118,95-104

EXPERIMENTALNEUROLOGY

(1992)

Abnormal Cerebellar Output in Rats with an Inherited Movement Disorder JOAN F. LORDEN,JACQUELINE Department

of Psychology

and the Neurobiology

Research

LUTES, VICTORIA L. MICHELA,ANDJEFFERVIN Center,

University

of Alabama

at Birmingham,

Birmingham,

Alabama

35294

consistent differences from normal in absolute glucose utilization. The DCN are also the only sites at which significant alterations in glutamic acid decarboxylase (GAD) activity and GABA receptor number have been identified in comparisons between dystonic and normal rats (5). The cells of the DCN project widely to brainstem, thalamus, and motor cortex (53). Abnormal function of the cells of the DCN could trigger a cascade of events that might contribute significantly to the movement disorder displayed by the mutant. Studies of glucose utilization are useful in identifying sites of abnormality, but do not specify the nature of the abnormality. The DCN receive a massive inhibitory input from the Purkinje cells, the output neurons of the cerebellar cortex (26). Although the Purkinje cell projection to the DCN is inhibitory, the cells of the DCN also receive excitatory collateral fibers from the climbing fiber and mossy fiber projections to the cerebellar cortex (17,35,52). The abnormal increase in glucose utilization found in the DCN of the mutants (9) is assumed to be presynaptic (29) but may represent either increased inhibition or increased excitation. Thus, we have recorded spontaneous activity from cells in the medial and interpositus nuclei of normal and dystonic rats. In order to assessthe significance of any differences in DCN neuronal activity for transmission of information through the cerebellum, the response of DCN cells to harmaline administration was also recorded in normal and dystonic rats. The genetically dystonic rat is known to be insensitive to the tremorogenic effects of harmaline, but not other tremor-causing agents (33,39). The importance of this observation lies in the fact that harmaline is thought to induce tremors by activating the cells of the inferior olive (14, 30, 31). The olivary neurons act as a pacemaker for this tremor. Activation of the climbing fiber pathway from the inferior olive to the cerebellar cortex changes the slow, irregular firing of Purkinje cell complex spikes to a faster, regular pattern of activity. Simple spike activity is suppressed, particularly in the vermis, and sustained rhythmic bursts of activity are detected in the DCN and the bulbar reticular formation (14, 30, 31). The selective insensitivity to harmaline displayed by the dystonic rat suggests the presence of a functional

Biochemical and metabolic mapping techniques have consistently identified the deep cerebellar nuclei (DCN) of the genetically dystonic rat as a site of abnormality. Extracellular single-unit recording techniques were used to assess the functional significance of these findings in affected rats and normal littermates between 16 and 25 days of age. Cells in the medial nucleus of the mutant rats had significantly increased spontaneous firing rates in comparison with cells from normal rats. In both the medial and the interpositus nuclei, cells from the mutants fired more rhythmically than those from the normal rats. When harmaline was administered systemically to activate the olivo-cerebellar system, in normal rats, increased firing rate and bursting patterns of activity were seen. There was no reliable change in the average firing rate or rhythmicity of cells in the medial nucleus of the dystonic rats, although previous studies have shown that harmaline activates neurons in the inferior olive in the mutants. It is likely that naturally stimulated olivary activity also fails to modulate cerebellar output in this model of inherited movement disorder. Anatomical studies did not reveal any consistent changes in the number of Purkinje cells, the volume of the DCN, or the soma size of DCN neurons. Since the electrophysiological findings cannot be ascribed to a loss of the Purkinje cells that normally provide an inhibitory input to the cerebellar nuclei, the results of this study indicate the presence of a functional defect in the control of cerebellar output in the dystonic rat that accounts for the failure of these animals to display harmaline tremor and which may be critical to the motor syndrome. 0 is92 Academic press, inc.

INTRODUCTION The movement disorder in the genetically dystonic rat includes twisting movements of the limbs and neck, hyperextension of the limbs, and hyperflexion of the trunk (32). Although the nervous system of the mutant appears to be morphologically intact at the light microscopic level, a recent study of glucose utilization identified the deep cerebellar nuclei (DCN) as a potential focus of abnormality (9). Of the 62 sites examined in that study, the DCN were among the few that showed

95 All

Copyright 0 1992 rights of reproduction

0014-48&3/92 $5.00 by Academic Press, Inc. in any form resewed.

