SUPPLEMENTARY MATERIAL AND METHODS Administration of the Y2 Receptor Agonist PYY3-36 in Mice Induces Multiple Behavioral Changes Relevant to Schizophrenia Ulrike Stadlbauer*, Wolfgang Langhans, Urs Meyer Physiology and Behavior Laboratory, Swiss Federal Institute of Technology (ETH) Zurich, Schorenstrasse 16, 8603 Schwerzenbach, Switzerland. *Correspondence: US (ulrike-stadlbauer@ethz.ch) Tel.: +41 44 655 74 86; Fax.: +41 44 655 72 06. Elevated Plus Maze Test Anxiety-related behavior was studied using a standard elevated plus maze test. The apparatus was made of a Plexiglas maze consisting of 4 arms (each 5 cm x 30 cm) radiating from a square center zone (5 cm x 5 cm). Two arms were open with a 3 mm border along the outer edges; the other pair of opposing arms was enclosed with opaque walls (15cm height) except for the center zone. The maze was positioned in a testing room with diffused light and elevated 70 cm above the floor. A camera, mounted above the maze, captured images and transmitted them to a PC running the Ethovision tracking system (Noldus Technology, Wageningen, Netherlands). At the beginning of a test session, animals were placed into the center zone facing one of the closed arms. After 10 min of exploring freely, animals were returned to their home 1 cages. After each test session, the maze was cleaned with water and dried before the next trial. To index anxiety-related behavior, the relative time spent in open arms and open arm entries were calculated using the formulas [(time spent in open arms) / (time spent in all arms)] x 100 and [(open arm entries) / (total arm entries)] x 100]. Total distance moved in the entire maze was additionally analyzed to assess general locomotor activity. Social Interaction Test We assessed the effects of PYY3-36 on social interaction using a social approach test in a modified Y-maze as established before (Vuillermot et al, 2011). The apparatus used was in the form of a Plexiglas Y-maze consisting of three identical arms (50cm x 9cm x 10cm, length x width x height), radiating from a triangle center zone (each side 8 cm). Two of the three arms contained rectangular wire grid cages, the third arm served as the start zone. Animals were habituated to the apparatus by being allowed to explore for 10 min on two days, which served to reduce novelty-induced locomotor hyperactivity. During the test, one wire cage contained an unfamiliar C57BL6 mouse, the other one contained a ‘dummy mouse’ made of black LEGO™ (Billund, Denmark). The allocation of the objects (live mouse versus dummy object) was counterbalanced across arms and treatments. At the beginning of a test session the animal was placed in the end of the start arm and allowed to explore freely for 5 min. A camera, mounted above the maze, captured images and transmitted them to a tracking system to assess general locomotor activity. Social interaction was defined as nose direction to the wire cage in a 6-cm interaction zone adjacent to the wire cage and was assessed by a trained experimenter blind to the treatments. After 5 min the animal was removed and the apparatus was cleaned. 2 Prepulse Inhibition Test Sensorimotor gating was assessed using the paradigm of prepulse inhibition (PPI) of the acoustic startle reflex. The test apparatus consisted of 4 startle chambers for mice (San Diego Instruments, San Diego, CA) as has been fully described elsewhere (Meyer et al, 2005). In the PPI test, subjects were presented with a series of discrete trials comprising a mixture of 4 trial types as fully described previously (Meyer et al, 2005). These included pulse-alone trials, prepulse-plus-pulse trials, prepulse-alone trials, and no-stimulus trials in which no discrete stimulus other than the constant background noise was presented. The pulse and prepulse stimuli employed were in the form of a sudden elevation in broadband white noise level (sustaining for 40 and 20 ms, respectively) from the background (65 dBA), with a rise time of 0.2–1.0 ms. The program consisted of a pulse (120 dBA) and 5 intensities of prepulse (69, 73, 77, 81 and 85 dBA, corresponding to 4, 8, 12, 16 and 20 dBA above background,). The stimulus-onset asynchrony of the prepulse and pulse stimuli in prepulse-plus-pulse trials was 100 ms. After a 2-min acclimatization period, the first 6 trials consisted of 6 startle-alone trials to habituate and stabilize the animals’ startle response and were not included in the analysis. Following the habituation phase each trial stimulus was presented 12 times in a random order with 15 ± 5 sec intertrial interval. The program finished with 6 consecutive pulse-alone trials. PPI was indexed by percent inhibition of the startle response obtained in the pulse-alone trials by the following expression: 100% × (1 – [mean reactivity on prepulse-plus-pulse trials/mean reactivity on pulse-alone trials]), for each subject. 