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Eco-HAB as a fully automated and ecologically relevant assessment of social impairments in mouse models of autism

  1. Alicja Puścian
  2. Szymon Łęski
  3. Grzegorz Kasprowicz
  4. Maciej Winiarski
  5. Joanna Borowska
  6. Tomasz Nikolaev
  7. Paweł M Boguszewski
  8. Hans-Peter Lipp
  9. Ewelina Knapska Is a corresponding author
  1. Nencki Institute of Experimental Biology of Polish Academy of Sciences, Poland
  2. Polish Academy of Sciences, Poland
  3. Warsaw University of Technology, Poland
  4. University of Zurich, Switzerland
  5. Kwazulu-Natal University Durban, Republic of South Africa
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Cite as: eLife 2016;5:e19532 doi: 10.7554/eLife.19532

Figures

Figure 1 with 6 supplements
A schematic representation of Eco-HAB system and data processing.

Eco-HAB consists of four housing compartments (a), tube-shaped inter-territorial passages (b), radio-frequency identification antennas (c), and impassable, perforated partitions behind which social and non-social (control) provenance stimuli may be presented (d, red/green dots). Food and water is available in housing compartments adjacent to those containing partitions. Eco-HAB is equipped with customized electronics and two software packages: Eco-HAB.rfid (for data acquisition and collection) and Eco-HAB.py (for filtering corrupted data segments and performing tailored analysis). For a detailed system and software description, see 'Materials and methods'.

https://doi.org/10.7554/eLife.19532.002
Figure 1—figure supplement 1
Block schematic diagram of customized electronic system for Eco-HAB.
https://doi.org/10.7554/eLife.19532.003
Figure 1—figure supplement 2
RFID antenna efficiency compared to video-based manual scoring.

We counted the number of RFID registrations per visually registered crossing under the antenna. The implemented system of coils recognizes subjects at a rate of at least one or more RFID registration per one video-recorded passing, a rate better than needed to record all events. Superfluous RFID readouts are later eliminated by Eco-HAB.py software that contains algorithms recognizing such events (see 'Materials and methods'). Due to the built-in internal synchronization system, RFID antennas run independently and do not disrupt one another. All eight coils may be activated simultaneously for unlimited time, which leads to highly effective animal recognition (less than 0.6% unidentified animals’ positions).

https://doi.org/10.7554/eLife.19532.004
Figure 1—figure supplement 3
Comparison of time (person-hours) needed for Eco-HAB testing versus three-chambered apparatus testing (stress reducing conditions) of a group of 12 mice.

For detailed description of behavioral protocols see 'Materials and methods'.

https://doi.org/10.7554/eLife.19532.005
Figure 1—figure supplement 4
Eco-HAB measures in-cohort sociability in mice.

(a) Detailed data regarding quantity of time spent by each mouse with every other animal in the group can be obtained in Eco-HAB. (b) Based on simultaneous territory occupation for each individual, we calculate the minimum time each given pair of subjects must spend together. (c) After subtracting expected time together from the acquired one, we obtained amount of time animals willfully spent together, as it cannot be attributed to the dispersal pattern within the system.

https://doi.org/10.7554/eLife.19532.006
Figure 1—figure supplement 5
Eco-HAB allows for a detailed analysis of subjects' preference to spend time with another mouse from a tested cohort.

Examples show (a) a pair of mice spending most of their time together, regardless of their position within the territory, and (b) a pair of mice spending time mostly in different areas of Eco-HAB. Bars represent presence of a mouse in one of four Eco-HAB compartments (numbered 1–4).

https://doi.org/10.7554/eLife.19532.007
Figure 1—figure supplement 6
Monitoring of subjects’ dispersal within Eco-HAB territory for exemplary cohorts of (a) C57BL/6 and (b) BALB/c mice.

Customized software provides easy access to data on the amount of time spent by a mouse in each Eco-HAB compartment. An example here shows mouse activity distribution in 12-hr dark phase during the adaptation stage.

https://doi.org/10.7554/eLife.19532.008
Main stressors interfering with reliable measurement of social behavior in rodents (a) and their effects on social preference scores in C57BL/6 and BALB/c mice in the conventional three-chambered test (b–e) under low- and high-stress conditions.

