J. Physiol. (1975), 248, pp. 231-246 With 6 text-figurew Printed in Great Britain
231
ELECTROPHYSIOLOGICAL PROPERTIES OF SPINAL MOTONEURONES OF NORMAL AND DYSTROPHIC MICE
BY P. HUIZAR, M. KUNO AND Y. MIYATA* From the Department of Physiology, University of North Carolina School of Medicine, Chapel Hill, N.C. 27514, U.S.A.
(Received 11 November 1974) SUMMARY
1. The properties of spinal motoneurones of normal and dystrophic mice (129/ReJ) were examined with intracellular electrodes. 2. The following parameters of spinal motoneurones showed no significant differences between normal and dystrophic mice: resting and action potentials, the amplitude and duration of after-hyperpolarization, rheobasic current for excitation, threshold for excitation of the somadendritic membrane (IS-SD inflexion) and input resistance. 3. The changes in motoneurone properties observed 13-16 days after section of the sciatic nerve (axotomy) were similar in both normal and dystrophic mice. 4. The axonal conduction velocity of motoneurones in dystrophic mice was about ten times slower than that in normal mice. The conduction velocity of the sciatic nerve was only about 25 % slower in dystrophic mice than in the normal animal. The estimated ventral root conduction velocity as well as the observed dorsal root conduction velocity in dystrophic mice was at least twenty times slower than that in normal mice. 5. In dystrophic mice, spinal motoneurones often showed multiple discharges in response to single, antidromic stimuli. The site of initiation of multiple discharge was located in the motor axon rather than in the motoneurone cell body. 6. In dystrophic mice, nerve impulses were transmitted from fibre to fibre ('cross-talk'). The site of impulse transmission among nerve fibres was near the distal portion of the spinal roots. 7. Synaptic potentials and peripheral reflex discharges evoked by stimulation of the dorsal roots showed a longer latency and were more prolonged in dystrophic mice than in the control mice. * Present address: Department of Pharmacology, Faculty of Medicine, Tokyo Medical and Dental University, Yushima, Bunkyo-ku, Tokyo, Japan.
P. HUIZAR, M. KUNO AND Y. MI YATA 232 8. The motoneurone properties of dystrophic mice showed no tendency of progressive changes with age ranging from 63 to 148 days. 9. It is concluded that the properties of motoneurone cell bodies examined in dystrophic mice are indistinguishable from those in normal mice and that the only abnormality in motoneurones of the former resides in the motor axon. 10. It is suggested that integrity of the discharge pattern of spinal motoneurones in dystrophic mice is interfered by anomalous impulse transmission in the motor axons and that the motoneurones in dystrophic mice are a homogeneous group rather than a mixture of 'normal' and
'abnormal' neurones. INTRODUCTION
A dystrophic mutation in mice originally described by Michelson, Russel & Harman (1955) is characterized by progressive, muscular atrophy and paralysis. The disorder in the dystrophic mice was considered to be primarily myogenic (Michelson et al..1955; West & Murphy, 1960; Pearce & Walton, 1963), but some abnormalities in the nervous system have subsequently been noticed. For example, the number of peripheral nerve fibres in these animals is significantly reduced over the whole range of axon diameters (Harris, Wallace & Wing, 1972; but cf. Bradley & Jenkison, 1973). Also, motor nerve terminals of dystrophic mice contain fewer synaptic vesicles, many of which are smaller in size and more elongated than those in normal mice (Ragab, 1971; Gilbert, Steinberg & Banker, 1973). Furthermore, the majority of fibres in the spinal roots (Bradley & Jenkison, 1973; Salafsky & Stirling, 1973) and cranial nerves (Biscoe, Caddy, Pallot, Pehrson & Stirling, 1974) of dystrophic mice are devoid of the myelin sheath. Salafsky (1971) has reported that the murine dystrophic muscle transplanted into a normal mouse regenerates with normal twitch characteristics, whereas the normal muscle transplanted into a dystrophic host achieves no functional regeneration (also, cf. Hironaka & Miyata, 1973). However, conflicting results have been observed by other investigators who used similar procedures (Laird & Timmer, 1965, 1966; Cosmos, 1973; Cosmos, Butler & Milhorat, 1973). Thus, sites of the primary disorder in dystrophic mice remain equivocal. It is known that the properties of skeletal muscles are determined, at least in part, by the properties of the innervating motoneurones (Buller, Eccles & Eccles, 1960; also, cf. Guth, 1968; Close, 1972). Morphological features of spinal motoneurones are similar in both normal and dystrophic mice (Papapetropoulos & Bradley, 1972; Bradley & Jenkison, 1973). However, no information has as yet been available concerning their
