The Pennsylvania State University

The Graduate School

Graduate Program in Cellular and Molecular Biology

ABNORMAL CEREBELLAR SIGNALING INDUCES

DYSTONIA IN MICE

A Thesis in

Cell and Molecular Biology

by

Carolyn E. Pizoli

Copyright 2003 Carolyn E. Pizoli

Submitted in Partial Fulfillment

of the Requirements

for the Degree of

Doctor of Philosophy

May 2003

We approve the thesis of Carolyn E. Pizoli.

Date of Signature

____________________________________ ______________________Ellen J. HessAssociate Professor of NeurologyThesis Co-AdvisorCo-Chair of Committee

____________________________________ ______________________Melvin L. BillingsleyProfessor of PharmacologyThesis Co-AdvisorCo-Chair of Committee

____________________________________ ______________________Robert LevensonProfessor of PharmacologyDirector, Cell and Molecular Biology Program

____________________________________ ______________________James ConnorProfessor of Neuroscience

____________________________________ ______________________Teresa WoodAssociate Professor of Neuroscience

iii

Abstract

Dystonia is a relatively common neurological syndrome characterized by twisting

movements or sustained abnormal postures. The pathophysiology of dystonia remains

poorly understood; however, recent evidence suggests that abnormal cerebellar signaling

contributes to the expression of dystonia. To study the role of the cerebellum in dystonia,

we first analyzed neurotransmission in the cerebellum of the genetically dystonic mouse,

tottering. A deficiency in excitatory but not inhibitory neurotransmission in tottering

mice was seen after superfusion of cerebellar synaptosomes with 60mM KCl. Further

analysis of the role of cerebellar Purkinje cells in the generation of tottering dystonia was

completed through breeding a transgene responsible for post-developmental Purkinje cell

death onto the tottering mouse. Prior to Purkinje cell degeneration, transgenic tottering

mice exhibited classical tottering dystonic events; however, the same animals failed to

exhibit dystonia after Purkinje cell loss had occurred in adulthood. The loss of the

dystonic phenotype in double mutant mice indicates that Purkinje cells and the cerebellar

cortex participate in the pathogenesis of dystonia in the tottering mouse. These data

support the theory that an abnormal signal from the cerebellar cortex of tottering mice is

responsible for the dystonic phenotype. To test this theory and examine the role of the

cerebellum in dystonia, we developed a novel mouse model of dystonia. Microinjection

of low-doses of the glutamate analogue kainic acid into the cerebellum of wild type mice

elicited reliable and reproducible dystonia. Transgenic mice lacking Purkinje cells

showed dramatically decreased dystonia after kainic acid injections, supporting the

theory that aberrant cerebellar excitation is sufficient to produce dystonia. Together these

data suggest that the cerebellum plays a role in the pathophysiology underlying dystonia.

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Table of Contents

Chapter Page

List of Tables vi

List of Figures vii

1 Introduction/Literature Review 11.1 Dystonia in Humans 1

Introduction 1Classification 2Pathophysiology 3Treatment 12Paroxysmal Dyskinesias 13

1.2 Tottering Mouse: Animal Model of Dystonia 19Introduction 19Calcium Channels 19Behavioral Phenotype 23Cellular Pathology 26

1.3 Chapter Summary 271.4 Hypotheses 28

2 Altered Neurotransmission in the Tottering Cerebellum 30Abstract 30Introduction 30Materials and Methods 35Results 37Discussion 40

3 Role of Purkinje Cells in the Expression of Tottering Dystonia 45Abstract 45Introduction 46Materials and Methods 48Results 51Discussion 58

4 Role of the Cerebellum in a Novel Animal Model of Dystoniain Wild Type Mice 63

Abstract 63Introduction 64Materials and Methods 65Results 71Discussion 86

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5 Discussion 875.1 Mouse Models of Dystonia 87

The Tottering Mouse 87Kainate-Induced Dystonia 101

5.2 The Cerebellum and Dystonia 104Cerebellar Circuitry 104Cerebellar Function 107Pathophysiology of Dystonia 110

REFERENCES 118

vi

List of Tables

Table Page

1.1 Familial dystonic syndromes for which 3gene loci have been identified

1.2 Subgrouping of the paroxysmal dyskinesias 14

1.3 Classification of neuronal high-voltage activated 22calcium channels

3.1 Genotypes of progeny generated in SV4+/-;+/tg X 53+/+;tg/tg crosses

5.1 Comparison of salient features of tottering dystonia and PNKD 98

5.2 Comparison of the salient features of the kainate model of 103dystonia and PKD

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List of Figures

Figure Page

1.1 Schematic depiction of brain regions and 9pathways theorized to malfunction in dystonia

1.2 Schematic depiction of a voltage-gated calcium channel 21

2.1 Schematic diagram of neuronal connections in the cerebellum 34

2.2 3H-Glutamate release from cerebellar synaptosomes 38

2.3 Comparison of tottering and wild type peak 3H-glutamate 38release

2.4 3H-GABA release from cerebellar synaptosomes 39

2.5 Comparison of tottering and wild type peak 3H-GABA 39release

3.1 PCR genotyping for presence of the SV40 transgene 53

3.2 Body weights of F7 generation progeny 54

3.3 Loss of dystonic phenotype in transgenic tottering mice 56over time

3.4 Regional loss of dystonic phenotype in F7 transgenic 57tottering mice

3.5 Calbindin mRNA in situ hybridization 58

4.1 Typical dystonic postures after cerebellar kainate injection 71in wild type mice

4.2 Dose-response and dose-recovery curves after cerebellar 73injection of kainic acid in wild type mice

4.3 c-fos in situ hybridization in wild type mice after cerebellar 75 kainate injection

4.4 Regional expression of c-fos mRNA after cerebellar kainate 76injection

4.5 Cerebellar injection site localization 77

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4.6 Dystonic severity in transgenic mice lacking Purkinje cells 81

4.7 Representative EEG recordings from wild type mice 83receiving cerebellar microinjections of kainic acid

4.8 Dystonic severity after cerebellar NBQX co-injection with 85kainic acid

4.9 Dystonic severity after cerebellar injection of domoic acid 86in wild type mice

5.1 Schematic diagram of pathways theorized to malfunction 108in cerebellar-induced dystonia

5.2 Comparison of release of inhibition treating dystonia in the 116basal ganglia and the cerebellum

Chapter 1. INTRODUCTION/Literature Review

1.1. DYSTONIA IN HUMANS

Introduction

The scientific advisory board to the Dystonia Medical Research Foundation defines

dystonia as a neurological syndrome characterized by the simultaneous co-contraction of

antagonistic muscles leading to twisting and repetitive movements or sustained abnormal

postures (Fahn S, 1987). However, a great deal of confusion exists because the term

dystonia refers to both the behavioral symptom of certain abnormal hyperkinetic

involuntary movements and to the syndrome or disease entity itself. In fact, the term

dystonia encompasses numerous heterogeneous disorders which share the characteristic

symptom of sustained contraction of antagonistic muscles. Taken together, the various

forms of dystonia represent a common neurological disorder which is the second most

commonly encountered disorder seen in movement disorder clinics after Parkinsonism

(Fahn S, 1995) and reaches an estimated prevalence of ¾ that of multiple sclerosis

(Richter A, 1998). Dystonic movements vary greatly in speed, amplitude, rhythmicity,

torsion, forcefulness, distribution, and initiating factors (Fahn S, 1988). Historically, the

wide range of characteristics involved in dystonia syndromes led to confusion and

misdiagnosis. Terms such as seizures, convulsions, myoclonus, ballism, and

choreoathetosis were often misused to describe dystonia. Currently, clinical neurology is

readily equipped to correctly diagnose and report dystonia, but the fields of laboratory

research and animal research in particular are still slow to recognize either the symptom

or syndrome of dystonia.

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Classification

Dystonia is an extremely heterogeneous disorder classified by age of onset,

distribution, and etiology. While age of onset and distribution are useful classifications

for prognostics and treatment strategies, etiologic classification is essential for

understanding the pathophysiology and prevention of disease (Fahn, S, 1998). Dystonia

is divided into two broad etiologic classifications, primary and secondary, each with

numerous sub-classifications. Primary dystonia is either of a familial or sporadic

etiology while secondary dystonia occurs symptomatically from a broad range of

neurological diseases and lesions.

Secondary dystonia is of particular interest because of the concomitant insight gained

to the basis of the dystonia, particularly the brain regions involved. Numerous

environmental factors may cause insults leading to secondary dystonia, including cerebral

palsy, encephalitis, stroke, tumors, drugs, and toxins. Heredodegenerative diseases,

marked by neuronal loss, also may cause secondary dystonia; these include Parkinson’s

disease, Wilson’s disease, and Huntington’s disease among others (Fahn, S, 1998). A

majority of secondary dystonias result from lesions in the basal ganglia and, to a lesser

degree, the thalamus. Therefore, these regions have historically been implicated in the

pathogenesis of dystonia syndromes.

To meet the clinical definition of primary dystonia, dystonia must be the sole

neurological sign other than tremor and no other exogenous, inherited, or degenerative

cause should be identified (Bressman, 1998). Primary dystonia accounts for 2/3 of

cases, including both the familial and sporadic forms. Currently, little is known about

sporadic dystonia, which remains the single largest category of patients. Familial

3

dystonias are being actively researched; the genes responsible for many of the

monogenetic inherited dystonias have been mapped and some of the gene products

identified. It is also important to note that many of these genetically determined

syndromes express symptoms in addition to dystonia and are thus classified as dystonia-

plus syndromes rather than primary dystonia (Fahn, S, 1998). Table 1.1 summarizes the

dystonic syndromes, both primary and dystonia-plus for which gene loci have been

established.

Gene Location Origin Syndrome Protein ReferencesDYT1 9q34.1 Ashkenazi jews

and non-jewishEuropean decent

Early-onset primarydystonia (Dystoniamusculorum deformans)

ATP-bindingprotein

Ozelius, 1989;Kramer 1990;Kwiatkowski,1991

DYT3 Xq13 Philippines Lubag (X-linkedParkinsonism-dystonia)

? Eidelberg, 1993

DYT5(DRD)

14q22.1 -- Dopa-responsivedystonia (DRD)

GTPcyclohydro-lase I gene, TH

Ichinose, 1994;Knappskog,1995

DYT6 8p21-q22 Mennonite/Amish Mixed: Childhood/Adultwith limb or cranialonset

? Almasy, 1997

DYT7 18p German kindred Adult onset torticollis ? Leube, 1996

-- 2q33-35 Polish-American Paroxysmal non-kinesegenic dyskinesia

? suspected Na+

channelFouad, 1996Fink, 1996

-- 1p German kindred Choreoathetosis/spasicity, episodic (CSE)

? suspected K+

channelAuburger, 1996

*italicized genes denote non-primary dystonia syndromes; TH, Tyrosine HydroxylaseTable 1.1. Familial dystonic syndromes for which gene loci have been identified.

Pathophysiology

Although the genetic basis of a few inherited dystonias, as well as the pathology

behind most secondary dystonias has been determined, little is known concerning the

pathophysiology of this disease. As etiologies continue to be discerned, research

4

elucidating central mechanisms involved in genetic, sporadic, or secondary dystonia will

provide insight to this broad and devastating group of disorders.

Medicine has traditionally viewed dystonia as a disorder of the basal ganglia. The

basis for this view rests largely in the knowledge that the basal ganglia is a common site

of pathology in many of the secondary dystonias caused by toxins, injuries, and various

heredodegenerative diseases. Furthermore, the majority of movement disorders are

considered to have an origin in the basal ganglia until proven otherwise. However, if

dystonia is indeed a disorder of the basal ganglia, it is the least well understood basal

ganglia disorder in terms of pathophysiology (Crossman AR, 1998). Gross and histologic

examination of brain tissue from patients with primary dystonia fail to demonstrate

morphological changes in the basal ganglia in contrast to lesions seen in secondary

dystonia. Alternatively, a biochemical abnormality may exist in the basal ganglia of

primary dystonia patients. The incidence of dystonia arising secondary to drug

treatments affecting monoamines in the striatum supports this theory (Fahn S, 1995);

however, no consistent changes have been documented. As with gross lesions, it appears

that dopaminergic dysfunction is readily discernable in secondary cases (Vidailhet M,

1999), while studies in primary dystonia fail to show consistent abnormalities in the

nigro-striatal dopaminergic pathway (Playford ED, 1993).

Numerous theories attempt to describe the pathophysiology behind dystonia; these

implicate (1) the basal ganglia, particularly the globus pallidus, and (2) the thalamus.

More recent work has also implicated (3) the sensorimotor cortex and (4) the cerebellum

as well. While these four regions may all be capable of independently causing dystonia,

the existence of a single common pathway affected by each of these regions is a more

5

likely and sought after explanation. The possibility of numerous independent pathways

resulting in various primary and secondary dystonia syndromes cannot be excluded

however.

Secondary dystonias and the dopa-responsive dystonia-plus syndrome have

historically maintained the basal ganglia at the center of dystonia research. Some of the

strongest evidence implicating the basal ganglia in dystonia comes from studies in drug-

induced primate models of dystonia and functional brain studies of drug-induced human

dystonia (and other hyperkinetic dyskinesias). These models implicate a causative role

for decreased basal ganglia output in dystonia. Decreased globus pallidus (internal

segment, Gpi) and substantia nigra (pars reticulata, SNr) output result in disinhibition of

the thalamic motor nuclei and increased excitatory input to the cerebral cortex. In theory,

decreased Gpi/SNr output can result from increased direct pathway inhibition of these

structures or decreased indirect pathway excitation (Berardelli A, 1998). It is precisely

this theory expressed by Vitek and Giroux as they describe dystonia as a hyperkinetic

movement disorder. They describe firing rates of Gpi being decreased, with altered

patterns and synchronization in dystonic states (Vitek JL, 2000).

A more complex theory was presented by Crossman and Brotchie however, as they

describe dystonia as both a hypokinetic and dyskinetic disorder (Crossman AR, 1998).

Because hypokinesias result from increased Gpi/SNr output and dyskinesias result from

decreased Gpi/SNr output, the authors concluded that temporal and/or spatial fluctuations

in Gpi/SNr activity are responsible for dystonia. Alternatively, the basal ganglia may not

be central to the production of dystonia; rather, inhibitory and excitatory influences of the

basal ganglia may fluctuate as a consequence of the dystonia. Components of the motor

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control circuitry may attempt to compensate for the activity resulting in the dystonia and

these changes are what researchers are identifying.

While lesions of the basal ganglia often result in dystonia (Munchau A, 2000;

Lehericy S, 1996; Kostic VS, 1995), other areas of the brain also induce dystonia when

lesioned, most commonly the thalamus (Lehericy S, 1996; Lee MS, 1994). Focal,

segmental, and generalized hemi-dystonia have been described after lesions of the

thalamus. In a review of movement disorders caused by thalamic lesions, Lee and

Marsden discuss two points which suggest that the mechanism of dystonia induced by

basal ganglia disorders and thalamic dysfunction are indeed separate. First, thalamic

lesions reported to induce dystonia were often restricted to the posterior, posterolateral,

and paramedian regions (Lee MS, 1994). These areas do not overlap with those receiving

input from the basal ganglia (ventrolateral, ventromedial, and ventroanterior regions).

Rather, these areas are largely associated with somatosensory input. Lehericy et al.

defined other regions of the thalamus involved in dystonia-producing lesions and they

also determined that the striatopallidal circuit was entirely unaffected. The ventral

intermediate and ventral caudal regions defined by the latter are involved in sensory and

cerebellar relays (Lehericy S, 1996). Thus, a direct connection cannot be drawn between

the pathway basal ganglia dysfunction affects in the thalamus and that affected in

dysfunction based within the thalamus itself. Secondly, most basal ganglia researchers

present a theory of pallidal disinhibition of the thalamus in dystonia. The resulting

excitation of the thalamus would therefore be in direct contradiction to the presumed

effect of thalamic lesion. Therefore, the overexcitation of the thalamus through

disordered basal ganglia function, and the defective output of the thalamus due to lesions

7

result in dystonia through what appears to be separate pathways (Lee MS, 1994; Lehericy

S, 1996). Although numerous separate circuits exist within the thalamus, the thalamus

acts as an integrating relay with considerable excitatory efferents reaching the

sensorimotor cortex. As research continues to define at least two separate mechanisms of

dystonia induction involving the thalamus, perhaps study of the resulting effects on

cortical function will merge these apparently divergent pathomechanisms.

Hallett proposed a broad deficiency of cortical inhibition as the central mechanism of

dystonia from a range of causes (Hallett M, 1998). Instead of the thalamus providing

excessive excitatory input, perhaps too little inhibitory input is given, or a combination of

both. Balance between excitation and inhibition is a crucial role the basal ganglia exerts

on the motor cortex through the thalamus, which itself influences the cortex in an

excitatory manner. Inhibition of areas adjacent to those activated ("center-surround") in

the somatatopically-organized motor cortex is necessary to prevent co-contraction of

antagonistic muscle groups. It is precisely co-contraction and overflow into adjacent

muscles that characterizes dystonia on EMG recordings. Therefore, any loss of surround

inhibition in the cortex could theoretically result in dystonia (Hallett M, 1998) and does

so when GABA antagonists are applied directly to the motor cortex (Matsumura M,

1991). Defective inhibition of the cortex could be endogenous to the area or due to

altered excitatory versus inhibitory input from the thalamus or other structures. The

thalamus in turn may be the original site of malfunction or may receive aberrant signals

from the basal ganglia or even the cerebellum, which has a large input to the motor

thalamus.

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The theory of impaired cortical inhibition also applies to repetitive-use induction of

dystonia, which results in larger cortical representation areas of the affected body parts

and that may affect inhibition (Hallett M, 1998). While many theories are currently being

discussed in the literature, very few are consistently supported by findings in dystonic

patients. This could mean that the current theories do not address the mechanisms central

to dystonia or perhaps no one common pathway is responsible for dystonic states. It is

plausible that multiple pathways could result in dystonia independently; however, it is

more likely that multiple causes merge into one common final pathway leading to

dystonia. If the latter is the case, theories concentrating on upstream components of the

generic or common pathway will not be supported by data collected from dystonic

patients or animals that have dystonic origin in another upstream branching pathway.

Thus, research on a particular cause or theory of dystonia will not show consistent results

if dystonias of different etiologies comprise the population being studied. However, a

potentially common downstream component of the dystonic pathway should consistently

show alterations in many forms of dystonia when studied. This theory of branching

etiologies is depicted schematically in figure 1.1.

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Figure 1.1. Schematic depiction of brain regions and pathways theorized to malfunctionin dystonia. There are four main regions currently implicated in the pathophysiology ofdystonia: (1) the basal ganglia, (2) the thalamus, (3) the sensorimotor cortex, and (4) thecerebellum.

While theories implicating altered basal ganglia function, thalamic dysfunction and

cortical disinhibition continue to be debated, studies from animal models and patients

with dystonia will help determine the mechanistic basis of dystonia. Despite new

evidence and the description of alternative plausible theories, such as the central broad

deficiency of cortical inhibition, the role of the basal ganglia continues to dominate the

field of dystonia. Concentration on and search for alterations in basal ganglia function in

MOTOR CORTEX

THALAMUS

Striatum

Gpi/SNr

SomatosensoryCortex

CEREBELLUM

DYSTONIA

BASALGANGLIA

1

2

3

4

10

patients with primary dystonia occurs perhaps at the expense of crucial new insights to

the yet undetermined pathophysiology of this disease.

Findings in animal models suggest that the main basal ganglia-thalamo-cortical

circuit is too restricted as the sole pathway examined in understanding the

pathophysiology of dystonia (Richter A, 1998). Numerous other brain regions and even

the spinal cord have been shown to be functionally altered in dystonic patients. Evidence

implicating sensory dysfunction and impaired reciprocal inhibition in dystonic patients

are two examples of consistent findings seemingly unrelated to the basal ganglia.

Perhaps the most consistent finding however comes from functional imaging studies of

dystonic patients representing a wide range of etiological origin. Blood flow analysis and

glucose utilization imaged in the CNS of dystonic patients consistently demonstrates

increased activity in the cerebellum. Patients with familial generalized idiopathic

dystonia due to the DYT1 mutation were studied and two patterns of altered glucose

metabolism were identified. One related to dystonic movements (movement related) and

the other unrelated to movement (movement free). The midbrain, cerebellum and

thalamus showed hypermetabolism in the movement related pattern of activity while the

lentiform nuclei, cerebellum and supplemental motor areas were hypermetabolic in non-

manifesting as well as dystonic patients carrying the DYT1 mutation in the movement

free pattern (Eidelberg D, 1998). Furthermore, the movement free pattern was also noted

during sleep, when involuntary movements are suppressed. Prior to this study, a single

patient with generalized dystonia was reported to have increased cerebral blood flow in

subcortical motor structures in addition to changes in the cerebellar blood flow.

Specifically, the was discordance between the blood flow in the right and left deep

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cerebellar nuclei (DCN) and abnormal correlation between right cerebellar cortical and

right DCN blood flow (LeDoux MS, 1995). As more imaging studies are reported, an

enormous amount of literature is accumulating, many demonstrating functional alteration

of the cerebellum in patients with dystonia.

Patients with writer's cramp, a form of focal dystonia, repeatedly demonstrate

increased activity in the contralateral primary sensorimotor and premotor corticies and

thalamus with ipsilateral hyperactivity in the cerebellum (Odergren T, 1998; Preibisch C,

2001). These regions form the cerebrocerebellar circuit and the regions of the cerebellum

and thalamus involved indeed correspond to the areas of efferent origin and termination

from the cerebellum respectively. Such over-activation in the cerebrocerebellar circuit

may be causative in initiating dystonia or may reflect an attempt to compensate for an

otherwise initiated dystonic signal. Similar activation of the cerebellum was also

demonstrated in patients with essential blepharospasm (EB), a focal dystonia affecting

eyelid closure. EB patients demonstrated increased metabolism in patterns analogous to

those described above for the movement-free pattern described for DYT1 dystonia. Their

movement related pattern however differed from that seen in DYT1 dystonia with

hypermetabolism in the cerebellum and pons being most notable (Hutchinson M, 2000).

