A disinhibitory circuit mediates motor integration in the somatosensory cortex

Abstract

The influence of motor activity on sensory processing is crucial for perception and motor execution. However, the underlying circuits are not known. To unravel the circuit by which activity in the primary vibrissal motor cortex (vM1) modulates sensory processing in the primary somatosensory barrel cortex (S1), we used optogenetics to examine the long-range inputs from vM1 to the various neuronal elements in S1. We found that S1-projecting vM1 pyramidal neurons strongly recruited vasointestinal peptide (VIP)-expressing GABAergic interneurons, a subset of serotonin receptor-expressing interneurons. These VIP interneurons preferentially inhibited somatostatin-expressing interneurons, neurons that target the distal dendrites of pyramidal cells. Consistent with this vM1-mediated disinhibitory circuit, the activity of VIP interneurons in vivo increased and that of somatostatin interneurons decreased during whisking. These changes in firing rates during whisking depended on vM1 activity. Our results suggest previously unknown circuitry by which inputs from motor cortex influence sensory processing in sensory cortex.

Figure 1

Long-range excitatory inputs from vM1 to different types of neurons in the superficial layers of S1. (a) Expression of ChR2-mCherry in vM1 pyramidal neurons. Left, expression of ChR2 in vM1 after injection of AAV-DIO-ChR2-mCherry to vM1 in Emx1-cre mice. Scale bar represents 500 μm. Right, axons of vM1 pyramidal neurons innervating S1 expressed ChR2-mCherry. Scale bar represents 100 μm. Note the strong expression of ChR2-mCherry in layers 1 and 2 in S1 (blue, DAPI). (b) Schematic of slice recording configuration. Red lines indicate the innervation patterns of vM1 axons. A specific interneuron type was simultaneously recorded with a nearby pyramidal neuron. (c–e) Photo-stimulation–evoked synaptic currents (left) and voltage responses (right) recorded in a fast-spiking (FS) interneuron and a pyramidal (Pyr) neuron (c), a 5HT3aR interneuron and a pyramidal neuron (d), and a SST interneuron and a pyramidal neuron (e). Gray traces represent individual sweeps and solid colored traces represent the average (red for fast spiking, orange for 5HT3aR, blue for SST and black for pyramidal neurons). Evoked synaptic currents were recorded at −70 mV in voltage-clamp mode. Voltage responses were recorded at the resting membrane potential (V_rest, indicated next to the voltage traces) under current-clamp mode. Light blue traces indicate photo-stimulation (LED, 470 nm, 5 ms) delivered at 5 Hz. (f,g) EPSCs (f) and EPSPs (g) of 5HT3aR (5HT), fast-spiking and SST interneurons normalized to simultaneously recorded nearby pyramidal neurons (normalized EPSCs: 5HT3aR, 25 cells, 18 slices, 8 mice, P < 0.001; fast spiking, 13 cells, 8 slices, 4 mice, P = 0.1; SST, 13 cells, 9 slices, 4 mice, P < 0.001; normalized EPSPs: 5HT3aR, 21 cells, 17 slices, 8 mice, P < 0.001; fast spiking, 6 cells, 4 slices, 2 mice, P = 0.18; SST, 6 cells, 4 slices, 2 mice, P = 0.01). Statistical significance was computed using Wilcoxon signed-rank test; *P < 0.05, **P < 0.005. Bars indicate mean.

