- Email: [email protected]
Swelling of frog dorsal root ganglion and spinal cord produced by afferent volley of impulses
360
Brain Research, 272 (19g.3) 361~-36~
Elsevier
Swelling of frog dorsal root ganglion and spinal cord produced by afferent volley of impulses ICHIJI TASAK1 and PAUL M. BYRNE National Institute of Mental Health, 9000Rockville Pike, Building36, Room 1D-02, Bethesda, MD 20205 (U. S.A.
(Accepted April 19th, 1983) Key words." swelling of neurons - - mechanical response - - dorsal root ganglion - - spinal cord
Electrical stimulation of the frog sciatic nerve was found to produce rapid, transient swelling of the 8th and 9th dorsal root ganglia followed by prolonged swelling of the spinal cord. Swelling of the ganglion is analogous to the rapid mechanical change observed in invertebrate axons during excitation. The mechanical change observed in the spinal cord is probably related to prolonged depolarization of the primary afferent fibers near their terminals.
It has been shown recently that production of an action potential in the squid and crab nerve fiber is accompanied by rapid, reversible mechanical changes in the fiber 4,12,~3. These mechanical changes observed in invertebrate nerve fibers are considered to be brought about by a profound alteration of the state of the macromolecules at and near the axon surface 12. The present communication describes the results of our experiments designed to test whether or not similar mechanical changes can be demonstrated in the sciatic nerve, dorsal root ganglion and spinal cord of the frog. Large grass-frogs, Rana pipiens, and bullfrogs, Rana catesbeiana, were used. Following decapitation, the spinal cord was surgically exposed from the dorsal side. In the first series of experiments, the 8th and 9th dorsal root ganglia were excised together with the sciatic nerve and the dorsal roots connected. Mechanical measurements were carried out by mounting the preparation in a plastic chamber with a convex bottom (see the diagram in Fig. 1). Electric shocks were delivered either to the sciatic nerve or to the dorsal root by means of silver electrodes placed across a plastic partition of the chamber. The action potentials were recorded extracellularly across another partiiion. In the second series of experiments, the spinal cord was left in the surgically opened vertebral column. When electric shocks were delivered to the sciatic nerve (see the diagram in Fig. 2), the 8th,
9th and 10th ventral roots were severed. When stimulation of the dorsal roots was required, the 8th and 9th dorsal roots were tied together with thread and were severed near the ganglia. As a rule, the spinal cord was kept submerged in oxygenated Ringer's solution containing glucoseS. Mechanical changes in the neural tissues were examined with a sensitive mechano-electric transducer constructed by using a film of polyvinylidene fluoride (PVDF) which was a generous gift of Kureha Chemicals in Tokyo. P V D F is a synthetic polymer which becomes highly piezo-electric when stretched at a high temperature in a high electric field 9. The film employed was 9 ~ m in thickness and had a 0.03/~m thick aluminum layer deposited on each surface. A strip of P V D F film (3 mm in width and 35 mm in length) was folded in the middle to make a piezo-electric device with bimorph structure 1-~. The outer aluminum layer was grounded and the inner layer was led to the input of a charge amplifier. The sensitivity of the detector was approximately 7 V/g. A small stylus (S in the figures) was used to transmit the force generated by the neural tissue to the detector. The output of the amplifier was connected to a signal averager after amplification by a factor of 100. The signal was averaged usually over 64 to 1024 trials. Fig. 1 shows an example of the records obtained from the dorsal root ganglia. The upward deflection of the upper trace represents a rise in the force tend-
361
S
E
i\ /////i//,//l'///#y[/'////////////////,////~/,
DORSAL
RIN~SER
I'
ROOT
l
GANGLION
!
5ms
Fig. 1. Left: schematic drawing of the experimental set-up used for demonstrating transient mechanical changes in the frog dorsal root ganglion. S, stylus attached to a piezo-electric transducer; E, stimulating electrodes; V, site of action potential recording. Right: swelling (rise in pressure) of a dorsal root ganglion produced by maximal stimulation of the sciatic nerve (top) and the action potential recorded from the dorsal root connected to the ganglion (bottom). The mechanical record was obtained by signal-averaging over 1024 trials repeated 10/s. The left end of the time marker represents the time of delivery of an electric shock. 21 °C.
E
S
CORD
_
RI
~,
~////////////////////
, I
.
,,.
•
v"~..-.]~
~
"-'Ix.-. , .....