Increased cerebellar perfusion was also seen in studies of patients with paroxysmal

exercise-induced dystonia with relative hypoperfusion of the frontal cortex and to a lesser

extent the basal ganglia (Kluge A, 1998). Thus, it appears that consistent findings of

altered cerebellar function affecting the cerebrocerebellar circuit are made in dystonias of

vastly different etiologies.

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An animal model of primary generalized dystonia, the dystonic rat shows a consistent

and necessary role of abnormal cerebellar activity in the dystonic phenotype, specifically

increased DCN output (Ledoux MS, 1995). The wriggle mouse sagami has also

demonstrated cerebellar abnormalities, mainly histologic changes within the molecular

layer (Ikeda M, 1989) and there exist lesions of mossy fibers within the cerebellum of the

dystonia musculorum mutant mouse (Sotelo C, 1988). The dystonic hamster, a rodent

model of paroxysmal nonkinesegenic dystonia, demonstrates cerebellar (DCN) along

with red nuclear and thalamic alterations in metabolism (reviewed in Richter A, 1995).

While rodent animal models and human functional brain studies implicate a role for

the cerebellum in numerous dystonic states, the basal ganglia continues to receive the

most attention in the field of dystonia research. In fact, literature pertaining to studies of

human dystonia frequently mention the lack of suitable animal models of dystonia other

than MPTP treated monkeys, which represent a drug-induced dystonia model of basal

ganglia origin. The restricted focus of dystonia research to pathologies of the basal

ganglia may severely impede progress in the field and retard potential benefit to the

patients who suffer from dystonia. Hopefully, the findings of consistently abnormal

cerebellar function in human studies will broaden the field of dystonia research and

expand the role rodent animal models play in understanding the pathophysiology of

dystonia.

Treatment

Given the lack of understanding of the pathophysiology of dystonia and the

heterogeneity of dystonic syndromes, no single treatment is effective in all patients. In

fact, treatment for the dystonic syndromes is only moderately effective overall. Initially,

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levodopa is administered to verify that a diagnosis of dopamine responsive dystonia has

not been overlooked (Adler CH, 2000; Fahn S, 1995). A series of drugs are reported to

have some effect in different forms of dystonia, and are administered on a trial -and-error

basis. These include anticholinergics (high-dose), baclofen (high-dose), benzodiazepines,

and anti-dopaminergics. Each of these classes of drugs works only in a minority of

patients and thus underscores the need for better understanding of the pathophysiology of

dystonia. Surgical thalamotomy is also used in severe refractive cases with variable and

often temporary success. A more recent central intervention is the use of deep brain

stimulation of the globus pallidus. This technique is in its infancy, but holds promise for

the future (Adler CH, 2000). A common treatment for focal dystonia is periodic injection

of botulinum toxin in the affected muscle groups. The majority of patients tolerate the

toxin well and benefit from the therapy. Surgical denervation of affected muscles is

sometimes used in intractable focal dystonias as well (Adler CH, 2000; Fahn S, 1995).

Paroxysmal Dyskinesias

Paroxysmal dyskinesias are a specific subgroup of dystonia defined by the

intermittent nature of the dystonic movements on a background of otherwise healthy

individuals. The episodic nature, involvement of motor behaviors, and other salient

features of this subgroup of dystonia historically resulted in misdiagnosis as reflex

epilepsy, movement-induced seizures, and subcortical epilepsy (Demirkiran M, 1995).

However, the dystonic nature of the movements, absence of any EEG correlates,

complete maintenance of consciousness, and lack of any postictal state refuted the theory

that the paroxysmal dyskinesias are a form of epilepsy (Demirkiran M, 1995).

Paroxysmal dyskinesias occupy a unique position in that they represent a crossroads

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between dystonia and episodic neurological disease. Consequently, advancement in the

study of episodic diseases will benefit research on the paroxysmal dystonias and therefore

on dystonia as a whole.

The paroxysmal dyskinesias are rare syndromes of intermittent dystonia subdivided

into four groups based on clinical characteristics: (1) Paroxysmal kinesigenic dyskinesia

(PKD), (2) Paroxysmal non-kinesigenic dyskinesia (PNKD), (3) Paroxysmal exercise-

induced dyskinesia (PED), and (4) Paroxysmal hypnogenic dyskinesia (PHD).

Definitions and salient features of the paroxysmal dyskinesias are presented in Table 1.2.

Further consideration will only be given to the first two forms, PKD and PNKD, due to

relevance here.

Subgroup CharacteristicsPKD (PKC) Brief duration (seconds to 5 minutes)

Always preceded by sudden initiation of movementPNKD (PDC) Prolonged duration (2 minutes to 4 hours)

Spontaneous or triggered by various stressorsPED Intermediate duration (5 to 30 minutes)

Precipitated by continuous movement (not sudden)PHD Occurs during sleep (ADNFLE in some kindreds)ADNFLE, Autosomal Dominant Nocturnal Frontal Lobe Epilepsy,PKC, Paroxysmal kinesigenic choreoathetosis, PDC, Paroxysmal DystonicChoreoathetosisTable 1.2. Subgrouping of the Paroxysmal Dyskinesias.

Paroxysmal dyskinesias like other forms of dystonia result from both primary and

secondary etiologies. Secondary paroxysmal dyskinesias are relatively uncommon and

have been associated with neurological diseases such as multiple sclerosis, cerebral palsy,

stroke, encephalitis, birth asphyxia, certain seizure disorders, or metabolic diseases such

as idiopathic hypoparathyroidism and thyrotoxicosis (Goodenough DJ, 1978; Demirkiran,

1995; Nardocci, 1989). In contrast to the primary paroxysmal dyskinesias, secondary

dyskinesias are accompanied by neurological findings and often abnormal EEG

15

recordings (Goodenough DJ, 1978). Symptomatic presentation of paroxysmal

dyskinesias is similar between primary and secondary forms; however, for ease of

presentation the remainder of discussion will concern primary paroxysmal dyskinesias.

Paroxysmal Kinesigenic Dyskinesia

PKD occurs more frequently than PNKD (Nardocci, 1989; Goodenough DJ, 1978).

PKD is characterized by attacks of dystonia precipitated by sudden movement lasting

from a few seconds to 5 minutes. The majority of patients experience 1 to 10 attacks per

day (Houser MK, 1999; Goodenough DJ, 1978; Nardocci, 1989; Bhatia KP, 1999). The

most common precipitating situation is standing from a sitting or lying position. In

general, sudden movements after a period of rest most commonly cause the attacks.

Startle is also occasionally associated and predisposing factors to increased sensitivity

include alcohol and exhaustion. The distribution of the dystonia varies widely, often

restricted to one side of the body but may be generalized. Some of the attacks display

mixed hyperkinetic involuntary movements of dystonia, choreoathetosis, and ballismus

(Demirkiran M, 1995; Goodenough DJ, 1978; Bhatia KP, 1999). Sensory aura preceding

the attacks is also common and described as "tingling" or stiffness in the limbs. Most

patients have learned to avoid the induction of attacks altogether by initiating movements

slowly or warming-up prior to initiation (Goodenough DJ, 1978; Bhatia KP, 1999). No

alteration in consciousness is ever reported and no postictal state is associated with the

attacks (Houser MK, 1999; Goodenough DJ, 1978; Bhatia KP, 1999). EEG recordings

are almost invariably normal during and between attacks in PKD patients. All other

physical and neurological exams and laboratory studies are also normal, confirming the

healthy background on which this episodic disorder occurs (Goodenough DJ, 1978). The

16

age of onset is typically in early adolescence or early adulthood with a male

preponderance often noted. The vast majority of described cases are of primary etiology

with nearly 1/2 to 2/3 being sporadic and the remainder of familial origin (Houser MK,

1999; Nardocci, 1989). Transmission in families with PKD occurs in an autosomal

dominant fashion with reduced penetrance (Goodenough DJ, 1978). The frequency of

the paroxysms decreases with age after peaking sometime in adolescence (Goodenough

DJ, 1978; Nardocci, 1989). Administration of anticonvulsants (e.g., phenytoin,

carbamazepine) causes marked reduction in attack frequency or elimination of the

paroxysms altogether.

Similar to other dystonias, the basal ganglia is also thought to be of central

importance in the paroxysmal dyskinesias because of the involuntary nature of the

movements, the absence of EEG abnormalities, and the presence of basal ganglia

pathology in conditions leading to secondary or symptomatic disease (Nardocci N, 1989).

Abnormal basal ganglia metabolism has been reported on PET scans of secondary PKD

patients during attacks and some have experienced a favorable response to levodopa

therapy (Goodenough DJ, 1978). Others have found thalamic lesions in the ventral

posterolateral nuclei in some secondary PKD cases (Sunohara N, 1884; Camac A, 1990;

Burguera JA, 1991; Nijssen PCG, 1992).

Paroxysmal Non-kinesigenic Dyskinesia

PNKD is characterized by attacks of dystonia that are spontaneous in nature. The

paroxysms are longer than those in PKD and last from 5 minutes to a few hours. Few

patients may have longer attacks but a majority last 30 minutes to one hour. Attack

frequency ranges widely as many as 2-3 per day to as few as 2-3 per year, with the

17

majority of patients experiencing a few attacks per month (Jarman PR, 2000;

Goodenough DJ, 1978; Nardocci, 1989; Bhatia KP, 1999). In general, the frequency of

attacks is higher in childhood and declines with increasing age and the duration of attacks

shortens with increasing age as well. PNKD is never associated with sudden initiation of

movement and is generally considered to be a spontaneous event. However, a number of

triggers have been identified that precipitate attacks in most patients. Common triggers

include caffeine, alcohol, stress, fatigue, hunger, cold, menstruation, intercurrent illness,

and excitement (Jarman PR, 2000; Goodenough DJ, 1978; Nardocci, 1989). In addition to

certain precipitating factors, there is a diurnal pattern with an increased frequency of

attacks in the late afternoon and evening compared to early in the day (Jarman PR, 2000).

Attacks always begin in a limb and progress to hemidystonia or generalized dystonia

consisting largely of sustained dystonic posturing (Jarman PR, 2000; Nardocci, 1989;

Bhatia KP, 1999). Sensory aura preceding the attacks is also common and described as

"tingling" in the skin or tightness in the muscles with a feeling of restlessness (Jarman

PR, 2000; Goodenough DJ, 1978; Bhatia KP, 1999). During the sensory prodrome and in

the initial portion of an attack, patients may interrupt the progression by going to sleep.

Just a few minutes of sleep will often abort the attack altogether if initiated early in the

course (Jarman PR, 2000). No alteration in consciousness is ever reported and no

postictal state is associated with the attacks. EEG recordings are almost invariably

normal during and between attacks in patients with PNKD. The age of onset is typically

in infancy or childhood, which is earlier than PKD. As with PKD a male preponderance

is noted, especially in the more common familial PNKD (Goodenough DJ, 1978;

Nardocci, 1989; Bhatia KP, 1999). An autosomal dominant mode of inheritance is seen

18

in families with PNKD. Administration of anticonvulsants is not an effective treatment in

PNKD, clearly separating this disorder from PKD in treatment strategies.

Benzodiazepines often have a modest degree of improvement, but not in all patients

(Jarman PR, 2000; Nardocci, 1989).

Linkage analysis of several families with PNKD has localized the causative gene in

the autosomal dominant form of the disease to chromosome 2q33-35 (Fouad GT, 1996;

Fink JK, 1996). Due to the episodic nature of the disorder and the association of episodic

disease and channelopathies, a candidate ion channel in this region has been identified

(Hofele K, 1996). A second gene locus has also been identified in a German kindred

with a variant of PNKD that is triggered by physical exercise in addition to the common

PNKD triggers. In addition, this form is often associated with headaches, diplopia

(which is also a rare symptom in some severe PNKD cases), perioral paresthesias, and

spastic paraplegia in some of the patients. The gene has mapped to an area of

chromosome 1p where several potassium channel genes reside and has been termed CSE

(choreoathetosis/spasicity, episodic) (Auburger G, 1996). As is typical with

channelopathies, occurrence of other episodic neurological diseases in patients with

PNKD has been reported. Migraine has most commonly been associated with

paroxysmal dyskinesias, along with epilepsy and an isolated case of episodic ataxia as

well (Hofele, 1997; Mayeux, 1982).

19

1.2. TOTTERING MOUSE: ANIMAL MODEL OF DYSTONIA

Introduction

The use of animal models in the study of human disease has proved invaluable in

defining pathophysiologies and testing treatment strategies. Models can be derived

through surgical, drug, or genetic manipulation as well as occur naturally through random

mutations. The tottering mouse is one such naturally occurring genetic animal model of

human disease. As a model for absence epilepsy and more recently, paroxysmal

dystonia, the tottering mouse has been studied extensively since its discovery in 1957 at

the Roscoe B. Jackson Memorial Laboratory in Maine. An abnormal, wobbly gait was

observed in three DBA/2J mice who subsequently were shown to be homozygous for a

new recessive mutation, which was named tottering (Green, 1962). The original mice

were fertile when out-crossed to C57Bl/10JGn and F1 intercrossing produced tottering F2

in expected ratios for autosomal recessive trait. Tottering (tg) was found to be closely

linked to Oligosyndactylism (Os) with crossover occurring between Os and tg rarely, if at

all. (Green, 1962) Through positional cloning, the tottering mutation was identified as a

C to T base substitution at position 1802 in the ? 1A subunit of the P/Q-type high-voltage

dependent calcium channel. The mutation results in a proline to leucine substitution at

amino acid 601 near the P-domain of repeat II between transmembrane segments S5-S6

(Fletcher, 1996).

Calcium Channels

Structure

Calcium channels are part of the cellular mechanism that tightly regulates calcium

concentration in and around the cell. Such tight regulation is necessary for the accurate

20

and timely execution of numerous calcium-dependent functions. Excitation-contraction

coupling in muscle cells, gene regulation, and second messenger system activation are all

important functions of various cells that occur in response to changes in local calcium

concentration. In neurons, additional processes dependent on dynamic local calcium

concentration include membrane excitability and neurotransmitter release. Neuronal

calcium channels pertinent to this discussion are the voltage-dependent calcium channels

(VDCC), comprised of a pore-forming ? 1 subunit and additional auxiliary subunits ? ,

? 2-?, and sometimes ?. Numerous genes encode ? 1 subunits, all of which share a

common structure of four repeated domains, each containing six transmembrane

segments (S1-S6). A P-domain in the extracellular space between S5-S6 transmembrane

segments is responsible for the pore environment and ion selectivity. In addition to

determining ion selectivity, the ? 1 subunit acts as the voltage sensor and determines the

kinetics of activation and inactivation. The cytoplasmic face of the protein also interacts

with G-protein ? ? subunits. The remaining auxiliary subunits of intact calcium channels

act to modulate gating and kinetics of the channel (Catterall WA, 1988; Walker D, 1998).

The subunits associate to form a complete channel as depicted in Figure 1.1.

21

Figure 1.2. Schematic depiction of voltage-gated calcium channel. The ? 1 subunit isthe main pore-forming subunit while ? and ? 2- ? subunits are auxiliary.

Function

VDCC function varies depending on the subunit composition as well as the

subcellular and tissue distribution. Five genes encode neuronal high-voltage activated

Ca2+ channel pore-forming ? 1 subunits (A-E). Four encode the ? subunit (1-4), two

genes are believed to encode the ? 2-?, and one for the neuronal ? subunit. The neuronal

high-voltage activated calcium channels are classified in Table 1.3 according to their

molecular biology, pharmacology, and functional characteristics. An enormous potential

for VDCC heterogeneity exists because of the numerous combinations of ? 1, ? , ? 2-? and

? subunits that may assemble and the number of splice variants of each subunit. High-

voltage activated calcium channels involved in neurotransmitter release (P, Q, N and R-

type) are located at synaptic terminals in close approximation to the docking and release

machinery of synaptic vesicles (Westenbroek RE, 1992; Ludwig A, 1997). As the

? 1

?

?

? 2? 2

extracellular

intracellular

22

depolarizing action potential reaches the axon terminal, the VDCC senses the change in

electrical potential and opens the channel pore if the potential is indeed great enough.

This allows calcium to rapidly enter the terminal by flowing down its concentration

gradient into the cell. The local calcium concentration increases tremendously and acts

as a trigger for release of synaptic vesicle contents into the synaptic cleft. L-type

channels play an entirely different role in neurons. These channels (? 1C and ? 1D) are

located on the proximal dendrites and somata of neurons and conduct calcium in response

to membrane depolarization leading to further excitability, gene transcription, and

activation of second messenger cascades (Ludwig A, 1997; Hell JW, 1993). Due to the

slight redundancy of VDCC subtype functions, it is plausible that one subtype may

compensate for altered activity of another similar subtype. For this sort of compensation

to occur however, a second subtype would have to be expressed in that region already or

expression would have to begin de novo.

Subunit Subtype Pharmacology Location Function? 1A-a P ? -agatoxin IVA

? 1A-b Q ? -conotoxin MVIIC

cerebellum, hippocampus,inferior colliculus olfactory

bulb, spinal cord

Neurotransmitterrelease

? 1B N ? -conotoxin MVIIA, ? -conotoxin GIVA

diffuse, hippocampus Neurotransmitterrelease

? 1C L

? 1D L

dihydropyridines,benzothiazepines,

and phenylalkylamines

diffuse, olfactory bulbs,hippocampus, superiorcolliculus, cerebellum

Electrical excitability,gene transcription

? 1E R cadmium, nickel olfactory bulb, habenula,cortex, hippocampus,

cerebellum

Neurotransmitterrelease

(Hillman D, 1991; Tanaka O, 1995; Westenbroek RE, 1992; Stea A, 1994; Ludwig A, 1997)Table1.3. Classification of neuronal high-voltage activated calcium channels.

23

Behavioral Phenotype

As with many disorders caused by mutations in ion channels, tottering mice display

several phenotypes and some are characterized as episodic or intermittent in nature.

Diseases caused by mutations in ion channel genes, termed channelopathies, are of

growing interest as they demonstrate causative roles in episodic neurological and

neuromuscular diseases such as migraine, ataxia, epilepsy, dystonia, and paralysis

(Ophoff RA, 1996; Grosson CLS, 1996; Browne DL, 1994; Fouad GT, 1996; Auburger

G, 1996; Steinlein OK, 1995). These diseases often share an overlapping clinical spectra

of symptoms, suggesting common pathomechanisms involving the ion channel gene

defects. Co-occurrence of multiple episodic phenotypes in a single disorder (e.g.,

migraine headache and hemi-paresis in Familial Hemiplegic Migraine) is common and

suggests a unique pathomechanism in channelopathies. It is likely that the expression of

mutated channels in multiple regions results in the seemingly unrelated concomitant

phenotypes, but the reason for the intermittent expression of the phenotypes is less clear.

Tottering mice display three distinct behavioral phenotypes as part of their neurological

syndrome, including polyspike discharges, ataxia and paroxysmal dystonia. Both the

polyspike discharges and the dystonic attacks are episodic in nature, while the ataxia is

always present.

Polyspike Recordings

Initial studies were aimed at defining an epileptiform activity associated with the

bizarre characteristic motor episodes of the tottering mouse that are described below

under 'intermittent dystonic attacks.' Instead of defining motor seizures however,

researchers discovered an unexpected phenotype of abnormal bursts of bilaterally

24

synchronous and symmetrical spike waves, six to seven per second, over the cerebral

hemispheres in tottering mice at rest. This activity constitutes ~10% of resting EEG

activity and is always accompanied by sudden arrest in movement, staring and often

twitching of the jaw. The bursts are 200-400?V in amplitude and last from 0.3-10

seconds. The spike-wave abnormality is fully developed in the 4-week old animal,

although the wave component decreases substantially with age (Noebels, 1979). These

polyspike bursts occurred paroxysmally during waking hours and within motor episodes,

but reliably during drowsiness. Sometimes a spike-wave appearance was appreciated,

resembling human absence seizures (3/s spike-wave activity) and postictal EEG

depression was never present, similar to absence epilepsy (Kaplan, 1979). This

serendipitous finding has led to the use of the tottering mouse as a genetic animal model

of absence epilepsy.

Ataxia

The most easily observed phenotype of the tottering mouse is that for which it was

named, an ataxic gait. Original reports on Os/tg stock at Jackson laboratories describe

increased toeing-out of the hind feet at 2-3 weeks and soon thereafter the trunk is held

closer to the ground and the mouse may lean while walking (Green, 1962). Analysis of

tottering gait patterns revealed decreased stride and step lengths and increased gait angle

compared to controls (Campbell DB, 1999). Although the ataxic phenotype of the

tottering mouse is useful in differentiating tottering animals from control littermates, little

research has been aimed at defining the basis for this finding.

