Calcium Sensitive Adenylyl Cyclases in Depression and Anxiety: Behavioral Consequences

Supplementary Material

Calcium Sensitive Adenylyl Cyclases in Depression and Anxiety: Behavioral and Biochemical Consequences of Isoform Targeting

Vaishnav Krishnan1, Ami Graham1, Michelle S. Mazei-Robison1, Diane C. Lagace1, Kyoung- Shim Kim2, Shari Birnbaum1, Amelia J. Eisch1, Pyung-Lim Han2, Daniel R. Storm3, Venetia Zachariou1,4, Eric J. Nestler1

1Departments of Psychiatry and Neuroscience, The University of Texas Southwestern Medical Center (UTSWMC), Dallas, TX; 2Division of Nanosciences and Brain Disease Research Institute, Ewha Womans University School of Medicine, Seoul, Republic of Korea; 3Department of Pharmacology, The University of Washington, Seattle, WA; 4Department of Basic Sciences, University of Crete, Heraklion, Crete, Greece

Corresponding Author: Eric J. Nestler, MD, PhD [email protected] Ph: 214 648 1111 Fax: 214 648 4947 SUPPLEMENTARY METHODS AND MATERIALS

Overview For each line of mice (AC5KO and AC1/8DKO), two approximately equal sized cohorts were serially run through a battery of behavioral tests in the following order: locomotor testing  open field  elevated plus maze  dark light  forced swim testing. We employed an inter-test rest period of approximately 3- 5 days. Separate cohorts of mice were employed for sucrose preference  social interaction testing. In experiments where the same testing apparatus was repeatedly utilized for multiple mice, the surface was wiped with a solution of quatricide to eliminate any possible odor cues left by previous subjects. Mice that were utilized for immunoblotting and BrDU immunohistochemistry experiments were not previously employed for behavioral analyses. The Grubbs outlier test was utilized to identify and discard outlier behavior. All experiments were performed in accordance with the UTSW Institutional Animal Care and Use Committee.

General Sensory and Motor Function Male and female mice from both strains (AC5 and AC1/8) were evaluated through a simple screen for general sensory and motor responses (1). Individual mice were placed in a clean cage and were allowed to freely explore the cage. Using the tip of a clean cotton swab, the eye blink reflex was tested by approaching one eye at a time, as well as the ear twitch and whisker-orienting reflex by approaching the ears or whiskers respectively. Next, the cage was agitated (sideways and up and down) to observe that mice extended all four legs to maintain an upright position (postural reflex). Mice were then forcibly placed on their backs to observe the speed at which they returned to an upright posture (righting reflex). Finally, by shining a penlight into one eye at a time, pupillary constriction and subsequent pupillary dilation was confirmed.

Elevated Plus Maze (2) The elevated plus maze apparatus was constructed in black Plexiglass fitted with white surfaces to provide contrast, and raised 65 cm above the ground. Two open arms (30 x 5 cm) and two closed arms (30 x 5 x 15 cm-high walls) were attached to the center “square”. The maze was set up under a video recorder, with each testing session lasting 5 min and carried out under an illumination of 4.6 lux. In each trial, mice were placed in the center of the maze, and their location within the maze was tracked by videotracking (Ethovision) and analyzed for crosses and durations within specific “zones”.

Dark Light Testing (3) The dark-light two chamber apparatus (Med Associates) was constructed in black and white Plexiglass, with each chamber measuring 25 x 26 x 25 cm. The “dark” chamber was not illuminated, while the “light” side was illuminated by a fluorescent lamp providing 2400 lux of illumination. Movement of the mouse within each chamber was measured by photocell beam breaks. Mice were placed in the dark side for 2 min, and then the automatic door between the compartments opened and they were allowed to freely explore both chambers for 10 min.

Open Field (2, 4) Open field testing was performed in a white square shaped arena (44 x 44 x 30 cm-high walls) made of wood. 5 min long trials were carried out under 3lux of illumination. In each trial, mice were placed in the periphery of the arena, and their location was tracked by videotracking (Ethovision) and analyzed for duration of time spent in the center (defined as a concentric 34 x 34 cm square).

Forced Swim testing (2, 4)

Krishnan et al. 2 For forced swim testing, mice were placed in a 4 L beaker containing approximately 3 L of tap water (241oC). All sessions were videotaped and scored by an experienced blind observer who recorded the duration of immobility during the last 4 min of a 6 min swim test. Immobility was defined as floating or a single paddling hind limb directed exclusively to maintain the head above the water. Between sessions, tap water within the beaker was replaced.

Sucrose Preference (2) For sucrose preference testing, mice were socially isolated. Two 50ml bottles fitted with two-balled sipper tubes (Ancare) were positioned on the food rack. While bottle “B” always contained pure tap water, bottle “A” was filled with increasing concentrations of sucrose ranging from 0% to 5%. The fluid level on both bottles was measured daily, and bottle position was switched daily to avoid side biases. For each concentration tested, total fluid intake and sucrose preference was measured and averaged across two days of testing. Sucrose preference was calculated as the volume of fluid consumed from bottle “A” divided by the total volume consumed (A/[A+B]).

