Ancestral Exposure to Stress Generates New Behavioral Traits and a Functional Hemispheric Dominance Shift

Abstract

In a continuously stressful environment, the effects of recurrent prenatal stress (PS) accumulate across generations and generate new behavioral traits in the absence of genetic variation. Here, we investigated if PS or multigenerational PS across 4 generations differentially affect behavioral traits, laterality, and hemispheric dominance in male and female rats. Using skilled reaching and skilled walking tasks, 3 findings support the formation of new behavioral traits and shifted laterality by multigenerational stress. First, while PS in the F1 generation did not alter paw preference, multigenerational stress in the F4 generation shifted paw preference to favor left-handedness only in males. Second, multigenerational stress impaired skilled reaching and skilled walking movement abilities in males, while improving these abilities in females beyond the levels of controls. Third, the shift toward left-handedness in multigenerationally stressed males was accompanied by increased dendritic complexity and greater spine density in the right parietal cortex. Thus, cumulative multigenerational stress generates sexually dimorphic left-handedness and dominance shift toward the right hemisphere in males. These findings explain the origins of apparently heritable behavioral traits and handedness in the absence of DNA sequence variations while proposing epigenetic mechanisms.

Introduction

Early life experience, such as prenatal stress (PS), modifies the developing brain and behavior with potentially life-long consequences. Severe PS may result in reduced hippocampal volume (Lemaire et al. 2000) and altered function of prefrontal structures such as anterior cingulate cortex (ACC) and orbital frontal cortex (OFC) (Murmu et al. 2006; Muhammad et al. 2012; Mychasiuk et al. 2012). PS animals often show reduced dendritic spine density and dendritic atrophy in both ACC and OFC (Murmu et al. 2006). These anatomical changes induced by PS are associated with cognitive impairments, such as learning and memory deficits (Lemaire et al. 2000; Welberg and Seckl 2001; Bowman et al. 2004; Weinstock 2008; Glover 2011; Harris and Seckl 2011), delayed motor reflex development (Patin et al. 2004), and reduced motor ability and strength in later life (Kofman 2002; Canu et al. 2007; Cao et al. 2014) in both experimental animals and humans.

Growing evidence suggests that programming by PS may affect subsequent generations of offspring. Studies involving the Dutch famine birth cohorts have suggested that prenatal under nutrition can significantly affect the health of children and grandchildren (Roseboom et al. 2001; Katti et al. 2002, 2007; Roseboom and Watson 2012). Furthermore, prenatal toxins and steroid hormones may alter behavioral functions across several subsequent generations (Anway et al. 2006; Skinner et al. 2011; Vyssotski 2011; Iqbal et al. 2012; Matthews and Phillips 2012). Recently, our laboratory has shown that stress during pregnancy alters maternal behavior and newborn development across several generations, with pronounced changes induced by cumulative effects of recurrent stress (Yao et al. 2014). Our findings indicated that multigenerational adverse exposure may result in behavioral traits that may vary across generations (Ward et al. 2013). Behavioral variations across generations may be explained by epigenetic mechanisms (Skinner 2008; Meaney 2010; Dunn et al. 2011; Migicovsky and Kovalchuk 2011; Skinner et al. 2011; Babenko et al. 2012) that are transmitted and potentially accumulated across generations. Thus, through epigenetic regulation, a continuously stressful environment may result in multigenerational behavioral changes and potentially generate new behavioral traits through altered neuroplasticity.

A prominent behavioral trait with potentially ancestral origins is handedness. Handedness reflects cerebral asymmetry and has been linked to specific gene functions (Scerri et al. 2011). Genetic models have been used to explain greater incidence of left-handedness in males (McManus 1991). However, a recent genome-wide association study found no genetic linkages (Armour et al. 2014). Notably, left-handedness seems to be passed on through the maternal lineage rather than the paternal lineage (McManus 1991; Annett et al. 1996). Transgenerational epigenetic programing by adverse maternal experience may therefore provide a mechanism for apparently heritable patterns in handedness and laterality in the absence of genetic changes.

Here, we tested if a single versus a continuously stressful environment, induced by exposure to multigenerational PS across 4 generations, can lead to changes in motor laterality, cerebral asymmetry, and altered neuronal morphology in rats. The experiment was designed to investigate stress-induced alterations in fine sensorimotor skills, paw preference, and structural plasticity of parietal cortex (Par 1) in adult male and female F1 generations, exposed to PS, versus the effects of transgenerational and multigenerational PS in the F4 generation. The findings revealed that multigenerational exposure to PS exceeded the effects of single generation PS and promoted new behavioral traits. Importantly, multigenerational exposure to recurrent PS induces a shift of brain lateralization toward the right hemisphere and left-handedness in males, but not in females. These functional changes were accompanied by neuromorphological changes in the right parietal cortex.

