Investigating the role of Foxp1 in motor coordination and vocal behavior

Enrique De Alba, Ashley Anderson, and Genevieve KonopkaDepartment of Neuroscience, University of Texas Southwestern Medical Center, Dallas, Texas

IntroductionFOXP1, a member of the forkhead box protein family oftranscription factors, is an important regulator of manydevelopmental processes. Mutations in FOXP1 have recentlybeen linked to cases of autism spectrum disorder (ASD),intellectual disability, and significant language impairments.FOXP1 expression is highest within dopamine 1 (Drd1) and 2receptor (Drd2) expressing medium spiny projection neurons ofthe striatum. The striatum is an area of the brain associated withlanguage processing, motor control, and reward-based learning.FOXP2, the most homologous member within the FOXP family toFOXP1, is also highly expressed in the striatum and has beenlinked to patients with severe language impairments. Mice with aloss of Foxp2 cannot produce ultrasonic vocalizations (USVs)and our lab has shown neonatal mice heterozygous for Foxp1(Foxp1+/-) have reduced USVs. FOXP1 and FOXP2 are known toheterodimerize and have overlapping expression patterns in thestriatum. In this study, we examine the role of FOXP1 in vocalcommunication and motor coordination in mice by knocking outFoxp1 distinct striatal cell-types. Using cre-lox technology, wegenerated Foxp1 conditional knockout mice (cKO) that expresscre-recombinase in two distinct subpopulations of striatalneurons; Drd1a, Drd2, or both. We tested the motor coordinationof these mice using a rotorod behavioral test to determine howlong the mice could stay on the accelerating rotating rod (latencyto fall). We also measured vocal behavior in adult male miceusing specialized recording equipment to record USVs and thenanalyzed various parameters like number of calls, call slope, andmean frequency. We also examined these behaviors in Foxp1+/-mice.

Conclusions• Foxp1 has a significant role in motor coordination. Drd2 and

DDcKO mice show significant decreased motor coordination.There is no significant motor coordination difference betweenFoxp1+/- and Foxp1+/+ mice.

• Foxp1 expression in specific dopamine receptor populationsseems to affect vocal behavior, however more animals need tobe tested.

• Foxp1+/- adult mice appear to have significantly increasedvocalizations compared to the control mice.

• Although cKOs have a lower number of calls on day 1 comparedto the WT mice, on day 2 cKOs appear to have increasedvocalizations, especially in Drd2-Cre mice.

• There appears to be a trend for the call slope of the Drd2-Cremice to be positive and increasing from day 1 to day 2 as well asa trend of negative call slopes for the WT mice and positive callslopes for the other cKO mice.

• Drd2-Cre mice show a significant increase in number of bouts.There is also a trend of increasing number of bouts for Drd1a-Cre mice.

• DDcKO mice show significant increased mean frequency, butthis could be due to variability because of DDcKO’s low Nnumber.

Future directions• Test more mice for USVs recordings and rotorod experiments.• Use better sound proof lids for USVs to minimize possible noise

pollution.• Obtain a larger number of DDcKO mice to reduce variability.• Record adult USVs for 3 or 4 days to get a better understanding

of the changes in their vocalization as they could be interpretedas learning behaviors.

• Test the effects of inserting back Foxp1 or some pivotal gene thatFoxp1 regulates into mutant mice.

Literature cited1. Holy TE, Guo Z (2005) Ultrasonic Songs of Male Mice. PLoS Biol 3(12): e386.2. Ferland, R.J. et al., 2003. Characterization of Foxp2 and Foxp1 mRNA and protein inthe developing and mature brain. The Journal of comparative neurology, 460(2), pp.266–279.3. . Neuroscience, 124(2), pp.261–267.4. Lai, C.S.L. et al., 2001. A forkhead-domain gene is mutated in a severe speech andlanguage disorder. Nature, 413(6855), pp.519–523.5. Shu, W. et al., 2005. Altered ultrasonic vocalization in mice with a disruption in theFoxp2 gene. Proceedings of the National Academy of Sciences of the United States ofAmerica, 102(27), pp.9643–9648.