3 Latent Inhibition Test Selective associative learning was measured using the paradigm of latent inhibition (LI), in which non-reinforced pre-exposures to a to-be-conditioned stimulus (CS) retards subsequent conditioning between the same CS and the unconditioned stimulus (US) (Lubow, 2005). LI was assessed in a Pavlovian fear conditioning paradigm, in which a tone served as the CS and electric foot shock as the US. The apparatus comprised four test chambers consisting of four metal operant boxes (30 × 25 × 29 cm; Model E10-10, Coulbourn Instruments, Allentown, PA, USA), each installed in a ventilated and soundinsulated Coulbourn Instruments chest. The animal was confined to a rectangular enclosure (17.5 × 13 cm) in the middle of the operant box. The construction of the test chambers as well as the image analysis software used to evaluate freezing behavior have been fully described previously (Richetto et al, 2013). The test procedures of the LI test followed protocols established before (Richetto et al, 2013) and consisted of three phases: pre-exposure, conditioning and tone test: Pre-exposure phase: The animals were placed in the appropriate test chamber: Subjects assigned to conditioned stimulus (CS) pre-exposure (PE) condition received 50 presentations of a 30-s tone stimulus at a variable inter-stimulus interval of 40 ± 30 s; nonpre-exposed (NPE) subjects were confined to the chamber for an equivalent period of time without any stimulus presentation. Conditioning: Conditioning commenced immediately at the end of the pre-exposure phase without removing the animals from the chambers. Conditioning comprised three discrete trials of pairing between the CS and an unconditioned stimulus (US): Each trial began with the 30 s tone stimulus (identical to the one used during pre-exposure) followed 4 immediately by the delivery of a 1-s foot-shock set at 0.3 mA (= US). Each trial was preceded and followed by a 180 s interval. Test: The test of conditioned responding to the tone CS was conducted another 24 h later, when the animals were placed in the same test chambers again. Following an initial period of 360 s acclimatization, the tone CS was delivered and remained on for 90s, in which the time of conditioned freezing to the tone stimulus was evaluated. Conditioned freezing was expressed as percent time freezing and data were analyzed separately for conditioning and test phase. Spatial Working Memory Test We evaluated the effects of PYY3-36 on spatial working memory using an established matching-to-position working memory paradigm in the Morris water maze (Meyer et al, 2010). The apparatus consisted of a white fiberglass tank (1 m diameter), positioned in a well-lit room enriched with distal special cues and contained a Plexiglas cylinder (7 cm in diameter) to provide an escape platform, which was 6 mm under the water. The working memory test was based on the matching-to-position paradigm, in which animals were required to learn the novel position of a hidden platform located on the first trial of each day in order to navigate effectively to the same location in the subsequent trial. The platform remained at the same position across trials on a given day, but took a new position on each habituation and test day. To begin each trial, an animal was placed gently in the water maze against the maze wall at one of the four cardinal positions (N, E, S, W) and was allowed a maximum of 60 s to locate the platform. If it failed to reach the platform, it was then guided to it by the experimenter. Each animal was allowed to spend 10 sec on 5 the platform before it was removed from it and placed into a waiting box containing home cage sawdust for another 10 sec. The animal was then placed into the watermaze again to commence the second trial. Hence, the