Male mice were 5–7 weeks old. Wild type (wt) C57BL/6 mice were purchased from Charles River Laboratories (Kingston, NY). Transgenic mice expressing thy1
-derived GFP (Feng et al., 2000
) enabled visualization of dendritic spines. Our final experiment utilized GFP-expressing p190RhoGAP-deficient mice (p190rhogap
+/−), which have ~32–40% less p190RhoGAP protein (Brouns et al., 2000
). All were bred on a C57BL/6 background, maintained on a 12-hour light cycle (0700 on), and provided food and water ad libitum
unless otherwise indicated. Procedures were Yale and Emory IACUC-approved.
CORT (4-pregnen-11β-21-DIOL-3-20-DIONE-21-hemisuccinate; Steraloids, Newport, RI) was dissolved in water and administered for 20 days (25 µg/ml free-base, translating to ~4.97 mg/kg/day). This protocol recapitulates blood CORT levels in mice exposed to chronic restraint stress (Gourley et al., 2008
). Mice were euthanized at 20 days or 20 days + a 1-week washout period.
In a final experiment using GFP-expressing p190rhograp+/− and p190rhograp+/+ mice, 10 µg/ml was used as a subthreshold CORT concentration. This dose is described in text as a “subthreshold CORT.”
Biocytin injection of hippocampal neurons
Wild type mice were deeply anaesthetized with pentobarbital, and as previously described (Sfakianos et al., 2007
), hippocampal slices (400 µm) were prepared and maintained in a standard interface chamber at 33°C. Individual CA1 pyramidal neurons were injected with 4% biocytin solution in 2M sodium acetate solution, pH 7.5. Neurons were injected with 100–300 ms current injections of 5 nA at 1 Hz for 20 min. Only neurons that maintained a membrane potential and fired action potentials during this entire period were analyzed. After 10 min of recovery, injected neurons were fixed in 4% paraformaldehyde overnight, cryoprotected in 30% sucrose, then resectioned at 40 µm, and visualized using standard avidin-horseradish peroxidase (HRP) staining (Vectastain Elite ABC; Vector Laboratories, Burlingame, CA).
Morphometric analysis of dendrites
Z-stack series of individual biocytin-labeled neurons were considered complete only when clean dendrite-free sections were detectable on the far +Z and −Z margins. The 5–8 serial sections containing dye-filled neurons were traced sequentially starting at the cell body and moving in + and – directions under 100X magnification using a light microscope with a Z drive. Cells were then reconstructed using Neurolucida software (MicroBrightField, Williston, VT). As is standard practice, sections were apposed using landmarks and aligned at high magnification by joining interrupted primary and secondary branches based on position, orientation, and dendrite thickness. Sholl analysis, total dendrite length, and branch point number were determined using NeuroExplorer (MicroBrightField). Neurons were traced by an experimenter blind to group. 2–4 neurons were sampled from each mouse; each mouse (n=9–11/group) was considered an independent sample.
Dendritic spine capture and enumeration
As described (Gourley et al., 2012
), fresh GFP-expressing brains were submerged in 4% paraformaldehyde for 48 hours, then transferred to 30% w/v sucrose, followed by sectioning into 40 µm-thick sections on a microtome held at −15°C. Unobstructed dendritic segments running parallel to the surface of the section were imaged on a spinning disk confocal (VisiTech International, Sunderland, UK) on a Leica microscope. Z-stacks were taken with a 100x 1.4NA objective using a 0.1 µm step size, sampling above and below the dendrite. After imaging, we confirmed at 10X that the image was collected from the intended subregions.
Collapsed z-stacks were analyzed using NIH ImageJ: Each protrusion ≤4 µm was considered a spine (Peters and Kaiserman-Abramof, 1970
). Individual planes were evaluated to detect protrusions perpendicular to the z-stack. Bifurcated spines were considered singular units. To generate density values, spine number for each segment was normalized to the length of the segment.
From cortical and amygdalar neurons, 6–8 independent segments from secondary and tertiary dendritic branches within 50–150 µm of the soma were collected. Each group contained 6–8 mice, with each animal contributing a single density value to statistical analyses. Due to the stellate appearance of amygdalar and oPFC neurons (Liston et al., 2006
; Kolb et al., 2008
), apical vs
. basal branches were not distinguished. In the ILc, apical branches were evaluated.
In the hippocampus, CA1 neuronal alignment allows for unambiguous sampling as a function of distance from the soma. Thus, 6–8 independent basal neurons/mouse were identified, and 1 dendrite/neuron/30-µm window radiating from the soma was sampled. A single blinded rater scored spines.
Semi-automated dendritic spine reconstruction
To evaluate both spine density and head diameter in GFP-expressing p190rhogap
+/− samples, 3D reconstructions were accomplished with the FilamentTracer module of Imaris (Bitplane AG, Zurich, Switzerland) as described (Swanger et al., 2011
). A dendritic segment ~25 µm in length sampled from the oPFC (as above) or basal CA1 (60–90 µm from the soma) was drawn using the AutoDepth function. FilamentTracer processing algorithms centered the segment and determined dendrite diameter. The autodepth function drew dendritic spines along the dendrite. Each spine was then reconstructed in 3D using the FilamentTracer algorithm. A single blinded rater processed all images.
We utilized a model of anhedonia: 1% (w/v) sucrose replaced regular drinking water for 2 days starting 2 days after CORT exposure. Animals were then habituated to water restriction by removing the water bottle for 19 hours. Next, mice were again water-restricted overnight, and each mouse was allowed 1-hour access to the sucrose solution in its home cage while cagemates were housed in a clean cage in a quiet room. Liquid consumption was recorded, and the next mouse was tested. This approach allows us to evaluate sucrose consumption in the home cage in individual mice while still maintaining standard laboratory group housing (2–5 mice/cage) (Gourley et al., 2008
). The average water restriction period for each cage was 16 hours. The test was repeated the following day with water to confirm that general fluid consumption did not differ between groups.
In the first experiment (), mice were wild type (control vs. CORT, n=6/group), in the second (), GFP-expressing p190rhogap+/− and GFP-expressing littermate controls exposed to exogenous CORT or CORT-naïve (4 groups, n=7–14/group depending on litter composition). These mice were euthanized after test for dendritic spine capture.
Chronic corticosteroid exposure has regionally-selective effects on dendritic spine density
p190RhoGAP determines the oPFC response to CORT
Sucrose consumption and morphometric measures were analyzed by 1- or 2-factor ANOVA as appropriate, with repeated measures when values were analyzed as a function of distance from the soma. Post-hoc comparisons were made using Tukey’s t-tests, and when significant, results are indicated graphically. When two groups were compared, 2-tailed t-tests were used. To highlight whether genotype determined dendritic spine sensitivity to CORT, percent change from baseline (meaning the mean value of CORT-naïve mice of the same genotype) was calculated and compared to 0 (no change) by location t-test. p<0.05 was considered significant, and outliers were excluded.
Spine head diameters were analyzed by Kolmogorov-Smirnov (K-S) comparisons. Because of the high degree of statistical power generated by K-S tests, only p<0.001 was considered significant.