Page 1 Normal Cerebellar Conditioning in DYT1 and DYT6 Dystonia

Page 1 Normal cerebellar conditioning in DYT1 and DYT6 dystonia

Title Page

(1) Title

All in the blink of any eye: insights into the pathophysiology of DYT1 and DYT6 dystonia

(2) Authors

Sadnicka A1, Teo J, Kojovic M, Kassavetis P1, Pareés I1, Saifee TA1, Swingenschuh P, Bhatia KP1, Rothwell JC1, Edwards MJ1

1. Sobell Department of Motor Neuroscience and Movement Disorders, Institute of Neurology, University College London, UK

(3) Corresponding Author

Dr Mark J Edwards

Senior Lecturer and Honorary Consultant Neurologist

UCL Institute of Neurology and National Hospital for Neurology and Neurosurgery

Postal Address: Box 146, National Hospital for Neurology and Neurosurgery, Queen Square, London, WC1N 3BG.

0044 203 4488749

(4) Word count

(5) Running title

Normal cerebellar conditioning in DYT1 and DYT6 dystonia

(6) Key words:

Dystonia

DYT1

DYT6

Eyeblink conditioning

Cerebellum

7) Financial Disclosure/Conflict of Interest

Nil

(8) Funding sources for study.

Nil

Structured Abstract

Background

Methods

Results

Conclusions

Our data suggest that we should be alert to different contributions of the key neuroanatomical constituents between the dystonia subtypes.

JNNP …

Short reports

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NEUROLOGY

Word count ~ 1600

Introduction

Dystonia is a challenging group of syndromes to define pathophysiologically. While t Traditionally linked to dysfunction of the basal ganglia, more recent research defines dystonia as a network disorder within which the cerebellum may be an important node [1-3]. DYT1 and DYT6 are generalised genetic dystonias with identified genes and proteins (Torsin and THAP1) and it is suggested that these genes may play a role in neurodevelopment of neural cicruits, particularly the basal ganglia and cerebellum (Zhao et al., 2011; Niethammer et al, 2011).[JT1]The case for cerebellar involvement is perhaps particularly strong in DYT1 dystonia due to the ability to investigate the disease using animal models which are increasing refined in their ability to probe and implicate the cerebellum[4]. [JT2]

In humans, neuroimagingHuman imaging datasuggests that both DYT1 and DYT6 have reduced integrity of the cerebello-thalomo-cortical tract and metabolic cerebellar abnormalities have been identified in functional imaging studies [5]. Eyeblink conditioning (EBC) is a form of associative learning critically dependent on the cerebellum and olive[6]. Patients with cervical dystonia and focal hand dystonia have lower rates of conditioning compared to controls [7], and this is perhaps the most direct evidence in humans that there is cerebellar dysfunction in focal dystonia.

In this study wehave examined 11 patients with DYT1 dystonia and 5 patients with DYT6 dystonia to determine if these patients have impairments in EBC or changes in the blink reflex recovery cycle (BRR, a marker of brainstem hyperexcitability). These two paradigms providea unique window into the function of the brainstem and cerebellum in these genetic dystonias.

Method

11 patients with DYT1 dystonia and 5 patients with DYT6 dystonia were recruited from the National Hospital for Neurology and Neurosurgery, London (table 1[JT3]). 16aged age-matched controls were also studied. The study was approved by the local ethics committee and written informed consent was obtained.

Electrical stimulation (square wave pulse of200μs)of the supraorbital nerve was applied using chloride disc surface electrodes (cathode: right supraorbital foramen, anode: 2 cm above). Eye blinks were captured by surface EMG electrodes over right and left orbicularis oculi (OO) muscles and signal was amplified (gain 2000x), bandpass filtered (20Hz–30,000Hz), digitised (5kHz), and stored for offline analysis using Cambridge Electronic Design (CED) 1401 hardware and Signal software (CED).

The EBC paradigm was identical to that used by Teo et al., 2009 [7]. The CS was a loud (∼70 dB) 2000 Hz tone of 400 ms played via binaural headphones. The US was an electrical stimulus (200μsec, 5 times sensory threshold) to the supraorbital nerve 400ms after the CS, to elicit a blink reflex (US). Repeated pairs of CS and US yielded conditioned blink responses (CRs) occurring within the 200 ms before the US (figure 1a). Six blocks of 11 trials (9 x CS-US, 1 x US and 1 x CS) were performed. The US only trial was for detecting spontaneous blinks and the CS only trial was to confirm that CRs were being acquired, independent of the US. A seventh block consisted of 11 CS only trials to measure extinction. EMG bursts were regarded as CRs if latency was >200 ms after the CS but before the US. For the CS only trials, EMG bursts occurring 200–600 ms after the CS were considered CRs.

