MEK inhibition ameliorates social behavior phenotypes in a Spred1 knockout mouse model for RASopathy disorders

Spred1-/- mice were generated as previously described [39]. Spred1+/−  mice were backcrossed at least 10 times onto the C57BL/6 J (B6) background prior to breeding to generate experimental animals. In two separate cohorts of mice for the automated tube test and open field (see Table 1), Spred1+/−  mice on B6 background were crossed with 129T2/SvEmJ mice, and F2 hybrids (referred to as B6;129T2 from hereon) from these crosses were used for experiments. Mice were genotyped by PCR of DNA extracted from neonatal toe biopsy. For all behavior experiments analyzing Spred1-/- mice, Spred1 + / + wildtype mice (henceforth referred to as WT), which were littermates from the Spred1+/− x Spred1+/−  breeding, were used as controls. Mice were housed under standard laboratory conditions on a 12 h dark/light cycle and all behavior testing was carried out in the light cycle. Bedding was Lignocel® BK 8/15, and standard laboratory food and water were available ad libitum. The details on the cohorts of mice for all behavior tests are summarized in Table 1, with information on background strain, sex, age, housing and any drug treatment. All experiments were reviewed and approved by the Animal Ethics Committee of KU Leuven or the Animal Ethical Committee of Erasmus MC Rotterdam in accordance with the European Union Directive 2010/63/EU.

Mice were weaned at 3–4 weeks and housed in same sex pairs, with one WT and one Spred1-/- mouse together (Table 1). Occasionally when weaning, a correct genotype/sex partner was not immediately available. In that case, the mouse was group housed with same sex littermates until the next litter was weaned, and then paired appropriately. If this housing rearrangement was done, it was done no later than 6 weeks of age. Testing was carried out in adult mice in age matched cohorts, starting at 2–3 months of age. Mice were behaviorally naïve before tube test experiments and were not used for other behavior experiments, so as not to interfere with social hierarchy. The automated tube test apparatus (Benedictus Systems, Rotterdam, The Netherlands) has been previously described [40, 41]. In brief, it is a transparent 50 cm long fiberglass tube with a center door, connected at both ends to fiberglass boxes with automated entrance doors, and an infrared tracking system. Training and tournament protocols were carried out based on previous studies [40]. A 5-day training protocol was first performed for all mice. On day 1 mice received 2 habituation trials with doors open, allowing free exploration of the tube for a maximum of 180 s, or until the mice walked through the tube into the other box. On days 2–5, mice had a limit of 30 s to walk through the tube, after which they were assisted manually. If the mice remained in the starting box for more than 5 s, they received an air puff to assist them entering the tube. They received 6 trials total each day, starting from alternate boxes. Two days after training was complete, the tournament protocol was started and lasted for 5 days. On each tournament day, mice received 2 training trials, 45 min before the tournament matches started. In the matches, a mouse was placed in each box, and the doors automatically opened simultaneously, allowing mice to move into the tube. If the mouse remained in the box for more than 5 s, it received a puff of air. The center door opened automatically when both mice were within 4 cm of the door. The match was complete when one mouse had retreated with all 4 paws into its starting box (assigned the “loser”), and the other mice was assigned as the “winner”. The apparatus was cleaned after every training trial and tournament match with a 70% ethanol solution. For all tournaments, cohorts of 4–6 cages of same sex pairs (n = 4–6 mice/genotype) played against each other. Both male and female cohorts were tested on B6 and B6;129T2 F2 hybrid backgrounds. For cohorts on the B6;129T2 background, matches were played between all mice and WT-Spred1-/- matches are analyzed and presented. For all other cohorts including MEK inhibitor studies, matches were played only between WT and Spred1-/- mice in each cohort. For MEK inhibitor studies, cohorts of both male and female mice were used. On each tournament day, the same matches were played, but with a pseudorandomized order, with starting box alternated and inter-match interval balanced. Experiments were repeated on independent cohorts to verify reproducibility.

