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. 2017 Apr 4:8:14912.
doi: 10.1038/ncomms14912.

Genetic silencing of olivocerebellar synapses causes dystonia-like behaviour in mice

Joshua J White  1   2   3 , Roy V Sillitoe  1   2   3   4
Affiliations

Genetic silencing of olivocerebellar synapses causes dystonia-like behaviour in mice

Joshua J White et al. Nat Commun. .

Abstract

Theories of cerebellar function place the inferior olive to cerebellum connection at the centre of motor behaviour. One possible implication of this is that disruption of olivocerebellar signalling could play a major role in initiating motor disease. To test this, we devised a mouse genetics approach to silence glutamatergic signalling only at olivocerebellar synapses. The resulting mice had a severe neurological condition that mimicked the early-onset twisting, stiff limbs and tremor that is observed in dystonia, a debilitating movement disease. By blocking olivocerebellar excitatory neurotransmission, we eliminated Purkinje cell complex spikes and induced aberrant cerebellar nuclear activity. Pharmacologically inhibiting the erratic output of the cerebellar nuclei in the mutant mice improved movement. Furthermore, deep brain stimulation directed to the interposed cerebellar nuclei reduced dystonia-like postures in these mice. Collectively, our data uncover a neural mechanism by which olivocerebellar dysfunction promotes motor disease phenotypes and identify the cerebellar nuclei as a therapeutic target for surgical intervention.

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Conflict of interest statement

The authors declare no competing financial interests.