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defect in the olivo-cerebellar system of the mutants. In addition, this pharmacological observation offers a means of testing the functional integrity of the olivocerebellar system. In previous studies, harmaline administration in the dystonic rat has been shown to produce a reliable increase in both the frequency and rhythmicity of single-unit activity in the caudal medial accessory olive (cMA0) (48). This effect is indistinguishable from the response of cells from normal rats. These observations indicate that in dystonic rats, harmaline is reaching its central nervous system site of action in sufficient quantities to appropriately activate a population of cells critical to the generation of the tremor. The response of the dystonic rats to harmaline that is evident in the inferior olive appears to deteriorate at the level of the cerebellar cortex. Because each Purkinje cell receives input from a single olivary axon, in normal animals there is a one-to-one relationship between the complex spike activity of the Purkinje cells and the activity of climbing fibers from the inferior olive (16). In normal rats, harmaline administration significantly increases the frequency and rhythmicity of complex spikes in 7080% of the cells in the vermis (6,49). The average firing rate for complex spikes closely matches that of cells in the cMA0 (48). In dystonic rats, although olivary neurons are activated, relatively few (10%) verma1 Purkinje cells were identified that showed a normal response to harmaline (49). This suggests that there is a failure in neurotransmission between the inferior olive and the Purkinje cells of the mutant rats that could affect the output of the cerebellum. The present study evaluated this idea directly by asking whether a normal change in firing in DCN cells could be detected in response to harmaline in the dystonic rat. Since the results of the electrophysiological study suggested increased activity in the cells of the mutant DCN, quantitative anatomical techniques were used to provide a more detailed evaluation of the morphological integrity of the cerebella-nuclear projection. In normal and dystonic rats, Purkinje cells were counted to determine whether a partial loss of cells projecting to the DCN might account for the increase in firing rate observed in the mutant rats. In addition, the DCN were reconstructed on the assumption that a reduction in inhibitory terminals might alter the volume of the nuclei. Finally, the soma size of cells in the DCN was measured. Studies in mice with mutations affecting the cerebellum have shown that these measures are sensitive to changes in afferent input to the DCN (12,23,40,44,51). MATERIALS

AND

METHODS

Animals used in these experiments came from a colony of Sprague-Dawley-derived rats at the University of Alabama at Birmingham. Dystonic rats were obtained by breeding heterozygotes. Both males and fe-

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males were used. The phenotype of the rats can be unequivocally determined by Postnatal Day 12 and the disease progresses rapidly. By 20-25 days of age, the clinical signs are severe. In order to avoid the use of animals in deteriorating general health, only rats between 16 and 25 days of age were studied.

Electrophysiology. Studies were conducted in 33 laboratory-bred dystonic and 31 normal littermate control rats. Parylene-coated stainless steel microelectrodes with impedances of approximately 2 Mohm (Microprobe, Inc.) were used to record single-unit activity from urethane-anesthetized rats (1.9 mg/g). Body temperature was maintained with Deltaphase heating pads (Braintree Scientific). For recordings in the DCN, a small hole (l-2 mm diameter) was drilled over the cerebellum at predetermined stereotaxic coordinates and the dura was punctured. An electrode was lowered through the cerebellar cortex with a hydraulic microdrive to a depth of approximately 3500 pm from the surface of the brain. Neural activity was monitored as the electrode passed through layers of Purkinje cells. DCN cells were identified by their location and the absence of complex spike activity. Unit activity was amplified by a BAK A-l high impedance amplifier and monitored on an oscilloscope and an audio monitor. Unit activity was digitized (Neuro Data Instruments Corp. Neurocorder) and stored on VHS tape for offline analysis. When a cell was isolated, lo-15 min of predrug activity was recorded. Harmaline (15 mg/kg) was then injected through a previously implanted Silastic jugular catheter. This dose was well in excess of that needed to activate rhythmic firing of cells in the cMA0 of dystonic rats (48). Recording continued for an additional 60-90 min. Pre- and postdrug data were obtained for one cell per rat. After the recording session was completed, a small marking lesion was made in order to localize the recording site. Frozen sections were cut through the cerebellum and stained with cresyl violet for histological verification of electrode placement. Unit data were analyzed offline using R. C. Electronics Computerscope software. For all cells reported, ratemeter histograms, interspike interval histograms, and autocorrelograms were obtained for 10 min of baseline activity during the predrug period and for the period from 15 to 25 min after drug administration. Unanesthetized normal animals administered harmaline show a clear and virtually continuous tremor at this postdrug interval. The autocorrelograms were used to examine the regularity with which a cell fired. As in previous studies (48, 49), a rhythmicity index was calculated from autocorrelograms by dividing the number of counts at the first peak of the autocorrelogram by the bin width and the total number of counts and multiplying by 1000. This measure was devised to normalize the peak height according to the average impulse rate on which the peak is super-

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imposed. Autocorrelograms were based on 3000-4000 spikes. Unless otherwise indicated, electrophysiological data were analyzed by a three-way (phenotype X nucleus X drug) analysis of variance with repeated measures on the third factor (i.e., preharmaline vs postharmaline). Cells were sampled in the medial and interpositus nuclei.

