PfliJgers Archiv
PflfigersArch. 369, 187- 192 (1977)
EuropeanJoumal of Physiology
9 by Springer-Verlag1977
Bilateral Dorsal Root Potentials in the Lower Sacral Spinal Cord KRYSTYNA LUPA and ANDRZEJ NIECHAJ Department of Human Physiology,MedicalSchool, Dymitrowa11, PL-20-080 Lublin,Poland
Summary. Bilateral dorsal root potentials (DRPs) evoked in the $3 dorsal roots by stimulation of the $2 and L6 dorsal roots and the cutaneous afferents entering the spinal cord in the lumbar segments have been studied in spinal cats. Stimulation of all these afferents produces DRPs which have the same amplitude on both sides of the spinal cord. During longlasting repetitive stimulation the negativity of the ipsilateral dorsal root is maintained only when this stimulation is applied to the neighbouring dorsal root. Depression of the testing DRPs produced by preceding single volleys or repetitive stimulation is only slightly larger on the contralateral side of the cord. The difference between depression of the DRPs on both sides of the cord is significantly smaller in the $3 than in the L7 segment. Following conditioning tetanization both ipsi- and contralateral DRPs undergo depression. The pattern of bilateral DRPs in the $3 segment significantly differs from that observed in the L7 segment and these differences correspond to the already known distinct arrangement of the substantia gelatinosa in the two parts of the cord. Key words: Bilateral dorsal root potentials - Presynaptic inhibition - Spinal cord - Sacral cord.
INTRODUCTION The dorsal root potentials (DRPs) indicate the depolarization of primary afferent terminals produced by stimulation of the dorsal roots or peripheral nerves. In the cat they were studied mainly in the lower lumbar segments in close neighbourhood to the place where an afferent volley enters the spinal cord. These DRPs have latencies of a few milliseconds and show temporal Send offprint request to: A. Niechaj at the above address
facilitation and depression during repetitive activation which are features characterizing polysynaptic pathways [4,5,7,8]. The potential changes may also be recorded on the contralateral side of the cord where they are smaller, appear after longer delay and display more pronounced depression in interaction experiments [2,10,11,13,14,16]. The difference between the DRPs on both sides of the spinal cord has been attributed to the properties of the pathway producing contralateral depolarization of the primary afferent fibres [13]. The DRPs are generated by the activity of the substantia gelatinosa reflecting on to primary afferent fibres [23]. It was found, however, that both these structures display distinct arrangement in the lumbar and sacral segments. Following section of the lumbar dorsal roots the degeneration of afferent fibres is unilateral. After severing the $3 dorsal root this degeneration is seen on both sides of the cord and stimulation experiments demonstrated continuity of fibres from the dorsal roots to the contralateral grey matter [12,19, 21,25]. The substantia gelatinosa in the lumbar enlargement lies separately in each half of the cord. In the sacral segments it extends across the midline and forms a continuous sheet spreading from one side of the cord to the other [23, 24]. The above data suggest that the DRPs in the lower sacral cord may have properties different from these displayed by the potential changes in the lumbar segments. The present study is an investigation of the bilateral DRPs in the third sacral segment of the spinal cord.
METHODS The experimentswere performed on 32 adult cats weighing2.03.8 kg lightlyanaesthetizedwith thiamylalsodium(Surital, ParkeDavis, 30 mg/kg given i.p.) or pentobarbitone sodium(Nembutal, Abbott Lab., 40 mg/kg i.p.). Animals were immobilized with gallamine triethiodide(Flaxedil, Specia, repeated doses of 10 mg/
188 kg, given i.v.) and set on artificial respiration. The spinal cord was transected at a low thoracic level. A laminectomy was performed from the third lumbar to the middle caudal segments. The dorsal root filaments of the right and left $3 were traced to their entrance into the spinal cord. Two filaments entering the cord from both sides at the same level and being approximately the same diameter were cut distally. In the same way the L7 dorsal rootlets were prepared. These filaments were used for recording the bilateral DRPs at the two segmental levels. The dorsal rootlets of the $2 and L6 and two peripheral nerves: the superficial peroneal and posterior tibial were dissected on one side and cut peripherally for electric stimulation. The S1, $2 and the remaining part of the $3 dorsal root were cut. This procedure confined the zone of entry of impulses produced by stimulation of the peripheral nerves to the lumbar segments of the cord. It thus made it possible to compare the DRPs produced by stimulation of the cutaneous nerves with potentials resulting from stimulation of the L6 dorsal root. The ventral roots were severed from L6 to $3. Hooked platinum electrodes were used for stimulation and recording. The DRPs were recorded with bipolar electrodes on both sides of the cord. The interelectrode distance was about 15 mm. The spinal cord and peripheral nerves were prevented from drying by mineral oil in pools formed from skin and muscle flaps. The temperature of the oil was maintained between 37 and 38~C by an infrared lamp. Rectal temperature was kept at the same level by heating the ventral body surface. For producing the DRPs single pulses of a width of 0.1 ms and repetitive stimulation at a frequency of 250 c/s lasting 500 ms were used. The strength of stimuli was 4 times the threshold strength. The DRPs were evoked every 8 - 1 0 s. In experiments concerning the post-tetantic changes of the D RPs they we}'eevoked every 2 - 4 s. The recording electrodes were connected to amplifiers with time constant of 1 s. When the prolonged negatively of the dorsal root during repetitive stimulation had to be evaluated, directcoupled amplification was employed.