Materials and Methods

Animals

This study involved generations of Long-Evans hooded rats bred and raised at the Canadian Centre for Behavioural Neuroscience vivarium. The 152 adult rats subject to this study were housed in groups (males in pairs, females 3 per cage) under a 12:12 h light/dark cycle with light starting at 07:30 h and the room temperature set at 22°C. Prior to behavioral training, rats were food deprived to reach 90–95% of their baseline weight. To maintain this weight, rats received standard chow food in their home cages 5 h after completion of daily skilled reaching training sessions. Rats were weighed daily. All procedures were approved by the University of Lethbridge Animal Care Committee in compliance with the guidelines by the Canadian Council on Animal Care.

Experimental Design

Two different lineages of rats were bred under standardized conditions (Fig. 1), where S refers to prenatal stress and N refers to nonstress conditions. Parental female rats were either stressed during late gestation (S) or remained unstressed (N). In the multigenerational stress lineage, their pregnant F1 daughters (F1-S), F2 granddaughters (F2-SS), and F3 great-granddaughters (F3-SSS) were stressed again during pregnancy (Skelin et al. 2015; Erickson et al. 2014; Yao et al. 2014). A lineage of yoked controls was bred with each generation (nonstress pregnant F0, F1-N, F2-NN, F3-NNN). Depending on litter size, a maximum of 6 offspring per litter of each sex were randomly selected to be included in the experiments. Each experimental group included offspring from 4 different litters.

Figure 1.

Mutigenerational and transgenerational stress model. Pregnant dams (P0) were stressed to generate F1 prenatally stressed offspring. The transgenerational stress lineage was produced from the prenatally stressed F1 generation followed by nonstress pregnancies to generate a lineage in which only the maternal great–great-grandmother had been stressed. The multigenerational stress lineage was produced by stressing pregnant mothers in 4 consecutive generations (F0, F1, F2, F3) to produce the stressed F4 generation. Maternal stress (red arrow, red letters) occurred during gestational days 12–18. The present experiment involved the F1 generation of nonstressed (F1-N) and prenatally stressed (F1-S) offspring, and the F4 generation of nonstressed offspring (F4-NNNN), transgenerationally stressed offspring (F4-SNNN), and multigenerationally stressed offspring (F4-SSSS).

The F1 generation included “prenatally stressed” (F1-S) animals and nonstressed controls (F1-N), and the F4 generation included “transgenerationally prenatally stressed” rats with stress limited to the F0 generation (F4-SNNN) and “multigenerationally prenatally stressed” rats in which stress had occurred in 4 successive generations (F4-SSSS) to investigate the influence of prenatal, transgenerational, and multigenerational stress on motor function and brain laterality. The experiments included the male F1 generation (males: n = 41 [F1-S = 20, F1-N = 21]) and males and females from the F4 generation (males: n = 55 [F4-NNNN = 21, F4-SNNN = 12, F4-SSSS = 22]; females: n = 56 [F4-NNNN = 24, F4-SNNN = 8, F4-SSSS = 24]). The groups were trained and tested in a skilled reaching and a skilled walking task to determine dexterity and paw preference. Since the multigenerational stress (F4-SSSS) showed the most significant difference for paw preference and laterality, the behavioral and neuronal analysis focused on this generation. Follow-up studies involved behavioral tests of male and female animals exposed to multigenerational stress at the age of 6 months (male n = 21[F4-NNNN = 11, F4-SSSS = 10]; female n = 24 [F4-NNNN = 13, F4-SSSS = 11]). Animals were then randomly assigned to neuromorphological analyses (male n = 12 [F4-NNNN = 6, F4-SSSS = 6]; female n = 12 [F4-NNNN = 6, F4-SSSS = 6]).

Prenatal Stress

From gestational days 12–18, pregnant dams were subjected daily to restraint in a Plexiglas cylinder for 20 min and forced swimming in warm water (22°C) for 5 min (Yao et al. 2014). The animals received both stress procedures each day in a semirandom order either in the morning or afternoon hours.

Behavioral Testing

Skilled Reaching Task

Animals were trained and tested in skilled reaching according to protocols described previously (Metz and Whishaw 2000). Briefly, rats were pretrained to reach asymptote levels in skilled reaching success.

Reaching success scores

Percent success was recorded for 14 consecutive days. In each session, rats were allowed to reach for 20 food pellets (45 mg, Bioserv, Inc., NJ, USA). The success percent (%) was calculated using the following formula:

$$\text{Success percent}=\frac{\text{number of successes}}{\text{number of attempts}}\times 100$$

On day 15 of behavioral testing, the performance was videotaped with a digital video camcorder (Panasonic WV-BP330, Panasonic, Minato-ku, Tokyo, Japan), and an experimenter blind to the experimental conditions performed the qualitative analysis of video recordings.

Qualitative reaching performance was based on 11 main movement components and 35 subcomponents (Metz and Whishaw, 2000). Three successful reaches per each rat were analyzed and averaged to assess the qualitative features of movements. The maximum reaching movement score was 35 points.

Paw preference scores

Paw dominance was determined by preferred paw use when reaching for a food pellet. Right paw dominance was recorded if an animal used the right paw to in >90% of reaches, left paw dominance was recorded for usage of the left paw in >90% of reaches. Ambidextrous animals used either right or left paw for 40–60% of the time.