BRR was measured by applying pairs of electric stimuli at 5 times sensory threshold to the supraorbital nerve at interstimulus intervals (ISI) of 200, 300, 400 and 1000 ms [8] in a pseudorandomised manner. The bilateral R2 component of the EMG response to the second stimulusis typically suppressed at ISIs of 200, 300 and 400 ms [8]. For each trial, EMG data from the non-stimulated left orbicularis occuliwere rectified and the area ratio of the first and second R2 responses was calculated following subtraction of the mean prestimulus background activity (R2 duration*mean backgroundactivity). This was necessary to compensate for the higher levels of resting EMG in patients with dystonia of the face. Mean values were calculated from the 8 trials for each ISI.

Rates of CRs during EBC were assessed using rmANOVA (SPSS for Windows, USA) with “block” aswithin within-subject (block1:6) and “group” as between between-subjectsfactor (dystonia, normal).Extinction rates insubjects who successfully conditioned (defined as >40% CRs in any block) were examined usingpaired student t-tests comparing the number of CRs in block 6 to the number of CR in the extinction block. BRR was assessed using rmANOVA with “ISI” as within subject factor (200,300,400,1000) and “group” (dystonia, normal) as between subjects factor. The Greenhouse-Geisser method was used to correct for non-sphericity and theBonferonni correction was used with multiple comparisons. Otherwise statistical significance was defined as p.05.

Results

Two patients (one in each group) and two controls completed EBC only as they found supraorbital nerve stimulation uncomfortable. [JT4]

Patients with DYT1 dystonia had comparable rates of conditioning to controls(rmANOVA: effect of “block”F(3.16,63.3) = 11.62, p<.001; but no“block*group” interactionF(3.16, 63.3) = 1.17, p = .33) or effect of “group”F(1,20) = .128, p = .725. Seven patients and nine DYT1 controls acquired the CR to a level of 40%. Both patients (student’s paired t test, p =.0068) and controls (student’s paired t test, p=.0094) exhibited extinction.

EBC was also similar in patients with DYT6 dystonia and their age-matched controls (rmANOVA: “block”F(5,40) = 15.4, p < .001; “block*group”F(5,40) = .752, p =.60;“group” F(1,8) = 0.16, p = .903)(figure 1E).The fact that all patients with DYT6 conditioned to high levels, despite the small group numbers, suggests that there is no impairment in acquisition of EBC in this genetic group either. AlthoughEBC appears to differ between DYT1 and DYT6,this may be due to the age difference between them (DYT6, 35yrs; DYT1, 50yrs).[JT5]All DYT6 patients and their controls acquired the CR to a level of 40%. Unlike the controls (student’s paired t test, p =.030), DYT6 patients failed to showsignificant extinction (student’s paired t test, p = .525).

BRR differed between controls and DYT1 patients (“ISI”F(1.56,25.0) = 12.0, p =.001, “ISI*Group”, F(1.56,25.0)=3.83, p=.045, but no effect of Group (F(1,16) = 2.11, p=.166)). Post hoc Student’s t-tests were not significant at any ISI. DYT6 patients and controls had similar BRR curves to their age-matched controls (significant main effect of“ISI”F(1,41,9.86) = 5.15, p =.0380), but not“ISI*Group”F(1.41, 9.85) = .828, p =.425 or“Group”F(1,7) = .432, p=.532.

Discussion

In this study we have demonstrated that patients with DYT1 and DYT6 have EBC rates comparable to controls. We have also shown that patients with DYT1 have a different profile of BRR,suggesting reducedinhibition within brainstem circuits,which was not seen in DYT6.

Eyeblink conditioning is critically dependent on intact olivo-cerebellar function, and is abnormal in patients with focal hand and cervical dystonia [7, 9]. The normal EBC seen in DYT1 and DYT6 is in contrast to these focal and segmental dystoniasand suggests that the different forms of dystonia may have different neuroanatomical patternsfocal and generalised dystonias may not share similar pathophysiology. It is intriguing to hypothesise what this signifies. Most obviously our data may have their origins in the phenotypical variances associated with subtypes of dystonia. Most focal dystonias are still considered sporadic acquired disorders, with a greater influence of environment factors, and occur later in life. Perhaps the cerebellum takes a greater compensatory role in focal dystonia to counteract the dystonic motor activity and due to competing demands on its net function this impairs the ability of the cerebellar networks to acquire CRs. It is unknown why DYT1 and DYT6 patients would not have the same increasing competing demands in EBC when there is evidence of increased sensorimotor network activity in other tasks (Carbon et al., 2010).

