An excitatory peri-tegmental reticular nucleus circuit for wake maintenance

Abstract

Sleep is a necessity for our survival, but its regulation remains incompletely understood. Here, we used a human sleep duration gene to identify a population of cells in the peri-tegmental reticular nucleus (pTRN^ADRB1) that regulate sleep-wake, uncovering a role for a poorly understood brain area. Although initial ablation in mice led to increased wakefulness, further validation revealed that pTRN^ADRB1 neuron stimulation strongly promotes wakefulness, even after stimulation offset. Using combinatorial genetics, we found that excitatory pTRN^ADRB1 neurons promote wakefulness. pTRN neurons can be characterized as anterior- or posterior-projecting neurons based on multiplexed analysis of projections by sequencing (MAPseq) analysis. Finally, we found that pTRN^ADRB1 neurons promote wakefulness, in part, through projections to the lateral hypothalamus. Thus, human genetic information from a human sleep trait allowed us to identify a role for the pTRN in sleep-wake regulation.

Keywords: FNSS; pTRN; short sleep; sleep; wake.

Fig. 1.

Discovery of the pTRN region in sleep–wake regulation. (A) Total mobile time averaged over 4 consecutive days with video tracking for ADRB1-Cre (n = 4 to 5) and WT (n = 4 to 5) littermate mice injected with AAV1.hSyn.Flex.taCasp3.TEVp into the indicated regions. (WT vs. Cre, P = 0.0225, random sampling with replacement bootstrap, 10,000 iterations, ns P > 0.05, *P < 0.05). See also SI Appendix, Fig. S1. (B) Schematic showing the coronal location of the pTRN. The Inset shows pTRN^ADRB1 neurons from an ADRB1-Cre mouse crossed to an L10a reporter. (Scale bar, 400 µm.) (C) Experimental schematic for caspase ablation combined with EEG/EMG recording. (D) Hour-by-hour quantification of wake (Left), NREM (Middle), or REM (Right) over a 24-h period from ADRB1-Cre (n = 5) or WT littermate (n = 6) mice averaged over 2 consecutive days. The lights-on period (12 h) is denoted by a yellow bar, while the lights-off period is denoted by a black bar. (WT vs. Cre, two-way RM ANOVA with post hoc Sidak’s multiple comparisons test, *P < 0.05, **P < 0.01, ***P < 0.001). See also SI Appendix, Fig. S3. (E) Total time spent in wake (Left), NREM (Middle), or REM (Right) from D. (WT vs. Cre, wake: P = 0.0002, NREM: P = 0.0002, REM: P = 0.044, Student’s two-sided t test, *P < 0.05, ***P < 0.001). (F and G) Representative in situ staining from a caspase-injected WT (F) or ADRB1-Cre (G) mouse. (Scale bar, 60 µm.) (H) Quantification of cells positive for Adrb1 using in situ staining from ADRB1-Cre (n = 5) or WT littermate (n = 6) mice from D (WT vs. Cre, P = 0.0001, Student’s two-sided t test, ***P < 0.001). (I) Quantification of sleep–wake epoch transition changes from ADRB1-Cre (n = 5) or WT littermate (n = 6) mice. There was an increase in wake-to-wake epoch transition probability and a decrease in wake-to-NREM epoch transition probability for ADRB1-Cre vs. WT mice. Thicker lines imply increases in state change, while thinner lines imply decreases in state change in ablated mice (WT vs. Cre, wake-to-wake transition: P = 0.0006, wake-to-NREM transition: P = 0.0006, Student’s two-sided t test, ***P < 0.001). (J) Quantification of wake, NREM, and REM bout number from D (WT vs. Cre, wake: P = 0.0032, NREM: P = 0.0013, REM: P = 0.2214, Student’s two-sided t test, ns P > 0.05, **P < 0.01). (K) Quantification of wake, NREM, and REM bout length from D (WT vs. Cre, wake: P = 0.0076, NREM: P = 0.627, REM: P = 0.9816, Student’s two-sided t test, ns P > 0.05, **P < 0.01). BLA, basolateral amygdala; DC, dorsal cochlea; dscp, superior cerebellar peduncle decussation; LH, lateral hypothalamus; mcp, middle cerebellar peduncle; pTRN, peri-tegmental reticular nucleus; PG, pontine gray; PGG, paragigantocellular nucleus; PN, pedunculopontine nucleus; PZ, parafacial nucleus; RN, nucleus of reunions. Error bars represent ± SEM for D, J, and K. For A, E, and H, the boxes show the quartiles, and the whiskers show the rest of the distribution.