25

Intermittent dystonic attacks

Tottering mice display striking episodic attacks of severe motor dysfunction that were

originally described as seizures. Quite stereotypical in nature, the attacks typically begin

with a hind limb being held tight against the trunk or abducted with some paddling in the

air. Forelimb involvement follows with the limb again being held tight against the trunk

or with paddling. Progression to include both hindlimbs, abducted at the hip and knee

into the air, results in the abdomen resting on the cage bottom and the trunk flattens. The

back next becomes stiff and arched such that the perineum is pressed against the cage

bottom. As forelimbs become more prominently involved in the next phase, the neck

also flexes severely and the ears fall flat against the back and the jaw and eyelids may

move repetitively (Green, 1962). The attacks typically last 30-60 minutes and occur

spontaneously or in response to several known stressors and pharmacological agents with

no discernable refractory period. Although the described attack is most common, there

are other characteristic postures adopted by dystonic tottering mice and attacks do vary in

course, duration, and severity within and between mice. There is no reliable abnormal

EEG recording during these attacks (Kaplan, 1979). Therefore, the term ‘seizure’ is

rapidly falling out of favor as the preferred descriptor of this behavior. Convulsions,

motor seizures, motor episodes, and myoclonic-like movement disorder are all still used

in the literature to describe this phenomenon but we believe that the best and most

accurate descriptor is paroxysmal dystonia. The remainder of this work will deal almost

exclusively with the phenotype of intermittent dystonia and defining the neuronal

networks responsible for its occurrence.

26

Cellular Pathology

Locus ceruleus hyperarborization

Using the tottering mouse as a model for absence epilepsy, researchers investigated

catecholaminergic innervation in the mutant. Histochemical analysis showed a

significant increase in the number of noradrenergic axons in terminal fields innervated by

the nucleus locus ceruleus (LC) when compared to wild type. A concomitant 100-200%

rise in norepinephrine (NE) levels is found in the same areas, including hippocampus,

cerebellum, and dorsal lateral geniculate. These changes were indeed due solely to

hyperarborization as the size and number of LC neuronal somata were unchanged (Levitt,

1981). 6-hydroxydopamine lesioning of noradrenergic fibers innervating the neocortex

and hippocampus resulted in loss of the polyspike phenotype (Noebels JL, 1984). In

these experiments the dystonic phenotype and LC innervation to cerebellar and brainstem

structures remained however. Therefore, locus ceruleus axons were lesioned with the

neurotoxin, DSP-4 in the tottering mouse to test the hypothesis that the noradrenergic

hyperinnervation (to the cerebellum in particular) was responsible for the intermittent

dystonic phenotype. After successful lesioning of LC innervation to the cerebellum,

tottering dystonic attacks remained unchanged further supporting the independence of

these two phenotypes (Campbell DB, 1999).

Aberrant Tyrosine Hydroxylase Expression

The discovery of noradrenergic hyperinnervation also led researchers to examine the

expression patterns of the rate-limiting enzyme in catecholamine synthesis, tyrosine

hydroxylase (TH). TH mRNA and protein expression is normal in the major

catecholaminergic nuclei, however, expression was altered in the cerebellum. TH mRNA

27

and protein are transiently expressed in Purkinje cells (PC) of normal and heterozygous

mice during development at postnatal days P21-P35. Expression is aberrantly maintained

in posterior cerebellar PC and expressed de novo in the anterior cerebellum throughout

adulthood in tottering mice, indicating compromised gene regulation in the mutant mouse

(Hess EJ, 1991; Fureman, 2001).

Purkinje Cell abnormalities

Determination of the direct effect the tottering mutation has on the P/Q-type VDCC

has been achieved using electrophysiological analysis. Dissociated mutant PC from 18-

30 day old tottering pups show a 40% reduction in total calcium current density

compared to wild type without changes in cell size. Recombinant tottering channel

protein expressed in baby hamster kidney cells also showed decreased current density

(Wakamori M, 1998). Thus, the mutation does indeed alter the basic function of the P/Q-

type calcium channel as expected.

1.3. CHAPTER SUMMARY

Dystonia is a relatively common neurological disorder with an estimated prevalence

of 30 per 100,000 in the population (Nutt JG, 1988). Despite the discovery of genes

involved in a few of the monogenetic inherited dystonias and knowledge of the pathology

present in secondary dystonias, the pathophysiology resulting in dystonia remains poorly

understood. Although knowledge of the neuronal basis of dystonia has grown

tremendously, conflicting results and heavy reliance on human studies underscore the

need for better utilization and discovery of animal models. Animal models provide

unique opportunities to study and manipulate in vivo neurological systems, allowing great

strides in the understanding of the pathophysiological basis of disease. Current rodent

28

animal models are limited in usefulness because the primary gene defect resulting in the

phenotype is unknown. The tottering mouse, therefore has a unique advantage as a

genetic rodent model of dystonia because of the well-defined nature of the genetic

mutation and a growing understanding of the resultant behavioral and cellular

phenotypes. Discovery of the neuronal basis of tottering mouse dystonia can further

define the neuronal networks capable of producing dystonia of other etiologies. The next

step in the use of animal models of dystonia would then be to replicate the dystonia-

producing signals defined in the tottering mouse in genetically normal animals.

Replication of the abnormal dystonic activation in wild type animals acts to eliminate

confounding variables introduced by the wide spread neurological effects in the tottering

mouse. This rationale guided the study of the cerebellar role in tottering dystonia and the

subsequent discovery of a novel animal model suited to the study of neuronal networks

capable of causing dystonia in wild type mice.

1.4 HYPOTHESES

The cerebellum plays a key role in the expression of dystonia in a mouse model with a

defined genetic background, tottering, and in a novel model using kainic acid in wild type

mice. Together, this information supports the theory that aberrant cerebellar cortical

activity leads to profound dystonic attacks.

1. Neurotransmission in the cerebellum of tottering mice is altered compared to controls.

Tottering mice harbor a mutation in the ? 1A pore-forming subunit of P/Q-type

calcium channels that function at presynaptic terminals to trigger neurotransmitter

release. Cerebellar granule and Purkinje cells in particular have a high density of P/Q-

29

type calcium channels; therefore, it is theorized that tottering cerebella may have

disrupted neurotransmitter release as a functional consequence of the mutated gene.

2. Purkinje cells are a necessary component in the expression of tottering dystonia.

Cerebellar Purkinje cells contain a high density of P/Q-type calcium channels, the

channel subtype mutated in tottering mice. The cerebellum is intensely activated during

tottering dystonic attacks and the Purkinje cell is the sole output source for the cerebellar

cortex. The role of the Purkinje cell in the cerebellar circuitry, the high level of P/Q-type

channel expression and the degree of cerebellar activation during tottering dystonia all

support the theory that the cerebellar Purkinje cell is central to the expression of tottering

dystonia.

3. Specific localized excitation of the cerebellum with kainic acid in wild type mice

causes dystonia.

Aberrant activation of the cerebellar cortex in tottering mice is involved during

initiation of dystonic attacks. Exogenous excitation of the cerebellar cortex in wild type

mice will also induce dystonia.

4. Kainic acid excitation in mice lacking Purkinje cells fails to cause dystonia.

The cerebellar cortex is able to activate pathways in excess, leading to dystonic

attacks. As is the case with the tottering mouse, it is theorized that the Purkinje cell is an

essential component of these pathways in the kainate-induced dystonia model and

dystonia cannot be elicited in animals lacking Purkinje cells.

30

Chapter 2. ALTERED NEUROTRANSMISSION IN THETOTTERING CEREBELLUM

Abstract

P/Q-type calcium channels are involved in several neuronal functions including

neurotransmitter release. Tottering mice have a mutation in the ? 1A pore-forming

subunit of P/Q-type calcium channels. This calcium channel subtype is expressed

abundantly in the cerebellum. Furthermore, the tottering mouse behavioral phenotypes of

generalized ataxia and intermittent dystonic episodes are largely cerebellar in origin.

Because P/Q-type calcium channels have been implicated in calcium-dependent

neurotransmitter release, glutamate and GABA release was investigated in the tottering

mouse cerebellum as a functional consequence of the channel mutation. Cerebellar

synaptosomes from wild type and tottering mice were preloaded with 3H-glutamate or

3H-GABA and then superfused with Earle’s balanced salt solution. Neurotransmitter

release was induced by depolarization with 60mM KCl. Potassium-stimulated release of

3H-glutamate and 3H-GABA in wild type and tottering mouse cerebellar synaptosomes

was calcium-dependent. Potassium stimulated calcium-dependent 3H-glutamate release

was significantly decreased in tottering cerebella compared to controls while 3H-GABA

release remained unchanged. These data indicate a deficiency in excitatory but not

inhibitory neurotransmission in the tottering cerebellum.

Introduction

Tottering mice harbor a mutation in the ? 1A pore-forming subunit of P/Q-type

voltage dependent calcium channels (VDCCs). P/Q-type calcium channels are high-

voltage activated channels located at presynaptic terminals where influx of calcium

31

through P/Q-type channels triggers neurotransmitter release. Therefore, it is theorized

that tottering mice may have disrupted neurotransmitter release in regions of high P/Q-

type calcium channel expression as a functional consequence of the mutated gene.

Furthermore, alterations in neurotransmitter release may in part be responsible for the

tottering mouse phenotypes of six- hertz polyspike discharges (absence seizures), ataxia,

and paroxysmal dystonia. In effort to examine neurotransmitter release in the tottering

mouse, numerous researchers have employed various techniques to investigate

neurotransmission in this mutant.

The tottering neuromuscular junction (NMJ) was investigated by Plomp et al. because

P-type calcium channels are expressed at the NMJ and these synapses are relatively easy

to study. Miniature endplate potential (MEPP) and low frequency evoked endplate

potential (EPP) amplitudes were not different between tottering homozygotes, tottering

heterozygotes, or wild type controls. However, EPP amplitude run down was

significantly increased in tottering mice and tottering muscles were paralyzed at lower

concentrations of the acetylcholine receptor antagonist, tubocurine, than were controls.

These data indicate a smaller 'safety factor' in tottering mice, which is the ratio between

EPP size and the depolarization necessary for muscle firing. The safety factor is typically

substantial in the NMJ to prevent small changes in acetylcholine release from failing to

elicit the all-or-none requirement for muscle activation. A second change noted in the

tottering NMJ was an increase in spontaneous MEPP frequency in both tottering homo-

and hetero-zygotes (Plomp JJ, 2000). Although it is difficult to extrapolate the

significance of these NMJ changes on CNS neurotransmission, the increased spontaneous

32

release of quanta and the increased run down of release after high frequency stimulation

suggest a deficit in the control of neurotransmitter release in tottering mice.

In the CNS, neurotransmission studies are usually separated into excitatory and

inhibitory categories with glutamate and GABA being representative of each group

respectively. Ayata et al. showed decreased glutamate and GABA release in the

frontoparietal cortex (neocortex) of adult male and female tottering mice using in vivo

microdialysis after 100mM KCl stimulation. A 2-fold decrease in glutamate release and

a 3-fold decrease in GABA release was measured in tottering mice compared to controls

(Ayata C, 2000). Different results were obtained in thalamus when Caddick et al.

measured excitatory post-synaptic potentials (EPSPs) and inhibitory post-synaptic

potentials (IPSPs) using whole cell recordings of single neurons in the somatosensory

thalamus (ventrobasal nucleus) of P14-28 male tottering mice. Stimulus-evoked EPSPs

recorded after electrical stimulation of adjacent neurons revealed significantly smaller

EPSPs in tottering animals compared to wild type controls. In contrast, tottering and

control maximal evoked IPSPs were not significantly different. These data support a

defect in excitatory neurotransmission in tottering thalami with no change in inhibitory

neurotransmission (Caddick SJ, 1999).

Tottering mouse neurotransmission at the hippocampal Schaffer collateral synapse

was studied by Qian and Noebels. Both Ca2+ influx and field excitatory post-synaptic

potentials (fEPSPs) were recorded after electrical stimulation. Although Ca2+ influx and

fEPSPs were at normal levels, tottering animals have an increased requirement for N-type

calcium channel function compared to controls (Qian J, 2000). Similar results were

obtained by Jun et al. working with the ? 1A subunit knockout mouse (Jun K, 1999). In

33

these animals, neurotransmission at the hippocampal Schaffer collateral synapse was

solely dependent on N-type channels in comparison to controls, which rely on both N-,

and P/Q-type channels. Barium currents were also studied in these knockout animals in

the excitatory cerebellar granule cells and in inhibitory Purkinje cells (PCs). Total

current was decreased in both cell types; however, only PCs showed a compensatory

increase in current through N- and L- type channels. Campbell and Hess reported the

original evidence for alternative calcium channel subunit compensation for deficits in the

tottering mouse, with increased L-type calcium channel expression in the tottering

cerebellum (Campbell DB, 1999). All of these data suggest that some regions of the

nervous system may better be able to compensate for impaired P/Q-type calcium channel

function than other areas. Regions with both N- and P/Q-type channel dependent release

may more easily compensate for the P/Q-type deficiency than neurons where N-type (or

R-type) channels are not typically expressed. In fact the ability to compensate for

decreased P/Q-type dependent release may not only be regionally restricted, but also

restricted by neuronal sets, such as excitatory versus inhibitory.

The 6-Hz polyspike phenotype of the tottering mouse is often studied as a model for

absence epilepsy. Therefore, brain regions tested for altered neurotransmission often are

chosen due to their relationship to seizure generation. A less studied phenotype of the

tottering mouse is the characteristic intermittent dystonic attacks. The cerebellum has

been associated with tottering dystonic attacks through in situ studies of c-fos activation

following an attack and through genetic ablation of cerebellar PCs in double mutant

pcd/tottering mice (Campbell DB, 1998; Campbell DB, 1999). In addition, the calcium

channel subunit mutated in tottering mice, ? 1A, is abundantly expressed in cerebellar

34

PCs and cerebellar granule cells. Together these data suggest that the cerebellum is a

likely site for functional alterations from the tottering mutation and such changes may

directly relate to dystonic attacks in this mutant.

We therefore examined neurotransmitter release in the cerebellum, a region not yet

studied. GABA and glutamate are the most prevalent neurotransmitters in the

cerebellum. Mossy fibers and climbing fibers are the extracerebellar input to the

cerebellar cortex and these connections excite granule cells and PCs, respectively,

through glutamatergic synapses. Glutamate is also released by granule cells to stimulate

Purkinje, basket, stellate, and golgi cells. The inhibitory neurotransmitter, GABA, is

released by Purkinje, basket, stellate, and golgi cells to their respective targets as

illustrated in Figure 2.1. Superfusion of cerebellar synaptosomes preloaded with tritiated

neurotransmitters was used to test the hypothesis that neurotransmitter release is altered

in the cerebellum of the tottering mouse as a functional consequence of the mutation in

the P/Q-type calcium channel.

Figure 2.1. Schematic diagram of neuronal connections in the cerebellum. Arrowheadsindicate excitatory neurotransmission of glutamate and flat bars represent GABAergicinhibitory neurotransmission. GC, granule cell; PC, Purkinje cell; G, golgi cell; S,stellate cell; B, basket cell; DCN, deep cerebellar nuclei.

GC PC

ExtracerebellarNuclei

DCN

Climbing FiberMossy

Fiber

G

B

S

35

Materials and Methods

Mice

Originally obtained from Jackson laboratories, tottering mice and control C57Bl/6J

mice were maintained at the Pennsylvania State University College of Medicine vivarium

on a 12-hour light cycle with access to food and water ad libitum. Tottering progeny

were rapidly identified by lack of oligosyndactylism from crosses between tottering

heterozygotes carrying the oligosyndatylism allele in repulsion to the tottering allele.

Male and female mice used in these experiments were between 8-10 weeks of age.

Synaptosome Preparation

Mice were sacrificed by carbon dioxide asphyxiation followed by decapitation and

brains were removed on ice. Cerebella were homogenized in 10 volumes of 0.32 M

sucrose. The homogenate was centrifuged for 10 minutes at 3,300 rpm at 4oC. The

supernatant was then centrifuged for 45 minutes at 13,500 rpm at 4oC to yield a pellet

containing the crude synaptosomal fraction. The pellet was resuspended in 1.2 ml of

balanced Earles salt solution (1.8mM CaCl2, 5.3mM KCl, 0.8mM MgSO4, 117mM NaCl,

26mM NaHCO3, 1mM NaH2PO4-H2O, 5.6mM glucose). The synaptosomal preparation

was equilibrated to 37oC for 10 minutes. Tritiated glutamate or GABA was added to a

concentration of 150 nM or 200 nM respectively. The synaptosomes were incubated for

20 minutes at 37oC to allow incorporation of the exogenous neurotransmitter into

synaptic vesicles.

Release Assay

200? l of the preloaded synaptosomes were aliquoted into a 12-chamber Brandel

superfusion apparatus between two GF/C filters (Brandel, Gaithersburg, MD). The

36

synaptosomes were rinsed with Earles buffer without CaCl2 for 45 minutes at 0.5 ml/min.

The buffer was continually oxygenated with 95%oxygen/5%carbon dioxide gas. Three

baseline fractions were collected for 3 minutes each before the perfusate was changed to

the stimulation buffers containing 60 mM KCl with or without 1.8 mM CaCl2 for 2 min.

The buffer was returned to basal Earles buffer without CaCl2 for the remainder of the

fraction collection. Following collection, water perfused the synaptosomes to release any

remaining neurotransmitter by osmotic lysis. Three milliliters of ScintiVerse (Fisher,

Pittsburgh, PA) was added to each of the 1.5 ml fractions (3 min collection at 0.5 ml/min)

and mixed prior to liquid scintillation spectroscopy at an efficiency of 35-45%.

Data Analysis

The neurotransmitter collected in each fraction is expressed as a percentage of the

total neurotransmitter available in that chamber at the time of fraction collection. Percent

fractional release is calculated using the following formula (Snyder DL, 1992):

% Fractional = DPM of Fraction x 100 Release (Total DPM – DPM collected in prior fractions)

This conversion allows comparison between samples that may contain differing amounts

of tritiated neurotransmitter. The release in each of the two fractions following KCl

stimulation was subtracted from baseline and summed for every chamber. Each animal

provided 2 experimental (60mM KCl, 1.8mM CaCl2) and 2 control (60mM KCl; 0mM

CaCl2) samples. The duplicate samples from each animal were then averaged after

correction for baseline to generate a peak %-fractional release value per animal. The

peak % fractional release was then averaged and compared between genotypes and tested

for statistical significance using the Student's t-test. Data greater than two and a half

37

standard deviations from the mean were excluded from analysis. Occasionally a given

chamber failed to show release of any kind indicated by extremely low DPM upon liquid

scintillation spectroscopy; these chambers were excluded from further analysis.

Results

3H-Glutamate release from cerebellar synaptosomes

Synaptosomes exposed to stimulation buffer containing 60mM KCl and 1.8mM

CaCl2 released 3H-glutamate in the two fractions following stimulation. Synaptosomes

exposed to 60mM KCl in the absence of CaCl2 did not release 3H-glutamate, verifying

the calcium-dependence of release in these experiments (Figure 2.2). The average peak

%-fractional release of 3H-glutamate from tottering synaptosomes was 33% reduced

compared to controls representing a significant reduction (p<0.005) in excitatory

neurotransmission (Figure 2.3).

3H-GABA release from cerebellar synaptosomes

Cerebellar synaptosomes from wild type and tottering animals released 3H-GABA

after perfusion with stimulation buffer containing 60mM KCl only in the presence of

1.8mM CaCl2. Samples exposed to 60mM KCl in the absence of CaCl2 did not

demonstrate neurotransmitter release, again indicating the calcium dependence of release

in this assay (Figure 2.4). No difference was observed in the peak % fractional release

between wild type and tottering mice (Figure 2.5). Together these data suggest a

decrease in excitatory neurotransmission in the tottering cerebellum with no change in

inhibitory neurotransmission.

38

Figure 2.2. 3H-Glutamate Relesase From Cerebellar Synaptosomes. Buffers wereperfused over cerebellar synaptosomes loaded with 3H-glutamate at a rate of 0.5ml/minand collected in 9 three-minute fractions. The horizontal line in fraction 3 indicates timeof stimulation buffer perfusion containing 60mM KCl/1.8mM CaCl2 in closed symbolsand 60mM KCl without CaCl2 in open symbols. Wild type animals (n=9) are representedby squares and tottering animals (n=9) by triangles.

0

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40

45

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wt tg

Ave

rage

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ctio

nal R

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se

**

Figure 2.3. Comparison of Tottering and Wild Type Peak 3H-Glutamate Release.Percent fractional release was summed over two fractions after correction for basal 3H-glutamate release for every animal. The peak release was then averaged for the 9 animalsin each group and compared using the Student's t-test. Data represent mean %-fractionalrelease + SEM. ** indicates significant decrease in tottering release, p<0.005.

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1 2 3 4 5 6 7 8 9Fraction

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ract

iona

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tg +K/+Cawt +K/-Catg +K/-Ca

39

Figure 2.4. 3H-GABA Relesase From Cerebellar Synaptosomes. Buffers were perfusedover cerebellar synaptosomes loaded with 3H-GABA at a rate of 0.5ml/min and collectedin 9 three-minute fractions. The horizontal line in fraction 3 indicates time of stimulationbuffer perfusion containing 60mM KCl/1.8mM CaCl2 in closed symbols and 60mM KClwithout CaCl2 in open symbols. Wild type animals (n=6) are represented by squares andtottering animals (n=6) by triangles.

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wt tg

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Figure 2.5. Comparison of Tottering and Wild Type Peak 3H-GABA Release. Percentfractional release was summed over two fractions after correction for basal 3H-GABArelease for every animal. The peak release was then averaged for the 6 animals in eachgroup and compared using the Student's t-test, p>0.05. Data represent mean %-fractionalrelease + SEM.

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40

Discussion

Multiple VDCC subtypes regulate neurotransmission in the mammalian CNS,

including P/Q-, N- and R-type channels. Co-expression of VDCC subtypes occurs within

the same neuron and within the same terminal where several VDCCs are involved in

neurotransmission. The proportions of VDCC subtypes expressed in a given terminal

vary depending on the region of the CNS and the type of neuron (Dunlap K, 1995).