Anatomy. Anatomical measures were carried out in 20-day-old normal and dystonic rats. For Purkinje cell counts, three normal and three dystonic rats were deeply anesthetized with sodium pentobarbital and perfused through the heart with isotonic saline followed by 10% neutral buffered formalin. The brains were removed and placed in formalin overnight. The cerebellum from each animal was cut in the midsagittal plane and each hemicerebellum embedded in paraffin. The paraffin blocks were cut sagittally at 10 pm and the sections were mounted and stained with cresyl violet. The total length of the Purkinje cell layer was measured in sections 200 pm apart beginning at the midline. A section known to be near the midline was selected as a starting point when the length of the Purkinje cell layer in that section exceeded that of adjacent sections. In the same sections, every Purkinje cell with a visible nucleus was counted. In order to determine a correction factor for estimating Purkinje cell numbers, the minor diameter of all counted Purkinje cells was measured in a section lying in a plane approximately halfway through the hemicerebellum. Counted cells were plotted as a function of the distance from the midline. Using the method described by Wetts and Herrup (55), the total area under the curve was used to estimate the total number of Purkinje cells. Since counts such as this are always considered to be an overestimate, the correction method described by Abercrombie (1) was then applied. The density of Purkinje cells was computed by dividing the corrected number of cells in each section by the length of the Purkinje cell layer in that section. A mean density for each rat was determined using all counted sections. All measurements were made with a Bioquant Image Analysis System (R&M Biometrics). Statistical comparisons between groups were made with t tests for independent means. The volume of the DCN was calculated using cerebellar sections collected from six normal and five dystonic rats perfused with 0.1 M phosphate buffer followed by Stefanini’s picric acid-formaldehyde-phosphate buffer fixative (47). The cerebellum was hemisected and 30-pm thick coronal sections were sliced on a Vibratome. Every section was mounted and stained with thionin. Individual nuclei were drawn and area measurements were made throughout the extent of the DCN. Nuclei were then reconstructed using an Evans and Sutherland three-dimensional reconstruction system. The remaining hemicerebellum was used for measures of soma size.

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97

A multivariate analysis of variance was used to compare normal and dystonic rats with the volume measurements for each nucleus treated as separate dependent measures. For cell size measurements, sagittal sections 30 pm thick were cut on a Vibratome and every fourth section was mounted and stained with thionin. Sections spaced evenly throughout the mediolateral extent of the DCN were analyzed in each animal (N = 41group). The crosssectional area of all cells with a minor diameter of ~=6 pm and a visible nucleolus were measured. Similar numbers of cells were counted for each group of rats in each nucleus. Measurements were made at a final magnification of 3575~ using a Bioquant Image Analysis System. Separate groups of animals were perfused with a zinc salicylate fixative containing 10% formalin at 25 days of age. Soma sizes were measured as described for the 20day-old rats. For all groups, mean cross-sectional area was computed for each nucleus in each rat. Frequency distributions for soma size were also constructed for the group data. Data were analyzed with a multivariate analysis of variance. RESULTS

Complete pre- and postdrug data Electrophysiology. sets were collected for cells from 17 normal and 20 dystonic rats with histologically verified electrode placements. The average age of the dystonic rats was 21.1 days (SD = 2.7) and of the normal rats, 21.3 days + 2.1. Thirteen cells from dystonic rats and eight from normal rats were located in the nucleus interpositus. The remaining cells were in the medial nucleus. Predrug data only were obtained from an additional six cells from normal rats and five cells from dystonic rats. All of these cells were located in the medial nucleus. Analysis of pre- and postdrug firing rate for all cells for which complete data were available indicated that harmaline significantly increased the firing rate of DCN cells (F(1,33) = 8.959, P < 0.005). These data are presented in Figs. 1A and 1B. As is evident in Fig. 1, the effect of harmaline was qualified by a significant phenotype X drug interaction (F(1,33) = 4.263, P < 0.047). Harmaline only increased the firing rate of cells from normal rats. The pre- and postdrug firing rates of cells from dystonic rats were similar and did not differ from the postdrug firing rates of normal rats. Figure 1 also shows that the differences noted between normal and dystonic rats were due mainly to the contribution of cells from the medial nucleus. Planned comparisons between cell means were made with t tests and indicated that in the medial nucleus, the spontaneous firing rate of cells from dystonic rats was higher than that of normal rats, but that only normal rats showed a significant increase in firing rate after harmaline. Analysis of the predrug data from all medial nucleus cells, including