Fig. 2.

Endogenous activity of pTRN neurons. (A) Experimental schematic for simultaneous 32-channel electrophysiology and EEG/EMG recording. (B) Histology showing silicon probe placement stained with Cd11b (green), a marker for microglia, which are activated near the probe track. (Scale bar, 200 µm.) (C) Trace of 20 spikes from a representative unit. (D) The Z-scored firing rate of the isolated units between sleep states from WT mice (n = 3 mice, n = 34 units) (wake vs. NREM: P < 0.0001, wake vs. REM: P < 0.0001, NREM vs. REM: P = 0.0002, repeated measures one-way ANOVA with post hoc Sidak’s multiple comparisons test, ***P < 0.001, ****P < 0.0001). See also SI Appendix, Fig. S4. (E) REM–NREM activity difference vs. wake–NREM activity difference from D. Each circle represents one unit. (F) Experimental schematic for imaging bulk calcium fluorescence from pTRN^ADRB1 neurons. (Scale bar, 50 µm.) (G) Histology showing the probe track (outlined in yellow) combined with GCaMP signal. (Scale bar, 150 µm.) (H) Z-scored fluorescence vs. sleep–wake state from ADRB1-Cre mice (n = 4) injected with AAV1.Syn.Flex.GCaMP6s (wake vs. NREM: P = 0.0310, wake vs. REM: P = 0.6024, NREM vs. REM: P = 0.0310, repeated measures one-way ANOVA with post hoc Sidak’s multiple comparisons test, *P < 0.05). (I) Z-scored calcium fluorescence peristimulus time histograms for NREM-to-wake (n = 191), wake-to-NREM (n = 107), NREM-to-REM (n = 114), and REM-to-wake (n = 73) transitions averaged over all trials across n = 4 mice. Gray bars represent SEM. (J) Z-scored heatmaps from I. The intensity of blue corresponds with the ΔF/F value. (K) Experimental setup for imaging pTRN^ADRB1 neurons using a miniscope. (L) The probe track (outlined in yellow) above pTRN^ADRB1 neurons expressing GCaMP7f. (Scale bar, 200 µm.) (M) REM–NREM activity difference vs. wake–NREM activity difference. Each circle represents one cell (n = 4 mice, n = 38 cells). For D and H, the boxes show the quartiles, and the whiskers show the rest of the distribution. PG, pontine gray; mcp, middle cerebellar peduncle.

Fig. 3.