DO YOU NEED SOME REFERENCE AND MENTION SOMEWHERE ABOUT KOJOVIC 2013 PAPER ON PRIMARY SEGMENTAL VERSUS SECONDARY DYSTONIAS?[JT6]

Our results are surprising as evidence in support of cerebellar deficits in DYT1 dystonia, in particular, is stronger than for focal dystonia. While histological studies do not demonstrate clear structural abnormalities of the cerebellum in humans with DYT1 dystonia [13] or in rodent models [4],subtle microstructural defects (such as thinner dendrites and fewer dendritic spines) are observed in the Purkinje cells of DYT1 mouse models [10] and reducing cerebellar output by knocking out Purkinje cells improves motor symptoms in a DYT1 knock in animal model [11]. As yet there are no animal models of DYT6. The gene is widely expressed in the central nervous system including cerebellar neurones [5] and interestingly neuropathological changes including reduced Purkinje cell linear density in post-mortem human brains of subjects with cervical dystonia have been linked to THAP1 sequence variations [12].In humans imaging studies using fractional anisotropy demonstrates reduced integrity of the cerebello-thalamo-cortical (CbTC) pathway in patients with DYT1 and DYT6 dystonia. Other studies have examined cerebellar functional activity during the motor task of sequence learning. Interestingly a genotypic effect was found, such that subjects with DYT1 dystonia had impaired sequence learning and an associated excessive activation of the left lateral cerebellar cortex whereas subjects with DYT6 dystonia did not demonstrate the impairment in sequence learning or the abnormal cerebellar activation.

The absence of clinical signs of cerebellar dysfunction in patients with primary dystonia highlights that if the cerebellum is implicated in the pathophysiology it is likely to be a selective impairment of a pertinent feature of postural control. Our patients with genetic dystonia, at least in the circuits essential to EBC, seem to have normal cerebellar function. Furthermore this type of associative learning with its clear dependency on recognising salient sensory inputs within millisecond timing intervals is not impaired.

The BRR in DYT1 patients showed hyperexcitability to controls in a similar fashion to focal cervical dystonia (Teo et al., 2009), but DYT6 patients did not differ from controls (Figure 1G).

The BRR, a measure of the excitability of brainstem circuits, had a different time course in DYT1 compared to a control group. Patients with DYT6 do not demonstrate any clear differences in BRR compared to controls. This variability heterogeneity in findings across subgroups of dystonia again highlights potentially important differences in the pathophysiology between subtypes of dystonia. Recently the BRR has shown surprising ability to dissociate between tremor subtypes that have been first classified clinically as dystonic tremor or essential tremor (100% sensitivity and specificity) [13] and has been proposed as a potential test for dystonic tremor. These data suggest caution in proposing a single electrophysiological abnormality as diagnostic of dystonia.

Our study was limited by the small number of available subjects with DYT6 dystonia, which reflects the lower prevalence of this genetic dystonia.

Conclusions

The cerebellum is receivinghas received increasing attention as an important neuroanatomical structure involved in the pathophysiology of dystonia. However this research is still at an early stage and it remains difficult to obtain direct evidence in humans to specifically implicate the cerebellum in this enigmatic diseasedystonia. Our data suggest that the circuits involved with EBC within the cerebellumin the cerebellum and olive maintain normal function in DYT1 and DYT6 dystonia. Thus,current disease models discussing dystonia from a neuroanatomical perspective need to incorporate emerging differences between subtypes of primary dystonia and be able to explain any results that currently appear at odds across animal and human research modalities.

Acknowledgment

We would like to thank the patients and control subjects that gave their time to participate in this study.

Author Roles

Sadnicka A

Research project: A. Conception, B. Organization, C. Execution;

Statistical Analysis: A. Design, B. Execution, C. Review and Critique;

Manuscript: A. Writing of the first draft, B. Review and Critique.

Teo JT

Research project: C. Execution (Data sharing);

Statistical Analysis: C. Review and Critique;

Manuscript: B. Review and Critique

Kassavetis P

Research project: C. Execution;

Statistical Analysis: C. Review and Critique;