Fmr1KO synapses have more docked SVs than WT synapses and they are insensitive to isoproterenol

We recently found an enhanced SV docking at cerebrocortical Fmr1 KO synapses that prevents a further increase by the activation of β-ARs with isoproterenol [13]. First, we tested whether these changes also occur at PF-PC synapses. We observed an increasing trend in the number of docked vesicles at PF-PC WT synapses (those located within 10 nm of the AZ membrane) after the exposure to isoproterenol (Generalized Linear Mixed Model, GLMM, P = 0.048 for interaction genotype-treatment; followed by Tukey HSD multiple comparisons test, P = 0.059, Fig. 1a, c, d) at the expense of SVs within 10–20 nm, which decreased in number (***P < 0.001, Fig. 1a). Notably, we also observed a increasing trend in the number of docked SVs at PF-PC Fmr1KO synapses in the basal state compared to WT synapses (P = 0.072) and isoproterenol failed to significantly increase this parameter (P = 0.997, Fig. 1e, g, h). Moreover, isoproterenol did not change the SVs at a distance of 10–20 nm from the AZ membrane in Fmr1KO synapses either (P = 0.988, Fig. 1f).

Loosely docked and primed SVs located 8 nm from the AZ membrane due to the partial zippering of SNARE complexes were distinguished from the tightly docked and fully primed SVs in which SNARE complex zippering has progressed much further, bringing the SVs closer to the AZ membrane (0–5 nm) [40,41,42,43]. There were more SVs within 0–5 nm of the AZ membrane in WT slices following exposure to isoproterenol (GLMM, P = 0.004 for interaction genotype-treatment; followed by Tukey HSD multiple comparisons test (***P < 0.001). Moreover, Fmr1KO synapses had more SVs within 5 nm than WT synapses (##P < 0.01, Fig. 1f) and isoproterenol failed to further increase this parameter (P = 0.951). No change in the active zone (AZ) length was observed between WT and Fmr1 KO synapses, [unpaired Student’s t test, t(913) = 0.43, P > 0.05, Fig. 1i]. Thus, Fmr1 KO synapses have more tightly docked and fully primed SVs than WT synapses, and the ability of β-ARs to further increase this parameter is prevented.

The lack of FMRP increases aEPSC frequency and prevents isoproterenol induced potentiation despite normal β-AR expression and cAMP generation

We asked whether enhanced SV docking results in an increase in spontaneous neurotransmitter release. However, as it is not possible to distinguish PF from climbing fiber miniature excitatory postsynaptic currents (mEPSCs) [44] we measured asynchronous release evoked by PF stimulation in the presence of Sr2+. Under these conditions, asynchronous release represents single release events from PF [44]. In WT slices, isoproterenol increased synchronous release (two-way ANOVA followed by Tukey’s multiple comparison test, F(3, 26) = 18.92, ***P < 0.001, Fig. 2a, b) and the frequency (two-way ANOVA followed by Tukey’s multiple comparison test, F(3, 26) = 8.49, ***P < 0.001, Fig. 2c–e) but not the amplitude (two-way ANOVA followed by Tukey’s multiple comparison test, F(3, 26) = 0.30, P > 0.05, Fig. 2c, f, g) of asynchronous release. However, isoproterenol failed to enhance sEPSCs at Fmr1KO slices (P > 0.05, Fig. 2a, b), and as the frequency of aEPSCs in Fmr1KO mice was higher than in WT slices (#P < 0.05, Fig. 2d), this further prevented the potentiation by isoproterenol (P > 0.05, Fig. 2d, e).

