Altered structural and functional synaptic plasticity with motor skill learning in a mouse model of fragile X syndrome

Abstract

Fragile X syndrome (FXS) is the most common inherited intellectual disability. FXS results from a mutation that causes silencing of the FMR1 gene, which encodes the fragile X mental retardation protein. Patients with FXS exhibit a range of neurological deficits, including motor skill deficits. Here, we have investigated motor skill learning and its synaptic correlates in the fmr1 knock-out (KO) mouse. We find that fmr1 KO mice have impaired motor skill learning of a forelimb-reaching task, compared with their wild-type (WT) littermate controls. Electrophysiological recordings from the forelimb region of the primary motor cortex demonstrated reduced, training-induced synaptic strengthening in the trained hemisphere. Moreover, long-term potentiation (LTP) is impaired in the fmr1 KO mouse, and motor skill training does not occlude LTP as it does in the WT mice. Whereas motor skill training induces an increase of synaptic AMPA-type glutamate receptor subunit 1 (GluA1), there is a delay in GluA1 increase in the trained hemisphere of the fmr1 KO mice. Using transcranial in vivo multiphoton microscopy, we find that fmr1 KO mice have similar spine density but increased dendritic spine turnover compared with WT mice. Finally, we report that motor skill training-induced formation of dendritic spines is impaired in fmr1 KO mice. We conclude that FMRP plays a role in motor skill learning and that reduced functional and structural synaptic plasticity might underlie the behavioral deficit in the fmr1 KO mouse.

Figure 1.

Impaired motor skill learning in the fmr1 KO mouse. A, An illustration of the training box with a mouse reaching out for a food pellet through a narrow slit. The trained forelimb and the contralateral-trained hemisphere are marked. B, Average success rates during training for WT controls (black, n = 36) and fmr1 KO (blue, n = 38) mice. Both genotype and days of training affect success rate (p < 0.01, two-way repeated-measures ANOVA). C, There was no difference in the number of reaches performed by the KO and WT mice at any of the training days. mean ± SEM. *p < 0.05, **p < 0.01.

Figure 2.

Reduced synaptic strengthening with motor skill learning in the fmr1 KO mouse. A, A schematic diagram of the slice recording from the forelimb M1 in the trained and untrained hemispheres. B, Input–output curves in the preferred (pr) and unpreferred (upr) M1 of untrained WT mice and in the 5 d trained (tr) and untrained (utr) M1 hemispheres from WT and KO mice. Inset, Representative evoked field potential responses in layer 2/3 M1. Scale bar: 0.4 mV, 5 ms. C, Mean interhemisphere ratio of FP amplitude at threshold factor 4 in the untrained WT mice (gray, n = 5), WT mice trained for 2 or 5 d (black, n = 4 and n = 10, respectively). D, Mean FP amplitude interhemisphere ratio after 5 d of training in the WT and fmr1 KO mice (blue, n = 16 respectively). Mean ± SEM; *p < 0.05, **p < 0.01.

Figure 3.

Absence of training-induced occlusion of cLTP in the M1 of the fmr1 KO mouse. A, Average time course of the change in FPs after cLTP. Representative FPs (average of 5 traces) with the black and gray traces taken before (1) and after (2) cLTP, respectively (time points indicated on the graph). In the WT mice, cLTP is occluded in the trained hemisphere (black, n = 5 slices) compared with the untrained hemisphere (gray). In the fmr1 KO mice, no occlusion of cLTP is detected in the trained hemisphere (dark blue, n = 6 slices) compared with the untrained hemisphere (light blue). Scale bars: 0.4 mV, 5 ms. B, Average FPs 30–40 min after initiation of cLTP induction. Mean ± SEM. The interaction between genotype and training on cLTP was significant (p = 0.022, two-way ANOVA); *p < 0.05, **p < 0.01.

Figure 4.

Learning induces a transient increase in synaptic GluA1 that is delayed in the fmr1 KO mouse. A, Western blots of synaptic GluA1 from forelimb M1 regions of preferred (pr) and unpreferred (upr) hemispheres from untrained (Utr) mice or from trained (tr) and untrained (utr) hemispheres of trained WT and fmr1 KO mice. B, Untrained WT mice (n = 10) were compared with trained WT mice after 2 h (n = 7), 1 d (n = 14), 2 d (n = 7), or 5 d (n = 8) of training. Untrained KO mice (n = 15) were compared with trained KO mice after 2 h (n = 7), 1 d (n = 8), 2 d (n = 9), or 5 d (n = 7) of training. For quantification, protein levels were normalized to GAPDH and interhemisphere ratios were normalized to untrained WT or KO mice. Mean ± SEM. The interaction between genotype and training on GluA1 interhemisphere ratio was significant (p = 0.013, two-way ANOVA); *p < 0.05, ***p < 0.001.

Figure 5.

Normal density but higher turnover rate of dendritic spines in the fmr1 KO mouse. A, Multiphoton in vivo imaging of dendritic spines in layer 1 of Thy1 YFP-H mice crossbred with fmr1 KO mice. Imaging was performed on two consecutive days in untrained mice. Yellow arrow points to a filopodia that was lost on the second day of imaging. Scale bar, 3 μm. B, Density of dendritic protrusion and percentage of filopodia were similar between the genotypes. C, Spine dynamics, measured as rates of spine formation, elimination, and TOR were higher in the KO mouse. WT (n = 7) and KO (n = 8) mice. Mean ± SEM. *p < 0.05.

Figure 6.

Lack of motor learning induced increase in the number of dendritic spines in the fmr1 KO mouse. A, Multiphoton in vivo imaging of dendritic spines in layer 1 of Thy1 YFP-H mice crossbred with fmr1 KO mice. Imaging was performed on two consecutive days with imaging performed 2–3 h after training on day 1. New spines and lost spines are marked with red and yellow arrows, respectively. Scale bar, 3 μm. B, After training the total spine number in the trained hemisphere of WT mice (black, n = 6) was higher than in the untrained hemisphere (gray, n = 8), whereas in the KO mice there was no difference between trained (dark blue, n = 8) and untrained hemispheres (light blue, n = 6). In the WT mouse, the number of spines formed in the trained hemisphere exceeded the number of spines formed in the untrained hemisphere, whereas no differences were detected in the trained KO. A trend toward reduction in the number of eliminated spines was observed in the trained hemispheres of WT and KO mice. Mean ± SEM. The interaction between genotype and training on total spines and spine formation was significant (p = 0.019 and p = 0.036, respectively, two-way ANOVA); *p < 0.05, ***p < 0.001.