Many studies of neurotransmitter release reveal marked inhibition of release after

application of a VDCC subtype-specific antagonist and further decrease in release with

the addition of a second antagonist specific to a second VDCC subclass (Takahashi T,

1993; Luebke JI, 1993). These studies support the participation of multiple independent

classes of VDCCs in triggering neurotransmitter release in the same neurons. Some

recent studies have demonstrated however, that VDCCs can also act cooperatively to

induce release rather than independently. In studies of dopamine release from rat striatal

synaptosomes, antagonists from two VDCC classes failed to reduce release when applied

separately but caused a marked reduction in release when co-administered. These data

also support the presence of multiple channel types within the same neurons and

furthermore, suggest that a single channel subtype can supply sufficient Ca2+ to support

maximal release under conditions of strong depolarization (Turner TJ, 1993). It is not yet

clear why VDCCs are coexpressed when one can support maximal secretion. Perhaps

selective pressure in environments with subclass specific VDCC toxins, differential

control of secretion under various conditions of stimulation, and subtype specific

modulation of activity all require co-expression of VDCC subtypes involved in

41

neurotransmission (Dunlap K, 1995). In the case of calcium channel mutations, this

redundancy may be the key to viability.

While multiple VDCCs involved in neurotransmitter release may be expressed

within the same neuron, differential expression and regulation of VDCCs occurs between

neurons. Within a given region of the brain, different VDCCs participate in triggering

neurotransmitter release. Patterns of subtype specific VDCC release may be further

subdivided within regions in excitatory versus inhibitory neurons. For instance, in the

rat cerebellum, parallel and climbing fibers form excitatory synapses with PCs and these

fibers rely on P/Q/N- type currents for neurotransmitter release. Alternatively, basket and

stellate cells forming inhibitory synapses on PCs rely on ‘resistent,’ or R-type VDCCs for

neurotransmitter release (Doroshenko PA, 1997). Studies in CA1 hippocampal neurons

demonstrate reduction of GABAergic inhibitory transmission by 85-90% with application

of the N-type VDCC antagonist ? -conotoxin-GVIA, with only 65-70% blockage of

excitatory glutamatergic transmission (Horne AL, 1991). These data suggest that within

this region, N-type VDCCs affect inhibitory transmission to a greater extent than

excitatory transmission.

While the aforementioned studies were performed in wild type rodents, these

concepts also apply to the tottering mouse. In fact, recent studies in tottering mice have

demonstrated compensation of P/Q-type deficient release with other VDCCs co-

expressed in the same neurons. Excitatory neurotransmission in the hippocampus occurs

at normal levels in the tottering mouse due to compensation for the deficient P/Q-type

current by the endogenous, co-expressed N-type VDCC (Qian J, 2000). These results are

not at all surprising due to the overlapping expression patterns of these channels in the

42

hippocampus and the redundancy of their functions. Earlier discovery of increased L-

type calcium channel expression in the tottering mouse cerebellum (Campbell DB, 1999)

was somewhat surprising because P/Q-type and L-type channels do not have redundant

functions.

L-type channels affect electrical excitability and other processes, however, they are

not traditionally involved in neurotransmitter release. Therefore, compensation of

deficient P/Q-type function directly by L-type channels seems unlikely. Some studies do

implicate L-type VDCCs in neurotransmitter release however. Momiyama and

Takahashi demonstrated decreased frequency of miniature inhibitory post-synaptic

currents (mIPSCs) after high-potassium stimulation in cerebellar DCN by 49% in the

presence of the L-type channel blocker, nicardipine. Frequency was also decreased by

83% in the presence of the P-type blockers, but unaffected by N-type blockade. Together

these data suggest that both L- and P-type VDCCs contribute to GABA release from

cerebellar Purkinje cells under potassium-stimulated conditions (Momiyama A, 1994).

In the studies presented here, potassium-stimulated glutamate release from tottering

cerebellar synaptosomes was decreased compared to control C57Bl/6J mice. In contrast,

GABA release was unchanged in tottering mice. Together these results suggest a defect

in excitatory but not inhibitory neurotransmission in the cerebellum of tottering mice.

These results are analogous to those obtained by Caddick et al. (1999) in the thalamus of

tottering mice and by Ayata et al. (2000) in the neocortex of leaner mice (allelic ? 1A

mutation).

As mentioned above, excitatory synapses within the cerebellum, including parallel

and climbing fiber inputs to PC, rely on P/Q- and N-type VDCCs although P-type

43

channels play a predominant role (Doroshenko PA, 1997). Therefore, the decreased

glutamatergic release seen here suggests that full compensation of the deficient P/Q-type

dependent release does not occur in these cell types. Inhibitory synapses however

probably rely on R-type channels at basket and stellate cell terminals (Doroshenko PA,

1997) and are therefore, likely to be unaffected in the tottering mouse. Inhibitory PC

input to DCN relies predominantly on P/Q-type channel function, with reports of N-, R-,

and L-type expression and release as well (Volsen SG, 1995; Momiyama A, 1994; Chung

YH, 2000; Stea A, 1994; Tanaka O, 1995). The up-regulation of L-type channels in the

tottering cerebellum (Campbell DB, 1999) and the effect of L-type antagonists on mIPSC

frequency in DCN after potassium stimulation (Momiyama A, 1994) suggests that

compensatory mechanisms may maintain PC neurotransmission in tottering mice. The

necessity of the PC in expression of tottering dystonia (Campbell DB, 1999; Chapter 3

Section 2) refutes this theory however. Therefore, a functional deficit in PC inhibitory

neurotransmission may still exist, and the whole cerebellar synaptosome preparation used

here simply diluted the PC effects with the GABA released from molecular layer

inhibitory synapses.

The mutation of the P/Q-type VDCC and the resultant decreased Ca2+ current density

(Wakamori M, 1998) suggest that P/Q-type dependent neurotransmitter release may be

altered functionally in the tottering mouse. The abundant expression and function of

P/Q-type VDCCs in the CNS suggest that profound deficits in neurotransmission are

likely to occur as a consequence of the mutation. However, the relatively mild phenotype

and results from studies of neurotransmission suggest a more moderate effect of the

tottering mutation on neurotransmitter release. In theory, such moderate effects can be

44

due to a less significant effect of the mutation on release than is expected, or may result

from compensation by other classes of VDCCs. It is possible that the phenotypes of the

tottering mouse are due largely to the moderate functional consequence of the P/Q-type

mutation in regions with little compensation by other channel subtypes. Conversely, the

lack of more widespread and severe consequences of the tottering mutation is likely due

to compensation by other VDCCs in other regions.

45

CHAPTER 3. ROLE OF PURKINJE CELLS IN THEEXPRESSION OF TOTTERING DYSTONIA

Abstract

The tottering mouse is a neurologic mouse mutant characterized by an ataxic gait,

polyspike EEG recordings, and spontaneous intermittent dystonic episodes. The

mutation responsible for the tottering mouse has been mapped to a missense mutation in

the ? 1A subunit of P/Q-type voltage dependent calcium channel (Fletcher CF, 1996).

The ? 1A subunit is highly expressed in the granule and Purkinje cells of the cerebellar

cortex. Furthermore, these cell types are rapidly and markedly activated during tottering

dystonic episodes as revealed by proto-oncogene, c-fos, expression (Campbell DB,

1998). It is hypothesized that output from the cerebellar cortex via Purkinje cells is an

absolute and necessary step in the expression of tottering dystonia. Transgenic mice that

express the SV40 T antigen specifically in Purkinje cells via the pcp-2 promoter have

been generated; the transgene causes the selective elimination of Purkinje cells post-

developmentally over many weeks (Fedderson RM, 1992). The pcp-2-SV40 transgene

was bred onto the tottering mouse strain. The double mutation appears additive with no

gross abnormalities. Prior to Purkinje cell degeneration, double mutant mice exhibit

classical tottering dystonic events; however, these same animals fail to exhibit dystonia

after Purkinje cell loss has occurred in adulthood. In this double mutant experiment,

every tottering mouse acted as its own control, eliminating genetic background as a

behavioral variable. The loss of the dystonic phenotype in double mutant mice indicates

that Purkinje cells and the cerebellar cortex participate in the pathogenesis of dystonia in

the tottering mouse.

46

Introduction

Tottering mice represent a unique opportunity to examine the pathophysiology of

dystonia, particularly paroxysmal dystonia. The genetic mutation responsible for the

tottering syndrome has been defined as a calcium channel mutation and thus places the

tottering mouse in a growing population of disorders due to ion channel mutations termed

channelopathies. Channelopathies are an extremely unique and interesting group of

disorders characterized by marked genetic heterogeneity and episodic manifestation of

phenotype in otherwise normal individuals. The tottering mouse displays a paroxysmal

dystonic phenotype strikingly similar to paroxysmal non-kinesigenic dyskinesia (PNKD)

in onset, movements, duration, frequency, triggers, and pharmacological treatment

profile. While it is likely that PNKD is caused by channelopathies, it is not necessarily

due to a calcium channel mutation per se. Mutations in different ion channels can cause

similar phenotypes and different mutations in the same ion channel can cause different

phenotypes. Furthermore, it is not suggested that dystonia as a whole is caused by

mutations in calcium channels, however, the tottering mouse may provide a model for

analysis of neuronal circuits involved in at least this form of dystonia. The utility of

dissecting the pathophysiology behind genetic animal models of dystonia is similar to the

usefulness of studying secondary dystonias. Determination of the brain regions and

neuronal circuits involved in dystonic states will identify generic or common pathways

affected and ultimately suggest appropriate steps in intervention of the disease process.

The neuronal circuitry activated during the course of tottering dystonic attacks was

assessed by Campbell et al. using immediate early gene, c-fos, mRNA expression as a

marker for neuronal activity. Their findings indicated that the cerebellum and principal

47

cerebellar relay nuclei are activated during tottering attacks and that the activation

preceded temporally a more modest activation in the cerebral cortex. Within the

cerebellum, granule cells, PC and DCN are all activated early in the course of an attack.

These data suggest a central role for the cerebellum in the initiation and/or maintenance

of tottering paroxysmal dystonia (Campbell DB, 1998). It is not at all surprising that the

cerebellum is involved in tottering mouse phenotypes due to the abundant expression of

the mutated P/Q-type channel in both cerebellar granule and Purkinje neurons. Together,

the abundant cerebellar expression of P/Q-type channels and the marked activation of the

cerebellum during tottering attacks led Campbell et al. to further investigate the role of

the cerebellar PC in the generation and/or maintenance of tottering dystonia.

To this end, the mutation pcd was used to lesion cerebellar PC. Purkinje cell

degeneration (pcd) is a mouse mutant characterized by the rapid death of PC at postnatal

days 15-29. Genetic lesioning of the cerebellar PC of tottering mice through the pcd

mutation eliminated the tottering dystonic attacks. These data suggest that cerebellar

output is necessary for the induction of tottering dystonic attacks.

Since the time these experiments were undertaken a slightly different approach to

address the same question was developed. Possible confounds of the above experiments

include phenotypic effects of the pcd mutation on the CNS of the double mutants. Until

the gene responsible for the pcd mutant is discovered, it is unlikely that confounding

effects of the mutation on tottering dystonic attacks can be ruled out completely. Further,

the genetics of this experiment prohibited the generation of completely appropriate

control mice. Therefore, a new approach to evaluate the role of PC in tottering dystonia

was devised using a more defined system and internal controls. Here, tottering mice were

48

bred with transgenic mice expressing the SV40 large T antigen (described in detail

below) to produce tottering mice that lose their PC slowly over time after cerebellar

development is complete. The advantage to this model system is that each mouse serves

as its own control; at early time points, tottering dystonia should be unaffected and over

time the effect of graded PC loss can be evaluated. Furthermore, the transgene results in

death of PC only, abrogating any confounding effects of the second 'mutant’. Through

the use of this model, the role of the PC as a necessary and essential component in the

expression of tottering dystonic attacks is evaluated.

Materials and Methods

Transgenic and mutant mice

Mice from the SV4 transgenic mouse line were obtained as a generous gift from Dr.

R.M. Fedderson.

SV4 mice were generated by driving expression of the SV40 T antigen in PCs using

the pcp-2 promoter. The pcp-2 protein is expressed exclusively in PCs and the simian

virus 40 T antigen is known to disrupt cell cycle regulation in post-mitotic cells.

Although cerebellar development proceeds normally in this line, PCs gradually die over

the course of several months beginning at ~P23. Very few PCs remain by 5 months of

age. This loss of PCs occurs relatively slowly over time and is generally reproducible

between mice. Only occasional PCs remain by P70; at all times, T antigen

immunoreactivity is restricted to PCs (Fedderson RM, 1992).

Transgenic mice received from Dr. Fedderson were on the FVB strain, derived from

the original SV4 line (Fedderson RM, 1992). Because tottering is on the C57Bl/6J

background, FVB transgenic mice were bred onto the C57Bl/6J background for 5

49

generations prior to breeding with tottering animals. F5 generation transgenic females

(SV4 +/-; +/+) were bred with tottering males (SV4 -/-; tg/tg) to produce F6 generation

transgenic animals heterozygous for the tottering mutation (SV4 +/-; +/tg). F6 female

transgenic animals were bred with tottering males again to produce F7 transgenic

tottering animals (SV4 +/-; tg/tg) and control genotypes (SV4 +/-; +/tg, SV4 -/-; tg/tg,

SV4 -/-; +/tg). While back-crossing the transgenic line onto C57Bl/6J for 5 generations,

a smaller group of animals was bred directly with tottering animals. In this group of

mice, the experimental animals were the F3 generation of transgenic animals onto

C57Bl/6J. These two genetically distinct groups will be referred to as F7 and F3,

respectively. All animals were housed on a 12-hour light/dark cycle in the vivarium at

Penn State University College of Medicine and had access to food and water ad libitum.

Genotyping

Presence of the SV40 transgene was determined by PCR analysis. Ear punch DNA

was prepared by adding 300? l of 0.05 M NaOH to the punch and heating at 95oC for 10

minutes. The punch was then vortexed, neutralized with 50? l of 1 M Tris pH8.0 and

vortexed again. Punch debris was spun down at 14,000rpm for 6 minutes and the

supernatant removed. One microliter of the unpurified supernatant was added to 10? l

PCR reactions containing 400nM each dNTP, 10?M each forward 5' (5'-

AGTACTGTCCCCCAAGAGATAGTAG-3') and reverse 3' (5'-CCATTCATCAGTTC

CATAGGTTGG-3') primers, 1X Vent (exo-) DNA polymerase buffer (10mM KCL,

10mM (NH4)2SO4, 20mM Tris-HCl pH8.8, 2mM MgSO4, 0.1% Triton X-100), and 1 to

1.5 Units Vent (exo-) DNA polymerase (New England Biolabs). The reactions were

denatured at 95oC for 5 min prior to undergoing 30 cycles consisting of 1 min at 95oC, 2

50

minutes at an annealing temperature of 57oC, and 2 min of extension at 72oC. A 7 min

extension at 72oC was added after the cycles completed. Reaction products were

separated on a 1.2% agarose gel by electrophoresis and viewed after ethidium bromide

staining under UV light. The amplified transgene segment migrates at 1076 bp size.

Dystonia assessment

Animals were brought to the laboratory every three weeks for dystonia assessment

two hours prior to testing. To induce dystonia, animals were injected subcutaneously

with 25mg/kg caffeine at 4 weeks of age and 15mg/kg caffeine at all other timepoints

prepared in 0.9% saline. Animals were assessed every three weeks and sacrificed either

after the initial 4 week assessment or at the age when signs of dystonia were lost. Four-

week-old animals appear slightly more resistant to caffeine induction of dystonia than

older mice and it was imperative that they be correctly phenotyped so the higher dose

was used initially. Animals were observed for one hour and scored every 10 minutes on

a general dystonia scale and according to body regions involved. The generalized

dystonia rating scale used is as follows (modified from Jinnah HA, 2000):

Score Behavioral PhenotypeD0 no motor abnormalitiesD1 slightly slowed or abnormal motor behaviorD2 rare, isolated dystonic postures seenD3 mild motor impairment due to dystonic posturesD4 moderate impairment due to dystonic posturesD5 severe immobility due to prolonged dystonic postures

Dystonic severity was also evaluated by noting the presence or absence of hindlimb,

forelimb, trunk, neck, and head involvement. Limb involvement was categorized as

isolated, tonic, or paddling. Trunk dystonia was described as flattened, twisted, hunched,

or rearing. Cranial dystonia was separated into neck flexion or extension and by ear, eye,

or jaw involvement.

51

Histologic analysis

After final behavioral testing, mice were sacrificed by carbon dioxide asphyxiation.

The brains were rapidly removed and frozen in isopentane chilled to -40oC and stored at -

70oC until processing. Brains were cut into 20?m coronal sections using a cryostat and

thaw-mounted onto Superfrost plus slides (Fisher, Pittsburgh, PA).

Purkinje cell analysis of F3 generation SV4+/-;tg/tg mice was completed using in situ

hybridization for calbindin mRNA. Calbindin is abundantly and specifically expressed in

Purkinje cells within the cerebellum. Age-matched +/+;tg/tg mice were used as controls

to determine basal Purkinje cell calbindin mRNA levels. The in situ hybridization

protocol is described in detail in the methods section of Chapter 4. cDNA template for

calbindin was obtained as a generous gift from Dr. T.L. Wood and subcloned into

pBluescript SK+. Plasmid was linearized and used to generate antisense single-stranded

radiolabeled RNA probes of 1.2kb in length.

Stereologic cell counting of PC in all folia of the cerebellum in the F7 generation

progeny will be completed in the future. This technique should allow for precise

correlation between numbers and regions of Purkinje cells necessary for the dystonic

attacks of tottering mice to occur.

Results

Generation of tottering mice carrying the SV40 transgene

All progeny were genotyped for presence of the SV40 T antigen transgene using ear

punch DNA (Figure 3.1). Tottering mice were phenotyped by their characteristic ataxic

gait at the time of weaning and by presence of dystonic attacks. Tottering mice carrying

the SV40 transgene and all control genotypes were generated in ratios expected for

52

autosomal independent segregation of the alleles in both the F3 and F7 groups of mutant

mice (Table 3.1). While tottering mice bearing the transgene were the smallest of the

genotypes, their weights did not differ dramatically from control genotypes (Figure 3.2)

and the animals appeared healthy and behaved normally in general.

53

Figure 3.1. PCR genotyping for presence of the SV40 transgene. Ear punch DNA wasused as template to amplify the 1076bp fragment of the transgene using primers specificfor the pcp-2 promotor and the SV40 T antigen gene. Lanes 1,2,5,6,7,and 9 contain theamplified transgene fragment; L denotes 1 kB ladder.

AGenotype Male Female TotalSV4+/-;tg/tg 2 4 6 (31.6)SV4-/-;tg/tg 3 3 6 (31.6)SV4+/-;+/tg 3 2 5 (26.3)SV4-/-;+/tg 1 1 2 (10.5)Total 9 10 19

BGenotype Male Female TotalSV4+/-;tg/tg 5 5 10 (24.4)SV4-/-;tg/tg 7 8 15 (36.6)SV4+/-;+/tg 3 8 11 (26.8)SV4-/-;+/tg 2 3 5 (12.2)Total 17 24 41

Table 3.1. Genotypes of progeny generated in SV4+/-;+/tg X +/+;tg/tg crosses.Summary of the genotypes and sexes for F3 (A) and F7 (B) generations determined byPCR of the SV40 transgene and phenotyping of the tottering homozygous state. Percentof total mice for each genotype is indicated in parentheses.

L 1 2 3 4 5 6 7 8 9

1 kB

54

Figure 3.2. Body weights of F7 generation progeny. Weights were averaged for all micein each genotype. Transgenic tottering mice (SV4/tg) were healthy and viable with bodyweights similar to control genotypes: wild type (wt), transgenic (SV4), and tottering (tg).Single-factor ANOVA analysis revealed no significant difference in weight betweengenotypes (p>0.05).

Loss of dystonic phenotype in transgenic tottering mice

In the F3 group of animals, all tottering mice carrying the SV40 T antigen transgene

displayed characteristic tottering dystonic attacks indistinguishable from control tottering

mice at the initial 4 week time point. At the 10 week timepoint, transgenic tottering

animals displayed an extremely mild version of dystonia, marked by involvement of the

limbs only while the head, neck and trunk were largely spared. As the weeks progressed,

individual transgenic tottering animals displayed absolutely no dystonia after caffeine

challenge (Figure 3.3A). In contrast, tottering mice lacking the transgene continued to

respond to caffeine with a dystonic attack. By 18-19 weeks, transgenic tottering animals

0

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4 7 10 13 16 19

Age (weeks)

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55

failed to display any dystonic phenotype. Female animals lost the dystonic phenotype an

average of one month earlier than male animals.

Before loss of dystonic phenotype was confirmed, animals were challenged with

caffeine a second time and by restraint as well. Similar results were obtained in the F7

group of animals (Figure 3.3B). This generation of animals showed somewhat more

delayed loss of the dystonic phenotype however. Head, neck and trunk dystonia were

again lost prior to dystonic limb involvement (Figure 3.4) and female transgenic tottering

animals lost the dystonic phenotype prior to males. Transgenic control animals that were

tottering heterozygotes never displayed any dystonia and control tottering animals

negative for the transgene displayed characteristic dystonic attacks throughout the entire

experiment. In both generations, transgenic tottering mice that had lost the dystonic

phenotype were still distinguishable from their littermates due to a unique ataxic gait,

which was different from that of a transgenic or tottering mouse alone.

