Effects of age and strain differences on the red nucleus of the mouse mutant Dystonia musculorum.код для вставкиСкачать
THE ANATOMICAL RECORD 206:313-318 (1983) Effects of Age and Strain Differences on the Red Nucleus of the Mouse Mutant Dystonia muscuiorum EDITH STANLEY, ANNE MESSER, AND NORMAN L. STROMINGER Birth Defects Institute, Center for Laboratories and Research, New York State Department of Health, Albany, N Y 12201 (E.S., A.M.) and Department ofAnatomy, Albany Medical College, Albany, N Y 12208 (E.S., A.M., N.L.S.) ABSTRACT The mouse mutant Dystonia musculorum exhibits pathological changes in the magnocellular neurons of the red nucleus. The present study shows that allelic differences occur in the age of onset and severity of this pathology. The magnocellular neurons of the Jackson allele (df')almost completely disappear prior to 4 weeks of age while some of these cells are retained in the adult of the However, acetylcholinesterase histochemistry suggests that Albany strain (dtAib). the remaining rubral neurons in are nonfunctional. This pathology may contribute to the severe locomotor disturbances seen in these animals. The mouse mutant Dystonia musculorum demonstrates a n autosomal recessive neurological syndrome. The first symptom, which appears at about 2 weeks of age, is an abnormal limb placement when the animal is held by the tail. Locomotor deficits progress rapidly, and by 4 weeks of age the animal demonstrates spasms, posturing of limbs and tail, writhing movements of the trunk, increased muscle tone, dragging or shaking of stiffened hind limbs, and adduction of the forelimbs. Degenerative changes in central and peripheral sensory nerve fibers were described in the original allele of the mutant (Duchen et al., 1964; Janota, 1972). Additional pathology has been reported in magnocellular neurons of the red nucleus of both the original allele maintained at the and a second allele Jackson Laboratories (df') of the mutant which arose spontaneously in a BALBicBy mouse stock maintained at the New York State Department of Health Laboratories in Albany, New York (dtALb)(Messer and Strominger,1980).Neurochemical changes have also been reported in the striatum and substantia nigra (reduced whole-tissue synthesis of gamma-aminobutyric acid (GABA), apparently due to failure of glutamate uptake; Messer and Gordon, 1979), and in the cerebellum (increased tyrosine hydroxylase and norepinepherine turnover, Riker et al., 1981; decreased acidic amino acid concentrations, Messer, 1982; both without the loss of a major cell population). No other brain area was found to be abnormal in a light microscopic survey (Messer and Strominger, 1980). 1963 ALAN R LISS, INC. In a n effort to assess the role of the red nucleus pathology on the motor dysfunction of this mutant, we compared the onset and severity of the pathologic changes in the red nucleus of the two alleles. Acetylcholinesterase (AChE) activity to the red nucleus was used as a histochemical measure of the functional capacity of rubral neurons in the Albany allele as acetylcholine is thought to be a major transmitter of the inputs to this nucleus (McGeer, P., personal communication). MATERIALS AND METHODS Both the BALBicBy dtAIband B6C3 dtJ stocks are maintained by brother-sister mating of animals heterozygous for the trait. Normal animals of the same age and inbred background are used as wild-type controls. Mice ranging from 2 to 8 weeks of age were anesthetized with ether and killed by transcardial perfusion of 0.9% buffered saline followed by 10% buffered formalin. At least two mutant and two control animals were examined a t each age. Brains were postfixed and embedded in plastic according to the protocol accompanying the JB4 embedding kit (Polysciences, Inc., Warrington, PA) with the addition of hardening at 60°C for 1 hour and 4°C for a n additional hour. Fivemicron sections were cut using a standard A 0 microtome with a metal knife. Every tenth section was stained with thionin and examined for pathologic changes. Additional animals were Received J u n e 16, 1982, accepted March 25, 1983 3 14 E. STANLEY, A. MESSER, AND N.L. STROMINGEK processed using the paraffin embedding method described previously (Messer and Strominger, 1980). In