Age- and sex-dependent impact of repeated social stress on morphology of rat prefrontal cortex pyramidal neurons

Abstract

Chronic stress can lead to psychiatric illness characterized by impairments of executive function, implicating the prefrontal cortex as a target of stress-related pathology. Previous studies have shown that various types of chronic stress paradigms reduce dendritic branching, length and spines of medial prefrontal cortex (mPFC) pyramidal neurons. However, these studies largely focused on layer II/III pyramidal neurons in adult male rats with less known about layer V, the site of projection neurons. Because the prefrontal cortex develops throughout adolescence, stress during adolescence may have a greater impact on structure and function than stress occurring during adulthood. Furthermore, females display greater risk of stress-related psychiatric disorders, indicating sex-specific responses to stress. In this study, male and female adolescent (42-48 days old, 4 rats per group) or adult (68-72 days old, 4 rats per group) Sprague-Dawley rats were exposed to 5 days of repeated social stress in the resident-intruder paradigm or control manipulation. We examined dendritic morphology of cells in the mPFC in both layer II/III and Layer V. Repeated social stress resulted in decreased dendritic branching in layer II/III apical dendrites regardless of sex or age. In apical layer V dendrites, stress increased branching in adult males but decreased it in all other groups. Stress resulted in a decrease in dendritic spines in layer V apical dendrites for male adolescents and female adults, and this was mostly due to a decrease in filopodial and mushroom spines for male adolescents, but stubby spines for female adults. In sum, these results suggest that repeated stress reduces complexity and synaptic connectivity in adolescents and female adults in both input and output layers of prelimbic mPFC, but not in male adults. These changes may represent a potential underlying mechanism as to why adolescents and females are more susceptible to the negative cognitive effects of repeated or chronic stress.

Keywords: Development; Golgi stain; Morphology; Prefrontal cortex; Sex; Stress.

Fig. 1

Dendritic Morphology. Dendritic branching in both apical and basal dendrites was assessed in adolescent and adult males and females in layer II/III and layer V. (* = p < 0.05, post-hoc 3-way interaction; ‡ = p < 0.05, age effect; # = p < 0.05, sex effect; † = p < 0.05, stress effect). A) Layer II/III.1. Basal Dendritic Branching. Five days of social stress decreased branching, regardless of age or sex. Furthermore, stress reduced branching in male adults. Group sizes were: n = 32 cells/4 rats for male adult controls, 25 cells/4 rats for male adult defeats, 28 cells/4 rats for male adolescent controls, 28 cells/4 rats for male adolescent defeats, 31 cells/4 rats for female adult controls, 36 cells/4 rats for female adult defeats, 25 cells/4 rats for female adolescent controls, 34 cells/4 rats for female adolescent defeats.2. Apical Dendritic Branching. Social stress decreased apical branching in adolescents regardless of sex. Group sizes were: n = 32 cells/4 rats for male adult controls, 25 cells/4 rats for male adult defeats, 28 cells/4 rats for male adolescent controls, 28 cells/4 rats for male adolescent defeats, 31 cells/4 rats for female adult controls, 36 cells/4 rats for female adult defeats, 25 cells/4 rats for female adolescent controls, 34 cells/4 rats for female adolescent defeats).* = p < 0.05.B) Layer V.1. Basal Dendritic Branching. Social stress increases dendritic branching in adult males. Group sizes were: n = 36 cells/4 rats for male adult controls, 37 cells/4 rats for male adult defeats, 34 cells/4 rats for male adolescent controls, 30 cells/4 rats for male adolescent defeats, 30 cells/4 rats for female adult controls, 30 cells/4 rats for female adult defeats, 31 cells/4 rats for female adolescent controls, 30 cells/4 rats for female adolescent defeats.2. Apical Dendritic Branching. Social stress increased branching in adult males but decreased branching in female adolescents, male adolescents, and female adults. Group sizes were: n = 36 cells/4 rats for male adult controls, 37 cells/4 rats for male adult defeats, 34 cells/4 rats for male adolescent controls, 30 cells/4 rats for male adolescent defeats, 30 cells/4 rats for female adult controls, 30 cells/4 rats for female adult defeats, 31 cells/4 rats for female adolescent controls, 30 cells/4 rats for female adolescent defeats.* = p < 0.05.