56

Figure 3.3. Loss of dystonic phenotype in transgenic tottering mice over time. In the F3generation (A), all transgenic tottering animals (blue circles) were non-dystonic by 19weeks of age. Similar results were obtained in the F7 generation (B), with a greaterdiscordance between age of phenotype loss between males (blue circles) and females (redsquares) however. Control tottering mice (pink diamonds) maintained a high level ofdystonia in both experiments (A and B). Linear regression lines are shown for eachgrouping.

B

A

0

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3 6 9 12 15 18 21 24

Age (weeks)

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57

Figure 3.4. Regional loss of dystonic phenotype in F7 transgenic tottering mice.Presence of dystonia was tallied (+ or -) over the one hour observational period (6 totaltime bins) and summed by region. Data are presented as number of bins positive fordystonia by age (weeks) for each region. Transgenic tottering mice (blue bars) lost thecharacteristic dystonic phenotype over time while control tottering littermates (pink bars)sustained dystonic involvement of all regions. Phenotypic loss of head involvement (A)occurred first, followed by neck (B) and trunk (C) involvement, and lastly by limb (D)involvement. Error bars represent SEM. Number of dystonic bins were comparedbetween genotypes using the Student's t-test at each age. Transgenic tottering mice weresacrificed after disappearance of all dystonia and these animals were given a minimalscore for subsequent timepoints. * indicates p<0.05, ** p<0.005, and *** p<0.0005.

Demonstration of Purkinje cell loss in transgenic tottering animals

Verification of PC ablation after the loss of the dystonic phenotype in all SV4+/-;tg/tg

animals of the F3 generation was performed using in situ hybridization to calbindin

mRNA. Mice were sacrificed shortly after cessation of any dystonic signs or allowed to

0

1

2

3

4

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4 7 10 13 16 19 22

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Head Neck

Trunk Limbs

Age (weeks)

Dys

toni

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ins

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***

***

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*** ******

58

a

b

c

d

e

f

g

h

age for a few more weeks to obtain a range of ages to compare degree of PC loss over

time. One mouse, the 15 week old female (figure 3.5f) still demonstrated some rare

isolated hindlimb dystonia with the rear limb occasionally being held up against the trunk

when sacrificed. Animals were between 15 and 29 weeks of age when sacrificed.

Results from calbindin mRNA in situ hybridization demonstrate profound loss of

cerebellar Purkinje cells in all transgenic tottering mice compared to age-matched control

tottering mice (Figure 3.5). Degree of PC loss increases substantially with age and with

female sex.

Figure 3.5. Calbindin mRNA in situ hybridization. Cerebella from F3 transgenictottering animals and two age-matched control tottering animals were hybridized withcalbindin antisense mRNA. Transgenic tottering animals (a-f) show marked loss of PC incomparison to control tottering animals (g-h). Within the transgenic group, increasingage and female sex are correlated with greater loss of PC: a, 23 week old female, b, 26week old male, c, 29 week old female, d, 18 week old female, e, 24 week old male, f, 15week old female. Tottering controls were 19 weeks old.

Discussion

The loss of the characteristic dystonic phenotype in tottering animals after widespread

PC loss demonstrates the necessary role of the cerebellar signaling in the expression of

59

tottering dystonia. Transgenic tottering mice in these experiments served as their own

controls, as initial development and expression of dystonia proceeded normally. Loss of

dystonia corresponded to the death of PC, as confirmed by calbindin in situ hybridization.

These data support those obtained by Campbell et al., in which the tottering dystonic

attacks were eliminated by the pcd mutation (Campbell DB, 1999). In the latter

experiments however, initial normal development of the attacks could not be

demonstrated due to rapid loss of PC (P15-P29) and unknown widespread effects of the

pcd mutation could not be excluded. Lesion of cerebellar PC specifically with the SV40

T antigen transgene circumvented these issues due to its inherent specificity and slow

progression of cell death.

Generation of healthy transgenic tottering mice proceeded without difficulty. These

animals displayed characteristic tottering dystonic attacks, indicating no gross phenotypic

alterations of the dystonia after cross-breeding. Tottering animals lacking PC were

relatively healthy and viable, suggesting that no genetic interference occurred between

the tottering gene and the SV40 T antigen transgene. In addition to the loss of the

dystonic phenotype however, transgenic tottering mice displayed a unique gait around the

time of dystonia loss that appeared more wobbly and uncoordinated than either transgenic

or tottering gaits alone.

In the initial F3 generation experiment, all transgenic tottering animals displayed

stereotypical generalized dystonia initially. By 8-10 weeks of age, the dystonia was

restricted to the limbs only. As the weeks progressed, the severity continued to lessen

until absolutely no dystonia was observed. Calbindin mRNA in situ hybridization

confirmed the loss of nearly all PC in transgenic tottering mice no longer expressing the

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dystonic phenotype. Behavioral data from the F7 generation animals was similar to that

from the F3 generation, indicating no gross effect of the heterogenous genetic

background in the original experiments. Characteristic tottering attacks were observed by

four weeks of age in the F7 generation and again at seven weeks of age. A greater time

range for the restriction of the dystonia to the limbs (10-16weeks) and for complete loss

of the phenotype (13-25weeks) was seen in the F7 generation however. This increased

time range is in direct contrast to what would be expected from the increased genetic

homogeneity in the F7 generation animals in comparison to those in the F3 generation.

Therefore, the time of phenotypic onset is more likely due to factors inherent to repetitive

breeding of the transgene as opposed to the genetic background of the mice per se.

Phenotypic differences were noted between male and female mice, with loss of the

dystonic phenotype occurring in female animals by age 14 weeks on average (range 11-

16) and in males by 19 weeks in the F3 generation. Females in the F7 generation also

exhibited a graded loss of the dystonic phenotype on average of 6 weeks prior to males.

Female transgenic mice appear to develop the characteristic ataxia before males,

indicating earlier PC loss in female transgenic animals in general. Although not

quantitative, the calbindin in situ hybridization data support this finding. The reasons for

this sex discrepancy are unknown with no sex differences described in the original report

(Fedderson RM, 1992).

PC loss in transgenic animals is reported to occur gradually during the timeframe of

these experiments (Fedderson RM, 1992). Therefore, the graded loss of the dystonic

phenotype in transgenic tottering animals is likely due to decreasing cerebellar cortical

output through remaining PC. The early restriction of the dystonia to include limbs-only

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rather than simple overall reduction in severity is interesting and suggests regional

control of dystonia through subpopulations of PC. Presumably, the cells lost early were

responsible for more proximal dystonia of the trunk, head, and neck while those PC more

resistant to death caused limb-only dystonia for a number of weeks longer. Future

analysis may indeed demonstrate a somatotopic map of PC loss corresponding to the

regions of the body no longer involved in the dystonic phenotype. Tissue from all F7

transgenic tottering and control genotypes has been collected and will be processed for

stereologic PC counting at timepoints throughout the graded range of dystonia to test this

theory. Initial PC counts in transgenic tottering animals exhibiting characteristic full

body dystonia are expected to be similar to control genotypes. The restricted focus of the

dystonia is expected to occur only after loss of consistent sets of PC corresponding to the

rough cerebellar somatotopic regions for the trunk and head. Further loss of PC in areas

associated with limb motor control are expected to be lost in transgenic tottering animals

exhibiting no dystonia. Thus, overall severity and regional expression of tottering

dystonia will likely correspond to somatotopically organized PC activity.

While the correspondence between regions of PC loss and body parts involved in

transgenic tottering dystonia remain to be established, the data presented here indicate a

role for cerebellar cortical output in the expression of tottering dystonia. Although the

cerebellum has traditionally been viewed as a secondary processor in motor coordination,

recent evidence has implicated more significant and widespread functions of this region.

Roles in learning, memory, and other cognitive functions have been described for the

cerebellum. In the case of dystonia, aberrant DCN activity has been implicated in

producing the dystonic phenotype of the genetically dystonic rat (LeDoux, 1993) and

62

hypermetabolism in the cerebella of human patients with dystonia syndromes is

frequently noted (Eidelberg D, 1998; Odergren T, 1998; Preibisch C, 2001; Hutchinson

M, 2000). In the tottering mouse, specific activation of the cerebellum and cerebellar

relay nuclei after induction of dystonia implicates this region in the production of

dystonic attacks (Campbell DB, 1998). These data and those presented here suggest an

integral role for the cerebellum in the pathophysiology of dystonia. Evidence supporting

an active role for the cerebellum in a process such as dystonia is intriguing as it upsets

traditional views of cerebellar function. However, traditional studies of the cerebellum

investigated disturbances due to decreased normal function and activity as opposed to the

potential effects of abnormal signals and increased activity within the cerebellum, as

implicated in the aforementioned dystonias.

63

Chapter 4. ROLE OF THE CEREBELLUM IN A NOVELANIMAL MODEL OF DYSTONIA IN WILD TYPE MICE

Abstract

Dystonia is a neurological syndrome characterized by twisting movements or

sustained abnormal postures. Recent evidence suggests that abnormal cerebellar

signaling contributes to the expression of dystonia. To study the role of the cerebellum in

dystonia, we have developed a novel mouse model. Microinjection of low-doses of

kainic acid into the cerebellar vermis of mice elicited reliable and reproducible dystonia

characterized by hindlimb abduction and extension and a severely flattened trunk. The

severity of the dystonia increased linearly with kainate dose from 0 to 235 picomoles.

Co-injection of the glutamatergic antagonist NBQX with kainic acid dramatically

decreased dystonia verifying that AMPA and/or kainate receptors participate in the

expression of dystonia in this model. The abnormal movements were not associated with

kainate-induced seizures, as EEG recordings showed no epileptiform activity during the

dystonic events. Further, neuronal activation, as assessed by in situ hybridization for c-

fos, revealed c-fos mRNA expression in the cerebellum, locus ceruleus and red nucleus.

In contrast, regions associated with seizures such as the hippocampus did not exhibit

increased c-fos expression after cerebellar kainate injection. Transgenic mice lacking

Purkinje cells were also microinjected with kainate. Transgenic mice show dramatically

decreased dystonia after kainic acid injection suggesting an important role for Purkinje

cells and the cerebellar cortex in this model of dystonia. Together these data suggest that

the cerebellum plays a role in the pathophysiology underlying dystonia.

64

Introduction

Dystonia is a relatively common neurological disorder characterized by co-

contraction of antagonistic muscles and spill over to adjacent muscle groups on EMG

recordings. These effects result in sustained abnormal muscle contractions resulting in

twisting and odd postures (Fahn S, 1987). The pathophysiology of dystonia is poorly

understood, but biochemical changes within the basal ganglia are thought to play an

important role. The hyperkinetic movements of dystonia are theorized to result from

decreased pallidal inhibition of the thalamus, which then in turn would send excessive

excitatory input to the premotor and motor cortices (Berardelli A, 1998; Vitek JL, 2000).

The basal ganglia is frequently the site of pathology in secondary dystonias, but no

consistent changes have been defined in cases of primary dystonia.

Alternative theories suggest however, that impaired inhibition of muscles antagonistic

to those activated occurs at the level of the cerebral cortex or that the cerebrocerebellar

circuit can also lead to excessive excitation of the motor cortex (Halett M, 1998; Richter

A, 1998). Over the last few decades, studies have implicated numerous structures in the

pathophysiology of dystonia including the basal ganglia, thalamus, sensorimotor cortex,

and cerebellum. Genetic rodent models of dystonia implicate the cerebellum in the

production of dystonia. The dystonic rat (LeDoux MS, 1993) and hamster (Richter A,

1998), the dystonia musculorum mouse (Sotelo C, 1988) and the wriggle mouse sagami

(Ikeda M, 1989) all display cerebellar defects to some extent (for review, see Richter A,

1998). Another mouse mutant, tottering, has recently been suggested as a model for

paroxysmal dystonia and supports a role for the cerebellum in the generation of dystonia

as well (Campbell DB, 1998; Campbell DB, 1999). While genetic animal models are

65

useful tools in defining the cellular defects and pathways involved in disease, they also

carry inherent limitations. It is difficult to prove that yet undetermined phenotypes of the

mutation are not involved in a particular phenomenon. Thus, genetic animal models are

most useful in suggesting regions involved in the pathophysiology of a disease that can

later be studied in genetically wild type animals.

Marked activation of the cerebellum occurs in tottering mice upon initiation of

dystonic attacks (Campbell DB, 1998) and increased DCN output is likely responsible for

the generalized dystonia in the dystonic rat (LeDoux MS, 1995). Furthermore, numerous

functional imaging studies in human dystonia patients demonstrate increased metabolic

activity within the cerebellum (Eidelberg D, 1998; Odergren T, 1998; Preibisch C, 2001;

Hutchinson M, 2000). To test the theory that overactivity of the cerebellum is sufficient

to produce a profound dystonic phenotype, local excitation of the cerebellum was

achieved in wild type mice through cerebellar microinjection of kainic acid.

Materials and Methods

Mice

Originally obtained from Jackson Laboratories, wild type C57Bl/6J mice were bred at

the Pennsylvania State University College of Medicine vivarium. Male and female mice

used in the experiments weighed 21-26 grams and were between 2 and 4 months of age.

SV4 transgenic mice were originally obtained as a generous gift from Dr. R.M.

Fedderson on an FVB background. Mice used in these experiments were back-crossed

onto a C57Bl/6J background for five generations before beginning experiments providing

an estimated 96.9% genetic identity between animals. Genotyping for the presence of the

SV40 T-antigen transgene proceeded as described above (Chapter 3) and originally in

66

Fedderson et al., 1992. Male and female transgenic mice used in these experiments were

over 6 months of age to ensure maximal Purkinje cell death prior to experimentation. All

animals were housed on a 12-hour light/dark cycle with access to food and water ad

libitum.

Injections

Kainic acid H2O was obtained from Tocris (Ballwin, MO) and suspended in 0.9%

saline. To visualize injection sites, 1:10 v/v of Trypan Blue (0.4%, Sigma, St. Louis,

MO) was also added to the kainic acid solution. NBQX (Sigma, St. Louis, MO) and

domoic acid (Sigma, St. Louis, MO) were also suspended in 0.9% saline. For the dose-

response and transgenic experiments, animals were anesthetized with methoxyflurane

(Metofane; Mallinckrodt Veterinary Inc., Mundelein, IL) inhalational anesthetic in a

small glass chamber. A midline incision was made over the skull and a small hole was

drilled with a 21-gauge needle over the anterior cerebellum in the midline. A hamilton

syringe with a needle cut to 2mm was placed in the hole and 0.5 ? l kainic acid solution

was delivered over 5 seconds (AP -6.5 mm bregma, Lat 0 mm, Vert -2 mm from skull).

The wound was re-approximated and sealed with Nexaband S/C topical skin closure

(Veterinary Products Laboratories, Phoenix, AZ). All other injections were done under

isofluorane (IsoSol; Vedco, Inc., St. Joseph, MO) anesthesia due to discontinued

production of methoxyfluorane. Anesthetization was induced under 3% isofluorane and

maintained at 1% during injection. Lateral cerebellar injections were made 1-2 mm right

or left of midline with a 2mm needle length (AP -6.5 mm bregma, Lat 1-2 mm, Vert -2

mm from skull). Lateral ventricle(AP –0.5 mm bregma, Lat 1.25 mm, Vert -3 mm from

skull) and striatal injections (AP +1 mm bregma, Lat 2 mm, Vert -3 mm from skull)

67

were also completed. Prior to changing anesthetic, behavioral outcomes were compared

under each anesthetic and found to be identical for wild type mice receiving kainate

injections (data not shown). Because the effects of kainic acid are immediate in this

paradigm, rapid recovery from anesthesia was necessary. Therefore, injections were

made freehand rather than with the use of a stereotaxic apparatus to limit the amount of

time mice remained under and recovered from the effects of anesthesia. Using this

technique, injection site locations were consistent and reproducible (Figure 4.5).

Behavioral Observation

After wound closure, animals were immediately placed in an empty cage and scored

(see below) for dystonia every 10 minutes after 1 minute of observation. If no

spontaneous dystonia was seen, mice were disturbed by touch. If no escape or dystonia

resulted, mice were lifted by the tail and placed down again to encourage movement,

which most often preceded the dystonia. The behavioral rating scale illustrated below

was used to score the motor behavior of injected mice. Note that scores of D2-D5 only

are within the range of observed dystonia.

Score Behavioral PhenotypeD0 no motor abnormalitiesD1 no impairment; slightly slowed or abnormal (but not

dystonic) motor behaviorD2 mild impairment; sometimes limited ambulation

unless disturbed, dystonic postures when disturbedD3 moderate impairment; frequent spontaneous dystonic

posturesD4 severe impairment; sustained dystonic postures and

limited ambulationD5 prolonged immobility in dystonic posturesD6 erratic running, "popping," and seizures

modified from Jinnah HA, 2000

68

In Situ Hybridization

After the two-hour observation period, mice were sacrificed by carbon dioxide

asphyxiation and the brain removed and frozen in isopentane chilled to –40oC. Frozen

tissue for in situ hybridization was prepared by slicing 20?M coronal sections from fresh

frozen brains stored at -70oC using a cryostat and thaw mounted on Superfrost Plus glass

slides (Fisher, Pittsburgh, PA). After drying, the slide-mounted sections were stored at -

70oC. cDNA template for murine c-fos was obtained as a generous gift from Dr. M.E.

Greenberg and subcloned into pBluescript II SK+. Plasmid was linearized to generate

either sense or antisense single-stranded radiolabled RNA probes of 2.2kb in size after in

vitro transcription.

Transcription reactions were incubated for 1-1/2 hr at 37oC in a 25 µl volume

containing 40 mM Tris, pH 7.9, 6 mM MgCl2, 2 mM dithiothreitol (DTT), 40 U RNase

inhibitor (Promega, Madison, WI), 400 µM each ATP, GTP and UTP, 10 µM [35S]CTP

(800 Ci/mmol), 1 µg linearized c-fos template plasmid (sense or antisense) and 20 U T3

or T7 RNA polymerase (Promega, Madison, WI). After transcription was completed,

DNA template was removed by RNase-free DNase (Promega, Madison, WI) digestion

for 30 min at 37oC and riboprobes were reduced to 100-200 bp in size with 0.2 M NaOH

for 45 minutes on ice. Probes were extracted with phenol:chloroform:isoamyl alcohol

(25:24:1) and separated on a G50 Sephadex Nick column (Pharmacia, Piscataway, NJ) to

remove unincorporated nucleotides.

Pretreatment of slide-mounted tissue consisted of fixation in buffered 4%

formaldehyde for 5 min at room temperature followed by a 5 min rinse in 0.1 M

phosphate buffered saline (PBS). Slides were treated with 0.25% acetic anhydride in 0.1

69

M triethanolamine-HCl/0.15 M NaCl (pH 8.0) for 10 min and rinsed in 2X standard

sodium citrate (1X SSC; 0.15 M NaCl, 0.015 M sodium citrate). Sections were

dehydrated in graded ethanols for 1 min each followed by 5 min incubation in

chloroform. After final one min incubations in 100% and 95% ethanol slides were air

dried. Slides were hybridized with 100 µl of hybridization buffer containing 7.5 ng

cRNA probe in 50% formamide, 0.75 M NaCl, 20 mM 1,4-piperazine diethane sulfonic

acid, pH 6.8, 10 mM EDTA, 10% dextran sulfate, 5X Denhardt’s solution (0.02% bovine

serum albumin, 0.02% ficoll, 0.02% polyvinylpyrolidone), 50 mM DTT, 0.2% sodium

dodecyl sulfate and 100 µg/ml each salmon sperm DNA and yeast tRNA. Slides were

coverslipped and hybridized for 16 hr at 56oC in a closed humid chamber.

Following hybridization, coverslips were removed in 4X SSC with 300 mM 2-

mercaptoethanol at room temperature. Slides were incubated in this solution for 15 min

followed by 15 min in 4X SSC alone. The slides were treated with 50 µg/ml pancreatic

RNase A in 0.5 M NaCl, 50 mM Tris, pH 8.0, 5 mM EDTA for 30 min at 37oC, washed

in graded salt solutions (2X, 1X, and 0.5X SSC each for 5 min at 56oC), and in 0.1X SSC

at 65oC for 30 min. Slides were dipped in 60% ethanol with 0.33 M ammonium acetate

and air dried.

Sections were exposed to x-ray film (DuPont Cronex) and analyzed using MCID

M5+ optical software package. Regions analyzed in detail included the cerebellum, red

nucleus, locus ceruleus, striatum, hippocampus, and motor cortex. After correction for

film background, density of c-fos mRNA hybridization signal was quantified for multiple

sections per animal and averaged. The mean density in a given area per animal was then

averaged for animals receiving the same dose of kainic acid and compared.

70

Injection Site Localization

Tissue was collected for needle localization in the same manner as described for in

situ hybridization. Tissue slices analyzed by in situ hybridization were also studied for

needle tract location with tract markings and peak c-fos intensity as indicators of

injection site. Tissue not processed for in situ hybridization underwent standard

hematoxylin and eosin staining to verify the injection site.

EEG Recordings

Under isofluorane anesthesia, two small machine screws (1/32" diameter by 1/16"

length; Small Parts Inc.) were placed through the skull of C57Bl/6J mice and wrapped

once with 30 gauge silver-plated copper wire attached to a plug or pin connector.

Colloidal silver paint was applied over the screw and wire to ensure good electrical

conductivity and the apparatus was cemented to the cleaned skull surface using loctite

glue and accelerator (Loctite, Rocky Hill, CT). After a minimum of 24 hours post-

surgery, electrode pins were connected to an electroencephalagraph (EEG) recording

apparatus and baseline EEG were recorded. The animal was again anesthetized and 0.5? l

kainic acid (235 pmoles) was injected into the midline anterior cerebellum at a depth of

2mm. The mice were immediately re-connected and EEG recordings were collected for

10 seconds at 5 minute intervals for 90 minutes while behavioral data were collected. In

addition, EEGs were recorded when particularly gross dystonic postures were observed.

