Figure 3.
Inhibitory inputs on the perisomatic region of pyramidal cells projecting to different brain regions. A, Schematic representation of the experimental procedure. Retrograde tracers were injected into the BA, cPFC, DS, and PAG, respectively. After 4–8 d of tracer injections, PFC-containing sections were prepared and immunolabeled. B, Multicolor confocal images taken from the mPFC showing the distribution of retrogradely labeled cells (magenta) in distinct layers. Scale bar, 100 µm. C, Layer-specific distribution of the somata of pyramidal neurons targeting distinct brain regions. Number of the quantified retrogradely labeled cells is as follows: BA, n = 240 in 6 slices from 2 mice; cPFC, n = 1249 in 9 slices from 3 mice; DS, n = 2158 in 9 slices from 3 mice; PAG, n = 70 in 6 slices from 2 mice. D, Multicolor maximum z-intensity projection confocal images taken from a section immunostained for Kv2.1, FG, CB1, and PV to visualize the perisomatic CB1 and PV inputs (arrows) on FG-labeled neurons. Scale bar, 5 µm. E, PV bouton density on the perisomatic surface of pyramidal neurons projecting to distinct brain regions. Red dots indicate cells located in layer (L)2/3, gray dots represent pyramidal cells located in L5a, and purple dots show pyramidal cells located in L5b. Each dot represents the PV bouton density on single pyramidal neurons. Black lines indicate means. F, CB1 bouton density on the perisomatic membrane surface of pyramidal cells projecting to distinct brain regions. Colors, lines, dots, and numbers are the same as in E. G, Perisomatic PV/CB1 ratio on pyramidal cells projecting to different brain areas and located in distinct layers. Red lines indicate means. Numbers of examined cells by layers and by projection brain areas are the following: L2/3, BA, n = 25; cPFC, n = 22; DS, n = 12; L5a, BA, n = 17; cPFC, n = 36; DS, n = 20; L5b, cPFC, n = 17; DS, n = 10; PAG, n = 24. BA-projecting cells were counted in 6 slices from 2 mice, cPFC-projecting cells were counted in 10 slices from 4 mice, DS-projecting cells were counted in 6 slices from 2 mice, and PAG-projecting cells were counted in 4 slices from 2 mice. K–W ANOVA, L2/3, p = 0.104; L5a, p = 0.633; and L5b, p = 0.026. In L5b M–W test showed the difference between cPFC- and PAG-projecting neurons; p = 0.012; DS-PAG, p = 0.121; cPFC-DS, p = 0.303. n.s, not significant.