Alterations in cerebellar physiology are associated with a stiff-legged gait in Atcay(ji-hes) mice

Abstract

Recent evidence suggests that dystonia, a movement disorder characterized by sustained involuntary muscle contractions, can be associated with cerebellar abnormalities. The basis for how functional changes in the cerebellum can cause dystonia is poorly understood. Here we identify alterations in physiology in Atcay(ji-hes) mice which in addition to ataxia, have an abnormal gait with hind limb extension and toe walking, reminiscent of human dystonic gait. No morphological abnormalities in the brain accompany the dystonia, but partial cerebellectomy causes resolution of the stiff-legged gait, suggesting that cerebellar dysfunction contributes to the dystonic gait of Atcay(ji-hes) mice. Recordings from Purkinje and deep cerebellar nuclear (DCN) neurons in acute brain slices were used to determine the physiological correlates of dystonia in the Atcay(ji-hes) mice. Approximately 50% of cerebellar Purkinje neurons fail to display the normal repetitive firing characteristic of these cells. In addition, DCN neurons exhibit increased intrinsic firing frequencies with a subset of neurons displaying bursts of action potentials. This increased intrinsic excitability of DCN neurons is accompanied by a reduction in after-hyperpolarization currents mediated by small-conductance calcium-activated potassium (SK) channels. An activator of SK channels reduces DCN neuron firing frequency in acute cerebellar slices and improves the dystonic gait of Atcay(ji-hes) mice. These results suggest that a combination of reduced Purkinje neuron activity and increased DCN intrinsic excitability can result in a combination of ataxia and a dystonia-like gait in mice.

Keywords: Ataxia; Cerebellum; Deep cerebellar nuclei; Dystonia; Electrophysiology; Mutant mice; Patch-clamp; Purkinje cells.

Figure 1. The cerebellum contributes to the stiff-legged dystonic gait of Atcay^ji-hes mice

A. Footprint pattern of Atcay^ji-hes mice and littermate controls. B. Atcay^ji-hes mice exhibit reduced stride length with a normal base width. C. Atcay^ji-hes mice exhibit significantly abnormal foot placement and have impaired performance on the rotarod (D). E.. Atcay^ji-hes mice have a stiff legged gait with abnormal hind limb extension F. Following a partial cerebellectomy, the same mouse has a broad-based ataxic gait without hind limb extension during gait (n = 4 mice). G. The site of the cerebellar lesion involving the DCN and overlying cerebellar cortex is shown (n = 4 mice). Arrow: DCN, Arrowheads: Cerebellar cortical destruction. Data in each graph are presented as mean ± SEM.

Figure 2. Dystonia in the Atcay^ji-hes mice develops in the absence of changes in cerebellar architecture or neurodegeneration

A. Top row. Nissl staining of brain sections from wild-type and Atcay^ji-hes mice show no evidence of neurodevelopmental abnormalities. Middle row: Calbindin staining for Purkinje neurons shows intact Purkinje neurons with normal molecular layer thickness. Purkinje neuron terminals in the DCN are also intact (middle panel in each row for each genotype). Bottom row: GFAP staining reveals no evidence for gliosis in the cerebellar cortex or DCN suggesting an absence of neurodegeneration. N=3 mice of each genotype. Scale bars = 250 μM. B. Higher magnification images of calbindin staining the Purkinje cell layer shows no evidence of Purkinje cell loss or reduction in dendritic arborization. The lack of changes in Purkinje neuron morphology is summarized in C. and D. Data in the graphs are presented as mean ± SEM.

Figure 3. Purkinje neurons from Atcay^ji-hes mice display an absence of repetitive spiking

A. Cell attached recording from a Purkinje neuron from a wild-type mouse showing normal repetitive spiking. B. A whole-cell recording from a Atcay^ji-hes mouse Purkinje neuron that displayed no repetitive spiking in the cell-attached configuration, showing a membrane potential close to the resting membrane potential of wild-type Purkinje neurons. C. Summary of patterns of firing in Atcay^ji-hes mice and littermate controls. Numbers within the bars represent numbers of cells from which recordings were made. D. Wild-type Purkinje neurons maintain repetitive spiking in response to injection of depolarizing current. E. Atcay^ji-hes Purkinje neurons that displayed no repetitive spiking cannot sustain repetitive spiking even with injection of depolarizing current. F. The average firing frequency of the ~ 50% Atcay^ji-hes Purkinje neurons that do display repetitive spiking is similar to wild-type littermate controls. G. The regularity of spiking is preserved in Atcay^ji-hes Purkinje neurons that display repetitive spiking. CV: Coefficient of variation. H. Expression of the voltage-gated sodium channel, Nav1.6, is not significantly different between Atcay^ji-hes and wild-type mice (n = 5 animals from each genotype). Data in the graphs are presented as mean ± SEM.

Figure 4. DCN neurons from Atcay^ji-hes mice exhibit increased intrinsic excitability due to reduced SK channel afterhyperpolarization (AHP) current

A. Cell attached recording from a DCN neuron from a wild-type mouse showing repetitive spiking. B. Cell attached recording from a DCN neuron from a Atcay^ji-hes mouse showing repetitive spiking at an increased firing frequency C. A subset of Atcay^ji-hes DCN neurons exhibit a burst pattern of firing. D. Summary of patterns of firing in DCN neurons in Atcay^ji-hes mice and wild-type littermate controls. E. DCN neurons from Atcay^ji-hes mice exhibit repetitive spiking at a significantly increased firing frequency. F. Although the spiking of Atcay^ji-hes mice appeared less regular, this was not statistically significant. G. The increase in firing frequency of Atcay^ji-hes DCN neurons persisted in the presence of inhibitors of synaptic transmission. H. An SK channel AHP could be elicited in wild-type DCN neurons. I. The AHP was significantly smaller in Atcay^ji-hes DCN neurons, and summarized in J. Data in the graphs are presented as mean ± SEM.

Figure 5. An activator of SK channels reduces DCN neuron excitability and improves the dystonic gait in Atcay^ji-hes mice

A. An Atcay^ji-hes DCN neuron that exhibited burst firing in a cerebellar slice was converted by perfusion of 10 μM SKA-31 into a tonic firing neuron with a reduced firing frequency (B.). C. SKA-31 reduces the firing frequency of both wild-type and Atcay^ji-hes DCN neurons. D,E. The dystonic phenotype in an Atcay^ji-hes mouse (D) is maximally improved 20 minutes following the intraperitoneal administration of 2.5 mg/kg SKA-31(E). F. The severity of dystonia was assessed by the degree of hind limb extension during gait. 20 minutes following administration of SKA-31, the degree of hind limb extension was significantly reduced (n = 4 animals). Data in the graphs are presented as mean ± SEM.