between two metal rungs, thus breaking the rhythmic stride of the animal. Whereas a half-slip would entail only the animal’s placement of a paw between the space of two metal rungs, thus its rhythmic stride is maintained. A slip ratio was then calculated by taking the score obtained in a session divided by the number of steps taken to cross the 40 cm portion of irregularly spaced rungs. Slip ratios for each of the five trials were then averaged.

The social preference of mice was tested based on methods described in Fairless et al with slight modifications [47]. A Plexiglas chamber that could be compartmentalized into three compartments by perforated dividers was constructed. The perforations were large enough to allow for the exchange of visual, auditory, odor, and tactile (part of head only) information. Animals were habituated to the chamber for 5 minutes each day, two days prior to testing. The lack of preference for either side of the chamber was determined by a 10 minute exploratory session post-habituation. To test preference, mice were placed in the center of the chamber and a novel same-sex conspecific or inanimate object (clear plastic cube) was placed on either end of the chamber. Animals were video recorded for 10 minutes, the duration of time spent investigating each side was quantified.

Prepulse inhibition was conducted in an apparatus from SR-LAB, San Diego Instruments, San Diego, CA, USA, which was connected to a computer to monitor the startle response of individual animals placed in the apparatus. Animals were acclimatized to the apparatus for 3 minute with 65 dB of noise. A habituation phase served to stabilize the animal’s startle response, which consisted of ten pulse trials (40 ms, 120 dB). Subsequently, ten blocks of trials were presented. Each block consisted of a) ten pulse-alone trials (40 ms, 120 dB); b) ten pre-pulse plus pulse trials (pre-pulse: 20 ms, 80 dB; pulse: 40ms, 120 dB) c) ten no pulse trials (40 ms, 65 dB). The 30 trials were presented in a pseudorandom order within each block, with a variable intertrial interval of a mean duration of 15 seconds. Percent pre-pulse inhibition was analyzed by converting the startle data into percent scores: (% PPI = 100%×(pulse alone – prepulse plus pulse)/pulse alone).

Behaviour in the water maze was recorded and quantified using an automated video tracking system. The water maze consisted of a circular swimming pool 1.55 m in diameter and 0.46 m in height, constructed of white fiberglass. The pool was located in a room with various spatial cues. The pool was filled with 18 C water (30 cm in depth) and made opaque with skim milk. A platform, serving as a refuge from the water, was located in an arbitrarily defined quadrant of the maze, and 2 cm below the surface of the water so that the mice could not see the platform when swimming. Mice were recorded from a video camera directly mounted above the pool and connected to a computer with an image tracking system (HVS Image, Hampton, UK) which recorded total distance taken to get to the hidden platform, latency to reach the platform, average swimming speed, percent time spent in each quadrant. The water maze task was conducted based on methods described in Saxe et al [44]. On the first day, mice had AM and PM training sessions separated by 4 hours. Each session consisted of four-trials, where mice were released from the north, east, south, and west points of the pool with the platform in the center of an arbitrarily defined quadrant. Between each release points, mice were placed back in their home cage for a 10 minute inter-trial period. In each of the trials, mice were given 60 seconds to locate the platform, once found, they would remain on the platform for 10 seconds and this would end the trial. Mice unable to find the platform within the 60 seconds were placed on the platform for 10 seconds, thus ending the trial. On the second and third day, the mice would repeat the trials as described, with the platform remaining in the same location, while varying the sequence of release. After three days of training, animals were given a probe trial. The platform would not be placed in the pool during the probe trial and the mice would swim for the entire 60 seconds in duration, then removed from the pool. To assess long-term spatial memory, mice would be tested again in the same manner as the probe trial 7 days later. Data from the tracking software was converted into Microsoft Excel format to allow further data analysis.

For passive avoidance, training and retention trials were conducted in a chamber which had internal dimensions of approximately 45 cm×15 cm×25 cm (Hamilton-Kinder, LLC, San Diego, CA). Mice were placed in one half of the chamber and allowed to habituate for 2 minutes, after the 2 minutes a gate would open, allowing the mouse access to the other side of the chamber. When the mouse stepped onto the metal rungs on the other side of the chamber it would receive a foot shock (0.7 mA for 1.5 seconds). The chamber is connected to a computer that measures the latency to cross from the side the mouse was originally placed to the opposite side. The chamber was cleaned with 70% ethanol between each session. 48 hours and 7 days after the initial training session, animals were tested for retention using the same procedure as described, however no shock was administered. The latency to cross to the other side was measured by the computer, with a maximum allotted time of 300 s to cross.

Fear conditioning was conducted based on methods outlined in Saxe et al [44]. Briefly, fear conditioning was conducted in a chamber with removable plastic walls and a metal rung floor 3 cm above the base of the apparatus (Hamilton-Kinder, LLC, San Diego, CA). The internal dimensions of the chamber were approximately 20 cm×15 cm×25 cm. The chamber was located inside a larger, insulated metal and plastic cabinet which provided protection from outside light and noise. The behaviour of the mice was recorded by a digital video camera directly mounted above the conditioning chamber. Amount of time spent freezing was quantified, defined by the complete absence of motion.

