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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. Hess Associate Professor of Neurology Thesis Co-Advisor Co-Chair of Committee ______________________ ____________________________________ Melvin L. Billingsley Professor of Pharmacology Thesis Co-Advisor Co-Chair of Committee ______________________ ____________________________________ Robert Levenson Professor of Pharmacology Director, Cell and Molecular Biology Program ______________________ ____________________________________ James Connor Professor of Neuroscience ______________________ ____________________________________ Teresa Wood Associate 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. iv Table of Contents Chapter Page List of Tables vi List of Figures vii 1 Introduction/Literature Review 1.1 Dystonia in Humans Introduction Classification Pathophysiology Treatment Paroxysmal Dyskinesias 1.2 Tottering Mouse: Animal Model of Dystonia Introduction Calcium Channels Behavioral Phenotype Cellular Pathology 1.3 Chapter Summary 1.4 Hypotheses 1 1 1 2 3 12 13 19 19 19 23 26 27 28 2 Altered Neurotransmission in the Tottering Cerebellum Abstract Introduction Materials and Methods Results Discussion 30 30 30 35 37 40 3 Role of Purkinje Cells in the Expression of Tottering Dystonia Abstract Introduction Materials and Methods Results Discussion 45 45 46 48 51 58 4 Role of the Cerebellum in a Novel Animal Model of Dystonia in Wild Type Mice Abstract Introduction Materials and Methods Results Discussion 63 63 64 65 71 86 v 5 Discussion 5.1 Mouse Models of Dystonia The Tottering Mouse Kainate-Induced Dystonia 5.2 The Cerebellum and Dystonia Cerebellar Circuitry Cerebellar Function Pathophysiology of Dystonia 87 87 87 101 104 104 107 110 REFERENCES 118 vi List of Tables Table Page 1.1 Familial dystonic syndromes for which gene loci have been identified 3 1.2 Subgrouping of the paroxysmal dyskinesias 14 1.3 Classification of neuronal high-voltage activated calcium channels 22 3.1 Genotypes of progeny generated in SV4+/-;+/tg X +/+;tg/tg crosses 53 5.1 Comparison of salient features of tottering dystonia and PNKD 98 5.2 Comparison of the salient features of the kainate model of dystonia and PKD 103 vii List of Figures Figure Page 1.1 Schematic depiction of brain regions and pathways theorized to malfunction in dystonia 9 1.2 Schematic depiction of a voltage-gated calcium channel 21 2.1 Schematic diagram of neuronal connections in the cerebellum 34 2.2 3 38 2.3 Comparison of tottering and wild type peak 3H-glutamate release 38 2.4 3 39 2.5 Comparison of tottering and wild type peak 3H-GABA release 39 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 over time 56 3.4 Regional loss of dystonic phenotype in F7 transgenic tottering mice 57 3.5 Calbindin mRNA in situ hybridization 58 4.1 Typical dystonic postures after cerebellar kainate injection in wild type mice 71 4.2 Dose-response and dose-recovery curves after cerebellar injection of kainic acid in wild type mice 73 4.3 c-fos in situ hybridization in wild type mice after cerebellar kainate injection 75 4.4 Regional expression of c-fos mRNA after cerebellar kainate injection 76 4.5 Cerebellar injection site localization 77 H-Glutamate release from cerebellar synaptosomes H-GABA release from cerebellar synaptosomes viii 4.6 Dystonic severity in transgenic mice lacking Purkinje cells 81 4.7 Representative EEG recordings from wild type mice receiving cerebellar microinjections of kainic acid 83 4.8 Dystonic severity after cerebellar NBQX co-injection with kainic acid 85 4.9 Dystonic severity after cerebellar injection of domoic acid in wild type mice 86 5.1 Schematic diagram of pathways theorized to malfunction in cerebellar-induced dystonia 108 5.2 Comparison of release of inhibition treating dystonia in the basal ganglia and the cerebellum 116 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. 2 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 dystoniaplus 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 DYT1 Location 9q34.1 Origin Ashkenazi jews and non-jewish European decent Syndrome Early-onset primary dystonia (Dystonia musculorum deformans) Protein ATP-binding protein DYT3 Xq13 Philippines Lubag (X-linked Parkinsonism-dystonia) ? DYT5 (DRD) 14q22.1 -- Dopa-responsive dystonia (DRD) GTPcyclohydrolase I gene, TH Ichinose, 1994; Knappskog, 1995 DYT6 8p21-q22 Mennonite/Amish Mixed: Childhood/Adult with limb or cranial onset ? Almasy, 1997 DYT7 18p German kindred Adult onset torticollis ? Leube, 1996 -- 2q33-35 Polish-American Paroxysmal nonkinesegenic dyskinesia ? suspected Na+ channel Fouad, 1996 Fink, 1996 Choreoathetosis/ ? suspected K+ spasicity, episodic (CSE) channel *italicized genes denote non-primary dystonia syndromes; TH, Tyrosine Hydroxylase -- 1p German kindred References Ozelius, 1989; Kramer 1990; Kwiatkowski, 1991 Eidelberg, 1993 Auburger, 1996 Table 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 druginduced 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 6 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. 