For pups, the day of birth was defined as postnatal day 0 (P0). Pups were evaluated for ultrasonic calls emitted at neonatal stages on P4, P6, P8, P10 and P12. These mice were a separate cohort not used for adult behavior assays. On each day of testing, pups were individually removed from the nest and placed in an empty plastic container located inside a sound attenuating Styrofoam box. An ultrasound microphone, sensitive to frequencies of 10–180 kHz (Avisoft UltrasoundGate condenser ultrasound microphone CM16, Avisoft Bioacoustics e.K., Glienicke/Nordbahn, Germany), was placed through a hole in the Styrofoam box approximately 20 cm above the pup. Ultrasonic vocalizations (USVs) were recorded for 3 min using Avisoft-RECORDER software, using a 250 kHz sampling rate and 16-bit format. After testing, pups were weighed and returned to the nest. After testing on P4, pups were also individually identified with a paw tattoo using a 22G syringe needle filled with Indian ink. Two independent cohorts were analyzed. In the second cohort, righting reflex was also assessed in mice on P4 and P8, after USV recording. Pups were placed on their back and the time taken to right onto all 4 paws was recorded. Data from both sexes was pooled as we saw no significant differences in vocalization between sexes (data not shown). We also excluded dam experience as a major factor influencing our data (data not shown), and present here data pooled from pups born to both naïve and experienced dams.

USVs during courtship behavior were measured in a separate cohort of adult male mice, aged 10-11 weeks, in response to unfamiliar female adult intruders. Male adult subject mice were housed overnight with an adult female B6 for sexual experience. They were then housed individually for 3 days. Adult female B6 mice (aged 2–3 months) that served as stimuli were different to the mice used for overnight housing, and were group housed [4–5 mice per cage]. Female mice did not have estrus induced, and estrous cycle was not tracked. On the day of the test, the male subject was placed in a clean testing cage, in a sound attenuating Styrofoam box with an ultrasound microphone above, as described above. The mice were habituated for 10 min to the novel environment. In the last 4 min, a baseline recording was made. Next, an unfamiliar female mouse was introduced into the cage and the mice were allowed to freely interact whilst USVs were recorded for 4 min. We did not determine whether the recorded USVs were from male or female mice, because in the context of a male–female encounter, likely representing courtship USVs, USVs are mainly produced by males [42, 43].

Acoustic properties of audio files were analyzed in Avisoft SASLab Pro software (version 5.2.10, Avisoft Bioacoustics e.K., Glienicke/Nordbahn, Germany). Spectrograms were generated with a Fourier transformation (FFT)-length of 1024 and time window overlap of 75% (100% frame, Hamming window). A high-pass filter at 30 kHz cut-off was applied to reduce background noise outside of the relevant frequency band. Call detection was by an automatic threshold-based algorithm and a hold-time mechanism (hold time 20 ms). An experienced user blinded to genotype checked accuracy of the call detection to ensure concordance between automated and observational detection. Parameters automatically extracted for each file included number of calls, the mean duration of calls, mean peak frequency (pitch, kHz) and mean peak amplitude (loudness, measured in decibels) [44]. For analysis of neonatal stages, no sex differences were observed (data not shown), so data from male and female pups were pooled together for analysis.

Female WT and Spred1-/- mice were tested at 20 weeks of age (Table 1). The apparatus was an enclosed rectangular transparent Plexiglas box with opaque floor (width x depth x height: 94 × 26 × 30 cm), divided into three chambers. The central chamber (42 × 26 cm) was connected to a left and right chamber (26 × 26 cm) via openings (6 × 8 cm) in that could be manually closed. The left and right chamber contained cylindrical wire cups (height x diameter: 11 × 10 cm). Access to the chambers was controlled by manually operated guillotine doors. The test consisted of three phases, conducted consecutively in a three-chambered test apparatus as previously described [45]. In the habituation phase [5 min], mice were placed in the central chamber to acclimatize. In the sociability phase (10 min), the animal’s interest in a social stimulus (a same sex conspecific, not used for behavior testing) versus an empty chamber were tested, with the social stimuli mouse constrained under a wire cage in one chamber, and an empty wire cage in the other chamber. In the final stage, preference for social novelty (10 min), interest in a familiar social stimulus versus a non-familiar social stimulus was assessed. The apparatus was cleaned in between subjects with 70% ethanol. Mice were recorded from above with a camera and their heads tracked using ANY-Maze software (Stoelting Europe, Ireland). Sniffing time (= time spent within 2 cm proximity to the wire cage) and pathlength were quantified. For the sociability phase, a social preference index was calculated as follows: Time_stranger1/(Time_stranger1 + Time_empty).

