Callosal Projections Drive Neuronal-Specific Responses in the Mouse Auditory Cortex

Photostimulation of callosal projections suppresses action potentials in CCort, but not CCol pyramidal neurons. A, Experimental paradigm for photostimulating ChR2-positive callosal projections while recording from CCort and CCol pyramidal neurons identified by anatomical retrograde labeling. B, Bright-field (left) and epifluorescence (right) images of a slice containing the AC showing expression of ChR2-tdTomato after injection of AAV-ChR2 into the right AC. C, Left trace, Response of a layer 5 CCort pyramidal neuron in the whole-cell current-clamp configuration to current injection (375 pA, 10 ms; n = 10; animals n = 6). Middle traces, Response to current injection with photostimulation of callosal projections (blue bar 5–10 ms) and to current injection with photostimulation of callosal projections and bath application of gabazine 20 mm. (n = 5; animals n = 5). Right, Summary of ChR2-mediated action potentials suppression of CCort pyramidal neurons (n = 10; animals n = 6) during current injection and current injection combined with photostimulation of the ChR2 callosal projections. D, Left trace, Response of a layer 5 CCol pyramidal neuron in the whole-cell current-clamp configuration to current injection (495 pA, 10 ms; n = 10; animals n = 6). Middle trace, Response to current injection with photostimulation of callosal projections (blue bar 5–10 ms). Right, Summary of ChR2-mediated action potential suppressions of CCol pyramidal neurons (n = 10; animals n = 6) during current injection and current injection combined with photostimulation of the ChR2 callosal projections.

Photostimulation of callosal projections elicits strong disynaptic inhibition onto CCort but not onto CCol pyramidal neurons. A, Experimental paradigm for photostimulating ChR2-positive callosal projections while recording excitatory and inhibitory synaptic inputs from CCort and CCol pyramidal neurons identified by anatomical retrograde labeling. B, Bright-field (left) images of a slice containing the AC showing the soma location of layer 5 CCort (right) and CCol (left) pyramidal neurons. Left plot shows the group average soma location (± SEM) of CCort (black) and CCol (magenta) pyramidal neurons. Different symbols mark the absolute distances from the pia to the soma (CCort: black circles, n = 14, animals n = 7; CCol: magenta circles, n = 14, animals n = 7). C, Examples of EPSCs (green trace) recorded at −70 mV and IPSCs (red trace) recorded at +10 mV from layer 5 CCort pyramidal neurons before and after application of ionotropic glutamate receptor antagonists (NBQX 10 μm, CPP 5 μm: dashed green and dashed red trace). D, Left, Plot of onset latencies recorded in CCort (n = 14; animals n = 7) pyramidal neurons for EPSCs (green circles) and IPSCs (red circles), including group averages (± SEM). Middle, Plot of EPSC and IPSC peaks calculated for individual EPSC-IPSC pairs for CCort pyramidal neurons, including group averages (± SEM). Right, Plot of EPSC and IPSC charge transfer calculated for individual EPSC-IPSC pairs for CCort pyramidal neurons, including group averages (± SEM). E, Same as in C but for CCol neurons (n = 14; animals n = 7). F, Same as in D but for CCol neurons (n = 14; animals n = 7). G, Summary of correlation between EPSC and IPSC peaks (left, middle left); EPSC and IPSC charge (right, middle right) calculated for individual pairs of CCort (n = 14; animals n = 7) and CCol (n = 14; animals n = 7) pyramidal neurons during photoactivation of callosal projections.

Callosal projections synaptically drive FS-PARV interneurons. A, Experimental paradigm for photostimulating ChR2-positive callosal projections while recording action potential from FS-PARV interneurons, CCort and CCol pyramidal neurons. B, Firing pattern (top left) and high resolution image of biocytin-labeled FS-PARV interneurons (middle left). Firing patterns (top middle) and high resolution image of biocytin-labeled CCort pyramidal neuron (middle center). Firing patterns (top right) and high-resolution image of biocytin-labeled CCol pyramidal neuron (middle right). Example of responses recorded in cell-attached mode during photoactivation of callosal projections (bottom left, middle, and right); FS-PARV interneurons (n = 15; animals n = 7), but not CCort (n = 9; animals n = 7) and CCol (n = 7; animals n = 7) pyramidal neurons, fired an action potential (arrow).

FS-PARV-ChR2-evoked inhibitory input onto CCort and CCol pyramidal neurons. A, Experimental paradigm for photostimulating FS-PARV ChR2-positive interneurons while recording IPSCs from CCort and CCol pyramidal neurons identified by anatomical retrograde labeling. B, Bright-field (left) and epifluorescence (right) images of a slice containing the AC showing expression of ChR2 in FS-PARV following injection of AAV-ChR2-flex into the left AC. C, Examples of IPSCs recorded at +10 mV from a layer 5 CCort (black) and CCol (magenta) pyramidal neuron. D, Plot of correlation between IPSC peaks (left) and IPSC charge (right) calculated for individual pairs of CCort and CCol pyramidal neurons while stimulating FS-PARV ChR2-positive interneurons.

Suppression of CCort pyramidal neurons by FS-PARV interneurons as a function of photoactivation of callosal projections. A, Experimental paradigm for photostimulating ChR2-positive callosal projections and photosilencing Halo-positive FS-PARV interneurons while recording from CCort pyramidal neurons identified by anatomical retrograde labeling after injection of AAV-ChR2 in the right AC and AAV-Halo-flex into left AC. B, Left, Example showing an action potential and its suppression recorded both in cell-attached (top left, n = 8; animals n = 8) and whole-cell mode (bottom left, n = 8; animals n = 8) from a FS-PARV-Halo interneuron during photoactivation of callosal projections and during simultaneous photoactivation of callosal projections and photosilencing of the same FS-PARV-Halo interneuron. Right, Example showing photosilencing of action potentials during a current-evoked barrage of action potentials in a FS-PARV-Halo interneuron by flashing amber light (565 nm) in the middle of the barrage of action potentials (n = 5; animals n = 5). C, Left, Examples of EPSCs recorded at −70 mV from layer 5 CCort pyramidal neurons during photoactivation of callosal projections (green trace, n = 8; animals n = 8) and during simultaneous photoactivation of callosal projections and photosilencing of FS-PARV-Halo interneurons (amber trace, n = 8; animals n = 8). Right, Examples of IPSCs recorded at +10 mV from layer 5 CCort pyramidal neurons during photoactivation of callosal projections (red trace, n = 10; animals n = 8) and during simultaneous photoactivation of callosal projections and photosilencing of FS-PARV-Halo interneurons (amber trace, n = 10; animals n = 8). D, Summary plot of EPSC (left, n = 8; animals n = 8) and IPSC (right, n = 10; animals n = 8) peaks recorded from layer 5 CCort pyramidal neurons during photoactivation of callosal projections and during simultaneous photoactivation of callosal projections and photosilencing of FS-PARV-Halo interneurons. E, Black trace, Response of a layer 5 CCort pyramidal neuron in the whole cell current-clamp configuration to current injection (385 pA, 10 ms; n = 5; animals n = 5). Blue trace, Response to current injection with photostimulation of callosal projections (blue bar 5–10 ms; n = 5; animals n = 5). Amber trace, Response to current injection with photostimulation of callosal projections and photosilencing of FS-PARV-Halo interneurons (blue bar: 5–10 ms, amber bar: 50–100 ms; n = 5; animals n = 5).