Increased vulnerability of the brain norepinephrine system of females to corticotropin-releasing factor overexpression

Abstract

Stress-related psychiatric disorders are more prevalent in women than men. As hypersecretion of the stress neuromediator, corticotropin-releasing factor (CRF) has been implicated in these disorders, sex differences in CRF sensitivity could underlie this disparity. Hyperarousal is a core symptom that is shared by stress-related disorders and this has been attributed to CRF regulation of the locus ceruleus (LC)-norepinephrine arousal system. We recently identified sex differences in CRF(1) receptor (CRF(1)) signaling and trafficking that render LC neurons of female rats more sensitive to CRF and potentially less able to adapt to excess CRF compared with male rats. The present study used a genetic model of CRF overexpression to test the hypothesis that females would be more vulnerable to LC dysregulation by conditions of excess CRF. In both male and female CRF overexpressing (CRF-OE) mice, the LC was more densely innervated by CRF compared with wild-type controls. Despite the equally dense CRF innervation of the LC in male and female CRF-OE mice, LC discharge rates recorded in slices in vitro were selectively elevated in female CRF-OE mice. Immunoelectron microscopy revealed that this sex difference resulted from differential CRF(1) trafficking. In male CRF-OE mice, CRF(1) immunolabeling was prominent in the cytoplasm of LC neurons, indicative of internalization, a process that would protect cells from excessive CRF. However, in female CRF-OE mice, CRF(1) labeling was more prominent on the plasma membrane, suggesting that the compensatory response of internalization was compromised. Together, the findings suggest that the LC-norepinephrine system of females will be particularly affected by conditions resulting in elevated CRF because of differences in receptor trafficking. As excessive LC activation has been implicated in the arousal components of stress-related psychiatric disorders, this may be a cellular mechanism that contributes to the increased incidence of these disorders in females.

Figure 1

CRF immunoreactivity in the LC region of CRF-OE and WT mice. (A) Photomicrographs of 30 µm coronal sections through the LC showing TH immunoreactive cell bodies of the LC (green) and CRF immunoreactive fibers (red). Sections from the WT mice (left panels) and CRF-OE mice (right panels) were photographed using the same exposure (50 ms). Dorsal is at top and medial is to the right. Scale bars=100 µm. (B,C) Bars show the mean optical density of CRF immunoreactivity in the core of the LC (B) or the dorsolateral peri-LC region (C) for the different groups. CRF immunoreactivity was denser in both the core and peri-LC in CRF-OE mice compared to WT mice as revealed by two main effects of genotype [F(1,12)= 15.5, p<0.01, F(1,12)= 25.9, p<0.001, respectively]. There were no significant effects of sex or interactions. Bars represent the mean (± SEM) of 3–5 mice/group.

Figure 2

Sex and genotype differences in electrophysiological properties of LC neurons. (A) Bars represent the mean firing rate (Hz) of LC neurons (n=12–16 cells/group). There was a significant sex by genotype interaction for firing rate [F(1,50)= 6.0, p<0.05]. Post-hocs revealed that neurons of female CRF-OE mice fired faster than all other groups (p<0.05). (B) The plot displays the number of APs elicited by increasing depolarizing current injections (n=12–16 cells/group). There was a significant sex by genotype by depolarizing current interaction [F(3,150)=2.7, p<0.05] and a significant sex by genotype interaction [F(1,50)=5.5, p<0.05]. Post-hocs revealed that male WT mice fired fewer APs than any other group (p<0.05), indicating that their cells were less excitable. Data are represented as the mean (± SEM). Asterisks indicate a significant difference from all other groups (p<0.05).

Figure 3

Sex and genotype differences in CRF₁ compartmentalization. (A) Electron photomicrographs of LC dendrites (d) from a male WT, male CRF-OE, female WT and female CRF-OE showing CRF immunogold labeling. CRF-immunogold labeling is seen on the plasma membrane (arrowheads) in male WT mice and female CRF-OE mice whereas it is in the cytoplasm in male CRF-OE mice and female WT mice. t=axon terminal. Scale bars=500 nm. (B) Bar graph comparing the mean ratio of immunolabeled gold particles localized within cytoplasm to the total number (percent internalized). There was a significant sex by genotype interaction [F(1,16)=144.9, p<0.001]. Post-hoc tests revealed that female WT mice had a significantly greater percentage of cytoplasmic CRF₁ labeling than male WT mice. However, the opposite effect was observed in CRF-OE mice where CRF₁ labeling is more prominent in the cytoplasm of male compared to female CRF-OE mice (p<0.001). Data are represented as the mean (± SEM) of 5 mice/group.

Figure 4

Sex and genotype differences in LC dendritic morphology. (A) Fluorescent photomicrograph of a biocytin labeled LC neuron (green) merged with TH labeling (red) from a male CRF-OE mouse. (B) Neurolucida tracing of the cell shown in A. (C) Bars represent the mean total dendritic length. There was a significant sex by genotype interaction for total dendrite length [F(1,69)=4.5, p<0.05]. Post-hoc tests revealed that there was a trend for male WT mice to have shorter dendrites than all other groups (#p< 0.09). (D) Bars depict the mean number of nodes (i.e., branchpoints). There was a significant sex by genotype interaction for the number of nodes [F(1,69)=9.2, p<0.01]. Female WT and male OE mice had significantly more nodes than male WT mice (*p<0.05). There was a trend for more nodes in female OE mice than male WT mice (p=0.06). (E) Bars show the mean number of ends. There was a significant sex by genotype interaction for end number [F(1,69)=8.4, p<0.01]. Compared to WT males, CRF-OE males had more ends (*p<0.01). There was a trend for female WT and OE mice to also have more ends than WT males (p=0.053, p=0.07, respectively). (F) The graph represents the number of branches broken down by branch order (i.e., 1st, 2nd, 3rd, 4th, and 5th). Although the sex by genotype by branch order interaction failed to reach significance [F(4,276)=2.2, p=0.07], there was a significant sex by genotype interaction [F(1,69)=7.8, p<0.01]. Male WT mice had fewer branches than any other group [F(1,69)=7.8, p<0.01, post hoc p<0.05]. Data are represented as the mean (± SEM) of 15–20 cells/group.