Fig. 2

Dendritic Arborization and Complexity in Adult Males. Sholl analysis was run on adult male neurons in layer II/III and layer V, using 20 μm concentric rings. Intersections of dendrites with each concentric ring were recorded. Group size = 34 cells/4 rats for controls, 30 cells/4 rats for defeats. A. Layer II/III.1. Basal Dendrites. Repeated social stress decreased the complexity as measured by number of intersections. 2. Apical Dendrites. There was no effect of stress. B. Layer V.1. Basal Dendrites. Repeated social stress increased the complexity as measured by number of intersections. 2. Apical Dendrites. Repeated social stress increased the complexity as measured by number of intersections.

Fig. 3

Total Dendritic Spine Densities. Dendritic spines were counted in 40 μm segments midway down the basal dendrites and at the distal termination, and in 50 μm segments midway and at the distal termination for apical dendrites and averaged to create a final spines/μm density for adolescent and adult males and females. (* = p < 0.05, post-hoc 3-way interaction; @ = p < 0.05, post-hoc 2-way interaction; ‡ = p < 0.05, age effect; # = p < 0.05, sex effect; † = p < 0.05, stress effect). A. Layer II/III.1. Basal Dendrites. Females had greater spine density than males regardless of age or stress. There is an interaction of Age*Sex*Stress such that stressed adolescent females have spine density greater than stressed adolescent males or stressed female adults. The group sizes were: n = 9 cells/4 rats for male adult controls, 10 cells/4 rats for stressed male adults, 9 cells/4 rats for male adolescent controls, 10 cells/4 rats for stressed male adolescents, 7 cells/4 rats for female adult controls, 14 cells/4 rats for stressed female adults, 11 cells/4 rats for female adolescent controls, 10 cells/4 rats for stressed female adolescents. 2. Apical Dendrites. There was no effect on apical dendritic spine density. The group sizes were: n = 9 cells/4 rats for male adult controls, 10 cells/4 rats for stressed male adults, 9 cells/4 rats for male adolescent controls, 10 cells/4 rats for stressed male adolescents, 7 cells/4 rats for female adult controls, 14 cells/4 rats for stressed female adults, 11 cells/4 rats for female adolescent controls, 10 cells/4 rats for stressed female adolescents]. B. Layer V.1. Basal Dendrites. Adult males had lower spine density than adult females or adolescent males regardless of stress. Adult male controls had lower spine density than adult female controls. Stress reduced dendritic spine density overall regardless of sex or age. Group sizes were: n = 10 cells/4 rats for male adult controls, 9 cells/4 rats for male adult defeats, 11 cells/4 rats for male adolescent controls, 10 cells/4 rats for male adolescent defeats, 9 cells/4 rats for female adult controls, 10 cells/4 rats for female adult defeats, 10 cells/4 rats for female adolescent controls, 11 cells/4 rats for female adolescent defeats. 2. Apical Dendrites. Stress decreased spine density overall regardless of age or sex. This was driven by adult females and adolescent males. Adult female controls had greater spine density than adolescent female controls. Group sizes were: n = 10 cells/4 rats for male adult controls, 9 cells/4 rats for stressed male adults, 11 cells/4 rats for male adolescent controls, 10 cells/4 rats for stressed male adolescents, 9 cells/4 rats for female adult controls, 10 cells/4 rats for stressed female adults, 10 cells/4 rats for female adolescent controls, 11 cells/4 rats for stressed female adolescents.