Figures

Figure 1
Figure 1. An in vivo conditional genetic strategy for silencing neurotransmission at excitatory olivocerebellar axon terminals in mice.
(a) Ptf1aCre can be used to target the inferior olivary nucleus as demonstrated by tdTomato expression. Scale bar, 200 μm. (b) Using the same Ptf1aCre driver, exon 2 of Vglut2 can be selectively removed and Vglut2 deleted from the inferior olive. (c) An antisense in situ probe for Vglut2 mRNA reveals its removal from the inferior olive by postnatal day 0. Scale bar, 200 μm. (d) Ptf1aCre-driven tdTomato expression compared with the distribution of NeuN-positive cells in the inferior olive, as schematized in the right panel. Scale bar, 100 μm. Schematic reproduced in part, with permission, from Reeber et al.. (e) Quantification of the recombination efficiency of Ptf1aCre by dividing the number of tdTomato-positive cells by the number of NeuN-positive cells in the inferior olive in Ptf1aCre;Rosalox-stop-lox-tdTomato mice; n=3 mice. (f) Quantification of VGLUT2 protein expression in the molecular layer of lobules in all four transverse zones of the cerebellum show a significant reduction in Ptf1aCre;Vglut2fx/fx mice (P=9.294 × 10−16; Student's unpaired t-test); n=4 mice of each genotype. ***P<0.001. (g) Examples of expression of VGLUT2 protein in the molecular layer of lobule VI in a Vglut2fx/fx and a Ptf1aCre;Vglut2fx/fx mouse. Scale bar, 50 μm. (h) CART expression in climbing fibres in the molecular layer of lobule IX in a Vglut2fx/fx and a Ptf1aCre;Vglut2fx/fx mouse. Note that although CART expression in lobules IX and X is predominant and heaviest in climbing fibres, in other lobules it does label a subset of mossy fibres and scattered beaded fibres. Arrowheads point to climbing fibres. Scale bar, 20 μm. (i) Electron microscopy showing the ultrastructure of climbing fibre synapses onto large, Purkinje cell dendritic branches in a Vglut2fx/fx and a Ptf1aCre;Vglut2fx/fx mouse. Scale bar, 200 nm; n=4 mice of each genotype. (j) Schematic depicting the outcome of genetically silencing climbing fibre terminals. CF, climbing fibre; gl, granular layer; pcl, Purkinje cell layer; ml, molecular layer; PCd, Purkinje cell dendrite; CART, cocaine- and amphetamine-related transcript.
Figure 2
Figure 2. Loss of VGLUT2 eliminates climbing fibre-driven complex spikes.
(a) A schematic showing a cerebellar recording in an anaesthetized mouse. Schematic adapted with permission, from drawings published in ref. . (b) A schematic depicting a single-unit recording from a Purkinje cell with its climbing fibre. Samples of a simple spike (SS) and a climbing fibre-driven complex spike (CS) are shown. (c) Molecular expression showing the one-to-one relationship between a climbing fibre and a Purkinje cell with VGLUT2 marking the climbing fibre terminals. Scale bar, 20 μm. (d) A schematic of the juxtacellular labelling process. Current pulses are used to find cells based on increased resistance. Once a cell is found, the recording step begins by loosely patching on the membrane of the cell, and recording its extracellular spiking activity. After recording for a sufficient period of time, the cell is filled by delivering neurobiotin using 500 ms pulses of positive current between 1 and 5 nA for ∼1 h. Schematic adapted with permission, from drawings published in ref. . (e) Examples of traces from 3-month-old adult Vglut2fx/fx and Ptf1aCre;Vglut2fx/fx Purkinje cells either using metal electrodes for extracellular recording or glass electrodes for juxtacellular recording. Complex spikes are labelled with asterisks. Note the lack of complex spikes in the mutant traces. (f) Examples of filled Purkinje cells in adult 3–5-month-old Vglut2fx/fx and Ptf1aCre;Vglut2fx/fx mice. n>4 cells for each genotype. Scale bar, 50 μm. (g) Quantification of the number of Purkinje cells with identifiable complex spikes in Vglut2fx/fx mice compared with recordings from Ptf1aCre;Vglut2fx/fx mice of all ages from postnatal day 19 to 8 months. This analysis includes data from all of our electrophysiology experiments. The Purkinje cells were recorded from all lobules of the vermis and from crusI and II of the hemispheres. gl, granular layer; pcl, Purkinje cell layer; ml, molecular layer; NPY, neuropeptide Y (in this case a transgenic allele drives GFP expression).
Figure 3
Figure 3. Blocking climbing fibre function causes severe dystonia-like behaviours.
(a) Serial stills of both early postnatal (with a control Vglut2fx/fx littermate shown) and adult Ptf1aCre;Vglut2fx/fxmice displaying dystonia-like postures. Mobility is severely impaired and their limbs are extended and stiff. Arrows identify extended, stiff limbs. (b) Weights of mice at different ages (postnatal day (P)7, P14, P21, P30–P60, P60–P90 and P90–P180) reveal no significant differences between control and mutant mice (at least 8 mice from each genotype for each age, gender-matched, Student's unpaired t-test). Note that although movement is abnormal in the mutants, they are still capable of eating, exploring the cage and mating. Error bars are defined as s.e.m. (c) Rotarod performance is severely impaired in the mutant mice; n=62 Vglut2fx/fx mice, 38 Ptf1aCre;Vglut2fx/fx mice. Performance of the last day is significantly different between