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OYSTONIC 0

NORMAL

PREDRUG

POSTDRUG

PREDRUG

POSTORUG

100 GO GO 40

20 0 I PREORUC

PDSTDRUG

I

PREDRUG

POSTORUG

FIG. 1. Mean firing rate (spikes/set + SE) in normal and dystonic rats before and after harmaline administration in the medial (A) and interpositus (B) nuclei. Bottom panels show the mean rhythmicity index (*SE) for the same data with the medial nucleus in C and the interpositus in D. Asterisks indicate significant differences between normal and dystonic rats under the same drug condition. Crosses indicate significant differences between pre- and postdrug conditions for animals of the same phenotype. P < 0.05 for all.

those for which only predrug data were available, indicated that the mean spontaneous firing rate in dystonic rats (N = 12) was 23.7 Hz + 11.8 (M f SD). This was significantly higher (t = 2.81, df = 25, P < 0.01) than the mean firing rate of medial nucleus cells in normal rats (12.8 f 8.2, N = 15). Analysis of the rhythmicity index, a measure of the regularity of firing, revealed a significant main effect of phenotype (F(1,33) = 6.272, P < 0.017). As can be seen in Figs. 1C and lD, the firing pattern of cells from dystonic rats was more regular than that of normal rats. The effect was seen in the spontaneous firing of cells in both the medial and interpositus nuclei. Analysis of the predrug rhythmicity data from the entire sample of medial nucleus cells, including those from which only predrug data were available, confirmed this finding (t = 5.96, df = 25, P < 0.001). A phenotype X drug interaction (F(1,33) = 7.676, P < 0.009) is also evident in the rhythmicity data. Only the normal rats show an increase in rhythmicity following harmaline treatment and this was significant only in the medial nucleus. In normal rats, harmaline induced a bursting pattern of activity in seven of nine medial nucleus cells and in one of eight interpositus cells. An example of a cell from

the medial nucleus of a normal rat is shown in Fig. 2. Prior to drug administration, this cell had an average firing rate of 13.5 Hz. Following harmaline, this cell showed an overall increase in frequency to 23.7 Hz. The firing of the cell, however, was organized into a pattern of bursts with 4-6 spikes per burst and approximately 4 bursts per second. The frequency of the bursts is close to the average firing rate of harmaline-stimulated olivary neurons and the average complex spike rate of Purkinje cells recorded under similar conditions (48, 49). The intervals between bursts are reflected in the appearance of a second peak in the postdrug interspike interval histogram for this cell. A cell from the medial nucleus of a dystonic rat is displayed in Fig. 3. This cell had an average firing rate (17 Hz) that was slightly higher than that of the cell shown in Fig. 2. The pattern of firing, however, was much more regular than that of the cell in Fig. 2 prior to drug administration. Little change in pattern was evident in the postdrug period, although the firing rate increased to 21 Hz. When cells from dystonic rats did show a change in firing pattern after the drug treatment, the most common response was a reduction in the rhythmicity of firing, rather than an increase as seen in

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1500

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DISORDER

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Z m 3; 5 ii ul

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INTERSPIKE

INTERVAL

(MSEC)

rB 4000 Z 00 \ E Y ii m

N10209

POSTORUC

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2000

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0

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3

64

INTERSPIKE

96 INTERVAL

126

1

(MSEC)

FIG. 2. A shows a predrug interspike interval histogram for a medial nucleus cell from a normal rat. A sample of the raw data is displayed to the right. B shows data from the same cell following administration of 15 mg/kg of harmaline. There is an increase in frequency and as shown in the raw data on the right, the firing of the cell is interrupted by regular pauses with a frequency of about 4 Hz.

normal animals. This was sometimes accompanied by a small decrease in firing rate. Because age dependent changes in firing rate of Purkinje cells have been reported in normal rat pups (13, 56) and because the severity of symptoms in the mutant rats increases with age, correlational analyses were conducted on the spontaneous activity of cells from the medial nucleus. In normal rats only, firing rate increased significantly with age (r = 0.46, P < 0.05). In the mutant rats, firing rate was high even in the youngest animals. The rhythmicity of firing did, however, increase with age in the dystonic group (r = 0.59, P < 0.05). This was not the case in normal animals. Anatomical measures. Purkinje cells were counted in three normal and three dystonic rats. There were no reliable differences in the number or density of cells. The mean number of cells (KSE)/mm of Purkinje cell layer was 11.67 + 0.47 in the normal rats and 11.69 + 0.58 in the mutants. A total of 116,352 + 4443 cells/ hemicerebellum was counted in normal rats in comparison with 107,383 f 4840 in mutant rats. In order to reduce variability introduced by dissection, cell numbers were examined in sections containing the DCN. This provided more accurate landmarks for starting and end-