After the recording period, mice were injected with 60mg/kg pentalinetetrazol i.p. to

induce generalized seizure activity.

71

Results

Behavioral response to cerebellar kainate microinjection

Wild type mice reliably and reproducibly displayed a dystonic phenotype in response

to low dose kainic acid injection into the cerebellum. Mice typically displayed the first

sign of dystonia 10-20 minutes after injection, with a hindlimb being held up tonically

against the trunk as the mouse was exploring. Within a few minutes, the entire trunk and

all four limbs were involved with the mouse flattened against the cage bottom with an

arched back and the perineum pressed down. The hindlimbs were abducted at the hip and

knee and held out above the base of the tail, often paddling in the air. The forelimbs were

typically held tightly against the trunk or exhibited paddling. The neck often flexed or

extended and the ears were held back against the fur and the eyes closed (Figure 4.1).

This was the most common dystonic posture seen and varied in severity and duration.

Mild and moderately affected mice ambulated or rested normally in between dystonic

attacks that lasted 2 to 15 seconds. Severely dystonic animals remained immobilized in a

tensely held dystonic posture for 2 to 20 minutes at a time. Mildly affected animals

showed dystonia only after being disturbed. Other mice were spontaneously dystonic and

this is reflected in the scoring system. In general, dystonic postures occurred during

initiation of movement. After being disturbed and attempting to escape, or upon

volitional initiation of movement, dystonia was consistently preceded by a change in

movement. A sudden ambient noise that startled the mice would also frequently incite a

dystonic attack. Severely affected mice immobilized in prolonged postures exhibited

exacerbations defined by further tensing of the muscles in the posture; initiation or

change in movement could not be appreciated in dystonia at this stage of severity.

72

Figure 4.1. Typical dystonic postures after cerebellar kainate injection in wild type mice.This mouse, injected in the cerebellum with 235 picomoles of kainic acid demonstrates aflattened, arched trunk with hindlimbs abducted and held out from the body. The photoon the left also shows some mild flexion of the neck while the photo on the rightillustrates outstretched forelimbs and flattened ears.

Severity of dystonia increases linearly with kainate dosage

The association of the dystonic phenotype with kainate injection was investigated

using a dose-response paradigm. Kainic acid concentrations used were 0-, 10-, 25-, 50-,

75-, 100-, and 150-? g/ml. In the 0.5 ? l delivered, this corresponds to 0-, 25-, 60-, 115-,

175-, 235-, and 350 picomoles kainic acid respectively. Within this dosage range, a full

spectrum of behavior was appreciated. Beginning with no motor abnormalities, the

behaviors continued through a range of dystonic severity and ended with seizure activity

at the highest dose. The severity of dystonia increased linearly with dose, providing a

dose-response correlation coefficient of 0.98 (Figure 4.2A). The behavior returned to

normal or near-normal in all dystonic animals within 2 hours of injection (Figure 4.2B).

Mice given a dose of 350 pmoles kainic acid developed wild and erratic running

behaviors, became seizurigenic and were sacrificed before the observational period

ended. These mice were given a maximal score of D6 for the remaining time in the data

presented in Figure 4.2.

73

Figure 4.2. Dose-response and dose-recovery curves after cerebellar injection of kainicacid in wild type mice. Figure A shows the increased severity of dystonia elicited byincreasing dosage of kainic acid. The average D-score was taken at 30 minutes afterinjection from 5-6 mice per dosage. Error bars represent SEM and R2 value of 0.98 isshown. Figure B illustrates the recovery of the mice to non-dystonic behavior within 2hours of injection (5-6 mice per dosage).

0

1

2

3

4

5

6

0 10 20 30 40 50 60 70 80 90 100 110 120minutes

D s

core

0pmol25pmol60pmol115pmol175pmol235pmol350pmol

R2 = 0.980

1

2

3

4

5

6

0 50 100 150 200 250 300 350

Dose KA (pmoles)

Ave

rage

D s

core

A

B

74

in situ hybridization of c-fos expression after cerebellar kainate injection

In situ hybridization of the immediate early gene, c-fos, was performed to determine

the neuronal pathways activated during kainate-induced dystonia. At the end of the two-

hour behavioral observation period, animals were sacrificed and tissue was collected for

in situ hybridization. Regions of c-fos hybridization analyzed were based on their

proposed relation to dystonia or kainate-induced behaviors. Representative sections from

c-fos in situ hybridization are shown for several regions and over a wide dose range in

Figure 4.3. Intense c-fos induction was seen in the cerebellum of animals receiving high

doses of kainic acid. The lower doses produced modest changes as would be expected

from the mechanical stress of injection. c-fos mRNA hybridization occurred in all

regions of the cerebellum with even more intense induction seen at the needle sites of

injection. Other regions demonstrating c-fos induction in a dose-dependent manner

included the red nucleus and the locus ceruleus. Regions believed to be involved in

dystonia, including the striatum and motor cortex failed to demonstrate substantial c-fos

hybridization. In fact a significant decrease in c-fos hybridization was seen in the

striatum. The hippocampus also failed to demonstrate c-fos induction and rather showed

significantly decreased expression, indicating that the behavior recorded was not a

kainate-induced seizure. In fact, intense c-fos hybridization in the hippocampus and

motor cortex was seen in one animal that did undergo behaviorally recognized seizures

after injection of kainate presumably too near the inferior colliculi (data not shown).

Mean optical density of c-fos signal was quantified in the aforementioned areas and is

presented in Figure 4.4.

75

Figure 4.3. c-fos in situ hybridization in wild type mice after cerebellar kainateinjection. Representative sections from regions of c-fos activation and other areas ofinterest are shown from mice receiving 0, 60, 115, or 235 picomoles kainic acid in avolume of 0.5? l injected into the anterior cerebellar vermis. Mice receiving kainic aciddisplayed dystonic behavior in a dose-dependent manner.

Cerebellum

Striatum andCortex

Hippo-campus

RedNucleus

LocusCeruleus

0pmoles 60pmoles 115pmoles 235pmoles

76

Figure 4.4. Regional expression of c-fos mRNA after cerebellar kainate injection. Insitu hybridization of c-fos mRNA was quantified two hours after injection into thecerebellum. Data presented are the mean of four animals at each dosage and error barsrepresent SEM. cb-ant, anterior cerebellum, cb-post, posterior cerebellum, rn, rednucleus, lc, locus ceruleus, str, striatum, hip, hippocampus, ctx, primary motor cortex.

0.00

0.05

0.10

0.15

0.20

0.25

0.30

0.35

0.40

0.45

ctx str hip rn lc cb-ant cb-post

Opt

ical

Den

sity

0pmol60pmol115pmol235pmol

77

Cerebellar needle localization

Confirmation of cerebellar needle tract location was performed to verify accuracy of

freehand injections. At the time of brain extraction, injection site was verified in the

anterior vermis of the cerebellum by gross analysis. Tissue processed for c-fos in situ

hybridization was also analyzed for needle tracts and regions of most intense c-fos

induction as markers for injection location within the cerebellum. Numerous sections

were analyzed for each animal and locations approximated by comparison with Franklin

and Paxanos stereotaxic atlas of the mouse brain (Figure 4.5)

Figure 4.5. Cerebellar injection site localization. Injection sites were localized andrepresented as symbols on representative sections modified from Franklin and Paxanos,1997. Millimeters from bregma are shown for each section. Kainate dosages shown inlegend are in picomoles.

Regionalized kainate injections

To verify that the cerebellum is indeed responsible for the dystonic phenotype seen

after kainate injection, several other brain regions were also injected using this protocol.

First, the cerebellum was injected 1-2 mm lateral from the midline, anterior and in the

vermis. Mice were injected either to the left or right of midline with 235 pmoles kainic

acid at a depth of 2 mm. Circling in the direction contralateral to injection was seen

78

shortly after recovery from anesthesia, immediately prior to and at the onset of dystonia.

Initial mild to moderate dystonia (D2-D3 level) was restricted to the ipsilateral side of the

injection site for 30 to 60 minutes. As the dystonic attacks progressed to more severe

stages (D4-D5), the body became bilaterally affected.

If the kainic acid injected into the cerebellum acted by diffusing to other regions,

injections of kainic acid into the lateral ventricle would also likely result in dystonia.

Therefore, the lateral ventricle was injected in 5-6 mice each with 0 pmoles, 60 pmoles,

115 pmoles, and 235 pmoles kainic acid. No behavioral effect was appreciated at the 0

pmoles and 60 pmoles doses. Mice receiving 115 pmoles showed a paucity of movement

with most of their time spent sitting still in the corner of the observational cage with a

few displaying a seizurigenic phenotype. Mice receiving the highest dose, 235 pmoles,

also remained still in the corner but 60% also had numerous (2+) seizures within the

observational period. The seizures would often begin with the mouse extending at the

neck and having gasping jaw movements in synchrony with the tail jerking above the

body toward the head. After a few seconds the forepaws would begin beating downward

and away from the body again in synchrony with the head and tail movements. Saliva

could often be noted coming out of the mouth and onto the fur. Often the body would

jerk severely enough to cause the mouse to fall to one side and lay on the cage bottom

while the seizure continued. One mouse died in a particularly severe seizure 44 minutes

after injection. In general the seizures lasted 5-15 seconds and the mice were extremely

still between seizures. By the end of the observational period, most seizurigenic mice

had improved and would sniff or explore a bit without as frequent seizure occurrence.

Dystonic postures were never observed after ventricular injection of kainic acid.

79

Because the basal ganglia has been implicated in nearly all movement disorders,

including dystonia, kainic acid was injected into the striatum of wild type mice to

determine if this region could elicit dystonia using this protocol. Mice were again

injected with 0-, 60-, 115-, and 235-pmole doses of kainic acid (5-6mice/dose). Mice

receiving saline typically would explore for the first hour then rest in the second hour.

The 60 pmole dose of kainic acid caused hyperactive grooming in the first hour followed

by resting in the second. Mice receiving 115 pmoles kainic acid never rested for long

periods, they actively groomed and explored for the entire observational period. Mice

would occasionally circle in a direction contralateral to the injection site while exploring.

Mice receiving the highest dose of kainic acid, 235 pmoles, were very actively grooming

and exploring with some circling and two mice had brief, isolated seizures at 1 and 2

hours after injection respectively. As in the ventricular injections, striatal injections

never resulted in dystonic posturing.

Standard hematoxylin and eosin stained sections from all mice receiving regional

kainic acid injections confirmed the position of the freehand injection to either the lateral

ventricle or striatum (data not shown).

Kainate injections in SV4 mice lacking Purkinje cells

To determine that cerebellar signaling is necessary for the kainate-induced dystonia,

transgenic mice lacking Purkinje cells were injected with kainic acid in the anterior

cerebellar vermis in the midline at a depth of ~2 mm. If the cerebellar cortex is indeed

necessary for the production and/or maintenance of dystonia in this model, mice lacking

Purkinje cells, the sole output of the cerebellar cortex, should not display a dystonic

phenotype after kainate injection. Conversely, if cerebellar cortical output is not vital to

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the production of dystonia, transgenic mice lacking Purkinje cells should display a

dystonic phenotype similar to controls. Fifteen aged transgenic mice were injected with

235pmoles kainic acid and most displayed no notable motor behavior in addition to the

characteristic transgenic ataxia (9/15). A few mice displayed brief and relatively mild

dystonic motor behavior (4/15) and the remaining mice developed brief wild

running/seizure behavior at 10-20 minutes post-injection (2/15). The seizing mice

quickly recovered and proceeded to maintain odd, stiff arching postures, which were not

typical of the transgenic phenotype. While these postures were also not typical to those

seen with kainate-induced dystonia, they caused the mice to be immobilized in odd

postures and were therefore scored as such (D5). It is also important to note however,

that this behavior remitted temporarily after disturbing the mice. This is in direct contrast

to the exacerabatory effect disturbance has on mice immobilized in typical dystonic

postures. Average D score over the two-hour observation period was significantly

reduced (p<0.0005) in SV4 mice receiving kainic acid compared to control mice

receiving kainic acid (figure 4.6).

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Figure 4.6. Dystonic severity in transgenic mice lacking Purkinje cells. Transgenic mice(n=15) were injected with 235 pmoles kainic acid in the midline anterior cerebellarvermis and observed for two hours for dystonic postures (SV4-235). Transgenic animalswere injected with saline (n=6) to demonstrate the basal level of motor disturbance inthese animals (SV4-0). D score was summed over the two-hour observational period andaveraged per group. Wild type data is illustrated for comparison and is the same aspresented above in figure 4.2 (saline n=5, WT-0; kainate n=6, WT-235). *** indicatessignificantly reduced dystonia in SV4 mice compared to wild type animals, p<0.0005using the Student's t-test.

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EEG recordings of dystonic mice after kainate injection into the cerebellum

Kainic acid is frequently used to induce seizures after i.p. or central injection in

experimental animals. For this reason and because dystonia is sometimes confused with

motor seizures in animal models, EEG recordings were collected during kainate induced

dystonia in mice. EEGs from five mice were recorded during kainate-induced dystonia

(235 pmoles). In order to distinguish the dystonic EEG from that of a seizure, 60 mg/kg

pentylenetetrazole was administered after 90 minutes to induce generalized seizures.

Numerous baseline recordings prior to kainate injection were also recorded and

representative traces of baseline, kainate-induced dystonia, and pentylenetetrazole seizure

are shown in figure 4.7. Eighty EEGs recorded during behavioral dystonia were analyzed

for the five mice and none were associated with epileptiform activity.

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A. Baseline

B. Kainate-Induced Dystonia

C. PTZ-induced seizure

Figure 4.7. Representative EEG recordings from wild type mice receiving cerebellarmicroinjections of kainic acid. EEGs shown were recorded before kainic acid injection(A), during profound dystonic posturing after kainic acid injection (B), or during seizuresinduced after pentylenetetrazole (PTZ) injection (C).

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NBQX antagonism of kainate-induced dystonia

Kainic acid is known to activate both kainate and AMPA ionotropic glutamate

receptors. In order to verify that kainate is acting locally within the cerebellum at these

receptor sites, NBQX, a kainate and AMPA receptor antagonist, was used to block the

kainate-induced dystonia. Mice were injected in the anterior cerebellar vermis with 0.5? l

of 2.35 nanomoles NBQX alone or 2.35 nmoles NBQX mixed in solution with either

115pmoles or 235pmoles kainic acid. Mice receiving NBQX alone displayed either

normal (2/5), hyperactive/jittery (2/5), or decreased (1/5) locomotor activity.

Combination of NBQX and 115pmoles kainic acid caused 4/5 mice to remain mostly still

during the observational period with slowed ambulation once disturbed. This phenotype

was indistinguishable from the decreased locomotor activity seen in one of the control

NBQX-only injected mice. The remaining NBQX/115 pmoles kainate-injected mouse

showed normal locomotor activity (1/5). Co-injection of NBQX with 115 pmoles kainic

acid never resulted in any dystonic postures. Injection of NBQX together with 235

pmoles kainic acid resulted in 1/5 mice behaving normally, 2/5 mice displaying

decreased activity (as described for both NBQX alone and NBQX/115 pmoles), and 2/5

mice demonstrating a relatively mild dystonic phenotype. Total D score was summed

over two hours of observation and averaged over five mice per group (Figure 4.8).

Statistically significant reduction in total D score was seen in mice injected with 235

pmoles kainic acid in combination with NBQX compared to control kainate-injected

mice (235 pmoles kainate alone).

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Figure 4.8. Dystonic severityafter cerebellar NBQX co-injection with kainic acid.Mice were injected with NBQX(2.35 nmoles) alone and incombination with 235pmoleskainic acid. Total D score wassummed over two hours ofobservation and averaged for 5-6 mice in each drug paradigm.** indicates p<0.005 betweenkainate with and withoutNBQX.

Domoic acid injection induces dystonia in wild type

As a complement to the antagonism provided by the NBQX experiments, domoic

acid was used to duplicate the dystonic phenotype through activation of glutamate

receptors. Similar to kainic acid, domoic acid is an agonist at AMPA and kainate

receptors. Activation of these glutamate receptor subtypes is presumed to underlie the

mechanism of dystonia induction by kainic acid described above. Wild type mice were

injected along the midline in the anterior cerebellar vermis and dystonic behavior

recorded. Similar to kainate-injected mice, domoate-injected mice displayed a

reproducible phenotype of generalized and intermittent dystonia within 10 to 20 minutes

after injection. Domoate-injected mice were observed to have flattened trunks with the

hindlimbs abducted and externally rotated, suspended in the air. Forelimb paddling and

facial movements were also consistently noted. These dystonic postures were identical to

those observed in mice after kainate injection into the cerebellum. The quality of the

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dystonia seen after 7.5 pmoles domoate injection was different from kainate-induced

dystonia in one regard however. Domoate mice more frequently remained in odd, semi-

flattened postures in between dystonic exacerbations than did kainate-injected mice. It is

important to note however, that not all domoate mice displayed this behavior and some

kainate mice also displayed this behavior. Domoate-injected mice displayed a level of

dystonia analogous to that of 235 pmole injected kainate mice (Figure 4.9) and also

recovered to near-normal behavior within two hours after injection (data not shown). As

with kainate, domoic acid induced seizures were seen infrequently when needle

localization was such that the inferior colliculi were likely exposed to concentrated drug.

Figure 4.9. Dystonic severityafter cerebellar injection ofdomoic acid in wild type mice.Domoate (7.5 pmoles) wasinjected into the anteriorcerebellar vermis of fiveC57Bl/6J mice and dystonicbehavior recorded. D score wassummed over two hoursimmediately following injectionand averaged for 5-6 mice pregroup. Saline and kainate (235pmoles) data is the same as thatpresented above (Figure 4.5).Error bars represent SEM.

Discussion

Movement disorders are by nature difficult to describe in rodent models. Limited

understanding of the clinical terminology by researchers further compounds this

difficulty making it exceedingly arduous to determine the exact behavior described in the

literature and comparing it to other behaviors seen or described. This is particularly true

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in the case of dystonia, which historically is misjudged as seizures, myoclonus, ballism,

and chorea. The use of kainic acid in this model of dystonia therefore had to be done

with special attention to the possibility of seizure induction. We believe that the motor

behavior described here represents dystonia and not seizures because of the quality of the

movements, the absence of seizure activity on EEG recordings, and the lack of c-fos

induction in the hippocampus or motor cortex. Furthermore, a few mice did respond to

kainic acid injection with generalized seizures and this was readily discernable from the

dystonic phenotype. In situ hybridization of a seizurigenic mouse showed marked c-fos

induction in the hippocampus and cerebral cortex. This confirmed molecularly what was

very apparent behaviorally and further distinguished the seizure-inducing effects of

kainic acid from the dystonia-producing effects described in this model.

Microinjection of 60 to 250 picomoles of kainic acid (in 0.5? l) into the anterior

cerebellar vermis of C57Bl/6J mice resulted in an acute and reproducible dystonic

phenotype. Similar to the neurotoxic and seizure-producing effects of kainic acid

(Lothman EW, 1981; Sperk G, 1985), kainate-induced dystonia was positively associated

with increasing kainic acid dosage. All body regions were involved in dystonic postures.

Paddling or tonic extension of the hindlimbs along with a severely flattened and extended

trunk hallmarked the most common dystonic posture. Face, head and neck were also

involved in dystonic postures, categorizing these attacks as a form of generalized

dystonia. Dystonic attacks began 10-20 minutes after kainate injection and lasted for 1-2

hours. While the most severely affected mice would remain immobilized in tense

dystonic postures for the much of the time, the majority of mice displayed short-lived

intermittent dystonic attacks initiated by movement or startle.

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The immediate early gene, c-fos, expression was studied to determine the neuronal

pathways activated during kainate-induced dystonia. Two hours after cerebellar kainic

acid injection, nearly the entire cerebellar cortex and the DCN show markedly increased

c-fos induction. Of the cerebellar nuclei targets, only the red nucleus demonstrated c-fos

activation in dystonic mice. The locus ceruleus also demonstrated c-fos induction in a

dose-dependent manner to kainate injection. These changes in c-fos expression

theoretically may have resulted from activation of pathways independent from that

involved in the production of the dystonia. However, the general paucity of neuronal

activation seen after cerebellar kainic acid injection increases the likelihood that these

regions were involved in the pathway responsible for the dystonic phenotype as well.

Cerebellar c-fos induction increased with kainate dosage. Dramatic activation in the

posterior and anterior cerebellum, both medially and laterally, was seen after mid- to

high- dose kainate injections. These dosages corresponded to animals demonstrating

profound dystonic behavior. Low doses of kainic acid resulted in relatively mild and

localized c-fos induction within the cerebellum, while saline injection produced very

slight induction around the needle tract, consistent with mechanical stress. The

difference between the intense broad activation seen at higher kainate doses compared to

the local mild activation at lower doses is quite remarkable and unlikely to result from

diffusion of kainic acid alone. Broad activation throughout all cerebellar folia despite the

small injection volume (0.5? l) suggests neuron to neuron activation played a role in

spreading as opposed to simple diffusion. It is more reasonable that neuron to neuron

activation played a role because of the distance and number of folia the kainate would

have traveled to activate the distal tissue directly. Furthermore, if diffusion to the CSF

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between folia occurred, neighboring regions such as the colliculi and brainstem would

show more background activation in animals receiving high kainate doses or generalized

seizure activity would result from high concentrations of kainate circulating in the CSF.