Skilled Walking Task

The horizontal ladder rung walking apparatus (Metz and Whishaw 2002) was used to assess skilled walking performance. Briefly, pretrained animals were tested 3 times in each test session and video recordings were collected. The recordings were analyzed by a blind experimenter according to our previously published rating scale (Metz and Whishaw 2002).

Skilled walking scores

The quantitative analysis included the average number of errors for left and right forelimbs and left and RHL as a ratio of the total number of steps. For qualitative analysis, the limb placement was scored on a scale ranging from 0 to 6, where 0 represented a total miss and 6 represented a correct limb placement. An error was defined as a limb placement that received score of 0, 1, or 2 points.

Paw preference scores

To confirm paw preference as determined in the skilled reaching task, paw usage was measured for initiation of stepping cycles. Right paw dominance in reaching was accompanied by initiating a stepping cycle with the right forelimb 90% of the time. Right paw preference in skilled reaching closely correlated with right paw dominance in stepping on the ladder rung walking task.

Histological Processing for Golgi–Cox Staining

After behavioral testing was completed the animals were treated with an overdose of pentobarbital and were intracardially perfused with 0.9% saline. The brains were removed and preserved in Golgi–Cox solution for 14 days in a dark location (Ramon-Moliner 1957). The brains were then placed in 30% sucrose for 28 days. A vibratome (Leica, Buffalo Grove, IL, USA) was used to cut the brains at 200 µm and the slices were mounted on gelatin-coated slides. In the final step, the brains were stained according to the procedure published by Gibb and Kolb (1998). According to our previously published procedures, the entire dendrites of individual neurons were stained, including the distal segments. Dendritic segments that met the criteria of being thoroughly stained and without overlap with another dendrite or blood vessel were included in the examination. Various types of spines were drawn and included in the analysis according to previous studies (Robinson and Kolb 1999; Kolb et al. 2000,  2003; Wellman 2001; Brown et al. 2005; Wilber et al. 2011).

Pyramidal cells from layer III of the parietal cortex (Par1; Fig. 2A) were selected for analysis. A camera lucida mounted on a microscope was used to trace individual neurons from the Golgi–Cox stained brains (Fig. 2B,C). A total of 10 cells (5 per hemisphere) were traced at ×200 magnification (Gibb and Kolb 1998). In the current study, 60 cells were traced per group (10 per animal, 6 animals per group) across 4 experimental groups (i.e., 240 cells in total). Neuromorphological measurements included apical and basilar Sholl analysis (an estimate of dendritic length derived from dendritic branches that intersect concentric circles spaced 25 µm apart), apical, and basilar dendritic branch order (an estimation of dendritic complexity based on the number of branch bifurcations) and spine density (the number of spine protrusions on a 50-µm segment of dendrite traced at ×1000 magnification; Gibb and Kolb 1998; Fig. 2D). The exact length of the segment was calculated and expressed as the number of spines per 10 µm (Mychasiuk et al. 2013).

Figure 2.

Morphometry of Golgi-stained pyramidal neurons in the parietal cortex Par1. (A) Schematic diagram of coronal sections through the right hemisphere illustrating the location of the parietal cortex Par1 (shaded area) according to standard cytoarchitectural criteria. Indicated is the position relative to bregma (Paxinos and Watson 1998); (B) Photomicrograph as seen through the camera lucida; (C) Representative pyramidal neuron showing apical and basilar dendrites; (D) High-power image of a dendritic segment with dendritic spines.

Statistical Analysis

Statistical analysis was performed using SPSS 20 for Windows 11.5.0 (IBM Corporation, Armonk, NY, USA). Three-way analysis of variances (ANOVAs) with Stress, Sex, and Hemisphere as factors were run, and revealed no main effect of Hemisphere, except for spine density. Thus, two-way ANOVAs with Stress and Sex as factors were run for behavioral tasks (pellet reaching task and skilled walking task) and neuromorphology of the parietal cortex (Par 1). Unpaired sample t-tests were used for all post hoc analyses. χ² tests were performed to reveal possible associations between treatment and paw preference. Exact binomial confidence limits and proportions were calculated for nominal variables (paw preference). Pearson's correlations were used to examine possible relationships between behavior and neuromorphology (spine density) based on hemispheric dominance (ipsilateral and contralateral hemisphere). Paw preference from each animal was used to determine hemispheric dominance, and therefore spine density from each hemisphere was assigned to the ipsilateral or contralateral side. For example, for an animal with right paw preference and left hemispheric dominance, the spine density of the left hemisphere was labeled contralateral (i.e., contralateral to the preferred limb). Reaching scores in individual animals were correlated to their contralateral and ipsilateral apical spine density measurement. The results are shown as the means ± standard error of the mean (SEM). Asterisks indicate significances: *P < 0.05, **P < 0.01, ***P < 0.001.

