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Direct contralateral inhibition in the lower sacral spinal cord
EXPERIMENTAL
NEUROLOGY
Direct
1,
28-43
Contralateral Sacral KARL
FRANK
(1959)
Inhibition in the Lower Spinal Cord AND
JAMES
M.
SPRAGUEI
Laboratory of Neurophysiology, National Institute of Neurological Blindness, National Institutes of Health, Bethesda, and the Department and Institute of Neurological Sciences, University of Pennsylvania School, Philadelphia, Pennsylvania Received
November
Diseases and of Anatomy Medical
10, 1958
The pcssibility of an interneuron in the so-called “direct” inhibitory pathway has been examined by studying postsynaptic potentials of motor horn cells in the SZ-S3 segments of cat’s spinal ccrd following excitatory and inhibitory volleys in ipsilateral and contralateral dorsal roots. Monosynaptic connections were demonstrated histologically from ipsilateral dorsal roots to motor horn cell somata and dendrites and from contralateral dorsal roots to motor horn cell dendrites alone. However, direct comparison of ipsilateral EPSP and contralateral IPSP latencies in the same unit showed an IPSP latency longer than the EPSP latency by 0.3 to 0.7 msec. Since no adequate explanation could be found to account for this extra latency on the basis of monosynaptic connections, the presence of an interneuron in the direct inhibitory pathway remains a definite possibility. Introduction
The first definition and measurement of the shortest latency reflex discharge and its “direct” inhibition in the spinal cord were obtained from recordings on ventral root and peripheral nerve (14, 19). The minimal latency of this excitation was such that it was thought to be monosynaptic and to be mediated by the reflex fibers shown by Cajal (5) to extend directly from dorsal funiculus to motoneurons. The striking similarities between short latency excitation and inhibition suggested rather strongly that “direct” inhibition was also monosynaptic. These similarities are seen in (a) the exponential decay of the two processes acting on a test reflex (16) ;” (b) the almost linear relationship between the 1 Supported in part by a research grant (B-461) from the National Institute Neurological Diseases and Blindness of the National Institutes of Health, U. Public Health Service. 2 However, see the conclusions of Bradley, Easton, and Ecdes (2). 28
of S.
DIRECT
INHIBITION
29
degree of inhibition or facilitation and the amplitude of the responsible dorsal root spike, beginning with the smallest measurable input (13, 1.5) ; and (c) the demonstration that the fibers in the dorsal root responsible for both actions have the same threshold (1.5). Precise measurement of the latency of “direct” inhibition of a test monosynaptic ventral root discharge is difficult because even the most synchronous discharge has a duration of about 1 msec. Microelectrode studies (10) show that the individual action potentials of the single ventral root fibers making up this discharge have a duration of 0.3 to 0.5 msec. Thus, the ventral root discharge reflects the time of initiation of only its earliest components, while the late units alone are inhibited when excitatory and inhibitory volleys approach synchrony (Fig. 1). Brock, Coombs, and Eccles (3) found by use of intracellular microelectrodes that the firing of a motoneuron is preceded by a depolarization of the cell membrane, the excitatory postsynaptic potential or EPSP, and is prevented by hyperpolarization, the inhibitory postsynaptic potential or IPSP. This technique permits a comparison of inhibitory and excitatory latencies, each measured between the time of the initiation of the afferent volley and the first evidence of its postsynaptic effect in the motoneuron. This comparison deals only with “direct” inhibition and does not apply to “remote” inhibition (12)) which acts without producing postsynaptic hyperpolarization. Eccles, Fatt, and Landgren (8) have challenged the conclusion that “direct” inhibition is monosynaptic. They found, using nerves innervating a variety of synergistic and antagonistic muscles of the hind leg, that IPSP trailed EPSP by minimal intervals of 0.5 to 1.0 msec. This difference in latency was believed by them to implicate an interneuron in the inhibitory pathway, an interpretation expanded into a general hypothesis that all central inhibitory pathways act through small interneurons