Animals engaging in operant testing were moved to a 9a-9p reversed light cycle to permit testing during

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1 Running title: Male-specific deficits in a mouse model of p. hemideletion Supplementary Information Supplementary Methods Operant testing Animals engaging in operant testing were moved to a a-p reversed light cycle to permit testing during the dark period. Food restriction was begun to maintain -% of free feeding weight. Animals were preexposed to the reinforcer (Yoohoo, Mott s, Plano TX USA) by providing an additional water bottle containing oz Yoohoo for h in the home cage and verifying consumption by all cagemates. Animals were tested in -hole mouse operant chambers (Lafayette Instruments, Lafayette IN USA) with peristaltic pumps to deliver liquid reward (ul for males, ul for females to accommodate smaller body size). The holes (operanda) are in the rear, and contain recessed lights and infrared beams that register nose pokes, while the liquid delivery magazine is at the front (See Figure a for schematic). There are also two infrared beams at the front (~cm from magazine) and back (- cm from holes), and a house light. Testing was run in darkened chambers. On days -, animals were exposed to daily minute sessions of pretraining, where free reward was delivered approximately once a minute in conjunction with illumination of the center hole of the -hole array for seconds, providing a Pavlovian cue predicting reward delivery. All animals learned the Pavlovian association between illumination of the hole and imminent reward delivery, as measured by approaches to the magazine during the second cue presentation on day (see Supplemental Figure ). Next, animals transitioned to a fixed ratio (FR) schedule. The center hole was lit and reinforcer could be earned by a single nose poke breaking the IR beam in the hole. Responses per day and days to criterion were logged for each animal, and analyzed using repeated measures ANOVA. The proportion of nosepokes delivered to the nonreinforced holes, from furthest from the center to closest to the center, was analyzed via chi-squared testing in comparison with wildtype males. Following acquisition of FR responding, animals were tested with two sessions of progressive ratio (PR) testing, week apart (the first test is shown in Figure as results were similar). The PR schedule was as previously described. Briefly,

2 Running title: Male-specific deficits in a mouse model of p. hemideletion the schedule incremented in an arithmetic sequence (,,,,,, n), and the number of responses needed to earn reinforcement increased every third trial, i.e.,,,,,,,...n. PR testing was terminated after an animal failed to make a nosepoke response at the active hole for minutes, or after a total of minutes of testing, whichever came first. The PR breakpoint was defined as the last trial where the ratio was completed and reinforcer was delivered. Following PR, animals were transitioned to an FR where a response at any hole was rewarded in preparation for -choice testing. Responses rapidly became distributed to all operanda. However, del/+ males took longer to distribute these responses amongst all the active holes than did wildtype males (see Supplemental Figure ). -CSRTT was administered as previously described. Schematic of the task can be seen in Figure. Errors were punished by time out (house light on for s, no reinforcer, operanda inactive). Magazine entry was necessary to initiate the next trial after correct and error trials. Briefly, animals began -CSRTT testing on the least challenging schedule, with a fixed second intertrial interval (ITI) and a second stimulus duration. Criteria for having learned and performed the task adequately were > correct responses, with >% correct, for consecutive days for that schedule. Performance for the training schedule and subsequent schedules was assessed on the second day of having met criteria for that schedule and analyzed by ANOVA within a schedule and by repeated measures ANOVAs across schedules. Time to learn the task on the least challenging schedule did not differ between groups (- days). Performance was assessed on the second day after meeting criterion, as in. This permits us to capture potential contributors to behavioral deficits in del/+ males that would impede performance once the task was understood, without the influence of overtraining with reinforcement to ameliorate performance deficits, as has been shown in rodents as well as in humans in the analogous Continuous Performance Task. Thus, we assess performance for each animal when each individual performs above criterion, when we can capture a snapshot of the remaining behavioral impairments that may temporarily prohibit del/+ males from performing as well as wildtype males (Supplemental Figure ).