Results

Multigenerational and Transgenerational Stress Promote Left Paw Preference

Based on skilled reaching performance, rats were tested for paw use in terms of left, right, and ambidextrous traits. The experience of PS in the F1 generation (F1-S) had no significant consequence on paw preference in male rats (n = 20) compared with nonstressed (F1-N) controls (n = 21; χ²(1) = 1.33, P = 0.25). However, the proportion of left paw preference in F1-S animals was slightly larger (35% or 0.35 with 95% confidence limits of 0.15 and 0.60) than in nonstressed controls (19% or 0.19 with confidence limits of 0.05 and 0.40; Fig. 3A). Recurrent PS across generations increased the proportion of left paw preference in F4-SSSS males from 35 to 50% (0.5 with 95% confidence limits of 0.28 and 0.717; χ²(1) = 0.03, P = 0.33; Fig. 3A). Interestingly, the experience of both transgenerational (F4-SNNN) and multigenerational (F4-SSSS) stress affected paw use (Fig. 3B) when compared with animals exposed to PS in the F1 generation. The ratio of paw preference in skilled reaching among male and female rats revealed a sexually dimorphic effect of multigenerational PS; multigenerational PS (F4-SSSS) significantly increased left paw preference in males (χ²(1) = 4.53, P < 0.05, Fig. 3C) but not in females (χ²(1) = 0.78, P = 0.37, Fig. 3C) in comparison with nonstressed control animals.

Figure 3.

Effects of prenatal stress across generations on handedness. Percentage of rats displaying paw preference in the reaching task. (A) Paw preference in males was slightly affected by both prenatal stress (F1-S) and transgenerational stress (F4-SNNN), and significantly altered by multigenerational stress (F4-SSSS) in comparison with nonstressed (F4-NNNN) animals. (B) Transgenerational stress (F4-SNNN) modified handedness to similar degrees in both males and females. (C) Multigenerational stress significantly shifted paw preference toward left-handedness in males; nonstressed males (n = 21) had 19% absolute left paw preference while multigenerationally stressed males (n = 22) preferred the left paw 50% of the time. However, multigerationally stressed females showed only a slight shift in left paw preference (34% in F4-NNNN to 46% in F4-SSSS).

Nonstressed (F4-NNNN) male rats (n = 21) were more likely to be right-handed (81% or 0.8 with 95% confidence limits of 0.58 and 0.95) than to be left-handed (19%; Fig. 3C). Multigenerational PS (F4-SSSS) decreased the proportion of right paw preference among males (n = 22) to 50%, compared with nonstressed rats (F4-NNNN, n = 21; Fig. 3C). Among nonstressed females (F4-NNNN, n = 24) the right:left ratio was 66% (0.66 with 95% confidence limits of 0.45 and 0.84) right-handed and 34% (0.34 with 95% confidence limits of 0.15 and 0.55) left-handed. Multigenerational PS in females (F4-SSSS; n = 24) changed this ratio to 54% (0.54 with 95% confidence limits of 0.45 and 0.84) right-handed and 46% (0.46 with 95% confidence limits of 0.15 and 0.55) left-handed. A notable but nonsignificant shift in left paw preference was observed in transgenerationally stressed (F4-SNNN) males (χ²(1) = 1.82, P = 0.178, and females (χ²(1) = 0.62, P = 0.40). Interestingly, F4-SNNN animals showed a shift in paw preference toward the left limb from 19 to 41% (0.41 with 95% confidence limit of 0.26 and 0.63), similar to F4-SSSS animals. F4-SNNN females were more likely to be left-handed (66% or 0.66 with 95% confidence limits of 0.22 and 0.95) than right-handed (34% or 0.34 with confidence limits of 0.04 and 0.77) in comparison with F4-NNNN females, which were rather right-handed (66%). As F4-SSSS males demonstrated the most significant shift in paw preference and laterality, this group was used for subsequent behavioral and neuromorphological examinations.

Multigenerational Stress had Sexually Dimorphic Effects on Skilled Limb use

Although there was no main effect of Sex, Stress, or any interaction for percent success, there were significant changes in movement trajectories, as revealed by qualitative analyses. The average qualitative reaching movement score for skilled reaching movements revealed a main effect of sex (F_1,35 = 50.37, P < 0.001), as females generally performed better than males (P ≤ 0.001; Fig. 4A). The average score in females was 30 points compared with an average of 24 in males. Furthermore, there was a significant Sex × Stress interaction (F_1,35 = 7.30, P < 0.05), as F4-SSSS females performed better than nonstressed F4-NNNN females (P ≤ 0.05). Furthermore, F4-SSSS males performed slightly worse than F4-NNNN males (P = 0.50; Fig. 4A). Notably, stress exerted sex-specific effects on the aim component. F4-SSSS males revealed impaired aim of the paw directed toward the pellet with a greater supination of the paw toward the reaching side (Fig. 4B,C). Furthermore, in comparison with F4-SSSS females, when initiating a reach F4-SSSS males displayed weight shift toward their reaching forelimb as if to use the body to aid in lifting the paw from the floor. This component was left intact in F4-SSSS females in comparison with controls (Fig. 4C).

Figure 4.