Activation of pTRN^ADRB1 neurons promotes wakefulness. (A, Top) Schematic to express Gq or mCherry in pTRN^ADRB1 neurons. (Bottom) Representative histology. (Scale bar, 400 µm.) (B) Hour-by-hour quantification of time spent in wake (Left), NREM (Middle), or REM (Right) in Gq (n = 7) or mCherry (n = 7) mice for 6 h post-CNO or saline injection at ZT1 (Gq-Sal vs. Gq-CNO, two-way RM ANOVA with post hoc Sidak’s multiple comparisons test, *P < 0.05, **P < 0.01, ****P < 0.0001). (C) Total time in the 6-h postinjection period spent in wake (Left), NREM (Middle), and REM (Right) from B (wake Gq-Sal vs. Gq-CNO: P = 0.0240, wake Gq-CNO vs. mCherry-CNO: P = 0.0053, wake mCherry-Sal vs. mCherry-CNO: P = 0.1219; NREM Gq-Sal vs. Gq-CNO: P = 0.0432, NREM Gq-CNO vs. mCherry-CNO: P = 0.0109, NREM mCherry-Sal vs. mCherry-CNO: P = 0.2933; REM Gq-Sal vs. Gq-CNO: P = 0.0247, REM Gq-CNO vs. mCherry-CNO: P = 0.0002, REM mCherry-Sal vs. mCherry-CNO: P = 0.7925, repeated measures one-way ANOVA with post hoc Sidak’s multiple comparisons test, ns P > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001). See also SI Appendix, Figs. S5 and S6. (D) Schematic showing the setup to express ChR2 or EYFP in pTRN^ADRB1 neurons. (E) Histology showing probe track (outlined in yellow) in the pTRN. (Scale bar, 400 µm.) (F) Effect of optogenetic activation (n = 6 mice, n = 300 trials). The blue stripe shows when the light-emitting diode (LED) is on (20 Hz, 90 s). Error bars represent the 95% confidence interval from 10,000 iterations. LED stimulation increased wake and decreased NREM and REM. Yellow and purple bars are for time periods analyzed in H (increase in wake: P < 0.0001, decrease in NREM: P < 0.0001, decrease in REM: P = 0.0001, bootstrap). See also SI Appendix, Fig. S7. (G) Same as in F but for EYFP-injected mice (n = 6 mice, n = 300 trials). There was no change in sleep–wake states when analyzed by bootstrapping (increase in wake: P = 0.0768, decrease in NREM: P = 0.2358, decrease in REM: P = 0.1225, bootstrap). (H) LED stimulation was able to increase the percentage of wakefulness 2 to 3 min after stimulation (purple) compared with 2 to 3 min before stimulation (yellow) from F. The time periods from F analyzed are denoted with yellow and purple bars (3 min before vs. 3 min after, P = 0.0063, Student’s two-sided t test, **P < 0.01). Error bars represent ± SEM for B. For C and H, the boxes show the quartiles, and the whiskers show the rest of the distribution. mcp, middle cerebellar peduncle; PG, pontine gray.

Fig. 4.

Excitatory pTRN^ADRB1 neurons promote wake. (A) Schematic showing the approach to express ChR2 in excitatory pTRN^ADRB1 neurons. (B) Effect of optogenetic activation (n = 6 mice, n = 275 trials). The blue stripe shows when the LED is on (20 Hz, 90 s). Error bars represent the 95% confidence interval from 10,000 iterations. LED stimulation increased wake, decreased NREM, and led to no change in REM. Yellow and purple bars are for time periods analyzed in C (increase in wake: P < 0.0001, decrease in NREM: P < 0.0001, decrease in REM: P = 0.40, bootstrap). See also SI Appendix, Fig. S9. (C) LED stimulation was able to increase the percentage of wakefulness 2 to 3 min after stimulation (purple) compared with 2 to 3 min before stimulation (yellow) from B. The time periods analyzed from B are denoted with yellow and purple bars (3 min before vs. 3 min after, P = 0.0203, Student’s two-sided t test, *P < 0.05). (D) Schematic showing the approach to activating inhibitory pTRN^ADRB1 neurons. (E) LED stimulation led to a slight increase in wake, a decrease in NREM, and no change in REM. Yellow and purple bars are for time periods analyzed in F (n = 6 mice, n = 300 trials) (wake: P = 0.0046, NREM: P = 0.0039, REM: P = 0.683, bootstrap). (F) Quantification of yellow and purple bar time periods from E (3 min before vs. 3 min after, P = 0.48, Student’s two-sided t test, ns P > 0.05). (G) Approach to stimulating all excitatory pTRN neurons as a control for A. (H) Effect of LED stimulation (n = 6 mice, n = 300 trials) for G. LED stimulation led to an increase in wake and decreases in NREM and REM (increase in wake: P < 0.0001, decrease in NREM: P < 0.0001, decrease in REM: P = 0.0217, bootstrap). Yellow and purple bars are for time periods analyzed in I. (I) Quantification of yellow and purple bar time periods from H (3 min before vs. 3 min after, P = 0.68, Student’s two-sided t test, ns P > 0.05). (J) Schematic showing the approach to expressing ChR2 in inhibitory non-ADRB1 pTRN neurons as a control for D. (K) Effect of LED stimulation (n = 6 mice, n = 295 trials) for J. LED stimulation led to no change in wake, NREM, or REM state (increase in wake: P = 0.4423, decrease in NREM: P = 0.2784, decrease in REM, P = 0.9159, bootstrap). Yellow and purple bars are for time periods analyzed in L. (L) Quantification of yellow and purple bar time periods from K (3 min before vs. 3 min after, P = 0.91, Student’s two-sided t test, ns P > 0.05). Error bars represent the 95% confidence interval from 10,000 iterations for B, E, H, and K. For C, F, I, and L the boxes show the quartiles, and the whiskers show the rest of the distribution.