Open field was performed in male and female mice on the B6;129T2 background. The open field was conducted in a circular arena with a diameter of 110 cm. Mice were placed gently in the center of the arena and allowed to freely explore for 10 min. The arena was cleaned with 70% ethanol in between subjects. Mice were recorded from above with a camera and their body position tracked with the SMART video tracking system (Panlab, Barcelona, Spain). For analysis, the arena was divided into three regions: inner region of 25 cm diameter, surrounded by a middle region 10 cm radius and then an outer region of 10 cm radius. Total path length and percentage of time spent in each region were calculated. No sex differences were observed, therefore data from both sexes was pooled for analysis.

Nest building was conducted based on a previously reported protocol [46]. One week before testing, mice were single housed in standard Makrolon 3108 plastic mouse cages, with 5 cm deep clean bedding material but without environmental enrichment items to habituate. The testing phase was a 2-day protocol. On day 1 mice received pressed cotton nestlets weighing a total of 3 g at noon. Nest building behavior was assessed on day 2, in the morning. For qualitative assessment, a 5-point rating scale was used as previously published [46], with a score of “1” representing largely untouched nesting materials (no shredding) and “5” indicating a perfect nest with high sides.

Mice were habituated for 10 min in a large Makrolon 3108 plastic mouse cage (375 × 215 × 140 mm) with 5 cm depth clean bedding material (Lignocel® BK 8/15). Subsequently, marble burying was tested for 40 min. During the test phase, cages contained 5 cm deep clean bedding material with 10 glass marbles (14 mm diameter) arranged in a 2 × 5 grid on top of the bedding. 10 marbles were used instead of 20 to reduce the impact of overexposure to simultaneous stimuli. The number of buried marbles was counted. A marble was consider buried when 2/3 of the marble was covered by bedding.

Burrowing is an innate behavior, described in this experiment as ‘digging a hole or tunnel in or through food pellets in a tube’. The experiment was carried out based on a previously reported protocol [47]. In the habituation phase on day 1, a full burrow tube is placed overnight into the group home cage of the mice, which are later individually tested. Each burrow is filled with 200 g food pellets normally supplied as diet. The testing phase started on day 2 around 5 pm, as mice are normally awake at this time and burrowing starts more slowly than during the dark phase when mice are very active. Mice were placed individually in Makrolon 3108 plastic mouse cages together with a filled burrow tube. Two hours after the start, a snapshot measurement was taken of the weight of the remaining food pellets; the burrow was emptied into a container and weights, then the pellets were replaced into the burrow and placed back into the cage. If the mouse was in the burrow, it was gently removed and placed in the cage. The weight of non-burrowed pellets was presented as a percentage of total weigh of 200 g food pellets. The second reading was taken the next morning at 9am, again weighing the remaining non-burrowed food pellets.