which produce a chemical inhibitory transmitter. Following Cajal’s (5, Fig. 113) demonstration that monosynaptic fibers passing from the dorsal funiculus to the motoneurons give off collaterals to a nearby interneuron nucleus, Eccles, Fatt, and Landgren (8) studied the activity of these or adjacent interneurons to stimulation of muscle nerves. They reported that many of these cells are activated specifically by group IA fibers from muscle, and in their opinion, their firing to this activation is such that most of the previously described characteristics of direct inhibition can be accounted for by such a disynaptic mechanism. One of the difficulties in interpreting the differences in latency between
30
FRANK
AND
SPRAGUE
excitatory (EPSP) and inhibitory postsynaptic potentials (IPSP) in the lumbosacral segments, is the fact that pathways of different lengths are usually involved. More satisfactory, in this respect, are the lower sacral segments (SZ-S3) in which ipsilateral monosynaptic reflexes (serving the
FIG. 1. Direct inhibition of S3 ventral root spike evoked by ipsilateral dorsal root 53, E, and inhibited by contralateral dorsal root S3, I. A: Mean of 10 traces stimulating ipsilateral dorsal root S3 twice at F and E. B: Mean of 10 traces stimulating ipsilateral dorsal root S3 at F and E and contralateral dorsal roct S3 at I. C: Difference between A and B. A large stable monosynaptic test reflex frequently requires two shccks to dorsal root S3. Note that even for this stimulus interval (0.4 msec) the late components of the spike are those which are selectively inhibited.
musclesof the tail) are inhibited “directly” by volleys in the contralateral dorsal roots ( 17, 22, and unpublished results of Lloyd, Sprague, and Edisen, 1955). After unilateral section of these dorsal roots, terminal degeneration was found on cell bodies and dendrites of ipsilateral motoneurons and on dendrites only of contralateral motoneurons. Thus, mono-
DIRECT
31
INHIBITION
synaptic connections exist between each S2-S3 dorsal root and the motoneurons of both sides of the cord, pathways of approximately equal length (Fig. 10). Wilson and Lloyd (22) recorded monosynaptic reflex discharges concurrently in the two S3 ventral roots, while changing the timing of one S3 dorsal root volley with respect to the other. With this arrangement, each dorsal root volley sets up a monosynaptic reflex on the ipsilateral side, and inhibits the reflex on the opposite side. They found that both reflexes may be inhibited for as much as 0.1 msec on either side of synchrony of dorsal root shocks, and concluded that no time is present for an interneuron in the inhibitory pathway. Because of the reservations about ventral root recording discussed earlier in this paper, and because of the above anatomical evidence for contralateral monosynaptic connections, it was felt that “direct” inhibition in the S2-S3 spinal segments should be investigated using intracellular microelectrodes. Methods
Ten cats were prepared by high spinal section, intercollicular decerebration, or by use of pentobarbital sodium (Nembutal), d-tubocurarine being used for immobilization in all animals. Dorsal and ventral roots of the second and third sacral segments, were exposed and cut bilaterally at equal distances from the cord for stimulation and recording. Ventral root reflexes were recorded following dorsal root shocks (up to 10X threshold) throughout the experiment to ascertain the presence of ipsilateral, monosynaptic responses, and their “direct” inhibition by near simultaneous stimulation of the contralateral dorsal roots. The crossed inhibition was not always found, and when present, it was usually in S3 segment. In those cases, where the lumbosacral plexus was subsequently dissected, the absence of crossed inhibition was correlated with a postfixed plexus (unpublished results of Lloyd, Sprague, and Edisen, 1955) in which these segments contributed to the innervation of the hind leg, and thus were not associated solely with crossed innervation of the tail muscles. Search was made in the S2-S3 area of the cord with a microelectrode for motoneurons which showed responses to ipsilateral and/or contralateral dorsal root stimulation. Excitatory and inhibitory postsynaptic potentials of the lower sacral 3 During the course of this investigation it was learned being conducted by Curtis, Kmjevic, and Miledi (7).
