DOI: 10.5061/dryad.8cz8w9h59
title: Inactive but awake behaviour as indicating a depression-like state in mice: Aetiological factors and association with adult hippocampal neurogenesis

description: In laboratory mice, ‘inactive but awake’ (IBA) home-cage behaviour
 involves animals being spontaneously motionless with eyes open, not
 interacting with their surroundings. Conventional (barren) housing
 typically triggers IBA more than comparatively enriched environments.
 Compellingly greater IBA is associated with some depression-like features
 in mice and we further explored this through three aims. First, we aimed
 to replicate previous results highlighting environmental and genetic
 (using two strains of mice: DBA/2J and C57BL/6J) aetiological contributors
 to IBA. Second, we explored whether the performance of IBA varied as the
 level of enrichment was either increased or reduced. Third, we
 opportunistically investigated whether elevated IBA predicted lower
 density of immature neurons in the dorsal (dDG) or ventral dentate gyrus
 (vDG) of the hippocampus. As expected, mice housed in conventional cages
 displayed more IBA than those in comparatively enriched cages and even
 more so in DBA/2J mice. As predicted enrichment loss generally increased
 IBA while enrichment gain decreased IBA. Unsurprisingly, immature neuron
 density was lower in conventional compared to enriched cages, although
 only for vDG. Elevated IBA predicted reduced immature neuron density in
 the dDG, and this effect tended to be stronger for C57BL/6Js.
description: # Inactive but awake behaviour as indicating a depression-like state in
 mice: Aetiological factors and association with adult hippocampal
 neurogenesis Dataset DOI:
 [10.5061/dryad.8cz8w9h59](10.5061/dryad.8cz8w9h59) ## Description of the
 data and file structure *Animals, housing and husbandry* Thirty-one female
 C57BL/6J (C57, hereafter) and 31 female DBA/2J (DBA, hereafter) mice were
 pair-housed post-weaning in either highly enriched (N = 15 cages) or
 non-enriched (N = 16 cages) transparent *Techniplast* cages. *Behavioural
 scan sampling of mice* The behaviour relevant to the hypothesis under test
 was being *inactive but awake* (IBA hereafter), defined  as ‘mouse
 motionless, muzzle in sight and eyes open, for at least 15s’. All
 observations were conducted during the animals’ dark (active) phase under
 red ambient light, for three weeks, four days per week, over four
 90-minute time blocks per day: 9:30-11:00, 11:30-13:00, 13:45-15:15,
 15:45-17:15. Behaviour was recorded *via* live scan-sampling, switching
 from scan to 15s focal sampling to allow for differentiation between
 behaviours characterised by a lack of movement (*e.g.* IBA *versus*
 sleeping). In total, 24 scan samples were taken per mouse each day, spread
 evenly across the four blocks (totalling 288 scan samples/mouse for this
 first phase of observation (IBA Observation 1, Pre-adjustment Phase)).
 Environmental adjustment, *i.e.* moving into the alternative environment,
 or staying in the initial environment for control groups, happened on the
 day following the last day of observation in the Pre-adjustment Phase.
 Home-cage behaviour was monitored again for three consecutive weeks
 starting 2 days after environmental adjustment (IBA Observation 2,
 Post-adjustment Phase), following the above-described procedure and also
 totalling 288 scan samples/mouse during this second phase of observation.
 Finally, we had the opportunity to observe IBA in the mice during the
 8^th^ week following environmental adjustment (IBA Observation 3,
 Post-adjustment Phase). This followed the procedure outlined for
 Observations 1 and 2 with the exception that behavioural observations took
 place over one week instead of three, totalling 96 scan samples/mouse
 during this observation. In the attached data files, the overall mean
 proportion of visible scans spent performing IBA has been calculated for
 each observation period. When analysing the change in IBA between
 observations, the change in mean IBA has been calculated. We also present
 the immature neuron density in the dorsal (dDG) and ventral (vDG) regions
 of the dentate gyrus in the hippocampus for each mouse (alongside its IBA
 during observation 3).  ### Files and variables #### File:
 Final_code_All_Observations_Absolute_values_Figure_5.R
 **Description:** The R script code used to analyse the mean proportion of
 visible time each mouse spent performing IBA during each of the 3
 observation periods and to generate Figure 5 in the manuscript. #### File:
 Proportion_of_time_spent_IBA_during_Observation_1.xlsx
 **Description:** The data used to analyse the mean proportion of visible
 time each mouse spent performing IBA during observation 1. ##### Variables
 * **Mouse** Unique Individual Mouse ID (range 1-62 mice) - **Cage**   
 Individual Cage ID (range 1-31 mice) - **Strain**  Strain of mouse: DBA =
 DBA/2J; C57 = C57BL/6J. One of each strain was housed in each cage -
 **Ttm**       Treatment: NE (housed initially in non-enriched condition
 i.e. for observation 1); EE (housed initially in enriched condition i.e.
