184
Brain Research, 106 (1976) 184 18,~ :t" Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands
Physiological evidence of formation of new synapses from cerebrum in the red nucleus neurons following cross-union of forelimb nerves
N. T S U K A H A R A AND Y. FUJ1TO
Department of Biophysical Engineering, Faculty of Engineering Science, Osaka University, Toyonaka, Osaka (Japan) (Accepted January 5th, 1976)
Histological 4-6 and physiological 4,9,10 investigations have shown that after partial denervation of the afferent input synaptic reorganization occurs m some central neurons of adult as well as of immature mammals. This indicates that the capacity for synaptogenesis is not limited to the ontogenetic period. To what extent this synaptic reorganization occurs in circumstances other than the denervation remains an important problem. Sperry 7,s investigated functional compensation of movement disorders following cross-innervation of nerves innervating flexor and extensor muscles in various species of mammals and found in monkey that m o t o r re-education occurs by inhibiting the reversed action and by learning the correct and smoothly coordinated movements. This has never been observed in similar experiments in rats. Eccles e t a l . 'e investigated the possible change of the synaptic connections of spinal motoneurons after cross-innervation of flexor and extensor nerves of hindlimbs. Although they found evidence suggesting the formation of new synapses on motoneurons after cross-innervation, the degree and extent of this reorganization was very limited. Since the synaptic plasticity in the higher level of the nervous system is expected to be larger than that in the lower level, it would be interesting to know whether the synaptic connections of the supraspinal neurons will receive modification after crossinnervation of flexor and extensor nerves. Red nucleus (RN) neurons provide an excellent substrate for investigating the synaptic modification by electrophysiological methods. The excitatory synapses from nucleus interpositus (IP) of the cerebellum make contact with the somatic membrane while corticorubral synapses are located on the peripheral dendrites 9. This synaptic organization characterizes several features of the excitatory postsynaptic potentials. Furthermore, efferent fibers of R N neurons innervate flexor and extensor muscles indirectly 3. The present investigation was undertaken to clarify with the physiological technique whether the formation of new synapses occurs in the R N neurons after crossinnervation of forelimb nerves. It wilt be shown that 2-5 months after cross-innervation the rising time course of the corticorubral EPSPs induced from the cerebral peduncle (CP) becomes shorter than that of normal cats. It will be suggested by analo-
185 1
A Jl__
2
D '~--
[ 0.5Kg
' 6 sec'
'2ms'
E
2ms
C F
:1 ~'~/ /
[ mv 2ms
2ms
Fig. I. A: isometric tension induced in the biceps muscle by direct (A1) stimulation as well as by stimulation o f radial nerve (A2) after 87 days o f cross-innervation. B - F : upper traces are intracellular responses in R N neurons, while the lower traces show the corresponding field potentials recorded at a just extracellular position. B and C illustrate a CP-EPSP and an IP-EPSP respectively (same cell) from a normal cat. D and E show a CP-EPSP and a S M - E P S P from a C cell o f cross-innervated cat (at 133 days after operation). F shows a C P - E P S P from a L cell o f cross-innervated cat (at 87 days after operation). Voltage calibration for all intra- and extracellular responses is shown at F. Time calibration in D also applies to E.
gy with the previous experimentsg, TM that new synaptic contacts are formed at the proximal portion of the soma-dendritic membrane of R N cells from corticorubral fibers which normally end at the peripheral dendrites. Adult cats were anesthetized with pentobarbitone sodium and paralyzed with gallamine and artificially respirated for acute experiments. Recording from RN neurons was essentially as reported previously°, 1°. The corticorubral fibers were stimulated at two levels: within the sensorimotor cortex (SM) and the cerebral peduncle (CP). For cross-union of the forelimb nerves, the musculocutaneous, median, radial and ulnar nerves of the right side (contralateral to the recording side from RN) were sectioned at the axillary region. The central stumps of the musculocutaneous, median and ulnar nerves were united to the peripheral stump of the radial nerve by suturing the nerve sheath with a fine silk thread. A similar procedure was applied to the central stump of the radial nerve which was united to the peripheral stumps of the musculocutaneous, median and ulnar nerves. For self-union of the forelimb nerves, the above mentioned nerves were sectioned and reunited without crossing by suturing the nerve sheath. All chronic operations were performed under aseptic conditions. After a postoperative period varying from 2 to 5 months, the cats were prepared for intracellular recording from R N contralateral to the nerve cross-union. The distal tendons of the muscles of triceps and biceps muscles were cut, and the distal portion of each muscle was freed of surrounding structures. The cut tendon was attached to a tension
186
N 10
A
N 15
5
0
TIME TO PEAK
msec
N 10-
N
B
-15
-10
-5
2
5
lIME TO PEAK
Z
0
g msec
Fig. 2. The frequency distribution of time to peak of CP-EPSPs in cross-innervated cats. A: C cells; B: L cells. Number of cells is shown on the ordinate and time to peak of CP-EPSPs in msec on the abscissa. The shaded columns in A and B illustrate the frequency distribution of time to peak in normal cats drawn from data given in ref. 10. The right-hand side scales of ordinates apply for normal cats and left-hand side ones for cross-innervated cats.
