Design and Methods - Mutations Affecting Motor Function
Primary screen for motor control deviants - Rationale. The number of identified independent mutations affecting motor systems falls far short of that described for their human disease counterparts and makes clear the need for more animal models. Our overall goal is to identify new mutants with cerebellar and motor dysfunction, which may, in turn, provide a means to determine the underlying molecular mechanisms of CNS development, neuronal function and neuronal degeneration. Moreover, there is a strong bias in spontaneous mutants with an early age of onset (often by two weeks of age) and overt pathology. Visible and early-onset phenotypes are useful, but they do not adequately represent the spectrum of disease that exists in the human population including slowly progressive myopathies or late-onset neurodenerative diseases such as ALS and late-onset ataxias. Mouse models with subtle or no visible defects, in particular those with ages of onset beyond a few months, are very poorly represented.
Observe weanlings for overt gait and posture abnormalities, muscle wasting or paralysis - All mice will be observed at weaning (when cerebellar development is nearly complete) for signs of cerebellar and neuromuscular dysfunction. Signs of cerebellar dysfunction include side to side swaying, falling on one side when walking, inability to right from a recumbent position and a wide-stanced gait. Neuromuscular signs include muscle wasting, stiff or slow gait, paralysis of fore- or hindlimbs, and spasticity of limbs to passive movement. Since cerebellar dysfunction is often accompanied by a fine tremor at rest or while walking, mice will also be observed for tremor. Cerebellar mutants are usually smaller than littermates, likely due to the impact of their behavioral problems on competition for food. Thus a reduction in size together with behavioral deficits in deviant mice would support possible cerebellar dysfunction. Aged mice will also be observed for signs of ataxia.
Battery of sensitive assays to detect more subtle defects - In addition to detection of obvious ataxias by simple observation, we will begin to screen mice for more subtle ataxias. Ataxic mice often have activity changes that can be easily detected in monitoring cages. Such changes may include increases or decreases in spontaneous activity, as well as changes in vertical activity. Thus we expect that mice with motor deficits will be detectable from the 2800 mice (i.e. 1400 8-wk old mice plus 1400 1-yr old mice) screened in the CCMS (see Section 3.2) [108, 109]. In contrast to rotarod, we find that pawprint analysis provides more reliable and sensitive detection of mutants. Examination of individual data points for Down's partial trisomy mice with non-visible gait defects revealed 10 of 20 mutants with stride lengths 3 s.d.shorter than normal controls and all but three were at least 2 s.d. from the normal mean [105] Appendix V). Further, all mutants had more stride length variation. We will continue to evaluate paw-print and rotorod side-by-side on ataxic mutants. However, the traditonal labor intensive paw-printing method is not ideal for high-throughput so we are currently working with corporate partners to develop automated mouse gait recording and analysis (e.g. see Appendix VI). We are confident that this, in combination with rearing behavior data from CCMS will detect most mice with subtle gait defects.
Grip Strength - The production of forelimb grip forces will be assessed using a procedure developed by co-investigator A. Costa [105] Appendix II) that utilizes a digital push-pull strain gauge (Grip Strength Meter, Columbus Instr) with a sandpaper-covered grasping ring. The mouse is allowed to grasp the ring with its forepaws and its grip strength is measured as the peak force generated when it is pulled from the ring using a gravity-driven system that produces a consistent 10-N downward force onto the animal's tail. The system is activated manually after the mouse grasps the bar firmly with both paws and then falls onto a soft cushion approximately 25 cm below. We anticipate screening 8000 mice per year with this high-throughput screen to detect single-gene mutations that affect performance by > 3 s.d. relative to B6 controls.
Defects in aging mice - The above tests will be done in a cohort of 1400 mice aged to 1 yr to detect slowly progressive, delayed onset disorders to model those found in human but underrepresented in mice.
Secondary screens to further characterize the nature of motor function defects - Rationale. Since locomotor movement is controlled by the integration of many systems, we will perform secondary tests to more precisely localize the primary defect. For example, neuromuscular disease categories include degenerative diseases of upper or lower motor-neuron cell bodies, peripheral neuropathy, conduction defects, or myopathic disease. Furthermore, abnormalities in these systems can be congenital or progressive and may be idiopathic or accompanied by obvious pathologies that require further classification.
Motor coordination mutants - To test ataxia progression, rotorod tests will be performed on cohorts of proven mutants. For ataxias detected at 1-year of age, cohorts will be tested at 3-month intervals for the onset of motor incoordination using pawprinting and 4-trial rotorod. To test for abnormalites in cerebellar development, descendants of mice identified as ataxic at 8 weeks will be examined for motor coordination and by histopathology at 21 days of age, when cerebellar development is largely completed; for overt abnormalities, pathology will be performed as early as obvious neurodysfunction is seen. Our expertise characterizing developmental ataxic mutants is shown by our characterization of the rcm mutation [110-111, 112].
Histopathology. These functional results will be correlated with pathological analysis. Briefly, animals will be anaesthetized, perfused with Bouin's fixative, and brains removed and post-fixed overnight. Hemotoxylin- and eosin-stained sagittal sections will be analyzed for cerebellar size, foliation, trilaminar structure, cell-density defects, and presence of the deep cerebellar nuclei. Immunohistochemistry will be performed with anti-calbindin D28 antibodies to examine Purkinje cell placement and orientation.
Neuromuscular mutants - In situ measurement of muscle contractile properties is used extensively in the characterization of mouse models of neuromuscular diseases [113-120]. Several measures are recorded in a single experiment and each may detect defects in one or more key processes involved in muscular force generation. In combination, these measures are sufficient to provide a starting point for hypothesis-driven examination of identified mutants.
Data collection and interpretation
General. The variability inherent in physiological measures will require testing groups of 4-6 mutant mice to obtain an initial indication of any aberrations. If none are evident, no further experiments will be performed. If aberrations are evident (i.e. all mice deviate in same direction in one or more measures) but statistical significance is not reached after 6 mice, up to 12 more mice may be added for confirmation. Measures will be compared to mice from the appropriate background strain (initially C57BL/6J, n=12).
Measures. Each experiment will measure: Peak twitch force (Pt), time to peak twitch force, half-relaxation time, potentiated twitch (following tetanic stimulation), nerve conduction velocity (delay between stimulus onset and Pt), tetanic force at various stimulation frequencies, tetanic fade and fatigue resistance. Measures of muscle electrical activity (EMG) will be made concomitantly with force measures. Collected measures provide indirect data related to contractile machinery, sarcoplasmic reticulum function, excitation-contraction coupling, calcium handling kinetics, neuromuscular junction function, and nerve conduction. Careful interpretation of data and the interrelationships between measures should allow possible location of defects to be narrowed to the point that hypotheses could be formulated for further targeted studies.
Histopathology. To build on functional results, nerve cross sections will be examined for nerve count, diameter, and myelination. Muscles will be examined for atrophy, necrosis, fiber type staining (skewing), and neuromuscular junction intactness (using abungarotoxin). The spinal cord itself will be examined for cell death, dorsal roots, dystrophic axons and gliosis. Immunostaining for appropriate cell markers may also be indicated.