Fear conditioning was conducted over three days. Day one, mice were placed in the conditioning chamber with black walls and received three pairings between a tone (20 s, 80 dB) and a coterminating shock (1 s, 0.5 mA). The inter-trial interval between each of the pairings was 2 minutes. Mice were habituated to the chamber for 2 minutes, after which the first tone-shock pairing was presented. The chamber was cleaned with 70% ethanol after each mouse. On the second day, the procedure and context were changed in several ways to test conditioned fear of the tone in the absence of contextual cues associated with the shock. The walls were made white; the chamber was scented with coconut extract; the experimenter wore different material gloves; a non-alcohol disinfected was used; and mice were kept in a different room before testing. Each mouse was placed in the chamber for approximately 6 minutes. A tone was presented twice, 120 seconds, and 260 seconds after being placed into the chamber. No shocks were administered. Freezing was scored 1 minute before the first presented tone (pre-tone) and during the 20 s of the first tone (tone). On day three, mice were tested for conditioned fear in the original training context. The testing procedure and context were identical to those used on day one, except that the shock was not presented. Each minute within the entire session was scored for freezing.

Parental Observations

Males and females in the three different parental environment conditions were left undisturbed during observations. Four litters were observed per parental environment condition. The four litters in which parental observations were performed per parental environment condition contributed to a subset of animals to the study. Additional males and females were used to generate the numbers of animals required to carry out the study to eliminate litter specific effects. The duration of time spent conducting the parental behaviours of: arch back nursing (female is in arched position over pups), licking and grooming, nest building, resting in nest and off nest, was recorded by an observer. Parental behaviours were observed once per minute in 15 minute increments four times each day (08∶00, 10∶30, 14∶00, 17∶30, with two observations during the light cycle and the other two during the dark cycle with red light illumination) from P0-P10. Visible ear notches were used to identify the virgin females and males in the maternal-virgin and maternal-paternal conditions, maternal females did not have their ears notched.

BrdU Administration

Mice were given BrdU (Sigma) (120 mg/kg, i.p. dissolved in 0.007% NaOH in phosphate buffer) every 2 hours for 10 hours and killed 16 hours after the final injection. Mice were given 10 hours of BrdU to label the entire constitutively proliferating population of cells in the subventricular zone, as previously outlined in Morshead et al [48]. To detect the phenotype of BrdU labeled cells in the dentate gyrus and corpus callosum BrdU was administered at the appropriate time points, then animals were allowed to survive for an additional 3–4 weeks or 12 days, if analyzing the dentate gyrus and corpus callosum, respectively. Animals would then be killed and tissue processed for immunohistochemistry analysis as outlined below.

Tissue Histology

These methods are detailed in previous studies [49], [50]. The animals were killed by anesthetic overdose of Somnotol and perfused transcardially with 4% paraformaldehyde in PBS, pH 7.2. Brains were post-fixed in the same paraformaldehyde solution overnight at 4°C, and cyroprotected for 24 hours in 20% sucrose in PBS. The brains were then embedded in Tissue Tek O.C.T. compound (Sakura Fineteck, Torrance, CA) before coronal sections of the subventricular zone, hippocampus, and spinal cord were cyrosectioned at 14 µm. Before immunocytochemistry, the sections were post-fixed with acetone for 30 seconds at room temperature and washed with PBS. For BrdU staining, the tissue was treated with 1 M HCl for 22 minutes at 60°C to denature the cellular DNA. Rat monoclonal anti-BrdU (1∶200, Oxford Biotechnology, Oxfordshire, UK) and rabbit polyclonal anti-Ki67 (1∶500, Novacastra, Newcastle upon Tyne, UK) were used to detect proliferating cells in the dentate gyrus. Sections were then incubated overnight at room temperature in primary antibody diluted in 0.3% PBS-T containing 10% NGS, washed with PBS, and then incubated with donkey or goat biotinylated secondary antibodies (all used at 1∶200, Jackson ImmunoResearch, Westgrove, PA, USA) for 1 hour at room temperature. This was followed with 45 min incubation at room temperature with streptavidin-Cy3 (1∶1500, Jackson ImmunoResearch) and Hoechst 33258 (0.015 mg/ml stock solution diluted to 0.001 mg/ml, Sigma) in PBS. After rinsing with ddH₂O, sections were mounted with Fluorosave (Calbiochem, San Diego, CA) and viewed with a Zeiss Axiophot fluorescence microscope. For double labeling in the corpus callosum, and dentate gyrus, sections were first processed for BrdU immunocytochemistry, using donkey anti-rat IgG conjugated to fluorescein as the secondary antibody. Subsequently, slides were processed with NeuN (1∶200; Santa Cruz Biotechnology, Santa Cruz, CA); platelet-derived growth factor receptor alpha (PDGFRa) (1∶10; R & D Systems, Minneapolis, MN), or for detection of glutathione S-transferase-π (GSTπ), sections were washed in PBS, then placed in boiling 0.01 M citrate buffer, pH 6.0 for 1.5 minutes prior to application of any antibody. Mouse anti-GSTπ (1∶50; BD Biosciences PharMingen, San Diego, CA) was used.