233 23 MOTONEURONES OF D YSTROPHIC MICE properties. The present study was undertaken to examine whether the electrophysiological properties of spinal motoneurones in dystrophic mice are different from those in normal mice. METHODS
The dystrophic mice (dy/dy), their phenotypically normal litter mates (?/ + ) and control mice (+ 1+) of the same strain (129/ReJ) were obtained from the Jackson laboratory, Bar Harbor, Maine. Animals of either sex ranging in age from 9 to 28 weeks were used in the present study. The body weight ranged from I11 to 18 g in dystrophic mice and from 18 to 26 g in the phenotypically normal litter mates and the control mice. In total, twelve control mice ( + / + ), sixteen phenotypically normal litter mates (?/ +) and thirty-one dystrophic (dy/dy) mice were used. The electrophysiological properties of the motoneurones in the first two groups of animals (+I/+ and ?/ + ) were indistinguishable. Therefore, the results obtained from these two groups were lumped as a control and compared with those obtained from dystrophic (dy/dy) mice. In preliminary experiments, attempts were made to maintain the animal on artificial respiration after decerebration or destruction of the brain rostral. to the medulla under ether or sodium pentobarbitone anaesthesia. The optimum rate of respiration seemed to be 160/mmn with a tidal volume of 0-2 ml., but all the animals expired within 3-4 hr. The results described in this report were therefore obtained from animals maintained on natural respiration. The mouse was initially anaesthetized by an i.r. injection of sodium pentobarbitone, 75 mg/kg, and a supplementary dose of 25 mg/kg (i.r.) was employed every 20-90 min throughout the experiment (of. McComas & Mossawy, 1965). At this level of anaesthesia, flexion reflexes to noxious stimuli were very weak, or absent, and respiratory movements could be minimized. After tracheostomy, the lumbar spinal cord was exposed by laminectomy with a pair of corneal scissors. The dorsal roots from the 4th to the 6th lumbar segments were cut on the left side. In a few experiments, the central ends of the cut dorsal roots were stimulated with a suction elec. trode to evoke synaptic responses in spinal motoneurones or reflex discharges in the peripheral nerve. In the left hind leg, the sciatic nerve was dissected. The tibial and common peroneal nerve branches were cut distally and prepared for stimulation or recording. Intracellular recording from spinal motoneurones was performed with glass micro-electrodes filled with 2 m potassium acetate. The resistance of the electrodes was between 8 and 30 MCiI. Recording from the motoneurone was identified by antidromic action potentials generated by stimuli applied to the central end of the cut tibial or common peroneal nerve. Exposed tissues in the hind leg were covered with a pool of paraffin oil, and the ik6~tal temperature was kept between 350 and 380 C by external heat.I The vertebral column was fixed by two spinous process clamps placed just above and below the laminectomy site. The vertebral bodies near the recording region were grasped with rigid steel wires from both sides. The entire exposed spinal cord was covered by 2 % agar dissolved in Hartmann's solution. Despite precautions taken to minimize movement of the spinal cord, most cells showed deterioration within a few minutes after penetration. Only in relatively few units was a stable membrane potential maintained for more than 30 min. At the end of the experiment, the spinal cord with attached ventral roots and peripheral nerves was excised to measure the conduction distance from the sites of stimulation to the recording segment. The conduction velocity was calculated from
234
P. HUIZAR, M. KUNO AND Y. MI YATA
the latency of antidromic action potentials evoked in the motoneurone at a stimulus intensity twice the threshold of each motor axon under study. The mean values of the results obtained from the control and dystrophic groups were statistically examined by two tail t tests with significance limit of 2P < 0 05.