Effects of multigenerational prenatal stress on skilled reaching performance. Multigenerational stress affected skilled reaching success and movement trajectories in a sexually dimorphic manner. (A) Females performed skilled reaching movements with overall higher success rates and higher qualitative movement scores. Multigenerational stress in males (n = 10) reduced success rates and qualitative movement scores compared with nonstressed males (n = 11). In contrast, multigenerational stress in females (n = 10) increased reaching success and qualitative movement scores compared with nonstressed females (n = 10). (B) The aim movement component was performed better by females than males. Additionally, multigenerational stress negatively affected males while it benefited females. (C) Photographs of representative aim movements for each sex and group. The illustration shows that ancestral stress caused the aim to be at a greater angular position toward the pellet in males (see arrow). Asterisks indicate significances: *P < 0.05. All data mean ± SEM.

Multigenerational Stress Increased Skilled Walking Errors Only in Males

The error rates in skilled walking revealed a significant main effect of Sex, as female rats overall made fewer forelimb (left fore limb: F_1,40 = 15.37, P < 0.001; right fore limb: F_1,40 = 22.67, P < 0.001) and hind limb [left hind limb: F_1,40 = 5.24, P < 0.05; right hind limb (RHL): F_1,40 = 8.78, P < 0.005] errors than males (Fig. 5). Furthermore, RHL errors revealed a main effect of Stress in the F4 generation (F_1,40 = 4.58, P < 0.05) and a significant Sex × Stress interaction for RHL errors (F_1,40 = 8.52, P < 0.01) and for left forelimb errors (F_1,40 = 4.32, P < 0.05). Multigenerational PS had sexually dimorphic effects on skilled walking in RHL errors as stressed F4-SSSS males made more errors than nonstressed F4-NNNN males (P ≤ 0.05). In contrast, the stressed F4-SSSS females made slightly fewer errors than nonstressed F4-NNNN females (P = 0.35; Fig. 5).

Figure 5.

Effects of multigenerational prenatal stress on skilled walking performance. Multigenerational prenatal stress altered skilled walking ability in a sexually dimorphic manner. Stressed females (F4-SSSS) showed higher foot placement accuracy than stressed males (F4-SSSS). A history of stress in males increased the error rates with reduced error rates errors in the RHL, indicating that their brain laterality was modified. In contrast, foot placement accuracy in stressed females (F4-SSSS) was not affected. All data mean ± SEM.

Multigenerational Stress Altered Spine Density and Dendritic Complexity in Parietal Cortex in a Sexually Dimorphic Manner

A family history of stress revealed to be a significant determinant of neuronal plasticity. A three-way ANOVA revealed a main effect of Stress (F_2,40 = 5.0, P < 0.001) and Sex (F_2,40 = 7.3, P < 0.001) but no main effect of Hemisphere (F_2,40 = 1.6, P > 0.05). In general, females had longer dendrites with more elaborate branching than males. Males, on the other hand, had greater spine density than females. Stress increased dendritic length and branching but decreased spine density. Notably, there was a significant effect of Hemisphere for spine density. The right hemisphere revealed a significantly higher spine density in both apical (F_1,40 = 4.9, P < 0.05), and basilar dendrites (F_1,40 = 4.0, P < 0.05). Using two-way ANOVAs, branch orders of the parietal cortex (Par 1) in the right and left apical fields revealed a significant main effect of Stress in the F4 generation (F_1,23 = 4.10, P ≤ 0.05). There also was an increase in dendritic branching in both right (F_1,20 = 7.39, P < 0.05) and left (F_1,20 = 7.44, P < 0.05) apical neurons of multigenerationally stress F4-SSSS male and female animals (Fig. 6A,B). In the basilar fields, females overall showed significantly longer dendrites in the right (F_1,20 = 9.93, P < 0.005) and left (F_1,20 = 5.96, P < 0.05) parietal cortex compared with males (Fig. 6A,C). Furthermore, females had significantly longer dendrites in the apical field in the right parietal cortex compared with males (F_1,20 = 8.96, P < 0.01).

Figure 6.

Neuroanatomical pathology of the parietal cortex in response to multigenerational prenatal stress. A history of multigenerational stress altered dendritic morphology in the parietal cortex. (A) Camera lucida drawing of apical and basilar pyramidal cells in the Par 1 of stressed F4-SSSS and nonstressed F4-NNNN male and female rats. (B) Dendritic branching, (C) dendritic length, and (D) Mean spine density in the Par1 region of both right and left hemispheres in male and female rats exposed to multigenerational stress. Note that multigenerational stress altered neuronal morphology of the parietal cortex in sex-specific manner. Asterisks indicate significances: *P < 0.05. All data mean ± SEM.