Fig. 5.

Projection patterns of pTRN neurons. (A) MAPseq overview. Briefly, a barcoded Sindbis virus was injected, and eight brain regions (gray circles) involved in sleep–wake regulation were dissected 42 h later for sequencing and subsequent analysis. (B) The cumulative percentages of neurons that project to the sleep–wake brain areas were analyzed. Nearly all neurons projected to four or fewer areas. (C) PCA of the projection neurons from a binarized projection matrix. (D) Heatmap of different areas to which the LH-projecting neurons also projected. (E) Heatmap of different areas to which the PZ-projecting neurons also projected. See also SI Appendix, Fig. S10. (F) The number of pTRN neurons projecting to each projection region. (G) Count number for pTRN projection motifs. (H, Top) Schematic of the approach for tracing pTRN^ADRB1 processes through the entire brain. (Bottom) Representative image of the pTRN injection site. (Scale bar, 400 µm.) (I) Example of a three-dimensional reconstruction of pTRN^ADRB1 input to the brain by signal intensity. (J) Quantification of the relative input from pTRN^ADRB1 neurons to different brain regions (n = 3 mice). Nomenclature adopted from the Allen Brain Atlas: ACA, anterior cingulate area; ADP, anterodorsal preoptic nucleus; AHN, anterior hypothalamic nucleus; AON, anterior olfactory nucleus; AT, anterior tegmental nucleus; ATN, anterior group of the dorsal thalamus; AUD, auditory areas; AVP, anteroventral preoptic nucleus; AVPV, anteroventral periventricular nucleus; BF, basal forebrain; BLA, basolateral amygdala; BMA, basomedial amygdala; CA, Ammon’s horn; CAL, claustrum; CN, cochlear nuclei; COA, cortical amygdala area; CS, superior central nucleus raphe; DG, dentate gyrus; DMH, dorsomedial hypothalamus; DN, dentate nucleus; DTN, dorsal tegmental nucleus; EP, endopiriform nucleus; EPI, epithalamus; EW, Edinger-Westphal nucleus; GENv, geniculate group, ventral thalamus; GRN, gigantocellular reticular nucleus; GU, gustatory areas; HEM, hemispheric regions; III, ocularmotor nucleus; ILM, intralaminar nuclei of the dorsal thalamus; LAT, lateral group of the dorsal thalamus; LDT, laterodosal tegmental nucleus; LH, lateral hypothalamus; LPO, lateral preoptic nucleus; LSX, lateral septal complex; MA3, medial accessory oculomotor nucleus; MBO, mammillary body; MED, medial group of the dorsal thalamus; MEPO, median preoptic nucleus; mcp, middle cerebellar peduncle; MO, somatomotor areas; MPO, medial preoptic nucleus; MRN, midbrain reticular nucleus; MT, medial terminal nucleus of the accessory optic tract; NI, nucleus incertus; NLOT, nucleus of the lateral olfactory tract; OLF, olfactory areas; ORB, orbital area; Pa4, paratrochlear nucleus; PAA, piriform amygdalar area; PAG, periaqueductal gray; PAR, parasubiculum; PDTg, posterodorsal tegmental nucleus; PeF, perifornical nucleus; PG, pontine gray; PGRN, paragigantocellular reticular nucleus; PH, posterior hypothalamic nucleus; PHY, perihypoglossal nuclei; PIR, piriform area; PMd, dorsal premammillary nucleus; PNG, paranigral nucleus; PPN, pedunculopontine nucleus; PRE, presubiculum; PRNc, pontine reticular nucleus-ventral part; PRNr, pontine reticular nucleus; PRT, pretectal region; PS, parastrial nucleus; PSTN, parasubthalamic nucleus; PVH, paraventricular hypothalamic nucleus; PVT, paraventricular nucleus of the thalamus; PZ, parafacial zone; RAmb, midbrain raphe nuclei; RN, red nucleus; RPO, nucleus raphe pontis; RR, midbrain reticular nucleus-retrorubral area; RSP, retrosplenial area; RT, reticular nucleus of the thalamus; sAMY, striatum-like amygdalar nuclei; SCm, superior colliculus, motor related; SG, supragenual nucleus; SLD, sublaterodorsal nucleus; SNc, substantia nigra—compact part; SNr, substantia nigra—reticular part; SPA, subparafascicular area; SPF, subparafascicular nucleus; SS, somatosensory areas; STN, subthalamic nucleus; STR, striatum; SUB, subiculum; TT, taenia tecta; TU, tuberal nucleus; VENT, ventral group of the dorsal thalamus; VERM, vermal regions; VIS, visual areas; VLPO, ventrolateral preoptic nucleus; VMH, ventromedial hypothalamic nucleus; VNC, vestibular nuclei; VTA, ventral tegmental area; VTN, ventral tegmental nucleus; ZI, zona incerta.