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231
ELECTROPHYSIOLOGICAL PROPERTIES OF SPINAL MOTONEURONES OF NORMAL AND DYSTROPHIC MICE
BY P. HUIZAR, M. KUNO AND Y. MIYATA* From the Department of Physiology, University of North Carolina School of Medicine, Chapel Hill, N.C. 27514, U.S.A.
(Received 11 November 1974) SUMMARY
1. The properties of spinal motoneurones of normal and dystrophic mice (129/ReJ) were examined with intracellular electrodes. 2. The following parameters of spinal motoneurones showed no significant differences between normal and dystrophic mice: resting and action potentials, the amplitude and duration of after-hyperpolarization, rheobasic current for excitation, threshold for excitation of the somadendritic membrane (IS-SD inflexion) and input resistance. 3. The changes in motoneurone properties observed 13-16 days after section of the sciatic nerve (axotomy) were similar in both normal and dystrophic mice. 4. The axonal conduction velocity of motoneurones in dystrophic mice was about ten times slower than that in normal mice. The conduction velocity of the sciatic nerve was only about 25 % slower in dystrophic mice than in the normal animal. The estimated ventral root conduction velocity as well as the observed dorsal root conduction velocity in dystrophic mice was at least twenty times slower than that in normal mice. 5. In dystrophic mice, spinal motoneurones often showed multiple discharges in response to single, antidromic stimuli. The site of initiation of multiple discharge was located in the motor axon rather than in the motoneurone cell body. 6. In dystrophic mice, nerve impulses were transmitted from fibre to fibre ('cross-talk'). The site of impulse transmission among nerve fibres was near the distal portion of the spinal roots. 7. Synaptic potentials and peripheral reflex discharges evoked by stimulation of the dorsal roots showed a longer latency and were more prolonged in dystrophic mice than in the control mice. * Present address: Department of Pharmacology, Faculty of Medicine, Tokyo Medical and Dental University, Yushima, Bunkyo-ku, Tokyo, Japan.
P. HUIZAR, M. KUNO AND Y. MI YATA 232 8. The motoneurone properties of dystrophic mice showed no tendency of progressive changes with age ranging from 63 to 148 days. 9. It is concluded that the properties of motoneurone cell bodies examined in dystrophic mice are indistinguishable from those in normal mice and that the only abnormality in motoneurones of the former resides in the motor axon. 10. It is suggested that integrity of the discharge pattern of spinal motoneurones in dystrophic mice is interfered by anomalous impulse transmission in the motor axons and that the motoneurones in dystrophic mice are a homogeneous group rather than a mixture of 'normal' and
'abnormal' neurones. INTRODUCTION
A dystrophic mutation in mice originally described by Michelson, Russel & Harman (1955) is characterized by progressive, muscular atrophy and paralysis. The disorder in the dystrophic mice was considered to be primarily myogenic (Michelson et al..1955; West & Murphy, 1960; Pearce & Walton, 1963), but some abnormalities in the nervous system have subsequently been noticed. For example, the number of peripheral nerve fibres in these animals is significantly reduced over the whole range of axon diameters (Harris, Wallace & Wing, 1972; but cf. Bradley & Jenkison, 1973). Also, motor nerve terminals of dystrophic mice contain fewer synaptic vesicles, many of which are smaller in size and more elongated than those in normal mice (Ragab, 1971; Gilbert, Steinberg & Banker, 1973). Furthermore, the majority of fibres in the spinal roots (Bradley & Jenkison, 1973; Salafsky & Stirling, 1973) and cranial nerves (Biscoe, Caddy, Pallot, Pehrson & Stirling, 1974) of dystrophic mice are devoid of the myelin sheath. Salafsky (1971) has reported that the murine dystrophic muscle transplanted into a normal mouse regenerates with normal twitch characteristics, whereas the normal muscle transplanted into a dystrophic host achieves no functional regeneration (also, cf. Hironaka & Miyata, 1973). However, conflicting results have been observed by other investigators who used similar procedures (Laird & Timmer, 1965, 1966; Cosmos, 1973; Cosmos, Butler & Milhorat, 1973). Thus, sites of the primary disorder in dystrophic mice remain equivocal. It is known that the properties of skeletal muscles are determined, at least in part, by the properties of the innervating motoneurones (Buller, Eccles & Eccles, 1960; also, cf. Guth, 1968; Close, 1972). Morphological features of spinal motoneurones are similar in both normal and dystrophic mice (Papapetropoulos & Bradley, 1972; Bradley & Jenkison, 1973). However, no information has as yet been available concerning their