8 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. 9 3 MOTOR CORTEX DYSTONIA Somatosensory Cortex 2 THALAMUS 1 BASAL GANGLIA Gpi/SNr 4 CEREBELLUM Striatum Figure 1.1. Schematic depiction of brain regions and pathways theorized to malfunction in dystonia. There are four main regions currently implicated in the pathophysiology of dystonia: (1) the basal ganglia, (2) the thalamus, (3) the sensorimotor cortex, and (4) the cerebellum. 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 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 nonmanifesting 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 11 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. 12 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, 13 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 14 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 exerciseinduced 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 PKD (PKC) Characteristics Brief duration (seconds to 5 minutes) Always preceded by sudden initiation of movement PNKD (PDC) Prolonged duration (2 minutes to 4 hours) Spontaneous or triggered by various stressors PED 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 Dystonic Choreoathetosis Table 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 ?2 ?2 ? extracellular ?1 intracellular ? Figure 1.2. Schematic depiction of voltage-gated calcium channel. The ? 1 subunit is the 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. Highvoltage activated calcium channels involved in neurotransmitter release (P, Q, N and Rtype) 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 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 ? 1A-a Subtype P Pharmacology ? -agatoxin IVA Location cerebellum, hippocampus, inferior colliculus olfactory bulb, spinal cord Function Neurotransmitter release ? 1A-b Q ? -conotoxin MVIIC ? 1B N ? -conotoxin MVIIA, ? conotoxin GIVA diffuse, hippocampus Neurotransmitter release ? 1C L diffuse, olfactory bulbs, hippocampus, superior colliculus, cerebellum Electrical excitability, gene transcription L dihydropyridines, benzothiazepines, and phenylalkylamines ? 1D ? 1E R cadmium, nickel olfactory bulb, habenula, Neurotransmitter cortex, hippocampus, release cerebellum (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 1830 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/Qtype 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 dystoniaproducing 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 THE TOTTERING 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 3 H-GABA and then superfused with Earle’s balanced salt solution. Neurotransmitter release was induced by depolarization with 60mM KCl. Potassium-stimulated release of 3 H-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 highvoltage 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/Qtype 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 homoand 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. Extracerebellar Nuclei Climbing Fiber Mossy Fiber GC PC DCN B S G Figure 2.1. Schematic diagram of neuronal connections in the cerebellum. Arrowheads indicate excitatory neurotransmission of glutamate and flat bars represent GABAergic inhibitory neurotransmission. GC, granule cell; PC, Purkinje cell; G, golgi cell; S, stellate cell; B, basket cell; DCN, deep cerebellar nuclei. 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 = Release DPM of Fraction (Total DPM – DPM collected in prior fractions) x 100 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 3 H-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). 3 H-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 40 Averege % Fractional Release 35 wt +K/+Ca tg +K/+Ca wt +K/-Ca tg +K/-Ca 30 25 20 15 10 5 0 1 2 3 4 5 Fraction 6 7 8 9 Figure 2.2. 3H-Glutamate Relesase From Cerebellar Synaptosomes. Buffers were perfused over cerebellar synaptosomes loaded with 3H-glutamate at a rate of 0.5ml/min and collected in 9 three-minute fractions. The horizontal line in fraction 3 indicates time of stimulation buffer perfusion containing 60mM KCl/1.8mM CaCl2 in closed symbols and 60mM KCl without CaCl2 in open symbols. Wild type animals (n=9) are represented by squares and tottering animals (n=9) by triangles. Average Peak % Fractional Release 50 45 40 ** 35 30 25 20 15 10 5 0 wt tg 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 3Hglutamate release for every animal. The peak release was then averaged for the 9 animals in each group and compared using the Student's t-test. Data represent mean %-fractional release + SEM. ** indicates significant decrease in tottering release, p<0.005. 