3 Running title: Male-specific deficits in a mouse model of p. hemideletion After acquisition, stimulus criteria became more stringent. As animals achieved criteria with these settings, the stimulus duration was progressively shortened and the ITI was made variable in the following pattern: s and s ITI; s stimulus and s ITI; stimulus and s ITI; stimulus and -s ITI; s stimulus and -s ITI. As shown previously and as seen in Supplemental Figure, animals rapidly improved in the task, and the initial deficits seen in animals that had recently acquired the basic - CSRTT were no longer apparent as animals gained more experience with the task demands, even with the addition of the variable ITI and shortened stimuli. All data was analyzed using GraphPad Prism. unless otherwise noted. Sucrose preference Preference for sweetened liquids was assessed in a separate cohort of animals from those used in operant testing. Animals were singly housed prior to sucrose testing. % sucrose was dissolved in deionized water and provided in the home cage using water bottles identical to those which provided standard water, next to the bottles providing standard water. Location of sucrose bottle was counterbalanced between animals. Sucrose bottles were provided in the home cage for hours. Consumption of sucrose and water was assessed daily by measuring bottle volumes, and data from the last three days of testing were calculated as a percentage of each liquid consumed over the total volume of liquid consumed. Average percent preferences were assessed by unpaired t-tests. Auditory brainstem response The hearing ability of p. hemideletion (del/+) mice and wildtype littermates was tested by recording auditory brainstem responses (ABR). Animals were housed with same-sex litter mates on a reversed - h/-h light/dark schedule. All mice had ad libitum access to food and water. We tested twelve del/+ and twelve wildtype mice ( males and females per group). All experiments were conducted at Washington State University Vancouver.

4 Running title: Male-specific deficits in a mouse model of p. hemideletion Mice were anesthetized using a ketamine and xylazine mixture ( mg/kg and mg/kg, i.p.), placed on a heating pad to maintain body temperature near C. ABRs were recorded in a sound attenuating chamber using standard subcutaneous needle electrodes (Grass Technologies, Warwick, RI, USA) with the active electrode parallel to the right mastoid and the reference electrode at the vertex of the skull. Pure tone stimuli were generated and ABR responses recorded with custom written software. Myogenic artifacts greater than µv, although rare, were rejected by the software. Brainstem responses were amplified and filtered ( khz; Hz- khz; Dagan Corporation, Minneapolis, MN, USA), band-pass filtered (-, Hz; Krohn-Hite, Brockton, MA, USA), passed through a spike signal enhancer (Fredrick Haer, Bowdoin, ME, USA) and then digitized (, samples/s; Microstar Laboratories, Bellevue, WA, USA). Pure tone stimuli ( ms duration, ms rise/fall time, repetition rate /s) were output via a high-speed data acquisition board (, samples/s; Microstar Laboratories, Bellevue, WA, USA) and presented free-field via an Infinity Emit leaf-tweeter speaker placed cm from the right ear and from midline. The speaker was regularly calibrated using a / inch calibrated microphone (Bruel and Kjaer model ) placed in the position normally occupied by the mouse s ear. Speaker output was flat between and khz. There was a gradual decrease in sound pressure of about. db per khz between to khz, with a maximum output of db SPL at khz. Distortion components were buried in the noise floor at least db SPL below the signal level. Hearing thresholds were determined from to khz in steps of khz and from to khz in steps of khz. Hearing threshold was defined as the lowest sound pressure level (SPL) in which a recognizable and repeatable waveform was present. Responses were sampled over a ms window with a ms stimulus onset delay. The recording window was averaged over repetitions for intensities at least db SPL above threshold and averaged over repetitions for intensities within db SPL of threshold. Each mouse was tested once and average thresholds and standard deviations were calculated