233 23 MOTONEURONES OF D YSTROPHIC MICE properties. The present study was undertaken to examine whether the electrophysiological properties of spinal motoneurones in dystrophic mice are different from those in normal mice. METHODS
The dystrophic mice (dy/dy), their phenotypically normal litter mates (?/ + ) and control mice (+ 1+) of the same strain (129/ReJ) were obtained from the Jackson laboratory, Bar Harbor, Maine. Animals of either sex ranging in age from 9 to 28 weeks were used in the present study. The body weight ranged from I11 to 18 g in dystrophic mice and from 18 to 26 g in the phenotypically normal litter mates and the control mice. In total, twelve control mice ( + / + ), sixteen phenotypically normal litter mates (?/ +) and thirty-one dystrophic (dy/dy) mice were used. The electrophysiological properties of the motoneurones in the first two groups of animals (+I/+ and ?/ + ) were indistinguishable. Therefore, the results obtained from these two groups were lumped as a control and compared with those obtained from dystrophic (dy/dy) mice. In preliminary experiments, attempts were made to maintain the animal on artificial respiration after decerebration or destruction of the brain rostral. to the medulla under ether or sodium pentobarbitone anaesthesia. The optimum rate of respiration seemed to be 160/mmn with a tidal volume of 0-2 ml., but all the animals expired within 3-4 hr. The results described in this report were therefore obtained from animals maintained on natural respiration. The mouse was initially anaesthetized by an i.r. injection of sodium pentobarbitone, 75 mg/kg, and a supplementary dose of 25 mg/kg (i.r.) was employed every 20-90 min throughout the experiment (of. McComas & Mossawy, 1965). At this level of anaesthesia, flexion reflexes to noxious stimuli were very weak, or absent, and respiratory movements could be minimized. After tracheostomy, the lumbar spinal cord was exposed by laminectomy with a pair of corneal scissors. The dorsal roots from the 4th to the 6th lumbar segments were cut on the left side. In a few experiments, the central ends of the cut dorsal roots were stimulated with a suction elec. trode to evoke synaptic responses in spinal motoneurones or reflex discharges in the peripheral nerve. In the left hind leg, the sciatic nerve was dissected. The tibial and common peroneal nerve branches were cut distally and prepared for stimulation or recording. Intracellular recording from spinal motoneurones was performed with glass micro-electrodes filled with 2 m potassium acetate. The resistance of the electrodes was between 8 and 30 MCiI. Recording from the motoneurone was identified by antidromic action potentials generated by stimuli applied to the central end of the cut tibial or common peroneal nerve. Exposed tissues in the hind leg were covered with a pool of paraffin oil, and the ik6~tal temperature was kept between 350 and 380 C by external heat.I The vertebral column was fixed by two spinous process clamps placed just above and below the laminectomy site. The vertebral bodies near the recording region were grasped with rigid steel wires from both sides. The entire exposed spinal cord was covered by 2 % agar dissolved in Hartmann's solution. Despite precautions taken to minimize movement of the spinal cord, most cells showed deterioration within a few minutes after penetration. Only in relatively few units was a stable membrane potential maintained for more than 30 min. At the end of the experiment, the spinal cord with attached ventral roots and peripheral nerves was excised to measure the conduction distance from the sites of stimulation to the recording segment. The conduction velocity was calculated from
234
P. HUIZAR, M. KUNO AND Y. MI YATA
the latency of antidromic action potentials evoked in the motoneurone at a stimulus intensity twice the threshold of each motor axon under study. The mean values of the results obtained from the control and dystrophic groups were statistically examined by two tail t tests with significance limit of 2P < 0 05.
A
B
C
'