High-stress conditions differ from low-stress conditions in the intensity of light and subjects' and mouse social objects' habituation to the experimenter and the experimental environment (for a detailed protocol see 'Materials and methods'). (b) BALB/c mice showed social preference only in low-stress conditions (n = 17), and they avoided social interactions when tested in a typical experimental setting (n = 11). (c) In contrast, C57BL/6 mice displayed social preference in both stressful (n = 11) and stress-reducing (n = 38) conditions. Social preference was calculated as the time spent in the chamber containing the social object compared to the time spent in the chamber with a non-social object. (d,e) Under stress, both tested strains of mice showed reduced locomotor activity. Data are median values and error bars represent IQR (interquartile range), *p<0.05, **p<0.01, ***p<0.001 (Mann-Whitney U-test).

https://doi.org/10.7554/eLife.19532.010
Figure 3 with 2 supplements
Sociability measurements in Eco-HAB and three-chambered apparatus social approach test performed under stress reducing conditions.

Figure (ad) depict tests involving C57BL/6 mice and (eh) tests involving BALB/c mice. (a) and (e) show social approach in the Eco-HAB system defined as the increase in proportion of time spent in the compartment with social odor during the first hour after its presentation, divided by the proportion of time spent in the compartment with non-social stimulus. For (a), VPA-treated n = 26 and CTRL n = 35. For (e), VPA-treated n = 18 and CTRL n = 20. (b) and (f) show social approach in the three-chambered test, defined as the increase in proportion of time spent in the compartment with an unfamiliar mouse, divided by the proportion of time spent in the compartment with unfamiliar inanimate object. For (b), VPA-treated n = 18, CTRL n = 27. For (f), VPA-treated n = 23 and CTRL n = 26. (c) and (g) show density plot matrices for Eco-HAB housed control and valproate-treated cohorts. Each small square, for which position in the matrix represents one pair of subjects, shows the total time spent together minus the time animals would spend together assuming independent exploration of the apparatus (see 'Materials and methods'). Histograms (d) and (h) show the distribution of this measure for all pairs of valproate-treated and control animals. Data are mean values and error bars represent SEM, *p<0.05, **p<0.01, ***p<0.001 (Mann-Whitney U-test).

https://doi.org/10.7554/eLife.19532.011
Figure 3—source data 1

Eco-HAB measured social approach and in-cohort sociability of valproate-treated and control C57BL/6 and BALB/c mice.

The names of the Excel sheets refer to corresponding figures and contain data used for analysis of the behavioral measures obtained by the implementation of Eco-HAB.py software (see 'Materials and methods').

https://doi.org/10.7554/eLife.19532.012
Figure 3—source data 2

Eco-HAB measured social approach and in-cohort sociability of valproate-treated and control C57BL/6 and BALB/c mice.

The names of the Excel sheets refer to corresponding figures and contain data used for analysis of the behavioral measures obtained by the implementation of Eco-HAB.py software (see Materials and methods).

https://doi.org/10.7554/eLife.19532.013
Figure 3—source data 3

Eco-HAB measured social approach and in-cohort sociability of valproate-treated and control C57BL/6 and BALB/c mice.

The names of the Excel sheets refer to corresponding figures and contain data used for analysis of the behavioral measures obtained by the implementation of Eco-HAB.py software (see Materials and methods).

https://doi.org/10.7554/eLife.19532.014
Figure 3—source data 4

Eco-HAB measured social approach and in-cohort sociability of valproate-treated and control C57BL/6 and BALB/c mice.

The names of the Excel sheets refer to corresponding figures and contain data used for analysis of the behavioral measures obtained by the implementation of Eco-HAB.py software (see Materials and methods).

https://doi.org/10.7554/eLife.19532.015
Figure 3—figure supplement 1
Three-chambered apparatus testing performed on group-housed valproate-treated C57BL/6 (VPA-treated n = 14, CTRL n = 38) (a) and BALB/c (n = VPA-treated n = 15, CTRL n = 17) (b) mice did not reveal any differences in sociability.

Data are represented as mean ± SEM.

https://doi.org/10.7554/eLife.19532.016
Figure 3—figure supplement 2
Eco-HAB allows for long-term monitoring of responses to social stimuli.

We assessed subjects approach to social odor in four different periods, namely 30’, 1 hr, 2 hr, and 4 hr after the presentation of olfactory stimuli, in order to see how their behavior changes with time. Approach to social odor in (a) valproate-treated C57BL/6 mice (n = 26) gradually decreases during first 4 hr of testing, whereas in (b) valproate-treated BALB/c mice (n = 18) it is high and stable during this time. In control C57BL/6 mice (n = 35) and BALB/c mice (n = 20) approach to social odor gradually decreases with a faster decrease rate in C57BL/6 mice. Data are represented as mean, error bars represent SEM, *p<0.05, **p<0.01, ***p<0.001 (Mann-Whitney U-test).

https://doi.org/10.7554/eLife.19532.017
Social impairment of Fmr1 knockout mice compared to wild-type control measured in Eco-HAB.