,
20ms Fig. 2. Top: schematic diagram of the experimental set-up for detecting rapid mechanical changes in the frog spinal cord. S, stylus; E, stimulating electrodes. Bottom: record of rise in pressure of the spinal cord produced by a volley of afferent impulses. The arrow indicates delivery of an electric shock (0.1 ms in duration). The record was taken after signal-averaging over 128 trials repeated at 2/s. 22 °C.
362 ing to move the stylus upwards. The maximum value of the force observed varied usually between 5 and 15 /~g. The action potential recorded extraceilularly across the partition located about 2 mm away from the ganglion is also shown. The peak of the action potential is seen to precede the peak of the mechanical change. Note that the ganglion contains roughly twice as many cell bodies that are connected to nonmyelinated nerve fibers as those connected to myelinated fibers 14. It is reasonable to assume that cell bodies connected to small fibers make a significant contribution to the mechanical change. The delay of the peak of the mechanical trace may by explained on the basis of this assumption. By using the same experimental set-up, attempts were made to record transient mechanical changes in the sciatic nerve following maximal stimulation of the nerve. No detectable mechanical change was observed. (Note that mechanical changes smaller than about 1/~g in amplitude cannot be detected under the present conditions.) The signal averager record furnished in Fig. 2 is an example of the mechanical changes taken from the spinal cord. Again the upward deflection of the trace indicates a rise in the force tending to move the stylus upwards. Following maximal stimulation of the sciatic nerve, the force is seen to rise rapidly and fall gradually. With the site of stimulation located at about 10 mm distal to the point of entry of the 9th root, the maximum of the mechanical change was reached between 2 and 8 ms after the shock. The maximum force developed was usually between 15 and 30 ~g. Since the flattened surface of the stylus, placed near the sites of entry of the 8th and 9th roots, had an area of about 0.01 cm 2, the pressure rise induced by invasion of an afferent volley was of the order of few dyne/cm 2. The duration of the falling phase of the mechanical change was variable. When electric shocks were delivered directly to the dorsal roots, the rise-time of the mechanical change was shorter. The dependence of the latency and the rise-time of the mechanical changes on the distance between the spinal cord and the site of stimulation indicates that the phenomenon under study is brought about by the afferent volley of impulses and not by spread of applied electric currents. The possible significance of the experimental findings described above is now considered. The phe-
nomenon of swelling of the dorsal root ganglion associated with excitation is in all probability similar to that described in the crab nerve and in the squid giant axon4.12~13. The nerve cells in the ganglion are known to be directly involved in the process of action potential production3.7.The force generated by the ganglion during excitation is of the same order of magnitude as the corresponding value in the crab nerve 4. A large dorsal root ganglion of the frog is known to contain 2000-3000 nerve cells 14. It is difficult, therefore, to study the temporal relationship between the mechanical change and the action potential. The difficulty of demonstrating rapid mechanical changes associated with excitation of the sciatic nerve is most probably related to the presence of many large myelinated nerve fibers. Since the nodal area occupies an extremely small fraction of the surface of a myelinated nerve fiber 12, mechanical changes associated with a propagated impulse are expected to be very small. We have known for some time that displacements of the surface of the sciatic nerve cannot be detected by our optical device which is capable of detecting a rapid displacement of a few nm in squid axons le. The interference method employed previously by Kayushin and Lyudkovskaya 6 did not produce any evidence of rapid movements of the sciatic nerve during excitation 10. Finally, the possible origin of the mechanical changes in the spinal cord produced by an afferent volley of impulses is considered. According to Joseph and Whitlock 5, the nerve fibers in the 9th dorsal root terminate in the ipsilateral dorsal and intermediate gray matter within 5 segments. Upon arrival of an afferent volley of impulses, the terminal portions of these primary afferent fibers, which are probably making extensive axo-axonic and axo-dendritic contacts 1, are thrown into a state of prolonged depolarization n. The primary mechanical events evoked by the afferent volley are probably associated with this prolonged presynaptic depolarization. Note that depolarization of the squid giant axon by a pulse of outwardly directed membrane current brings about swelling of the axon 13. In the following period, excitation of interneurons and motoneurons takes place2,U. Further studies are required to clarify the relationship between various electrophysiological events in the spinal cord and the observed mechanical changes.
363 We thank Mr, Araki and Mr. O b a r a of Kureha
Tokyo, for the gift of polyvinylidene fluoride film.