ing points for the cell counts. In these sections, the number of cells in normal rats was 70,989 f 3158 and in dystonic rats 69,692 + 3828. Plots of the distribution of cells from medial to lateral showed little variation from animal to animal. No statistically significant differences were obtained. Measurements of DCN volume revealed differences in volume between nuclei in both groups (Table 1). The interpositus nucleus was largest followed by the lateral nucleus and the medial nucleus in both groups. A multivariate analysis of variance conducted on the volume measurements did not indicate the presence of a statistically significant difference between normal and mutant rats (F(3,7) = 3.22, P < 0.09). Although the nuclei tended to be smaller in dystonic rats than in littermate controls, when the values for specific nuclei were compared, no reliable differences were obtained. The largest difference between groups was in the lateral nucleus (P < 0.12); however, power analysis indicated that a sample size of 15/group would be needed for a reliable difference at an (Y level of 0.05. Measurements of soma size were carried out in all three nuclei at two ages: 20 and 25 days (Table 2). Because of overall differences in values at the two ages, the

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012019

(t&EC)

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FIG. 3. the right. in normal

114

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INTERVAL

A shows a predrug interspike interval histogram from a medial nucleus cell in a dystonic rat. A sample of raw data is displayed to Postdrug data are shown in B. The cell showed an increase in frequency without the appearance of a bursting pattern of firing as seen rats.

data were converted to z-scores. Multivariate analysis of variance did not indicate the presence of a statistically significant difference between groups. When the nuclei were examined separately with t tests, the difference between normal and dystonic rats was largest in the medial nucleus (t = 1.795, df = 14, P < 0.09). In the interpositus and lateral nuclei, cell sizes in the two groups were similar (P > 0.5, for both). Examination of the distribution of cell sizes did not indicate any differences between groups at either age.

TABLE

1

Mean Deep Cerebellar Nuclei Volume (mm3+ SE) in Normal and Dystonic Rats Nucleus Group Normal Dystonic

Medial (6) (5)

Note. Numbers 20 days

190

(YSEC)

of age.

0.353 0.311

+ 0.035 + 0.018

in parentheses

Interpositus

Lateral

0.461 f 0.046 0.426 z 0.036 are sample

0.397 0.345

sizes. All animals

+ 0.020 + 0.022 were

DISCUSSION

Spontaneous

Activity

The results of the present study confirm the existence of abnormal function in the DCN of the genetically dystonic rat. In the medial nucleus of the mutant rats, the data suggest that the inhibition of DCN cells by Purkinje cells may be reduced. In normal rats, the firing rate of medial nucleus cells ranged from 3.8 to 25.6 spikes/s and the activity was irregular. This range of firing rates is similar to that previously reported in DCN slices from rats 17-25 days of age (21). The average spontaneous firing rate of medial nucleus cells in the mutant rats significantly exceeded that seen in normal controls. In addition, in both the medial nucleus and the nucleus interpositus, the firing pattern of cells was significantly more regular than that seen in normal controls. This variable was correlated with age and therefore, with increasing severity of symptoms in the mutant rats. It is possible that this increase in rhythmicity represents a gradual loss of control over DCN cells by Purkinje cells. Studies of the relationship between transmitter release and firing pattern in other systems

CEREBELLAR

TABLE

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2

Mean Cross-Sectional Area (pm’ f SE) of Cells in the Deep Cerebellar Nuclei of Normal and Dystonic Rats Nucleus Group 20 days old Normal (4) Dystonic (4) 25 days old Normal (4) Dystonic (4)

Medial

Interpositus

Lateral

294.9 267.9

f 11.5 f 4.6*

250.5 251.8

+ k

4.5 5.9

326.1 299.5

f 10.2 +- 16.7

301.4 295.6

k 9.6 f 13.2

283.8 272.4

f 18.6 +_ 6.9

326.0 f 12.5 314.6 k 16.0

Note. Numbers in parentheses are sample sizes. * Differs from normal controls of the same age, P i 0.05. Because of overall differences between the two age groups, data were also converted to z-scores for analysis. There were no significant differences between groups in any nuclei in the z-score analysis when animals from the two age groups were combined.

