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Decreased Cerebellar 3',5'-Cyclic Guanosine Monophosphate Levels

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Decreased Cerebellar 3',5'-Cyclic Guanosine Monophosphate Levels 0270.6474/65/0510-2616$02.00/O The Journal of Neuroscience Copyright 0 Society for Neuroscience Vol. 5. No. IO, pp. 2618-2625 Printed in U.S.A. October 1965 Decreased Cerebellar 3’,5’-Cyclic Guanosine Monophosphate Levels and Insensitivity to Harmaline in the Genetically Dystonic Rat (dt)’ JOAN F. LORDEN,* GARY A. OLTMANS,§ TINA W. McKEON,* JACQUELINE LUTES,* AND MITCHELL BEALESQ Departments of l Psychology and $ Anatomy, University of Alabama in Birmingham, Birmingham, Alabama 35294 and § Department of Pharmacology, Chicago Medical School, North Chicago, Illinois 60064 Abstract The dystonic rat (dt) is an autosomal recessive mutant abnormality in the central or peripheral nervous systems (Eldridge, displaying a complex motor syndrome that includes sus- 1970; Zeman, 1970; Lorden et al., 1984). tained axial twisting movements. The syndrome is correlated Investigations in motor and non-motor areas of the central nervous with increased glutamic acid decarboxylase activity in the system of the dt rat have yielded evidence of biochemical disturb- deep cerebellar nuclei and increased cerebellar norepineph- ances in the cerebellum. Animals displaying the motor syndrome rine levels in comparison with phenotypically normal litter- have increased glutamic acid decarboxylase (GAD) activity in the mates. Biochemical, behavioral, and anatomical techniques deep cerebellar nuclei (Oltmans et al., 1984) and elevated levels of were used to investigate the possibility that the abnormalities norepinephrine (NE) in the whole cerebellum (Lorden et al., 1984). noted in the cerebellum of the dt rat were indicative of altered Although it is not known whether these changes are unique to the function of the major projection neurons of the cerebellar cerebellum, they have not been detected in any of the other brain cortex, the Purkinje cells. Phenotypically normal rats showed regions studied to date. Specifically, GAD activity appears normal in tremor in response to harmaline, a drug that acts on the the caudate-putamen, and NE levels are not reliably different from inferior olive to produce bursting in the climbing fiber path- littermate control values in the neocortex and hippocampus. way. Dystonic rats were insensitive to the effects of harma- The cause of the biochemical changes found in the cerebellum line but did respond to oxotremorine. Levels of the cyclic of the dt rat is not known. Nevertheless, studies of the effects of nucleotide 3’,5’-cyclic guanosine monophosphate, a bio- lesions on GAD activity in other brain areas provide a framework for chemical marker for Purkinje cells, increased in response to developing hypotheses concerning the origin of the abnormalities in harmaline in normal rats but were significantly lower in dys- the dt rat. For example, in the red nucleus increases in GAD activity tonic rats under both basal and harmaline-stimulated condi- have been found following destruction of afferent pathways (Nieoul- tions. Purkinje cell soma size was reduced in the dystonic Ion and Dusticier, 1981). Since cerebellar GAD immunoreactivity has rats but no other morphological correlates of the behavioral been localized in the Purkinje cell terminals in the deep cerebellar or biochemical deficits were noted. Taken together with other nuclei (Chan-Palay, 1982) the increased GAD activity of the dystonic observations on this mutant, the results suggest an impair- rat may indicate a functional deafferentation of the Purkinje cells. ment in the cerebellum or in its connections with lower Decreased afferent stimulation could result from a reduction in either brainstem and spinal cord sites. climbing fiber or mossy fiber activity (Eccles et al., 1966a, b). To examine the status of the Purkinje cells and their response to the activation of afferent neurons directly, we have measured 3’,5’- The dystonic (dt) rat displays sustained twisting movements of cyclic guanosine monophosphate (cGMP) levels in the cerebella of the axial musculature during waking (Lorden et al., 1984). Hyperflex- dystonic rats and their normal littermates. This nucleotide is thought ion of the trunk, frequent falls to the side with rigid extension of the to be a relatively specific marker for Purkinje cells (Biggio et al., limbs, and poor limb placement during locomotion are also charac- 1977a, b, 1978; Nairn and Greengard, 1983) as cerebellar guanylate teristic of the disorder. The rat disease is inherited as an autosomal cyclase and cGMP have both been localized immunohistochemically recessive mutation and the clinical syndrome is observed beginning in Purkinje cells (Ariano et al., 1982). Furthermore, quantities of on postnatal days 9 to IO, following a period of apparently