39 50 45 wt +K/+Ca wt +K/-Ca tg +K/+Ca tg +K/-Ca % Fractional Release 40 35 30 25 20 15 10 5 0 1 2 3 4 5 6 7 8 9 Fraction Figure 2.4. 3H-GABA Relesase From Cerebellar Synaptosomes. Buffers were perfused over cerebellar synaptosomes loaded with 3H-GABA at a rate of 0.5ml/min and collected in 9 three-minute fractions. The horizontal line in fraction 3 indicates time of stimulation buffer perfusion containing 60mM KCl/1.8mM CaCl2 in closed symbols and 60mM KCl without CaCl2 in open symbols. Wild type animals (n=6) are represented by squares and tottering animals (n=6) by triangles. 60 Average Peak % Fractional Release 55 50 45 40 35 30 25 20 15 10 5 0 wt tg Figure 2.5. Comparison of Tottering and Wild Type Peak 3H-GABA Release. Percent fractional release was summed over two fractions after correction for basal 3H-GABA release for every animal. The peak release was then averaged for the 6 animals in each group and compared using the Student's t-test, p>0.05. Data represent mean %-fractional release + SEM. 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 coexpressed 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 Ltype 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 THE EXPRESSION 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 postdevelopmentally 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. Fourweek-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 D0 D1 D2 D3 D4 D5 Behavioral Phenotype no motor abnormalities slightly slowed or abnormal motor behavior rare, isolated dystonic postures seen mild motor impairment due to dystonic postures moderate impairment due to dystonic postures 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 L 1 2 3 4 5 6 7 8 9 1 kB Figure 3.1. PCR genotyping for presence of the SV40 transgene. Ear punch DNA was used as template to amplify the 1076bp fragment of the transgene using primers specific for the pcp-2 promotor and the SV40 T antigen gene. Lanes 1,2,5,6,7,and 9 contain the amplified transgene fragment; L denotes 1 kB ladder. A Genotype SV4+/-;tg/tg SV4-/-;tg/tg SV4+/-;+/tg SV4-/-;+/tg Total Male 2 3 3 1 9 Female 4 3 2 1 10 Total 6 (31.6) 6 (31.6) 5 (26.3) 2 (10.5) 19 Genotype SV4+/-;tg/tg SV4-/-;tg/tg SV4+/-;+/tg SV4-/-;+/tg Total Male 5 7 3 2 17 Female 5 8 8 3 24 Total 10 (24.4) 15 (36.6) 11 (26.8) 5 (12.2) 41 B 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 by PCR of the SV40 transgene and phenotyping of the tottering homozygous state. Percent of total mice for each genotype is indicated in parentheses. 54 35 30 Weight (grams) 25 20 15 SV4/tg SV4 tg wt 10 5 0 4 7 10 13 16 19 Age (weeks) Figure 3.2. Body weights of F7 generation progeny. Weights were averaged for all mice in each genotype. Transgenic tottering mice (SV4/tg) were healthy and viable with body weights similar to control genotypes: wild type (wt), transgenic (SV4), and tottering (tg). Single-factor ANOVA analysis revealed no significant difference in weight between genotypes (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 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 35 AA 30 Total D Score 25 20 15 10 5 0 3 6 9 12 15 18 21 24 Age (weeks) 35 BB 30 Total D Score 25 20 15 10 5 0 3 6 9 12 15 18 21 24 Age (weeks) Figure 3.3. Loss of dystonic phenotype in transgenic tottering mice over time. In the F3 generation (A), all transgenic tottering animals (blue circles) were non-dystonic by 19 weeks of age. Similar results were obtained in the F7 generation (B), with a greater discordance between age of phenotype loss between males (blue circles) and females (red squares) however. Control tottering mice (pink diamonds) maintained a high level of dystonia in both experiments (A and B). Linear regression lines are shown for each grouping. 