DOI: 10.5061/dryad.6djh9w19b
title: Central amygdala somatostatin neurons modulate stress-induced sleep-onset insomnia

description: Sleep-onset insomnia, characterized by difficulty falling asleep, is
 linked to increased health risks. Previous studies have shown that the
 central amygdala (CeA) plays a crucial role in stress regulation, with the
 somatostatin neurons in the CeA (CeASST+) involved in adaptive stress
 responses. However, the role of CeASST+ neurons in stress-induced
 sleep-onset insomnia remains unclear. In this study, we found that the
 activity of CeASST+ neurons is closely associated with stressful
 events using fiber photometry in mice. Acute optogenetic activation of
 CeASST+ neurons induced a rapid transition from non-rapid eye
 movement (NREM) sleep to wakefulness. Semi-chronic optogenetic and
 chemogenetic activation of CeASST+ neurons led to prolonged sleep-onset
 latency and increased wakefulness. Chemogenetic inhibition of these
 neurons ameliorated sleep-onset insomnia induced by stressful stimuli but
 did not affect sleep-wake behavior under physiological conditions.
 Collectively, our results suggested that CeASST+ neurons are a
 key neural substrate for modulating stress-induced sleep-onset insomnia,
 without influencing physiological sleep. These findings highlight CeASST+
 neurons as a promising target for treating stress-related sleep-onset
 insomnia in clinical practice.
description: # Central amygdala somatostatin neurons modulate stress-induced
 sleep-onset insomnia
 [https://doi.org/10.5061/dryad.6djh9w19b](https://doi.org/10.5061/dryad.6djh9w19b) We have submitted our raw data (**original_data.xlsx**). This dataset contains the data used to generate figures. In this study, we found that the activity of CeASST+ neurons is closely associated with stressful events using fiber photometry in mice. Acute optogenetic activation of CeASST+ neurons induced a rapid transition from non-rapid eye movement (NREM) sleep to wakefulness. Semi-chronic optogenetic and chemogenetic activation of CeASST+ neurons led to prolonged sleep-onset latency and increased wakefulness. Chemogenetic inhibition of these neurons ameliorated sleep-onset insomnia induced by stressful stimuli, but did not affect sleep-wake behavior under physiological conditions. Collectively, our results suggested that CeASST+ neurons are a key neural substrate for modulating stress-induced sleep-onset insomnia, without influencing physiological sleep. ## Description of the data and file structure This repository contains all the raw data used in the article. Some of the raw data were processed using MATLAB for Ca2+ activity of CeASST+ neurons. Statistical analyses were conducted primarily in GraphPad Prism. Users are provided with the processed datasets required for replication, so access to MATLAB is not necessary. Sheet names correspond to the plots for the major figures and supplementary figures in the manuscript, categorized below based on behavioral metrics and experimental conditions. ### **original data.xlsx**\_Data This excel workbook contains all data sheets. #### **figure 1E-1F** This sheet contains raw data for heatmap and peri-event plot of the average Ca2+ transients values associated with physical restraint. Number of variables: 2 Number of header rows: 1 Number of rows: 762 Variable list: *Time: Time records before and after the event, using the moment of the event as the reference point (Time = 0). *Trial: An experiment conducted under defined conditions. Data type: alphanumeric, numeric #### **figure 1H-1I** This sheet contains raw data for heatmap and peri-event plot of the average Ca2+ transients values associated with rat bedding stress. Number of variables: 2 Number of header rows: 1 Number of rows: 762 Variable list: *Time: Time records before and after the event, using the moment of the event as the reference point (Time = 0). *Trial: An experiment conducted under defined conditions. Data type: alphanumeric, numeric #### **figure 1K-1L** This sheet contains raw data for heatmap and peri-event plot of the average Ca2+ transients values associated with air puff stress. Number of variables: 2 Number