transducer in order to measure isometric tension induced by direct as well as nerve stimulation. The degree of functional reinnervation was roughly evaluated by the ratio of the maximal tension obtained by nerve stimulation (Fig. 1Az and Fig. 3A2) relative to that produced by direct stimulation of muscles (Fig. 1A1 and Fig. 3A1) 1. R N neurons were identified antidromically by stimulation of the contralateral C1 and L1 spinal segments. R N neurons activated antidromically from both C1 and L1 spinal segments were referred to as 'L cells' while those activated only from Ct were identified as 'C cells'. The typical slow CP-EPSP and fast IP-EPSP in a normal cat are illustrated in Fig. 1B and C, respectively. In contrast, the CP-EPSP of a C-cell of Fig. 1D from a cat with cross-union of the forelimb nerves has a much faster rise time than that seen in normal cats. Correspondingly, the spike potentials following CP stimulation were initiated with shorter latencies than in normal cats. Moreover, the fast-rising component of the CP-EPSP exhibited 'facilitation' of the synaptic transmission when tested by double CP stimuli just as observed in the normal corticorubral EPSPs (not illustrated). Similar fast-rising EPSPs could be induced by stimulation of the SM (Fig. 1E). On the contrary, the CP- and SM- EPSPs of L-cells had a much slower rise time than that in C-cells as shown in Fig. 1F.
187 I
2
A D
0.2Kg 5se¢
4 mV r
N 5¸
2ms
1 ~f
2 TIME
3
4
5 msec
TO P E A K
2ms
Fig. 3. A : isometric tension induced in the biceps muscle induced by direct (A1) stimulation as well as by stimulation of the musculocutaneous nerve (A2) after 84 days of self-union. B and C: upper traces are intracellular responses in RN neurons, while the lower traces show the extracellular controls. B and C illustrate CP-EPSPs of C-cells in self-union cat as in A. D: the frequency distribution of time to peak of CP-EPSPs of C-cells in a self-union cat. Numbers of cells on the ordinate and time to peak of CP-EPSPs in msec on the abscissa. Fig. 2A illustrates the frequency d i s t r i b u t i o n o f the time to peak of the CPEPSPs of C-cells of 9 cross-innervated cats as m e a s u r e d from the onset of the EPSP to the top after correcting for the extracellular field potentials. It is clearly seen that the time to peak of the CP-EPSPs in C-cells is m u c h faster t h a n in n o r m a l cats (shaded c o l u m n s o f Fig. 2 d r a w n from the previous experiments from our laboratory1°). In contrast, much slower time to peak of EPSPs was f o u n d in L-cells as shown in Fig. 2B. A similar experiment was performed in a cat with self-union of the forelimb nerves. As shown in Fig. 3B a n d D, the time to peak of the m a j o r i t y o f CP-EPSPs in C-cells is in the n o r m a l range, a l t h o u g h in some cells fast-rising CP-EPSPs could be induced in C-cells as exemplified in Fig. 3C. The most straightforward i n t e r p r e t a t i o n of the results is to assume that dendritic corticorubral synapses s p r o u t to form new synaptic contacts on the proximal portion of the soma-dendritic m e m b r a n e of R N cells. In view of the larger change of rise time of CP-EPSPs in cross-union experiments t h a n in self-union ones, this postulated synaptogenesis is considered to be caused by the reversed o u t p u t s after nerve crossing between flexors a n d extensors, although the possibility that this is due to a n o n specific effect after nerve sectioning c a n n o t completely be excluded. O u r results, however, suggest that in a d u l t cats the synaptic c o n n e c t i o n s of the intact R N n e u r o n s which are neither deafferented n o r deefferented can be altered.