Figure 2

VIP interneurons receive the strongest input from vM1. (a,c) Representative firing patterns of an irregular spiking neuron (a, VIP-positive 5HT3aR interneuron) and a late spiking neuron (c, non-VIP, 5HT3aR interneuron) produced by increasing step current injections (500 ms) in current-clamp mode. (b,d) Examples of morphologies of the irregular-spiking (b) and late-spiking (d) interneurons reconstructed using Neurolucida tracing. Dendrite and soma are shown in blue and axon in red. Scale bars represent 100 μm. (e,f) EPSCs evoked by photo-stimulation of vM1 axons, recorded in the irregular-spiking neuron (e) and the late-spiking neuron (f). Blue traces indicate photo-stimulation (LED, 470 nm, 5 ms) delivered at 5 Hz. (g) VIP interneurons received significantly stronger vM1 inputs compared with non-VIP interneurons in the 5HT3aR population. EPSCs of VIP and non-VIP interneurons were normalized to simultaneously recorded nearby pyramidal neurons. Bars indicate mean value (VIP interneurons: 15 cells, 12 slices, 8 mice, VIP to pyramidal ratio, P < 0.001; non-VIP interneurons: 10 cells, 7 slices, 6 mice, non-VIP to pyramidal ratio, P = 0.51). **P < 0.005, Wilcoxon signed-rank test. (h) We found no relationship between the recording depth (relative to pial surface) and the strength of the vM1 input. Recording depth is plotted against normalized EPSC. White circles represent non-VIP interneurons and gray circles represent VIP interneurons (correlation coefficient: non-VIP, r² = 0.078; VIP, r² = 0.001).

Figure 3

VIP interneurons most strongly inhibit SST interneurons in S1 superficial layers. (a) Specific expression of ChR2-mCherry in VIP interneurons. ChR2-mCherry expression in Vip-cre mice was confined to VIP neurons. Antibody-stained VIP neurons (left) showed nearly 100% overlap (right) with neurons expressing ChR2-mCherry (middle). Scale bar represents 100 μm. (b) VIP interneurons provided weak inhibition to fast-spiking interneurons. Photo-stimulation–evoked inhibitory synaptic currents recorded in a fast-spiking interneuron and in a pyramidal neuron in the Vip-cre B13 mouse are shown. (c) VIP interneurons provided strong inhibition to SST interneurons. Photo-stimulation–evoked inhibitory synaptic currents in a SST interneuron and a simultaneously recorded pyramidal neuron in a Vip-cre GIN mouse are shown. Cesium-based internal pipette solution was used to record inhibitory currents at 0 mV in voltage-clamp mode. Gray traces indicate individual sweeps and colored traces indicate the average (red, fast spiking; blue, SST; black, pyramidal neurons). Light blue traces indicate photo-stimulation (LED, 470 nm, 5 ms) delivered at 5 Hz. (d,e) IPSC amplitude (d) and total charge (e) in fast-spiking (14 cells, 8 slices, 4 mice) and SST (8 cells, 5 slices, 3 mice) interneurons normalized to the corresponding values in simultaneously recorded nearby pyramidal neurons. *P < 0.05, **P < 0.005, Wilcoxon signed-rank test. (f) Photo-stimulation–evoked total IPSC charge in fast-spiking interneurons (red) and SST interneurons (blue) plotted against total IPSC charge in simultaneously recorded pyramidal neurons. Dashed line indicates unity. + indicates mean (fast spiking to pyramidal ratio: amplitude, P = 0.02; charge, P < 0.001; SST to pyramidal ratio: amplitude, P = 0.01; charge, P < 0.001).

Figure 4

VIP interneurons mediate disynaptic feedforward inhibition of SST interneurons following vM1 activation. (a) Specific expression of NpHR-eYFP in VIP interneurons. AAV-ChR2-mCherry was injected to vM1 and AAV-DIO-NpHR-eYFP in S1 of a Vip-cre GIN mouse (left). Middle, expression of NpHR-eYFP in VIP interneurons in S1. NpHR-eYFP–expressing VIP interneurons (arrow) were easily distinguishable from GIN neurons (arrowhead) as a result of the preferential labeling of the periphery of the cells resulting from membrane expression of eYFP in VIP interneurons. Note the mCherry-expressing vM1 axons in layer 1 of S1. Scale bar represents 100 μm. Right, firing patterns of a NpHR-eYFP–expressing VIP interneuron and a GIN interneuron. (b) Activation of NpHR reduced vM1 activation-evoked spikes from NpHR-expressing VIP interneurons. Photo-stimulation of vM1 axons (blue light, 470 nm) elicited reliable spikes from a VIP interneuron in S1 (top). Photo-stimulation (yellow light, 590 nm) of NpHR sufficiently hyperpolarized the VIP interneuron such that simultaneous photo-stimulation (blue light, 470 nm) of ChR2-expressing vM1 axons evoked only subthreshold EPSPs in the VIP interneuron (bottom). (c) VIP interneurons mediated disynaptic feedforward inhibition of SST interneurons during vM1 activation. Left, schematic of slice recording and photo-stimulation configuration. Right, evoked EPSCs followed by IPSCs recorded from a SST interneuron to blue light (top) and blue light combined with yellow light (bottom). Inset, overlay of averaged postsynaptic currents traces to blue light only (blue trace) and blue light combined with yellow light (green). Gray traces represent individual sweeps, and black traces show the average. Evoked synaptic currents were recorded at −55 mV in voltage-clamp mode. (d) Population data from c. Blue and yellow light stimulation–evoked total IPSC charge was normalized to blue light only stimulation–evoked response in SST interneurons (9 cells, 6 slices, 3 mice; 470 nm + 590 nm to 470 nm ratio, charge P = 0.008). *P < 0.05, Wilcoxon signed-rank test.