Fmr1 knockouts, n = 22. Wild-type controls, n = 10. (a) Odor-based social preference in the Eco-HAB system defined as the increase in proportion of time spent in the compartment with social odor during the first hour after its presentation, divided by the proportion of time spent in the compartment with non-social stimulus. Histogram (b) shows the distribution of in-cohort sociability for all pairs of knockout and control animals. Data are mean values and error bars represent SEM, *p<0.05, **p<0.01, ***p<0.001 (Mann-Whitney U-test).

https://doi.org/10.7554/eLife.19532.018
Figure 4—source data 1

Eco-HAB measured social approach and in-cohort sociability of Fmr1 knockouts and wild-type controls.

The names of the Excel sheets refer to corresponding figures and contain data used for analysis of the behavioral measures obtained by the implementation of Eco-HAB.py software (see 'Materials and methods').

https://doi.org/10.7554/eLife.19532.019
Figure 4—source data 2

Eco-HAB measured social approach and in-cohort sociability of Fmr1 knockouts and wild-type controls.

The names of the Excel sheetsrefer to corresponding figures and contain data used for analysis of the behavioral measures obtained by the implementation of Eco-HAB.py software (see 'Materials and methods').

https://doi.org/10.7554/eLife.19532.020
Eco-HAB provides reproducible assessment of approach to social odor in group-housed mice.

Individual results of approach to social odor for all cohorts of (a) valproate-treated (n = 20) and control (n = 18) BALB/c subjects (4 cohorts), (b) valproate-treated (n = 26) and control (n = 35) C57BL/6 mice (6 cohorts) and (c) Fmr1 knockouts (n = 22) and wild-type (n = 18) animals (5 cohorts). Each column represents one cohort of animals, while data points (dots and squares) represent scores of particular mice. Since the measure of approach to social odor is a proportion (for detailed description see 'Materials and methods'), which may take values from 0 to +∞, we present logarithmic data to depict reproducibility of social preference and social avoidance in an unbiased manner. All analyses, including statistical testing, were performed on raw data. Average results of these data are presented in Figures 3A,E and 4A, respectively.

https://doi.org/10.7554/eLife.19532.021
Figure 5—source data 1

Eco-HAB measured social approach score for valproate-treated and control C57BL/6 and BALB/c mice and Fmr1 knockouts and wild-type controls.

These data are identical to Figure 3A—source data 1, Figure 3E—source data 3, Figure 4A—source data 1 with respect to Figures 3A,E and 4A and are available as a separate file for the readers’ convenience.

https://doi.org/10.7554/eLife.19532.022
Figure 5—source data 2

Eco-HAB measured social approach score for valproate-treated and control C57BL/6 and BALB/c mice and Fmr1 knockouts and wild-type controls.

These data are identical to Figure 3A—source data 1, Figure 3E—source data 3, Figure 4A—source data 1 with respect to Figures 3A,E and 4A and are available as a separate file for the readers’ convenience.

https://doi.org/10.7554/eLife.19532.023
Figure 5—source data 3

Eco-HAB measured social approach score for valproate-treated and control C57BL/6 and BALB/c mice and Fmr1 knockouts and wild-type controls.

These data are identical to Figure 3A—source data 1, Figure 3E—source data 3, Figure 4A—source data 1 with respect to Figures 3A,E and 4A and are available as a separate file for the readers’ convenience.

https://doi.org/10.7554/eLife.19532.024
Assessment of approach to social odor in Fmr1 knockouts and respective littermate controls performed in two different laboratories.

(a, Fmr1 knockout n = 22, wild-type control n = 18) vs. (b, Fmr1 knockout n = 11, wild-type control n = 9). Regardless of experimental environment, evaluation carried out in Eco-HAB revealed comparable impairment in Fmr1 knockouts. Presented data are logarithmic values.

https://doi.org/10.7554/eLife.19532.025
Figure 6—source data 1

We include source data for Figures 6, 7 and 8 concerning reproducibility results of both Eco-HAB measures.

The names of the Excel sheets refer to corresponding figures and contain data used for analysis of the behavioral measures obtained by the implementation of Eco-HAB.py software (see 'Materials and methods').

https://doi.org/10.7554/eLife.19532.026
Figure 6—source data 2

We include source data for Figures 6, 7 and 8 concerning reproducibility results of both Eco-HAB measures.

The names of the Excel sheetsrefer to corresponding figures and contain data used for analysis of the behavioral measures obtained by the implementation of Eco-HAB.py software (see Materials and methods).

https://doi.org/10.7554/eLife.19532.027
Evaluation of in-cohort sociability in Fmr1 knockouts and wild-type littermate controls undertaken in two different laboratories.