1 ECCLES,J. C., ECCLES, R. M., AND KOZAK,W., Further investigations on the influence of motoneurones on the speed of muscle contraction, J. Physiol. (Lond.), 163 (1962) 324~339. 2 ECCLES,J. C., ECCLES, R. M., SHEALY,C. N., AND WILLIS,W. D., Experiments utilizing monosynaptic excitatory action on motoneurons for testing hypothesis relating to specificity of neuronal connection, J. Neurophysiol., 25 (1962) 559-579. 3 GHEZ, C., Input-output relations of the red nucleus in the cat, Brain Research, 98 (1975) 93-108.
188 4 LYNCH, G., DEADWYLER, S., AND COTMAN, C., Postlesion axonal growth produces permanent functional connections, Science, 180 (1973) 1364-1366. 5 NAKAMURA,Y., MIZUNO, N., KONISHI, A., AND SATO, M., Synaptic reorganization of the red nucleus after chronic deafferentation from cerebellorubral fibers: an electron microscopic study in the cat, Bra#~ Research, 82 0974) 298-301. 6 RAISMAN,G., Neuronal plasticity in the septal nuclei of the adult rat, Brain Research, 14 (1969) 2548. 7 SPERRY, R. W., The problem of central nervous reorganization after nerve regeneration and muscle transposition, Quart. Rev. Biol., 20 (1945) 311-369. 8 SPERRY, R. W., Effects of crossing nerves to antagonistic limb muscles in the monkey, Arch. Neurol. Psychiat. (Chic.), 58 (1947) 452473. 9 TSUKAHARA,N., HULTBORN, H., AND MURAKAMI,F., Sprouting of corticorubral synapses in red nucleus neurones after destruction of the nucleus interpositus of the cerebellum, Experientia (Basel), 30 (1974) 57-58. l0 TSUKAHARA,N., HULTBORN, H., MURAKAMt, F., AND FUJ1TO, Y., Electrophysiological study of the formation of new synapses and collateral sprouting in the red nucleus neurons after partial denervation, J. Neurophysiol., 38 (1975) 1359-1372.
Brain Research, 106 (1976) 184 18,~ :t" Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands
Physiological evidence of formation of new synapses from cerebrum in the red nucleus neurons following cross-union of forelimb nerves
N. T S U K A H A R A AND Y. FUJ1TO
Department of Biophysical Engineering, Faculty of Engineering Science, Osaka University, Toyonaka, Osaka (Japan) (Accepted January 5th, 1976)
Histological 4-6 and physiological 4,9,10 investigations have shown that after partial denervation of the afferent input synaptic reorganization occurs m some central neurons of adult as well as of immature mammals. This indicates that the capacity for synaptogenesis is not limited to the ontogenetic period. To what extent this synaptic reorganization occurs in circumstances other than the denervation remains an important problem. Sperry 7,s investigated functional compensation of movement disorders following cross-innervation of nerves innervating flexor and extensor muscles in various species of mammals and found in monkey that m o t o r re-education occurs by inhibiting the reversed action and by learning the correct and smoothly coordinated movements. This has never been observed in similar experiments in rats. Eccles e t a l . 'e investigated the possible change of the synaptic connections of spinal motoneurons after cross-innervation of flexor and extensor nerves of hindlimbs. Although they found evidence suggesting the formation of new synapses on motoneurons after cross-innervation, the degree and extent of this reorganization was very limited. Since the synaptic plasticity in the higher level of the nervous system is expected to be larger than that in the lower level, it would be interesting to know whether the synaptic connections of the supraspinal neurons will receive modification after crossinnervation of flexor and extensor nerves. Red nucleus (RN) neurons provide an excellent substrate for investigating the synaptic modification by electrophysiological methods. The excitatory synapses from nucleus interpositus (IP) of the cerebellum make contact with the somatic membrane while corticorubral synapses are located on the peripheral dendrites 9. This synaptic organization characterizes several features of the excitatory postsynaptic potentials. Furthermore, efferent fibers of R N neurons innervate flexor and extensor muscles indirectly 3. The present investigation was undertaken to clarify with the physiological technique whether the formation of new synapses occurs in the R N neurons after crossinnervation of forelimb nerves. It wilt be shown that 2-5 months after cross-innervation the rising time course of the corticorubral EPSPs induced from the cerebral peduncle (CP) becomes shorter than that of normal cats. It will be suggested by analo-
185 1
A Jl__
2
D '~--
[ 0.5Kg
' 6 sec'
'2ms'
E
2ms
C F
:1 ~'~/ /
[ mv 2ms
2ms
Fig. I. A: isometric tension induced in the biceps muscle by direct (A1) stimulation as well as by stimulation o f radial nerve (A2) after 87 days o f cross-innervation. B - F : upper traces are intracellular responses in R N neurons, while the lower traces show the corresponding field potentials recorded at a just extracellular position. B and C illustrate a CP-EPSP and an IP-EPSP respectively (same cell) from a normal cat. D and E show a CP-EPSP and a S M - E P S P from a C cell o f cross-innervated cat (at 133 days after operation). F shows a C P - E P S P from a L cell o f cross-innervated cat (at 87 days after operation). Voltage calibration for all intra- and extracellular responses is shown at F. Time calibration in D also applies to E.