The dendritic length in the basilar field in the left parietal cortex revealed a significant Sex × Stress interaction (F_1,20 = 4.00, P < 0.05). Multigenerational PS reduced dendritic length in females and increased the same in males (Fig. 6A,C). A significant effect of Sex was found for dendritic spine density in the basilar field of the right (F_1,20 = 6.57, P < 0.05) and left (F_1,20 = 9.74, P < 0.05) parietal cortices; males generally had a larger spine density than females (P ≤ 0.05; Fig. 6D). Similarly, males showed a significantly greater spine density in the left parietal cortex apical field in comparison with females (F_1,20 = 12.23, P < 0.01). Importantly, multigenerational PS decreased spine density in the right (P < 0.05) and left parietal cortex apical fields in males (Fig. 6D). Figure 7 illustrates that, in contrast to nonstress F4-NNNN males, the multigenerationally stressed F4-SSSS males revealed significantly more complex dendritic branching patterns and lower dendritic spine density in the right parietal cortex.

Figure 7.

Computer-assisted reconstruction of Golgi-stained neurons and apical spines in layer III of the parietal 1 cortex (Par1). Images depict representative neurons in the left (A) and right (B) hemispheres of nonstressed F4-NNNN males; neurons in the left (C) and right (D) hemispheres of multigenerationally stressed F4-SSSS males. Note the increased dendritic complexity and decreased spine density in the right hemisphere of multigenerationally stressed males, which accompanied the shift toward left-handedness.

Multigenerational Stress is Associated with Significant Relationships Between Skilled Motor Ability and Spine Density in Males

Correlation analysis revealed a significant relationship between the spine density of the parietal contralateral hemisphere and skilled reaching performance among males in both the F4 multigenerational PS and control conditions. F4-NNNN rats with greater dendritic spine density in the contralateral hemisphere of the parietal cortex showed higher qualitative reaching scores (r = 0.76, P < 0.05; Fig. 8A). Additionally, there was a significant relationship between ipsilateral hemisphere and the reaching scores (r= 0.83, P < 0.05; Fig. 8A) in F4-NNNN. Importantly, there was a positive relationship between reaching movement scores and dendritic spine density in the contralateral hemisphere in F4-SSSS-stressed males (r = 0.82, P < 0.05; Fig. 8B). No relationship was found between the ipsilateral hemisphere and reaching movement performance in F4 SSSS animals. Thus, rats with higher spine density in the contralateral hemisphere showed better reaching performance. However, no significant correlation was found between the ipsilateral or contralateral hemispheres and reaching performance in females (Fig. 8C,D). Additionally, our results revealed a positive correlation between dendritic length in the right apical field and skilled walking error scores in F4-SSSS-stressed males (r = 0.88, P < 0.05; data not shown). These findings suggest that improved skilled reaching and walking ability of the left paw were associated with larger dendritic remodeling of the right hemisphere in males, further emphasizing pronounced laterality and hemispheric shift in multigenerationally stressed males.

Figure 8.

Relationship between skilled reaching performance and spine density. Correlation between mean spine density in the apical dendrites of the ipsilateral and contralateral hemispheres and mean qualitative movement score for (A) nonstressed (F4-NNNN) males, (B) stressed (F4-SSSS) males, (C) nonstressed (F4-NNNN) females, and (D) stressed (F4-SSSS) females. A strong positive correlation between qualitative reaching score and mean spine density was found for nonstressed males in the left hemisphere, and for stressed males in the right hemisphere. This shift indicates that multigenerational stress modified brain laterality in males. Additionally, nonstressed males reaching score and mean spine density of the ipsilateral (right) hemisphere were negatively correlated. No correlation was found in female rats. Asterisks indicate significances: *P < 0.05.

Discussion

PS represents a powerful impact on behavior and neuronal morphology. The present study expands this previous research by investigating if recurrent PS across 4 generations aggravates the consequences of PS in a single generation to promote the development of new behavioral traits by shifting behavioral laterality in a sexually dimorphic manner. The present study provides 3 main findings. First, multigenerational PS shifted paw dominance in males but not in females. Second, multigenerational PS compromised skilled movement trajectories and skilled walking ability in males. In contrast, a family history of PS rather improved these abilities in females. Third, cortical dendritic morphology indicated that multigenerational PS decreased multisynaptic plasticity in males but increased it in females. Notably, the shift toward left-handedness in stressed males was accompanied by increased dendritic complexity in the right parietal cortex. Therefore, the present data indicate that multigenerational PS promotes dysmasculinization in behavior and brain morphology among males.

The present study confirms the notion that altered behavioral traits caused by PS vary between males and females. PS and chronic stress exposure was shown to be associated with reduction in hippocampal (McEwen and Magarinos 1997) and cortical plasticity in males (Brown et al. 2005; Radley et al. 2006, 2013; Shansky et al. 2009) and an increase in females (Schmitz et al. 2002; Muhammad and Kolb 2011; Mychasiuk et al. 2012). Additionally, PS induces memory and learning impairments in males, while it improves these cognitive functions in females (Lemaire et al. 2000; Bowman et al. 2004; Son et al. 2006; Darnaudery and Maccari 2008). Human studies have reported greater motor deficits in male than the female PS offspring (Patin et al. 2004; Cao et al. 2014). Importantly, a particular outcome of PS may affect behavioral laterality in a sexually dimorphic manner, with males being more susceptible to laterality changes than females (Alonso et al. 1991, 1997; Tang and Verstynen 2002). These experimental data are supported by human findings where maternal stress at 18 weeks of pregnancy predicted atypical handedness that was more prominent in males (Glover et al. 2004). Specifically, left-handedness was observed to increase in response to PS exposure only in males (Elis 1991). The present findings expand these observations by suggesting that PS recurring across generations of the maternal lineage will program vulnerability to facilitate a shift in handedness and brain lateralization in the male offspring.