Fig. 2

An increased aEPSC frequency and absence of isoproterenol induced potentiation at Fmr1KO synapses. a Isoproterenol (100 μM, 10 min) enhances the sEPSCs recorded in the presence of Sr2+ (2.5 mM) in WT but not in Fmr1KO slices. b Quantification of the effects of isoproterenol on the sEPSC amplitude in WT (n = 9, ***P < 0.001 compared to control values) and in Fmr1KO slices (n = 6, P > 0.05). c Individual traces showing asynchronous release events in control (black) and isoproterenol exposed (red) WT and Fmr1KO slices. d, f Quantification of the isoproterenol induced changes in aEPSC frequency (d) in WT (n = 306 events/9 slices and, n = 502 events/9 slices: ***P < 0.001) and Fmr1KO slices (n = 305 events/6 and n = 312 events/6 slices: P > 0.05 comparing to control values; #P < 0.05 comparing to control aEPSC frequency in WT slices), as well as in amplitude (f) in WT (P > 0.05) and in Fmr1KO slices (P > 0.05). e, g Cumulative probability plots of isoproterenol induced changes in aEPSC frequency (inter event interval, IEI) (e) in WT (***P < 0.001) and Fmr1KO slices (P > 0.05), and amplitude (g) in WT (P > 0.05) and Fmr1KO slices (P > 0.05). Bar graphs show raw data and the mean. Scale bars in (a, c) represent 100 pA and 10 ms, and 25 pA and 10 ms, respectively. Two-way ANOVA followed by Tukey test in (b, d, f). Kolmogorov–Smirnov test in (e, g). Several slices per mice were prepared

We assessed whether the failure of isoproterenol to potentiate SV docking and neurotransmitter release at Fmr1KO synapses was a consequence of changes in β-AR expression and/or activity, which was examined in a preparation of cerebellar nerve terminals (synaptosomes). The spontaneous release of glutamate from WT cerebellar synaptosomes increased in the presence of isoproterenol (two-way ANOVA followed by Tukey’s multiple comparison test, F(3, 24) = 14.33, ***P < 0.001, Additional file 1: Figure S1A,C). However, Fmr1KO synaptosomes showed an increase in spontaneous release under basal conditions (#P < 0.05, Additional file 1: Figure S1C) but this was not further potentiated by isoproterenol (P > 0.05, Additional file 1: Figure S1B,C). Thus, Fmr1KO cerebellar synaptosomes recapitulate the occlusion phenotype evident in Fmr1KO slices.

The absence of β-AR mediated potentiation of spontaneous release in Fmr1KO synaptosomes could be due to weaker expression of this receptor. However, β1-AR expression was similar in Fmr1KO and WT synaptosomes when assessed in western blots (unpaired Student’s t test, t(4) = 0.61, P > 0.05, Additional file 1: Figure S1D,E). We also assessed β-AR expressing synaptosomes by immunofluorescence using antibodies against β1-AR and synaptophysin as a marker of SVs. There were a similar number of Fmr1KO and WT cerebellar synaptosomes labeled for synaptophysin that also expressed β1-AR synaptosomes (unpaired Student’s t test, t(136) = 1.29, P > 0.05, Additional file 1: Figure S1F,G,H). These data indicate that a change in β1-AR expression is not responsible for the failure of isoproterenol to potentiate spontaneous release in Fmr1 KO synaptosomes. β-ARs activate Adenylyl Cyclase (AC) and generate cAMP, which activates downstream signals to potentiate spontaneous release [45]. The lack of isoproterenol induced potentiation was not due to loss of receptor function as there was a similar β-AR mediated increase in cAMP in Fmr1KO synaptosomes as that in WT synaptosomes (unpaired Student’s t test, t(27) = 0.193, P > 0.05, Additional file 1: Figure S1I), reinforcing the idea that potentiation mediated by β-ARs is prevented at Fmr1 KO synapses.