57 6 6 Head 5 5 4 4 3 3 ** 2 2 1 Dystonic Bins Neck ** 1 ** 0 4 7 10 13 16 *** *** *** 19 4 5 4 4 * 2 *** 13 16 19 22 Limbs * 1 *** 0 10 13 2 1 7 10 3 ** 4 7 6 5 3 *** 0 22 Trunk 6 *** 16 19 0 22 4 7 10 13 16 19 22 Age (weeks) 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 total time bins) and summed by region. Data are presented as number of bins positive for dystonia by age (weeks) for each region. Transgenic tottering mice (blue bars) lost the characteristic 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 compared between genotypes using the Student's t-test at each age. Transgenic tottering mice were sacrificed after disappearance of all dystonia and these animals were given a minimal score 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 58 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. a c e g b d f h Figure 3.5. Calbindin mRNA in situ hybridization. Cerebella from F3 transgenic tottering animals and two age-matched control tottering animals were hybridized with calbindin antisense mRNA. Transgenic tottering animals (a-f) show marked loss of PC in comparison to control tottering animals (g-h). Within the transgenic group, increasing age and female sex are correlated with greater loss of PC: a, 23 week old female, b, 26 week old male, c, 29 week old female, d, 18 week old female, e, 24 week old male, f, 15 week 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 60 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 1116) 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 61 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 NOVEL ANIMAL 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 cfos, 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 cocontraction 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 doseresponse 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 D0 D1 D2 D3 D4 D5 D6 Behavioral Phenotype no motor abnormalities no impairment; slightly slowed or abnormal (but not dystonic) motor behavior mild impairment; sometimes limited ambulation unless disturbed, dystonic postures when disturbed moderate impairment; frequent spontaneous dystonic postures severe impairment; sustained dystonic postures and limited ambulation prolonged immobility in dystonic postures 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 2mercaptoethanol 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 postsurgery, 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 a flattened, arched trunk with hindlimbs abducted and held out from the body. The photo on the left also shows some mild flexion of the neck while the photo on the right illustrates 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 6 A Average D score 5 4 3 2 1 2 R = 0.98 0 0 50 100 150 200 250 300 350 Dose KA (pmoles) 6 B 0pmol 25pmol 60pmol 115pmol 175pmol 235pmol 350pmol 5 D score 4 3 2 1 0 0 10 20 30 40 50 60 70 minutes 80 90 100 110 120 Figure 4.2. Dose-response and dose-recovery curves after cerebellar injection of kainic acid in wild type mice. Figure A shows the increased severity of dystonia elicited by increasing dosage of kainic acid. The average D-score was taken at 30 minutes after injection from 5-6 mice per dosage. Error bars represent SEM and R2 value of 0.98 is shown. Figure B illustrates the recovery of the mice to non-dystonic behavior within 2 hours of injection (5-6 mice per dosage). 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 twohour 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 0pmoles 60pmoles 115pmoles 235pmoles Striatum and Cortex Hippocampus Red Nucleus Locus Ceruleus Cerebellum Figure 4.3. c-fos in situ hybridization in wild type mice after cerebellar kainate injection. Representative sections from regions of c-fos activation and other areas of interest are shown from mice receiving 0, 60, 115, or 235 picomoles kainic acid in a volume of 0.5? l injected into the anterior cerebellar vermis. Mice receiving kainic acid displayed dystonic behavior in a dose-dependent manner. 76 0.45 0pmol 60pmol 115pmol 235pmol 0.40 0.35 Optical Density 0.30 0.25 0.20 0.15 0.10 0.05 0.00 ctx str hip rn lc cb-ant cb-post Figure 4.4. Regional expression of c-fos mRNA after cerebellar kainate injection. In situ hybridization of c-fos mRNA was quantified two hours after injection into the cerebellum. Data presented are the mean of four animals at each dosage and error bars represent SEM. cb-ant, anterior cerebellum, cb-post, posterior cerebellum, rn, red nucleus, lc, locus ceruleus, str, striatum, hip, hippocampus, ctx, primary motor cortex. 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 and represented as symbols on representative sections modified from Franklin and Paxanos, 1997. Millimeters from bregma are shown for each section. Kainate dosages shown in legend 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 80 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). 81 45 40 35 Average Total D Score 30 25 *** 20 15 10 5 0 WT-0 WT-235 SV4-0 SV4-235 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 cerebellar vermis and observed for two hours for dystonic postures (SV4-235). Transgenic animals were injected with saline (n=6) to demonstrate the basal level of motor disturbance in these animals (SV4-0). D score was summed over the two-hour observational period and averaged per group. Wild type data is illustrated for comparison and is the same as presented above in figure 4.2 (saline n=5, WT-0; kainate n=6, WT-235). *** indicates significantly reduced dystonia in SV4 mice compared to wild type animals, p<0.0005 using the Student's t-test. 82 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. 