Acknowledgments Thank you to the STARS program for making this invaluable experiencepossible. Thank you to Genevieve Konopka for giving me the opportunity to bepart of her amazing research team and letting me do behavior testing. Thankyou to Ashley Anderson for being the best mentor ever and the whole Konopkalab for welcoming me as part of their family. Special thanks to Jayant Madugulafor creating an automated pipeline for our adult USVs analyses.

Materials and methodsAdult USVs: Each male mouse(>8W) was mated to single femalesfor at least a week before experimenting. Females wereseparated from the males 4 days before USVs recordings. USVsrecordings began after letting the males acclimate to the roomwith the styrofoam lid for 10 min.

Rotorod: Each mouse was gently placed onto the rotorod andreplaced if they fell before the experiment began. The rotorodassay began once all the mice were on the rods (maximum of 5mice/trial). Sensors were pressed if mice gripped the rod for a fullrotation. Intervals between trials were 20 min before beginning anew trial.

Analysis: Two-way ANOVA statistical tests were used to compareeach cKO or het data to their respective control with no matchingor repeated measures. Each cell mean was compared regardlessof rows and columns with the Tukey multiple comparisons test at95% confidence.

Recorded mice for 3 min withunscented (unsc.) bedding. Thenrecorded mice for 3 min withfemale scented (sc.) bedding.Then recorded mice for 3 min witha female.

Number of call bouts

Average number of bouts for Drd1a-Cre, Drd2-Cre, and DDcKO are approximatelythe same as those for WT on day 1. Drd1a-Cre and Drd2-Cre increase while the WTand DDcKO decrease on day 2. The number of bouts for Drd2-Cre mice issignificantly greater than that of the WT (p = 0.0010). [ ± SEM n = 2-8/group]

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Call slope

Average slope of calls for Drd1a-Cre, Drd2-Cre, and DDcKO are slightly higher thanthat for WT on day 1. The call slope for Drd2-Cre appears to be increasing on day 2(p=0.0526) whereas WT and Drd1a-Cre appear to have decreased call slopes. [± SEM n = 2-8/group]

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USV analysis in adult Foxp1+/- mice

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In contrast to what our lab had previously found in neonatal mice, Foxp1+/- adultmice have increased vocalizations compared to the control (Ctrl) mice. Foxp1+/- micehave statistically significant increases in number of calls, call duration, and theirfrequency range. [ ± SEM n = 6-8/group]

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There is no significant difference in the mice’s latency to fall between genotypes.Foxp1+/+ and Foxp1+/- mice may not have any differences in motor coordination.[ ± SEM n = 4-6/group]

No motor deficits in Foxp1+/- mice USV analysis in Foxp1 cKOs: Number of calls

Average number of calls for Drd1a-Cre, Drd2-Cre, and Drd1a;Drd2-Cre (DDcKO)are less than those for WT mice on day 1 but increase and supersede the WT onday 2. [ ± SEM n = 2-8/group]

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Mean frequency of calls

Mean frequency of calls for Drd1a-Cre and Drd2-Cre are slightly lower than that forWT on day 1 while mean frequency for DDcKO mice is significantly higher (p =0.0073). Drd1a-Cre and WT mean frequency slightly decrease on day 2, but Drd2-Cre and DDcKO both increase. [ ± SEM n = 2-8/group]

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Drd2 and DDcKO mice have significantly decreased motor coordination. Theforcegrip test shows that there is no significant difference between genotypes. Thisshows that there are no evident neuromuscular differences between WT and cKOmice, therefore the decreased latency to fall for the cKO mice is most likely due tomotor coordination deficits from the deletion of Foxp1 in the striatum.

Motor coordination deficits in Foxp1 cKO mice