Systemic pharmacological suppression of neural activity reverses learning impairment in a mouse model of Fragile X syndrome

The enhancement of associative synaptic plasticity often results in impaired rather than enhanced learning. Previously, we proposed that such learning impairments can result from saturation of the plasticity mechanism (Nguyen-Vu et al., 2017), or, more generally, from a history-dependent change in the threshold for plasticity. This hypothesis was based on experimental results from mice lacking two class I major histocompatibility molecules, MHCI H2-Kb and H2Db (MH-CI KbDb−/−), which have enhanced associative long-term depression at the parallel fiber-Purkinje cell synapses in the cerebellum (PF-Purkinje cell LTD). Here, we extend this work by testing predictions of the threshold metaplasticity hypothesis in a second mouse line with enhanced PF-Purkinje cell LTD, the Fmr1 knockout mouse model of Fragile X syndrome (FXS). Mice lacking Fmr1 gene expression in cerebellar Purkinje cells (L7-Fmr1 KO) were selectively impaired on two oculomotor learning tasks in which PF-Purkinje cell LTD has been implicated, with no impairment on LTD-independent oculomotor learning tasks. Consistent with the threshold metaplasticity hypothesis, behavioral pre-training designed to reverse LTD at the PF-Purkinje cell synapses eliminated the oculomotor learning deficit in the L7-Fmr1 KO mice, as previously reported in MHCI KbDb−/−mice. In addition, diazepam treatment to suppress neural activity and thereby limit the induction of associative LTD during the pre-training period also eliminated the learning deficits in L7-Fmr1 KO mice. These results support the hypothesis that cerebellar LTD-dependent learning is governed by an experience-dependent sliding threshold for plasticity. An increased threshold for LTD in response to elevated neural activity would tend to oppose firing rate stability, but could serve to stabilize synaptic weights and recently acquired memories. The metaplasticity perspective could inform the development of new clinical approaches for addressing learning impairments in autism and other disorders of the nervous system.


Figure 1 -
Figure 1 -figure supplement 1.Similar oculomotor learning impairments and efficacy of diazepam pretreatment in male and female L7-Fmr1 KO mice.(A) VOR-increase training.Top Left, In male L7-Fmr1 KO mice (red), VOR-increase learning

Figure 1 -
Figure 1-figure supplement 2. Baseline oculomotor performance of L7-Fmr1 KO mice was indistinguishable from WT.The gain of the eye movement responses (ratio of eye movement amplitude to vestibular stimulus amplitude; see Methods) of L7-Fmr1 KO mice (red) was not significantly different from that of WT mice (black) during baseline tests of the VOR in the dark before training (left; p= 0.95, two sample t-test) or during the first 45 sec of the paired presentation of visual and vestibular stimuli used for VOR-increase training (middle; p= 0.50, two sample t-test) or for VOR-decrease training (right; p= 0.76, two sample t-test).Number of mice tested is indicated in each bar.

Figure 2 -
Figure 2-figure supplement 1.Data from Figure 2 were subsampled to compare VOR-increase learning in subpopulations of mice matched for the mean learned decrease in the VOR during pre-training.Subsampling was done by eliminating the WT mice (black) with the smallest decrease and L7-Fmr1 KO mice (red) with the largest decrease in the VOR measured after 30 min of pre-training (just before the start of VOR-increase training), until the mean values in the two populations were within 2%.In these sub-sampled populations, the amount of VOR-increase learning was not significantly different between the L7-Fmr1 KO and WT mice after VOR-decrease pre-training (top; p=0.74,L7-Fmr1 KO mice vs. WT, 30 min, Tukey) or after Vestibular only pre-training (bottom; p=0.40,L7-Fmr1 KO mice vs. WT, 30 min, Tukey), as also observed in the full samples.

Figure 3 -
Figure 3 -figure supplement 1. Diazepam did not affect baseline VOR performance.The gain of the VOR (ratio of eye velocity to vestibular stimulus velocity) was measured in the dark in L7-Fmr1 KO (red) and WT (black) mice before (Pre), 2 hours after (Post-diazepam (2 hours)) and 18-24 hours after (Post-diazepam (18-24 hours)) an IP injection of diazepam (0.5 mg/kg).There was no effect of diazepam on the gain of the VOR in L7-Fmr1 KO mice (red; p=0.72,Pre vs. 2 hours Post Diazepam; p= 0.77, Pre vs. 18-24 hours Post-diazepam; Tukey) or WT mice (black; p=0.99,Pre vs. 2 hours Post diazepam; p= 0.36, Pre vs. 18-24 hours Post-diazepam; Tukey).Moreover, the gain of the VOR of L7-Fmr1 KO mice was not significantly different from that of WT mice during baseline tests of the VOR in the dark before diazepam administration Pre (left; p= 0.79, Tukey), Post-diazepam (2 hours) (middle; p= 0.77, Tukey) and Post-diazepam (18-24 hours) (right; p= 0.97, Tukey).The 2-hour and 18-24-hour VOR performance measurements were made just before the VOR-increase training sessions (training time = 0) shown in Fig. 3-figure supplement 2B, and Fig. 3B top, respectively.The Pre VOR-performance measurements were made just before the VOR-increase training sessions shown in Fig.1A, right for the subset of mice that were also tested 1 day after diazepam administration.

Figure 3 -
Figure 3 -figure supplement 2. The acute effect of diazepam was inhibition of VOR-increase learning.(A) Mice were given an intraperitoneal (IP) injection of diazepam (2.5 mg/kg or 0.4-0.5 mg/kg) and then returned to the home cage for 2 hours before VOR-increase training.When VOR-increase training was delivered two hours after IP injection of 2.5 mg/kg diazepam (B), 0.4-0.5 mg/kg diazepam (C), no learned increase in VOR amplitude was observed in L7-Fmr1 KO (red) or WT (black) mice.

Figure 5 -
Figure 5 -figure supplement 1. Baseline optokinetic reflex (OKR) performance normal in L7-Fmr1 KO mice and after diazepam pre-treatment.The baseline OKR was measured during the first three minutes of OKR adaptation training in L7-Fmr1 KO (red) was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.It is made