Calcium imaging of sleep-wake related neuronal activity in the dorsal pons

Abstract

The dorsal pons has long been implicated in the generation of rapid eye movement (REM) sleep, but the underlying circuit mechanisms remain poorly understood. Using cell-type-specific microendoscopic Ca(2+) imaging in and near the laterodorsal tegmental nucleus, we found that many glutamatergic neurons are maximally active during REM sleep (REM-max), while the majority of GABAergic neurons are maximally active during wakefulness (wake-max). Furthermore, the activity of glutamatergic neurons exhibits a medio-lateral spatial gradient, with medially located neurons more selectively active during REM sleep.

Figure 1. Calcium imaging from GABAergic neurons in the dorsal pons.

(a) Schematic of microendoscopic Ca²⁺ imaging and brain state recording. (b) An example imaging session from a GAD2-IRES-Cre mouse. Left panels: imaging field (upper) and identified ROIs corresponding to neuronal somas (lower). Grey masks and numbers indicate ROIs whose ΔF/F traces are plotted on the right. Scale bar, 100 μm. Right panels: EEG power spectrogram, EMG trace and ΔF/F traces. Brain states are indicated by coloured shading. Grey, wake; white, NREM; blue, REM (c) Z-scored ΔF/F activity during wakefulness, NREM and REM sleep averaged across all GABAergic neurons imaged in six mice (n=130). F_2,129=160.7, P=4.7 × 10⁻⁴⁶, one-way repeated measures ANOVA; **P<0.01, two-tailed Tukey's post hoc comparison. (d) Difference between REM and NREM activity versus difference between wake and NREM activity. Each symbol represents one neuron (n=130). Diagonal line: equal activity between wake and REM states. The majority of GABAergic neurons were more active during wake than REM states (below diagonal). (e) ΔF/F activity at brain state transitions, averaged across all GABAergic neurons. Grey shading, ±s.e.m.

Figure 2. Calcium imaging from glutamatergic neurons in the dorsal pons.

(a) Example imaging sessions from two VGLUT2-IRES-Cre mice. Shown in each example are imaging field, identified ROIs, EEG power spectrogram, EMG trace and ΔF/F traces of example ROIs across brain states (coloured shading). Grey, wake; white: NREM; blue: REM. Scale bars, 100 μm. (b) Z-scored ΔF/F activity during wakefulness, NREM sleep and REM sleep averaged across all glutamatergic neurons imaged in seven mice (n=104). F_2,103=234.6, P=8.0 × 10⁻⁵⁴, one-way repeated measures ANOVA; **P<0.01, two-tailed Tukey's post hoc comparisons. (c) Difference between REM and NREM activity versus difference between wake and NREM activity. Each symbol represents one neuron (n=104). Diagonal line: equal activity between wake and REM states. Most of the glutamatergic neurons were above the diagonal. (d) ΔF/F activity at brain state transitions averaged across all glutamatergic neurons. Grey shading, ±s.e.m. (e) Distributions of relative REM versus wake activity of GABAergic and glutamatergic neurons (>0, more active during REM than wakefulness).

Figure 3. Spatial distributions of different functional types of glutamatergic neurons.

(a) Scatter plot of both glutamatergic and GABAergic neurons in the space spanned by the first two principal components (PC1 versus PC2) of ΔF/F activity at brain state transitions (n=220). The cluster identity determined with k-means clustering is indicated by colour. The centroids of Clusters 1 and 2 are marked as c1 and c2. (b) REM–NREM versus wake–NREM activity of all glutamatergic and GABAergic neurons in the three clusters (cluster identity is colour-coded). Note that neurons in Clusters 1 and 3 are REM- and wake-max, respectively. Neurons in Cluster 2 showed weaker brain-state modulation than those in Clusters 1 and 3, but their average activity is wake-max. (c) The cell-type composition of each cluster. (d) ΔF/F activity at brain state transitions averaged across neurons within each cluster (cluster identity is colour-coded). Grey shading, ±s.e.m. (e) Spatial distribution of sleep-wake activity of glutamatergic neurons. Brain-state-dependent activity of each neuron is quantified by the relative distances from the centroids of Cluster 1 (c1 in a, REM-max) and Cluster 2 (c2, wake-max), coded by colour. Top panels: spatial organization of neuronal functional properties in two example VGLUT2-IRES-Cre mice. Scale bars, 100 μm. Lower panel: spatial organization of all glutamatergic neurons imaged in seven mice. Different symbols indicate cluster identity.