Project protocol — Contents
Workflow and sampling
Reagents, supplies, and solutions
Procedure for measuring social approach using an automated 3-chambered test box
Step Procedure performed Age (wks) Apparatus Time (min) Data collected 1 Mice are brought from the vivarium to the test room to acclimate before testing begins 6-7 - 30 - 2 A mouse is placed in the center chamber of the social test box and allowed to habituate to the testing conditions 6-7 social test box/ habituation 10 data not submitted 3 Following a period of habituation, the mouse is given a choice between proximity to an unfamiliar mouse or to a novel object; the time spent with the conspecific mouse is a measure of sociability 6-7 social test box/ sociability test 10 duration of chamber occupancy, number of entries into each chamber, and duration of sniffing each cage (occupied or empty) 4 After sociability test, the mouse is tested again for preference for social novelty with the addition of a second conspecific mouse 6-7 social test box/ preference for social novelty test 10 duration of chamber occupancy, number of entries into each chamber, and duration of sniffing each occupied cage 5 After the administration of sociability and social novelty preference tests, the testing chambers are cleaned for subsequent testing 6-7 social behavior test box - -
• Automated three-chambered apparatus (National Institutes of Mental Health, Bethesda, MD, USA) made of clear polycarbonate for social behavior test
Figure 1: Social behavior test box, where a mouse is given a choice between staying in the center chamber, spending time in the side chamber with an unfamiliar mouse (stranger 1), or spending time in the side chamber with either a novel object or a newly introduced mouse (stranger 2) during social preference tests. Stranger mice are enclosed in wire cages.
- Activity assessment system using photocells (National Institutes of Mental Health, Bethesda, MD, USA)
- Customized computer software developed by Dr. Josephine M. Johns, University of North Carolina, Chapel Hill, NC, and Dr. Larry W. Means, East Carolina University, Greenville, NC; used for scoring time spent sniffing each wire cage
- Disinfectant: 70% isopropanol used to clean the test chambers
- Paper towels
- Water bottle for cleaning test chambers
Acclimation to test conditions
Testing is conducted during the light phase under fluorescent laboratory lighting (320-340 lx). Mice are transported from the vivarium into the test room and are allowed to acclimate for at least 30 min and habituate to testing conditions for 10 min.
I. Pre-testing observations
a. Following arrival to the animal facility, the mice are subjected to a series of observations and a battery of behavioral tests for at least a week (see Moy et al. 2007-8).
b. Included in the series of observations are general health and neurological evaluations by a single observer (see Moy et al. 2007-8).
II. Testing for sociability and preference for social novelty using social behavior apparatus
a. An empty wire cage is placed within each of the side chamber, and a weighted cup is placed on top of it to prevent a mouse from climbing over the wire cage (see Figure 1).
b. With the doorways into the two side chambers opened, the test mouse is placed in the middle chamber and allowed to explore the apparatus for 10 min.
c. Embedded photocells within each doorway entrance are activated to automatically track the number of entries and duration spent in each chamber.
d. The sociability test is immediately conducted following the habituation phase.
Investigator's Notes: "To confirm the absence of a side preference bias for either of the two side chambers of the social test box, measures were taken of time spent in each side during the 10-min habituation period. None of the strains showed a significant preference for either the right or left side [no main effect of side; p > 0.05, within group repeated measures analysis for each strain]. In addition, separate groups of C57BL/6J mice were periodically evaluated in the social behavior task, to confirm that the environmental parameters for the assay had not changed and normal tendencies for social approach could still be observed. Stranger mice were adult male C57BL/6J (JAX®), and were housed in cages separate from and distant to the cages housing the subject mice, to avoid visual, auditory, and olfactory contact. Strangers have no previous physical contact with the subjects, and were kept in a separate location from the subjects on the day of testing. Several days before the start of social testing, the mice serving as strangers were habituated to the wire cages in the social apparatus for 5-10 min per day, for at least 5 days. Each stranger was used only once per day, and the strangers for the sociability test and the social novelty tests were taken from separate cages. Containing the stranger mouse in a wire cage served the purpose of preventing aggressive and sexual interactions, as well as ensuring that all social approach was initiated only by the subject mouse. Previous experiments indicated that the strain of the stranger did not change the social approach of the subject. The empty wire cage served as a control for the properties of the container, in addition to serving as the novel inanimate object with no social valence (see Moy et al. 2007)."
III. Sociability test
a. Shortly after the habituation period, the test mouse is enclosed in the center compartment of the social test box, and an unfamiliar mouse (stranger1; a C57BL/6J male) is enclosed in one of the wire cages and placed in one of the side chambers.
b. The location for stranger1 alternated between the left and right sides of the social test box across subjects.
c. Following placement of stranger 1, the doors are re-opened, and the mouse subject is again allowed to explore the entire social test box for another 10-min session.
d. Measurements are automatically recorded of the amount of time spent and the number of entries into each chamber by the automated testing system.
e. A human observer scored time spent sniffing each wire cage, using a computer keypad and customized software.
VI. Preference for social novelty test
a. Following the sociability test, each mouse is again tested in a third 10-min session to measure preference in spending time with a new stranger.
b. The test mouse is again briefly enclosed in the center compartment.
c. A new unfamiliar mouse (stranger2) is placed in the wire cage that had been empty during the previous 10-min session (sociability test).
d. The test mouse is given a choice between the first, already-investigated, now-familiar mouse (stranger1) and the novel unfamiliar mouse (stranger2).
e. As before, measurements are automatically recorded of the amount of time spent and the number of entries into each chamber by the automated testing system.
f. As above, a human observer scored time spent sniffing each wire cage in both side chambers, using a computer keypad and customized software.
g. At the end of each three-10 min sessions, the chambers are cleaned with water and paper towels before testing another mouse subject.
h. Each wire cage is used only once per day, and the three-chambered apparatus is cleaned and disinfected at the end of the day.Using an automated social behavior test box, the following measurements were obtained:
a, b, c or d: These measurements are similar measurements, but recorded under two distinct tests.
Measurement description Sociability test Preference for social novelty test Units Duration of occupancy in center-chamber yesa yesa s Time spent in chamber with empty cage (a novel object) yes not applicable s Time spent in chamber with stranger 1 (a novel mouse) yesb yesb s Time spent sniffing empty cage yes not applicable s Time spent sniffing cage with stranger 1 yesc yesc s Number of entries to chamber with empty cage yes not applicable n Number of entries to chamber with stranger 1 in cage yesd yesd n Time spent in chamber with stranger 2 (a second novel mouse) not applicable yes s Time spent sniffing cage with novel stranger 2 not applicable yes s Number of entries to chamber with novel stranger 2 in cage not applicable yes n
Moy SS, Nadler JJ, Young NB, Nonneman RJ, Grossman AW, Murphy DL, D'Ercole AJ, Crawley JN, Magnuson TR, Lauder JM. Social approach in genetically engineered mouse lines relevant to autism. Genes Brain Behav. 2009 Mar;8(2):129-42. Epub 2008 Nov 11.
Nadler JJ, Moy SS, Dold G, Trang D, Simmons N, Perez A, Young NB, Barbaro RP, Piven J, Magnuson TR, Crawley JN. Automated apparatus for quantitation of social approach behaviors in mice. Genes Brain Behav. 2004 Oct;3(5):303-14.