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Effects of age and strain differences on the red nucleus of the mouse mutant Dystonia musculorum.

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THE ANATOMICAL RECORD 206:313-318 (1983)
Effects of Age and Strain Differences on the Red
Nucleus of the Mouse Mutant Dystonia
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.)
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).
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).
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
processed using the paraffin embedding method
described previously (Messer and Strominger,
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.
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.
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
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.
3 15
3 16
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.
TABLE 1. Acetylcholinesterase in caudal red nucleus
us. Balb C'
(coarse neurons) of dtAlbldtAlb
DFP pretreatment
Balb C
1.5 mg/kg
5 mglkg
5 mgikg +
'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,
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
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
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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,
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.
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Can. J. Neurol., 9.185-188.
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red nucleus and striatum. Neuroscience, 5:543-549.
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of the rhesus monkey. J. Camp. Neurol.. 152:327-346.
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(Dystonia musculorum). Acta Neuropathol. (Ber1.1,
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musculorum, effect, strait, differences, red, mouse, dystonic, age, mutant, nucleus
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