Figure 7. SIn_ACh and M2_ACtx Synapses Converge on Single Auditory Cortical Neurons.

(A) Schematic of the experimental strategy. AAV-FLEX-GFP was injected into SIn of ChAT-Cre mice. AAV-tdTomato was injected into the M2 of the same mice. (B) Confocal micrograph showing GFP labeling at the injection side. Blue is DAPI. The inset shows the injection site, immunostained for ChAT (red). (C) tdTomato labeling at the M2 injection site. (D) Labeling from SIn_ACh (green) and M2_ACtx (red) axons in ACtx. The inset shows the fluorescence intensity of SIn_ACh and M2_ACtx labeling across layers (averaged measurements from several brain sections). (E) Higher magnification Z stack showing SIn_ACh and M2_ACtx axons near a putative cell body (asterisk). (F) Higher magnification image from (E). The Z stack was interpolated and rotated slightly. Nicotinic ACh receptors are labeled with ɑ-bungarotoxin (greyscale). (G) Another Z stack showing SIn_ACh and M2_ACtx axons near a cell body from a different mouse. DNA is labeled with DAPI. (H) Higher magnification Z stack from (G). (I) Schematic of the experimental strategy. AAV-FLEX-ChR2-GFP was injected into SIn of ChAT-Cre mice. AAVChR2-GFP was injected into the M2 of the same mice. Whole-cell voltage clamp recordings were made from acute coronal ACtx brain slices. (J) Confocal Z stack showing a pyramidal neuron labeled following whole-cell recording surrounded by ChR2-expressing axons. (K) Average traces of excitatory currents (5 neurons) in response to photostimulation of putative SIn_ACh and M2_ACtx axons in normal ACSF (blue) and following application of blockers (orange; the shading indicates SEM). Nicotinic transmission is preserved after blocking glutamatergic M2_ACtx currents. (L) Onset (blue) and offset (magenta) time constants of excitatory currents from (J).