Parallel fiber LTP is absent in PF-PC Fmr1KO synapses

An increase in the number of releasable vesicles at PF-PC Fmr1KO synapses should decrease the paired-pulse ratio (PPR). PPR was reduced at the interstimulus interval (ISI) of 20 ms (unpaired Welch test, t(9) = 2.35, *P < 0.05) and 40 ms (unpaired Welch test, t(8) = 2.49, *P < 0.05). Although no change was observed at higher ISIs (Fig. 3A). An increase in the number of releasable vesicles at PF-PC Fmr1 KO synapses should also increase the synaptic efficacy (averaged EPSC amplitude including failures) measured at different stimulation intensities. Fmr1KO synaptic efficacy was not altered at 50 μA (unpaired Welch test, t(5) = 1.09, P > 0.05) but increased at 100 μA (unpaired Welch test, t(5) = 2.64, *P < 0.05), 150 μA (unpaired Student’s t test, t(10) = 2.59, *P < 0.05) and 200 μA (unpaired Student’s t test, t(10) = 2.75, *P < 0.05, Fig. 3B) compared to WT.

Fig. 3

Fmr1 KO synapses have increased RRP size and lack cerebellar PF-PC LTP. a Changes in the PPR (mean of 6 consecutive pairs of stimuli delivered at 0.05 Hz) compared to WT at different inter-interval stimuli (ISI) (WT: n = 8 cells, 8 slices, 3 mice. Fmr1 KO: n = 9 cells, 9 slices, 3 mice. Scale bars: 100 pA, 20 ms), b changes in synaptic efficacy (mean of 6 consecutive EPSCs delivered at 0.05 Hz) compared to WT at different stimuli intensity (WT: n = 6 cells, 6 slices, 3 mice. Fmr1KO: n = 7 cells, 7 slices, 3 mice). Scale bars: 500 pA, 1 ms. c Changes in EPSC amplitude induced by a 10 Hz stimulation. d Quantification of EPSCs (mean of 6 consecutive EPSCs delivered at 0.05 Hz) 30 min after stimulation (2) compared to the respective values before stimulation (1): WT, (n = 9 cells/9 slices/5 mice); WT/propranolol (100 μM, 30 min, n = 10 cells/10 slices/6 mice); Fmr1 KO (n = 12 cells/12 slices/9 mice). Scale bars: 50 pA, 10 ms. e Changes in the PPR. f Quantification of the changes in PPR (mean of 6 consecutive pairs of stimuli delivered at 0.05 Hz) compared to the respective values before stimulation: WT (n = 9 cells/9 slices/5 mice); WT/propranolol (100 μM, 30 min, n = 10 cells/10 slices/6 mice); Fmr1 KO (n = 12 cells/12 slices/9 mice). Scale bars: 20pA, 10 ms. g, i Cumulative EPSC amplitudes in WT and Fmr1KO slices before (basal) and 30 min after LTP induction by 10 Hz stimulation (10 Hz). RRP size was calculated from the cumulative amplitude plots as the y-intercept from a linear fit of the steady-state level attained during a train of 100 stimuli at 40 Hz (as see in c). h, j Quantification of the RRP size: WT (n = 11 cells/11 slices/5 mice: **P < 0.01); Fmr1KO (n = 13 cells/13 slices/6 mice: P > 0.05, comparing 10 Hz vs basal values). RRP size WT basal vs Fmr1KO basal (#P < 0.05). Bar graphs show raw data and the mean. *P < 0.05, **P < 0.01, ***P < 0.001, #P < 0.05, unpaired Student’s t test or Welch test when the variances of the populations were significantly different

