Organization of the core respiratory network: Insights from optogenetic and modeling studies
Fig 3
Photostimulation causes membrane depolarization of rhythmically active ChR2-expressing pre-BötC inspiratory VGAT-positive neurons in vitro.
(A) Schematic of the in vitro rhythmic slice preparation from a neonatal VGAT-ChR2 transgenic mouse illustrating whole-cell current-clamp recordings from pre-BötC inspiratory VGAT-positive neurons with unilateral pre-BötC laser illumination (0.5–5 mW) and suction-electrode recordings from hypoglossal (XII) nerves to monitor inspiratory activity. NAsc, semi-compact division of nucleus ambiguus; V4, fourth ventricle; IO, inferior olivary nucleus. The pre-BötC regions are indicated by gray circles (~300 μm diameter). (B) Two-photon single-optical plane images of a pre-BötC inspiratory neuron targeted for whole-cell recording, showing VGAT-Cre driven tdTomato labeling (B1), ChR2-EYFP expression (B2) and confirmed co-expression in merged image (B3). (C) Current-clamp recording from a VGAT-positive inhibitory pre-BötC inspiratory neuron in B illustrating inspiratory spikes synchronized with integrated inspiratory XII nerve activity (∫XII). The membrane potential (Vm) of this neuron was depolarized by ~7 mV at 2 mW and by ~10 mV at 5 mW of laser power (spikes are truncated). The neuron was hyperpolarized from resting baseline potential to -64 mV by applied constant current in this example to reveal the magnitude of the light-induced membrane depolarization. The lower trace indicates the duration and amplitude of the laser stimulation. (D) Summary data (n = 8 neurons from 3 slice preparations, mean ± SEM) showing that ChR2-mediated membrane depolarization of VGAT-positive pre-BötC inspiratory neurons was laser power-dependent.