Figure 5

Spiking activity of VIP interneurons and SST interneurons in superficial layers of S1 during active whisking. (a) Image of an eGFP-positive and tdTomato-positive cell targeted for cell-attached in vivo recording in a Vip-cre tdTomato 5HT3aR^eGFP mouse. Arrow points to an eGFP-positive, tdTomato-negative interneuron (non-VIP interneuron) and the arrowhead points to an eGFP-positive, tdTomato-positive interneuron (VIP interneuron). Scale bars represent 50 μm. (b–d) Example recordings from a 5HT3aR-positive, VIP-positive interneuron (b), a 5HT3aR-positive, VIP-negative interneuron (c), and a SST interneuron (d). Whisking motion was computed from whisker video recordings to define whisking and non-whisking periods (top). Bottom, spikes in the corresponding interneuron type recorded under two-photon guidance in loose-patch configuration. (e–g) Spiking activity of VIP interneurons and SST interneurons in superficial layers of S1 during active whisking. Whisking onset triggered raster plots of representative VIP (e), non-VIP, 5HT3aR (f) and SST (g) interneurons. Bottom, population PSTHs of VIP, non-VIP, 5HT3aR and SST interneurons. Dotted vertical line at time 0 indicates whisking onset. Dark lines indicate smoothed PSTHs. (h) Firing rates of VIP (open circles, 11 cells, 5 mice), non-VIP (gray circles, 5 cells, 3 mice) and SST (black circles, 6 cells, 2 mice) interneurons during whisking plotted against their firing rates during non-whisking periods. Dashed line indicates unity. (i) Firing rates of VIP, non-VIP and SST interneurons during whisking (W) normalized to the firing rates during non-whisking (NW) periods. Each line indicates an individual neuron (VIP interneurons, P = 0.001; non-VIP interneurons, P = 0.94; SST interneurons, P = 0.02). *P < 0.05, **P < 0.005, Wilcoxon signed-rank test. (j) Summary of recording depths of VIP (open circle), non-VIP (gray) and SST (black) interneurons.

Figure 6

vM1 activity is responsible for the increased activity of VIP interneurons and decreased activity of SST interneurons in S1 during whisking. (a) TTX injection to vM1 silenced the activity of vM1, but did not affect S1 activity, as evidenced by LFPs recorded in S1 and vM1 before and after TTX injection to vM1. TTX injection and LFP recordings were conducted under anesthesia. (b,c) Representative recordings from a VIP interneuron (b) and an SST interneuron (c) during vM1 inactivation. Whisking motion was computed from whisker video recordings to define whisking (W) and non-whisking (NW) periods (top). Bottom, spikes in the corresponding interneuron type recorded under two-photon guidance in loose-patch configuration. (d) Firing rates of VIP (open circles) and SST (black circles) interneurons during whisking plotted against their firing rates during non-whisking periods after vM1 inactivation. Dashed line indicates unity. (e) Summary data comparing firing rates of VIP (9 cells, 2 mice) and SST (8 cells, 3 mice) interneurons during whisking and non-whisking periods after inactivation of vM1 with TTX injection (VIP interneurons, P = 0.08; SST interneurons, P = 0.2, Wilcoxon signed-rank test).