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. 2019 Oct 17:372:112068.
doi: 10.1016/j.bbr.2019.112068. Epub 2019 Jul 2.

Reversal of ultrasonic vocalization deficits in a mouse model of Fragile X Syndrome with minocycline treatment or genetic reduction of MMP-9

Maximiliano A Toledo  1 , Teresa H Wen  2 , Devin K Binder  3 , Iryna M Ethell  3 , Khaleel A Razak  4
Affiliations

Reversal of ultrasonic vocalization deficits in a mouse model of Fragile X Syndrome with minocycline treatment or genetic reduction of MMP-9

Maximiliano A Toledo et al. Behav Brain Res. .

Abstract

Fragile X Syndrome (FXS) is a leading genetic cause of autism and intellectual disabilities. The Fmr1 knockout (KO) mouse is a commonly studied pre-clinical model of FXS. Adult male Fmr1 KO mice produce fewer ultrasonic vocalizations (USVs) during mating, suggestive of abnormal social communication. Minocycline treatment for 2 months from birth alleviates a number of FXS phenotypes in mice, including USV call rate deficits. In the current study, we investigated if treatment initiated past the early developmental period would be effective, given that in many cases, individuals with FXS are treated during later developmental periods. Wildtype (WT) and Fmr1 KO mice were treated with minocycline between postnatal day (P) 30 and P58. Mating-related USVs were then recorded from these mice between P75 and P90 and analyzed for call rate, duration, bandwidth, and peak frequency. Untreated Fmr1 KO mice call at a significantly reduced rate compared to untreated WT mice. After minocycline treatment from 1 to 2 months of age, WT and Fmr1 KO mice exhibited similar call rates, due to an increase in calling in the latter group. Minocycline is thought to be effective in reducing FXS symptoms by lowering matrix-metalloproteinase-9 (MMP-9) levels. To determine whether abnormal MMP-9 levels underlie USV deficits, we characterized USVs in Fmr1 KO mice which were heterozygous for MMP-9 (MMP-9+/-/Fmr1 KO). The MMP-9+/-/Fmr1 KO mice were between P75 and P90 at the time of recording. MMP-9+/-/Fmr1 KO mice exhibited significantly increased USV call rates, at times even exceeding WT rates. Taken together, these results suggest that minocycline may reverse USV call rate deficits in Fmr1 KO mice through attenuation of MMP-9 levels. These data suggest targeting MMP-9, even in late development, may reduce FXS symptoms.

Keywords: Autism; Fragile X Syndrome; MMP-9; Minocycline; Social communication; Ultrasonic vocalization.

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Conflict of interest statement

Declarations of interest: none

Figures

Figure 1)
Figure 1)
Example spectrograms of ultrasonic vocalizations recorded in a mating context from a male WT (A), a male Fmr1 KO (B), and a male HET (C) mouse. In all three examples, the females were untreated, virgin and estrous induced Fmr1 KO mice. These example 10 second snippets were chosen to illustrate the point that Fmr1 KO mice produce calls at a reduced rate.
Figure 2)
Figure 2). Minocycline treatment (MT) between P30-P58 increases calling rate in Fmr1 KO mice to WT levels.
A) Minute-by-minute mean (+/− s.e.) USV call rate during the course of 10 minute courtship interactions in a 2 genotypes (WT, Fmr1 KO) x 2 treatments (regular water, MT (minocycline-treated) water) design. The graph shows that the calling rates tend to decrease over time in all 4 groups of mice. Over the first 5 minutes, WT mice call at a higher rate (calls/minute) than the Fmr1 KO mice. The MT mice (both genotypes) tend to call at intermediate rates. B) To quantify genotype and treatment effects, the mean calling rates during the first 5 minutes were compared in the 2 genotypes x 2 treatments design. For the first 5 minutes, a 2-way ANOVA showed a genotype effect F(l,23)= 5.02, p=0.035, but no significant effect of treatment (p=0.70) and no significant treatment x genotype interaction (p=0.10). Tukey post-hoc pairwise comparison revealed a WT vs. Fmr1 KO difference in the untreated group (*p=0.016), but not a difference in the MT treated group (p=0.60). C) For the second 5 minutes of pairing, a 2-way ANOVA showed no genotype effect F(l,23)= 0.63, p=0.44, no effect of treatment (p=0.69), and no significant treatment x genotype interaction (p=0.67).
Figure 3)
Figure 3). Spectrotemporal properties of USV calls are not grossly affected by minocycline treatment during the first 5 minutes of dyadic interactions.
A) Average duration of individual USV calls during the first 5 minutes showed no significant difference between the groups (2-way ANOVA, p=0.74). B) Average USV bandwidth during the first 5 minutes showed no significant difference between the groups (2-way ANOVA, p=0.073). C) Average USV peak frequency during the first 5 minutes showed a trending decrease in Fmr1 KO mice (2-way ANOVA, p=0.05). (D-F) Same data as in (A-C) during the second half (5 mins) of recording window when the mice called at a reduced rate.
Figure 4)
Figure 4). Genetic reduction of MMP-9 in Fmr1 KO (HET) mice increases USV calling rate to WT levels.
A) Minute by minute changes in average USV call rate during a 10 minute courtship window. The WT and Fmr1 KO data are the same as shown in Figure 2. Over the first 5 minutes, the HET mice showed call rates similar to the WT mice, and generally more calls than the Fmr1 KO mice. Interestingly, the HET mice continued to call at a relatively higher rate even during the last 5 minutes compared to the other genotypes. A 2-way ANOVA (genotype and time) (F(2,200)=21.64, p<0.001) shows that the Fmr1 KO mice exhibit significantly lower call rates compared to WT mice (Tukey test of main effects WT vs. KO **p<0.01) and genetic reduction of MMP-9 in HET mice restored call rates to WT levels (KO vs. HET ***p<0.001; WT vs. HET *p<0.05). The WT vs. HET difference is likely carried by the enhanced calling in the latter group throughout the recording. B) Analysis of just the first 5 minutes shows a genotype difference, likely carried by reduced call rate in the Fmr1 KO mice (1-way ANOVA: F(2,20)=3.74, p=0.042; Tukey test of main effects WT vs. KO p=0.07, HET vs. KO p=0.08, WT vs. HET p=1.00). C) Analysis of the second 5 minutes shows a genotype difference (One Way ANOVA: F(2,20)=5.89, p=0.01 : Tukey test of main effects HET vs. KO p=0.033).
Figure 5)
Figure 5). Call properties were not different in HET mice compared to WT and Fmr1 KO mice during the first 5 minutes of dyadic interactions.
A) Average duration of USV calls during the first 5 minutes was comparable across genotypes (1-way ANOVA, p=0.31). B) Average USV bandwidth during the first 5 minutes was comparable across genotypes (1-way ANOVA, p=0.49). C) Average USV call peak frequency during the first 5 minutes was comparable across genotypes (1-way ANOVA, p=0.39). Same data as in (A-C) during the second half (5 mins) of recording window.
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