To detect cell death using TUNEL staining, slides were re-hydrated in PBS for 10 minutes and then incubated in 3% hydrogen peroxide in methanol for 10 minutes at room temperature. Sections were washed in PBS and treated with 20 µg/µl proteinase K diluted 1∶1000 in 10 mM Tris-HCl (pH 8) for 2.5 minutes at room temperature and washed with PBS. Sections were treated with 100 µl of TUNEL reaction mixture (ROCHE In Situ Cell Death Kit Fluorscien (FITC)), as outlined by the manufacturer and incubated for 1.5 hrs at 37°C. Subsequent immuno-detection was done with anti-FITC (1∶400, Sigma) overnight at 4°C, followed by biotinylated goat anti-mouse (1∶200), streptavidin-FITC (1∶1000) (Jackson Immunoresearch) and Hoechst 33258 (1∶100).

Cell Quantification

For the subventricular zone, a one-in-ten series of coronal sections (14 µm) from the rostral tip of the lateral ventricle to 1400 µm caudal of the ventricles were collected. Cells labeled by both the antibody of interest and Hoechst 33258 staining were counted blind in the defining SVZ. Cells labeled with the antibody of interest, but not with Hoechst 33258 and cells not clearly labeled with the antibody of interest were not counted.

For the hippocampus, a one-in-ten series of every other coronal section (14 µm) from the rostral portion of the hippocampus to 2800 µm caudal was collected. Cells were counted blind in the dentate gyrus as defined by Hoechst 33258 staining. Cells labeled with the antibody of interest, but without Hoechst 33258 and cells not clearly labeled with the antibody of interest were not counted. In the case of BrdU/NeuN double labeled cells, cell counts were performed blind and only cells clearly labeled with BrdU and NeuN were counted. Cells within the dentate gyrus of each representative slide of an entire hippocampus (10 sections) were counted using confocal microscopy under a 40x objective with Olympus Microsuite tissue analysis software. The software created and complied the Z-stacks for labeled cells within the dentate gyrus so that all distinctly labeled cells would be counted while labeled artifacts were discounted. Stereology was conducted using StereoInvestigator software (Microbrightfield Bioscience) with 10 coronal sections immuno-stained with anti-BrdU. The entire dentate gyrus area was quantified by tracing the perimeter using a 2.5x objective in each of the coronal sections, the software calculated the total area. BrdU-labeled cells were counted using the StereoInvestigator software with the same tracings for the area measurements. BrdU-labeled cells were counted in a 50 µm×50 µm counting frame with a 100 µm×100 µm grid size.

For the corpus callosum, a two-in-ten series of coronal sections (14 µm) from the rostral tip of the corpus callosum to 2800 µm caudal was collected. BrdU, BrdU/PDGFRa, or BrdU/GSTπ -positive cells were counted from the lateral-medial regions of the corpus callosum blind, in both hemispheres. Only cells that were clearly BrdU, PDGFRa, GSTπ, and Hoeschst 33258 labeled were included in the quantification.

For the spinal cord, a two-in-eight series of coronal sections (14 µm) were cut from cervical, lumbar, and thoracic regions of the spinal cord. The representative regions of the spinal cord were pre-cut prior to embedding the tissue in OCT and sectioning. BrdU positively labeled cells, which co-labeled with Hoeschst 33258 were included in the quantification.

Imaging

All fluorescent images were acquired on a Zeiss Axiophot fluorescence microscope using 25×, 40×, and/or 63× objectives, with the Rhodamine, FITC, and/or DAPI filters. Images were obtained using the AxioVision 3.1 software program at a resolution of 2600×2060 in the basic adjustment RGB mode. Images were saved as TIFF files and then transferred into Adobe Photoshop7.0 for processing. Adobe Photoshop7.0 manipulations involved cropping and varying the brightness and contrast, where the latter was applied equally across the entire image.

Statistical Analysis

Data were analyzed statistically using GraphPad Prism (San Diego, CA) or SPSS (Armonk, NY). Analysis of significant differences was performed using a two-tailed student’s t-test when an experiment only had two groups to compare or one- or two- way ANOVA followed by Tukey’s post-hoc analysis for experiments with more than two groups, or repeated measures ANOVA when similar data was collected from groups of animals over a number of days. P-value was set at 0.05.

