Fig. 1: PrL GABAergic projection neurons target mainly L1 in the Cg1/2.
a Schematic of the injection for anterograde tracing. AAV1-CAG-Flox-eGFP-WPR6 and AAV1-CAMKIIa-hChR2(h134a)-mCherry were injected into the PrL of GADCre mice. b Schematic of the injection site into the PrL and c the projection site in the Cg1/2 in a coronal section. d Representative confocal image of Cre-dependent eGFP (green) and CAMKII-promoter dependent mCherry (magenta) expression at the injection site in the PrL in a DAPI stained (blue) coronal section. e Cre-dependent eGFP expression in GABAergic neurons. f mCherry expression in excitatory neurons. g Representative confocal image of a DAPI stained coronal section showing eGFP and mCherry at the projection site, i.e., the Cg1/2. h PrL GABAergic neuron-derived GFP expressing axons in the Cg1/2. i PrL excitatory neuron-derived mCherry-expressing axons in the Cg1/2. j–l Magnified view of the boxed area in (g). m Density of GABAergic axons (L1 vs. L2/3, p = 0.0166, L1 vs. L5, p = 0.0122, L1 vs. L6, p = 0.0297, DF = 8, two-sided Dunnett’s test) and n excitatory axons in the indicated layers of the Cg1/2 (L1 vs. L2/3, p = 0.974, L1 vs. L5, p = 0.996, L1 vs. L6, p = 0.932, DF = 8, two-sided Dunnett’s test). Open circles represent mean values from individual mice. o GABAergic and excitatory axons calculated as percentage of total axons in L1a (p = 0.000216, DF = 4, two-sided t-test). Red asterisks and n.s. above bars refer to a one-sample t-test of the percentage of GABAergic or excitatory neuron axons in L1a versus 50% (GABAergic, p = 0.00337, DF = 2, glutamatergic, p = 0.0783, DF = 2). d–o Images and data were obtained from nine slices from three mice. m–o Data are presented as mean ± S.E.M. Scale bar in (d–f), 500 μm, in (g–i), 200 μm, in (j–l), 50 μm. PrL prelimbic cortex, Cg1/2 cingulate area 1 and 2, eGFP enhanced green fluorescent protein, DAPI 4’,6-diamidino-2-phenylindole. n.s. not significant, *p < 0.05, **p < 0.01, ***p < 0.001, DF degree of freedom.
