Results: 4

1.
Figure 1

Figure 1. From: Sex Differences in Corticotropin-Releasing Factor Receptor Signaling and Trafficking: Potential Role in Female Vulnerability to Stress-Related Psychopathology.

The role of cAMP signaling in the sex- and stress-related differences in LC neuronal activation by CRF. (A-D) LC activation by local CRF after pretreatment with Rp-cAMP-S (600 ng in 120 nl, intra-LC) or ACSF (120 nl) is shown for unstressed male (n=5-8) and female rats (n=4-6) and male (n=6-8) and female rats 24 h after swim stress (n=4-6). Bars depict the average response to CRF following ACSF (black) or Rp-cAMP-S (dark gray). Light gray bars represent the cAMP-mediated component (calculated by taking the difference between the vehicle and Rp-cAMP-S treated groups). In the unstressed state, a relatively low CRF dose activated LC neurons in females only [t(6)=3.56, p<0.05], and this response was completely cAMP-dependent. For both males and females, neuronal responses to the higher dose (30 ng) were mediated by cAMP-dependent and independent processes [F(1,26)=12.88, p<0.05] . Swim stress changed the cAMP signaling profile in males {stress×dose×drug interaction [F(1,46)=4.45, p<0.05]}, but not in females [F(1,39)=1.87, p>0.05].

Debra A. Bangasser, et al. Mol Psychiatry. ;15(9):877-904.
2.
Figure 3

Figure 3. From: Sex Differences in Corticotropin-Releasing Factor Receptor Signaling and Trafficking: Potential Role in Female Vulnerability to Stress-Related Psychopathology.

Sex differences in proteins involved in CRFr internalization processes. (A) Blots represent the phosphothreonine band (red), CRFr band (green) and the merged image (yellow) indicating that both label the same protein (i.e., phosphorylated CRFr , MW=52 kDa). Protein for this blot was collected 24 h after stressor exposure. (B1-3) Bar graphs show the mean ratio of phosphothreonine:CRFr for each condition from tissue collected immediately [F(3,12)=0.39, p>0.05], 1 h [F(3,20)=0.58, p>0.05]or 24 h [F(3,20)=0.66, p>0.05] post-stress (n=4-6 determinations, pooled 3 rats per determination). (C) The Western blot shows the β-arrestin2 band (MW = 54 kDa) and the CRFr band 24 h after stressor exposure or handling. (D1-3) Graphs illustrate the ratio of β–arrestin2:CRFr for rats sacrificed immediately [F(3,12)=0.52, p>0.05], 1 h [F(3,16)=4.74, p<0.05] or 24 h post-stress [F(5,30)=5.77, p<0.05] (n=4-6 determinations, pooled 3 rats per determination). At both 1 and 24 h after stress, β–arrestin2 association with the CRFr was significantly increased in males (p<0.05) but not in females (p>0.05). Cycling females were included for an additional comparison at the 24 h timepoint, and there was no statistically significant difference in CRFr-β-arrestin2 association between ovariectomized and cycling females in either the unstressed or stressed condition (p>0.05). Data are represented as the mean (±SEM). Asterisks indicate a significant effect of stress compared to unstressed same sex control (p<0.05).

Debra A. Bangasser, et al. Mol Psychiatry. ;15(9):877-904.
3.
Figure 2

Figure 2. From: Sex Differences in Corticotropin-Releasing Factor Receptor Signaling and Trafficking: Potential Role in Female Vulnerability to Stress-Related Psychopathology.

Sex- and stress-related differences in CRFr association with different G proteins. (A-C) Representative blots of immunoprecipitated CRFr (green, MW=52kD) from different groups and (A) the Gs protein (red, MW=48kDa), (B) the Go protein (red, MW=40kDa) and (C) the Gq/11 protein (red, MW=40kDa). So that the presentation in A, matched that in B and C, a lane containing the molecular weight marker that was between female and male samples on the same gel was deleted and the image of male samples that were to the right of this lane were moved to the left of female samples. (D-F) Graphs show the mean ratio of the integrated intensity of each band of G proteins to the corresponding band of CRFr from the same samples (n=4-6 determinations, pooled 3 rats per determination). CRFr-Gs coupling was greater in unstressed ovariectomized and intact females compared to unstressed males [F(5,26)=2.56, p<0.05, post-hocs p<0.05]. Stress increased coupling in males (p<0.05) to a level comparable to that of unstressed females (p>0.05), but had no further effect on females (regardless of hormonal status; p>0.05). There were no significant differences in coupling of the CRFr to Go [F(3,20)=0.55, p>0.05] or Gq/11 [F(3,12)=0.55, p>0.05] (top band quantified). Data are represented as the mean (±SEM). Number sign indicates sex difference under basal (unstressed) conditions (i.e., greater coupling in unstressed females vs. unstressed males; p<0.05). Asterisks indicate a significant stress-induced increase compared to the unstressed same sex control (p<0.05).

Debra A. Bangasser, et al. Mol Psychiatry. ;15(9):877-904.
4.
Figure 4

Figure 4. From: Sex Differences in Corticotropin-Releasing Factor Receptor Signaling and Trafficking: Potential Role in Female Vulnerability to Stress-Related Psychopathology.

Electron microscopic visualization of CRFr compartmentalization and stress-induced trafficking in LC dendrites. A-C are electron photomicrographs of sections through the LC. (A1) LC dendritic profile (d) in an unstressed male rat with immunogold-silver labeling for the CRFr along the plasma membrane (arrowheads). The dendrite receives synaptic contacts from axon terminals (t). (A2) Dendrite from a male rat 24 h following swim stress. CRFr labeling shifts from the plasma membrane to the cytoplasm. (B1) Dendrite from an unstressed female rat shows that CRFr is prominent in the cytoplasm. (B2) Dendrite from a female rat 24 h following swim stress shows that CRFr labeling shifts from the cytoplasm to the plasma membrane. (C1-2) TH-immunoperoxidase-labeled dendrites containing immunogold-silver labeling for CRFr (CRFr+TH) in a female control (C1) and a stressed rat (C2). Arrowheads point to CRFr on the plasma membrane in C2. Arrows point to immunoperoxidase reaction product. (D) Bar graph indicating the percentage of internalized receptors for each condition (n=3, mean per rat generated from at least 125 dendritic profiles). Unstressed females had a significantly greater percentage of cytoplasmic receptors than unstressed males [F(1,8)=45.3, p<0.05, post-hoc, p<0.05]. Swim stress increased the percentage of CRFr in cytoplasm in males rats (p<0.05). In contrast, swim stress decreased the percentage of cytoplasmic CRFr in females (p<0.05). Data are represented as the mean (± SEM). Number sign indicates sex difference under unstressed conditions (p<0.05). Asterisks indicate a significant effect of stress compared to the unstressed same sex control (p<0.05). Scale bars=500 nm (A-C).

Debra A. Bangasser, et al. Mol Psychiatry. ;15(9):877-904.

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