that
a similar
study
was
32
FRANK
AND
SPRAGUE
motoneurons were recorded with 3 M KCl-filled micropipettes according to the technique of Brock, Coombs, and Eccles (3) and Frank and Fuortes (10). It was commonly found that hyperpolarizing synaptic potentials reversed their sign soon after penetration of the motoneuron, presumably due to leakage of Cl- from the micropipette (6)) which shifts the ionic balance and thus raises the IPSP equilibrium potential of the membrane. Identification as IPSPs of such reversed potentials was accomplishedby passing depolarizing current through the electrode using the bridge technique of Araki and Otani (1) and Frank and Fuortes (10). Application of the depolarizing current changes the resting membrane potential, so it is again above the shifted IPSP equilibrium potential, and the IPSP reappears as a membrane hyperpolarization. The fact that the EPSP depolarization is not so reversed by either ion leakage or moderate depolarization makes this a convenient method of identification. Figure 2 shows the difference in effect of applied currents on EPSP (+) and IPSP (0) in sacral motoneurons. Note that the IPSP is depolarizing at zero current, but is correctly identified by the effect of the applied current.
+ + +
+ FIRING
“0: z i;j E I: Q: p:: t! %
0 ,-2
?
0 0
HYPERPOLARIZING
CURRENT
0 0
-4
0
CEPOLARIZING
CURRENT
FIG. 2. Effects of polarizing currents on postsynaptic potentials (PSP) of two S3 motoneurons. EPSP’s (+) are unaffected by positive and negative currents which alter and reverse IPSP’s (0). Ordinate: amplitude of PSP; reversal occurs at 0. Abscissa: amplitude of applied current; firing for EPSP occurs at arrow.
DIRECT
33
INHIBITION
Latencies for PSP’s were measured from the shock artifact rather than from the arrival of the volley at the cord surface. This is justified by measurements showing that within the range of stimuli used in these experiments (up to 10X threshold), the arrival time of the volley at the cord varied by not more than 0.06 msec. The relatively small number of cells recorded is explained by the diffrculty in penetrating motoneurons in the small part of the spinal cord. Results
Ipsilaterd PSP’s. Many motor horn cells showed EPSP’s to ipsilateral S2-S3 dorsal root stimulation which were typically monosynaptic in time course (11). The minimum latencies of 11 such units ranged from 0.65
FIG. 3. Variation of latency of EPSP with subthreshold single S3 motoneuron. A-C: increasing stimulus strength S3; D shows beginning of antidromic firing of same unit.
stimulus strength to ipsilateral dorsal Time: msec.
in a root
to 1.2 msec. For any one of these units, EPSP latency was distinctly a function of stimulus strength. Figure 3 shows that in a single unit the EPSP latency can be varied by as much as 0.6 msec. The maximum latency is difficult to determine because with weak shocks the PSP is lost in the noise level, but 3 cells had maximum latencies which were at least 1.0, 1.4, and 1.6 msec. The latter is illustrated in Fig. 3A. This EPSP has a time course which appears monosynaptic but could have a plurisynaptic contribution. The unit of Fig. 3 could not be fired within the range of stimuli used. On other units suprathreshold stimuli further reduced the EPSP latency as seen in Fig. 4. Threshold monosynaptic EPSP’s in lower sacral motoneurons usually rise to maximum in about 0.9 to 1.0 msec. Firing in
34
FRANK
AND
SPRAGUE
these units for threshold excitation shows a total latency equal to the sum of this EPSP rise time plus the time required for the presynaptic volley to initiate the EPSP. As illustrated in Fig. 4, a strong afferent volley can produce maximal reduction of both of these times: the EPSP rise time from 1 msec down to 0.45 msec, and EPSP latency from 0.95 down to 0.65 msec, so that total firing latency is reduced from 2.0 to 1.1 msec. Thus, in the spectrum of action potentials contributing to ventral root discharge, there will be a range of latencies equal to the sum of at least these two effects.
