Figure 3. Adolescent thalamic inhibition leads to a persistent reduction in the density of thalamo-prefrontal, but not BLA-prefrontal, projections.
(a) Experimental timeline and schematic. At P70, a retrograde tracer, GFP, was injected into the mPFC, before perfusion 3 weeks later. Created with Biorender.com. (b) Representative confocal images illustrating GFP staining in the mPFC (left), thalamus (middle), and basolateral amygdala (BLA, right) in control (top) and hM4D (bottom) animals. Outlines were determined using DAPI staining. For each animal, 6 slices were used for each of the 3 regions. Control: n=6 animals, hM4D: n=7 animals. (c) Stereology was conducted using DAPI staining for outlines of regions and GFP staining for cell counting. Quantification of GFP-positive cell density showed a significant decrease in thalamo-mPFC projecting cells in adolescent-inhibited hM4D animals compared to controls (top, Control: n=6 animals, 453.2±61.3 cells/mm2, hM4D: n=7 animals, 260.3±56.1 cells/mm2; two-sided unpaired t-test, t=2.326, df=11, *p=0.0401). Stereological estimates showed no difference in overall thalamic area (bottom, Control: 5.539±0.232 mm2, hM4D: 5.429±0.178 mm2; two-sided unpaired t-test, t=0.3834, df=11, p=0.7087). (d) Stereology in the BLA showed no differences in either GFP-positive cell density (top, Control: 602.4±61.1 cells/mm2, hM4D: 578.5±61.0 cells/mm2; two-sided unpaired t-test, t=0.2749, df=11, p=0.7885) or BLA area (bottom, Control: 5.687±0.211 mm2, hM4D: 5.432±0.163 mm2; two-sided unpaired t-test, t=0.9713, df=11, p=0.3523). (e) The ratio of thalamic to BLA projection cell densities showed a significant reduction in adolescent-inhibited hM4D animals compared to controls (top, Control: 0.742±0.065, hM4D: 0.467±0.091; two-sided unpaired t-test, t=2.376, df=11, *p=0.0368) but no change in region area (bottom, Control: 0.981±0.055, hM4D: 1.006±0.049; two-sided unpaired t-test, t=0.3471, df=11, p=0.7351). Dots represent individual animals; lines represent mean ± SEM. *p<0.05