Gross Brain Myelin Staining, Electron Microscopy and Quantification

A one-in-six series (14 µm) of sagittal sections starting medial and advancing 112 µm lateral was collected. Eriochrome cyanine staining was performed to visualize myelin, whereas the gray matter was counterstained with Eosin. This staining procedure is outlined by Kiernan [51]. Briefly, slides were placed in the staining solution (0.21 M aqueous ferric chloride (Sigma-Aldrich), 1.0 g Eriochrome cyanine R (Sigma-Aldrich), 2.5 ml sulphuric acid (Sigma-Aldrich), made up to 500 ml with dH₂O). The intensity of staining was monitored periodically, after which slides were placed in dH₂O and differentiated in 0.21 M aqueous ferric chloride. After differentiation, the gray matter was counterstained in Eosin then rinsed in dH₂O. Slides were dehydrated in ethanol, cleared with xylene, and then mounted with mounting medium. Slides were viewed with a light microscope, digital images of the region of interest were taken and the area of corpus callosum present within the region of analysis was assessed with ImageJ software.

Electron Microscopy

Female mice were perfused with saline containing 100 IU/ml heparin, followed by 10 minutes of 3% glutaraldehyde in Sorenson’s buffer. Brains were extracted from the animals and cut down the midline, then post-fixed for 1 hour in 3% glutaraldehyde in Sorenson’s buffer. Sections were then cut at 80 µm and the region of analysis (genu of corpus callosum) was dissected out (∼1 mm³ in size). The tissue was placed in 2.5% glutaraldehyde in 0.1 M cacodylate buffer overnight at 4C. The tissue was then transferred into a new vial containing 0.1 M cacodylate buffer and washed 3 times. Tissue was left in 0.1 M cacodylate buffer overnight at 4C and subsequently placed in 1% osmium tetroxide/0.1 M cacodylate buffer for ∼1 hr with gentle rotation. Tissue was then rinse with ddH₂O, serially dehydrated in ethanol, and subsequently serially infiltrated with Epon resin. Tissue was then placed in a capsule with Epon resin and polymerize at 60C for 24 hours. Ultrathin sections were collected on grids and subsequently processed with 5% uranyl acetate solution and Reynolds lead citrate solution prior to imaging. All solutions used for electron microscopy analysis were obtained from Electron Microscopy Sciences, USA. Fifteen photographs were randomly taken from each genu of corpus callosum at 5000×. The number of myelinated axons per field was counted based on methods previously outlined in Gregg et al [49].

Bi-parentally Raised Males Display Enhanced Neurogenesis, which is Passed onto the Next Generation of Male Offspring

Male and female mice raised in the three different parental conditions were injected with bromodeoxyuridine (BrdU) to label and quantify the number of proliferating cells within the adult brain. We found a main effect of sex (F_(1,23) = 12.79, p = 0.002) regarding the number of BrdU labeled cells in the dentate gyrus. This led us to investigate the effect bi-parental care has on cell proliferation in the adult male brain. We found that adult males raised in the maternal-virgin (MV males) and maternal-paternal (MP males) conditions had approximately 2.5 times more BrdU-labeled cells in the dentate gyrus compared to males raised in the maternal-only condition (MO males) (Fig. 1A) (F_(2,14) = 24.88, p<0.0001, posthoc: MO vs MV or MP, p<0.05). This was confirmed by quantifying the number of Ki67-labeled cells in the dentate gyrus (Fig. S4A) (F_(2,9) = 52.91, p<0.0001, posthoc: MO vs MV or MP, p<0.05). Further analysis of the dentate gyrus revealed no area differences between MO, MV, and MP males (Fig. S4B). Stereological assessment of the number of BrdU-labeled cells in the dentate gyrus further confirmed that MV and MP males have more BrdU-labeled cells in the dentate gyrus compared to MO males (Fig. S4C) (F_(2.7) = 8.649, p = 0.0128, posthoc: MO vs MV or MP, p<0.05). TUNEL analysis revealed no differences in apoptosis in males raised in the three different parental conditions (average number of cells±SEM, n = 6 per group; MO: 6.57±2.44, MV: 14.00±4.48, MP: 10.67±3.31). Surprisingly, cell proliferation in the adult female dentate gyrus was similar regardless of early life parental experience, and also greater than males raised in the maternal-only condition (Fig. 1A) (as previously observed in mice [52]). Moreover, there were no differences in SVZ cell proliferation between MO, MV, and MP males (Fig. 1B).

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Figure 1. Bi-parentally raised males display enhanced neurogenesis, which is transmitted to the next generation of male offspring.

