Fig. 5
From: Neurons in the Nucleus papilio contribute to the control of eye movements during REM sleep
Genetic ablation and optogenetic silencing of NPCalb neurons block EMs during REM sleep but not wakefulness. a Schematic representation of the genetic targeting of the diphtheria toxin receptor (DTR) expression to NPCalb neurons and implantation of optical fibre. b Experimental timeline. c Representative micrographs depicting immunoreactivity for Calb protein in a non-transduced (left) and a DTR-transduced (right) mouse, both i.p.-injected with diphtheria toxin (DTX). Note the remnants of disintegrating neurons in the extracellular space displaying some immunoreactivity for Calb (right). 4V: fourth ventricle; mlf: medial longitudinal fasciculus; NP: Nucleus papilio. d The number of Calb-expressing neurons is dramatically reduced upon DTX injection in DTR-transduced (n = 3) as compared with a wild-type animal that received DTX alone. e Representative experiment showing detected EMs, EOG, EEG and EMG signals, and time frequency heat-map of EEG spectral power from animals transduced with DTR and injected with DTX (upper set of traces) and control mice injected with DTX alone (lower set of traces) during REM sleep. f, g Averaged number of EMs during REM sleep f and wakefulness g, before and after DTX injection (n = 7 DTR+/DTX+ and four control animals; Wakefulness: P = 0.523, F (4, 36) = 0.8171; REM sleep: P ≤ 0.001, f (4,36) = 19.2). ***P < 0.0001, two-way ANOVA followed by Sidak’s post hoc tests). Genetic ablation of NPCalb neurons does not significantly decrease the number of EMs during wakefulness but eradicate EMs during REM sleep. h Schematic representation of the genetic targeting of AAV2-Ef1a-DIO-ArchT3.0-EYFP inhibitory opsin expression to NPCalb neurons and implantation of optical fibre. i Representative micrograph showing ArchT-YFP expression (green) in Calb-immunoreactive neurons (red). j Experimental strategy for optical silencing of NPCalb neurons selectively during REM sleep (top). Detected EMs, EOG, EEG and EMG traces and time frequency heat-map of EEG spectral power (bottom). Orange bar represents the duration of the optical silencing. k Averaged number of EMs during wakefulness and REM sleep in the control (n = 3) and ArchT3.0 (n = 4) transduced mice. **, P = 0.11, two-way-ANOVA followed by Sidak’s multiple comparison test. Error bars represent SEM values. l Duration of REM sleep episodes under conditions in which pulses of light were either not delivered (unstim.) or delivered (stim.) to silence the NPCalb neurons, in control and transduced mice. Error bars represent SEM values