5 Running title: Male-specific deficits in a mouse model of p. hemideletion for del/+ and wildtype mice in each group. Because these data were non-normally distributed (Shapiro- Wilk test), we used a Kruskal-Wallis test to compare the group means for all frequencies. Ex vivo dopamine and dopamine metabolites High-performance liquid chromatography (HPLC) was used to measure the content of DA and its metabolites in the mesolimbic reward areas of the brain (n = -). Whole brains were rapidly extracted and snap-frozen in liquid nitrogen. Striatal and cortical tissue were dissected out of serial slices for subsequent analysis. The tissue was prepared for analysis by homogenization in. N perchloric acid, centrifuged at, rpm, and the supernatant filtered. Samples were analyzed by a Bioanalytical Systems HPLC (West Lafayette, IN) using a LC-C electrochemical detector. Samples were injected onto a reverse phase microbore column. Separation for DA and DA metabolites was accomplished by a mobile phase consisting of -mm sodium acetate, -mm citric acid,.-mm ethylenediamine tetraacetic acid,.-mm sodium octyl sulfate, and % methanol v/v at a ph of.. Peak heights of samples were measured and compared with standards for DA and its metabolites,-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA). Data were analyzed as t-tests between genotypes.

6 Running title: Male-specific deficits in a mouse model of p. hemideletion Supplementary Figure Captions Supplemental Figure. Body weight reduction caused by p. hemideletion did not differ by sex. Mean body weights from animals undergoing operant testing were assessed at least twice a week during both the ad libitum feeding period prior to operant training, as well as the calorie-restricted feeding period during operant training (animals were restricted to -% of their free-feeding weights; see Supplemental Methods). Both male and female del/+ mice weighed significantly less than wildtypes of the same sex during both the ad libitum feeding period and the restricted period (males: main effect of genotype F(,)=., p=.; females: main effect of genotype F(,)=., p=.). In addition, all groups experienced a slight but significant drop in weight as a result of the food restriction paradigm (males: main effect of restriction F(,)=., p<.; females: main effect of restriction F(,)=., p=.), indicating that all groups were equally impacted by the caloric restriction. No significant interactions were observed (all p>.) corroborating that the impact of caloric restriction did not differ as a function of genotype. Ns are the same as Figure b and. indicates significant effect of genotype. # indicates significant effect of food restriction. All figures depict mean +/- SEM. Supplemental Figure. Activity patterns of animals during FR training reveal unique deficits in del/+ males in nonreinforced responses, but not general activity levels. A) The number of nonreinforced responses across FR training is significantly lower in del/+ males. The number of responses made to any hole other than hole during the first days of FR training was significantly higher in wildtype males than in del/+ males (-way ANOVA on the males, main effect of genotype: F(,)=., p=.), and that this did not significantly change over the course of the day training period (main effect of day: F(,)=., p=.), despite the fact that by day del/+ males were making an equal number of reinforced responses to the wildtype males. This suggests that wildtype males benefitted from making more responses, as well as from targeting these responses more rapidly to the area

7 Running title: Male-specific deficits in a mouse model of p. hemideletion around hole (Figure ). In contrast to the males, female del/+ and wildtype animals did not differ in their rate of nonreinforced responses (main effect of gene p=.), consistent with a lack of operant deficits in del/+ females. B) The number of magazine entries across FR training was not impacted by hemideletion of p., though the number of entries was reduced over the training period (main effect of day in males: F(,)=., p<.; females: F(,)=., p=.). C) Beam breaks at the front of the chamber, where the food magazine was located, did not differ as a result of genotype in either males or females (both main effects of gene not significant). D) Beam breaks at the rear of the chamber, where the -hole array was located, did not differ as a result of genotype in either males or females (both main effects of gene not significant). Ns are the same as Figure b and. indicates significant effect of genotype. All figures depict mean +/- SEM. Supplemental Figure. Del/+ animals are able to form normal Pavlovian approach responses in a conditioned approach task. To assess whether reinforcement learning deficits in del/+ males were generalized to Pavlovian appetitive tasks, we assessed animals in a conditioned approach task (see Suppemental Methods). Briefly, during the first days of operant chamber testing, male and female wildtype and p. del/+ mice were exposed to trials a day where the center hole was lit for seconds (the cue ) prior to the free delivery of the sweetened reward. Approach data to the reward magazine (top row), to the front of the chamber where the magazine was located (middle row), and to the rear of the chamber where the cue was located (bottom row) was assessed via infrared beam breaks measured during the s cue (a in males, c in females), as well as during the s prior to the cue start as a control (b in males, d in females). We found no significant differences between wildtype and del/+ males or females on any of the approach measures (as measured by t-tests, all p>.). However, -way ANOVA on magazine entries (the top row of data) for males during the cue (column a) versus before the cue (column b) showed a strongly significant approach to the food magazine during the cue over the rate of approach before the cue was on (main effect of cue: F(,)=., p<.). A -way ANOVA on magazine entries in females showed a similar significant