gy with the previous experimentsg, TM that new synaptic contacts are formed at the proximal portion of the soma-dendritic membrane of R N cells from corticorubral fibers which normally end at the peripheral dendrites. Adult cats were anesthetized with pentobarbitone sodium and paralyzed with gallamine and artificially respirated for acute experiments. Recording from RN neurons was essentially as reported previously°, 1°. The corticorubral fibers were stimulated at two levels: within the sensorimotor cortex (SM) and the cerebral peduncle (CP). For cross-union of the forelimb nerves, the musculocutaneous, median, radial and ulnar nerves of the right side (contralateral to the recording side from RN) were sectioned at the axillary region. The central stumps of the musculocutaneous, median and ulnar nerves were united to the peripheral stump of the radial nerve by suturing the nerve sheath with a fine silk thread. A similar procedure was applied to the central stump of the radial nerve which was united to the peripheral stumps of the musculocutaneous, median and ulnar nerves. For self-union of the forelimb nerves, the above mentioned nerves were sectioned and reunited without crossing by suturing the nerve sheath. All chronic operations were performed under aseptic conditions. After a postoperative period varying from 2 to 5 months, the cats were prepared for intracellular recording from R N contralateral to the nerve cross-union. The distal tendons of the muscles of triceps and biceps muscles were cut, and the distal portion of each muscle was freed of surrounding structures. The cut tendon was attached to a tension
186
N 10
A
N 15
5
0
TIME TO PEAK
msec
N 10-
N
B
-15
-10
-5
2
5
lIME TO PEAK
Z
0
g msec
Fig. 2. The frequency distribution of time to peak of CP-EPSPs in cross-innervated cats. A: C cells; B: L cells. Number of cells is shown on the ordinate and time to peak of CP-EPSPs in msec on the abscissa. The shaded columns in A and B illustrate the frequency distribution of time to peak in normal cats drawn from data given in ref. 10. The right-hand side scales of ordinates apply for normal cats and left-hand side ones for cross-innervated cats.
transducer in order to measure isometric tension induced by direct as well as nerve stimulation. The degree of functional reinnervation was roughly evaluated by the ratio of the maximal tension obtained by nerve stimulation (Fig. 1Az and Fig. 3A2) relative to that produced by direct stimulation of muscles (Fig. 1A1 and Fig. 3A1) 1. R N neurons were identified antidromically by stimulation of the contralateral C1 and L1 spinal segments. R N neurons activated antidromically from both C1 and L1 spinal segments were referred to as 'L cells' while those activated only from Ct were identified as 'C cells'. The typical slow CP-EPSP and fast IP-EPSP in a normal cat are illustrated in Fig. 1B and C, respectively. In contrast, the CP-EPSP of a C-cell of Fig. 1D from a cat with cross-union of the forelimb nerves has a much faster rise time than that seen in normal cats. Correspondingly, the spike potentials following CP stimulation were initiated with shorter latencies than in normal cats. Moreover, the fast-rising component of the CP-EPSP exhibited 'facilitation' of the synaptic transmission when tested by double CP stimuli just as observed in the normal corticorubral EPSPs (not illustrated). Similar fast-rising EPSPs could be induced by stimulation of the SM (Fig. 1E). On the contrary, the CP- and SM- EPSPs of L-cells had a much slower rise time than that in C-cells as shown in Fig. 1F.