(A) Offspring raised in maternal-only (MO), maternal-virgin (MV) and maternal-paternal (MP) conditions display sexually dimorphic enhancement of adult dentate gyrus (DG) cell proliferation (mean±SEM), where MV and MP males have more BrdU-labeled cells in the DG compared to MO males (n = 5 per group). (B) Cell proliferation in the SVZ is no different among adult male and female offspring raised in different parental conditions (mean±SEM). (C and D) Representative fluorescent micrographs of BrdU-NeuN double labeled cells in the DG of males raised in a single parent environment and bi-parental (MV) environment, respectively. Bars represent 50 µm. (E) MV and MP males show enhanced neurogenesis in the DG (mean±SEM) (n = 5 per group). (F) Males raised in a maternal-only (MO) environment, but sired by MV or MP fathers have more BrdU-labeled cells (mean±SEM) versus MO males sired by MO fathers (n = 5 per group). (G) Males raised in a MO environment, but sired by MV or MP fathers have more BrdU-NeuN double-labeled cells in the DG (mean±SEM) (n = 4 per group).

https://doi.org/10.1371/journal.pone.0062701.g001

To determine the differentiated neuronal fate of the newly generated cells in MV and MP males, the number of BrdU-NeuN double-labeled cells in the dentate gyrus was quantified three weeks after BrdU administration. MV and MP males had approximately 2.5 times more BrdU-NeuN double-labeled cells in the dentate gyrus than MO males (Fig. 1C,D,E, Fig. S4D,E) (F_(2,11) = 16.84, p = 0.0004, posthoc: MO vs MV or MP, p<0.05). TUNEL analysis revealed no differences in apoptosis in males raised in the three different parental conditions (average number of cells±SEM, n = 5 per group; MO: 3.4±0.87, MV: 2.0±0.71, MP: 2.2±0.73).

We next asked whether increased dentate gyrus neurogenesis in MV and MP males is transmitted to the next generation of offspring. MV and MP males were mated with MO females. Pups were raised in a single parent environment and BrdU was administered at 8 weeks of age. Surprisingly, we found that MO males with MV or MP fathers had approximately 2 times more BrdU-labeled cells (Fig. 1F) (F_(2,11) = 10.73, p = 0.0026, posthoc: MO (P: MO female×MO male) vs MO (P: MO female×MV male) or MO (P: MO female×MP male), p<0.05), which was further confirmed using stereological analyses (Fig. S4F) (F_(2,9) = 19.94, p = 0.0013, posthoc: MO (P: MO female×MO male) vs MO (P: MO female×MV male) or MO (P: MO female×MP male), p<0.05). Area analysis of the dentate gyrus revealed no difference between MO males with MO, MV, or MP fathers (Fig. S4G). MO males with MV or MP fathers also displayed 1.5 times more BrdU-NeuN double-labeled cells (Fig. 1G) in the dentate gyrus, than MO males with MO fathers (F_(2,10) = 6.14, p = 0.0182, posthoc: MO (P: MO female×MO male) vs MO (P: MO female×MV male) or MO (P: MO female×MP male), p<0.05). No differences were observed in the females sired by MO, MV, or MP fathers (average number of cells±SEM, n = 4 per group; MO females (MO female×MO male): 340.00±20.70, MO females (MO female×MV male): 422.75±52.30, MO females (MO female×MP male): 410.25±17.72).

Bi-parentally Raised Males Display Greater Freezing during Contextual Fear Conditioning

Previous studies have shown that newly generated neurons in the dentate gyrus play a role in certain hippocampal-dependent learning and memory tasks [44], [53], [54]. Therefore, we performed various hippocampal learning and memory tasks to assess the behavioural phenotype of MV and MP males, compared to MO males. Adult MO, MV and MP males and females were trained in the cue and contextual fear conditioning protocol outlined in Saxe et al [44]. We found that males and females raised within the three different parental conditions acquired the pairing of a neutral conditioned stimulus (tone) with an aversive unconditioned stimulus (shock) equally (Fig. 2A,C). We found no significant main effect of sex regarding the performance of males and females on cue conditioning. Although there appears to be a difference in the percentage of freezing during the pre-tone phase between MO, MV and MP groups, this is not significant (F_(2,52) = 3.676, p = 0.049). We next determined whether the acquisition of this pairing in a distinctive context would differ between MO, MV, and MP males and females. We found a significant main effect of sex (F_(1,52) = 4.131, p = 0.037) where further analyses revealed that both MV and MP males displayed significantly more freezing than MO males in the test of context-specific fear (Fig. 2B) (F_(2,52) = 7.809, p = 0.01, posthoc: MO vs MV p<0.01 for each of the minutes 3,4; MO vs MP p<0.0001 for each of the minutes 2,3,4), suggesting that enhanced dentate gyrus neurogenesis gives rise to a significant improvement in contextual fear conditioning. These results are similar to those reported for male mice reared in the double-mothering parental environment [15]. Moreover, adult MO, MV and MP females, which do not display differences in dentate gyrus neurogenesis, perform equally in cue and contextual fear conditioning (Fig. 2C,D). Interestingly, when MO males, sired by MV or MP fathers, were tested on cue and contextual fear conditioning, there were no differences observed in cue fear conditioning (Fig. 2E). However, MO males sired by MV or MP fathers displayed a greater degree of contextual fear conditioning (Fig. 2F) (F_(2,69) = 2.568, p = 0.0265, posthoc: MO (P: MO female×MO male) vs MO (P: MO female×MV male) p<0.05 for each of the minutes 2,3,4; MO (P: MO female×MO male) vs MO (P: MO female×MP male) p<0.05 for each of the minutes 3,4).