8 Running title: Male-specific deficits in a mouse model of p. hemideletion approach during the cue (main effect of cue: F(,)=., p<.). Thus, all animals were able to distinguish the cue-on versus the cue-off periods and displayed Pavlovian approach behavior to the magazine during the cue. These data suggest that the ability to form appetitive Pavlovian associations is maintained in p. del/+ animals of both sexes. All figures depict mean +/- SEM. Supplemental Figure. Del/+ males and females are able to form and express normal Pavlovian contextual fear memory. To assess whether reinforcement learning deficits in del/+ males were generalized to other forms of learning, we tested a cohort of male and female wildtype and del/+ mice in a Pavlovian contextual fear task (see Supplemental Methods). We examined levels of freezing before the shock was delivered on the training day ( Pre condition) as well as in the shock context hours later ( Post condition). Both male and female del/+ mice were completely unimpaired in forming and expressing Pavlovian fear associations (main effect of conditioning: males: F(,)=., p<.; females F(,)=., p=.). No differences in either pre shock or post shock freezing levels driven by del/+ were detected (main effect of genotype and genotype x conditioning interactions not significant in both sexes, all p>.). Two way ANOVAs conducted on just the pre-shock freezing levels between males and females of both genotypes revealed significantly higher pre-shock freezing in females (main effect sex F(,)=., p=.), but no significant effects of genotype and no interaction effects (all p>.). A similar cross-sex ANOVA on post-training freezing levels found no significant effects of sex, genotype, or sex x genotype interaction (all p>.). Thus, we did not find that p. hemideletion impacted any measure of Pavlovian contextual fear conditioning. # indicates significant effect of conditioning. indicates main effect of sex. All figures depict mean +/- SEM.

9 Running title: Male-specific deficits in a mouse model of p. hemideletion Supplemental Figure. When a greater variety of responses are associated with reinforcement, del/+ males earn the same number of reinforcers as wildtype, but from a less diverse selection of responses. A) In the process of transitioning animals from the FR task to the -CSRTT, the number of holes that could now elicit a reinforcer was increased from only the center hole to any odd-numbered hole. We examined the number and pattern of responses made by male and female wildtype and del/+ animals during the days they were exposed to this task. B) The number of reinforced responses made overall did not differ between male wildtype and del/+, or between females of either genotype (-way ANOVA within each sex; main effect of genotype and genotype x day interaction not significant). C) On the first day of reinforcement from any odd-numbered hole, del/+ males made a greater number of their reinforced responses at hole than wildtype males (χ ()=., p=.). No difference in response distribution was noted between female genotypes (χ p=.). D) On the third day of reinforcement from any oddnumbered hole, del/+ males were still performing a greater number of reinforced responses at hole as compared to wildtype males (χ ()=., p=.), with no difference in distribution in the females. E) By the fifth day of reinforcement from a response at any odd-numbered hole, del/+ males were now distributing their responses amongst the odd-numbered holes at a rate comparable to wildtype males (χ p=.). F) The number of nonreinforced responses (those delivered to the even-numbered holes) tended to be higher in wildtype males than del/+ males (main effect genotype F(,)=., p=., but did not differ between genotypes in females. In this task, where a greater number of responses are associated with reinforcement, del/+ males take longer to distribute their responses amongst the possibilities while still performing an equal number of reinforced trials, suggesting that deficits in learning to perform novel responses for reinforcement is uniquely impaired in del/+ males as compared to wildtype males. indicates significant effect of genotype. All figures depict mean +/- SEM.