Fig. 4

Filopodial Spine Densities. Filopodial spines were counted in 40 μm segments midway down the basal dendrites and at the distal termination, and in 50 μm segments midway and at the distal termination for apical dendrites and averaged to create a final spines/μm density for adolescent and adult males and females. (* = p < 0.05, post-hoc 3-way interaction; ‡ = p < 0.05, age effect; # = p < 0.05, sex effect; † = p < 0.05, stress effect). A. Layer II/III.1. Basal Dendrites. Spine density increased with age overall regardless of stress or sex. Females had more filopodial spines than males regardless of age or stress. There was an interaction of Age*Sex*Stress such that female stressed adolescents had greater spine density than stressed male adolescents, and female adult controls had greater spine density than male adult controls and adolescent male controls. Group sizes were: n = 9 cells/4 rats for male adult controls, 10 cells/4 rats for stressed male adults, 9 cells/4 rats for male adolescent controls, 10 cells/4 rats for stressed male adolescents, 7 cells/4 rats for female adult controls, 14 cells/4 rats for stressed female adults, 11 cells/4 rats for female adolescent controls, 10 cells/4 rats for stressed female adolescents. 2. Apical Dendrites. Females had greater spine density than males regardless of age or stress. Group sizes were: n = 9 cells/4 rats for male adult controls, 10 cells/4 rats for stressed male adults, 9 cells/4 rats for male adolescent controls, 10 cells/4 rats for stressed male adolescents, 7 cells/4 rats for female adult controls, 14 cells/4 rats for stressed female adults, 11 cells/4 rats for female adolescent controls, 10 cells/4 rats for stressed female adolescents. B. Layer V.1. Basal Dendrites. There was no effect on filopodial spine density. Group sizes were: n = 10 cells/4 rats for male adult controls, 9 cells/4 rats for stressed male adults, 11 cells/4 rats for male adolescent controls, 10 cells/4 rats for stressed male adolescents, 9 cells/4 rats for female adult controls, 10 cells/4 rats for stressed female adults, 10 cells/4 rats for female adolescent controls, 11 cells/4 rats for stressed female adolescents. 2. Apical Dendrites. Male controls had greater spine density than female controls and stressed males regardless of age. Stress decreased filopodial spine density in male adolescents. Male adolescent controls have greater spine density than female adolescent controls. Group sizes were: n = 10 cells/4 rats for male adult controls, 9 cells/4 rats for stressed male adults, 11 cells/4 rats for male adolescent controls, 10 cells/4 rats for stressed male adolescents, 9 cells/4 rats for female adult controls, 10 cells/4 rats for stressed female adults, 10 cells/4 rats for female adolescent controls, 11 cells/4 rats for stressed female adolescents.

Fig. 5

Mushroom Spine Densities. Mushroom spines were counted in 40 μm segments midway down the basal dendrites and at the distal termination, and in 50 μm segments midway and at the distal termination for apical dendrites and averaged to create a final spines/μm density for adolescent and adult males and females. (* = p < 0.05, post-hoc 3-way interaction; ‡ = p < 0.05, age effect; # = p < 0.05, sex effect; † = p < 0.05, stress effect). A. Layer II/III.1. Basal Dendrites. Age decreased spine density. There was an interaction of Age*Sex*Stress such that stressed female adolescents had greater spine density than stressed male adolescents, stressed female adults, female adult controls, adult male controls and stressed adolescent males. Stress reduced spine density in male adolescents. Group sizes were: n = 9 cells/4 rats for male adult controls, 10 cells/4 rats for stressed male adults, 9 cells/4 rats for male adolescent controls, 10 cells/4 rats for stressed male adolescents, 7 cells/4 rats for female adult controls, 14 cells/4 rats for stressed female adults, 11 cells/4 rats for female adolescent controls, 10 cells/4 rats for stressed female adolescents]. 2. Apical Dendrites. There was no effect on mushroom spine density. Group sizes were: n = 9 cells/4 rats for male adult controls, 10 cells/4 rats for stressed male adults, 9 cells/4 rats for male adolescent controls, 10 cells/4 rats for stressed male adolescents, 7 cells/4 rats for female adult controls, 14 cells/4 rats for stressed female adults, 11 cells/4 rats for female adolescent controls, 10 cells/4 rats for stressed female adolescents. B. Layer V.1. Basal Dendrites. Spine density increased with age overall regardless of sex or stress. Stress decreased spine density. Group sizes were: n = 10 cells/4 rats for male adult controls, 9 cells/4 rats for stressed male adults, 11 cells/4 rats for male adolescent controls, 10 cells/4 rats for stressed male adolescents, 9 cells/4 rats for female adult controls, 10 cells/4 rats for stressed female adults, 10 cells/4 rats for female adolescent controls, 11 cells/4 rats for stressed female adolescents. 2. Apical Dendrites. Females had greater spine density than males regardless of stress or age. Stress also decreased spine density regardless of age or sex. This was driven by stress decreasing spine density in male adolescents and female adults. Adult female controls had greater spine density than adult male controls or adolescent female controls. Group sizes were: n = 10 cells/4 rats for male adult controls, 9 cells/4 rats for stressed male adults, 11 cells/4 rats for male adolescent controls, 10 cells/4 rats for stressed male adolescents, 9 cells/4 rats for female adult controls, 10 cells/4 rats for stressed female adults, 10 cells/4 rats for female adolescent controls, 11 cells/4 rats for stressed female adolescents.