of header rows: 1 Number of rows: 762 Variable list: *Time: Time records before and after the event, using the moment of the event as the reference point (Time = 0). *Trial: An experiment conducted under defined conditions. Data type: alphanumeric, numeric #### **figure 1N-1O** This sheet contains raw data for heatmap and peri-event plot of the average Ca2+ transients values associated with cage change challenge. Number of variables: 2 Number of header rows: 1 Number of rows: 7200 Variable list: *Time: Time records before and after the event, using the moment of the event as the reference point (Time = 0). *Trial: An experiment conducted under defined conditions. Data type: alphanumeric, numeric #### **figure 2E-2F** This sheet contains raw data on NREM sleep, REM sleep and wakefulness before, during and after yellow or blue light stimulation in AAV-EF1a-DIO-hChR2(H134R)-mCherry mice. Number of variables: 6 Number of header rows: 4 Number of rows: 68 Variable list: *Epoch: A short, fixed-time segment of EEG (electroencephalogram) data. *Yellow light: Optogenetic stimulation using 589 nm yellow light. The time periods (rows 18 to 21) of stimulation are highlighted in yellow. *Blue light: Optogenetic stimulation using 473 nm blue light. The time periods (rows 55 to 58) of stimulation are highlighted in blue. *W=1: Defining the awake state as a numerical value of 1. *R=2: Defining the REM state as a numerical value of 2. *S=3: Defining the NREM state as a numerical value of 3. Data type: alphanumeric, numeric #### **figure 2H** This sheet contains raw data on sleep latency after yellow or blue light stimulation. Number of variables: 2 Number of header rows: 1 Number of rows: 10 Variable list: *Yellow light: Sleep latency after optogenetic stimulation using 589 nm yellow light. *Blue light: Sleep latency after optogenetic stimulation using 473 nm blue light. Data type: alphanumeric, numeric #### **figure 2J-2K** This sheet contains raw data on time course of wakefulness, NREM sleep, and REM sleep following yellow and blue light stimulation of the CeASST+ neurons from ZT 2 to ZT 3, and total time spent in each stage during the 1-hour exposure to yellow or blue light stimulation. Number of variables: 5 Number of header rows: 4 Number of rows: 54 Variable list: *Wake: The duration of wakefulness. *REM: The duration of REM (Rapid Eye Movement). *NREM: The duration of NREM (Non-Rapid Eye Movement). *Yellow light: Total time course after optogenetic stimulation using 589 nm yellow light. *Blue light: Total time course after optogenetic stimulation using 473 nm blue light. Data type: alphanumeric, numeric #### **figure 3B** This sheet contains raw data on sleep latency after yellow or blue light stimulation. Number of variables: 2 Number of header rows: 1 Number of rows: 9 Variable list: *Yellow light: Sleep latency after optogenetic stimulation using 589 nm yellow light. *Blue light: Sleep latency after optogenetic stimulation using 473 nm blue light. Data type: alphanumeric, numeric #### **figure 3C** This sheet contains raw data on time course of wakefulness, NREM sleep, and REM sleep following yellow or blue light stimulation during the ZT 2 - 5. Number of variables: 5 Number of header rows: 4 Number of rows: 36 Variable list: *Yellow light: Optogenetic stimulation using 589 nm yellow light. *Blue light: Optogenetic stimulation using 473 nm blue light. *Wake: The duration of wakefulness. *REM: The duration of REM (Rapid Eye Movement). *NREM: The duration of NREM (Non-Rapid Eye Movement). Data type: alphanumeric, numeric #### **figure 3D-3E** This sheet contains raw data on total time spent in each stage during the ZT 2 - 3 and ZT 3 - 5 exposure to yellow or blue light stimulation. Number of variables: 7 Number of header rows: 4 Number of rows: 36 Variable list: *ZT 2 - 3: The time period from 2 to 3 hours after the onset of the light period. *ZT 3 - 5: The time period from 3 to 5 hours after the onset of the light period. *Yellow light: Optogenetic stimulation using 589 nm yellow light. *Blue light: Optogenetic stimulation using 473 nm blue light. *Wake: The total time spent in wakefulness at each stage. *REM: The total time spent in REM (Rapid Eye Movement) at each stage. *NREM: The total time spent in NREM (Non-Rapid Eye Movement) at each stage. Data type: alphanumeric, numeric #### **figure 3F-3I** This sheet contains raw data on numbers of different episode lengths including wakefulness, REM sleep, and NREM sleep during ZT 3 - 5 exposure to blue or yellow light stimulation. Number of variables: 3 Number of header rows: 3 Number of rows: 18 Variable list: *Yellow light: Optogenetic stimulation using 589 nm yellow light. *Blue light: Optogenetic stimulation using 473 nm blue light. *Wake: The numbers of different episode lengths including wakefulness during ZT 3 - 5 exposure to optogenetic stimulation. *REM: The numbers of different episode lengths including REM sleep during ZT 3 - 5 exposure to optogenetic stimulation. *NREM: The numbers of different episode lengths including NREM sleep during ZT 3 - 5 exposure to optogenetic stimulation. Data type: alphanumeric, numeric #### **figure 4D** This sheet contains raw data on sleep latency after saline or 1mg/kg CNO injection. Number of variables: 2 Number of header rows: 1 Number of rows: 9 Variable list: *Vehicle: Sleep latency after saline injection. *CNO (1 mg/kg): Sleep latency after 1mg/kg CNO injection. CNO = clozapine N-oxide. Data type: alphanumeric, numeric #### **figure 4F-4G** This sheet contains raw data on time course of wakefulness, NREM sleep, and REM sleep following vehicle or CNO injection at ZT 2, and total time spent in each stage during the ZT 3 - 6 after intraperitoneal injection of vehicle or CNO. Number of variables: 5 Number of header rows: 4 Number of rows: 36 Variable list: *Wake: The duration of wakefulness. *REM: The duration of REM (Rapid Eye Movement). *NREM: The duration of NREM (Non-Rapid Eye Movement). *Vehicle: Total time course after saline injection. *CNO: Total time course after 1mg/kg CNO injection. Data type: alphanumeric, numeric #### **figure 4H-4K** This sheet contains raw data on numbers of different episode lengths including wakefulness, REM sleep, and NREM sleep during ZT 3 - 6 after vehicle and CNO (1 mg/kg) injection. Number of variables: 5 Number of header rows: 3 Number of rows: 18 Variable list: *Vehicle: Saline injection. *CNO: 1mg/kg CNO injection. *Wake: The numbers of different episode lengths including wakefulness during ZT 3 - 6 exposure to intraperitoneal injection. *REM: The numbers of different episode lengths including REM sleep during ZT 3 - 6 exposure to intraperitoneal injection. *NREM: The numbers of different episode lengths including NREM sleep during ZT 3 - 6 exposure to intraperitoneal injection. Data type: alphanumeric, numeric #### **figure 5C** This sheet contains raw data on Ca2+ activity (ΔF/F) peaks during wakefulness, NREM sleep, and REM sleep. Number of variables: 3 Number of header rows: 1 Number of rows: 40 Variable list: *Wake: Ca2+ activity (ΔF/F) peaks during wakefulness. *REM: Ca2+ activity (ΔF/F) peaks during REM sleep. *NREM: Ca2+ activity (ΔF/F) peaks during NREM sleep. Data type: alphanumeric, numeric #### **figure 5D** This sheet contains raw data on the Ca2+ activity of CeASST+ neurons during transitions from wakefulness to NREM. Number of variables: 2 Number of header rows: 1 Number of rows: 3050 Variable list: *Time: Time records before and after the event, using the moment of the event as the reference point (Time = 0). *Trial: An experiment conducted under defined conditions. Data type: alphanumeric, numeric #### **figure 5E** This sheet contains raw data on the Ca2+ activity of CeASST+ neurons during transitions from NREM to wakefulness. Number of variables: 2 Number of header rows: 1 Number of rows: 3050 Variable list: *Time: Time records before and after the event, using the moment of the event as the reference point (Time = 0). *Trial: An experiment conducted under defined conditions. Data type: alphanumeric, numeric #### **figure 5F** This sheet contains raw data on the Ca2+ activity of CeASST+ neurons during transitions from NREM to REM. Number of variables: 2 Number of header rows: 1 Number of rows: 3050 Variable list: *Time: Time records before and after the