For a long time, lateralized brains were believed to be a uniquely human feature (Diamond et al. 1981; Camp et al. 1984; Alonso et al. 1991; Tang and Verstynen 2002). However, asymmetries in brain and behavior were described in many other species, such as rat, cat, rabbit, and nonhuman primates (Kolb et al. 1982; Camp et al. 1984; Alonso et al. 1991; Rogers 2000; Tang and Verstynen 2002; Gao and Zhang 2008). For example, rat studies described behavioral deficits associated with right hemisphere lesions, which were not present in animals with a left hemisphere lesion (Robinson and Coyle 1980; Kolb et al. 1982; Kirkland et al. 2008). Therapeutic benefits after brain lesion also differ depending on hemispheric dominance (Nikkhah et al. 2001; Vyazovskiy and Tobler 2008). Furthermore, both humans and rats display a high degree of hemispheric specialization within the parietal cortex (Rushworth et al. 1997, 2001, 2003; Schluter et al. 2001; Culham et al. 2006; Mento et al. 2010). This lateralization difference supports the overall preference of the right upper limbs (Kolb and Whishaw 2003; Whishaw et al. 2003).

Paw preference and handedness reflect more precise use and individual preference of one limb over the other, which is ideally tested in skilled reaching or object manipulation tasks. Using standardized reaching tasks, early studies by Tsai and Maurer (1930) reported that rats generally prefer the right paw over the left. Distribution of rat paw preference is similar to that of human handedness in males (right 80% vs. 20% left), whereas in females this distribution appears rather equally distributed (Tsai and Maurer 1930; Tang and Verstynen 2002; Guven et al. 2003). However, there are inconsistent results in the literature regarding handedness. According to Tang and Verstynen (2002), these inconsistencies can be explained in terms of differences among testing methods. Furthermore, environment and early life experiences may modify handedness and associated neuromorphology.

Changes in handedness are usually reflected by altered neuronal morphology. For example, chronic stress and moderate PS exposure were shown to decrease spine density in the medial prefrontal and orbitofrontal cortices (Brown et al. 2005; Radley et al. 2006,  2013). In contrast, mild PS decreased spine density in medial prefrontal cortex and did not affect spine density in the orbitofrontal cortex (Kolb et al. 2012; Muhammad et al. 2012). Furthermore, Radley et al. (2013) reported that chronic variable stress (CVS) after 14 days in males induced synaptic destabilization in the hypothalamo-pituitary-adrenal (HPA) inhibitory prefrontal circuit. They found that CVS diminished the density of stubby spines in basilar dendrites and thin spines in apical and overall dendrites of the anterior bed nuclei of the stria terminalis (aBST)-projecting prelimbic area neurons (Radley et al. 2013). This was accompanied by a selective depletion of mature mushroom spines in aBST-projecting neurons (Radley et al. 2013). However, Bloss et al. 2011 reported that mature mushroom spines are the most stable during environmental challenges such as stress. Since this study analyzed only one time point following the chronic stress exposure, it is not clear if the elimination or shrinkage of mushroom spines is responsible for these alterations (Radley et al. 2013). The literature suggests that glucocorticoid-dependent mechanisms influenced by environmental factors are behind the elimination of mushroom spines in aBST-projecting prelimbic area neurons and the observed morphological shift (Radley et al. 2013).

A variety of environmental and experiential factors can also shift the ratio of handedness and hemispheric dominance. Cortical development in particular is susceptible to a variety of environmental factors (Geschwind et al. 2002; Kolb et al. 2012). The maturation of hemispheric dominance is influenced by the time of exposure, intensity of the experience, developmental stage, and sex (Michelsen et al. 2007; Muhammad and Kolb 2011; Kolb et al. 2012). The present data confirm previous observations that stress alters brain dominance and hand preference. The most profound modifications are generally found in males, where their absolute hand preference may decrease from 80:20 right-hand dominance in the control population to 60:40 (Tsai and Maurer et al. 1930; Tang and Verstynen 2002; Guven et al. 2003; Gao and Zhang 2008). In contrast, in rats the female paw dominance tends to be smaller with 60:40 right paw dominance (Tsai and Maurer 1930; Guven et al. 2003), which is also observed in the human population (Alonso et al. 1991; Tang and Verstynen 2002).