RESULTS
Bilateral DRPs Evoked by Single Volleys and by Repetitive Stimulation. Figure 1 A - C displays the DRPs produced in the $3 dorsal roots by single volleys in various afferent fibres. The most important observation is that in all instances the ipsilateral and contralateral depolarizations are of equal size. For comparison the DRP recorded in the L7 dorsal roots (Fig. 1 D) is much larger on the ipsi- than on the contralateral side of the cord. The sizes of the DRPs recorded on both sides of the cord in the $3 and L7 dorsal roots during stimulation of the superficial peroneal nerve were measured in 22 preparations. In the lower sacral segment the mean size of the ipsilateral DRP was 88.7 +_ 30.8 pV (this and all subsequent arithmetic means are followed by S.D, of an observation) and of the contralateral potential was 90.2 + 36.5 ~tV. The difference between means was statistically non-significant (P > 0.05). On the other hand in the L7 dorsal roots the size of the ipsilateral DRP was 461.3 + 51.3 ~tV and was significantly larger than of the contralateral potential which amounted to 82.1 + 17.5 gV (P < 0.01), It may be seen that the DRPs in the $3 dorsal roots evoked by stimulation
Pflfigers Arch. 369 (1977)
A
B
:~C
D
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100ms
Fig. 1. Dorsal root potentials evoked in the $3 dorsal roots by single volleys in the superficial peroneal nerve (A), L6 dorsal root (B), and $2 dorsal root (C). In D the DRPs produced by stimulation of the superficial peroneal nerve are recorded from the L7 dorsal roots. Upper traces of each record show ipsilateral and lower traces contralateral DRPs
A
B
~C
D
500ms Fig. 2. Dorsal root potentials produced in the $3 dorsal roots by long-lasting repelitive stimulation of the superficial peroneal nerve (A), L6 dorsal root (B), and $2 dorsal root (C). The DRPs in D are evoked in the L7 dorsal roots during repetitive stimulation of the superficial peroneal nerve. Upper traces show ipsi- and lower traces contralateral DRPs
of various afferent fibres are very similar to each other. The DRPs produced by stimulation of the superficial peroneal nerve or of the L6 dorsal root are practically identical (Fig. 1 A and B). The DRPs evoked from the posterior tibial nerve are also very similar (not illustrated). However, they are smaller and have longer time to peak than the DRPs elicited by volleys in the $2 dorsal root (Fig. 1 C). Only the latencies of the DRPs evoked in the $3 dorsal roots by stimulation of the neighbouring $2 dorsal rootlet were systematically measured. It was found that the latency of the ipsilateral DRP was 3.19 _+ 0.43 ms (n = 15) and of the contralateral DRP was 4.69 + 1.18 ms (n = 14). The comparable latencies of the DRPs in the L7 dorsal roots produced from the L6 dorsal rootlet were 3.22 + 0.59 ms (n = 14) and 4.13 +_ 0.82ms (n = 14). The latter values were similar to the results obtained in the same rootlets during stimulation of the superficial peroneal nerve [13]. These data indicate that the spread of depolarization on the contralateral side of the lower sacral cord occurs in the same way as in the L7 segment involving at least one synaptic relay. The use of repetitive stimulation for producing the DRPs does not change the relationship between the size of depolarizations recorded in both $3 dorsal roots. During stimulation of various afferent fibres the magnitude of the bilateral DRPs remains the same (Fig. 2 A - C ) . The measurements of the depolariza-
K. Lupa and A. Niecha]: Dorsal R.oot Potentials in Lower Sacral Cord
CON
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200
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80
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0
1000
~
~0~- o,4,~, 401t tt'w9 9 2O~L~ 0
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Fig. 3. Depression of ipsi- and contralateral dorsal root potentials by preceding afferent volleys. In A the first record shows the control DRPs (CON) evoked by a single volley in the superficial peroneal nerve. In the next records the testing stimulus was preceded by a single volley in the same nerve at the indicated intervals (in ms). In B the amplitudes of the testing DRPs expressed as percentages of the mean control values are plotted against the testing intervals. Open circles represent ipsilateral DRPs, filled circles the contralateral ones. Each point is arithmetic mean of eight to ten records
tions produced in 22 animals in the $3 dorsal roots by stimulation of the superficial peroneal nerve showed that the mean size of the ipsilateral potential was 258.2 __ 28.3 gV and that of the contralateral potential was 234.9 4- 30.1 gV. The difference between the means was statistically non-significant (P > 0.05). In the same preparations the mean size of the ipsilateral DRP recorded in the L7 dorsal root was 494.3 +_ 29.2 gV. On the contralateral side of the cord depolarization reached 191.7 + 26.8 ~tV. The difference between these values was statistically significant (P < 0.01). The pattern of the depolarization in the lower sacral cord depended on the place where afferent stimuli entered the spinal cord. Figure 2A and B shows that the DRPs evoked by repetitive stimulation of the cutaneous nerve and of the L6 dorsal root are very similar to one another but they profoundly differ from the DRPs produced by stimulation of the $2 dorsal root (Fig. 2C). The two former potentials rapidly rise to maximum and then fall to zero being very similar to the depolarizations produced in these fibres by single volleys. They also resemble much smaller but equally transient depolarization in the contralateral L7 dorsal root (Fig. 2D). On the other hand the DRPs produced by repetitive stimulation of the $2 dorsal root (Fig. 2C) after rapid rise to maximum decrease much slower. The contralateral depolarization drops to zero during the period of the stimulation while the ipsilateral one is maintained at some level till the end of the stimulation. The pattern
189
of the ipsilateral depolarization is very similar to the analogous potential change recorded in the L7 dorsal root (Fig. 2 D).