suggest that the pattern of activity may be an important variable in determining the functional efficacy of neuronal activity (22). The observations on the firing rate of cells in the DCN are in contrast to the results of recording studies in both the inferior olive and the cerebellar cortex of the dystonic rat (48,49). In both of these sites, the spontaneous firing rate of neurons in the dystonic rats was significantly slower than normal. The results of recordings in the DCN indicate that a slow spontaneous firing rate is not a uniform feature of cells in the mutant rat. We have previously suggested that the defect in the olivo-cerebellar system of the dystonic rat that accounts for the behavioral insensitivity of rats to harmaline is located in the cerebellar cortex (48). This was based on the dissociation between the firing rate of the olivary neurons and the complex spike rate of Purkinje cells in the mutant rats (48). The Purkinje cells fail to follow the increase in olivary activity induced by harmaline administration. In normal rats, there is an inverse relationship between the firing rate of cells in the DCN and that of Purkinje cells in the cerebellar cortex when large numbers of Purkinje cells are activated or inactivated (8). This relationship is maintained in the dystonic rats. The simple spike rate of Purkinje cells in the dystonic rat is 66% lower than that seen in normal rats and cells in the vermis were, on average, slower than those in more lateral regions of the cortex (49). This abnormally low Purkinje cell firing rate is associated with an increased firing rate of DCN cells, at least in the medial nucleus. Thus, the cells in the medial nucleus of the dystonic rat appear to show a normal response to an abnormal input. The contribution of a postsynaptic defect in the DCN cannot, however, be ruled out by these data. In rats with lesions of the inferior olive, there is a long-term increase in DCN cell activity and a decrease

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in the sensitivity of DCN neurons to inhibitory effects of GABA (7). The anatomical results support earlier nonquantitative assessments of the cerebellum that suggested that the structure was morphologically intact in the dystonic rat (32). Although negative results must be interpreted with caution, the results of Purkinje cell counts, measures of DCN volume, and DCN soma size all suggest that the difference in firing rate and pattern between DCN cells in dystonic and normal rats is due to a functional defect in the system and not to a deafferentation of the DCN cells.

Harmaline

Responses

In addition to differences between normal and dystonic rats in the rate and pattern of spontaneous activity in the cells of the DCN, there was a significant difference in the response of cells in the medial nucleus to harmaline. Following administration of harmaline, rhythmic bursts of activity were induced in seven of nine cells recorded in the medial nucleus of normal rats and one of eight cells in the nucleus interpositus. In dystonic rats, however, harmaline had no consistent effect. Although some modest harmaline effects were observed in recordings from Purkinje cells in dystonic rats (49), the lack of a response in the DCN shows that any effect on the Purkinje cells is not sufficient to produce a rhythmic cerebellar output. These findings account for the failure of the dystonic rat to display a harmaline tremor. Harmaline is thought to activate the cells of the DCN indirectly by way of the olivo-cerebellar pathway. In metabolic mapping and single-unit recording studies in normal rats, regional differences in sensitivity to harmaline have been observed with the caudal medial accessory olive (cMA0) being the region most affected (2, 6, 48). This is similar to earlier findings in the cat (14). In the rat, as in other species, specific regions of the inferior olivary complex send fibers to longitudinally organized zones of the cerebellar cortex (10,ll). In addition, there is a topographically organized projection from the cerebellar cortex to the DCN. Thus, injections of horseradish peroxidase into the medially located sagittal zone of the cerebellar vermis label both cells in the cMA0 and terminals in the medial nucleus (10). Injections into the more lateral cerebellar cortical subzones, however, label cells in the rostra1 medial accessory olive and dorsal accessory olive and terminals in the nucleus interpositus (11). Because of these anatomical relationships in the olivo-cerebella-nuclear circuit, activation of the climbing fibers from the cMA0 results in increased complex spike activity in the Purkinje cells of the vermis and bursting in the medial nucleus (6,14). This was observed in normal but not dystonic rats (49). In the present study, cells in the medial nucleus of normal rats

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showed a greater change in activity after harmaline than those in the interpositus. In addition, the differences between normal and dystonic rats were most pronounced in the medial nucleus. The DCN also receive a significant input from climbing fiber collaterals (52). Although the cells of the cMA0 are activated by harmaline in the dystonic rat (48), the olivary projection to the DCN cells is not sufficient to generate rhythmic activity typical of the harmaline response. This is similar to an early observation on the role of cerebellar cortex in harmaline tremor. Recording from motoneurons in cats, Llinas and Volkind (31) observed that the regular lo-Hz activity obtained after harmaline administration was disorganized by cooling of the vermis. They concluded that the integrity of the cerebellar cortex was necessary to produce the sharp bursts of motoneuron activation characteristic of harmaline intoxication in the intact animal. Since activation of the inferior olive of the mutant rats with harmaline fails to induce reliable responses in the DCN, we assume that at least in the medial nucleus, the output of the cerebellum in the dystonic rat is not modulated by the more subtle changes in climbing fiber activity produced by natural stimuli (37).