The MEK inhibitor PD325901 (Selleckchem, Munich, Germany) was dissolved in dimethyl sulfoxide (DMSO) to a final concentration of 5 mg/ml and frozen into aliquots, which were defrosted as needed immediately prior to injection. For all studies, animals in the treated group received 5 mg/kg PD325901, whilst the vehicle group received DMSO alone. This dose was chosen based on its effectiveness in attenuating ERK phosphorylation in mice [48, 49] and at reversing other phenotypes in RASopathy mouse models [50, 51]. Injection volumes were 1 µl per g (mouse weight). Mice were injected intraperitoneally on alternating sides each day with 0.3 ml insulin syringes (BD Micro-Fine). For automated tube test experiments, drugs were injected daily starting 3 days prior to the first day of training and continuing throughout training and tournaments, and injections were 5–6 h before behavior testing started each day. This regimen was based on previous studies that successfully reversed behavioral phenotypes in RASopathy mice [52, 53]. For each different tube test experiment (WT + DMSO vs Spred1-/- + DMSO; Spred1-/- + DMSO vs Spred1-/- + PD325901; WT + DMSO vs Spred1-/- + PD325901 and WT + DMSO vs WT + PD325901), a new, separate cohort of mice was used that were naïve to the automated tube test. Mice were always housed in pairs, with one mouse from each ‘treatment’ per cage, as outlined in Table 1. For nest building experiments, mice were injected for 3 days and the nesting protocol initiated 5–6 h after last injection, as described above. After the initial nesting test, mice were left untreated for 3 weeks for drug washout, and then re-tested for nest building. Tube test training/testing each day was started 5–6 h after injection.

For cohorts of mice used for the MEK inhibition study in the automated tube test, hippocampal lysates were collected for western blot analysis. 1 h after the last automated tube test tournament on day 5, mice were euthanized by cervical dislocation, the brain removed and hippocampi rapidly dissected out and snap frozen in liquid nitrogen. Tissue was homogenized in RIPA buffer (150 mM NaCl, 50 mM Tris–HCl, 1 mM EDTA, 1 mM EGTA, 0.1% Triton X-100, 0.1% Na-deoxycholate) with protease inhibitors (Complete, Roche) and phosphatase inhibitors (PhosphoSTOP, Roche) added. Protein concentration was adjusted to 1 µg/µL and 10 µg protein was loaded onto a NuPAGE Bis–Tris 4–12% mini gel (Invitrogen). The blot was probed with primary antibodies for pERK (Phospho-p44/42 MAPK #4370, anti-rabbit, Cell Signaling Technology) and ERK (p44/42 MAPK #9107, anti-mouse, Cell Signaling Technology). Secondary antibodies were Dylight IR 680 and IR 800 (Thermo Scientific) and were visualized using an Amersham Typhoon Biomolecular Imager. The blot was stripped with Restore™ PLUS Western Blot Stripping Buffer (Thermo Scientific) and re-probed with beta-actin (A5316, anti-mouse, Sigma-Aldrich) as loading control. Quantitative image analysis was performed in ImageJ (v1.51n, NIH) using the Gels function. The ratio of pERK to total ERK was calculated, normalized to a sample from the WT-Vehicle group from DMSO-only treated group (Additional file 1: Supplementary Fig. 4A), and expressed as arbitrary units (a.u.).

For automated tube test experiments, including MEK inhibitor experiments, each day of the tube test was analyzed with two-tailed binomial distribution, with the null hypothesis that if both groups were similar, 50% of matches are won by each group (i.e. chance level of winning). For most other data, including male USV, marble burying, burrowing and western blotting, normality of data was tested with the D’Agostino-Pearson test. If data were normally distributed, a parametric unpaired independent samples t-test was used. If data were not normally distributed, non-parametric Mann–Whitney test was performed. Neonatal USVs were analyzed with 2-way ANOVA with repeated measures, with age and genotype as the main factors. Nest building: for naïve mice, an unpaired independent t-test was used. For MEK inhibitor experiments assessing nesting, 2-way ANOVA with treatment and genotype as factors was performed. For sociability and social preference, sniff time was analyzed with 2-way ANOVA, using chamber and genotype as factors. Preference index for sociability was analyzed with an unpaired independent t-test. A significance level of 0.05 was used for all statistical analyses. Statistical analysis was performed in GraphPad Prism 8 (GraphPad Software, Inc., La Jolla, CA, USA).