Chemical Industry, 1-9 Horidome-cho, Nihonbashi,
1 Charlton, B. T. and Gray, E. G., Comparative electron microscopy of synapses in the vertebrate spinal cord, J. Cell Sci., 1 (1966) 67-80. 2 Hughes, J. and Gasser, H. S., The response of the spinal cord to two afferent volleys, Amer. J. Physiol., 108 (1934) 307-321. 3 Ito, M., The electrical activity of spinal ganglion cells investigated with intracellular microelectrodes, Jap. J. Physiol., 7 (1957) 297-323. 4 Iwasa, K., Tasaki, I. and Gibbons, R. C., Swellingof nerve fibers associated with action potentials, Science, 210 (1980) 338-339. 5 Joseph, B. S. and Whitlock, D. G., Central projections of selected spinal dorsal roots in anuran amphibians, Anat. Rec., 160 (1968) 279-288. 6 Kayushin, L. P. and Lyudkovskaya, R. G., Study of the elastic-volumetric changes in a nerve on stimulation by the interference method, Doklady Akad. nauk SSSR, 95 (1954) 253-255. 7 Koketsu, K., Cerf, J. A. and Nishi, S., Effect of quaternary ammonium ions on electrical activity of spinal ganglion cells
in frogs, J. Neurophysiol., 22 (1959) 177-194. 8 Machne, X., Fadiga, E. and Brookhart, J. M., Antidromic and synaptic activation of frog motor neurons, J. Neurophysiol., 22 (1959) 483-503. 9 Murayama, N., Nakamura, K., Obara, H., and Segawa, M., The strong piezoelectricity in polyvinylidene fluoride (PVDF), Ultrasonics., January (1976) 15-23. 10 Sandlin, R., Lerman, L., Barry, W. and Tasaki, I., Application of laser interferometry to physiological studies of excitable tissues, Nature (Lond.), 217 (1968) 575-576. 11 Schmidt, R. F., Presynaptic inhibition in the vertebrate central nervous system, Ergebn. Physiol., 63 (1971) 20--101. 12 Tasaki, I., Physiology and Electrochemistry of Nerve Fibers, Academic Press, New York, 1982, 350 pp. 13 Tasaki, I. and Iwasa, K., Further studies of rapid mechanical changes in squid guid axon associated with action potential production, Jap. J. Physiol., 32 (1982) 505--518, 14 Wilhelm, G. B. and Coggeshall, R. E., An electron microscopic analysis of the dorsal root of the frog, J, comp. Neurol., 196 (1981) 421-429.
Brain Research, 272 (19g.3) 361~-36~
Elsevier
Swelling of frog dorsal root ganglion and spinal cord produced by afferent volley of impulses ICHIJI TASAK1 and PAUL M. BYRNE National Institute of Mental Health, 9000Rockville Pike, Building36, Room 1D-02, Bethesda, MD 20205 (U. S.A.
(Accepted April 19th, 1983) Key words." swelling of neurons - - mechanical response - - dorsal root ganglion - - spinal cord
Electrical stimulation of the frog sciatic nerve was found to produce rapid, transient swelling of the 8th and 9th dorsal root ganglia followed by prolonged swelling of the spinal cord. Swelling of the ganglion is analogous to the rapid mechanical change observed in invertebrate axons during excitation. The mechanical change observed in the spinal cord is probably related to prolonged depolarization of the primary afferent fibers near their terminals.