Relationship

to Metabolic

and Biochmical

Findings

The results reported here suggest that increased glucose utilization previously reported in the DCN of the dystonic rat (9) may be the result of activity in recurrent axon collaterals of the DCN cells. In other systems, changes in glucose utilization measured with the 2deoxy-D-glucose technique have been found to represent either excitatory or inhibitory activity in axon terminals (29). Two sources of input to the DCN, the Purkinje cells and the inferior olivary neurons, have low spontaneous firing rates in the dystonic rat (48, 49). Thus, it is unlikely that either of these afferents is the source of increased glucose utilization in the DCN of the mutants. Mossy fiber collaterals are also assumed to provide an excitatory input to the DCN (21). We have no direct measure of the activity of cells contributing to the mossy fiber pathway. Measures of glucose utilization in the cerebellar cortex did not, however, show any indication of increased mossy fiber activity at that level (9). On the basis of in vitro studies of the DCN, Jahnsen (27, 28) has suggested that the tonic activity of DCN cells is intrinsically generated. Furthermore, in Golgistained preparations in the rat medial nucleus, large or medium-sized projection cells have been found to give off one or more recurrent axon collaterals that terminated within the nucleus (36). The finding of increased activity in cells in the medial nucleus suggests that the activity of these nuclear cells may contribute to altered glucose utilization in the DCN in the mutants. At least at a superficial level, the pattern of neuronal activity seen in the cells of the DCN in dystonic rats

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appears inconsistent with the increases in GAD activity previously reported in the DCN of dystonic rats in comparison with littermate controls (5,40,43). In the DCN, GAD is localized primarily in the terminals of Purkinje cells (20,54). Thus, increased GAD activity suggests an increased demand for transmitter in the Purkinje cells of the mutant rats. A similar increase in GAD activity in the DCN is obtained by destroying the inferior olive with the toxin 3-acetylpyridine (3AP) (42). This treatment is known to produce an immediate increase in Purkinje cell activity (3, 6). The interpretation of the biochemical studies may not, however, be straightforward. Studies in rats that have sustained 3AP lesions demonstrate that measures of total GAD activity in the DCN are poor predictors of Purkinje cell activity. In the immediate postlesion period, GAD activity and Purkinje cell activity are both high and DCN cell activity is strongly inhibited (3, 6). With time, Purkinje cell activity declines and DCN activity increases (4, 8). GAD activity, however, remains elevated (50). This apparent dissociation between enzyme activity and neuronal firing rate appears to occur in the dystonic rat as well. Since the single-unit recording studies in the mutant rats have been done under anesthesia, the possibility must be considered that the dissociation between the biochemical and electrophysiological effects is due to a masking of Purkinje cell hyperactivity by anesthesia. There are several arguments against this. First, the studies of SAP-treated rats, in which the same dissociation between electrical activity and measures of GAD occurs, show that depending on the time after the lesion both increases and decreases in DCN activity can be recorded (4, 50). Second, several correlates of the immediate effects of 3AP treatment have been examined in the dystonic rat. An initial increase in GABA levels and a temporary increase in the size of GAD-immunoreactive puncta that occur in the DCN during the period of Purkinje cell hyperactivity in SAP-treated rats are not evident in dystonic rats (34).

Relationship

to the Movement

Disorder

The medial nucleus of the cerebellum has long been recognized as a site related to the maintenance of posture and muscle tone (45,46). More recent studies in the rat have shown that unilateral axon-sparing lesions and local injections of both GABA agonists and antagonists in the medial nucleus produce postural abnormalities and changes in limb tonus (24). A functional defect in the midline cerebellum altering the output of the medial nucleus could alter the modulation of tonic neck reflexes through pathways from the medial nucleus to the reticulospinal or vestibulospinal systems (15, 25). This could contribute to some of the distinctive behavioral features of the mutant, such as twisting of the neck and

CEREBELLAR

OUTPUT

IN

INHERITED

rigid extension of the limbs. Recording studies in behaving rats will be needed to critically test this hypothesis. Because of the repetitive twisting movements that characterize the motor syndrome of the genetically dystonic rat, this mutant has been investigated as a model of inherited dystonia in man (32). As in the human disease, the dystonic rat has a period of apparently normal motor development that precedes the onset of clinical signs (32). The lack of any significant morphological defect in the dystonic rat constitutes another important point of similarity with the human disease. Dystonias are generally considered disorders of the basal ganglia, because of frequent neuropathological findings in these regions in patients with secondary dystonias (18). The neuroanatomical basis of inherited dystonia, however, remains unclear. There are no consistent positive findings in postmortem studies of the brains of dystonic patients, although recent studies suggest the potential involvement of a variety of regions including the cerebellum and other brain stem structures (e.g., 19,57,58). It has been noted by McGeer and McGeer (38) that lesions of the basal ganglia do not always lead to dystonia and in some cases, many years pass between basal ganglia insult and the onset of symptoms. One interpretation of these observations is that additional changes in brain function may be needed to cause dystonias. The finding of a functional defect in the cerebellum of the genetically dystonic rat suggests that this structure may also play a role in analogous human diseases.