 for observation 1) - **IBA**        Proportion of visible scans the mouse
 spent displaying Inactive but awake (IBA) behaviour during observation 1
 #### File:
 Proportion_of_time_spent_IBA_while_visible_all_obs_Figure_5.xlsx
 **Description:** The data used to analyse the mean proportion of visible
 time each mouse spent performing IBA during each of the 3 observation
 periods and to generate Figure 5 in the manuscript. ##### Variables *
 **Mouse** Unique Individual Mouse ID (range 1-62 mice) - **Cage**   
 Individual Cage ID (range 1-31 mice) - **Strain**  Strain of mouse: DBA =
 DBA/2J; C57 = C57BL/6J. One of each strain was housed in each cage -
 **Observation**  a=Observation 1 (Days 4-22: During the first 3 weeks of
 housing following a 3 day acclimatisation); b=Observation 2 (Days 25-41:
 During the 3 weeks immediately following the environmental adjustment,
 where the environment was adjusted); c=Observation 3 (Days 74-79: During
 the 8th week following the environmental adjustment) - **Ttm**      
 Treatment: NE-NE (stably housed in non-enriched condition throughout
 experiment); EE-EE (stably housed in enriched condition throughout the
 experiment); NE-EE (started in the non-enriched condition and moved to the
 enriched housing when the environmental adjustment occurred); EE-NE
 (started in the enriched condition and moved to the non-enriched housing
 when the environmental adjustment occurred) - **IBA**        Proportion of
 visible scans the mouse spent displaying Inactive but awake (IBA)
 behaviour #### File:
 Proportion_of_time_spent_IBA_while_visible_obs_3_plus_cells_per_mm_in_vDG_and_dDG.xlsx **Description:** The data used to analyse the association between the mean proportion of visible time each mouse spent performing IBA during observation 3 and the immature neuron density (cells/mm) in the ventral (vDG) and dorsal (dDG) regions of the dentate gyrus in the hippocampus. ##### Variables * **Mouse**  Unique Individual Mouse ID (range 1-62 mice) - **Cage**    Individual Cage ID (range 1-31 mice) * **Strain**  Strain of mouse: DBA = DBA/2J; C57 = C57BL/6J. One of each strain was housed in each cage - **Ttm**       Treatment: NE-NE (stably housed in non-enriched condition throughout experiment); EE-EE (stably housed in enriched condition throughout the experiment) * **IBA**        Proportion of visible scans the mouse spent displaying Inactive but awake (IBA) behaviour during observation 3 - **Dorsal** Cells per mm in the Dorsal region of the dentate gyrus in the hippocampus. Measure taken at end of life. * **Ventral** Cells per mm in the Ventral region of the dentate gyrus in the hippocampus. Measure taken at end of life. #### File: Proportion_of_time_spent_IBA_while_visible_change_between_obs_1-2.xlsx **Description:** The data used to analyse the change in the mean proportion of visible time each mouse spent performing IBA between observations 1 and 2. ##### Variables * **Mouse** Unique Individual Mouse ID (range 1-62 mice) * **Cage**    Individual Cage ID (range 1-31 mice) * **Strain**  Strain of mouse: DBA = DBA/2J; C57 = C57BL/6J. One of each strain was housed in each cage * **Ttm**       Treatment: NE-NE (stably housed in non-enriched condition throughout experiment); EE-EE (stably housed in enriched condition throughout the experiment); NE-EE (started in the non-enriched condition and moved to the enriched housing when the environmental adjustment occurred); EE-NE (started in the enriched condition and moved to the non-enriched housing when the environmental adjustment occurred) * **IBA**        Proportion of visible scans the mouse spent displaying Inactive but awake (IBA) behaviour #### File: Proportion_of_time_spent_IBA_while_visible_change_between_obs_2-3.xlsx **Description:** The data used to analyse the change in the mean proportion of visible time each mouse spent performing IBA between observations 2 and 3. ##### Variables * **Mouse** Unique Individual Mouse ID (range 1-62 mice) * **Cage**    Individual Cage ID (range 1-31 mice) * **Strain**  Strain of mouse: DBA = DBA/2J; C57 = C57BL/6J. One of each strain was housed in each cage * **Ttm**       Treatment: NE-NE (stably housed in non-enriched condition throughout experiment); EE-EE (stably housed in enriched condition throughout