10 Running title: Male-specific deficits in a mouse model of p. hemideletion Supplemental Figure. Early deficits shown by del/+ males in the -Choice Serial Reaction Time Task are mitigated with additional training, even as the stimulus durations are shortened. Because the deficits in the -CSRTT shown by del/+ males were limited to incorrect responses, which would be consistent with deficits in forming action-outcome associations rather than attention or impulse control errors, we continued to test animals on the -CSRTT on a standard training protocol of increasing difficulty. The data shown in Figure is shown here as the leftmost dots on each graph, when the stimulus was s long and the intertrial interval was a fixed s (see Supplemental Methods). A) Del/+ animals of both sexes performed the later stages of the -CSRTT with a comparable number of correct responses as wildtype animals. Both sexes increased their correct response rate over time (main effect time: males: F(,)=., p<.; females: F(,)=., p=.), consistent with our prior observations of improvement in animal models of neurodevelopmental disorder. B) Premature response rates decreased substantially in all animals with more training in the task, despite decreased stimulus durations and the introduction of variable ITIs (main effect time: males: F(,)=., p<.; females: F(,)=., p<.). C) Likewise, all animals performed fewer incorrect responses with increased exposure to the task (main effect time: males: F(,)=., p<.; females: F(,)=., p<.). However, del/+ males did perform significantly more incorrect responses at the point of just having learned the CSRTT (males: genotype x time interaction: F(,)=., p=.), consistent with a deficit in discriminating the outcomes of an incorrect and correct response. D) With shortening stimulus durations, the number of trials omitted by all animals increased, revealing the impact of this task demand on performance (main effect time: males: F(,)=., p<.; females: F(,)=., p<. indicates a significant gene x training interaction. E) Correct performance in the -CSRTT improves abruptly with acquisition of the task. Because animals acquire the C task at different rates, in Figure and in panels A-D of this Figure we compared behavior between groups normalized to the day each individual animal met acquisition criteria for the task. (Acquisition criteria were greater than % correct trials, see Methods and Supplementary Methods for additional detail.) This was done to ensure that we

11 Running title: Male-specific deficits in a mouse model of p. hemideletion were comparing deficits in animals that had demonstrated a fundamental understanding of the task. However, this can make it difficult to see how the animals are performing during acquisition, shown here. Correct performance during choice acquisition in wildtype and del/+ males improved abruptly over the course of the first days of training, with animals improving performance due to task acquisition beginning on training day. There was a trend towards a main effect of decreased percent correct performance in del/+ males across this period, but it did not reach significance (p=.). All figures depict mean +/- SEM. Supplementary Figure. Del/+ male and female animals exhibit normal hearing. The hearing ability of p. hemideletion (del/+) mice and wildtype littermates were tested by recording auditory brainstem responses (ABR) under anesthesia. Solid lines indicate that an ABR was detected at that intensity; dashed lines indicate that no ABR was detected at the highest intensity tested. Both wildtype and del/+ mice had normal auditory thresholds, regardless of sex. Average thresholds for wildtype and del/+ mice are shown for each frequency tested. No mice had auditory responses to khz or higher, even at maximum output intensity (indicated by dashed lines, colored as wildtypes). A) p. hemideletion had no significant effect on the sensitivity of hearing in males, and no significant effect on the range of maximum and minimum auditory sensitivity. B) Similarly, auditory sensitivity was not affected in del/+ females compared to wildtype females. n= per group. A and B depict mean +/- standard deviation. Supplementary Figure. Striatal gene expression for all genes in the p. region are reduced by half in both males and females. All del/+ animals are missing one copy of the genes in the p. region, but gene expression does not necessarily follow gene dosage. Thus, we analyzed baseline expression of all genes from the p. region in wildtype and del/+ male and female animals using a Fluidigm Biomark x system for high throughput gene expression and commercially available Taqman primers. Nearly all genes in the p.