Therefore, these data suggest that neuronal activation of the cerebellum by local kainic

acid injection results in amplification of the signal to result in broad activation of most of

the cerebellum. Whether this phenomenon participates in the production of dystonia or

results from the dystonia-producing signal remains to be seen. c-fos in situ hybridization

studies in transgenic mice lacking Purkinje cells and failing to demonstrate dystonia after

cerebellar kainate injection would address both of these questions. c-fos studies in

transgenic mice would also determine whether induction in the red nucleus and locus

ceruleus is part of the pathway causing dystonia or if these events are independent of one

another.

The red nucleus was the only target of DCN efferents clearly demonstrating c-fos

induction after cerebellar kainate injection. Because efferents of the DCN to the red

nucleus likely represent collaterals of axons destined for the motor thalamus, it is

surprising that these nuclei failed to demonstrate marked activation as well. The

theorized importance of the motor thalamus in the pathophysiology of dystonia makes

this finding particularly puzzling. The red nucleus is one of the major efferent targets of

the DCN, receiving excitatory input from the contralateral interpositus and dentate nuclei.

The red nucleus also receives afferents from other major motor centers in the brain,

including the motor and premotor cortices, the posterior thalamic nucleus, the basal

ganglia, and the spinal cord. This structure lies in the midbrain of limbed vertebrates and

gives rise to the fibers of the rubrospinal tract. The descending rubrospinal tract

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originates in somatotopically organized neurons of the red nucleus and terminates on

interneurons in the ventral gray column of the spinal cord. The other major efferents of

the red nucleus form a recurrent loop to the cerebellum. Direct contralateral fibers

synapse directly on the DCN while ipsilateral efferents of the red nucleus indirectly reach

the cerebellar cortex through the precerebellar nuclei. As with the cerebellum, a large

body of evidence is accumulating that suggests a role for the red nucleus in dystonia as

well. In a review of 7 patients with midbrain lesions resulting in dystonia, lesions in 7/7

patients involved the red nucleus, rubro-thalamic, and/or dentate-rubral fibers (Vidailhet

M, 1999). Induction of neck dystonia in rats after intrarubral injection of compounds that

bind opiate sigma receptors implicate involvement of the red nucleus in dystonia (Walker

JM, 1988; Matsumoto RR, 1990). These effects have been determined to act through the

sigma2 receptor subtype (Nakazawa M, 1999). Furthermore, the use of antipsychotics

with sigma receptor activity may induce motor side effects through these receptors rather

than presumed dopaminergic activity (Walker JM, 1988). Numerous abnormal findings

in genetic rodent models also support a role for the red nucleus in the pathophysiology of

dystonia. Magnocellular neurons in the red nucleus of the dystonia musculorum mouse

show pathological features consistently (Messer A, 1980; Stanley E, 1983). The dystonic

rat shows decreased metabolic activity within the red nucleus during dystonic posturing

only (Brown LL, 1989). Alternatively, greatly increased activity was seen in the red

nucleus of dystonic hamsters, again only in the presence of dystonia. Depth electrode

recordings from the red nucleus in these animals also suggests abnormalities in the red

nucleus (reviewed in Richter A, 1998). If the cerebrocerebellar circuit is hyperactive or

otherwise deranged in dystonia, cerebellar efferents to the red nucleus may play a part in

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expression of the dystonic program. Activation of muscles via the rubrospinal tract may

occur in discordance with activation/inhibition of muscles via the corticospinal tract

resulting in disordered movement as a direct consequence of cerebellar signaling.

Similar to the red nucleus, the locus ceruleus (LC) demonstrated marked activation in

a dose-dependent manner after cerebellar kainate injection. The locus ceruleus is a

relatively small pair of brainstem nuclei that project noradrenergic (NE) terminals widely

throughout the brain. As with the cerebellum and the red nucleus, substantial evidence

supporting a role for the locus ceruleus in the pathophysiology of dystonia continues to

accumulate (reviewed in Adams LM, 1988). Regions involved in motor control receive

substantial LC innervation including the motor cortex, thalamus, and the cerebellum.

Within the cerebellum and elsewhere, norepinephrine released from LC terminals enables

more effective transmission of both excitatory and inhibitory systems converging on the

same target neurons during periods of simultaneous activity (Bloom FE, 1979).

Facilitation of afferent excitatory and inhibitory activity while reducing endogenous

background activity allows LC-NE enhancement of target area function (Woodward DJ,

1979). For example, mossy fiber afferent stimulation of cerebellar granule cells results in

parallel fiber excitation of Purkinje cells lying along the length of the parallel fiber. The

same parallel fiber activates stellate and basket cells to inhibit adjacent Purkinje cells that

lie off that parallel fiber. In this situation, LC-NE input potentiates these effects and

therefore increases the precision of this inhibitory surround mechanism for incoming

excitatory signals (Eccles J, 1967; Woodward DJ, 1979; Moises HC, 1983). It is

interesting that failure of such center-surround inhibition at the level of the motor cortex

is theorized to result in dystonia (Halett M, 1998). While evidence implicating these

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functions of the LC in dystonia has not been defined, numerous lines of research have

demonstrated more general involvement of the LC in dystonia.

Biochemical analysis of two human dystonia musculorum deformans samples

revealed marked changes in NE levels in numerous LC targets (Hornykiewicz O, 1986).

Animal models of dystonia also support a role for the LC in the pathophysiology of

dystonia. In an experimental model of dystonia, microinjection of adrenocorticotropin

hormone (ACTH) fragments into the LC of rats caused ipsilateral leaning and postures

reminiscent of human dystonia for a number days depending on dosage. These results

were determined to result from the direct action of NE at ? -adrenergic receptors on

cerebellar PC (Jacquet YF, 1982; Jacquet YF, 1988). Further experimentation revealed

that the action of ACTH likely occurs through modulation of endogenous opiate peptides

in the LC (Bertolini A, 1986). The dystonic rat, dystonia musculorum mouse, tottering

mouse, and dystonic hamster all demonstrate altered NE concentrations, particularly in

the cerebellum (Riker DK, 1981; Richter 1998; Levitt P, 1981). As with the red nucleus,

it seems unlikely that the locus ceruleus alone is responsible for the production of

dystonia. However, LC activity may modulate motor center functions as a primary

component of the dystonia program or a secondary reaction to dystonic activity.

Despite the localization of the cerebellar injection sites, the broad and intense

cerebellar c-fos induction, and the activation of the red nucleus, it is still arguable that the

kainic acid acted to induce dystonia at another site. Therefore, microinjection of kainic

acid in other regions was attempted to verify the inciting role of the cerebellum in this

model of dystonia. First, we lateralized the cerebellar injections from the midline of the

anterior vermis to ~2mm lateral, in the paravermis. Dystonia in these mice remained

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ipsilateral to the injection site initially during moderate dystonic impairment, eventually

generalizing to both sides of the body as the severity of dystonia increased.

Displacement of the previously bilateral expression of dystonia through lateralization of

the cerebellar injection site strongly supports the importance of cerebellar activation in

the production of dystonia in this model. Injections of 0, 60, 115, or 235 picomoles

kainic acid into the striatum and the lateral ventricles never resulted in dystonia,

emphasizing the specificity of local excitation of the cerebellum in the induction of

dystonia with kainic acid microinjection.

To further illustrate the function of the cerebellum in the production of dystonia,

cerebellar kainic acid microinjection was done in transgenic mice lacking PCs. SV4

transgenic mice lose PCs over a period of weeks post-developmentally with no other

abnormalities reported, cerebellar or otherwise. Kainate injection in these animals

resulted in a highly significant decrease in average total dystonia. The appearance of

relatively mild dystonic postures in a few of the mice was likely due to the remaining PC

activity at the time of the experiments. The F5 generation of the transgene bred onto

C57Bl/6J background appears phenotypically delayed in comparison to the original

reports (Fedderson R, 1992) and to prior generations. Thus, despite the advanced age of

the transgenic mice used in these experiments (six months old), sparse PCs may have

remained in some animals, resulting in dystonic posturing after kainic acid injection. In

addition to cementing the necessary and inciting role the cerebellum plays in the

induction of dystonia, failure of mice lacking PCs to demonstrate dystonia directly

implies a role for cerebellar cortical output in the production of dystonia. These results

are in agreement with those obtained by Campbell et al., and data presented in this work

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(Chapter 3) using the tottering mouse as a model of dystonia. In both of these

experiments, selective destruction of PCs in the dystonic tottering mutant mouse resulted

in loss of the dystonic phenotype (Campbell DB, 1999; Chapter 3). Together these data

suggest that dystonia of cerebellar origin occurs through activation of a circuit involving

the cerebellar PC. Furthermore, the PC is a necessary component of the pathways

involved in the pathophysiology of dystonia in these two very different mouse models.

Data presented here indicate that kainic acid injection into the cerebellar vermis of

wild type mice produces a dystonic phenotype and c-fos induction throughout the

cerebellum, red nucleus, and locus ceruleus in a dose-dependent manner. The cellular

mechanism of kainate-induced dystonia remains to be uncovered. Kainic acid is a

glutamate analogue and an agonist at both kainate and AMPA receptors. AMPA (along

with NMDA) receptors are thought to mediate the majority of fast excitatory

neurotransmission in the CNS while kainate receptors contribute a relatively minor

component. A second presynaptic function of kainate receptors has been suggested with

modulation (depression) of GABAergic transmission being demonstrated (Rodriguez-

Moreno A, 1997). Antagonism of kainate-induced dystonia with co-injection of NBQX

and replication of the behavior with cerebellar domoic acid injections verify the notion

that kainic acid induces dystonia through activity at these receptors.

In summary, microinjection of kainic acid into the cerebellar vermis is sufficient to

produce profound dystonia in wild type mice. These data illustrate the potentially

powerful role the cerebellum may play in the production of dystonia because no

abnormalities in any other brain region existed at the time of injection. While a

contributory role for the red nucleus and the locus ceruleus remain to be established,

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these brainstem nuclei showed marked activation after cerebellar kainate injection in

dystonic animals. The well-defined and genetically normal background of these mice

eliminates confounding factors in the examination of dystonia pathophysiology in this

animal model. Surgical and pharmacological manipulations of this model may provide

profound insight not only to the pathophysiology of dystonia, but to possible

interventions as well. The production of profound and reproducible dystonia in wild type

animals after localized excitation of the cerebellum suggests that this region no longer be

overlooked in the pathophysiology of this devastating neurological disease.

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Chapter 5. Discussion

5.1. MOUSE MODELS OF DYSTONIA

Animal models of human disease represent powerful tools to examine

pathophysiology of disease and to identify possible interventions in the disease process.

Limitations of models arise from the innate differences between the physiologies of

humans versus laboratory animals; however, tremendous amounts of information

pertaining to the human disease state may be discovered through animal research.

Demonstration of similar findings in multiple models, each with different strengths and

limitations, lends support to the validity of the results, reducing the likelihood that they

represent an idiosyncrasy of an individual model.

Primary dystonia represents a common neurological disease of unknown etiology.

Numerous neuroanatomical regions have been implicated in the pathophysiology of this

disorder, however none have been definitively identified as responsible for this disease

process. Therefore, studies in current animal models and development of novel models

of dystonia can provide crucial insight to the pathophysiology and potential interventions

of this devastating disorder.

The tottering mouse

Current rodent models of dystonia are limited in usefulness because the primary gene

defect resulting in the phenotype is unknown. The tottering mouse, therefore has a

unique advantage as a rodent model of dystonia because of the well-defined nature of the

genetic mutation and a growing understanding of the resultant behavioral and cellular

phenotypes. Discovery of the neuronal basis of tottering mouse dystonia can further

define the neuronal networks capable of producing dystonia of other etiologies.

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Description

Tottering mice display striking episodic attacks of severe motor dysfunction

remarkably similar to human generalized dystonia. Described in detail in Chapter 1.2,

the attacks typically last 30-60 minutes and occur spontaneously or in response to several

known stressors and pharmacological agents with no discernable refractory period.

Characterization of attack induction after exposure to various environmental stimuli and

compounds has greatly increased the utility of this model in the study of dystonia.

Caffeine, alcohol, and stress (e.g., restraint, environmental novelty) reliably trigger

attacks in ~60-100% of tottering mice (Campbell DB, 1998; Fureman BE, 2001).

Furthermore, administration of common anticonvulsants not only fails to block attacks,

but some actually induce attacks in tottering mice (Syapin PJ, 1983). Conversely,

administration of L-type calcium channel antagonists (Campbell DB, 1999; Fureman BE,

2001) and benzodiazepines (Syapin PJ, 1983) reduces tottering dystonic attack frequency.

The phenotypic characteristics and pharmacology of tottering dystonia strikingly

resemble those of human PNKD (Chapter 1.1) and are highlighted in Table 5.1.

Characteristic Tottering Mouse PNKDAttack Duration 30-60 minutes 30-60 minutesAttack Frequency Unknown 2-3/month (range from 2-3/day to 2-

3/year)Occurrence Spontaneous SpontaneousCommon Triggers Stress, caffeine, alcohol Stress, caffeine, alcohol, fatigueOther Severity may wax and wane during

attacksNo discernable refractory period

Severity may wax and wane duringattacksNo discernable refractory period

Drug Response + L-type Ca2+ channel antagonists+ benzodiazepines- anticonvulsants

+ benzodiazepines (moderate effect)- anticonvulsants

Genetics ? utosomal recessive inheritance? 1A VDCC subunit mutation

Autosomal dominant inheritanceLinkage to 2q33-35, in area of suspectedion channel

Pathophysiology Strong evidence for role of aberrantcerebellar signaling

Unknown

Table 5.1. Comparison of salient features of tottering dystonia and PNKD.

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Role of the cerebellum in tottering dystonia

The tottering mouse has been studied as a model of dystonia with particular relevance

to PNKD (Fureman BE, 2001). Previous reports have implicated the cerebellum in the

production of tottering dystonia. Increased cerebellar L-type VDCC expression and

suppression of attacks with L-type calcium channel antagonists (Campbell DB, 1999)

lend circumstantial evidence for a role of the cerebellum in tottering dystonia. Intense c-

fos induction within the cerebellum during attacks also supports a role for this region in

the pathophysiology of tottering dystonia (Campbell DB, 1998). Elimination of the

phenotype on the pcd background provides even stronger evidence that the cerebellum,

and the cerebellar PC in particular, induce and/or maintain the dystonic phenotype of the

tottering mouse (Campbell DB, 1999). In addition, surgical removal of small portions of

the anterior cerebellar vermis reduce the duration and number of attacks while lesions of

the posterior cerebellar vermis show no differences. Similar results are obtained after

excitotoxic lesions of the same areas. (Abbott LC, 2000). Data presented in this work

further define an important role for the cerebellum in the generation of tottering dystonia

through the elimination of the dystonic phenotype after transgene-induced PC death

(Chapter 3). Future experiments in these mice will define subsets of PC responsible for

the regional expression of tottering dystonia.

Other data presented here demonstrate decreased excitatory neurotransmission in the

tottering cerebellum with no concomitant change in inhibitory transmission (Chapter 2).

These findings suggest an imbalance in the overall signaling in the tottering cerebellum

and provide evidence of a potential link between the genetic mutation and the behavioral

phenotype, as they represent a molecular consequence in a region defined in the

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pathophysiology of the disease. Future experiments designed to analyze

neurotransmission of single cell types in the tottering cerebellum would further define the

transmitter imbalance and dissect out cell types involved in the production of dystonia in

this mutant.

Future Studies

The PC has already been identified as an absolute and necessary component of the

dystonia-producing substrate in tottering mice (Campbell DB, 1999; Chapter 3). The

function of the PC as the sole output of the cerebellar cortex therefore indicates this

region in the pathophysiology of dystonia. Components of the circuitry upstream of PC

function need to be evaluated to determine whether the dystonia-producing signal

originates in the PC or if this cell type simply represents a crucial link between the origin

and expression of dystonia. Purkinje cells receive excitatory input from climbing fibers

and parallel fibers while inhibitory input is derived from stellate and basket cells.

Because PCs are of central importance to the generation of tottering dystonic attacks,

disruption of the PC spike rate through climbing fiber denervation was attempted in the

tottering mouse. Lesioning of climbing fibers through ablation of the inferior olive (IO)

with 3-acetylpyridine (3-AP) injection was not achieved however, even after

administration of high doses of 3-AP (300mg/kg; data not shown). Therefore, other

methods or further modifications of 3-AP intoxication should be attempted to evaluate

the role of climbing fiber excitation of PC in the expression of tottering dystonia.

Mossy fibers represent a second extracerebellar input to the cerebellar cortex and act

to excite PC through relay with granule cells. Lesioning of mossy fibers or granule cells

would therefore elucidate the role these inputs have in tottering dystonia. Breeding

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genetic mouse mutants that develop lesions of mossy fibers, granule cells, and other

combinations of cell types involved in the cerebellar circuitry onto the tottering mouse

would determine the effects these inputs have on the expression of tottering dystonia.

However, epigenetic effects and the contribution of extraneous phenotypes of the second

mutant may be difficult to evaluate.

The effect of GABAergic innervation on relatively small areas of PCs can be

evaluated through local application of GABA agonists/antagonists followed by dystonia

induction in tottering mice. Analysis of tritiated drug diffusion could verify the

distribution in the cerebellar cortex, eliminating the concern that the drugs affected the

DCN directly. Spatial restriction of locally applied drugs to relatively few PCs may limit

their potential response. However, small areas of the anterior cerebellar cortex

profoundly affect expression of dystonia in both ablative studies (Abbott LC, 2000) and

local excitation (Chapter 4) using glutamate agonists, suggesting that this region may be

sufficient to elicit a response. Microinjection of GABA agonists/antagonists in the

anterior cerebellar vermis in tottering mice can be done rapidly under isofluorane

anesthesia and the effect on dystonia evaluated almost immediately. In fact,

administration of GABA agonists or antagonists through an indwelling catheter to the

cerebellar cortex during a tottering dystonic attack can directly assess the effect inhibition

or release of PC activity has on the expression of dystonia. Resultant effects on dystonia

after PC GABA-receptor activation/inhibition would implicate a role for basket and/or

stellate cells in the modulation of dystonia-producing PC activity. These data would

furthermore reveal whether PC firing patterns are likely increased or decreased in

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tottering dystonia. This is an intriguing and complicated question and is discussed further

below in section 5.2.

In summary, the tottering mouse represents a unique animal model of dystonia that

closely resembles PNKD. Analysis of the neuroanatomical and functional consequences

of the P/Q-type VDCC mutation has determined that the cerebellar cortex is vital in the

pathophysiology of dystonia in this mutant. Further delineation of the dystonic origin

and the circuits involved in the expression of dystonia in tottering mice will greatly

expand current understanding of the pathomechanisms capable of producing dystonia.

Kainate-induced dystonia

Limitations of the tottering mouse as an animal model of dystonia arise from wide

spread neurological effects of the mutation. Despite limitations, consistent findings of

abnormal cerebellar structure and function in genetic rodent models of dystonia lend

strong support to a role of the cerebellum in dystonia. The next step in the use of animal

models of dystonia would then be to use genetically normal animals to replicate the

dystonia-producing signals implicated in the more limited genetic models of dystonia.

We present here, a novel animal model suited to the study of neuronal networks capable

of causing dystonia in wild type mice using a well-defined excitatory amino acid

microinjection protocol.

Description

The kainate-induced dystonia described here represents a novel drug-induced animal

model of human dystonia (Chapter 4). Microinjection of low-doses of kainic acid into

the cerebellar vermis of mice elicited reliable and reproducible generalized dystonia that

began 10-20 minutes after kainate injection and lasted for less than two hours. While the

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most severely affected mice remained immobilized in tense dystonic postures for the

much of the time, the majority of mice displayed short-lived intermittent dystonic attacks

initiated by movement or startle. The clinical spectrum of kainate-induced dystonia was

noted to somewhat resemble that of human PKD specifically (see Chapter 1.1).

Characteristics of these two forms of dystonia are compared in Table 5.2.

Characteristic Kainate Model PKDAttack Duration Seconds to minutes Seconds to minutesAttack Frequency ~10-100 attacks in 2 hours after injection 1-10/day (up to 100/day reported)Occurrence Initiation of movement, startle Initiation of movement, startleDrug Response Not tested + anticonvulsantsPathophysiology Aberrant cerebellar excitation UnknownTable 5.2. Comparison of salient features of the kainate model of dystonia and PKD.

Role of the cerebellum in kainate-induced dystonia

Kainic acid is a glutamate agonist at both kainate and AMPA ionotropic glutamate

receptors. Microinjection of kainic acid into the cerebellum of mice elicited dystonia

through actions at these receptor subtypes, as co-injection with NBQX antagonized these

effects and administration of domoic acid replicated the results. Furthermore, neuronal

activation, as assessed by in situ hybridization for c-fos mRNA, revealed specific patterns

of activity in the cerebellum, locus ceruleus and red nucleus. The specificity of the

neuronal induction occurring with kainate-induced dystonia suggests that localized

cerebellar effects were indeed responsible for the phenotype. Failure to induce dystonia

after microinjection of the striatum or lateral ventricle and lateralization of the phenotype

after paravermal cerebellar injection also suggest that kainate is acting locally within the

cerebellum to induce dystonia. Transgenic mice lacking Purkinje cells show dramatically

decreased dystonia after kainic acid injection suggesting an important role for Purkinje

cells and the cerebellar cortex in this model of dystonia as well. Together, these data

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suggest that aberrant cerebellar excitation is sufficient to produce dystonia and the

cerebellar cortex plays an essential role in generation of the dystonic signal or circuitry.