Conditioning of DRPs by Preceding Stimulation. Two series of conditioning were carried out. In the first single volleys preceded the testing stimulation at various intervals. In the second the repetitive stimulation lasting 500 ms was used for conditioning. In both series the testing DRPs were produced by single volleys. The conditioning and testing stimulations were applied to the same fibres. The course of conditioning of the DRPs preceded by single volleys is shown in Figure 3. At short intervals between stimulations bilateral testing DRPs were completely abolished. At the testing interval of 100 ms ipsilateral depolarization reached 20.3 % and the contralateral one attained 10.5% of the initial level. With lengthening of the testing interval depression of the bilateral DRPs rapidly decreased and when the interval was 400 ms the DRPs on both sides attained 8 0 - 9 0 % of the control. The differences between depression of the DRPs on both sides of the cord were very small but they persisted in all studied testing intervals. The same curves of conditioning were obtained in another 7 experiments and also with conditioning of the DRPs produced by stimulation of the $2 dorsal root. In order to compare directly the depression of the bilateral DRPs in the sacral and lumbar segments in the same preparations the course of conditioning in the L7 dorsal root was determined. In accordance with with previous findings [13] the depression in the L7 was more profound on the contralateral side of the cord. In the cat which was used for conditioning of the DRPs in the $3 dorsal roots (illustrated in Fig. 3) at the testing interval of 100 ms the ipsilateral D R P was depressed to 65.7~ and the contralateral D R P to 42 %. Comparable differences were maintained also at longer testing intervals. For evaluating the differences between depressions of the DRPs on both sides of the cord we have summed up differences between depressions of the ipsi- and contralateral depolarizations calculated for each testing interval. For the lower sacral cord (data from the experiment illustrated in Fig. 3) the resulting sum was 6.2 + 2.6%. In the same preparation the sum of differences between depressions of the DRPs in the L7 amounted to 17.5 4- 8.2 %. Difference between these values was statistically significant (P < 0.01). Similar results were obtained in another 7 preparations. The use of the conditioning repetitive stimulation only slightly increased depression of the testing DRPs on both sides of the sacral cord. However, in the L7
190
PflfigersArch. 369 (1977) CON
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28
48
80
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Fig. 4. Post-tetanicdepression of bilateral dorsal root potentials. A showsthe DRPs recorded from the $3 dorsal roots and evoked by single volleysin the $2 dorsal root. The strength of stimulation was adjustedto produce the DRPs attaining 60 ~ of their maximal amplitude. After the first specimen record (CON) the $2 dorsal root was stimulated for 15 s at 300/s. DRPs were then evoked at 4 s intervals and the specimenrecords shown were recorded at the intervals (in s) specifiedabove each record. The full time course of the post-tetanic depression is plotted in B. Open circles represent ipsi- and filledcircles contralateral DRPs respectively
DRP is slightly deeper than that of the ipsilateral potentials. The size of the post-tetanic depression of the DRPs depended on their initial amplitude. It was found that on both sides of the cord the decrease of depolarization was inversely proportional to their initial size. For this reason the post-tetanic changes of the DRPs evoked by stimulation of the L6 dorsal root or the cutaneous afferents could only be studied in preparations displaying very large depolarizations. These potentials showed considerable variability in the control conditions and then following prolonged tetanization. Nevertheless it was found that the initial depression of the depolarizations did not differ from that seen in the DRPs evoked from the $2 dorsal rootlet but the full recovery was shorter and occurred during 6 0 - 70 s. The post-tetanic changes of the bilateral DRPs produced in the L7 segment by stimulation of the cutaneous afferents were recently described [15]. They consisted in depression of the ipsilateral DRPs similar to that found in the $3 segment. On the contralateral side of the cord the DRPs were at first depressed but then they exhibited marked delayed potentiation. DISCUSSION
dorsal roots it much more enhanced depression of the contralateral than of the ipsilateral testing DRPs. When both conditionings were performed in the same preparation calculations of the sum of differences between depressions of the DRPs on both sides of the cord gave the following results. In the lower sacral cord this value amounted to 7.4 _+ 3.9 ~ . In the L7 segment it was significantly larger and reached 38.0 + 11.4~o (P < 0.01). The same results were obtained in another 7 preparations. These data indicate that irrespective of the kind of the conditioning stimuli the differences between depressions of the DRPs on both sides of the sacral cord are significantly smaller than these occurring in the L7 segment.
Post-Tetanic Depression of the DRPs. The DRPs recorded in the $3 dorsal roots were invariably depressed following conditioning tetanization of afferent fibres used for evoking the testing responses. Figure 4 shows the course of the post-tetanic changes of the DRPs evoked by stimulation of the $2 dorsal root. Just after the end of the tetanus both ipsi- and contralateral depolarizations are profoundly depressed attaining about 20 ~o of the initial level. A gradual increase of potentials is rather slow and during 80 s they reach 8 3 ~ of the control. The initial depression and the pattern of recovery are very similar on both sides of the cord. However, it may be seen that in 16 out of 21 successive records depression of the contralateral
The spread of the DRPs on the contralateral side of the lower sacral cord occurs via synaptic relays. Since following both single volleys and repetitive stimulation the contralateral DRP has the same amplitude as the ipsilateral DRP, it is suggested that in contrast to the L7 segment the spread of the depolarization on the contralateral side of the sacral cord occurs without appreciable depression. The above pattern of the DRPs is not affected by changing the place in which an afferent volley enters the spinal cord. This may indicate that the mechanisms deciding the size of the contralateral depolarizations are situated in or very closely to the third sacral segment. In accordance with observations of Barron and Matthews [1] it was found that the DRPs produced by volleys in the neighbouring dorsal roots are larger than these evoked by stimulation of afferent fibres entering the cord in more distant segments. The place in which an afferent volley enters the cord has profound effect on the depolarization of the ipsilateral dorsal root during long-lasting repetitive stimulation. Since this depolarization is maintained only during stimulation of the neighbouring root it is suggested that the prolonged negativity may be produced in the close proximity of a place in which an afferent volley enters the spinal cord. Experiments with preceding conditioning disclosed much smaller differences between depressions of the
K. Lupa and A. Niechaj : Dorsal Root Potentials in Lower Sacral Cord ipsi- and contralateral D R P s in the $3 than in the L7 segment. These results provide further evidence of the functional differentiation between these two segments. Following repetitive conditioning stimulation the depression of the testing D R P s in enhanced in the $3 and L7 dorsal roots. It should be r e m e m b e r e d that such a stimulation of the cutaneous afferents produces the p r o l o n g e d depolarization of the L7 dorsal root while in the $3 the potential change is short-lasting. It m a y be thus inferred that the shape of the depolarization evoked by the conditioning stimulation is not the only factor deciding the depth of depression of the testing D R P . Post-tetanic depression of the bilateral D R P s in the $3 segment results f r o m stimulation of b o t h the $2 dorsal root and the cutaneous afferents. In the l u m b a r cord stimulation o f the whole dorsal root produces the post-tetanic potentiation of the ipsilateral D R P s [18, 26]. Following tetanization of the cutaneous nerve the potentiation of the ipsilateral D R P is m u c h smaller and less frequently encountered the m o s t c o m m o n change being the depression of ipsi- and potentiation of the contralateral potential [15]. The increase of the depolarization o f the prim a r y afferent terminals is due to the after-hyperpolarization of the fibres consequent u p o n their preceding stimulation [6,9, 17]. It was postulated that the small post-tetanic potentiation of transmission accross the first cutaneous synapse is caused by a simultaneous potentiation of inhibition [24]. In the lower sacral cord inhibition would prevail over potentiation resulting in the clear-cut depression of the DRPs. The D R P s are the electrophysiological correlate of presynaptic inhibition (cf. 3, 20). H e n c e the results of our experiments show the properties of the presynaptic inhibition in the lower sacral cord and reveal that in contrast to the lower l u m b a r cord its pattern is u n i f o r m in the two halves of the $3 segment. T h e differences between both segments m o s t p r o b a b l y depend on distinct a r r a n g e m e n t o f the structures generating in t h e m the p r i m a r y afferent depolarization. In .the l u m b a r segments the depolarization of the p r i m a r y afferent fibres is at first p r o d u c e d by the substantia gelatinosa situated in the ipsilateral half of the cord. Then it spreads t h r o u g h the posterior c o m m i s s u r e to the substantia gelatinosa of the other side of the cord [22], We m a y suppose that the neurons f o r m i n g this connection are the m a i n source of depression of the contralateral D R P in the L7 segment. The u n i f o r m p a t t e r n of the bilateral presynaptic inhibition in the $3 segment is p r o b a b l y related to the fact that at this level the whole spread of the activity producing the p r i m a r y afferent depolarization occurs within one h o m o g e n e o u s structure [23, 24].