ABc-
II FIG. 4. Variation of latency and rise time of EPSP ipsilateral dorsal root S3 in a single S3 motoneuron. strength. Trace discontinued after beginning of spike in cate range of latency of IPSP in same cell (see Fig. 5).
with stimulus strength to A-D: decreasing stimulus A-C. Vertical lines indiTime: msec.
In one cat two cells showed hyperpolarizing PSP following ipsilateral stimulation, but their latencies were 3 and 4 msec respectively-clearly plurisynaptic. Contralateral PSP’s. The PSP’s which were recorded from motor horn cells following contralateral dorsal root volleys were usually initially hyperpolarizing potentials which were often followed by depolarization. Minimum latencies of these IPSP’s ranged from 1.1 to 1.6 msec in 6 units. Due to the difficulty of finding and holding these units (see Methods) the effect of stimulus strength on the latency was not carefully studied, but data on hand suggest that the variation of latency with stimulus strength was much less (ca. 0.2 msec) than that of the ipsilateral EPSP (ca. 0.6 msec). Figure 5 shows an example. The depolarization which often followed hyperpolarization in response to contralateral stimulation appeared to rise out of the IPSP more or less abruptly as seen in Fig. 6.
DIRECT
INHIBITION
35
The range of latencies for this contralateral EPSP was 1.8 to 2.3 msec in 7 cells (range 0.3 msec for 6 of the 7 cells). Polarizing currents applied through the microelectrode, as described in the section on Methods, served to distinguish between EPSP’s and IPSP’s which had been reversed pre-
FIG. 5. Variation of latency of IPSP with decreasing stimulus strength, A-E, to contralateral dorsal root S3 in the same S3 motoneuron as in Fig. 4. Note that stronger stimulus strengths in A-B bring in late depolarization, curtailing full development of early IPSP. Time: msec.
A-A FIG. 6. IPSP interrupted by EPSP in an S3 motoneuron following stimulation of contralateral dorsal root S3 (A). EPSP for the same motoneuron following stimulation of ipsilateral dorsal root S3 (B). Time: msec.
sumably due to leak of Cl- ions. Figure 7 shows an example. It may be seen that such applied currents do not affect the latencies of the PSP’s. Three cells in this study showed only brief latency EPSP’s to contralateral stimulation. Their latencies were 0.9, 1.0, and 1.4 msec-comparable to ipsilateral EPSP latencies. Comparison of EPSP and IPSP L&en&es. While the ranges of minimal latencies for ipsilateral EPSP’s and contralateral IPSP’s in different cells are broad enough to actually overlap, the more significant comparisons are obtained when both types of PSP’s are seen in the same cell. Five cells satisfied this condition and showed a longer IPSP than EPSP
36
FRANK
AND
SPRAGUE
latency. Differences in the two latencies were: 0.3, 0.5, 0.6, 0.65, 0.7 msec, mean 0.55 msec. Figure 8 shows tracings of the extremes of this range.
FIG. 7. Left. Reversal of IPSP with applied currents in a single S3 motoneuron stimulated by contralateral volley in dorsal root S3 (A-B) ; hyperpolarizing current EPSP in same cell following ipsilateral volley in (A), depolarizing current (B). dorsal root S3 (C-E) ; depolarizing current (C), hyperpolarizing current (D), no current (E). Cell apparently failing between C and D. Latencies of PSP’s unaffected by applied currents or cell deterioration. Time: msec. FIG. 8. Right. Latency differences for EPSP and IPSP in S3 motoneurons illustrating minimum (A, B) and maximum (C, D) differences. Stimulus to ipsilateral (A, C) and contralateral (B, D) dorsal root S3. Time: msec.