To examine whether social behaviors were altered in Spred1-/- mice, we evaluated mice in the automated tube test, which measures social dominance [40]. Spred1-/- mice on a B6;129T2 background won a majority of matches against wildtype (WT) opponents, a phenotype stable across 5 days of tournaments (Fig. 1A, B). This social dominance phenotype was seen in both male (Fig. 1A) and female (Fig. 1B) Spred1-/- mice against WT controls (Males—two tailed binomial test compared to chance (50%): day 1 p < 0.0001; day 2 p < 0.0001; day 3 p = 0.011; day 4 p < 0. 0001; Females—two tailed binomial test compared to chance (50%): day 1 p = 0.0011; day 3 p = 0.0011; day 4 p = 0.0251; day 5 p = 0.01). As background strain can influence behavior, we verified that this phenotype was robust to background strain, observing that the same social dominance phenotype was observed Spred1-/- mice when backcrossed more than 10 generations onto the B6 background in both male (Additional file 1: Supplementary Fig. 4A; two tailed binomial test compared to chance (50%): day 2 p = 0.0213; day 3 p = 0.0213; day 4 p = 0.0213; day 5 p = 0.0005) and female cohorts (Additional file 1: Supplementary Fig. 1A; two tailed binomial test compared to chance (50%): day 2 p = 0.0013; day 3 p < 0.0001; day 4 p < 0.0001; day 5 p < 0.0001). No genotype differences were seen in time to reach the center door in the training phase of the tube test (Additional file 1: Supplementary Fig. 1B, B6 background; 2 Way ANOVA: effect of day F(8,64) = 3.296, p = 0.0033; no effect of genotype, no interaction), and body weights were stable throughout the experiment (Additional file 1: Supplementary Fig. 1C, B6 background). The social dominance phenotype was not seen Spred1+/−  male mice when playing matches against WT controls in the automated tube test (Additional file 1: Supplementary Fig. 1D–F; Two tailed binomial test compared to chance (50%), days 1–5 p > 0.1). We examined whether the social dominance phenotype in male Spred1-/- mice was linked to aggression by performing the resident intruder test in a separate cohort of mice. There was no difference in either latency to attack or the total number of attacks that Spred1-/- male mice made onto intruders compared to WT littermates (Additional file 1: Supplementary Fig. 1G–H; latency—unpaired t-test, t = 1.794, df = 16, p = 0.0918; total attacks—unpaired t-test, t = 1.386, df = 16, p = 0.1848). In an open field test Spred1-/- mice displayed normal thigmotaxis, suggesting they were not less anxious than WT controls (Additional file 1: Supplementary Fig. 3C). Social approach and social preference in the 3-chamber social interaction test were normal in Spred1-/- mice (Additional file 1: Supplementary Fig. 2A–C; sociability phase preference index—unpaired t-test, t = 0.6898, df = 25, p = 0.4967. Social preference—2-way ANOVA, effect of chamber side: F(1,25) = 6.396, p = 0.0181; no effect of genotype). There was a significant reduction in the number of transitions between compartments made by Spred1-/- mice in both phases of the test (Additional file 1: Supplementary Fig. 2D–E. Sociability phase—unpaired t-test, t = 2.805, df = 25, p = 0.0096. Social preference phase—unpaired t-test, t = 2.061, df = 25, p = 0.0499), however locomotor activity in the open field was normal (Additional file 1: Supplementary Fig. 3D). This phenotype is similar to the slightly lower locomotor activity observed in Spred1-/- mice in operant chambers [36].