191 The lower sacral cord controls activity o f the midline structures. This function requires the c o n f o r m action o f the two Sides of the cord. Since the described pattern o f the presynaptic inhibition fulfills this requirement it m a y be considered to have functional significance.
REFERENCES 1. Barton, D. H., Matthews, B. H.: The interpretation of potential changes in the spinal cord. J. Physiol. (Lond.) 92, 276- 321 (1938) 2. Devanandan, M, S., Holmqvist, B., Yokota, T.: Presynaptic depolarization of group 1 muscle afferents by contralateral afferent volleys. Acta Physiol. Scand. 63, 46-54 (1965) 3. Eccles, J. C.: Presynaptic inhibition in the spinal cord. In: Physiology of spinal neurones, vol. 12, Progress in brain research (J. C. Eccles and J.P. Schad6, eds.), pp. 65-89. Amsterdam-London-New York: Elsevier 1964 4. Eccles, J. C., Eccles, R. M., Magni, F.: Central inhibitory action attributable to presynaptic depolarization produced by muscle afferent volleys. J. Physiol. (Lond.) 159, 147-166 (1961) 5. Eccles, J. C., Kostyuk, P. G., Schmidt, R. F.: Central pathways responsible for depolarization of primary afferent fibres. J. Physiol. (Lond.) 161, 237-257 (1962) 6. Eccles, J. C., Krnjevid, K. : Potential changes recorded inside primary afferent fibres within the spinal cord. J. Physiol. (Lond.) 149, 250-273 (1959) 7. Eccles, J. C., Magni, F., Willis, W. D. : Depolarization of central terminals of group I afferent fibres from muscle. J. Physiol. (Lond.) 160, 62-93 (1961) 8. Eccles, J. C., Schmidt, R. F., Willis, W. D. : The location and the mode of action of the presynaptic inhibitory pathways on to group I afferent fibres from muscle. J. Neurophysiol. 26, 506-522 (1963) 9. Eccles, J. C., Schmidt, R.F., Willis, W. D. : The mode of operation of the synaptic mechanism producing presynaptic inhibition. J. Neurophysiol. 26, 523- 536 (1963) 10. Eccles, R. M., Holmqvist, B., Voorhoeve, P. E.: Presynaptic inhibition from contralateral cutaneous afferent fibres. Acta Physiol. Scan& 62, 464-473 (1964) 1l. Eccles, R. M., Holmqvist, B., Voorhoeve, P. E.: Presynaptic depolarization of cutaneous afferents by volleys in contralateral muscle afferents. Acta Physiol. Scand. 62, 474-484 (1964) 12. Edisen, A. E. U. : Primary afferent fibres of contralateral origin in the lower spinal cord of cat. Exp. Neurol. 18, 38-48 (1967) 13. Hotobut, W., Niechaj, A. : The dorsal root potentials produced on both sides of the spinal cord by long-lasting stimulation of the cutaneous afferents. J. Physiol. (Lond.) 230, 1 5 - 2 7 (1973) 14. Holobut, W., Niechaj, A.: Properties of the dorsal root potentials produced on both sides of the spinal cord by stimulation of the cutaneous afferents. Acta Neurobiol. Exp. (Warsz.) 34, 629-643 (1974) 15. Hotobut, W., Niechaj, A.: Post-tetanic changes of bilateral dorsal root potentials evoked by stimulation of the cutaneous afferents. Experientia 31, 1294--1295 (1975) 16. Jfinig, W., Zimmermann, M.: Presynaptic depolarization of myelinated afferent fibres evoked by stimulation of cutaneous C fibres. J. Physiol. (Lond.) 214, 29-50 (1971) 17. Koketsu, K. : Intracetlular slow potential of dorsal root fibers. Am. J. Physiol. 184, 338-344 (1956)
192 18. Lloyd, D . P . C . : Electrotonus in dorsal nerve roots. Cold Spring Harbor. Symp. Quant. Biol. 17, 203-219 (1952) 19. Lloyd, D. P. C., Wilson, V. : Functional organization in the terminal segments of the spinal cord with a consideration of central excitatory and inhibitory latencies in monosynaptic reflex systems. J. Gen. Physiol. 42, 1219-1231 (1959) 20. Schmidt, R. F.: Presynaptic inhibition in the vertebrate central nervous system. Ergeb. Physiol. 63, 20-101 (1971) 21. Sprague, J. M. : The distribution of dorsal root fibres on motor cells in the lumbosacrat spinal cord of the cat and the site of excitatory and inhibitory terminals in monosynaptic pathways. Proc. R. Soc. (Biol. London) 149, 534-556 (1958) 22. Szent~tgothai, J. : Neuronal and synaptic arrangement in the substantia gelatinosa Rolandi. J. Comp. Neurol. 122, 219-240 (1964)
Pflttgers Arch. 369 (1977) 23. Wall, P. D. : The origin of a spinal-cord slow potential. J. Physiol. (Lond.) 164, 5 0 8 - 526 (1962) 24. Wall, P. D. : Presynaptic control of impulses at the first central synapse in the cutaneous pathway. In: Physiology of spinal neurones, vol. 12, Progress in brain research (J. C. Eccles and J. P. Schad6, eds.), pp. 98-112. Amsterdam-London-New York: Elsevier 1964 25. Wilson, V. J., Lloyd, D. P. C. : Bilateral spinal excitatory and inhibitory actions. Am. J. Physiol. 187, 641 (1956) 26. Woolsey, C. N., Larrabee, M. G. : Potential changes and prolonged reflex facilitation following stimulation of dorsal spinal roots. Am. J. Physiol. 129, 501P (1940)
Received January 27, 1977
PflfigersArch. 369, 187- 192 (1977)
EuropeanJoumal of Physiology
9 by Springer-Verlag1977
Bilateral Dorsal Root Potentials in the Lower Sacral Spinal Cord KRYSTYNA LUPA and ANDRZEJ NIECHAJ Department of Human Physiology,MedicalSchool, Dymitrowa11, PL-20-080 Lublin,Poland
Summary. Bilateral dorsal root potentials (DRPs) evoked in the $3 dorsal roots by stimulation of the $2 and L6 dorsal roots and the cutaneous afferents entering the spinal cord in the lumbar segments have been studied in spinal cats. Stimulation of all these afferents produces DRPs which have the same amplitude on both sides of the spinal cord. During longlasting repetitive stimulation the negativity of the ipsilateral dorsal root is maintained only when this stimulation is applied to the neighbouring dorsal root. Depression of the testing DRPs produced by preceding single volleys or repetitive stimulation is only slightly larger on the contralateral side of the cord. The difference between depression of the DRPs on both sides of the cord is significantly smaller in the $3 than in the L7 segment. Following conditioning tetanization both ipsi- and contralateral DRPs undergo depression. The pattern of bilateral DRPs in the $3 segment significantly differs from that observed in the L7 segment and these differences correspond to the already known distinct arrangement of the substantia gelatinosa in the two parts of the cord. Key words: Bilateral dorsal root potentials - Presynaptic inhibition - Spinal cord - Sacral cord.
