Ventral medullary control of rapid eye movement sleep and atonia

Abstract

Discrete populations of neurons at multiple levels of the brainstem control rapid eye movement (REM) sleep and the accompanying loss of postural muscle tone, or atonia. The specific contributions of these brainstem cell populations to REM sleep control remains incompletely understood. Here we show in rats that viral vector-based lesions of the ventromedial medulla at a level rostral to the inferior olive (pSOM) produced violent myoclonic twitches and abnormal electromyographic spikes, but not complete loss of tonic atonia, during REM sleep. Motor tone during non-REM (NREM) sleep was unaffected in these same animals. Acute chemogenetic activation of pSOM neurons in rats robustly and selectively suppressed REM sleep but not NREM sleep. Similar lesions targeting the more rostral ventromedial medulla (RVM) did not affect sleep or atonia, while chemogenetic stimulation of the RVM produced wakefulness and reduced sleep. Finally, selective activation of vesicular GABA transporter (VGAT) pSOM neurons in mice produced complete suppression of REM sleep whereas their loss increased EMG spikes during REM sleep. These results reveal a key contribution of the pSOM and specifically the VGAT+ neurons in this region in REM sleep and motor control.

Keywords: AAV; DREADD; GABA; REM arousal; VGAT.

Fig. 1

Injection of AAV-DTA into the RVM (A) and pSOM (B) of the rat medulla results in near complete cell loss along the midline, whereas injections of saline into the RVM (C) and pSOM (D) do not. High-magnification images of the insets (E–F) show cell loss in the lesion areas (left panels) compared to cell preservation in the saline injections (right panels). Overlay of lesion center of mass is shown at the multiple levels (G–H).

Fig. 2

Lesions of the pSOM result in EMG and behavior during REM sleep. Control animals have a flat, monotonous EMG during REM sleep (A), while pSOM DTA animals have numerous EMG spikes during REM sleep (B). Color-coded video analysis of movement (C–D) shows body movements overlaid as colors over a black and white still image. The colored regions show the extent of the body movements, with a color bar legend overlaid with a timescale to show when the movement–and corresponding color–occurred, relative to the EEG and EMG; e.g. in Panel C, the red-colored movements overlaid on the video occurred 5 s into the presented EEG/EMG data (C). Panel D shows a series of myoclonic twitches during REM sleep, in which multiple movements, colored blue to green to orange, occur from 2 to 3.5 s into the presented EEG/EMG data (D). pSOM lesioned animals, but not RVM lesioned animals, have significant increases in EMG spikes per REM epoch (E) and the percentage of REM epochs containing at least one EMG spike (F). Data are represented as mean ± SEM.

Fig. 3

Injection site of an AAV mixture into the RVM (A) and pSOM (B) regions are delimited by a DAB-based stain against mCherry. Following CNO injections (IP, 0.2 mg/kg), most transduced cells within the RVM or pSOM sites expressed c-Fos, demonstrating strong cellular activation by CNO (C). After CNO injection, both c-Fos positive double-stained neurons (black/teal arrows) are visible, as well as c-Fos negative, non-double-stained neurons (yellow-black arrows). In contrast, saline injections did not elicit c-Fos expression in transduced ventromedial medulla neurons (D), with no double-staining (yellow-black arrows). Overlays of the center of RVM injections (E) and pSOM injections (F) at multiple levels show the extent of the transduced area, with sporadic neurons in more caudal sections (colored dots).

Fig. 4

Injection (IP; 0.2 mg/kg) of CNO activates RVM neurons and increases wakefulness for several hours (A). Over the 6 h period of highest CNO effect, RVM virus animals have significantly higher wakefulness and lower NREM and REM sleep after CNO injection than after saline injection (B). Each animal experienced an increase in wakefulness in the 6 h following CNO injection compared to the same time period following saline injection (C). An example 4-hour hypnogram is shown for a single animal after injection with saline (D) or CNO (E). FFT delta power, a marker of NREM sleep, is plotted against time, with scored stages shown. Saline-injected animals alternated between wake, NREM, and REM sleep, while CNO-injected animals fail to initiate NREM or REM sleep episodes and exhibited low delta power.

Fig. 5

Injection (IP; 0.2 mg/kg) of CNO activated pSOM neurons and resulted in a reduction of REM sleep that lasted approximately 6 h (A). Following the REM suppression, total REM sleep time normalizes within a 24 h period, suggesting homeostatic regulation following CNO washout. Over the 6 h period of highest CNO effect, pSOM virus animals have significantly lower REM sleep after CNO injection than after saline injection (B), with no significant changes in wake or NREM sleep. Each animal experienced a steep decline in REM sleep in the 6 h following CNO injection compared to the same time period following saline injection (C). An example 4-hour hypnogram is shown for a single animal after injection with saline (D) or CNO (E). The theta/delta ratio, one marker of REM sleep, is plotted against time, with scored stages shown. Saline-injected animals alternate between wake, NREM, and REM sleep, while CNO-injected animals failed to initiate REM sleep episodes but continued to cycle between wake and NREM sleep.

Fig. 6

Injection of AAV-DTA into the pSOM level of Vgat-IRES-cre mice (A). In Vgat-IRES-cre mice, VGAT neurons express GFP; while AAV-DTA transduced neurons express dsRed. Injections in the pSOM result in complete loss of GFP+ VGAT neurons, while sparing non-GABA/glycine neurons in the same region. Overlay of lesion center of mass is shown at the multiple levels (B–C).

Fig. 7

Injection of an AAV mixture into the pSOM level of Vgat-IRES-cre mice are delimited by a DAB-based stain against mCherry (A). Administration of CNO (IP, 0.3 mg/kg) produced c-Fos expression in most cells, demonstrating strong cellular activation (B). Overlays of the center of injection mass at the pSOM level and adjoining levels (C) show the extent of the transduced area, with sporadic neurons in more caudal sections (colored dots).

Fig. 8

CNO activation of glycine/GABA neurons in the pSOM suppressed REM sleep. After injection with CNO, Vgat-cre mice experience a reduction of REM sleep lasting 6 h, similar to rats (A). Over the first 6 h after CNO injection, mice have reduced REM but no changes in wake or NREM sleep compared to the same period after saline injection. Each mouse experienced a decline in REM sleep time over 6 h after CNO injection compared to after saline injection. An example 3-hour hypnogram is shown for a single animal after injection with saline (D) or CNO (E). The theta/delta ratio, one marker of REM sleep, is plotted against time, with scored stages shown. Saline-injected animals alternate between wake, NREM, and REM sleep, while CNO-injected animals fail to initiate REM sleep episodes but continue to cycle between wake and NREM sleep.