Presynaptic LTP at PF-PC synapses depends on a Ca2+ mediated increase in presynaptic cAMP [30, 46] and on the RIM1α protein, a Rab-3 interacting molecule [47]. We recently found that PF-PC LTP requires a β-adrenergic receptor (β-AR) dependent increase in SV docking and an increase in the RRP size [48], events that contribute to enhanced neurotransmitter release [45, 48]. As presynaptic responses of β-ARs are absent at Fmr1KO PF-PC synapses, we assessed whether PF-PC LTP was lost at these synapses. PF-PC LTP can be induced by stimulating PFs at 10 Hz for 10 s [47] and it is expressed as a long lasting increase in EPSC amplitude (unpaired Student’s t test, t(16) = 5.04, ***P < 0.001, Fig. 3C,D). The presynaptic origin of this LTP was evident by the decreased paired-pulse ratio (PPR; unpaired Welch test, t(11) = 2.46, *P < 0.05, Fig. 3E,F), and the β-AR receptor antagonist propranolol prevented PF-PC LTP in WT slices (unpaired Welch test, t(14) = 0.38, P > 0.05, Fig. 3C,D). However, stimulation of PFs at 10 Hz for 10 s failed to induce LTP at PF-PC Fmr1KO synapses (unpaired Welch test, t(17) = 0.13, P > 0.05, compared to the baseline, Fig. 3C,D).

PF-PC LTP involves an increase in the RRP size [48] and hence, we determined if a change in the RRP size could explain the lack of LTP at Fmr1KO PF-PC synapses. An increase in the RRP size was evident 30 min after LTP induction at WT PF-PC synapses (unpaired Student’s t test, t(23) = 3.59, **P < 0.01, Fig. 3G,H). However, the basal RRP was larger in Fmr1 KO synapses than in WT slices (unpaired Student’s t test, t(22) = 2.54, *P < 0.05) and it was not further enhanced by stimulation at 10 Hz (unpaired Student’s t test, t(25) = 0.49, P > 0.05, Fig. 3G,H,I,J). As such, Fmr1KO synapses have a larger RRP under basal conditions that impedes LTP.

Decreasing extracellular Ca2+ reduces asynchronous release and rescues parallel fiber LTP in Fmr1KO slices

One presynaptic change at Fmr1KO synapses is the loss of functional Ca2+-activated K+ channels, with the result of action potential, AP, broadening and enhanced presynaptic Ca2+ influx [12]. Thus, we tested whether reducing [Ca2+]e from 2.5 to 1 mM re-established isoproterenol mediated potentiation in Fmr1KO synapses. When slices were maintained at 1 mM [Ca2+]e, isoproterenol increased the sEPSCs amplitude (unpaired Student’s t test, t(18) = 3.56, **P < 0.01, Fig. 4A,B). Moreover, the aEPSC frequency of Fmr1KO synapses decreased to values similar to those of WT synapses in 2.5 mM [Ca2+]e (two-way ANOVA followed by Tukey’s multiple comparison test, F(3, 34) = 7.17, P > 0.05, Fig. 4D), and consequently, exposure to isoproterenol increased the aEPSC frequency (*P < 0.05, Fig. 4C,D,E) with no change in aEPSC amplitude (unpaired Student’s t test, t(18) = 0.02, P > 0.05, Fig. 4C,F,G). When the [Ca2+]e was lowered to 1 mM, PF-PC LTP was also re-established at Fmr1KO slices (unpaired Student’s t test, t(26) = 4.49, ***P < 0.001, Fig. 4H,I), even though LTP was not supported in WT slices at this [Ca2+]e (unpaired Student’s t test, t(18) = 0.05, P > 0.05, Fig. 4H,I), consistent with earlier reports on the sensitivity of PF-PC LTP to decreases in [Ca2+]e [32]. Interestingly, the rescued LTP in Fmr1KO slices also exhibited other features of PF-PC LTP seen in WT synapses [48], such as its sensitivity to the β-AR antagonist propranolol (unpaired Welch test, t(10) = 0.09, P > 0.05, Fig. 4H,I) and the increase in RRP size (unpaired Welch test, t(15) = 4.96, ***P < 0.001, Fig. 4J,K). Indeed, reduced [Ca2+]e counteracted the changes that led to increased SV docking and the enhanced RRP in Fmr1KO PF-PC synapses, decreasing asynchronous release, rescuing the presynaptic potentiation by β-ARs and PF-PC LTP.