addition, 20-pm frozen sections were preand control mice pared from a total of 16 aged 8-12 weeks old. Every other section was reacted for AChE activity by the method of Geneser-Jensen and Blackstad (1971). To irreversibly inactivate existing AChE, mice were pretreated with diisopropylflurophosphate (DFP), 1.5 or 5.0 mgikg injected 8-25 hours before perfusion. Two controls and two mutants were used for each level of DFP treatment. Ten sections through the red nucleus of each brain were examined. Mipofax (1.0 mM) was added to the reaction mixture of the highest DFP level as a control to demonstrate the specificity of the reaction (Aldridge, 1953). The intensity of AChE staining of magnocellular neurons in the red nucleus of normal mice untreated with DFP was determined and used as a reference. A scale of zero to four was used as a quantitative approximation; zero indicated a complete lack of AChE staining and four denoted an intense brown reaction product. Scaling was assigned independently by all three authors and scores were averaged. Sections from mutants untreated with DFP, as well as sections from both mutants and controls pretreated with this irreversible AChE inhibitor, were compared to the standard. RESULTS appears morphologically more intact than that of dP. The results of histochemical examination of newly synthesized AChE activity, a measure of residual function of remaining rubral neurons in dtAlb,shown in Table 1, suggest that this difference in appearance may not be significant. Although some AChE activity is present in rubral neurons of the mutant, a considerable decrease in staining occurs. In animals pretreated with DFP the magnocellular neurons in the adult dtAlbshow little if any staining in contrast to the controls, which show substantial staining (Fig. 3). The pretreatment with DFP does not appear to affect survival or exacerbate the motor abnormality of the mutants. &*Ib CONCLUSION In summary, the age of onset of the most severe neuronal pathology correlates closely with that of the most severe locomotor abnormalities. Degenerative changes in the red nucleus are first apparent a t 2-3 weeks of age as motor deficits are becoming progressively worse. This observation is in contrast to the degenerative changes noted in peripheral nerve fibers and dorsal root ganglia a t birth, before the appearance of any clinical symptoms (Janota, 1972). The red nucleus of dtAibappears morphologically more intact than that of dtJ when measured with standard light microscopic histology. Acetylcholinesterase activity is one approximation of the functional integrity of this nucleus. The dramatic loss of newly synthesized AChE activity (Lehmann et al., 1979) of those large neurons which remain in the red nucleus of dtAIbsuggests that they have greatly decreased residual function, and that the red nucleus dysfunction in the two alleles may be similar. Since these neurons form the rubrospinal tract with projections to the lateral reticular nucleus and hence to the cerebellum (Miller and Strominger, 1973; Murray and Gurule, 19791, their degeneration may contribute significantly to Pathologic changes in the red nucleus of each allele differ both in severity and the age a t which they can first be observed. In normal mice the magnocellular neurons of the red nucleus are multangular, with centrally located nuclei and large discrete Nissl bodies. In both strains of the mutant this neuronal population exhibits distorted shapes, eccentric nuclei, and various degrees of chromatolysis. Such cellular pathology is first seen in a few cells of &*Ib at about 2 weeks of age. By 3 weeks a substantial fraction of the magnocellular neurons are pathological, and by 4 weeks there is a reduction in the total number of these neurons, with all remaining ones affected (Fig. 1).In contrast, similar rubral pathology occurs in a larger fraction of the cells as early as 2 weeks of age Fig. 1. Coronal sections (plastic, 5 km) near the caudal in the Jackson allele. By 3 weeks the degen- pole of the red nucleus in dt"Ib (A,C,E) and Balb controls erative changes are quite severe. In d2J ani- (B,D,F) of different ages. At 2 weeks IA,B) magnocellular mals studied at 4 and 8 weeks of age almost neurons appear similar in the mutant and