Discussion Intracellular potentials from motoneurons show that excitation is preceded by a depolarizing synaptic potential (EPSP) and the beginning of the potential is the first indication of the arrival of the afferent volley at the postsynaptic cell. Similarly, direct inhibition in this cell is indicated by the generation of a hyperpolarizing synaptic potential. There seems to be no reasonable doubt that these changes in membrane polarization are causally related to the reflex excitation and inhibition of the cell (see, however, 22). The measurements made in this study clearly demonstrate that the time required for a contralateral dorsal root volley
DIRECT
37
INHIBITION
to produce an IPSP in a motor horn cell in the lower sacral cord of the cat is 0.3 to 0.7 msec longer than the time for an ipsilateral dorsal root volley to produce an EPSP in the same cell. RANGE
OF EFFECTIVE
EXCITATORY
AND
INHIBITORY
LATENCIES
An excitatory afferent volley produces different degrees and latencies of depolarization (EPSP) in different motor cells. Many are depolarized subliminally, others reach threshold after maximum build-up of 1 msec, and some are so strongly depolarized that they reach threshold in less than 0.5 msec. The latencies of these EPSP’s vary directly (not necessarily linearly) with their build-up times (Figs. 3 and 4). The resulting mofrosynaptic reflex volley will be made up of action potentials in the ventral root fibers showing this same total temporal dispersion. The motoneuron of Fig. 4 shows a range of firing latency with varying stimulus strength similar to the temporal dispersion found among different cells contributing to a monosynaptic discharge elicited by a single stimulus. If an inhibitory volley to the same motoneurons is to be effective it must arrive in time to prevent some of the cells from being depolarized to their threshold levels. The tracings of Fig. 4 at different stimulus strengths are taken to represent the range in EPSP’s which occurs in different cells for a single afferent volley. It will be seen that an inhibitory volley, to be effective, can arrive as late as 1 msec after the beginning of a just threshold EPSP, or as late as 0.35 msec after the beginning of the strongest EPSP’s. The latencies for contralateral IPSP’s in the same cell are shown in Fig. 5 and the limits are indicated in Fig. 4 by the two vertical lines for synchronous delivery of ipsilateral and contralateral shocks. For this unit under these conditions, the inhibitory IPSP would arrive in time to prevent excitation by all but the strongest excitatory volleys. Thus, the contralateral inhibitory volley could have been delivered 0.6 msec later than synchrony and would still have been effective in blocking firing of the cell by weak excitatory volleys. THE
FALLACY
OF RENSHAW’S
CENTRAL
LATENCY
MEASUREMENTS
Renshaw (19) attempted to measure the central latencies for excitation and inhibition of motoneurons in the sixth and seventh lumbar segments. Figure 9 is a diagram showing the temporal relations of Renshaw’s findings to the potentials recorded with an intracellular microelectrode. He first measured the time between arrival of the excitatory volley at the
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SPRAGUE
L6 segment of the cord (Fig. 9, E) and the beginning of the reflex response on a branch of the crural nerve. From this time he subtracted the conduction time between direct excitation of these quadriceps motoneurons (by stimulation through a 50 u electrode in their midst) to the beginning of the action potential on the crural nerve. Since both of these measurements sampled the most rapidly conducting axons of the lowest threshold cells, his difference gives the central time (from arrival at the cord to beginning of spike) for the first cells to be fired by the excitatory volley
L
FIG. 9. Diagram
cf intracellular postsynaptic potentials in different cells to illustrate the fallacy in Renshaw’s measurements of central inhibitory latencies. Responses in 5 cells (I-5) to an excitatory volley arriving at the L6 segment at E ; (l-3) fire at inflection points, the earliest (1) at F. Responses in 5 cells (6-10, not necessarily l-5) to an inhibitory volley arriving at the L6 segment at I, 0.2 msec before E. Dashed line indicates the inhibition of firing in cell 3 resulting from the earliest IPSP (10).