Fig. 4

Reducing extracellular Ca2+ reduces asynchronous release and rescues isoproterenol-induced potentiation and PF-PC LTP at Fmr1KO slices. a Isoproterenol (100 μM, 10 min) enhances the sEPSC amplitude recorded in the presence of Sr2+ (1.0 mM). b Quantification of the effects of isoproterenol on sEPSC amplitude: (n = 6: **P < 0.01, unpaired Student’s t test). c Individual traces showing asynchronous release events in control (black) and after exposure to isoproterenol (red). d, f Quantification of the isoproterenol induced changes in aEPSC frequency (d) (n = 305 events/10 slices and n = 474 events/10 slices: two-way ANOVA followed by Tukey, *P < 0.05) and in aEPSC amplitude (f) (P > 0.05, unpaired Student’s t test). e, g Cumulative probability plots of isoproterenol induced changes in aEPSC frequency (inter event interval, IEI) (e) (***P < 0.001) and aEPSC amplitude (g) (P > 0.05, Kolmogorov–Smirnov test). h PF LTP was re-established in Fmr1KO slices, yet no LTP was induced in WT slices in the presence of 1 mM Ca2+. Experiments in Fmr1KO slices were also performed in the presence of the β-AR antagonist propranolol (100 µM, added 30 min prior to LTP induction). i Changes in EPSC amplitude (mean of 6 consecutive EPSCs delivered at 0.05 Hz) 30 min after stimulation (2) compared to basal (1): in Fmr1KO (n = 14 cells/14 slices/7 mice: ***P < 0.001, unpaired Student’s t test); in Fmr1KO slices treated with propranolol (n = 9 cells/9 slices/4 mice: P > 0.05, Welch test); and in WT (n = 10 cells/10 slices/4 mice: P > 0.05, unpaired Student’s t test). j Cumulative EPSC amplitudes (in the presence of 1 mM Ca2+) before and 30 min after LTP induction in Fmr1KO slices. RRP size was calculated from the cumulative amplitude plots as the y-intercept from a linear fit of the steady-state level attained during a train of 100 stimuli at 40 Hz. k Quantification of the RRP in Fmr1KO slices (n = 12 cells/12 slices/4 mice and n = 12 cells/12 slices/5 mice, before and after LTP induction: ***P < 0.001, Welch test). Bar graphs show raw data and the mean. Scale bar in a, c 10 pA and 100 ms; and in h 50 pA and 30 ms

The mGluR4 PAM VU0155041 rescues parallel fiber LTP at Fmr1 KO slices

Since decreasing [Ca2+]e re-establishes LTP at Fmr1KO synapses, it may be possible to rescue LTP using pharmacological tools that reduce Ca2+ influx at PF synaptic boutons, for example through the activation of G protein coupled receptors (GPCRs). Significantly, mGluR4 reduces Ca2+ influx and synaptic transmission at PF synaptic boutons [49, 50], where mGluR4 is the only group III mGluR present [51]. When mGluR4s were activated by the group III mGluR agonist, L-2-amino-4-phosphonobutyric acid, L-AP4, (40 μM) there was a reduction in the EPSC amplitude and once a new baseline was established, 10 Hz stimulation induced a strong and sustained increase in the EPSC amplitude (unpaired Student’s t test, t(18) = 11.73, ***P < 0.001, Fig. 5A,B). We also tested whether VU 0155041, a potent and selective PAM of mGluR4s that is active in vivo [21, 22] rescued PF-PC LTP. VU 0155041 reduced the EPSC amplitude and after 5 min, a 10 Hz stimulation provoked a sustained increase in EPSC amplitude (unpaired Student’s t test, t(20) = 18.16, ***P < 0.001, Fig. 5A,B). The rescued LTP at Fmr1KO synapses required β-AR activation, as does WT LTP, and indeed, the β-AR antagonist propranolol prevented this rescue by VU 0155041 (unpaired Student’s t test, t(18) = 1.61, P > 0.05, Fig. 5A,B). That a mGluR4 PAM rescues PF-PC LTP does not necessarily mean that the function of this receptor is altered in Fmr1 KO synapses. As such, we have found that the reduction of the EPSC amplitude caused by VU0155041 (100 μM) (35.6 ± 3.7%, ***P < 0.001, Welch test, t = 5.36, d.f. = 12) is similar to that in Fmr1 KO mice (32.5 ± 2.7%, ***P < 0.00, t = 5.01, d.f. = 20, unpaired Student’s t test) (Additional file 2: Figure S2) suggesting no changes in the expression of mGluR4 in the Fmr1 KO. Then, the rescue by VU0155041 of PF-PC LTP could result from normalization of other parameters essential for PF-PC synaptic potentiation. Thus, VU 0155041 reduced the RRP size in Fmr1KO synapses under basal conditions (unpaired Student’s t test, t(20) = 2.84, *P < 0.05, Fig. 5C,D) and permitted a further increase by 10 Hz stimulation (unpaired Student’s t test, t(18) = 3.76, **P < 0.01, compared with VU 0155041 in basal conditions). Thus, the mGluR4 PAM VU 0155041 rescued normal RRP size and PF LTP in Fmr1KO slices.