event, using the moment of the event as the reference point (Time = 0). *Trial: An experiment conducted under defined conditions. Data type: alphanumeric, numeric #### **figure 5G** This sheet contains raw data on the Ca2+ activity of CeASST+ neurons during transitions from REM to wakefulness. Number of variables: 2 Number of header rows: 1 Number of rows: 3050 Variable list: *Time: Time records before and after the event, using the moment of the event as the reference point (Time = 0). *Trial: An experiment conducted under defined conditions. Data type: alphanumeric, numeric #### **figure 6C** This sheet contains raw data on sleep latency after saline or CNO injection. Number of variables: 2 Number of header rows: 1 Number of rows: 8 Variable list: *Vehicle: Sleep latency after saline injection. *CNO (1 mg/kg): Sleep latency after 1 mg/kg CNO injection. Data type: alphanumeric, numeric #### **figure 6E-6F** This sheet contains raw data on time course of wakefulness, NREM sleep, and REM sleep following vehicle or CNO injection at ZT 2, and total time spent in each stage 2 hours after intraperitoneal injection of vehicle or CNO. Number of variables: 5 Number of header rows: 4 Number of rows: 36 Variable list: *Wake: The duration of wakefulness. *REM: The duration of REM (Rapid Eye Movement). *NREM: The duration of NREM (Non-Rapid Eye Movement). *Vehicle: Total time course after saline injection. *CNO: Total time course after 1 mg/kg CNO injection. Data type: alphanumeric, numeric #### **figure 7B** This sheet contains raw data on sleep latency after saline or CNO injection, following cage change. The cage is lined with soiled bedding from other mice. Number of variables: 2 Number of header rows: 1 Number of rows: 8 Variable list: *Dirty cage+Vehicle: Sleep latency after saline injection, following cage change. *Dirty cage+CNO (1 mg/kg): Sleep latency after 1 mg/kg CNO injection, following cage change. Data type: alphanumeric, numeric #### **figure 7D** This sheet contains raw data on time course of wakefulness, NREM sleep, and REM sleep after vehicle or CNO injection in cage change challenge. Number of variables: 5 Number of header rows: 4 Number of rows: 36 Variable list: *Dirty cage+Vehicle: Cage change challenge after saline injection. *Dirty cage+CNO (1 mg/kg): Cage change challenge after 1 mg/kg CNO injection. *Wake: The duration of wakefulness. *REM: The duration of REM (Rapid Eye Movement). *NREM: The duration of NREM (Non-Rapid Eye Movement). Data type: alphanumeric, numeric #### **Supplementary 1** This sheet contains raw data on the quantitative statistics of ΔF/F peaks during exposure to acute stress. ΔF = fractional change in fluorescence, and F = baseline fluorescence. Number of variables: 5 Number of header rows: 1 Number of rows: 35 Variable list: *Baseline: ΔF/F before the experimental operation. *Restraint: ΔF/F (fractional change in fluorescence) peaks after restraint. *Air puff: ΔF/F (fractional change in fluorescence) peaks during air puff. *Rat bedding: ΔF/F (fractional change in fluorescence) peaks while staying on the rat bedding. *Dirty cage: ΔF/F (fractional change in fluorescence) peaks following cage change. Data type: alphanumeric, numeric #### **Supplementary 2C-2D** This sheet contains raw data on normalized pupil size before, during and after blue light stimulation. Number of variables: 2 Number of header rows: 1 Number of rows: 7200 Variable list: *ChR2: Normalized pupil size before, during and after blue light stimulation in experimental mice. *Control: Normalized pupil size before, during and after blue light stimulation in control mice. Data type: alphanumeric, numeric #### **Supplementary 2F** This sheet contains raw data on the quantitative statistics of respiratory rate before and during blue light stimulation. Number of variables: 4 Number of header rows: 2 Number of rows: 7 Variable list: *ChR2: Quantitative statistics of respiratory rate before and during blue light stimulation in experimental mice. *Control: Quantitative statistics of respiratory rate before and during blue light stimulation in control mice. *Pre: Pre-light stimulation. *Stim: Light stimulation experiment. Data