(Fig. 9, E to F). He then determined the time of arrival at the samesegmentof the cord of the latest inhibitory volley in dorsal root L7 (Fig. 9, I) which produced a barely discernible reduction in the reflex. The time between arrival of this latest effective inhibitory volley and the production of a spike in the earliest firing motoneurons (I to F) was taken as the central inhibitory time. Actually this inhibitory volley blocked not the earliest but only the latest firing motoneurons (Fig. 9, 3). His figure of 0 to 0.2 msec for the difference in central times for excitation and inhibition is thus in error by the temporal spread in firing time described in the previous section. In addition, his difference takes no
DIRECT
39
INHIBITION
account of the fact that even the most strongly excited motoneurons require nearly 0.5 msec from the beginning of the EPSP to initiation of the action potential. It should be pointed out, of course, that at the time Renshaw’s paper was published it was not known that motoneuron excitation is the result of synaptic depolarization to a threshold level. ORIGIN
OF LONGER
INHIBITORY
LATENCY
Differences in Conduction Time. The present experiments, as well as those of Wilson and Lloyd (22) and Curtis, Krnjevic, and Miledi (7), were designed specifically to use excitatory and inhibitory pathways which were as similar as possible in order to minimize the uncertainty introduced by difference in conduction paths. The extramedullary pathways in the dorsal roots of the same segment are of identical length and the cord is so small in the S2-S3 region that intramedullary paths could scarcely differ by as much as 1 mm. It is considered unlikely that any marked difference exists in conduction velocity between the excitatory and inhibitory fibers since a corresponding difference in threshold would then be expected and no such difference was found. However, the possibility remains that dorsal root fibers with low thresholds and high conduction velocities were excitatory to ipsilateral motoneurons while those with higher thresholds and lower conduction velocities were inhibitory across the cord. Monitoring the arrival of the inhibitory volley at the cord does not reveal difference in coduction velocity, since a slightly later arrival of inhibitory impulses would be masked by the earlier arriving excitatory impulses. A comparison of latency differences between afferent volleys initiated at different distances from the cord would permit the measurement of conduction velocities of excitatory and inhibitory fibers; this was not considered practical in the present study. Where this type of experiment has been done (15)) no such conduction velocity differences were found. It is therefore assumed that similar fibers are responsible for “direct” inhibition in the lower sacral cord and that volleys arrive at the terminals of both types of afferents with substantially the same latencies. Differences in Synaptic Times. All measurements in this study are made to the beginning of the postsynaptic potential so that the buildup times for these potentials are not involved, at least for the motoneurons. But, it has been suggested that the longer time from the arrival of the inhibitory volley at the afferent terminals until the beginning of the IPSP in the motor horn cell might be due to a slower synaptic process than that
40
FRANK
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SPRAGUE
involved in excitation. No evidence bearing on this point is available from the present experiments, but Eccles, Fatt, and Landgren (8) have recorded, in the biceps-semitendinosus nucleus (L7), a presynaptic spike evoked by stimulation of the quadriceps nerve about 1.1 msec after its cord entry at dorsal root L6. Subtracting this figure from the mean IPSP delay (1.57 msec) recorded from 25 biceps-semitendinosus motoneurons, leaves a figure of 0.47 msec for the mean inhibitory synaptic delay. This is reasonably comparable to the 0.3 msec synaptic delay for excitation reported by Brock, Coombs, and Eccles (3) and the 0.3 to 0.45 msec of Brooks and Eccles (4). Although the identification of the presynaptic spike as inhibitory would have been more satisfying had it and the postsynaptic IPSP been measured simultaneously in the same preparation, there is no contrary evidence that the excitatory and inhibitory synaptic delays are not approximately equal. Dendritic Conduction Time. The anatomical findings of Sprague (20, 2 1) demonstrate monosynaptic endings on the cell bodies and dendrites of ipsilateral motoneurons and on the dendrites only of contralateral motoneurons in the S2-S3 region of the cat’s cord (Fig. 10). Either of two PSP responses in the motoneurons to contralateral stimulation could be related to these monosynaptic dendritic endings. These endings might be producing the short latency contralateral EPSP’s seen in three motor horn cells in this study and also observed by Curtis, Krnjevic, and Miledi (7). The latencies for these EPSP’s are comparable to those of ipsilateral EPSP’s and support the view that conduction times for excitatory and inhibitory afferent volleys are equally comparable. Alternatively, the contralateral, monosynaptic, dendritic endings might be responsible for the contralaterally evoked IPSP. In this case, it would be necessary to postulate a delayed electrotonic conduction of IPSP down the dendrites to the microelectrode in the soma. Three facts argue against this explanation: (a) The IPSP’s recorded in this study have an abrupt beginning and at least as rapid an initial slope as do the EPSP’s. This is qualitatively the kind of behavior to be expected of local synaptic activity rather than that conducted electrotonically over a distance sufficient to delay its beginning by 0.3 to 0.7 msec (18). (b) Contralateral IPSP’s are very sensitive to polarizing currents applied through the microelectrode as would be expected were they originating close to the microelectrode. (c) Insofar as the “direct inhibition” of the lumbar cord is similar to that in the lower sacral segments, the observations of Eccles, Fatt, and Landgren (8, see above) suggest that “extra time” has not
DIRECT
INHIBITION
41
been spent between the arrival of the presynaptic spike at the motoneuron and the appearance of an IPSP at the microelectrode. A third possibility is that the contralateral monosynaptic endings are not active under the conditions of these experiments and so do not contribute to the potentials recorded by intracellular microelectrodes. This would not be in accord with the fact that low threshold afferent fibers are thought to be those with monosynaptic connections ( 15).