Fig. 5

Activation of mGluR4 rescues PF-PC LTP in Fmr1KO slices. a LAP4 (40 μM) and the mGluR4 PAM VU 0155041 (100 μM) rescues PF-PC LTP in Fmr1KO slices. The β-AR antagonist propranolol (100 µM) was added 30 min prior to LTP induction. b Quantification of EPSC amplitude (mean of 6 consecutive EPSCs delivered at 0.05 Hz) 30 min after stimulation (2) compared to the respective values before stimulation (1): LAP4 (n = 10 cells/10 slices/5 mice); VU (n = 11 cells/11 slices/6 mice); propranolol + VU (n = 10 cells, 10 slices/6 mice). c Cumulative EPSC amplitudes in Fmr1KO slices in the presence or absence (basal) of VU (100 μM, 10 min) and before (VU) and 30 min after 10 Hz stimulation (VU + 10 Hz). d Quantification of the RRP size in the above conditions: Basal (n = 10 cells/10 slices/7 mice); VU (n = 12 cells/10 slices/7 mice: **P < 0.01 compared to basal); VU plus 10 Hz (n = 8 cells/8 slices/4 mice: ##P < 0.01, compared to VU alone). e VU restores Ca2+ dynamics to Fmr1KO cerebellar synaptosomes. Changes in the cytoplasmic free Ca2+ concentration ([Ca2+]c) in the presence and absence of VU (100 µM) added at least 5 min prior to KCl. f Quantification of the changes in [Ca2+]c: KCl/WT (n = 18/5 preparations); KCl/Fmr1 KO (n = 16/5 preparations: ###P < 0.001 compared to KCl/WT); VU/KCl/WT (n = 11/4 preparations); and VU/KCl/Fmr1KO (n = 12/4 preparations). g Scheme showing the preparation of cerebellar slices from VU or saline injected Fmr1KO mice. h Response to a 10 Hz stimulation in slices from VU and saline injected Fmr1KO mice i amplitude (mean of 6 consecutive EPSCs delivered at 0.05 Hz) 30 min after stimulation (2) compared to the respective values before stimulation (1): VU (5 mg/Kg) injected Fmr1KO mice (n = 14 cells/14 slices/10 mice); saline injected Fmr1KO mice (n = 10 cells/10 slices/8 mice). Bar graphs show raw data and the mean. Scale bars in (a, h) 100 pA and 10 ms. Unpaired Student’s t test in (b, d). Two-way ANOVA followed by Tukey in (f). Welch test in (i). *P < 0.05, **P < 0.01, ***P < 0.001