INTRODUCTION The dorsal root potentials (DRPs) indicate the depolarization of primary afferent terminals produced by stimulation of the dorsal roots or peripheral nerves. In the cat they were studied mainly in the lower lumbar segments in close neighbourhood to the place where an afferent volley enters the spinal cord. These DRPs have latencies of a few milliseconds and show temporal Send offprint request to: A. Niechaj at the above address
facilitation and depression during repetitive activation which are features characterizing polysynaptic pathways [4,5,7,8]. The potential changes may also be recorded on the contralateral side of the cord where they are smaller, appear after longer delay and display more pronounced depression in interaction experiments [2,10,11,13,14,16]. The difference between the DRPs on both sides of the spinal cord has been attributed to the properties of the pathway producing contralateral depolarization of the primary afferent fibres [13]. The DRPs are generated by the activity of the substantia gelatinosa reflecting on to primary afferent fibres [23]. It was found, however, that both these structures display distinct arrangement in the lumbar and sacral segments. Following section of the lumbar dorsal roots the degeneration of afferent fibres is unilateral. After severing the $3 dorsal root this degeneration is seen on both sides of the cord and stimulation experiments demonstrated continuity of fibres from the dorsal roots to the contralateral grey matter [12,19, 21,25]. The substantia gelatinosa in the lumbar enlargement lies separately in each half of the cord. In the sacral segments it extends across the midline and forms a continuous sheet spreading from one side of the cord to the other [23, 24]. The above data suggest that the DRPs in the lower sacral cord may have properties different from these displayed by the potential changes in the lumbar segments. The present study is an investigation of the bilateral DRPs in the third sacral segment of the spinal cord.
METHODS The experimentswere performed on 32 adult cats weighing2.03.8 kg lightlyanaesthetizedwith thiamylalsodium(Surital, ParkeDavis, 30 mg/kg given i.p.) or pentobarbitone sodium(Nembutal, Abbott Lab., 40 mg/kg i.p.). Animals were immobilized with gallamine triethiodide(Flaxedil, Specia, repeated doses of 10 mg/
188 kg, given i.v.) and set on artificial respiration. The spinal cord was transected at a low thoracic level. A laminectomy was performed from the third lumbar to the middle caudal segments. The dorsal root filaments of the right and left $3 were traced to their entrance into the spinal cord. Two filaments entering the cord from both sides at the same level and being approximately the same diameter were cut distally. In the same way the L7 dorsal rootlets were prepared. These filaments were used for recording the bilateral DRPs at the two segmental levels. The dorsal rootlets of the $2 and L6 and two peripheral nerves: the superficial peroneal and posterior tibial were dissected on one side and cut peripherally for electric stimulation. The S1, $2 and the remaining part of the $3 dorsal root were cut. This procedure confined the zone of entry of impulses produced by stimulation of the peripheral nerves to the lumbar segments of the cord. It thus made it possible to compare the DRPs produced by stimulation of the cutaneous nerves with potentials resulting from stimulation of the L6 dorsal root. The ventral roots were severed from L6 to $3. Hooked platinum electrodes were used for stimulation and recording. The DRPs were recorded with bipolar electrodes on both sides of the cord. The interelectrode distance was about 15 mm. The spinal cord and peripheral nerves were prevented from drying by mineral oil in pools formed from skin and muscle flaps. The temperature of the oil was maintained between 37 and 38~C by an infrared lamp. Rectal temperature was kept at the same level by heating the ventral body surface. For producing the DRPs single pulses of a width of 0.1 ms and repetitive stimulation at a frequency of 250 c/s lasting 500 ms were used. The strength of stimuli was 4 times the threshold strength. The DRPs were evoked every 8 - 1 0 s. In experiments concerning the post-tetantic changes of the D RPs they we}'eevoked every 2 - 4 s. The recording electrodes were connected to amplifiers with time constant of 1 s. When the prolonged negatively of the dorsal root during repetitive stimulation had to be evaluated, directcoupled amplification was employed.