It has been shown recently that production of an action potential in the squid and crab nerve fiber is accompanied by rapid, reversible mechanical changes in the fiber 4,12,~3. These mechanical changes observed in invertebrate nerve fibers are considered to be brought about by a profound alteration of the state of the macromolecules at and near the axon surface 12. The present communication describes the results of our experiments designed to test whether or not similar mechanical changes can be demonstrated in the sciatic nerve, dorsal root ganglion and spinal cord of the frog. Large grass-frogs, Rana pipiens, and bullfrogs, Rana catesbeiana, were used. Following decapitation, the spinal cord was surgically exposed from the dorsal side. In the first series of experiments, the 8th and 9th dorsal root ganglia were excised together with the sciatic nerve and the dorsal roots connected. Mechanical measurements were carried out by mounting the preparation in a plastic chamber with a convex bottom (see the diagram in Fig. 1). Electric shocks were delivered either to the sciatic nerve or to the dorsal root by means of silver electrodes placed across a plastic partition of the chamber. The action potentials were recorded extracellularly across another partiiion. In the second series of experiments, the spinal cord was left in the surgically opened vertebral column. When electric shocks were delivered to the sciatic nerve (see the diagram in Fig. 2), the 8th,
9th and 10th ventral roots were severed. When stimulation of the dorsal roots was required, the 8th and 9th dorsal roots were tied together with thread and were severed near the ganglia. As a rule, the spinal cord was kept submerged in oxygenated Ringer's solution containing glucoseS. Mechanical changes in the neural tissues were examined with a sensitive mechano-electric transducer constructed by using a film of polyvinylidene fluoride (PVDF) which was a generous gift of Kureha Chemicals in Tokyo. P V D F is a synthetic polymer which becomes highly piezo-electric when stretched at a high temperature in a high electric field 9. The film employed was 9 ~ m in thickness and had a 0.03/~m thick aluminum layer deposited on each surface. A strip of P V D F film (3 mm in width and 35 mm in length) was folded in the middle to make a piezo-electric device with bimorph structure 1-~. The outer aluminum layer was grounded and the inner layer was led to the input of a charge amplifier. The sensitivity of the detector was approximately 7 V/g. A small stylus (S in the figures) was used to transmit the force generated by the neural tissue to the detector. The output of the amplifier was connected to a signal averager after amplification by a factor of 100. The signal was averaged usually over 64 to 1024 trials. Fig. 1 shows an example of the records obtained from the dorsal root ganglia. The upward deflection of the upper trace represents a rise in the force tend-
361
S
E
i\ /////i//,//l'///#y[/'////////////////,////~/,
DORSAL
RIN~SER
I'
ROOT
l
GANGLION
!
5ms
Fig. 1. Left: schematic drawing of the experimental set-up used for demonstrating transient mechanical changes in the frog dorsal root ganglion. S, stylus attached to a piezo-electric transducer; E, stimulating electrodes; V, site of action potential recording. Right: swelling (rise in pressure) of a dorsal root ganglion produced by maximal stimulation of the sciatic nerve (top) and the action potential recorded from the dorsal root connected to the ganglion (bottom). The mechanical record was obtained by signal-averaging over 1024 trials repeated 10/s. The left end of the time marker represents the time of delivery of an electric shock. 21 °C.
E
S
CORD
_
RI
~,
~////////////////////
, I
.
,,.
•
v"~..-.]~
~
"-'Ix.-. , .....
,
20ms Fig. 2. Top: schematic diagram of the experimental set-up for detecting rapid mechanical changes in the frog spinal cord. S, stylus; E, stimulating electrodes. Bottom: record of rise in pressure of the spinal cord produced by a volley of afferent impulses. The arrow indicates delivery of an electric shock (0.1 ms in duration). The record was taken after signal-averaging over 128 trials repeated at 2/s. 22 °C.
362 ing to move the stylus upwards. The maximum value of the force observed varied usually between 5 and 15 /~g. The action potential recorded extraceilularly across the partition located about 2 mm away from the ganglion is also shown. The peak of the action potential is seen to precede the peak of the mechanical change. Note that the ganglion contains roughly twice as many cell bodies that are connected to nonmyelinated nerve fibers as those connected to myelinated fibers 14. It is reasonable to assume that cell bodies connected to small fibers make a significant contribution to the mechanical change. The delay of the peak of the mechanical trace may by explained on the basis of this assumption. By using the same experimental set-up, attempts were made to record transient mechanical changes in the sciatic nerve following maximal stimulation of the nerve. No detectable mechanical change was observed. (Note that mechanical changes smaller than about 1/~g in amplitude cannot be detected under the present conditions.) The signal averager record furnished in Fig. 2 is an example of the mechanical changes taken from the spinal cord. Again the upward deflection of the trace indicates a rise in the force tending to move the stylus upwards. Following maximal stimulation of the sciatic nerve, the force is seen to rise rapidly and fall gradually. With the site of stimulation located at about 10 mm distal