Future Studies

Further development of this model of dystonia using wild type and various mutant

mice can dissect out specific cellular components of the dystonia-producing pathway and

also determine possible interventions to the process. Mice lacking specific subsets of

neurons involved in cerebellar circuitry can more fully evaluate the neuroanatomical

substrate of kainate-induced dystonia. Lesioning of other brain regions implicated in the

pathophysiology of dystonia can determine whether these regions are essential in the

expression of dystonia using this model. For example, dopamine-depleted or dopamine

receptor knockout mice can be used in this microinjection protocol to determine whether

this dystonia of cerebellar origin acts in isolation from motor pattern modulation by the

basal ganglia. Lack of basal ganglia c-fos activation already suggests independence of

these two pathways in kainite-induced dystonia.

In summary, kainate-induced dystonia in wild type mice implicates the cerebellum as

an important component in the pathophysiology of dystonia. The cerebellar cortex plays

an essential role in the production of dystonia in this model. Traditional views of

cerebellar function would not predict such a phenotype being of cerebellar origin.

However, traditional studies of the cerebellum investigated disturbances due to decreased

normal function and activity as opposed to the potential effects of abnormal signals and

increased activity, as implicated in the rodent models of dystonia. As animal models and

human imaging studies continue to demonstrate abnormal cerebellar activity, the

perception of the cerebellum in health and disease may necessitate reevaluation.

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5.2. THE CEREBELLUM AND DYSTONIA

In addition to the data discussed in detail throughout this work, other aspects of

cerebellar anatomy and function raise provocative questions concerning the role of this

region in dystonia. Therefore, discussion of cerebellar circuitry and aspects of cerebellar

function pertinent to dystonia follow.

Cerebellar Circuitry

Intrinsic Cerebellar Circuitry

The intrinsic circuitry of the cerebellum is relatively simple and illustrated in Figure

2.1. Cerebellar PCs supply the sole output from the cerebellar cortex and form inhibitory

synapses directly on the DCN. This corticonuclear projection is the principal input to the

DCN and remains strictly ipsilateral. PCs maintain a roughly radial arrangement in their

projections to DCN with vermal PCs (medial) projecting to the fastigial nuclei while

paravermal (intermediate) and hemispheral PCs (lateral) project mainly to the interpositus

and dentate nuclei respectively (Fundamental Neuroscience, Development of the

Cerebellar System, and Principles of Neural Science).

Afferent Cerebellar Circuitry

Afferents into the cerebellum consist of two main types, climbing fibers from the

inferior olive and mossy fibers from various other precerebellar nuclei. Climbing fibers

exert a powerful excitatory influence on PCs, which then act to inhibit the DCN. Mossy

fibers arising from precerebellar nuclei such as spinocerebellar nuclei, lateral reticular

nucleus, prepositus nucleus, vestibular nuclei, pontine gray nucleus, and the pontine

reticulo-tegmental nucleus all form excitatory synapses with granule cells which in turn

excite PCs. A third, less well defined type of input to the cerebellum, consists of

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nonlaminar afferents from the locus ceruleus, raphe nuclei, and other undetermined

brainstem structures. These afferents synapse on PCs and throughout the cerebellum,

releasing NE, serotonin, and acetylcholine respectively (Fundamental Neuroscience,

Development of the Cerebellar System, and Principles of Neural Science).

Efferent Cerebellar Circuitry

The cerebellar DCN represent the output of the cerebellum and send efferents to

every major region involved in motor control except for the basal ganglia. DCN efferents

are excitatory with the exception of those reaching the inferior olive. Fibers originating

from the interpositus and dentate synapse in the contralateral red nucleus, superior

colliculus, and other visually related structures in the mesencephalon. Fibers terminating

in the diencephalon do so largely in the ventrolateral complex of the motor thalamus.

The interpositus and dentate nuclei also send contralateral fibers to the pontine grey

nuclei, the reticulotegmental nucleus, and the inferior olive, while the ipsilateral fibers

terminate in the reticular formation. All of these structures reached by the descending

branch of cerebellar efferents are involved in motor behaviors and are also precerebellar

nuclei that send mossy fibers into the cerebellum. Efferent fibers from the fastigial

nucleus synapse on the vestibular nuclei and numerous other precerebellar nuclei with a

few fibers reaching the spinal cord. Fibers originating in the fastigial nucleus also

terminate in the superior colliculus, visual nuclei, and the ventrolateral complex of the

motor thalamus. All projections to the red nucleus and to the motor thalamus maintain a

rough somatopographic organization, not as clearly defined as that of the cerebral motor

and sensory cortices.

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Cerebellar efferents affect the production and control of movement largely through

input to the cortex via the thalamus and to the red nucleus. Each of these areas in turn

send afferents back to the cerebellum to create upper and lower transcerebellar loops

respectively (Figure 5.1). The motor thalamus receives the largest component of

cerebellar efferents originating in the interpositus and dentate nuclei. The fastigial nuclei

also send a smaller bundle of fibers to this area of thalamus. The premotor and motor

areas of the cortex are the ultimate targets of the cerebellothalamic pathway. In addition

to the corticospinal tract, the cortex in turn sends descending fibers to the pontine gray

nucleus and other precerebellar nuclei that project mossy fibers back to the cerebellar

cortex, finishing this upper transcerebellar loop.

The red nucleus is the second major efferent target of the DCN that substantially

affects motor control. Contralateral fibers originating from the interpositus nucleus

synapse in the phylogenetically older magnocellular portion of the red nucleus, while

those from the dentate nucleus synapse with the parvicellular portion. It is believed that

these axons are collaterals of those ascending to the motor thalamus and are excitatory in

nature. The red nucleus also receives inputs from other structures, including large

somatotopically organized input from the premotor and motor cortex to the parvicellular

portion. Efferents of the red nucleus include the rubrospinal tract, arising largely from

the magnocellular neurons in a somatotopically-organized fashion. This tract is a motor

output pathway that terminates on interneurons in the ventral grey column of the spinal

cord. The parvicellular portion of the red nucleus sends efferents back to the cerebellum

via a direct contralateral pathway and an indirect ipsilateral pathway via precerebellar

nuclei. This circuit represents the lower transcerebellar loop (Fundamental Neuroscience,

108

Development of the Cerebellar System, and Principles of Neural Science). Altered

cerebellar output may act through the thalmo-cortical or rubrospinal paths to affect

muscle function. Each of the cerebellar loops may also serve to augment or spread a

locally aberrant signal within the cerebellum to include larger somatotopically organized

cerebellar regions.

Figure 5.1. Schematic diagram of pathways theorized to malfunction in cerebellar-induced dystonia. As depicted previously, there are four main regions currentlyimplicated in the pathophysiology of dystonia: (1) the basal ganglia, (2) the thalamus, (3)the sensorimotor cortex, and (4) the cerebellum. Additional regions involved incerebellar circuitry are included as work presented here implicates disordered cerebellaractivity may induce dystonia through connections with these regions. The uppercerebellar loop (A) is depicted in red and the lower cerebellar loop (B) in blue.

MOTOR CORTEX

SomatosensoryCortex

BasalGanglia

Thalamus

CEREBELLUM

InferiorOlive

Precerebellarnuclei

REDNUCLEUS

DYSTONIA

A

B1

23

4

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Cerebellar Function

Traditional views implicate the cerebellum in the timing and coordination of

movements. Studies in people and animal models after cerebellar lesioning typically

demonstrate relatively mild motor dysfunctions hallmarked by symptoms such as ataxia,

tremor, dysmetria, and other signs of general incoordination. A role in motor learning is

also attributed to the cerebellum and more recently, even cognitive functions have been

attributed to the cerebellum. The overall function of the cerebellum is excitation of

motor pathways after integration of tremendous amounts of afferent information derived

from motor, perceptual, cognitive, and sensory input from the length of the neuraxis.

Cerebellar output through the DCN is tonically active with a resting frequency of

firing at 40-50Hz. This frequency can either increase or decrease during initiation and

execution of movements, modulating the activity of motor pattern generators. Each of

the three DCN divisions acts in different aspects of motor function with slight overlap

occurring. The medial fastigial nuclei are largely involved with postural control and gait.

The interpositus nuclei act in stretch, contact, and other reflexes suggesting functional

involvement in compensatory or corrective movements. The dentate nuclei participate in

initiation of voluntary movements (Fundamental Neuroscience).

Interpositus nuclei

The interpositus nuclei seem most likely to be involved in dystonia as they function in

co-contraction of antagonistic muscles, reciprocal inhibition in reflexes, and motor

response to sensory input. It is important to note however that the interpositus is one of

many regions involved in each of these functions, as would be expected from the

extensive circuitry involved in generation and execution of motor programs. One

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connection between the interpositus nuclei and dystonia is the involvement of the

interpositus in somesthetic behaviors and the abnormal sensory findings in patients with

dystonia. Interpositus neurons modulate their activity in response to sensory feedback.

Studies of tremor accompanying movement demonstrate efferent control of the antagonist

muscle by the interpositus to dampen the tremor. This finding is consistent with the idea

that the interpositus is involved in somesthetic behaviors (Fundamental Neuroscience).

In dystonia, common sensory prodromata may trigger abnormal interpositus activity thus

inducing a dystonia-producing activity. Sensory tricks used to arrest development of

dystonia may also act at the interpositus, which receives prominent somatosensory and

proprioceptive input before sending efferent signals. Alterations of dystonic postures by

various sensory stimuli led the renowned neurologist, Denny-Brown to theorize that

dystonia resulted from an imbalance of reflex responses to natural stimulation, both

tonically and phasically (Denny-Brown D, 1965).

Other work suggests that the interpositus contributes to stretch reflex excitability by

controlling the discharge of gamma motor neurons. Gamma motor neurons modulate

overall sensitivity to stretch through activation of intrafusal muscle fibers, which in turn

affect the tension on the muscle spindle (Fundamental Neuroscience). Impaired

reciprocal inhibition during such reflexes is a common and possibly pathoneumonic sign

in dystonia (Rothwell JC, 1988). While these reflexes are controlled at the spinal,

brainstem, and central levels, it is believed that central disturbance is responsible for the

decreased inhibition of antagonistic muscles after reflex-inducing activation of the

agonistic muscles. Interpositus control of gamma motor neurons therefore may

contribute to the weakened reciprocal inhibition seen in dystonic patients.

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Control of agonist and antagonist muscle contraction around joints is another role of

the interpositus potentially deranged in dystonia. Interpositous ablation causes tremor

supporting the idea that the interpositus is most concerned with balancing the agonist-

antagonist muscle activity of a limb as it moves. Lesioning of the interpositus nucleus

also causes a deficit in the ability to bring a limb back to its hold position after being

disturbed. Interpositus neurons fire when the holding position of a limb is perturbed,

activating antagonist muscles that prevent overshooting the position by contraction of the

agonistic muscle. Thus, the interpositus neurons are involved in the timing of

antagonistic muscle contractions around joints (Fundamental Neuroscience). The

interpositus also acts to determine whether the muscles around a joint will undergo

reciprocal activation or co-contraction. During fine motor behaviors that involve co-

contraction, interpositus neurons fire as if activating both agonist and antagonist muscles,

and PC are silent (Wetts R, 1985). Conversely, the interpositus and PCs fire in similar

patterns during behaviors where agonists and antagonists are sequentially active.

Patterned PC inhibition of interpositus neuron firing results in a similar pattern of activity

in the interpositus neurons, which in turn produces the alternation between contraction of

agonist and antagonist muscles. These data suggest that the cerebellum plays an

important role in switching excitation and inhibition between antagonist muscles (Wetts

R, 1985). The mechanism of such control was presented in review by Richter and

Loscher:

"Parts of the excitatory efferences from the DCN (particularly theinterpositus nucleus) projecting to the ventrolateral thalamus form theinterposito-thalamiccortical path (Mori et al., 1995). There seems to exista reciprocal relationship between the activities of interpositus neurons andcorticospinal neurons. Thus, the activity of interpositus neurons, whichdischarge tonically in the absence of movements and rhythmically during

112

stepping movements, has been found to be precisely in opposite to that ofcorticospinal neurons. It has been concluded that the interpositus nucleusefferents which activate a particular muscle via the rubrospinal pathwaysimultaneously inhibit via the interposito-thalamocortical pathcorticospinal neurons that control the antagonistic muscle (Mori et al.,1995)." (Richter A, 1998)

While these three aspects of interpositus function potentially relate to the

pathophysiology of dystonia, other regions, namely the basal ganglia and motor cortex

have also been implicated in these functions and in dystonia as well. It is unclear under

what circumstances one of these regions dominates the others or how conflicting motor

programs are resolved, if at all. Therefore, the ability of each of these regions to induce

co-contraction, perhaps through the ultimate loss of center-surround inhibition at each

level, truly augments the difficulty of determining the pathophysiology underlying

dystonia.

Pathophysiology of Dystonia

In contrast to the basal ganglia, the cerebellum produces tonic excitatory rather than

inhibitory output. This explains why lesions of the basal ganglia frequently produce

positive motor signs while cerebellar lesions produce negative motor signs. As ablation

of normal activity represents the most common form of neuropathology, these regions are

largely associated positive and negative motor signs respectively. While deficits in

neuronal function are more common, increased activity in a region of the brain can be

produced by selective destruction of inhibitory interneurons or an ectopic focus of

possibly synchronized neuronal activity such as in epilepsy. The work presented here

suggests that an abnormal focus of activity involving the cerebellar cortex produces the

positive motor signs characteristic of dystonia.

Increased or decreased PC activity?

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I believe that within the tottering mouse cerebellum periods of disturbed

neurotransmitter release result in abnormal cerebellar cortical activity that in turn changes

DCN firing patterns to those that produce dystonia. Similarly, profound excitation of the

anterior cerebellar vermis though kainate microinjection also changes cerebellar cortical

activity such that DCN firing patterns elicit dystonia.

This theory becomes more complicated when the activity of the PC is theorized. As

described above, interpositus neurons fire as if activating both agonist and antagonist

muscles and PC are silent during fine motor behaviors that involve co-contraction (Wetts

R, 1985). Therefore, theoretical strong net inhibition of PCs would silence their activity,

allowing interpositus neurons to activate both agonist and antagonist muscles

simultaneously rather than sequentially. Decreased excitatory versus inhibitory input to

PCs in tottering mice may cause sufficient net inhibition and is suggested by data

presented in Chapter 2. In line with this theory, ablation of PCs would also release DCN

from inhibition and cause co-contraction. However, the exact opposite occurs and these

animals fail to demonstrate co-contraction. Failure to induce co-contraction after PC

ablation in the kainate model is also observed. Together these data suggest that either

silencing of PCs is not central to the induction of interpositus neuron firing patterns

causing co-contraction or deafferentation of interpositus neurons in transgenic animals

lacking PCs changes the inherent activity of these cells. PC death may also result in

pathological changes in presynaptic climbing fibers, which send excitatory collaterals to

DCN as they coarse through the granule cell layer. The balance between afferent

collateral excitation and PC inhibition is thought to set the basal rate of DCN discharge.

Therefore, deafferentation effects on the DCN directly or through potential alterations in

114

climbing fibers may reset the basal activity of the DCN. It seems more likely however,

that silencing of PCs is not complete and/or sufficient to induce co-contraction. A

particular motor program may need to be initiated through PC activity initially or for the

duration of DCN output. Thus, PC activity early in the dystonic program or specific

patterns of PC activity throughout may be necessary in the induction of DCN based co-

contraction.

Determining the activity of PCs during tottering and kainate-induced dystonia would

address this theory. While direct measure of PC activity would be ideal, recording such

activity in live mice is difficult. Prominent activation of both PCs and DCN in both

tottering mice and kainate-injected mice during dystonia supports the presence of

patterned activity between PCs and DCN rather than PC silencing. It also seems unlikely

that basket and stellate cell activation after kainate injection could fully overcome direct

excitation of PCs to cause PC silencing in the kainate model of dystonia. An initial

approach in determining whether PC firing patterns are likely increased or decreased in

tottering dystonia was discussed above under "future studies" (Chapter 5.1). Effects on

tottering dystonia after administration of GABA agonists or antagonists through an

indwelling catheter to the cerebellar cortex would reveal whether PC firing patterns are

likely increased or decreased during attacks. Cessation of an ongoing attack after

application of a GABA agonist would suggest the necessity of PC activity during the

expression of dystonia. Conversely, termination of an attack due to GABA antagonist

application would imply a need for silencing PC activity during dystonia.

The confusion over why inhibitory cerebellar cortical output through the PC is

necessary for the production of dystonia when disinhibition of the target DCN is likely

115

involved in the expression of the dystonia is paralleled within the basal ganglia circuitry.

The Gpi/SNr output of the basal ganglia is one of tonic inhibition on the thalamus,

analogous to the effect PC output has on DCN. Disinhibition of the thalamus by

pathologically decreased output of the basal ganglia is traditionally thought to cause

dystonia. However, surgically-induced GPi lesions can improve dystonia symptoms.

Improvement of dystonia after removal of the inhibitory structure (PC or Gpi) when the

activity level of that structure is theorized to be reduced by the disease process itself is

perplexing.

A more complex theory was presented in the introduction with the conclusion that

temporal and/or spatial fluctuations in Gpi/SNr activity are responsible for dystonia

(Crossman AR, 1998). Similarly, the effect of the PC derangement on DCN output

resulting in dystonia likely represents disordered activity rather than simply decreased

activity as well. This theoretical analogy is depicted in Figure 5.2. In the cerebellar-

induced dystonia systems presented in this work, removal of the cerebellar PC output

through transgenic loss of PC in both tottering and kainate models restores the normal

state. This is similar to the effect surgical Gpi ablation has on the analogous basal

ganglia system.

116

Basal Ganglia Cerebellum

Gpi Gpi Gpi PC PC PC

Thalamus Thalamus Thalamus DCN DCN DCN

Normal Dystonic Treated Normal Dystonic Treated

Figure 5.2. Comparison of release of inhibition treating dystonia in the basal ganglia andthe cerebellum. Depiction of globus pallidus (Gpi) inhibition of tonically active thalamusin dystonia and Purkinje cell (PC) inhibition of tonically active deep cerebellar nuclei(DCN) in dystonia. Destruction of somehow aberrant inhibitory signals returns eachsystem to its normal state.

Summary

The work presented here suggests that an abnormal focus of activity involving the

cerebellar cortex produces the positive motor signs characteristic of dystonia..

Furthermore, it is theorized that the resultant DCN activity, particularly that of the

interpositus, acts to induce involuntary co-contraction of antagonistic muscles by

disordered activation of opposing muscles through the red nucleus and cerebral cortex.

The DCN may fail to coordinate activity of agonistic and antagonistic muscles controlled

by the rubrospinal and corticospinal tracts respectively. The mechanism within the

cerebellar cortex resulting in pathologic DCN firing probably involves failure of center-

surround inhibition of PCs. The locus ceruleus, in turn, may attempt to correct ongoing

?

?

?

?

117

co-contraction through NE- mediated focusing of the center-surround mechanism, but to

no avail. Similar derangements occurring in the basal ganglia and motor cortex may

result in dystonia. However, the abundance and consistency of cerebellar findings in

studies of dystonia in addition to the role of the cerebellum in normal motor function,

suggests a role for this structure in the expression if not the origin of the dystonia-

producing signal.

118

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1

Carolyn E. Pizoli

EDUCATIONPennsylvania State University College of Medicine, Hershey, Pennsylvania 1996-2003M.D./Ph.D. degree candidate, anticipated graduation date: May 2003

Goucher College, Towson, Maryland 1992-1996B.A. in Biological Sciences and Chemistry earned in 1996Degree with Distinction and Honors in Biological Sciences and Chemistry

RESEARCHPenn State University College of Medicine 1998-2001Graduate Thesis Research. Conducted research in the neuroscience laboratory of Dr. Ellen Hessto determine the pathophysiology of dystonia. The role of the cerebellum in a genetic mousemodel of dystonia, tottering, and in a novel model using wild type mice was analyzed usingbiochemical, cellular, pharmacological and behavioral approaches.

Goucher College 1995-1996Independent Research. Conducted senior independent research project to determine effects ofDNA conformation on RNA polymerase kinetics.

Johns Hopkins University Summer 1995Research Fellow. Planned and conducted experiments analyzing RNA polymerase kinetics undera Howard Hughes research fellowship.

University of Maryland School of Medicine Summer 1994Research Fellow. Conducted experiments to clone the sodium/calcium exchanger gene in squidunder a Summer Undergraduate Research Fellowship.

HONORS/AWARDS The Stimson-Duvall Fellowship 1996

The Gairdner Moment Prize in Biology 1996The Louise Kelly Prize in Chemistry 1996Grace T. Lewis Merit Scholarship 1992-1996

PUBLICATIONS / ABSTRACTSCarolyn Pizoli, Hyder Jinnah, Melvin Billingsley, and Ellen Hess. Abnormal Cerebellar SignalingInduces Dystonia in Mice. Journal of Neuroscience. Sept. 1, 2002, 22(17): 7825-7833.

Carolyn Pizoli, Corey Hart, Steven Dear, Melvin Billingsley, and Ellen Hess. CerebellarActivation Induces Dystonia in Mice. Society for Neuroscience Abstracts. 2001, 27: #294.12

Carolyn Pizoli and Ellen Hess. Selective Elimination of Purkinje Cells in the Tottering Mouse.Society for Neuroscience Abstracts. 2000, 26:#135.6

Carolyn Pizoli and Ellen Hess. Potassium Stimulated Release of 3H-Glutamate from TotteringMouse Cerebellar Slices. Society for Neuroscience Abstracts. 1999, 25:#286.6

ACTIVITIES

Physician Scientist Student Association, co-president (1997-1999) 1996-presentChildlife volunteer, Hershey Medical Center Summer 1997

Shock Trauma Unit volunteer, University of Maryland Hospital Summer 1995Varsity Swim Team, Goucher College 1992-1993Special Olympics Swim Coach, Pittsburgh, PA 1990-1991