FMRP interacts with the Ca2+ activated K+ channels that control the duration of action potentials (APs) and thus, the loss of FMRP leads to AP broadening and an ensuing increase in Ca2+ influx [12]. As Ca2+ homeostasis is altered in Fmr1KO mice [52] we tested whether VU 0155041 might rescue Ca2+ dynamics in Fmr1KO mice by measuring the depolarization induced change in the cytosolic Ca2+ concentrations ([Ca2+]c) of fura-2 loaded cerebellar synaptosomes. Synaptosomes were depolarized with a low KCl concentrations (10 mM KCl) to induce synaptic events involving Na+, K+ and Ca2+ channel firing which are compatible with the generation of action potentials [29]. The KCl-induced increase in [Ca2+]c was larger in Fmr1KO than in WT synaptosomes (two-way ANOVA followed by Tukey’s multiple comparisons test, F(3, 56) = 17.04, ###P < 0.001, Fig. 5E,F), compatible with the prolonged action potentials at Fmr1KO synapses [12]. VU 0155041 reduced the KCl-induced change in [Ca2+]c in WT synaptosomes (*P < 0.05, Fig. 5E,F) and it restored the KCl-induced increase in [Ca2+]c in Fmr1KO to the levels of WT synaptosomes (P > 0.05 Fig. 5E,F). Together, these data indicate that Ca2+ homeostasis is deregulated in Fmr1KO cerebellar synaptosomes but can be restored with the mGluR4 PAM VU 0155041.

We also tested whether VU 0155041 injected “in vivo” rescue PF-PC LTP in cerebellar slices. Fmr1KO mice were injected (i.p.) with VU 0155041 (or the saline vehicle alone) and cerebellar slices were prepared 2 h later. PF-PC LTP was rescued in slices from VU 0155041 injected Fmr1KO mice (unpaired Welch’s test, t(13) = 2.932, *P < 0.05, compared to the baseline Fig. 5G,H,I) but not in those from Fmr1KO mice injected with saline alone (unpaired Welch’s test, t(10) = 0.37, P > 0.05, Fig. 5G,H,I). Similarly, VU 0155041 injected “in vivo” in adult mice (≥ 3 months) also rescued PF-PC LTP in cerebellar slices (unpaired Welch’s test, t(6) = 4.80, **P = 0.003, compared to baseline, Additional file 3: Figure S3A,B).

VU0155041 ameliorates the motor learning and social deficits of Fmr1KO mice

We evaluated motor coordination and learning in the rotarod. Fmr1KO mice showed no defects in this test that measures the time that each animal remained on the rod of and an accelerating rotarod treadmill (time to fall) (Fig. 6A), or in the elevated path that measures the time spent by a mouse placed in the center on an elevated bar to reach one of the two platforms (Fig. 6B). In both tests, all comparisons to WT sal were not significant, (Kruskal–Wallis followed by Dunn’s multiple comparison test, P > 0.05). Fmr1KO VU compared to Fmr1KO sal was also not significant (Kruskal–Wallis followed by Dunn’s multiple comparison test, P > 0.05). In order to assess the behavioral consequences of changes in basal synaptic transmission and the loss of PF-PC LTP we tested the performance of Fmr1KO mice in tests that evaluate motor learning. Fmr1 KO mice display impaired motor learning in a forelimb-reaching task [20]. In this test, mice are trained to use their forelimbs to grasp and retrieve food pellets through a narrow slit (Fig. 6C), and the cerebellum contributes substantially to the coordination of the skilled movements that require speed, smoothness and precision, such as reaching to grasp movements [53, 54]. We tested whether rescuing the PF-PC LTP with VU 0155041 improved skilled reaching. After two days of habituation, the animal’s efficiency (number of pellets retrieved/number of attempts) was measured over 5 days and compared to WT sal on the same day. A deficit in skilled reaching was observed in Fmr1KO that receive saline injection (two-way repeated measures ANOVA followed LSD’s multiple comparisons test, F(3, 123) = 2.67; day 3: *P < 0.05, day4: **P < 0.01, day 5:**P < 0.01, compared to WT sal Fig. 6D). Interestingly, VU 0155041 administration slightly improved this task in Fmr1KO mice (Fmr1KO VU) (day 3: P > 0.05, day 4: *P < 0.05, day 5: *P < 0.05, compared to WT sal, Fig. 6D), while it did not alter the performance of WT mice (WT VU) (day 3: P > 0.05, day 4: P > 0.05, day 5: P > 0.05, compared to WT sal, Fig. 6D).