to the point of entry of the 9th root, the maximum of the mechanical change was reached between 2 and 8 ms after the shock. The maximum force developed was usually between 15 and 30 ~g. Since the flattened surface of the stylus, placed near the sites of entry of the 8th and 9th roots, had an area of about 0.01 cm 2, the pressure rise induced by invasion of an afferent volley was of the order of few dyne/cm 2. The duration of the falling phase of the mechanical change was variable. When electric shocks were delivered directly to the dorsal roots, the rise-time of the mechanical change was shorter. The dependence of the latency and the rise-time of the mechanical changes on the distance between the spinal cord and the site of stimulation indicates that the phenomenon under study is brought about by the afferent volley of impulses and not by spread of applied electric currents. The possible significance of the experimental findings described above is now considered. The phe-
nomenon of swelling of the dorsal root ganglion associated with excitation is in all probability similar to that described in the crab nerve and in the squid giant axon4.12~13. The nerve cells in the ganglion are known to be directly involved in the process of action potential production3.7.The force generated by the ganglion during excitation is of the same order of magnitude as the corresponding value in the crab nerve 4. A large dorsal root ganglion of the frog is known to contain 2000-3000 nerve cells 14. It is difficult, therefore, to study the temporal relationship between the mechanical change and the action potential. The difficulty of demonstrating rapid mechanical changes associated with excitation of the sciatic nerve is most probably related to the presence of many large myelinated nerve fibers. Since the nodal area occupies an extremely small fraction of the surface of a myelinated nerve fiber 12, mechanical changes associated with a propagated impulse are expected to be very small. We have known for some time that displacements of the surface of the sciatic nerve cannot be detected by our optical device which is capable of detecting a rapid displacement of a few nm in squid axons le. The interference method employed previously by Kayushin and Lyudkovskaya 6 did not produce any evidence of rapid movements of the sciatic nerve during excitation 10. Finally, the possible origin of the mechanical changes in the spinal cord produced by an afferent volley of impulses is considered. According to Joseph and Whitlock 5, the nerve fibers in the 9th dorsal root terminate in the ipsilateral dorsal and intermediate gray matter within 5 segments. Upon arrival of an afferent volley of impulses, the terminal portions of these primary afferent fibers, which are probably making extensive axo-axonic and axo-dendritic contacts 1, are thrown into a state of prolonged depolarization n. The primary mechanical events evoked by the afferent volley are probably associated with this prolonged presynaptic depolarization. Note that depolarization of the squid giant axon by a pulse of outwardly directed membrane current brings about swelling of the axon 13. In the following period, excitation of interneurons and motoneurons takes place2,U. Further studies are required to clarify the relationship between various electrophysiological events in the spinal cord and the observed mechanical changes.
363 We thank Mr, Araki and Mr. O b a r a of Kureha
Tokyo, for the gift of polyvinylidene fluoride film.
Chemical Industry, 1-9 Horidome-cho, Nihonbashi,
1 Charlton, B. T. and Gray, E. G., Comparative electron microscopy of synapses in the vertebrate spinal cord, J. Cell Sci., 1 (1966) 67-80. 2 Hughes, J. and Gasser, H. S., The response of the spinal cord to two afferent volleys, Amer. J. Physiol., 108 (1934) 307-321. 3 Ito, M., The electrical activity of spinal ganglion cells investigated with intracellular microelectrodes, Jap. J. Physiol., 7 (1957) 297-323. 4 Iwasa, K., Tasaki, I. and Gibbons, R. C., Swellingof nerve fibers associated with action potentials, Science, 210 (1980) 338-339. 5 Joseph, B. S. and Whitlock, D. G., Central projections of selected spinal dorsal roots in anuran amphibians, Anat. Rec., 160 (1968) 279-288. 6 Kayushin, L. P. and Lyudkovskaya, R. G., Study of the elastic-volumetric changes in a nerve on stimulation by the interference method, Doklady Akad. nauk SSSR, 95 (1954) 253-255. 7 Koketsu, K., Cerf, J. A. and Nishi, S., Effect of quaternary ammonium ions on electrical activity of spinal ganglion cells
in frogs, J. Neurophysiol., 22 (1959) 177-194. 8 Machne, X., Fadiga, E. and Brookhart, J. M., Antidromic and synaptic activation of frog motor neurons, J. Neurophysiol., 22 (1959) 483-503. 9 Murayama, N., Nakamura, K., Obara, H., and Segawa, M., The strong piezoelectricity in polyvinylidene fluoride (PVDF), Ultrasonics., January (1976) 15-23. 10 Sandlin, R., Lerman, L., Barry, W. and Tasaki, I., Application of laser interferometry to physiological studies of excitable tissues, Nature (Lond.), 217 (1968) 575-576. 11 Schmidt, R. F., Presynaptic inhibition in the vertebrate central nervous system, Ergebn. Physiol., 63 (1971) 20--101. 12 Tasaki, I., Physiology and Electrochemistry of Nerve Fibers, Academic Press, New York, 1982, 350 pp. 13 Tasaki, I. and Iwasa, K., Further studies of rapid mechanical changes in squid guid axon associated with action potential production, Jap. J. Physiol., 32 (1982) 505--518, 14 Wilhelm, G. B. and Coggeshall, R. E., An electron microscopic analysis of the dorsal root of the frog, J, comp. Neurol., 196 (1981) 421-429.