83 A. Baseline B. Kainate-Induced Dystonia C. PTZ-induced seizure Figure 4.7. Representative EEG recordings from wild type mice receiving cerebellar microinjections of kainic acid. EEGs shown were recorded before kainic acid injection (A), during profound dystonic posturing after kainic acid injection (B), or during seizures induced after pentylenetetrazole (PTZ) injection (C). 84 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). 85 50 Average Total D Score 40 30 ** 20 10 0 NBQX 235 KA Figure 4.8. Dystonic severity after cerebellar NBQX coinjection with kainic acid. Mice were injected with NBQX (2.35 nmoles) alone and in combination with 235pmoles kainic acid. Total D score was summed over two hours of observation and averaged for 56 mice in each drug paradigm. ** indicates p<0.005 between kainate with and without NBQX. NBQX+235KA 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 86 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, semiflattened 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. Average Total D Score 50 40 30 20 10 0 saline kainate domoate Figure 4.9. Dystonic severity after cerebellar injection of domoic acid in wild type mice. Domoate (7.5 pmoles) was injected into the anterior cerebellar vermis of five C57Bl/6J mice and dystonic behavior recorded. D score was summed over two hours immediately following injection and averaged for 5-6 mice pre group. Saline and kainate (235 pmoles) data is the same as that presented 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 87 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. 88 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 89 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 90 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 91 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 92 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 93 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 94 (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 (RodriguezMoreno 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, 95 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. 97 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. 98 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 Attack Duration Attack Frequency Tottering Mouse 30-60 minutes Unknown Occurrence Common Triggers Other Spontaneous Stress, caffeine, alcohol Severity may wax and wane during attacks No discernable refractory period + L-type Ca2+ channel antagonists + benzodiazepines - anticonvulsants ? utosomal recessive inheritance ? 1A VDCC subunit mutation Drug Response Genetics Pathophysiology Strong evidence for role of aberrant cerebellar signaling PNKD 30-60 minutes 2-3/month (range from 2-3/day to 23/year) Spontaneous Stress, caffeine, alcohol, fatigue Severity may wax and wane during attacks No discernable refractory period + benzodiazepines (moderate effect) - anticonvulsants Autosomal dominant inheritance Linkage to 2q33-35, in area of suspected ion channel Unknown Table 5.1. Comparison of salient features of tottering dystonia and PNKD. 99 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 cfos 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 100 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 101 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 102 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 103 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 Attack Duration Attack Frequency Occurrence Drug Response Pathophysiology Kainate Model Seconds to minutes ~10-100 attacks in 2 hours after injection Initiation of movement, startle Not tested Aberrant cerebellar excitation PKD Seconds to minutes 1-10/day (up to 100/day reported) Initiation of movement, startle + anticonvulsants Unknown Table 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 104 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. 105 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 106 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. 