Fig. 6

Stubby Spine Densities. Stubby spines were counted in 40 μm segments midway down the basal dendrites and at the distal termination, and in 50 μm segments midway and at the distal termination for apical dendrites and averaged to create a final spines/μm density for adolescent and adult males and females. (* = p < 0.05, post-hoc 3-way interaction; @ = p < 0.05, post-hoc 2-way interaction; ‡ = p < 0.05, age effect; # = p < 0.05, sex effect; † = p < 0.05, stress effect). A. Layer II/III.1. Basal Dendrites. Stress decreased spine density in female adults, and these stressed female adults had lower spine density than female stressed adolescents and male adult stressed rats. Group sizes were: n = 9 cells/4 rats for male adult controls, 10 cells/4 rats for stressed male adults, 9 cells/4 rats for male adolescent controls, 10 cells/4 rats for stressed male adolescents, 7 cells/4 rats for female adult controls, 14 cells/4 rats for stressed female adults, 11 cells/4 rats for female adolescent controls, 10 cells/4 rats for stressed female adolescents. 2. Apical Dendrites. There was no effect on stubby spine density. Group sizes were: n = 9 cells/4 rats for male adult controls, 10 cells/4 rats for stressed male adults, 9 cells/4 rats for male adolescent controls, 10 cells/4 rats for stressed male adolescents, 7 cells/4 rats for female adult controls, 14 cells/4 rats for stressed female adults, 11 cells/4 rats for female adolescent controls, 10 cells/4 rats for stressed female adolescents. B. Layer V.1. Basal Dendrites. Overall, males have greater spine density than females regardless of age or stress. Stress decreases spine density regardless of sex or age. Group sizes were: n = 10 cells/4 rats for male adult controls, 9 cells/4 rats for male adult defeats, 11 cells/4 rats for male adolescent controls, 10 cells/4 rats for stressed male adolescents, 9 cells/4 rats for female adult controls, 10 cells/4 rats for stressed female adults, 10 cells/4 rats for female adolescent controls, 11 cells/4 rats for stressed female adolescents. 2. Apical Dendrites. Stress decreased spine density overall and this was driven by significant effects of stress in adult males and females but not in adolescents. Stress decreases spine density in adults. Group sizes were: n = 10 cells/4 rats for male adult controls, 9 cells/4 rats for stressed male adults, 11 cells/4 rats for male adolescent controls, 10 cells/4 rats for stressed male adolescents, 9 cells/4 rats for female adult controls, 10 cells/4 rats for stressed female adults, 10 cells/4 rats for female adolescent controls, 11 cells/4 rats for stressed female adolescents.