Species and Sex Differences in the Morphogenic Response of Primary Rodent Neurons to 3,3'-Dichlorobiphenyl (PCB 11)

Abstract

PCB 11 is an emerging global pollutant that we recently showed promotes axonal and dendritic growth in primary rat neuronal cell cultures. Here, we address the influence of sex and species on neuronal responses to PCB 11. Neuronal morphology was quantified in sex-specific primary hippocampal and cortical neuron-glia co-cultures derived from neonatal C57BL/6J mice and Sprague Dawley rats exposed for 48 h to vehicle (0.1% DMSO) or PCB 11 at concentrations ranging from 1 fM to 1 nM. Total axonal length was quantified in tau-1 immunoreactive neurons at day in vitro (DIV) 2; dendritic arborization was assessed by Sholl analysis at DIV 9 in neurons transfected with MAP2B-FusRed. In mouse cultures, PCB 11 enhanced dendritic arborization in female, but not male, hippocampal neurons and male, but not female, cortical neurons. In rat cultures, PCB 11 promoted dendritic arborization in male and female hippocampal and cortical neurons. PCB 11 also increased axonal growth in mouse and rat neurons of both sexes and neuronal cell types. These data demonstrate that PCB 11 exerts sex-specific effects on neuronal morphogenesis that vary depending on species, neurite type, and neuronal cell type. These findings have significant implications for risk assessment of this emerging developmental neurotoxicant.

Keywords: PCB 11; axons; dendrites; developmental neurotoxicity; in vitro; neuronal morphogenesis; sex bias.

Figure 1

PCB 11 increases dendritic arborization in female, but not male, mouse hippocampal neurons. (A) Representative photomicrographs of DIV 9 FusRed+ hippocampal neurons in male and female mouse neuron-glia cocultures exposed to vehicle or 1 pM PCB 11 for 48 h. (B) Sholl plot illustrating sex differences in dendritic arborization in vehicle control mouse hippocampal neurons. Dendritic arborization in male (C,D) and female (E,F) mouse hippocampal neurons exposed to vehicle (0.1% DMSO) or varying concentrations of PCB 11 as quantified by Sholl analysis of dendritic complexity (C,E) and the number of dendritic tips per neuron (D,F). Data are presented as mean ± SE (n > 60 neurons from 3–4 independent dissections). * Significantly different from vehicle control at p < 0.05, ** p < 0.01, as determined using a nonparametric one-way ANOVA (p < 0.05) followed by Dunnett’s post hoc test.

Figure 2

PCB 11 enhances dendritic arborization in male, but not female, mouse cortical neurons. (A) Representative photomicrographs of DIV 9 FusRed+ cortical neurons in male and female mouse neuron-glia cocultures exposed to vehicle or 1 nM PCB 11 for 48 h. (B) Sholl plot illustrating sex differences in dendritic arborization in vehicle control mouse cortical neurons. Dendritic arborization in male (C,D) and female (E,F) mouse hippocampal neurons exposed to vehicle (0.1% DMSO) or varying concentrations of PCB 11 as quantified by Sholl analysis of dendritic complexity (C,E) and the number of dendritic tips per neuron (D,F). Data are presented as mean ± SE (n > 80 neurons from 3–4 independent dissections). * Significantly different from vehicle control at p < 0.05, ** p < 0.01, as determined using a nonparametric one-way ANOVA (p < 0.05) followed by Dunnett’s post hoc test.

Figure 3

PCB 11 enhances dendritic arborization in both male and female rat hippocampal neurons. (A) Representative photomicrographs of DIV 9 FusRed+ hippocampal neurons in male and female rat neuron-glia cocultures exposed to vehicle or 1 nM PCB 11 for 48 h. (B) Sholl plot illustrating lack of sex differences in dendritic arborization in vehicle control rat hippocampal neurons. Dendritic arborization in male (C,D) and female (E,F) rat hippocampal neurons exposed to vehicle or varying concentrations of PCB 11 as quantified by Sholl analysis (C,E) and the number of dendritic tips (D,F). Data are presented as mean ± SE (n > 100 neurons from three–four independent dissections). * Significantly different from vehicle control at p < 0.05, *** p < 0.001 as determined using a nonparametric one-way ANOVA (p < 0.05) followed by Dunnett’s post hoc test.

Figure 4

PCB 11-induced dendritic arborization in rat cortical neurons is not sex-specific. (A) Representative photomicrographs of DIV 9 FusRed+ cortical neurons in male and female rat neuron-glia cocultures exposed to vehicle or 1 nM PCB 11 for 48 h. (B) Sholl plot illustrating lack of sex differences in dendritic arborization in vehicle control rat cortical neurons. Dendritic arborization in male (C,D) and female (E,F) rat cortical neurons exposed to vehicle or varying concentrations of PCB 11 as quantified by Sholl analysis (C,E) and the number of dendritic tips (D,F). Data are presented as mean ± SE (n > 100 neurons from three–four independent dissections). * Significantly different from vehicle control at p < 0.05, ** p < 0.01, *** p < 0.001 as determined using a nonparametric one-way ANOVA (p < 0.05) followed by Dunnett’s post hoc test.

Figure 5

PCB 11 increases axonal growth in mouse neurons regardless of sex or cell type. (A,B) Representative photomicrographs of DIV 2 hippocampal neurons in male and female mouse neuron-glia cocultures exposed to vehicle or 1 pM PCB 11 for 48 h. Axonal length was quantified in tau-1 immunopositive male (C,D) and female (E,F) hippocampal (C,E) and cortical (D,F) mouse neurons exposed to vehicle or varying concentrations of PCB 11. Data are presented as mean ± SE (n = 90–100 neurons from three independent dissections). * Significantly different from vehicle control at p < 0.05, ** p < 0.01, *** p < 0.001, as determined using a nonparametric one-way ANOVA (p < 0.05) followed by Dunnett’s post hoc test.

Figure 6

PCB 11 increases axonal growth in rat neurons regardless of sex or cell type. Representative photomicrographs of DIV 2 hippocampal neurons in male (A) and female (B) rat neuron-glia cocultures exposed to vehicle or 1 pM PCB 11 for 48 h. Axonal length was quantified in male (C,D) and female (E,F) hippocampal (C,E) and cortical (D,F) rat neurons exposed to vehicle or varying concentrations of PCB 11. Data are presented as mean ± SE (n = 90–100 neurons from three independent dissections). * Significantly different from vehicle control at p < 0.05, *** p < 0.001, as determined using a nonparametric one-way ANOVA (p < 0.05) followed by Dunnett’s post hoc test.