107 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. DYSTONIA MOTOR CORTEX 3 2 Somatosensory Cortex Thalamus 1 A Precerebellar nuclei B Basal Ganglia 4 RED NUCLEUS CEREBELLUM Inferior Olive Figure 5.1. Schematic diagram of pathways theorized to malfunction in cerebellarinduced dystonia. As depicted previously, there are four main regions currently implicated in the pathophysiology of dystonia: (1) the basal ganglia, (2) the thalamus, (3) the sensorimotor cortex, and (4) the cerebellum. Additional regions involved in cerebellar circuitry are included as work presented here implicates disordered cerebellar activity may induce dystonia through connections with these regions. The upper cerebellar loop (A) is depicted in red and the lower cerebellar loop (B) in blue. 109 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 110 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. 111 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 agonistantagonist 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 cocontraction, 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 the interpositus nucleus) projecting to the ventrolateral thalamus form the interposito-thalamiccortical path (Mori et al., 1995). There seems to exist a reciprocal relationship between the activities of interpositus neurons and corticospinal neurons. Thus, the activity of interpositus neurons, which discharge tonically in the absence of movements and rhythmically during 112 stepping movements, has been found to be precisely in opposite to that of corticospinal neurons. It has been concluded that the interpositus nucleus efferents which activate a particular muscle via the rubrospinal pathway simultaneously inhibit via the interposito-thalamocortical path corticospinal 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? 113 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 cocontraction. 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 cerebellarinduced 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 Gpi Gpi Cerebellum Gpi PC Thalamus Thalamus DCN ? Normal Dystonic PC ? ? Thalamus PC DCN DCN ? Treated Normal Dystonic Treated Figure 5.2. Comparison of release of inhibition treating dystonia in the basal ganglia and the cerebellum. Depiction of globus pallidus (Gpi) inhibition of tonically active thalamus in dystonia and Purkinje cell (PC) inhibition of tonically active deep cerebellar nuclei (DCN) in dystonia. Destruction of somehow aberrant inhibitory signals returns each system 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. 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Wetts R, Kalaska JF, and Smith AM. 1985. Cerebellar nuclear cell activity during antagonistic cocontraction and reciprocal inhibition of forearm muscles. Journal of Neurophysiology. 54(2):231-244. Carolyn E. Pizoli EDUCATION Pennsylvania State University College of Medicine, Hershey, Pennsylvania M.D./Ph.D. degree candidate, anticipated graduation date: May 2003 Goucher College, Towson, Maryland B.A. in Biological Sciences and Chemistry earned in 1996 Degree with Distinction and Honors in Biological Sciences and Chemistry 1996-2003 1992-1996 RESEARCH Penn State University College of Medicine 1998-2001 Graduate Thesis Research. Conducted research in the neuroscience laboratory of Dr. Ellen Hess to determine the pathophysiology of dystonia. The role of the cerebellum in a genetic mouse model of dystonia, tottering, and in a novel model using wild type mice was analyzed using biochemical, cellular, pharmacological and behavioral approaches. Goucher College 1995-1996 Independent Research. Conducted senior independent research project to determine effects of DNA conformation on RNA polymerase kinetics. Johns Hopkins University Summer 1995 Research Fellow. Planned and conducted experiments analyzing RNA polymerase kinetics under a Howard Hughes research fellowship. University of Maryland School of Medicine Summer 1994 Research Fellow. Conducted experiments to clone the sodium/calcium exchanger gene in squid under a Summer Undergraduate Research Fellowship. HONORS/AWARDS The Stimson-Duvall Fellowship The Gairdner Moment Prize in Biology The Louise Kelly Prize in Chemistry Grace T. Lewis Merit Scholarship 1996 1996 1996 1992-1996 PUBLICATIONS / ABSTRACTS Carolyn Pizoli, Hyder Jinnah, Melvin Billingsley, and Ellen Hess. Abnormal Cerebellar Signaling Induces Dystonia in Mice. Journal of Neuroscience. Sept. 1, 2002, 22(17): 7825-7833. Carolyn Pizoli, Corey Hart, Steven Dear, Melvin Billingsley, and Ellen Hess. Cerebellar Activation 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 Tottering Mouse Cerebellar Slices. Society for Neuroscience Abstracts. 1999, 25:#286.6 ACTIVITIES Physician Scientist Student Association, co-president (1997-1999) Childlife volunteer, Hershey Medical Center Shock Trauma Unit volunteer, University of Maryland Hospital Varsity Swim Team, Goucher College Special Olympics Swim Coach, Pittsburgh, PA 1 1996-present Summer 1997 Summer 1995 1992-1993 1990-1991