control, although all of the magnocellular neurons are absent a few exhibit early degenerative changes. Degeneration is more severe by 3 weeks (C,D), and by 4 weeks the remaining (Fig. 2). magnocellular neurons are grossly abnormal (El while the Although both alleles manifest similar lo- control red nucleus contains many healthy cells (F).Arrows comotor impairments, the adult red nucleus of indicate pathological cells. Bar = 100 pm. DYSTONIA MOUSE RED NUCLEUS 3 15 3 16 E. STANLEY, A. MESSER, AND N.L. STROMINGER Fig. 2. Coronal sections near the caudal pole of the red nucleus in dt' (A,C,E)and B6C3 controls (B,D,F)ofdifferent ages. At 2 weeks a substantial fraction of the magnocellular neurons of the mutant are already abnormal (A1 with pro- gressive involvement a t 3 weeks (C), leading to almost complete absence of this cell population a t 4 weeks (E). Controls a t 2 (B), 3 (DI, and 4 (F)weeks are shown for comparison. Arrows indicate pathological cells. Bar = 100 Fm. 317 DYSTONIA MOUSE RED NUCLEUS TABLE 1. Acetylcholinesterase in caudal red nucleus us. Balb C' (coarse neurons) of dtAlbldtAlb ~~~ ~~~~~~~ DFP pretreatment dtAIb Balb C None 1.5 mg/kg 5 mglkg 5 mgikg + Mipofax 2.5 1.5 0.5 4 0 0.5 3.5 3.0 'AChE activity was observed in frozen sections as described, with DFP pretreatment to highlight the capacity for new synthesis. Survival time was 24 hours for the 1 5-mg dose, and 6-10 hours for the 5-mg dose. A total of 16 animals were used. Fig. 3. Coronal sections showing relative amounts of acetylcholinesterase in the perikarya ofd@ vs. control caudal red nucleus cells. Animals were pretreated with DFP (5 mg/ kg) and perfused 8-12 hours later. A) Mutant with substrate; B) control with substrate; C) A + Mipofax inhibitor; D) B + Mipofax inhibitor. Bar = 50 km, ACKNOWLEDGMENTS the observed motor symptoms. The relationship between those changes, and less dramatic abnormalities in the cerebellum (Riker et al., This work was supported by a grant from the 19811, striatum (Messer and Gordon, 19'791,and Dystonia Medical Research Foundation. We peripheral motor neurons (Moss, 19811, re- thank Marilyn Dockum and Paul Maskin for technical assistance. mains to be elucidated. 3 18 E. STANLEY, A. MESSEK, AND N.L. S’rROMINCtER LITERATURE CITED Aldridge, W.N. (1953) Serum esterases I. Two types of esterase (A and B) hydrolysing p-nitrophenyl acetate, propionate and hutyrate, and a method for their determination. Biochem. J., 53:llO-116. Duchen, L.W., S.T. Strich, and D.S. Falconer (1964) Clinical and pathological studies of a n hereditary neuropathy in mice, dystonia musculorum, Brain, 87:367-378. Geneser-Jensen, F.A. and T.W. Blackstad (1971) Distribution of acetyl cholinesterase in the hippocampal region of the guinea pig. Zellforsch. Mikrosk. Anat., 114:460-481. Janota, I. (1972) Ultrastructural studies of an hereditary sensory neuropathy in mice. dystonia musculorum. Brain, 95:529-536. Lehmann, J., H.C. E’ihiger, and L.L. Butcher (1979) The localization ofacetylcholinesterase in the corpus striatum and substantia nigra of the rat following kainic acid lesion of the corpus striatum: A biochemical and histochemical study. Neuroscience, 4.217-225. Messer, A. 11982)Amino acid changes in the mouse mutant dystonia musculorum similar to those in Friedreichs ataxia. Can. J. Neurol., 9.185-188. Messer, A., and D. Gordon (1979) Changes in whole tissue biosynthesis of y-arninobutyric acid (GABA) in basal ganglia of the dystonia (&Ib) mouse. Life Sci., 252217-2221. Messer, A., and N.L. Strominger (1980) .4n allele of the mouse mutant dystonia musculorum exhibits lesions in red nucleus and striatum. Neuroscience, 5:543-549. Miller, R.A., and N.L. Strominger (1973) Efferent connections of the red nucleus in the brainstem and spinal cord of the rhesus monkey. J. Camp. Neurol.. 152:327-346. Moss, T.H. (19811 Segmental deniyelination in the peripheral nerves of mice affected by hereditary neuropathy (Dystonia musculorum). Acta Neuropathol. (Ber1.1, ,5-?:51-56. Murray, H.M., and M.E. Gurule (19791 Origin of the ruhrospinal tract of the rat. Neurosci. Lett., 14:19-23. Riker, D.K., A. Messer. and R.H. Roth (1981)Increased noradrenergic metabolism in the cerebellum of the mouse mutant dystoriza musculorum. J. Neurochem., 37:649454.