Fig. 4: N₂O fails to recruit synapses and dendrites to drive L5 activity.

a Representative two-photon GCaMP6 image of L5 apical dendritic segment (DS; teal box) containing 4 spines (S1, S2, S3, S4) in 2-D imaging plane. Spontaneous spine activity (middle image showing peak signal of single spine activation) is occasionally observed in min recordings under room air conditions. N₂O fails to recruit spine activity (n = 66 spines from 9 mice, two-sided Wilcoxon matched-pairs signed rank test: P = 0.22). Scale bar, 5 μm. b Left, two-photon z-stack of an individual L5 neuron coexpressing GCaMP6 and tdTomato. Middle, multiple 2-D imaging planes across L5 neuron from apical tuft dendrites to soma corresponding to the teal boxes located on z-stack. ROI labeled D for apical dendrite, P for parent dendrite, T for trunk. Right, GCaMP6 traces for ROIs under room air, N₂O inhalation, and following N₂O exhalation. N₂O fails to recruit superficial dendrites during treatment. Dendritic activity is found deep, in close proximity to soma. Scale bar, 20 μm. c Summary of calcium responses from across different L5 dendritic compartments (dendritic segments: n = 170 from 21 mice, two-sided Wilcoxon matched-pairs signed rank test: P = 0.19; deep dendrites/trunks: n = 34 from 14 mice, Wilcoxon matched-pairs signed rank test: P = 0.03) and soma (n = 47 from 19 mice, Wilcoxon matched-pairs signed rank test: P = 2.2 × 10⁻⁵). d Left, Z-stack of L5 neuron coexpressing GCaMP6 and tdTomato subject to dendritomies by 2-photon laser pulses. Right, GCaMP6 traces of dendritic ROIs and soma under wakefulness, N₂O, and following dendritomies. L5 soma and dendrites were activated by N₂O as compared to wakefulness. GCaMP images of two parents dendrites corresponding to teal box in Z-stack (ROI 1 and 2 at 136 μm) are targeted and sequentially cut using laser pulses resulting in a baseline fluorescence bump coupled with the elimination of transients. L5 activity persists following dendritomies despite loss of dendritic activity. Scale bar, 20 μm. e Top, Summary of individual L5 calcium activity following N₂O exposure and apical dendritomies (n = 5 L5 neurons from 5 mice). Bottom, L5 neurons under same conditions exposed to focal two-photon laser pulses directed at neuropil (control; n = 7 L5 neurons from 7 mice). L5 responses following neuropil pulses were not significantly different than those directed at dendrites (two-sided Mann Whitney rank sum (13): P = 0.53). Error bars show s.e.m.