MOVEMENT

7. BILLARD,

J. M., AND C. BATINI. 1991. Decreased sensitivity of cerebellar nuclei neurons to GABA and taurine: Effects of longterm inferior olive destruction in the rat. Neurosci. Res. 9: 246-

256. 8.

BILLARD, J. M., AND H. DANIEL. 1988. Persistent reduction of Purkinje cell inhibition on neurones of the cerebellar nuclei after climbing fibre deafferentation. Neurosci. I&t. 88: 21-26.

9.

BROWN, L. L., AND J. F. LORDEN. 1989. Local cerebral glucose utilization reveals widespread abnormalities in the motor system of the rat mutant dystonic. J. Neurosci. 9: 4033-4041.

10.

BUISSERET-DELMAS, C. 1988. Sagittal organization of the olivocerebellonuclear pathway in the rat. I. Connections with the nucleus fastigii and the nucleus vestibularis lateralis. Neurosci. Res. 6: 475-493.

11.

BUISSERET-DELMAS, C. 1988. Sagittal organization of the olivocerebellonuclear pathway in the rat. II. Connections with the nucleus interpositus. Neurosci. Res. 6: 494-512.

12.

CADDY, K. W. T., AND T. J. BISCOE. 1979. Structural and quantitative studies on the normal C3H and Lurcher mutant mouse. Philos. Trans. R. Sot. Land. Biol. 287: 167-201.

13.

CREPEL, F. 1972. Maturation of the cerebellar Purkinje Postnatal evolution of the Purkinje cell spontaneous the rat. Exp. Brain Res. 14: 463-471.

14.

DEMONTIGNY, C., AND Y. LAMARRE. 1973. Rhythmic activity induced by harmaline in the olivo-cerebella-bulbar system of the cat. Brain Res. 63: 81-95.

15.

DENOTH, F., P. C. MAGHERINI, 0. POMPEIANO, AND M. STANOJEVIC. 1980. Responses of Purkinje cells of cerebellar vermis to sinusoidal rotation of neck. J. Neurophysiol. 43: 46-59.

16.

ECCLES, J., R. LLINAS, aptic action of climbing lum. J. Physiol. 182:

17.

ECCLES, J., M. ITO, AND J. SZENTAGOTHAI. as a Neuronal Machine. Springer, Berlin.

ACKNOWLEDGMENTS

18. FAHN, This work cal Research 90-10187).

was supported in part by grants from the Dystonia Foundation and the National Science Foundation

1.

19.

ABERCROMBIE, M. 1946. Estimation microtome sections. Anat. Rec. 94:

of nuclear 239-247.

population

5.

6.

AND K. SASAKI. 1966. The excitatory synfibres on the Purkinje cells of the cerebel-

268-296. 1967. The Cerebellum

S. 1989. Dystonia: Where next? In Disorders of Moue(N. P. Quinn, and P. G. Jenner, Eds.), pp. 349-357. AcaPress, New York.

FLETCHER, N. A., R. STELL, A. E. HARDING, AND C. D. MARSDEN. 1988. Degenerative cerebellar ataxia and focal dystonia. Movement Disorders 3: 336-342. F., J. STORM-MAT~IISEN, AND F. WALEERG. 1970. Glutamate decarboxylase in inhibitory neurons: A study of the enzyme in Purkinje cell axons and boutons in the cat. Brain Res.

from

J. M., C. BATINI, J. M. BILLARD, C. BUISSERET-DELMAS, M. CONRATH-VERRIER, AND N. CORVAJA. 1983. Cerebellar output regulation by the climbing and mossy fibers with and without the inferior olive. J. Comp. Neural. 213: 464-477. BATINI, C., AND J. M. BILLARD. 1985. Release of cerebellar inhibition by climbing fiber deafferentation. Erp. Brain Res. 67: 370-

380. 4. BATINI,

cells. I. firing in

20. FONNUM,

2. BARDIN,

3.

ment, demic

Medi(BNS

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