control and mutant mice (P=1.746 × 10−14; Student's unpaired t-test). Error bars are defined as s.e.m. (d) Examples of open field activity from three different Vglut2fx/fx controls and three different Ptf1aCre;Vglut2fx/fx mutant mice showing the range of performance for both genotypes. Scale bar, 5 cm. (e) Quantification of Vglut2fx/fx (n=28 mice) and Ptf1aCre;Vglut2fx/fx (n=29) open field mobility over a 30 min period. The left graph shows a decrease in the total distance travelled in the mutants (P=9.62 × 10−4; Student's unpaired t-test) and the right graph shows a lower total distance travelled divided by the number of ambulatory events in the mutant mice (P=7.20 × 10−6; Student's unpaired t-test). Although the mutant mice exhibit periods of twisting with limited movement and sustained muscle contractions (see EMG in Supplementary Fig. 4d), they are typically capable of moving around albeit with impaired motion and altered posture (Supplementary Movie 3). (f) Tremor is significantly increased in Ptf1aCre;Vglut2fx/fx mutant mice. Raw waveforms are passed through a fast Fourier transform and the results graphed as power versus frequency; n=139 Vglut2fx/fx and 129 Ptf1aCre;Vglut2fx/fx mice. Tremor in all mice was of similar frequency (P=0.705; Student's unpaired t-test). Amplitude is significantly higher in Ptf1aCre;Vglut2fx/fx mice (P=1.05 × 10−6; Student's unpaired t-test). (f′) Raw tremor waveforms from Vglut2fx/fx and Ptf1aCre;Vglut2fx/fxmice. Error bars are defined as s.e.m. ***P<0.001.
Figure 4
Figure 4. Purkinje cell simple spike firing is abnormal in postnatal developing Ptf1aCre;Vglut2fx/fx mutants but normalizes by adulthood.
(a) A schematized image of the experimental procedure for extracellular recording of single units in an anaesthetized (Anaesth.) mouse and a schematized image of the electrode placement near a Purkinje cell. Schematic adapted with permission, from drawings published in refs , . (b) Low power traces illustrating the firing properties of postnatal Purkinje cells (PC) (ages P19–P21). A complex spike is labelled with an asterisk. Scale bar 500 ms. (c) The P19–P21 Ptf1aCre;Vglut2fx/fx Purkinje cells (12 cells from 3 mice) fire slowly (frequency; P=4.1 × 10−2; Student's unpaired t-test), with more overall regularity (CV, P=3.7 × 10−3; Student's unpaired t-test) as compared with Vglut2fx/fx cells (7 cells from 2 mice), but with no significant difference in local regularity (CV2, P=0.593; Student's unpaired t-test). Error bars are defined as s.e.m. *P<0.05 and **P<0.01. (d) A schematized image of the experimental procedure in an awake mouse and a schematized image of the electrode placement near a Purkinje cell. (e) Low power traces of awake Purkinje cell recordings in adult 5–8-month-old Vglut2fx/fx and Ptf1aCre;Vglut2fx/fx mice. Complex spikes are labelled with asterisks. Scale bar, 100 ms. (f) High power traces of awake Purkinje cell recordings in adult 5–8-month-old Vglut2fx/fx and Ptf1aCre;Vglut2fx/fx mice. A complex spike is labelled with an asterisk. Scale bar 10 ms. (g) Sample waveform averages of simple spikes (SS) from adult 5–8-month-old Vglut2fx/fx and Ptf1aCre;Vglut2fx/fx mice and complex spikes (CS) from Vglut2fx/fx mice. (h) Simple spike firing was unchanged in terms of frequency, CV and CV2 in awake, adult Ptf1aCre;Vglut2fx/fx mice as compared with Vglut2fx/fx mice (respectively: P=0.695; P=0.622; P=0.204; ages 5–8 months; n=18 Vglut2fx/fx cells from 7 mice and 17 Ptf1aCre;Vglut2fx/fx cells from 11 mice; Student's unpaired t-test). Error bars are defined as s.e.m.
Figure 5
Figure 5. Molecular layer thickness is abnormal in Ptf1aCre;Vglut2fx/fx mutant cerebella during the early postnatal ages but recovers with age.
(a) At postnatal day 0, the Purkinje cell layer is thinner in Ptf1aCre;Vglut2fx/fx mutants and VGLUT2 expression is not present in the Purkinje cell layer. (b) At postnatal day 7, the molecular layer is significantly thinner in the mutants and there is little to no VGLUT2 expression in the Purkinje cell layer (P=2.3 × 10−4). (c) At postnatal day 14, the Purkinje cell layer in Ptf1aCre;Vglut2fx/fx mutants is only slightly thinner than the molecular layer of Vglut2fx/fx controls and VGLUT2 expression is largely absent in the Purkinje cell layer. (d) At postnatal day 21, the Purkinje cell layer in Vglut2fx/fx mice is nearly the same thickness as that observed in Ptf1aCre;Vglut2fx/fx mice even though VGLUT2 expression is eliminated from the Purkinje cell layer. egl, external granular layer; pcl, Purkinje cell layer; igl, internal granular layer; ml, molecular layer; gl, granular layer. Scale bars (a''' and b'''), 20 μm. Scale bars (c''' and d'''), 50 μm.
Figure 6
Figure 6. Cerebellar output function is abnormal in dystonic Ptf1aCre;Vglut2fx/fx mice.