inter-trial interval was ~20 sec for all trials and training/test days. Following a 2-day pre-training session with the platform visible in the center of the maze, animals were first tested without any discrete manipulation using the platform made invisible for two consecutive days. On the next two consecutive days, all animals were injected with vehicle (VEH) solution only before working memory assessment. This served to stabilize the animals’ performance after exposure to additional stress by injections. Animals that exhibited excessive floating or did not display any improvement from trial 1 to 2 during the VEH-injection phase were excluded from the subsequent test phase, which took place one day after the last VEH-injection. During the test day, one third of the animals received VEH solution again, while the remaining animals were treated with PYY3-36 at low (1 μg/100g BW) or high (20 μg/100g BW) dose. Escape latencies were calculated for each trial. Working memory was indexed by the reduction in time spent to find the platform in trial 2 compared with trial 1 (when the position of the platform was unknown to the animals). Data collection was achieved using the Ethovision tracking system as described before (Meyer et al, 2010). Statistical Analyses All data were analyzed by parametric analysis of variance (ANOVA) followed by Fisher’s least significant difference (LSD) post-hoc comparisons or restricted ANOVAs whenever 6 appropriate. Statistical significance was set at P < 0.05. All statistical analyses were performed using the statistical software StatView (version 5.0). In the elevated plus maze test, percent (%) open arm time and frequency were analyzed using ANOVA. In the social interaction test, exploration time was analyzed using a 3 × 2 (PYY treatment × object) ANOVA, followed by one-way ANOVA restricted to each experimental group. In the first test of PPI, % PPI and the reactivity to prepulse-alone trials were analyzed using 3 × 5 (PYY treatment × prepulse levels) ANOVAs, and the reactivity to pulse-alone trials was analyzed using one-way ANOVA. In the second test of PPI, % PPI and the reactivity to prepulse-alone trials were analyzed using 4 × 5 (group × prepulse levels) ANOVAs, and the reactivity to pulse-alone trials was analyzed using one-way ANOVA. In the LI test, conditioned freezing toward the critical tone-CS was assessed 24 h following conditioning: Percentage time freezing during the 90-sec period of the tone presentation was analyzed using a 3 × 2 (PYY treatment × pre-exposure) ANOVA, followed by one-way ANOVA restricted to each PYY treatment condition. Percentage time freezing during the initial conditioning phase was analyzed using a 3 × 2 × 3 (PYY treatment × pre-exposure × trials) repeated-measure ANOVA. In the water maze matching-to-position paradigm of working memory, the latency to find the submerged platform served as the critical test read-out and was analyzed using a 3 × 2 × 2 (group × trials × days) repeated-measures ANOVA for the initial SAL treatment phase, and by a 3 × 2 (PYY treatment × trials) repeated-measures ANOVA for the subsequent test phase. In addition, the latency improvement scores (i.e., [time in trial 1] – [time in trial 2]) were analyzed in the water maze working memory test using one-way ANOVAs. 7 References Lubow RE (2005). Construct validity of the animal latent inhibition model of selective attention deficits in schizophrenia. Schiz Bull 31(1): 139-153. Meyer U, Feldon J, Schedlowski M, Yee BK (2005). Towards an immuno-precipitated neurodevelopmental animal model of schizophrenia. Neurosci Biobehav Rev 29(6): 913947. Meyer U, Knuesel I, Nyffeler M, Feldon J (2010). Chronic clozapine treatment improves prenatal infection-induced working memory deficits without influencing adult hippocampal neurogenesis. Psychopharmacology 208(4): 531-543. Richetto J, Feldon J, Riva MA, Meyer U (2013). Comparison of the long-term consequences of withdrawal from repeated amphetamine exposure in adolescence and adulthood on information processing and locomotor sensitization in mice. Eur Neuropsychopharmacol 23(2): 160-170. Vuillermot S, Joodmardi E, Perlmann T, Ove Ogren S, Feldon J, Meyer U (2011). Schizophrenia-relevant behaviors in a genetic mouse model of constitutive Nurr1 deficiency. Genes Brain Behav 10(5): 589-603. 8