normal cGMP and cGMP-dependent protein kinase, a biochemical marker motor development. The mutant’ has been proposed as a model for found solely in Purkinje cells (DeCamilli et al., 1984) are markedly dystonia musculorum deformans (DMD), an inherited neurological reduced in mutant mice lacking Purkinje cells (Mao et al., 1975b; disease of unknown pathogenesis. As in the human disease, the Schlichter et al., 1980). The finding that cerebellar levels of cGMP syndrome in the mutant rat is not associated with any morphological are increased when excitatory input to the Purkinje cells is enhanced indicates that cGMP reflects Purkinje cell activity (Biggio et al., 1977a, Received October 5, 1984; Revised March 18, 1985; b). Accepted April 10, 1985 Measurements of cGMP were made in dt rats both under unstim- ulated conditions, to examine basal levels of cyclic nucleotide, and ’ This work was supported by Grant NS18062 from the National Institute following treatment with harmaline, to assess the effects of afferent of Neurological and Communicative Disorders and Stroke. We would like to activation. In normal animals harmaline produces a generalized motor thank Dr. Paula Hoffman for the use of her microwave oven and Jeanette tremor and an increase in cerebellar cGMP levels (Lamarre et al., Millrgan for her care of the breeding colony. 1971; Mao et al., 1975a). Electrophysiological recording from Pur- *To whom correspondence should be addressed. kinje cells shows that the frequency of simple spikes decreases and 2618 The Journal of Neuroscience Cerebellar cGMP in dt Rats 2619 a bursting pattern of complex spikes is elicited (Lamarre et al., 1971). sodium pentobarbital and perfused intracardially with 0.9% sodium chloride These effects are thought to be caused by harmaline’s induction of followed by 10% neutral buffered formalin. Brains were removed and post- a synchronous firing pattern in the cells of the inferior olive which in flxed In buffered formalin. Before sectioning, the tissue was placed in 30% sucrose in buffered formalin and allowed to sink. The brains were then turn drives the Purkinje cells by way of the climbing fiber pathway serially sectioned rn the sagittal plane (30 Grn thickness) on a freezing (Lamarre and Mercier, 1971; Lamarre et al., 1971; Biscos et al., mtcrotome and stained with cresyl violet. 1973; De Montigny and Lamarre, 1973; Llinas and Volkind, 1973; Cerebellar sections from dt and normal rats were qualitatively evaluated Lamarre and Puil, 1974; Guidotti et al., 1975). The Purkinje cells in for differences in foliation and cell arrangement. For both cell size and layer turn drive a cerebellobulbar pathway that co-activates (Y- and y- thickness measurements, midvermian and paravermian sections were used. motoneurons to produce tremor (Lamarre and Weiss, 1973). The continuity of the superior cerebellar peduncle with the cerebellar medulla In addition to measuring the effects of harmaline on cerebellar served as a landmark for paravermian sections. A total of 24 Purkinje cells cGMP levels, we measured the effects of harmaline on gross In cerebellar folia V and VI from each animal were traced using a Leitz locomotor activity and monitored both cft rats and their normal microscope with camera lucida attachment. An equal number of cells meeting littermates for the appearance of tremor following treatment with the nucleolar criterion were traced from all animals. As a control for the specificity of any effects seen in the cerebellum, 20 pyramidal cells in area harmaline or oxotremorine. The tremorogenic actions of oxotremo- CA4 of the hippocampus were also traced In each animal. rine are believed to be mediated by systems other than the climbing Layer thickness was assessed by tracing the depths of the molecular and fibers (Guidotti et al., 1975). In addition to the biochemical and granular layers from the crowns of cerebellar folia V and VI in six different behavioral measurements, the Purkinje cells and the projection from sections from each animal. For each set of measurements, a scale bar was the inferior olivary complex were examined using anatomical tech- traced for calibration purposes. Measurements were made using a Houston niques. Instruments digitizing pad interfaced to an Apple II Plus computer. R & M Biometrics (Nashville, TN) software was used to calculate areas and lengths. Materials and Methods Statistical comparisons were made between normal and dystonic rats with t tests. Each region was analyzed separately. Animals. Dystonic rats and their phenotypically normal littermates were To examine Purkinje cell dendritic morphology, 11 dt and 12 normal 20. obtained from the colony at the University of Alabama in Birmingham at 16 day-old animals were anesthetized with ketamine and ether. A 0.5.~1 Hamilton to 20 days of age. Both males and females were used. Mutants were syringe
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