type: alphanumeric, numeric #### **Supplementary 3C** This sheet contains raw data on the quantification of time spent in center and periphery, and total distance traveled in the open field after yellow and blue light stimulation. Number of variables: 5 Number of header rows: 2 Number of rows: 8 Variable list: *Yellow light: Optogenetic stimulation using 589 nm yellow light. *Blue light: Optogenetic stimulation using 473 nm blue light. *In center: The duration of stay in the center during the open field. *In periphery: The duration of stay in the periphery during the open field. *Total distance: Total distance in the open field. Data type: alphanumeric, numeric #### **Supplementary 3F** This sheet contains raw data on the quantification of time spent in open arm and close arm and total distance traveled in the elevated plus maze after yellow and blue light stimulation. Number of variables: 5 Number of header rows: 2 Number of rows: 8 Variable list: *Yellow light: Optogenetic stimulation using 589 nm yellow light. *Blue light: Optogenetic stimulation using 473 nm blue light. *In open arms: The duration of stay in the open arms during the elevated plus maze. *In close arms: The duration of stay in the close arms during the elevated plus maze. *Total distance: Total distance in the elevated plus maze. Data type: alphanumeric, numeric #### **Supplementary figure 5A** This sheet contains raw data on the latencies of transition from NREM sleep to wakefulness upon yellow or blue light stimulation at different frequencies. Number of variables: 7 Number of header rows: 2 Number of rows: 10 Variable list: *Yellow light (589nm): Latencies of transition from NREM sleep to wakefulness upon yellow light stimulation at different frequencies. *Blue light (473nm): Latencies of transition from NREM sleep to wakefulness upon blue light stimulation at different frequencies. *Base: The light stimulation frequency parameter is set at 0 Hz as a baseline. *5: The light stimulation frequency parameter is set at 5 Hz. *10: The light stimulation frequency parameter is set at 10 Hz. *20: The light stimulation frequency parameter is set at 20 Hz. *40: The light stimulation frequency parameter is set at 40 Hz. Data type: alphanumeric, numeric #### **Supplementary figure 5B** This sheet contains raw data on the latencies of transition from NREM sleep to wakefulness upon yellow or blue light stimulation at different frequencies. Number of variables: 7 Number of header rows: 10 Number of rows: 10 Variable list: *Yellow light (589nm): Latencies of transition from NREM sleep to wakefulness upon yellow light stimulation at different frequencies. *Blue light (473nm): Latencies of transition from NREM sleep to wakefulness upon blue light stimulation at different frequencies. *Base: The light stimulation frequency parameter is set at 0 Hz as a baseline. *5Hz: The light stimulation frequency parameter is set at 5 Hz. *10Hz: The light stimulation frequency parameter is set at 10 Hz. *20Hz: The light stimulation frequency parameter is set at 20 Hz. *40Hz: The light stimulation frequency parameter is set at 40 Hz. Data type: alphanumeric, numeric #### **Supplementary figure 7B-7C** This sheet contains raw data on time course of wakefulness, NREM sleep, and REM sleep following vehicle or CNO injection at ZT 2 in controlmice, and total time spent in each stage 2 hours after intraperitoneal injection of vehicle or CNO, as a control experiment for figure 4. Number of variables: 5 Number of header rows: 4 Number of rows: 36 Variable list: *Wake: The duration of wakefulness. *REM: The duration of REM (Rapid Eye Movement). *NREM: The duration of NREM (Non-Rapid Eye Movement). *Vehicle: Total time course after saline injection. *CNO: Total time course after 1 mg/kg CNO injection. Data type: alphanumeric, numeric #### **Supplementary figure 8B-8C** This sheet contains raw data on time course of wakefulness, NREM sleep, and REM sleep following vehicle or CNO injection at ZT 2 in controlmice, and total time spent in each stage 2 hours after intraperitoneal injection of vehicle or CNO, as a control experiment for figure 6. Number of variables: 5 Number