the experiment); NE-EE (started in the non-enriched condition and moved to the enriched housing when the environmental adjustment occurred); EE-NE (started in the enriched condition and moved to the non-enriched housing when the environmental adjustment occurred) * **IBA**        Proportion of visible scans the mouse spent displaying Inactive but awake (IBA) behaviour #### File: Proportion_of_time_spent_IBA_while_visible_change_between_obs_1-3.xlsx **Description:** The data used to analyse the change in the mean proportion of visible time each mouse spent performing IBA between observations 1 and 3. ##### Variables * **Mouse** Unique Individual Mouse ID (range 1-62 mice) * **Cage**    Individual Cage ID (range 1-31 mice) * **Strain**  Strain of mouse: DBA = DBA/2J; C57 = C57BL/6J. One of each strain was housed in each cage * **Ttm**       Treatment: NE-NE (stably housed in non-enriched condition throughout experiment); EE-EE (stably housed in enriched condition throughout the experiment); NE-EE (started in the non-enriched condition and moved to the enriched housing when the environmental adjustment occurred); EE-NE (started in the enriched condition and moved to the non-enriched housing when the environmental adjustment occurred) * **IBA**        Proportion of visible scans the mouse spent displaying Inactive but awake (IBA) behaviour #### File: Trevarthen_et_al_All_data.xlsx **Description:** All data included in all analyses  ##### Variables * **Mouse**  Unique Individual Mouse ID (range 1-62 mice) - **Cage**    Individual Cage ID (range 1-31 mice) * **Strain**  Strain of mouse: DBA = DBA/2J; C57 = C57BL/6J. One of each strain was housed in each cage - **Ttm**       Treatment: NE-NE (stably housed in non-enriched condition throughout experiment); EE-EE (stably housed in enriched condition throughout the experiment); NE-EE (started in the non-enriched condition and moved to the enriched housing when the environmental adjustment occurred); EE-NE (started in the enriched condition and moved to the non-enriched housing when the environmental adjustment occurred) * **IBA**        Proportion of visible scans the mouse spent displaying Inactive but awake (IBA) behaviour during observation 3 - **Dorsal** Cells per mm in the Dorsal region of the dentate gyrus in the hippocampus. Measure taken at end of life. * **Ventral** Cells per mm in the Ventral region of the dentate gyrus in the hippocampus. Measure taken at end of life. #### File: Trevarthen_et_al_Final_code_to_analyse_change_in_IBA_between_Observations_1_and_2.R **Description:** The R script file and code used to analyse the change in the mean proportion of visible time each mouse spent performing IBA between observations 1 and 2. #### File: Trevarthen_et_al_Final_code_to_analyse_change_in_IBA_between_Observations_1_and_3.R **Description:** The R script file and code used to analyse the change in the mean proportion of visible time each mouse spent performing IBA between observations 1 and 3. #### File: Trevarthen_et_al_Final_code_to_analyse_vDG_dDG_and_IBA_obs_3.R **Description:** The R script file and code used to analyse the association between the mean proportion of visible time each mouse spent performing IBA during observation 3 and the immature neuron density (cells/mm) in the ventral (vDG) and dorsal (dDG) regions of the dentate gyrus in the hippocampus. #### File: Trevarthen_et_al_Final_code_to_analyse_change_in_IBA_between_Observations_2_and_3.R **Description:** The R script file and code used to analyse the change in the mean proportion of visible time each mouse spent performing IBA between observations 2 and 3. ## Code/software R Studio required install.packages("readr") install.packages("multcomp") install.packages("emmeans") install.packages("scales") install.packages("ggExtra") install.packages("ggsignif") install.packages("nlme") install.packages("lme") install.packages(lme4) install.packages(ggplot2) install.packages(dplyr) install.packages(MASS) install.packages(multcomp) install.packages(emmeans) install.packages("statmod") install.packages("tweedie") install.packages("lsmeans") install.packages("nlme") install.packages(scales) install.packages(stringr) install.packages('ggExtra') install.packages("ggsignif") install.packages("effectsize")