FIG. 10. Diagrammatic cross section of third sacral segment of the spinal cord of the cat showing distribution of degenerating moncsynaptic dcrsal root collaterals (broken line) to both sides of the cord. A, B and C indicate dorsal, lateral and medial dendrites of the motoneurons. Note pathway of collaterals through intermediate nucleus of Cajal (crosshatched area).
In view of the foregoing arguments, the first of these three alternatives is the most attractive, i.e., the brief latency contralaterally evoked EPSP’s are the result of excitation arriving monosynaptically over the dendritic synapsesobserved by Sprague (20, 21). The Inhibitory Interneuron. Three possible mechanisms accounting for the extra latency of the inhibitory pathway have been discussedand tentatively discarded. A fourth possibility is the existence of an inter-
42
FRANK
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SPRAGUE
neuron in the inhibitory pathway. This possibility was proposed by Eccles, Fatt, and Landgren (S), opposed by Wilson and Lloyd (22), and supported by Curtis, Krnjevic, and Miledi (7). The present study shows that in the cat’s lower sacral cord the latency for the beginning of the IPSP is 0.3 to 0.7 msec longer than that for the EPSP. It is the view of the present authors that the lower limit of 0.3 msec is probably too short for the introduction of an interneuron but that the mean of 0.55 msec, or certainly Eccles’ value of 0.7 msec, does not preclude such a possibility. Eccles, Fatt, and Landgren (8) recognized that such a difference in latency does not in itself establish the presence of an interneuron nucleus in the inhibitory pathway. The cells of such a nucleus must have the anatomical connections and the physiological properties which satisfy the well known characteristics of “direct” inhibition, Both Eccles, Fatt, and Landgren (8) and Curtis, Krnjevic, and Miledi (7) have studied spinal interneurons whose behavior was consistent in certain respects with these characteristics.4 One of the difficulties in accepting the interneuron in the direct inhibitory pathway is the demonstration (13, 15) of the nearly identical and linear development of facilitation and inhibition of a test reflex with stimuli of increasing strength beginning with the smallest measurable volleys. Both Eccles, Fatt, and Landgren (8) and R. Eccles and Lundberg (9) have found evidence of a subliminal fringe in the relevant interneuron pool. This evidence of spatial summation in the development of the IPSP supports the interneuron hypothesis but does not agree with the observations on direct inhibition. This discrepancy has not yet been resolved. The suggestion of Eccles, Fatt, and Landgren (8) and Eccles and Lundberg (9) that depth of anesthesia might be responsible should be tested. 1. 2.
3.
References ARAKI, T., and T. OTANI, Response of single motoneurone to direct stimulation in toad’s spinal cord. J. Neurophysiol. 18: 472-485, 1955. BRADLEY, K., D. M. EASTON, and J. C. ECCLES, An investigation of primary or direct inhibition. J. Physiol., Lond. 122: 474-488, 1953. BROCK, L. G., J. S. COOMBS, and J. C. ECCLES, The recording of potentials from motoneurones with an intracellular electrode. J. Physiol., Lond. 117: 431-460, 1952.