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Figure 2. Males with enhanced dentate gyrus neurogenesis display increased freezing during contextual fear conditioning.

(A) MO, MV, and MP males show no difference in cue fear conditioning (mean±SEM). (B) MV and MP males display enhanced freezing in contextual fear conditioning, compared to MO males (mean±SEM) (n = 12 per group). (C and D) MO, MV, and MP females, which display no difference in dentate gyrus neurogenesis, show no difference in cue fear conditioning (mean±SEM) or contextual fear conditioning (mean±SEM), respectively. (E) MO males sired by MO, MV, or MP fathers display no difference in cue fear conditioning (mean±SEM). (F) However, MO males sired by MV or MP fathers display a greater percentage of freezing in contextual fear conditioning (mean±SEM) (MO males (P: MO male×MO female) (n = 10), MO males (P: MV male×MO female) (n = 7), and MO males (MP male×MO female) (n = 9)).

https://doi.org/10.1371/journal.pone.0062701.g002

Given the lower percentage of freezing during the pre-tone phase in the cue conditioning task, it may be viewed that MO males have less anxiety than MV and MP males. Therefore, to ensure that increased context-specific freezing behaviour observed in MV and MP males was not due to an increase in anxiety, we examined male mice in the open field, elevated-plus maze and the light-dark choice task [44]. In the open field we found that MV and MP males travel a greater distance than MO males (Fig. S5A) (indicating increased activity and is similar to [55]) (F_(2,30) = 7.121, p = 0.0029, posthoc: MO vs MV p<0.001; MO vs MP p<0.05) and display a greater degree of thigmotaxis, as MO males ventured into the core of the arena more than MV and MP males (Fig. S5B) (F_(2,60) = 7.997, p = 0.0001, posthoc: MO vs MV core and periphery p<0.01; MO vs MP core and periphery p<0.01). However, the calculated anxiety ratio based on time spent in the open versus closed arms of the elevated-plus maze, as well as the time spent in the light or dark chambers in the light-dark choice task, was no different between the three groups (Fig. S5C,D). Therefore, using the above behavioural measures for anxiety, it appears that anxiety does not differ between MO, MV, and MP males, which is similar to the effect enriched tactile stimulation during early development has on anxiety behaviours [56].

Increased activity has been shown to enhance neurogenesis in the dentate gyrus [20]. Given that MV and MP males traveled a greater distance in the open field, we asked whether there were any differences in home cage activity. By using telemetry to monitor home cage activity over a one-week period, we found no differences in locomotor activity between MO, MV, and MP males (Fig. S5E). We next determined whether enhanced neurogenesis in MV and MP males would give rise to spatial memory differences using the Morris water maze and the Y maze (a place recognition task). We found that MO, MV, and MP males displayed equal ability to acquire where the hidden platform was placed in the water maze using extra-maze spatial cues (Fig. S5F), and displayed similar memory for its location in probe trials conducted one day (Fig. S5G) and one week (Fig. S5H) after acquisition. Moreover, in the Y-maze, males displayed an equal preference to explore the novel arm of the maze (Fig. S5I). Therefore, enhanced adult dentate gyrus neurogenesis in MV and MP males gives rise to improved performance on specific hippocampal-dependent behavioural tasks.

Bi-parentally Raised Females Display Enhanced White Matter Oligodendrocyte Production, which is Passed onto the Next Generation of Female Offspring

Given that bi-parental care can modulate adult neurogenesis, we next asked whether the generation of new myelinating oligodendrocytes, is also modulated by bi-parental care. We found a main effect of sex (F_(1,15) = 66.794, p = 0.00001), which led us to further investigate the effect bi-parental care has on the female brain. We found that MV and MP females had approximately 2 times more BrdU-labeled cells in the corpus callosum than MO females (Fig. 3A,B) (F_(2,9) = 11.90, p = 0.0030, posthoc: MO vs MV or MP, p<0.05). Surprisingly, cell proliferation in the male corpus callosum was similar regardless of early life parental care experience and approximately 2.5 times greater than MO females (Fig. 3B). Moreover, there were no differences in the number of TUNEL-expressing cells in the corpus callosum between MO, MV, and MP females (average number of cells±SEM, n = 6; MO: 13.5±3.96, MV: 18.33±1.71, MP: 11.00±2.11). To investigate whether increased cell proliferation persists in another central nervous system white matter region of MV and MP females, we quantified the number of BrdU-labeled cells in the spinal cord. MV and MP females had approximately 1.5 times more BrdU-labeled cells in the spinal cord than MO females (Fig. S6A,B) (F_(2,15) = 7.389, p = 0.0058, posthoc: MO vs MV or MP, p<0.05).