Fig. 6

VU 155041 ameliorates skilled reaching and classical eyeblink conditioning deficits of Fmr1KO. a Latency to fall of the mouse in an accelerating rotarod. Trials 1 and 2 were performed in day 1, and 3 and 4 in days 2 and 3, respectively. WT sal (n = 11), Fmr1KO sal (n = 14), WT VU (n = 10) and Fmr1KO VU (n = 11). All comparisons to WT sal were not significant (Kruskal–Wallis followed by Dunn’s test; trial 1: H(3) = 7.538, P < 0.05; trial 2: H(3) = 0.915, P > 0.05; trial 3: H(3) = 1.754, P < 0.05: trial 4: H(3) = 1.101, P < 0.05). Fmr1KO VU compared to Fmr1KO sal was not significant at in any trials (Kruskal–Wallis followed by Dunn’s test, P < 0.05). b In the elevated path the time spent to walk from the center of a 5 cm wide bar to one of its ends is measured. WT sal (n = 11), Fmr1KO sal (n = 14), WT VU (n = 10) and Fmr1KO VU (n = 11). All comparisons to WT sal were not significant (Kruskal–Wallis followed by Dunn’s test, H(3) = 6.125, P > 0.05). Fmr1KO VU compared to Fmr1KO sal was not significant (Kruskal–Wallis followed by Dunn’s test, P < 0.05). c Skilled reaching test. Mice use their forelimbs to grasp and retrieve food pellets through a narrow slit. d Efficiency in test performance (number of pellets retrieved per attempt) in the four experimental groups: WT sal (n = 31); Fmr1KO sal (n = 33); WT VU (n = 31); and Fmr1KO VU (n = 32) during 5 days. e Classical eyeblink conditioning was evoked with a conditioning stimulus (CS) consisting of a 350 ms tone (2.4 kHz, 85 dB) supplied by a loudspeaker located 50 cm in front of the animal’s head. The unconditioned stimulus (US) was presented at the end of the CS, and consisted of an electrical shock (a square, cathodal pulse, lasting for 0.5 ms) presented to the left supraorbital nerve. Conditioned responses (CRs) were determined from the EMG activity of the orbicularis oculi (O.O.) muscle ipsilateral to US presentations. f Examples of EMG recordings collected from representative WT sal and Fmr1KO sal mice during the 8th conditioning session. Note the presence of a noticeable CR in the WT sal mouse and its absence in the Fmr1KO sal animal. g CRs after 10 conditioning sessions of WT sal (n = 10), Fmr1KO sal (n = 10), WT VU (n = 10) and Fmr1KO VU (n = 10). *P < 0.05, **P < 0.01, ***P < 0.001. All post-hoc comparisons were to WT sal. Fmr1KO VU and Fmr1KO sal were also compared. Two-way repeated measures ANOVA followed LSD (d) or by Holm-Sidak’s (g). The data represent the mean ± S.E.M (a, d, g) and raw data and the mean (b). n is the number of mice used

We also tested classical eyeblink conditioning and the VOR, two paradigms that specifically evaluate cerebellar-dependent motor learning related to plasticity at PF-PC synapses [17, 19, 55]. Fmr1KO mice show deficits in classical eyeblink conditioning [56]. In the classical eyeblink conditioning the mouse learn to associate a conditioned stimulus (CS), such as a tone, with an unconditioned stimulus (US) such as a mild electric shock to the supraorbital nerve, which evokes eyeblinks (Fig. 6E,F). As a result of the CS-US association during tr