187 I
2
A D
0.2Kg 5se¢
4 mV r
N 5¸
2ms
1 ~f
2 TIME
3
4
5 msec
TO P E A K
2ms
Fig. 3. A : isometric tension induced in the biceps muscle induced by direct (A1) stimulation as well as by stimulation of the musculocutaneous nerve (A2) after 84 days of self-union. B and C: upper traces are intracellular responses in RN neurons, while the lower traces show the extracellular controls. B and C illustrate CP-EPSPs of C-cells in self-union cat as in A. D: the frequency distribution of time to peak of CP-EPSPs of C-cells in a self-union cat. Numbers of cells on the ordinate and time to peak of CP-EPSPs in msec on the abscissa. Fig. 2A illustrates the frequency d i s t r i b u t i o n o f the time to peak of the CPEPSPs of C-cells of 9 cross-innervated cats as m e a s u r e d from the onset of the EPSP to the top after correcting for the extracellular field potentials. It is clearly seen that the time to peak of the CP-EPSPs in C-cells is m u c h faster t h a n in n o r m a l cats (shaded c o l u m n s o f Fig. 2 d r a w n from the previous experiments from our laboratory1°). In contrast, much slower time to peak of EPSPs was f o u n d in L-cells as shown in Fig. 2B. A similar experiment was performed in a cat with self-union of the forelimb nerves. As shown in Fig. 3B a n d D, the time to peak of the m a j o r i t y o f CP-EPSPs in C-cells is in the n o r m a l range, a l t h o u g h in some cells fast-rising CP-EPSPs could be induced in C-cells as exemplified in Fig. 3C. The most straightforward i n t e r p r e t a t i o n of the results is to assume that dendritic corticorubral synapses s p r o u t to form new synaptic contacts on the proximal portion of the soma-dendritic m e m b r a n e of R N cells. In view of the larger change of rise time of CP-EPSPs in cross-union experiments t h a n in self-union ones, this postulated synaptogenesis is considered to be caused by the reversed o u t p u t s after nerve crossing between flexors a n d extensors, although the possibility that this is due to a n o n specific effect after nerve sectioning c a n n o t completely be excluded. O u r results, however, suggest that in a d u l t cats the synaptic c o n n e c t i o n s of the intact R N n e u r o n s which are neither deafferented n o r deefferented can be altered.
1 ECCLES,J. C., ECCLES, R. M., AND KOZAK,W., Further investigations on the influence of motoneurones on the speed of muscle contraction, J. Physiol. (Lond.), 163 (1962) 324~339. 2 ECCLES,J. C., ECCLES, R. M., SHEALY,C. N., AND WILLIS,W. D., Experiments utilizing monosynaptic excitatory action on motoneurons for testing hypothesis relating to specificity of neuronal connection, J. Neurophysiol., 25 (1962) 559-579. 3 GHEZ, C., Input-output relations of the red nucleus in the cat, Brain Research, 98 (1975) 93-108.
188 4 LYNCH, G., DEADWYLER, S., AND COTMAN, C., Postlesion axonal growth produces permanent functional connections, Science, 180 (1973) 1364-1366. 5 NAKAMURA,Y., MIZUNO, N., KONISHI, A., AND SATO, M., Synaptic reorganization of the red nucleus after chronic deafferentation from cerebellorubral fibers: an electron microscopic study in the cat, Bra#~ Research, 82 0974) 298-301. 6 RAISMAN,G., Neuronal plasticity in the septal nuclei of the adult rat, Brain Research, 14 (1969) 2548. 7 SPERRY, R. W., The problem of central nervous reorganization after nerve regeneration and muscle transposition, Quart. Rev. Biol., 20 (1945) 311-369. 8 SPERRY, R. W., Effects of crossing nerves to antagonistic limb muscles in the monkey, Arch. Neurol. Psychiat. (Chic.), 58 (1947) 452473. 9 TSUKAHARA,N., HULTBORN, H., AND MURAKAMI,F., Sprouting of corticorubral synapses in red nucleus neurones after destruction of the nucleus interpositus of the cerebellum, Experientia (Basel), 30 (1974) 57-58. l0 TSUKAHARA,N., HULTBORN, H., MURAKAMt, F., AND FUJ1TO, Y., Electrophysiological study of the formation of new synapses and collateral sprouting in the red nucleus neurons after partial denervation, J. Neurophysiol., 38 (1975) 1359-1372.
