A 2-way ANOVA revealed significant effects of both estrogen and stress on percent weight gain (OVX + veh control vs. stress: 21.2 ± 1.96% vs. 13.6 ± 1.46%; OVX + E control vs. stress: 8.33 ± 1.69% vs. 2.08 ± 0.93%. Effect of estrogen: F1,23 = 67.28, P < 0.0001; effect of stress: F1,23 = 21.56, P = 0.0001; ).
Dendritic Parameters: “Unlabeled” Neurons
Mean branch points, overall apical dendritic length, and Sholl analyses are shown for OVX + veh and OVX + E groups in , respectively. Two-way ANOVAs revealed no significant effects of stress or estrogen on unlabeled neurons’ branch points or on total apical dendritic length, nor were there any estrogen–stress interactions. Bonferroni post hoc tests did not reveal any localized effects of stress at any distance from the soma in either group. N = 8, 7 and 8, 7 for OVX + veh control, stress, and OVX + E control, stress, respectively.
Dendritic Parameters: BLA-Projecting Neurons
Images of representative Neurons and Neurolucida tracings of BLA-projecting IL neurons from the OVX + E control and stress groups are shown in , respectively. Mean branch points, overall apical dendritic length, and Sholl analyses are shown for OVX + veh and OVX + E groups in , respectively. Two-way ANOVAs of total branch points did not reveal a significant interaction of estrogen and stress, but Bonferroni post hoc tests showed a significant stress-related increase in branch points in OVX + E group only (control vs. stress: 15.4 ± 0.58 vs. 18.1 ± 0.93, P < 0.02). Two-way ANOVAs of total apical dendritic length did not reveal a significant interaction of estrogen and stress. However, post hoc tests revealed a near-significant effect of stress in OVX + E group only (control vs. stress: 1697.0 ± 52.9 vs. 1921.5 ± 105.0, P = 0.06), with stress causing a 13% increase in overall apical dendritic length in this group. Sholl analysis revealed no localized effects of stress for OVX + veh group, but OVX + E group showed a significant stress × distance interaction (F1,11 = 1.935, P = 0.04), with Bonferroni post hoc tests revealing significant effects of stress at 120 and 150 μm from the soma (control vs. stress: 283.9 ± 14.8 μm vs. 351.4 ± 16.8 μm, P < 0.05; 220.7 ± 7.5 μm vs. 284.2 ± 34.4 μm, P < 0.05). N = 7, 5, 7, and 6 for OVX + veh control, stress, and OVX + E control, stress, respectively.
Visualization and analysis of neurons. Representative Lucifer Yellow-filled BLA-projecting neurons and corresponding Neurolucida tracings from OVX + E stressed (A and B) and control (C and D) animals. Scale bar = 50 μm.
Representative deconvolved images of dendrite segments from OVX + veh control and stress groups are shown in , respectively. In unlabeled neurons, a mixed model F-test revealed an overall interaction of stress and estrogen on spine density (OVX + veh control vs. stress: 1.29 ± 0.13 vs. 1.84 ± 0.13 spines per micrometer, OVX + E control vs. stress: 1.48 ± 0.15 vs. 1.41 ± 0.19 spines per micrometer; F1,9 = 7.18; P < 0.03), with stress causing a 42% increase in spine density in OVX + veh group (post hoc test, P < 0.01) but no change in OVX + E group ().
In BLA-projecting neurons (), F-test revealed significant effects of both estrogen and stress on spine density but no interaction (OVX + veh control vs. stress: 1.31 ± 0.16 vs. 2.20 ± 0.15 spines per micrometer, OVX + E control vs. stress: 1.71 ± 0.16 vs. 2.19 ± −0.08 spines per micrometer. Effect of estrogen, F1,8 = 5.52, P < 0.05; effect of stress, F1,8 = 10.56, P = 0.01). Post hoc tests revealed significant effects of stress in both OVX + veh (69% increase in spine density, P < 0.001) and OVX + E (29% increase in spine density, P < 0.05) groups as well as a significant effect of estrogen in control animals (31% increase in spine density, P < 0.05).
In control animals, no differences were seen between BLA-projecting and unlabeled neurons in any of the parameters (F1,64
= 0.005, P
= 0.942), suggesting that there are no underlying baseline morphological differences between these 2 neuron populations. A summary of all findings combined with recent findings in male rats (Shansky et al. 2009
) is shown in .
Summary of morphological effects of stress effects in IL neurons in males and OVX + veh and OVX + E animals
The neurons used in the spine analysis represent a subset of the total neurons used for the dendritic arbor analyses, and thus, there is a smaller number (3–4 animals per group). However, the results are in many cases highly significant.