Social Interaction (2) Prior to social interaction testing, mice were socially isolated for 2-3 weeks. The social interaction task consisted of two 2.5 min long trials performed in darkness. An infrared lamp and an infrared-sensitive videocamera allowed for tracking during darkness. In the first trial, the mouse is placed in an open field arena (44 x 44 x 30 cm) with a wire mesh cage (10 x 6 x 30 cm) placed along the center of one wall (Supplementary Figure 3A). During the second trial, the mouse is placed back into the same arena, which now possesses an unfamiliar target mouse within the wire mesh cage. Videotracking (Ethovision) was employed to measure the distance moved and location of the mouse within the arena. The “interaction zone” was defined as a 15x30 cm rectangular area surrounding the wire mesh. The social cue/target mouse was always a sex-matched c57BL/6 mouse (Jackson labs).

SUPPLEMENTARY FIGURE LEGENDS

Figure S1 A: In a 5 min long open field test, male and female AC5KO mice exhibited reduced locomotor activity

(genotype main effect, F1,40 = 21.77, p<0.0001). B: The total number of transitions/crosses between chambers within the dark light apparatus was not altered in male or female AC5KO mice (genotype x sex interaction, F1,34 = 0.23, p>0.5). C: During the first five minutes of the dark light test, AC5KO mice explored the light compartment significantly more than their WT controls (genotype main effect, F1,34 = 10.53, p<0.01). D: In the elevated plus maze test (EPM), the total number of open arm entries was unchanged by sex or genotype (genotype x sex interaction, F1,39 = 2.41, p>0.1). E: Male and female AC5KO mice entered the maze’s closed arms significantly less frequently (F1,40 = 148.2, p<0.0001). F: When normalized to closed arm duration, male and female AC5KO mice spent significantly greater duration of time in the open arm (F1,40 = 6.28, p<0.0001). Data are presented as means + SEM (sample size denoted within bars), with * indicating significant post-hoc differences (*:p<0.05, ***:p<0.001).

Figure S2 A: In a 5 min long open field test, AC1/8 DKO and WT mice exhibited comparable locomotor activity

(genotype x sex interaction, F1,31 = 0.93, p>0.3). B: The total number of transitions/crosses between chambers within the dark light apparatus was not altered in male or female AC1/8 DKO mice (genotype x sex interaction, F1,31 = 2.84, p>0.1). C: During the first five minutes of the dark light test, AC1/8DKO did not explore the light compartment significantly more than their WT controls (genotype x sex interaction,

F1,31 = 2.773, p>0.1). D: In the elevated plus maze test (EPM), the total number of arm entries was unchanged by sex or genotype (genotype x sex interaction, F1,31 = 2.39, p>0.1). E: AC1/8 DKO mice entered the EPM’s closed arms more frequently than WT mice (genotype main effect, F1,31 = 12.24, p<0.01). F: When normalized to closed arm duration, male and female AC1/8 DKO and WT mice

Krishnan et al. 3 displayed comparable durations of time in the open arm (F1,31 = 0.26, p>0.5). Data are presented as means + SEM (sample size denoted within bars), with * indicating significant post-hoc differences (*:p<0.05, ***:p<0.001).

Figure S3 A: Photograph of the square arena (44 x 44 x 30 cm) and wire mesh cage (10 x 6 x 30 cm) employed for social interaction testing. The “interaction zone” is a 15 x 30 cm rectangle that surrounds the target enclosure. B,C: Total distance moved within the social interaction arena during “target absent” and “target present” conditions for the AC5 (B) and AC1/8 (C) lines of mice. D: Bar graph depicting average weights of male and female, AC1/8 DKO and WT mice at various ages (n30-40). AC1/8 DKO mice consistently weighed less than WT. Data are presented as means + SEM.

References

1. Crawley JN (2000): What's Wrong With My Mouse?: Behavioral Phenotyping of Transgenic and Knockout Mice. New York:: Wiley-Liss Press. 2. Krishnan V, Han M-H, Graham DL, Berton O, Renthal W, Russo SJ, et al. (2007): Molecular Adaptations Underlying Susceptibility and Resistance to Social Defeat in Brain Reward Regions. Cell. 131:391-404. 3. Powell CM, Schoch S, Monteggia L, Barrot M, Matos MF, Feldmann N, et al. (2004): The presynaptic active zone protein RIM1alpha is critical for normal learning and memory. Neuron. 42:143-153. 4. Monteggia LM, Luikart B, Barrot M, Theobold D, Malkovska I, Nef S, et al. (2007): Brain- derived neurotrophic factor conditional knockouts show gender differences in depression- related behaviors. Biological Psychiatry. 61:187-197.

Krishnan et al. 4 Supplementary Figure 1

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