ChCs receive input from L5 PyCs and innervate L2/3 PyCs
(A) Schematic with viral strategy for selective monosynaptic retrograde rabies tracing of L2/3 ChCs.
(B) Overview of superficial V1 region (top) with starter ChCs (yellow), non-starter ChCs (green) and presynaptic partners (red). Scale bar, 50 µm. Bottom: example images of RSC (left; scale bar, 100 µm) and dLGN (right; scale bar, 200 µm) containing input cells in red. Number of starter ChCs = 7.5 +- 3.8 (mean +- SEM, with a total of 30 starter ChCs from 4 mice).
(C) Quantification of input sources to ChCs (n = 4 mice) represented as percentage (mean +- SEM) of the total number of presynaptic neurons observed brain wide. LM, lateromedial visual area; dLGN, dorsal lateral geniculate nucleus; PL, posterolateral visual area; RSC, retrosplenial cortex; S1, primary somatosensory area; LD, lateral dorsal nucleus of the thalamus; LPN, lateral posterior nucleus of the thalamus; RL, rostrolateral area; AL, anterolateral visual area. The image shows the distribution of input neurons selectively within V1. Scale bar, 200 µm.
(D) Schematic with viral strategy for optogenetic activation of RSC inputs to L2 ChCs. PyCs in RSC were labeled with ChR2-eYFP, ChCs in V1 were labeled with the red fluorophore mCyRFP1.
(E) Confocal images showing the ChR2-eYFP (cyan) injection location in RSC (bottom) and their projections to L1 in V1 (top). Scale bar, 500 µm.
(F) Confocal images of the biocytin fill (red) of mCyRFP+ neurons revealed L2 ChC identity. Insets depict putative RSC inputs on apical dendrites of ChC in layer 1 (cyan, top) as well as characteristic rows of ChC bouton cartridges (bottom). Yellow arrow indicates soma. Scale bars, 50 µm.
(G) Schematic of whole-cell patch-clamp recordings from mCyRFP+ neurons. Current injections evoked firing patterns characteristic of ChCs. Scale bars, 10 mV, 100 ms. Optogenetic activation of RSC boutons evoked inward currents of on average 29.8 pA (n = 11/13 ChCs from 13 slices in 5 mice). Bar shows mean and SEM, dots individual cells. Scale bars, 20 ms, 10 pA.
(H) TTX/4-AP bath application confirmed monosynaptic RSC (470 nm optogenetically evoked, blue) inputs in ChCs. RM ANOVA **p = 0.0035, Holm-Šídák’s multiple comparisons test, *p = 0.012, **p = 0.008. Bar shows mean +- SEM, dots represent individual cells, n = 11 cells from 11 slices in 5 mice. Scale bars, 1 mV, 100 ms.
(I) Optogenetic stimulation at 20 Hz revealed a reduction in postsynaptic potential amplitudes. Circles show mean +- SEM. Scale bars, 2 mV, 50 ms. N = 8 cells from 8 slices in 3 mice.
(J) Voltage responses to a current injection steps in ChCs and PyCs during simultaneous recordings. Scale bars, 100 ms, 20 mV.
(K) Action potentials were generated by brief current injections in ChCs (left) or PyCs (right). In n = 5 out of 11 pairs, ChC stimulation generated postsynaptic responses in PyCs. In n = 0/11 PyC were projecting back onto ChC. Scale bars 10 ms, 20 mV, and 0.5 mV for subthreshold responses, 11 pairs from 11 slices in 6 mice.