RESULTS
Bilateral DRPs Evoked by Single Volleys and by Repetitive Stimulation. Figure 1 A - C displays the DRPs produced in the $3 dorsal roots by single volleys in various afferent fibres. The most important observation is that in all instances the ipsilateral and contralateral depolarizations are of equal size. For comparison the DRP recorded in the L7 dorsal roots (Fig. 1 D) is much larger on the ipsi- than on the contralateral side of the cord. The sizes of the DRPs recorded on both sides of the cord in the $3 and L7 dorsal roots during stimulation of the superficial peroneal nerve were measured in 22 preparations. In the lower sacral segment the mean size of the ipsilateral DRP was 88.7 +_ 30.8 pV (this and all subsequent arithmetic means are followed by S.D, of an observation) and of the contralateral potential was 90.2 + 36.5 ~tV. The difference between means was statistically non-significant (P > 0.05). On the other hand in the L7 dorsal roots the size of the ipsilateral DRP was 461.3 + 51.3 ~tV and was significantly larger than of the contralateral potential which amounted to 82.1 + 17.5 gV (P < 0.01), It may be seen that the DRPs in the $3 dorsal roots evoked by stimulation
Pflfigers Arch. 369 (1977)
A
B
:~C
D
I
I
100ms
Fig. 1. Dorsal root potentials evoked in the $3 dorsal roots by single volleys in the superficial peroneal nerve (A), L6 dorsal root (B), and $2 dorsal root (C). In D the DRPs produced by stimulation of the superficial peroneal nerve are recorded from the L7 dorsal roots. Upper traces of each record show ipsilateral and lower traces contralateral DRPs
A
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~C
D
500ms Fig. 2. Dorsal root potentials produced in the $3 dorsal roots by long-lasting repelitive stimulation of the superficial peroneal nerve (A), L6 dorsal root (B), and $2 dorsal root (C). The DRPs in D are evoked in the L7 dorsal roots during repetitive stimulation of the superficial peroneal nerve. Upper traces show ipsi- and lower traces contralateral DRPs
of various afferent fibres are very similar to each other. The DRPs produced by stimulation of the superficial peroneal nerve or of the L6 dorsal root are practically identical (Fig. 1 A and B). The DRPs evoked from the posterior tibial nerve are also very similar (not illustrated). However, they are smaller and have longer time to peak than the DRPs elicited by volleys in the $2 dorsal root (Fig. 1 C). Only the latencies of the DRPs evoked in the $3 dorsal roots by stimulation of the neighbouring $2 dorsal rootlet were systematically measured. It was found that the latency of the ipsilateral DRP was 3.19 _+ 0.43 ms (n = 15) and of the contralateral DRP was 4.69 + 1.18 ms (n = 14). The comparable latencies of the DRPs in the L7 dorsal roots produced from the L6 dorsal rootlet were 3.22 + 0.59 ms (n = 14) and 4.13 +_ 0.82ms (n = 14). The latter values were similar to the results obtained in the same rootlets during stimulation of the superficial peroneal nerve [13]. These data indicate that the spread of depolarization on the contralateral side of the lower sacral cord occurs in the same way as in the L7 segment involving at least one synaptic relay. The use of repetitive stimulation for producing the DRPs does not change the relationship between the size of depolarizations recorded in both $3 dorsal roots. During stimulation of various afferent fibres the magnitude of the bilateral DRPs remains the same (Fig. 2 A - C ) . The measurements of the depolariza-
K. Lupa and A. Niecha]: Dorsal R.oot Potentials in Lower Sacral Cord
CON
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200
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80
700
0
1000
~
~0~- o,4,~, 401t tt'w9 9 2O~L~ 0
I 200
I &O0
L 600
I 800
• 1000
J 120~"ms
Fig. 3. Depression of ipsi- and contralateral dorsal root potentials by preceding afferent volleys. In A the first record shows the control DRPs (CON) evoked by a single volley in the superficial peroneal nerve. In the next records the testing stimulus was preceded by a single volley in the same nerve at the indicated intervals (in ms). In B the amplitudes of the testing DRPs expressed as percentages of the mean control values are plotted against the testing intervals. Open circles represent ipsilateral DRPs, filled circles the contralateral ones. Each point is arithmetic mean of eight to ten records
tions produced in 22 animals in the $3 dorsal roots by stimulation of the superficial peroneal nerve showed that the mean size of the ipsilateral potential was 258.2 __ 28.3 gV and that of the contralateral potential was 234.9 4- 30.1 gV. The difference between the means was statistically non-significant (P > 0.05). In the same preparations the mean size of the ipsilateral DRP recorded in the L7 dorsal root was 494.3 +_ 29.2 gV. On the contralateral side of the cord depolarization reached 191.7 + 26.8 ~tV. The difference between these values was statistically significant (P < 0.01). The pattern of the depolarization in the lower sacral cord depended on the place where afferent stimuli entered the spinal cord. Figure 2A and B shows that the DRPs evoked by repetitive stimulation of the cutaneous nerve and of the L6 dorsal root are very similar to one another but they profoundly differ from the DRPs produced by stimulation of the $2 dorsal root (Fig. 2C). The two former potentials rapidly rise to maximum and then fall to zero being very similar to the depolarizations produced in these fibres by single volleys. They also resemble much smaller but equally transient depolarization in the contralateral L7 dorsal root (Fig. 2D). On the other hand the DRPs produced by repetitive stimulation of the $2 dorsal root (Fig. 2C) after rapid rise to maximum decrease much slower. The contralateral depolarization drops to zero during the period of the stimulation while the ipsilateral one is maintained at some level till the end of the stimulation. The pattern
189
of the ipsilateral depolarization is very similar to the analogous potential change recorded in the L7 dorsal root (Fig. 2 D).