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Figure 3. Females raised in a bi-parental environment have more newly generated myelinating oligodendrocytes and myelinated axons in the corpus callosum, which is transmitted to the next generation of female offspring.

(A) Offspring raised in maternal-only (MO), maternal-virgin (MV) and maternal-paternal (MP) conditions, display sexually dimorphic enhancement of adult corpus callosum (CC) cell proliferation. Fluorescent micrograph of BrdU-labeled cells in the CC. Bar represents 50 µm. (B) MV and MP females have increased numbers of BrdU-labeled cells in the CC compared to MO females (mean±SEM) (n = 4 per group). (C) Fluorescent micrograph of PDGFRa-BrdU double-labeled cells in the CC. Bar represents 50 µm. (D) MV and MP females display more PDGFRa-BrdU double-labeled cells in the CC than MO females (mean±SEM) (n = 4 per group). (E) Fluorescent micrograph of GSTπ-BrdU double-labeled cells in the CC. Bar represents 50 µm. (F) MV and MP females display enhanced oligodendrogliogenesis in the CC compared to MO females (mean±SEM) (n = 4 per group). (G) Electron micrograph of axons in the genu of CC. (H) There are more myelinated axons in the genu of CC in MV and MP females than MO females (mean±SEM) (n = 42 (MO images), n = 56 (MV images) and n = 56 (MP images)). (I) Females raised in a MO environment, but sired by MV or MP mothers have more BrdU-labeled cells (mean±SEM) (MO females (n = 4 per group). (J) Females raised in a MO environment, but sired by MV or MP mothers have more BrdU-GSTπ double-labeled cells in the corpus callosum (mean±SEM) (n = 4 per group).

https://doi.org/10.1371/journal.pone.0062701.g003

To determine the differentiated phenotype of newly generated cells in the female corpus callosum, the number of BrdU-platelet derived growth factor receptor alpha (PDGFRa) double-labeled cells were quantified, where PDGFRa is a marker for immature oligodendrocytes. There were approximately 1.5 times more BrdU-PDGFRa double-labeled cells in the corpus callosum of MV and MP females, compared to MO females (Fig. 3C,D) (F_(2,9) = 9.260, p = 0.0065, posthoc: MO vs MV or MP, p<0.05). To determine whether MV and MP females had increased number of mature oligodendrocytes in the corpus callosum, we counted the number of BrdU-glutathione S-transferase-π (GSTπ) double-labeled cells 12 days after administering BrdU. We found that both MV and MP females had 2 times more BrdU-GSTπ double-labeled cells in the corpus callosum than MO females (Fig. 3E,F) (F_(2,9) = 10.98, p = 0.0039, posthoc: MO vs MV or MP, p<0.05). Given that newly generated oligodendrocytes have the potential to contribute to myelination, we asked whether more myelinated axons were present in the genu of the corpus callosum of MV and MP females, compared to MO females. We first determined whether the area of the genu of corpus callosum was different between MO, MV, and MP females by staining white matter in the brain with eriochrome cyanine. Using ImageJ to quantify the area of interest, we found no gross anatomical white matter differences between MO, MV, and MP females (Fig. S6C,D). Using electron microscopy we found that the number of myelinated axons was 1.4 times greater in MV and MP females than MO females (Fig. 3G,H) (F_(2,151) = 67.39, p = 0.0001, posthoc: MO vs MV or MP, p<0.05). Therefore, bi-parental care increases the number of new myelinating oligodendrocytes and myelinated axons in the adult female corpus callosum.

Given that enhance dentate gyrus neurogenesis in males is transmitted to the next generation of male offspring, we asked whether increased numbers of new myelinating oligodendrocytes in the corpus callosum of MV and MP females is transmitted to the next generation of female offspring. Females sired by MV or MP mothers had approximately 1.5 times more BrdU-labeled cells (Fig. 3I) (F_(2,13) = 16.05, p = 0.0003, posthoc: MO (P: MO female×MO male) vs MO (P: MV female×MO male) or MO (P: MP female×MO male), p<0.05) and BrdU-GSTπ double-labeled cells (Fig. 3J) (F_(2,13) = 6.187, p = 0.0178, posthoc: MO (P: MO female×MO male) vs MO (P: MV female×MO male) or MO (P: MP female×MO male), p<0.05) in the corpus callosum than MO females sired by MO mothers. No differences were observed in males sired by MO, MV, or MP mothers (average number of cells±SEM, n = 5; MO males (MO female×MO male): 520.00±33.87, MO males (MV female×MO male): 621.20±36.48, MO males (MP female×MO male): 524.20±30.27).