(a, Fmr1 knockout n = 22, wild-type control n = 18) vs. (b, Fmr1 knockout n = 11, wild-type control n = 9) – gives corresponding results. A histogram illustrating score of Fmr1 knockouts is shifted to the left as compared to that for wild-type control, signifying less time voluntarily spent together with other subjects within a tested cohort.

https://doi.org/10.7554/eLife.19532.028
Figure 7—source data 1

We include source data for Figures 6, 7 and 8 concerning reproducibility results of both Eco-HAB measures.

The names of the Excel sheetsrefer to corresponding figures and contain data used for analysis of the behavioral measures obtained by the implementation of Eco-HAB.py software (see Materials and methods).

https://doi.org/10.7554/eLife.19532.029
Figure 7—source data 2

We include source data for Figures 6, 7 and 8 concerning reproducibility results of both Eco-HAB measures.

The names of the Excel sheetsrefer to corresponding figures and contain data used for analysis of the behavioral measures obtained by the implementation of Eco-HAB.py software (see Materials and methods).

https://doi.org/10.7554/eLife.19532.030
Eco-HAB allows remarkably reproducible assessment of approach to social odor in both (a) wild-type mice (n = 9) and (b) Fmr1 knockouts (n = 11).

Evaluation of social behavior of subjects was repeated twice in identical Eco-HAB experiments, separated by a 10-day period of regular housing. Each aligned dot and square encircled by an oval represent individual score of approach to social odor for each tested mouse, measured in two subsequent experimental repetitions. Dots are data, while the ovals serve to guide the eye. Data presented are logarithmic values.

https://doi.org/10.7554/eLife.19532.031
Figure 8—source data 1

We include source data for Figures 6, 7 and 8 concerning reproducibility results of both Eco-HAB measures.

The names of the Excel sheetsrefer to corresponding figures and contain data used for analysis of the behavioral measures obtained by the implementation of Eco-HAB.py software (see Materials and methods).

https://doi.org/10.7554/eLife.19532.032
Figure 8—source data 2

We include source data for Figures 6, 7 and 8 concerning reproducibility results of both Eco-HAB measures.

The names of the Excel sheetsrefer to corresponding figures and contain data used for analysis of the behavioral measures obtained by the implementation of Eco-HAB.py software (see Materials and methods).

https://doi.org/10.7554/eLife.19532.033
Figure 9 with 1 supplement
Tube dominance score of an animal does not correlate with overall activity in Eco-HAB apparatus.

Dominance is expressed as percentage of won encounters ('winning score percent') in the U-tube dominance test (see 'Materials and methods'). Activity in Eco-HAB is defined as the number of visits to all of its compartments during the first 12 hr of the habituation period. Social hierarchy does not correlate with exploration of the territory in either of the tested groups: (a) control (n = 23) or (b) valproate-treated C57BL/6 mice (n = 26), (c) control (n = 32) or (d) valproate-treated BALB/c mice (n = 19). Dependence between two variables tested by Pearson product-moment correlation coefficient.

https://doi.org/10.7554/eLife.19532.034
Figure 9—source data 1

Raw data from U-tube dominance test and Eco-HAB measured activity (number of visits to all compartments of the apparatus during 1st 12-hr period of adaptation).

Figure 9 depicts correlation between those two variables.

https://doi.org/10.7554/eLife.19532.035
Figure 9—source data 2

Raw data from U-tube dominance test and Eco-HAB measured activity (number of visits to all compartments of the apparatus during 1st 12 hr period of adaptation).

Figure 9 depicts correlation between those two variables.

https://doi.org/10.7554/eLife.19532.036
Figure 9—figure supplement 1
Aggressive interactions during testing in Eco-HAB are rare regardless of the tested strain.

Number of episodes and duration of aggressive behaviors in VPA-treated and control BALB/c (a, b), VPA-treated and control C57BL/6 (c, d) and Fmr1 knockout and wild-type mice (e, f) during first 6 hr of adaptation phase, as counted per each pair of animals within a tested cohort. Aggressive encounters, namely fighting, chasing and biting were quantified by manual video-based scoring and then divided by the number of mouse pairs in a given cohort.

https://doi.org/10.7554/eLife.19532.037

Videos

Video 1
Top view of the working Eco-HAB.

Flashing lights indicate activation of RFID antennas – sensors of the individual recognition system. The clip presents a 30 s period at the beginning of the adaptation phase, when animals are eagerly exploring new territory.

https://doi.org/10.7554/eLife.19532.009

Additional files

Supplementary file 1

Eco-HAB.py scripts with sample data enabling their execution.

https://doi.org/10.7554/eLife.19532.038

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