Conditioning of DRPs by Preceding Stimulation. Two series of conditioning were carried out. In the first single volleys preceded the testing stimulation at various intervals. In the second the repetitive stimulation lasting 500 ms was used for conditioning. In both series the testing DRPs were produced by single volleys. The conditioning and testing stimulations were applied to the same fibres. The course of conditioning of the DRPs preceded by single volleys is shown in Figure 3. At short intervals between stimulations bilateral testing DRPs were completely abolished. At the testing interval of 100 ms ipsilateral depolarization reached 20.3 % and the contralateral one attained 10.5% of the initial level. With lengthening of the testing interval depression of the bilateral DRPs rapidly decreased and when the interval was 400 ms the DRPs on both sides attained 8 0 - 9 0 % of the control. The differences between depression of the DRPs on both sides of the cord were very small but they persisted in all studied testing intervals. The same curves of conditioning were obtained in another 7 experiments and also with conditioning of the DRPs produced by stimulation of the $2 dorsal root. In order to compare directly the depression of the bilateral DRPs in the sacral and lumbar segments in the same preparations the course of conditioning in the L7 dorsal root was determined. In accordance with with previous findings [13] the depression in the L7 was more profound on the contralateral side of the cord. In the cat which was used for conditioning of the DRPs in the $3 dorsal roots (illustrated in Fig. 3) at the testing interval of 100 ms the ipsilateral D R P was depressed to 65.7~ and the contralateral D R P to 42 %. Comparable differences were maintained also at longer testing intervals. For evaluating the differences between depressions of the DRPs on both sides of the cord we have summed up differences between depressions of the ipsi- and contralateral depolarizations calculated for each testing interval. For the lower sacral cord (data from the experiment illustrated in Fig. 3) the resulting sum was 6.2 + 2.6%. In the same preparation the sum of differences between depressions of the DRPs in the L7 amounted to 17.5 4- 8.2 %. Difference between these values was statistically significant (P < 0.01). Similar results were obtained in another 7 preparations. The use of the conditioning repetitive stimulation only slightly increased depression of the testing DRPs on both sides of the sacral cord. However, in the L7
190
PflfigersArch. 369 (1977) CON
4
28
48
80
;00ms' % Ioo
B
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_a.~ ~8~0
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9
80
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60
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0
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9
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I 20
I 40
I 60
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Fig. 4. Post-tetanicdepression of bilateral dorsal root potentials. A showsthe DRPs recorded from the $3 dorsal roots and evoked by single volleysin the $2 dorsal root. The strength of stimulation was adjustedto produce the DRPs attaining 60 ~ of their maximal amplitude. After the first specimen record (CON) the $2 dorsal root was stimulated for 15 s at 300/s. DRPs were then evoked at 4 s intervals and the specimenrecords shown were recorded at the intervals (in s) specifiedabove each record. The full time course of the post-tetanic depression is plotted in B. Open circles represent ipsi- and filledcircles contralateral DRPs respectively
DRP is slightly deeper than that of the ipsilateral potentials. The size of the post-tetanic depression of the DRPs depended on their initial amplitude. It was found that on both sides of the cord the decrease of depolarization was inversely proportional to their initial size. For this reason the post-tetanic changes of the DRPs evoked by stimulation of the L6 dorsal root or the cutaneous afferents could only be studied in preparations displaying very large depolarizations. These potentials showed considerable variability in the control conditions and then following prolonged tetanization. Nevertheless it was found that the initial depression of the depolarizations did not differ from that seen in the DRPs evoked from the $2 dorsal rootlet but the full recovery was shorter and occurred during 6 0 - 70 s. The post-tetanic changes of the bilateral DRPs produced in the L7 segment by stimulation of the cutaneous afferents were recently described [15]. They consisted in depression of the ipsilateral DRPs similar to that found in the $3 segment. On the contralateral side of the cord the DRPs were at first depressed but then they exhibited marked delayed potentiation. DISCUSSION
dorsal roots it much more enhanced depression of the contralateral than of the ipsilateral testing DRPs. When both conditionings were performed in the same preparation calculations of the sum of differences between depressions of the DRPs on both sides of the cord gave the following results. In the lower sacral cord this value amounted to 7.4 _+ 3.9 ~ . In the L7 segment it was significantly larger and reached 38.0 + 11.4~o (P < 0.01). The same results were obtained in another 7 preparations. These data indicate that irrespective of the kind of the conditioning stimuli the differences between depressions of the DRPs on both sides of the sacral cord are significantly smaller than these occurring in the L7 segment.
Post-Tetanic Depression of the DRPs. The DRPs recorded in the $3 dorsal roots were invariably depressed following conditioning tetanization of afferent fibres used for evoking the testing responses. Figure 4 shows the course of the post-tetanic changes of the DRPs evoked by stimulation of the $2 dorsal root. Just after the end of the tetanus both ipsi- and contralateral depolarizations are profoundly depressed attaining about 20 ~o of the initial level. A gradual increase of potentials is rather slow and during 80 s they reach 8 3 ~ of the control. The initial depression and the pattern of recovery are very similar on both sides of the cord. However, it may be seen that in 16 out of 21 successive records depression of the contralateral
The spread of the DRPs on the contralateral side of the lower sacral cord occurs via synaptic relays. Since following both single volleys and repetitive stimulation the contralateral DRP has the same amplitude as the ipsilateral DRP, it is suggested that in contrast to the L7 segment the spread of the depolarization on the contralateral side of the sacral cord occurs without appreciable depression. The above pattern of the DRPs is not affected by changing the place in which an afferent volley enters the spinal cord. This may indicate that the mechanisms deciding the size of the contralateral depolarizations are situated in or very closely to the third sacral segment. In accordance with observations of Barron and Matthews [1] it was found that the DRPs produced by volleys in the neighbouring dorsal roots are larger than these evoked by stimulation of afferent fibres entering the cord in more distant segments. The place in which an afferent volley enters the cord has profound effect on the depolarization of the ipsilateral dorsal root during long-lasting repetitive stimulation. Since this depolarization is maintained only during stimulation of the neighbouring root it is suggested that the prolonged negativity may be produced in the close proximity of a place in which an afferent volley enters the spinal cord. Experiments with preceding conditioning disclosed much smaller differences between depressions of the
K. Lupa and A. Niechaj : Dorsal Root Potentials in Lower Sacral Cord ipsi- and contralateral D R P s in the $3 than in the L7 segment. These results provide further evidence of the functional differentiation between these two segments. Following repetitive conditioning stimulation the depression of the testing D R P s in enhanced in the $3 and L7 dorsal roots. It should be r e m e m b e r e d that such a stimulation of the cutaneous afferents produces the p r o l o n g e d depolarization of the L7 dorsal root while in the $3 the potential change is short-lasting. It m a y be thus inferred that the shape of the depolarization evoked by the conditioning stimulation is not the only factor deciding the depth of depression of the testing D R P . Post-tetanic depression of the bilateral D R P s in the $3 segment results f r o m stimulation of b o t h the $2 dorsal root and the cutaneous afferents. In the l u m b a r cord stimulation o f the whole dorsal root produces the post-tetanic potentiation of the ipsilateral D R P s [18, 26]. Following tetanization of the cutaneous nerve the potentiation of the ipsilateral D R P is m u c h smaller and less frequently encountered the m o s t c o m m o n change being the depression of ipsi- and potentiation of the contralateral potential [15]. The increase of the depolarization o f the prim a r y afferent terminals is due to the after-hyperpolarization of the fibres consequent u p o n their preceding stimulation [6,9, 17]. It was postulated that the small post-tetanic potentiation of transmission accross the first cutaneous synapse is caused by a simultaneous potentiation of inhibition [24]. In the lower sacral cord inhibition would prevail over potentiation resulting in the clear-cut depression of the DRPs. The D R P s are the electrophysiological correlate of presynaptic inhibition (cf. 3, 20). H e n c e the results of our experiments show the properties of the presynaptic inhibition in the lower sacral cord and reveal that in contrast to the lower l u m b a r cord its pattern is u n i f o r m in the two halves of the $3 segment. T h e differences between both segments m o s t p r o b a b l y depend on distinct a r r a n g e m e n t o f the structures generating in t h e m the p r i m a r y afferent depolarization. In .the l u m b a r segments the depolarization of the p r i m a r y afferent fibres is at first p r o d u c e d by the substantia gelatinosa situated in the ipsilateral half of the cord. Then it spreads t h r o u g h the posterior c o m m i s s u r e to the substantia gelatinosa of the other side of the cord [22], We m a y suppose that the neurons f o r m i n g this connection are the m a i n source of depression of the contralateral D R P in the L7 segment. The u n i f o r m p a t t e r n of the bilateral presynaptic inhibition in the $3 segment is p r o b a b l y related to the fact that at this level the whole spread of the activity producing the p r i m a r y afferent depolarization occurs within one h o m o g e n e o u s structure [23, 24].