Bi-parentally Raised Females Display Greater Bilateral Motor Coordination and Preference for Social Investigation

The corpus callosum is thought to be involved in establishing cerebral laterality and mediating interhemispheric crosstalk and spatial coupling between the limbs [57]. Further, individuals with autism spectrum disorders exhibit white matter deficiencies and thus, the corpus callosum is also thought, in part, to be involved in social behaviours [57], [58]. Interestingly, enriched environments increase myelination of the corpus callosum [59], whereas social deprivation leads to less complex oligodendrocyte morphology and reduced myelination in the prefrontal cortex [60]. Therefore, we asked whether a greater number of oligodendrocytes and myelinated axons in the corpus callosum would give rise to differences in bilateral coordination and social behaviour. Adult female mice were trained to walk along the horizontal ladder rung with evenly spaced rungs and subsequently tested for their ability to walk along unevenly spaced rungs. A slip ratio was calculated based on performance. We determined that MV and MP females were more proficient at walking along the unevenly spaced rungs than MO females (Fig. 4A) (F_(2,22) = 33.206, p = 0.01, posthoc: MO vs MV p<0.01 on days 1,2,3; MO vs MP p<0.01 on days 1,2,3). Interestingly, there was also a main effect of sex on ladder rung performance (F_(1,42) = 21.562, p = 0.00034), where females outperformed males. The preference for social investigation of females was then examined based on their duration of investigating an inanimate object versus a same-sex, age-matched, novel conspecific (presented simultaneously). MV and MP females showed a greater degree of interest in the novel conspecific than the MO females (Fig. 4B) (F_(2,23) = 17.831, p = 0.0021), whereas MO females showed a greater degree of investigation for the inanimate object (F_(2,23) = 7.505, p = 0.003). MO, MV, and MP males tested for bilateral coordination and social preference displayed equal performance on both tasks (Fig. 4C,D), which would be expected as no differences in the number of newly generated myelinated oligodendrocytes were observed in males. Moreover, MO females sired by MV or MP mothers also displayed greater bilateral coordination (Fig. 4E) (F_(2,29) = 2.756, p = 0.0370, posthoc: MO (P: MO female×MO male) vs MO (P: MV female×MO male) on day 3 p<0.01; MO (P: MO female×MO male) vs MO (P: MP female×MO male) on day 3 p<0.01). MO females sired by MO mothers displayed a greater degree of investigation for the inanimate object (F_(2,31) = 24.947, p = 0.00001) (Fig. 4F). MO females sired by MO, MV or MP mothers display an equal degree of investigation for the conspecific. However, MO females sired by MV and MP mothers show more investigation towards the conspecific than the inanimate object compared to MO females sired by MO mothers (posthoc: MO (P: MV female×MO male) and MO (P:MP female×MO male) females inanimate vs conspecific p<0.05).

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Figure 4. Females with enhanced numbers of newly generated myelinating oligodendrocytes and myelinated axons in the corpus callosum display greater bilateral motor coordination and preference for social investigation.

(A) MV and MP females display less slipping on the horizontal ladder rung task than MO females (n = 11 per group). (B) MV and MP females also show a greater preference for social investigation as they spend more time investigating a novel conspecific than an inanimate object (mean±SEM) (n = 10 per group). (C and D) MO, MV, and MP males, which do not display enhanced oligodendrogliogenesis, show no difference in performance on the horizontal ladder rung. MO, MV, and MP males all demonstrate a greater preference for investigating the novel conspecific versus the inanimate object (mean±SEM) (n = 8 per group). (E) MO females sired by MV or MP mothers demonstrate less slipping on the horizontal ladder rung over time (mean±SEM) (n = 10 per group). (F) MO females sired by MV or MP mothers display greater investigation towards a novel conspecific (mean±SEM) (MO females (P: MO female×MO male) (n = 10), MO females (MV female×MO male) (n = 11) and MO females (MP female×MO male) (n = 13)).

https://doi.org/10.1371/journal.pone.0062701.g004

To rule out any differences in general anxiety between MO, MV, and MP females (which may affect performance on the ladder rung and sociability tasks), behaviour in the open field was observed. We found that females display equal levels of thigmotaxic behaviour (Fig. S7A). Interhemispheric crosstalk through the corpus callosum may be important for tasks involving higher cognitive functioning [57], [58]. Therefore, we sought to determine whether differences in the Morris water maze, passive-avoidance, and pre-pulse inhibition tasks would be observed between MO, MV, and MP females. We found that there was no difference in acquiring where the hidden platform was placed in the water maze using extra-maze spatial cues (Fig. S7B), and all females displayed similar memory for where the hidden platform was during the probe trial (Fig. S7C). Similarly, MO, MV, and MP females performed equally on the passive-avoidance task, (which assesses short- and long- term memory) (Fig. S7D). Further, females displayed no differences in sensorimotor gating when using the prepulse inhibition task (Fig. S7E). Therefore, MO, MV, and MP females display similar levels of anxiety and sensorimotor gating, and performed equally on various learning and memory tasks.