12 Running title: Male-specific deficits in a mouse model of p. hemideletion region were expressed in the striatum. Two genes from the p. region, Zg and Gdpd, are not displayed; Zg expression was not detectable in the striatum, and Gdpd expression was highly variable in both wildtypes and del/+, suggesting the primer may have amplified multiple products. A) Expression of all genes in the p. region in male wildtype and del/+ animals, normalized to wildtype males. All measureable genes were reduced in expression % in del/+ males. B) Expression of all genes in the p. region in female wildtype and del/+ animals, normalized to wildtype females. All measureable genes were reduced in expression % in del/+ females. C) The Mapk (ERK) gene located within the p. region is closely related to another ERK isoform, Mapk (ERK). mrna expression of Mapk/ERK was not altered in either male or female del/+. n=- per group. All panels depict mean +/- SEM. indicates significant difference from wildtype from the same sex. Supplementary Figure. p. hemideletion does not alter dopamine levels or dopamine turnover. The changes in dopamine receptor gene expression and ERK activity led us to question whether dopamine synthesis or turnover were significantly impacted by the hemideletion. Dopamine (DA) and its major metabolites DOPAC and HVA were analyzed in wildtype and del/+ males in dorsal striatum (A and B), the nucleus accumbens and globus pallidus (C and D), and the medial prefrontal cortex (E and F). No changes in total DA, DOPAC, or HVA, or DA:DOPAC or DA: HVA ratios were seen in any tissue. n= in wildtype males and = in del/+ males. All panels depict mean +/- SEM. Supplemental Figure. Hemideletion of the p. region does not alter protein levels or activation of the ERK kinase MEK. The elevated ERK phosphorylation seen at baseline and especially following sucrose in del/+ males suggested that in del/+ males either there was a hyperactivation of the ERK kinase MEK in the striatum, or a deficit in the function of phosphatase activity for ERK (Figure ). We measured phosphorylation of

13 Running title: Male-specific deficits in a mouse model of p. hemideletion MEK as well as total MEK protein levels in the striatum of male wildtype and del/+ animals at baseline and following sucrose consumption. A) Consumption of sucrose led to elevated MEK phosphorylation in both wildtype and del/+ males normalized to total MEK levels (main effect sucrose F(,)=., p=.). B) Elevated MEK phosphorylation was also significant compared to GAPDH used as a loading control (main effect sucrose F(,)=., p=.). C) Neither loss of p. nor sucrose caused changes in the total amount of MEK in the striatum. D) Representative western blot images. Western blot images are displayed as a heat map, ranging from high protein levels (red) to low protein levels (blue). B indicates basal samples, S indicates samples from animals that consumed sucrose, and L indicates a ladder lane. n= per group. A-C depict mean +/- SEM. # indicates significant difference from basal animals of the same genotype. Supplementary Figure. p. hemideletion does not significantly alter the activation of StEP, only the total levels in males. Phosphorylation of StEP at Ser reduces its ability to form protein-protein interactions and thus reduces its catalytic activity. Because the total amount of StEP in striatal homogenates was reduced in del/+ males, we investigated whether there were also changes in the active pool of StEP protein using an antibody specific for the nonphosphorylated form of StEP. A) There were no significant differences between any groups in nonphosphorylated StEP normalized to the loading control b-tubulin, however the overall trend was the same as the total StEP blots seen in Figure. B) There were no differences in amount of nonphosphorylated StEP normalized to the total amount of StEP in the striatum. C) Representative western blot images. Western blot images are displayed as a heat map, ranging from high protein levels (red/yellow) to low protein levels (blue/black). n=- per group. A and B depict mean +/- SEM.