191 The lower sacral cord controls activity o f the midline structures. This function requires the c o n f o r m action o f the two Sides of the cord. Since the described pattern o f the presynaptic inhibition fulfills this requirement it m a y be considered to have functional significance.
REFERENCES 1. Barton, D. H., Matthews, B. H.: The interpretation of potential changes in the spinal cord. J. Physiol. (Lond.) 92, 276- 321 (1938) 2. Devanandan, M, S., Holmqvist, B., Yokota, T.: Presynaptic depolarization of group 1 muscle afferents by contralateral afferent volleys. Acta Physiol. Scand. 63, 46-54 (1965) 3. Eccles, J. C.: Presynaptic inhibition in the spinal cord. In: Physiology of spinal neurones, vol. 12, Progress in brain research (J. C. Eccles and J.P. Schad6, eds.), pp. 65-89. Amsterdam-London-New York: Elsevier 1964 4. Eccles, J. C., Eccles, R. M., Magni, F.: Central inhibitory action attributable to presynaptic depolarization produced by muscle afferent volleys. J. Physiol. (Lond.) 159, 147-166 (1961) 5. Eccles, J. C., Kostyuk, P. G., Schmidt, R. F.: Central pathways responsible for depolarization of primary afferent fibres. J. Physiol. (Lond.) 161, 237-257 (1962) 6. Eccles, J. C., Krnjevid, K. : Potential changes recorded inside primary afferent fibres within the spinal cord. J. Physiol. (Lond.) 149, 250-273 (1959) 7. Eccles, J. C., Magni, F., Willis, W. D. : Depolarization of central terminals of group I afferent fibres from muscle. J. Physiol. (Lond.) 160, 62-93 (1961) 8. Eccles, J. C., Schmidt, R. F., Willis, W. D. : The location and the mode of action of the presynaptic inhibitory pathways on to group I afferent fibres from muscle. J. Neurophysiol. 26, 506-522 (1963) 9. Eccles, J. C., Schmidt, R.F., Willis, W. D. : The mode of operation of the synaptic mechanism producing presynaptic inhibition. J. Neurophysiol. 26, 523- 536 (1963) 10. Eccles, R. M., Holmqvist, B., Voorhoeve, P. E.: Presynaptic inhibition from contralateral cutaneous afferent fibres. Acta Physiol. Scan& 62, 464-473 (1964) 1l. Eccles, R. M., Holmqvist, B., Voorhoeve, P. E.: Presynaptic depolarization of cutaneous afferents by volleys in contralateral muscle afferents. Acta Physiol. Scand. 62, 474-484 (1964) 12. Edisen, A. E. U. : Primary afferent fibres of contralateral origin in the lower spinal cord of cat. Exp. Neurol. 18, 38-48 (1967) 13. Hotobut, W., Niechaj, A. : The dorsal root potentials produced on both sides of the spinal cord by long-lasting stimulation of the cutaneous afferents. J. Physiol. (Lond.) 230, 1 5 - 2 7 (1973) 14. Holobut, W., Niechaj, A.: Properties of the dorsal root potentials produced on both sides of the spinal cord by stimulation of the cutaneous afferents. Acta Neurobiol. Exp. (Warsz.) 34, 629-643 (1974) 15. Hotobut, W., Niechaj, A.: Post-tetanic changes of bilateral dorsal root potentials evoked by stimulation of the cutaneous afferents. Experientia 31, 1294--1295 (1975) 16. Jfinig, W., Zimmermann, M.: Presynaptic depolarization of myelinated afferent fibres evoked by stimulation of cutaneous C fibres. J. Physiol. (Lond.) 214, 29-50 (1971) 17. Koketsu, K. : Intracetlular slow potential of dorsal root fibers. Am. J. Physiol. 184, 338-344 (1956)
192 18. Lloyd, D . P . C . : Electrotonus in dorsal nerve roots. Cold Spring Harbor. Symp. Quant. Biol. 17, 203-219 (1952) 19. Lloyd, D. P. C., Wilson, V. : Functional organization in the terminal segments of the spinal cord with a consideration of central excitatory and inhibitory latencies in monosynaptic reflex systems. J. Gen. Physiol. 42, 1219-1231 (1959) 20. Schmidt, R. F.: Presynaptic inhibition in the vertebrate central nervous system. Ergeb. Physiol. 63, 20-101 (1971) 21. Sprague, J. M. : The distribution of dorsal root fibres on motor cells in the lumbosacrat spinal cord of the cat and the site of excitatory and inhibitory terminals in monosynaptic pathways. Proc. R. Soc. (Biol. London) 149, 534-556 (1958) 22. Szent~tgothai, J. : Neuronal and synaptic arrangement in the substantia gelatinosa Rolandi. J. Comp. Neurol. 122, 219-240 (1964)
Pflttgers Arch. 369 (1977) 23. Wall, P. D. : The origin of a spinal-cord slow potential. J. Physiol. (Lond.) 164, 5 0 8 - 526 (1962) 24. Wall, P. D. : Presynaptic control of impulses at the first central synapse in the cutaneous pathway. In: Physiology of spinal neurones, vol. 12, Progress in brain research (J. C. Eccles and J. P. Schad6, eds.), pp. 98-112. Amsterdam-London-New York: Elsevier 1964 25. Wilson, V. J., Lloyd, D. P. C. : Bilateral spinal excitatory and inhibitory actions. Am. J. Physiol. 187, 641 (1956) 26. Woolsey, C. N., Larrabee, M. G. : Potential changes and prolonged reflex facilitation following stimulation of dorsal spinal roots. Am. J. Physiol. 129, 501P (1940)
Received January 27, 1977
