Extended Data Fig. 5: PVMVN→DTN neurons exert null control on non-PV neurons within the ipsilateral or contralateral MVN.
a, Schematic of using optogenetics-coupled whole-cell patch-clamp recording to assess whether PVMVN→DTN neurons directly innervate non-PV neurons in ipsilateral and contralateral MVN. PV-Cre mice were bilaterally injected with AAV5-DIO-FLP-mCherry in MVN to label PV neurons and unilateral injected with rAAV-fDIO-ChR2-EGFP in DTN to express optogenetic vector ChR2 in PVMVN→DTN neurons. b, Left, a representative image of DTN showing the injection site with rAAV-fDIO-ChR2-EGFP (green). Right, representative images of MVN showing PV neurons infected with both AAV5-DIO-mCherry (red) and rAAV-fDIO-ChR2-EGFP (green). c, Left, epifluorescence, bright-field and overlay images showing the configuration of whole-cell patch-clamp recording for DTN-projecting neurons in MVN with ChR2 expression (scale bar, 10 μm). Right, a representative current-clamp tracing from the sampled neuron (displayed in left) with ChR2 expression showed time-locked responses upon 1 Hz blue light stimulation. (c-e, representative recording traces from 5 neurons from 3 mice). d-e, Epifluorescence, bright-field and overlay images showing the configuration of whole-cell patch-clamp recording for non-PV neurons in the ipsilateral (d, left) and contralateral (e, left) MVN without mCherry expression (scale bar, 10 μm). Representative voltage-clamp tracings recorded from the sampled neuron (displayed in left) at 0 and −70 mV demonstrated absence of oIPSCs and oEPSCs responses upon 1 Hz blue light stimulation, indicating null recruitment from PVMVN→DTN neurons on non-PV neurons in the ipsilateral (d, right) and contralateral (e, right) MVN. Blue bars, optical stimulation.