Brain organization and motor functions that depend on handedness may be differentially expressed in males and females (Culham and Valyear 2006; Culham et al. 2006; Gardener et al. 2009; Tomasi and Volkow 2012). Evidence suggests that PS regulates fundamental epigenetic mechanisms and endocrine patterns across generations to promote demasculinization (Morgan and Bale 2011), and these factors may also shift lateralization in behavior and brain function. It is possible that early life adversity modifies cerebral asymmetry trough general neuromodulatory systems (Tang and Verstynen 2002). In particular, testosterone is a likely candidate to influence cortical lateralization and paw preference (Fleming et al. 1986; Stewart and Kolb 1988; Wisniewski 1998; Guven et al. 2003). Thus, the multigenerational PS in the present study may alter early cerebral development via reduction in testosterone levels, thus affecting male paw preference more severely than female laterality (Stewart and Kolb 1988; Geschwind et al. 2002; Culham et al. 2006). Similar mechanisms may be responsible for reducing male skilled reaching performance as well.

The present results revealed that multigenerational PS affected male and female skilled reaching and walking abilities differently. While multigenerational PS reduced skilled reaching performance in males, it seemed to promote performance among the stressed females. Notably, the impairments in skilled walking were greater for the nondominant limb. It is possible that successful skilled reaching motor performance among females is linked to improved recovery from stress (Jadavji and Metz 2008; Knieling et al. 2009), or to coping with PS (Bowman et al. 2004; Marrocco et al. 2012). Recurrent PS across generations may particularly favor the development of stress tolerance or resilience. Furthermore, females in general seem more resistant than males to stress-induced impairment (Zuena et al. 2008). Improved stress tolerance or resilience in females may promote both sensory and motor aspects of skilled movement performance.

The sexually dimorphic changes in sensorimotor performance were accompanied by neuromorphological adaptations in a functionally meaningful area, the parietal cortex. Although there are no previous data on the effects of multigenerational PS on cortical neuromorphology, earlier studies in PS confirm the present findings. Muhammad et al. (2012) reported that PS caused a decrease in basilar orbitofrontal cortex (angular insular cortex, dorsal part) in both males and females (Muhammad et al. 2012). Furthermore, PS diminished dendritic growth in males (Bustamante et al. 2013) with reduction in branching of the apical dendrites in layer II/III pyramidal neurons of the parietal cortex (Bustamante et al. 2013) and increased cortical spine density (Muhammad and Kolb 2011, 2012). Accordingly, cortical spine density in the right hemisphere and skilled reaching movement performance were positively correlated in stressed males but not in females. Other studies have shown that PS exposure modifies the neuronal morphology of hippocampus and nucleus accumbens as well (Mychasiuk et al. 2012). Thus, the effects of multigenerational stress may not be limited to the parietal cortex and may affect a variety of other areas and behaviors.

Alterations in neuronal plasticity may be due to toxic effects of excess circulating glucocorticoids (McEwen and Magarinos 1997; Takahashi 1998; Wellman 2001; Cook and Wellman 2004; Seckl and Meaney 2004; Brown et al. 2005) and prolonged HPA axis response (Koenig et al. 2005; Shansky et al. 2009; Radley et al. 2013). Mechanisms have been proposed that contribute to neuronal damage after exposure to PS. First, increased corticosterone circulation is associated with stress-induced decrease in neurotrophins, particularly brain-derived neurotrophic factor in various brain regions (Smith et al. 1995; Murakami et al. 2005; Uysal et al. 2011; Bustamante et al. 2013). Second, PS may alter microtubule-associated protein 2 synthesis in the brain (Barros et al. 2006), resulting in impaired synaptogenesis and neurite outgrowth (Bustamante et al. 2013). Additionally, the mechanism that explains sex differences may involve suppression of late prenatal testicular androgen secretion in males (Bowman et al. 2004). Alterations in androgen hormones during early development have profound effects on brain development, feminizing males and masculinizing females (Bowman et al. 2004; Zuena et al. 2008). Since recent genome-wide sequencing failed to identify any conclusive linkage to a specific locus associated with handedness (Armour et al. 2014), the importance of environmental factors and associated epigenetic programing, such as gene expression regulation through microRNAs and DNA methylation (Relton and Davey-Smith 2010; Yao et al. 2014; Babenko et al. 2015), of sex-specific brain development is further emphasized. The present study therefore supports the observation that left-handedness is passed on through the maternal lineage (McManus 1991; Annett et al. 1996) in the absence of DNA modifications (Armour et al. 2014).

The present study suggests that 4 generations of direct exposure to PS is associated with a hemispheric dominance shift to favor left-handedness in males, thus generating a momentum to develop new behavioral traits. These traits may be passed on to subsequent generations through the maternal lineage even in the absence of recognizable PS and in the absence of DNA sequence changes. Thus, transgenerational epigenetic inheritance may provide a primary mechanism for heritable patterns in handedness and laterality.

Funding

The authors acknowledge support by the Alberta Innovates-Health Solutions Interdisciplinary Team Grant #200700595 “Preterm Birth and Healthy Outcomes” (G.A.S.M.), grants from Alberta Innovates-Health Solutions (G.A.S.M.), the Natural Sciences and Engineering Research Council of Canada (B.K., G.A.S.M.), and the Canadian Institutes of Health Research grant #102652 (G.A.S.M.). G.A.S.M. is an AHFMR Senior Scholar.