Fig. 6.

Output circuits of pTRN^ADRB1 neurons. (A) Schematic for imaging pTRN^ADRB1→LH axons. (B) Histology showing the probe track (outlined in white) above the pTRN^ADRB1 processes (green). (Scale bar, 200 µm.) (C) Z-scored ΔF/F of pTRN^ADRB1→LH terminals. The box shows the quartiles, and the whiskers show the rest of the distribution (wake vs. NREM: P = 0.023, wake vs. REM: P = 0.0264, repeated measures one-way ANOVA with post hoc Sidak’s multiple comparisons test, *P < 0.05). (D) Z-scored calcium fluorescence peristimulus time histograms for NREM-to-wake, wake-to-NREM, NREM-to-REM, and REM-to-wake transitions, averaged over all trials. Gray bars represent SEM. (E) Z-scored heatmaps from D. The intensity of blue corresponds to the ΔF/F value. (F) Schematic showing the strategy to activate pTRN^ADRB1 axons in the LH. (G) Histology showing the probe track (outlined in white) near the pTRN^ADRB1 axons (red). (Scale bar, 200 µm.) (H) Effect of optogenetic activation (n = 6 mice, n = 300 trials). The blue stripe shows when the LED is on (20 Hz, 90 s). Error bars represent the 95% confidence interval from 10,000 iterations. LED stimulation increased wake and decreased NREM and REM (increase in wake: P < 0.0001, decrease in NREM: P < 0.0001, decrease in REM: P = 0.0003, bootstrap). See also SI Appendix, Figs. S11 and S12. (I, Top) pTRN^ADRB1 neurons were injected with ChR2, and an acute slice was taken from the LH. In the example trace of a patched neuron from an acute LH slice, blue denotes when the blue LED was on, and the inward current from the patched neuron is shown in red. The black trace is the same neuron but with the excitatory blockers CNQX (20 µM) and APV (50 µM). (Middle) The latency between LED stimulation and inward current deflection for the 21 connected neurons. (Bottom) Example gel for the single-cell RT-PCR for the four genes examined. (J, Top) Percentages of each of the connected cells expressing each gene (n = 3 mice, n = 21 cells). (Bottom) A matrix showing the expression and overlap of each gene for each individual connected cell. See also SI Appendix, Fig. S13. (K) Schematic showing the strategy to activate pTRN^ADRB1 terminals in the PZ. (L) Example of a histology image of the probe track (outlined in white) and fibers (red) in the PZ. (Scale bar, 200 µm.) (M) Effect of optogenetic activation (n = 6 mice, n = 300 trials). LED stimulation increased wake and REM and decreased NREM (increase in wake: P < 0.0001, decrease in NREM: P < 0.0001, increase in REM: P = 0.0002, bootstrap).