of header rows: 4 Number of rows: 36 Variable list: *Wake: The duration of wakefulness. *REM: The duration of REM (Rapid Eye Movement). *NREM: The duration of NREM (Non-Rapid Eye Movement). *Vehicle: Total time course after saline injection. *CNO: Total time course after 1 mg/kg CNO injection. Data type: alphanumeric, numeric #### **Supplementary figure 9C** This sheet contains raw data on sleep latency before the experimental operation and following cage change. Number of variables: 2 Number of header rows: 1 Number of rows: 10 Variable list: *Baseline: Sleep latency before the experimental operation. *Dirty cage: Sleep latency following cage change. Data type: alphanumeric, numeric #### **Supplementary figure 9E** This sheet contains raw data on time course of wakefulness, NREM sleep, and REM sleep in cage change challenge. Number of variables: 5 Number of header rows: 4 Number of rows: 36 Variable list: *Baseline: Before the experimental operation. *Dirty cage: Mice were transferred into the cage filled with dirty bedding. *Wake: The duration of wakefulness. *REM: The duration of REM (Rapid Eye Movement). *NREM: The duration of NREM (Non-Rapid Eye Movement). Data type: alphanumeric, numeric #### **Supplementary figure 10B** This sheet contains raw data on sleep latency following cage change. Number of variables: 2 Number of header rows: 1 Number of rows: 10 Variable list: *Dirty cage+Vehicle: Sleep latency after saline injection, following cage change. *Dirty cage+CNO (1 mg/kg): Sleep latency after 1 mg/kg CNO injection, following cage change. Data type: alphanumeric, numeric #### **Supplementary figure 10D** This sheet contains raw data on time course of wakefulness, NREM sleep, and REM sleep after vehicle or CNO injection in cage change challenge, as a control experiment for figure 7. Number of variables: 5 Number of header rows: 4 Number of rows: 36 Variable list: *Dirty cage+Vehicle: Cage change challenge after saline injection. *Dirty cage+CNO (1 mg/kg): Cage change challenge after 1 mg/kg CNO injection. *Wake: The duration of wakefulness. *REM: The duration of REM (Rapid Eye Movement). *NREM: The duration of NREM (Non-Rapid Eye Movement). Data type: alphanumeric, numeric #### **Supplementary figure 11C** This sheet contains raw data on sleep latency before the experimental operation and after restraint. Number of variables: 2 Number of header rows: 1 Number of rows: 10 Variable list: *Baseline: Sleep latency before the experimental operation. *Restraint: Sleep latency after restraint. Data type: alphanumeric, numeric #### **Supplementary figure 11E** This sheet contains raw data on time course of wakefulness, NREM sleep, and REM sleep in restraint experiment. Number of variables: 5 Number of header rows: 4 Number of rows: 36 Variable list: *Baseline: Before the experimental operation. *Restraint: Mice were confined to a 50 mL centrifuge tube for 1 hour. *Wake: The duration of wakefulness. *REM: The duration of REM (Rapid Eye Movement). *NREM: The duration of NREM (Non-Rapid Eye Movement). Data type: alphanumeric, numeric #### **Supplementary figure 12B** This sheet contains raw data on sleep latency after saline or CNO injection and restraint. Number of variables: 2 Number of header rows: 1 Number of rows: 10 Variable list: *Restraint+Vehicle: Sleep latency after saline injection and restraint. *Restraint+CNO (1 mg/kg): Sleep latency after 1 mg/kg CNO injection and restraint. Data type: alphanumeric, numeric #### **Supplementary figure 12D** This sheet contains raw data on time course of wakefulness, NREM sleep, and REM sleep after vehicle or CNO injection in restraint experiment, as a control experiment for figure 7. Number of variables: 5 Number of header rows: 4 Number of rows: 36 Variable list: *Restraint+Vehicle: Restrained in a centrifuge tube for 1 hour after saline injection. *Restraint+CNO (1 mg/kg): Restrained in a centrifuge tube for 1 hour after 1 mg/kg CNO injection. *Wake: The duration of wakefulness. *REM: The duration of REM (Rapid Eye Movement). *NREM: The duration of NREM (Non-Rapid Eye Movement). Data type: alphanumeric, numeric ##### **Software** GraphPad Prism 8.0 MATLAB R2019b