To study social communication in Spred1-/- and WT mice, male ultrasonic vocalization (USV) during an encounter with a novel B6 female conspecific was measured. Male Spred1-/- mice (B6 background) emitted significantly fewer USVs in response to a female compared to WT littermate controls (Fig. 1C; unpaired t-test, t = 4.481, df = 27 p = 0.0001). They also emitted on average shorter duration calls compared to WT (Fig. 1D; Mann Whitney test, p = 0.0002). The average pitch (mean peak frequency) and amplitude of calls (mean peak amplitude) was not significantly altered (Fig. 1E-F; frequency—unpaired t-test, t = 1.001, df = 27, p = 0.3258; amplitude—unpaired t-test, t = 0.8998, df = 27, p = 0.3762). Innate behaviors such as nest-building contribute to wellbeing and support social functions in mice [54], therefore nest building behavior was examined in adult female Spred1-/- and wildtype littermate controls. Spred1-/- had reduced nesting behavior, with a significantly reduced nesting score compared to WT mice, an indication of the quality of nest building (Fig. 1G; unpaired t-test, t = 3.496, df = 34, p = 0.0013). To investigate repetitive behaviors in this mouse line, behaviors that require digging were assessed, as digging has been interpreted as a repetitive behavior. No genotype differences were seen in the percentage of marbles buried between WT and Spred1-/- mice (Additional file 1: Supplementary Fig. 2F. Males: unpaired t-test, t = 0.4545, df = 14, p = 0.6564. Females: unpaired t-test, t = 0.8402, df = 20, p = 0.4107). Burrowing behavior was assessed as a measure with more ecological validity than marble burying, as burrowing behavior is seen in wild rodents [47, 54]. The amount of food pellets burrowed after either 2 h or overnight, was also unchanged in Spred1-/- mice (Additional file 1: Supplementary Fig. 2G. 2-way ANOVA with repeated measures, F < 1.00; p > 0.1). Thus, Spred1-/- have impaired social behavior and communication but no digging phenotypes that would be representative of changes in repetitive behaviors.

We next asked whether USV deficits are already present in early neonatal development in Spred1-/- mice. Isolation-induced ultrasonic vocalizations in Spred1-/- and WT pups (on the B6 background) were measured from P4-12. There were no significant differences between sexes at any of the time points, therefore data from male and female pups were pooled together for analysis. As previously reported in the literature for B6 mice [55], the rate of vocalization peaked at P8 in WT mice, and then declined to low levels again by P12 (Fig. 2A. 2-way ANOVA with repeated measures: main effect of age, F(4,156) = 12.28, p < 0.0001, no effect of genotype). No main effect of genotype was seen on call rate (Fig. 2A) or call duration (Fig. 2B; 2-way ANOVA with repeated measures: effect of age, F(4,156) = 7.347, p < 0.0001; no effect of genotype). Analysis of spectral properties of Spred1-/- and WT USVs revealed a main effect of genotype on call pitch (Fig. 2C; 2-way ANOVA with repeated measures: effect of age F(4,150) = 5.866, p = 0.0002; effect of genotype F(1,150) = 55.13, p < 0.0001), with post-hoc comparisons revealing significant decreases in mean peak frequency in calls from Spred1-/- pups compared to WT from P8-P12. There was also a main effect of genotype on the loudness in decibels of the call in Spred1-/- compared to WT, with post-hoc analysis revealing significant increase in amplitude at P10 (Fig. 2D; 2-way ANOVA with repeated measures: effect of age F(4,205) = 10.77, p < 0.0001, effect of genotype F(1,205) = 9.181, p = 0.0028). No main effect of genotype was seen on body weight (Additional file 1: Supplementary Fig. 3A; 2-way ANOVA with repeated measures: main effect of age, F(4,159) = 1325, p < 0.0001, no effect of genotype). No genotype differences were observed in righting reflex at P4 or P8 (Additional file 1: Supplementary Fig. 3B; 2-way ANOVA with repeated measures: main effect of age, F(1,29) = 77.15, p < 0.0001; no effect of genotype), indicating intact motor abilities in Spred1-/- pups. Together these data indicate altered spectral vocalization properties in neonatal Spred1-/- mice that primarily emerge in the second neonatal week.