14 Supplemental Figure : Body weights in wildtype and p. hemideleted animals b # # ad libitum () () restricted body weight (g) body weight (g) a # # ad libitum () () restricted

15 Supplemental Figure : Activity during FR acquisition a all nonreinforced responses magazine entries front beam breaks c rear beam breaks d wildtype () p del/+ () Days of FR training Days of FR training Days of FR training Days of FR training wildtype () p del/+ () b Days of FR training Days of FR training Days of FR training Days of FR training

16 Supplemental Figure : Pavlovian Conditioned Approach b a number of trials contact made at magazine during cue on s prior to cue number of trials contact made during cue on () () () number of trials contact made at front of chamber s prior to cue () at rear of chamber d c

17 Supplemental Figure : Contextual Fear Conditioning # # # % freezing # shock: pre post pre post wt () del/+ () pre post pre post wt () del/+ ()

18 Supplemental Figure : FR to odd-numbered holes a all reinforced responses reinforced responses c proportion of reinforced responses d wildtype () p del/+ () wt proportion of reinforced responses e nonreinforced responses proportion of reinforced responses all nonreinforced responses Days of hole FR training del/+ wt f Day del/+ wildtype () p del/+ () Days of hole FR training Day Days of hole FR training Day reinforced responses reinforced responses b Days of hole FR training

19 Supplemental Figure : CSRTT performance across time a Correct CSRTT Performance wildtype () p del/+ () Correct b stim s s ITI fixed s c Intertrial Interval stim s s ITI fixed s d stim s s ITI fixed s stim s s ITI fixed s e stim s s ITI fixed s s var -s stim s s ITI fixed s wt del s var -s s var -s s var -s % omitted % incorrect Trial End s var -s Omitted percent correct responses % premature stim s s ITI fixed s s var -s s var -s wildtype () p del/+ () % omitted % incorrect % premature Incorrect stim s s ITI fixed s Stimulus On Premature % correct Stimulus Trial End On % correct Intertrial Interval Days of c training s var -s

20 Supplemental Figure : Auditory brainstem responses Threshold (db SPL) a wildtype () p del/+ () Frequency (khz) Threshold (db SPL) b Frequency (khz) wildtype () p del/+ ()

21 .. c... fold change from wt fold change from wt fold change from wt Al Sp n Q prt Ki f M az Pr Pa rt gr a M v C p di Se pt z As l ph K d Tm ctd em Ta ok H iri In p o D e oc a Fa l m Al b do Pp A p Tb c x ER K Ype /M l ap C k or o Bo a la. n Q prt Ki f M az Pr Pa rt gr a M v C p di Se pt z As l ph K d Tm ctd em Ta ok H iri In p o D e oc a Fa l m Al b do Pp A p Tb c ER Y x K pe /M l a C pk or o Bo a la b. Al fold change from wt a Sp Supplemental Figure : Striatal gene expression of genes in p. region wildtype () p del/+ ().. ERK/ Mapk.... ERK/ Mapk. wildtype () p del/+ ()..

22 Supplemental Figure : Dopamine and metabolites total protein levels monoamines (ng/mg protein) c DA DA HVA DOPAC Prefrontal Cortex.. d... DA HVA DOPAC Dorsal Striatum.. DOPAC:DA HVA:DA Nucleus Accumbens..... f metabolite:da (ng/mg).. DOPAC. b metabolite:da (ng/mg) monoamines (ng/mg protein) HVA Nucleus Accumbens monoamines (ng/mg protein) wildtype () p del/+ () e Dorsal Striatum metabolite:da (ng/mg) a dopamine turnover DOPAC:DA HVA:DA Prefrontal Cortex DOPAC:DA HVA:DA

23 Supplemental Figure :MEK phosphorylation following sucrose in males # b # c # # MEK:GAPDH basal sucrose pmek:gapdh pmek:total MEK a wildtype p del/+ () () () () wildtype p del/+ d wt B phospho MEK total MEK GAPDH S L B del/+ S wildtype p del/+

24 Supplemental Figure : Nonphosphorylated (active) StEP in striatum b nonphospho StEP: total StEP nonphospho StEP: Tubulin a () () c wt nonphosphostep StEP βtubulin () () del/+ wt del/+