(a) A schematic example of a single-unit extracellular recording in the cerebellum of an awake mouse. (b) A schematic example of the electrode position relative to the neurons of the cerebellar nuclei (CN). (c) WGA-Alexa 555 injection marking the interposed cerebellar nucleus (In). In this example the medial portion of the In was recorded, although in other recordings more lateral In cells were also examined. The fastigial nucleus (Fn), which is the most medially located cerebellar nucleus, is also shown. The surrounding vermis lobules are indicated by Roman numerals. Scale bar 200 μm. (d) Low power examples of P19–P21 juvenile Vglut2fx/fx and Ptf1aCre;Vglut2fx/fx cerebellar nuclei neural firing patterns by binning instantaneous firing rate at 400 ms. (e) High power traces from neurons in the cerebellar nuclei of juvenile Vglut2fx/fx and Ptf1aCre;Vglut2fx/fx mice. Scale bar, 10 ms. (f) Quantifications of juvenile cerebellar nuclear neuron frequency (P=1.35 × 10−2; Student's unpaired t-test), CV (P=0.98; Student's unpaired t-test) and CV2 (P=0.053; Student's unpaired t-test); n=8 Vglut2fx/fx cells (from 3 mice), 11 Ptf1aCre;Vglut2fx/fx cells (from 3 mice). Error bars are defined as s.e.m. ***P<0.001. (g) Low power examples of adult Vglut2fx/fx and Ptf1aCre;Vglut2fx/fx cerebellar nuclei neural firing patterns showing instantaneous firing rate binned at 400 ms. (h) High power traces of neurons of the cerebellar nuclei in adult Vglut2fx/fx and Ptf1aCre;Vglut2fx/fx mice. Scale bar, 10 ms. (i) Quantifications of adult cerebellar nuclear neuron frequency (P=3.594 × 10−5; Student's unpaired t-test), CV (P=5.6 × 10−5; Student's unpaired t-test) and CV2 (P=6.56 × 10−4; Student's unpaired t-test); n=33 Vglut2fx/fx cells (from 14 mice), 27 Ptf1aCre;Vglut2fx/fx cells (from 10 mice). Error bars are defined as s.e.m.
Figure 7
Figure 7. Lidocaine delivery to the interposed cerebellar nuclei eliminates tremor and improves movement in Ptf1aCre;Vglut2fx/fx mutant mice.
(a) A schematic example of delivery of lidocaine via osmotic pumps to a mouse cerebellum. Schematic adapted with permission, from drawings published in ref. . (b) Anatomical verification of surgical targeting of the cannula to the cerebellar nuclei. In the higher power view of where the cannula tip was located (b′), the methylene blue staining approximates the local impact of the lidocaine injection and (b”) the lidocaine immunodetection was used to examine the local spread of lidocaine within the cerebellar nuclei. Scale bar in (b), 1 mm. Scale bar in (b' and b”), 100 μm. (c) Atlas schematic demonstrating the location of the cerebellar nuclei (highlighted in red) and the bilateral infusion of lidocaine into the interposed nuclei (highlighted in blue). Schematic reproduced in part, with permission, from Paxinos and Franklin. (d) Stills of videos of a dystonic mouse before, during and after lidocaine infusion. Arrows point to stiff extended limbs and tail. (e) Tremor power/frequency graph of Vglut2fx/fx and Ptf1aCre;Vglut2fx/fx mice before, during and after lidocaine infusion. Error bars are defined as s.e.m. (f) Quantification of peak power of Vglut2fx/fx (pre versus during: Student's paired t-test P value=0.1117), Ptf1aCre;Vglut2fx/fx (pre versus during: Student's paired t-test P value=0.0001) and Ptf1aCre;Vglut2fx/fx sham (pre versus during: Student's paired t-test P value=0.0068; during versus post: Student's paired t-test P value=0.2941). Vglut2fx/fx during versus Ptf1aCre;Vglut2fx/fx during Student's unpaired t-test P value=0.131; n=7 Vglut2fx/fx mice with lidocaine, 14 Ptf1aCre;Vglut2fx/fx mice with lidocaine, 4 Ptf1aCre;Vglut2fx/fx mice with saline. Error bars are defined as s.e.m. Either paired or unpaired Student's t-tests were used as noted. *P<0.05, **P<0.01 and ***P<0.001.
Figure 8
Figure 8. Deep brain stimulation of the interposed cerebellar nuclei restores mobility in severely dystonic mice.
(a) A schematic example of DBS targeting into the cerebellum. (b) Dystonia rating before and during DBS stimulation of the interposed cerebellar nucleus in naive Ptf1aCre;Vglut2fx/fx mutant mice (P=0.0014; n=7 mice; Student's paired t-test). There is no significant difference between pre-DBS and post-DBS suggesting limited residual effects once the DBS is turned off (day 1 pre-DBS (3.8±0.49) versus post-DBS (3.075±0.63) Student's paired t-test P value=0.1099). Error bars are defined as s.e.m. (c) Comparison of pre-DBS and during DBS dystonia ratings for each of the 5 days shows no significant difference between days in the pre-DBS dystonic rating or during DBS dystonic measures (pre-DBS days 1–5: F(2.454, 14.72)=2.762; P=0.0875; during DBS days 1–5: F(2.647, 15.88)=1.380; P=0.2841). A one-way ANOVA shows a between-treatment difference based on averages of pre-DBS, during DBS and post-DBS over 5 days of stimulation: F(2, 12)=31.42 (P<0.0001) (see also Supplementary Movie 8 for pre-, during and post-DBS effects). Control mice were not affected by DBS (please see Supplementary Movie 8, n=8 Vglut2fx/fx stimulated and 4 Ptf1aCre;Vglut2fx/fx shams). Error bars are defined as s.e.m. **P<0.01 and ***P<0.001. (d) Stills of a Ptf1aCre;Vglut2fx/fx mouse before and during stimulation. (e) Anatomical verification of surgical targeting of the DBS electrodes to the interposed cerebellar nuclei. Scale bar, 500 μm. (e') Haematoxylin counterstain (deep blue/purple) showing the site of electrode in the cerebellar nuclei. Scale bar, 200 μm. (e'') Compared with the sham, cFos expression (brown) is not upregulated by stimulation. Scale bar, 100 μm. Cb, cerebellum; a, anterior; p, posterior; CN, cerebellar nuclei; Fn, fastigial nucleus; In, interposed nucleus; Dn, dentate nucleus; asterisks, electrode tracks.
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