Overactivation of Ras-MAPK signaling is a hallmark of RASopathy disorders. We next asked whether inhibition of downstream MAPK signaling could restore social behaviors in Spred1-/- mice. PD325901 is a potent MEK inhibitor that has been demonstrated to shrink neurofibromas both in Nf1 mouse models [50] and early stage clinical trials in human NF1 patients [56]. PD325901 crosses the blood–brain barrier, as evidenced by reports of human neurotoxicity [57], and further confirmation has been seen in mice, where peripheral administration of PD325901 has been demonstrated to abolish cocaine-induced ERK activation in the striatum [48] and to reduce hyperactive ERK phosphorylation in the hippocampus in a HRas^G12V RASopathy mouse model [49]. To test the effect of PD325901 on social behavior in adulthood, tube test experiments were performed in Spred1-/- and WT mice on the B6 background acutely treated with either PD325901 or DMSO vehicle control. All cohorts learned to run through the tube in training sessions (Additional file 1: Supplementary Fig. 4B, Additional file 1: Supplementary Fig. 4E, Additional file 1: Supplementary Fig. 5 A–C). When Spred1-/- mice treated with PD325901 played matches against WT mice receiving vehicle, the dominant winning phenotype of Spred1-/- mice in the tube test was abolished, with winning at chance level (Fig. 3A). Confirming that PD325901 normalizes social dominance behavior of Spred1-/- mice under different social conditions, we saw that Spred1-/- mice treated with PD325901 lost significantly more matches against Spred1-/- receiving vehicle, an effect that was stable across the first 3 days of tournaments (Fig. 3B; two tailed binomial test compared to chance (50%): day 1 p = 0.0011; day 2 p = 0.0011; day 3 p = 0.025). Tube test tournaments when both Spred1-/- and WT mice received vehicle control recapitulated the phenotype of naïve Spred1-/- mice over WT mice (Additional file 1: Supplementary Fig. 4A, 4D. Males—two tailed binomial test compared to chance (50%): day 2 p = 0.0213; day 3 p = 0.0213; day 4 p = 0.0213; day 5 p = 0.0005. Females—two tailed binomial test compared to chance (50%): day 2 p < 0.0001; Day 4 p = 0.005; Day 5 p = 0.0042), demonstrating that treatment with vehicle alone did not significantly influence behavior. Notably, WT mice were treated with PD325901, they won significantly more matches against WT mice treated with vehicle (Fig. 3C. Two tailed binomial tests compared to chance (50%): day 2 p = 0.0003; day 3 p = 0.0003; day 4 p < 0.0001; day 5 p < 0.0001). This indicated a drug x genotype interaction, whereby in Spred1-/- mice the effect of PD325901 was to reduce winning, but in WT mice PD325901 had the opposite effect, increasing winning. No differences between groups were observed in weight across the experiment (Additional file 1: Supplementary Fig. 4C, F; Additional file 1: Supplementary Fig. 5 A–C), and we confirmed that in all cohort configurations, PD325901 treatment significantly attenuated hippocampal ERK activation compared to vehicle treatment, verifying the specificity of drug effect (Additional file 1: Supplementary Fig. 6A–D. WT + vehicle vs Spred1-/- + vehicle cohort—unpaired t-test, t = 0.1777, df = 8, p = 0.8634. WT + vehicle vs Spred1-/- + PD325901 cohort—unpaired t-test, t = 19.69, df = 6, p < 0.0001. Spred1-/- + vehicle vs Spred1-/- + PD325901 cohort—unpaired t-test, t = 3.846, df = 10, p = 0.0032. WT + vehicle vs WT + PD325901—unpaired t-test, t = 6.957, df = 6, p = 0.0004). In accordance with previous studies [32, 36], we do not observe significant differences in total hippocampal pERK levels in Spred1-/- mice compared to WT. Acute MEK inhibition also significantly improved nesting behavior in Spred1-/- mice, increasing nest building scores to levels similar to WT mice (Fig. 4A; 2-way ANOVA: effect of PD325901 treatment F(1,26) = 28.81, p < 0.0001; effect of genotype F(1,26) = 6.679, p = 0.0157; no interaction). After a 3-week washout the effect of PD325901 on Spred1-/- nesting behavior was largely abolished, further demonstrating the causality of the MEK inhibition on the behavioral rescue (Fig. 4B; 2-way ANOVA: no effect of treatment, effect of genotype F(1,26) = 15.99, p = 0.0005; no interaction). Together these results show that acute treatment with PD325901 reverses social behavior phenotypes in adult Spred1-/- mice.