4 Such interneurons were not seen in the present study, but they were not specifically sought due to the difficuhy of identification described by Frank and Fuortes (11) and because it is not clear how such interneurons are to be distinguished from excitatory interneurons in the same nucleus.
DIRECT
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INHIBITION
4. BROOKS, C. McC., and J. C. ECCLES, Electrical investigation of the monosynaptic pathway through the spinal cord. J. Neurophysiol. 10: 251-273, 1947. 5. CAJAL, RAMON Y, “Histologie du Systeme Nerveux de 1’Homme et des Vertebres,” Paris, Maloine, 1911. 6. COOMBS,J. S., J. C. ECCLES,and P. FATT, The specific ionic conductances and the ionic movements acrcss the motoneuronal membrane that produce the inhibitory postsynaptic potential. J. Physiol., Lond. 130: 326-373, 1955. 7. CURTIS, D. R., K. KRNJEVIC, and R. MILEDI, Crossed inhibition of sacral motoneurones. J. Neurophysiol. 21: 319-326, 1958. 8. ECCLES, J. C., P. FATT, and S. LANDGREN, The central pathway for the direct inhibitory action of impulses in the largest afferent fibers of muscle. J. Neurophysiol.
16: 75-98,
1956.
9. ECCLES, R. M., and A. LUNDBERG, Spatial facilitation in the direct inhibitory pathway. Nature, Lond. 179: 1305-1306, 19.57. 10. FRANK, K., and M. G. F. FUORTES, Potentials recorded from the spinal corl with microelectrodes. J. Physiol., Lond. 130: 625-654, 1955. 11. FRANK, K., and M. G. F. FUORTES, Stimulation of spinal motoneurones with intracellular electrodes. J. Physiol., Lond. 134: 451-470, 1956. 12. FRANK, K., and M. G. F. FUORTES, Presynaptic and postsynaptic inhibition of monosynaptic reflexes. Fed. PYOC., Bait. 16: 39-40, 19.57. 13. HUNT, C. C., Monosynaptic reflex response of spinal motoneurones to graded afferent stimulation. J. Gen. Physiol. 66: 813-852, 1955. 14. LLOYD, D. P. C., A direct central inhibitory action of dromically conducted impulses. J. Neurophysiol. 4: 184-190, 1941. 15. LLOYD, D. P. C., Reflex action in relation to pattern and peripheral source of afferent stimulation. J. Neuuophysiol. 6: 111-119, 1943. 16. LLOYD, D. P. C., Functional organization of the spinal cord. Physiol. Rev. 24: l-17, 1944. 17. LLOYD, D. P. C., Facilitation and inhibition of spinal motoneurons. J. Neurophysiol. 6: 421-438, 1946. 18. RALL, W., Membrane time constant of motoneurons. Science 125: 454, 1957. 19. RENSHAW, B., Reflex discharges in branches of the crural nerve. J. Neurophysiol.
6: 487-498,
1942.
20. SPRAGUE, J. M., Site of spinal monosynaptic 15: 176-177,
inhibition.
Fed.
PYOC.,
Bolt.
1956.
21. SPRAGUE,J. M., The distribution of dorsal root fibers on motor cells in the lumbosacral spinal cord of the cat, and the site of excitatory and inhibitory terminals of monosynaptic pathways. Proc. R. Sot., Ser. B, Biol. SC. Lond. 937: 534, 1958. 22. WILSON, V., and D. P. C. LLOYD, Bilateral spinal excitatory and inhibitory actions. Am. J. Physiol. 167: 641, 1956.
NEUROLOGY
Direct
1,
28-43
Contralateral Sacral KARL
FRANK
(1959)
Inhibition in the Lower Spinal Cord AND
JAMES
M.
SPRAGUEI
Laboratory of Neurophysiology, National Institute of Neurological Blindness, National Institutes of Health, Bethesda, and the Department and Institute of Neurological Sciences, University of Pennsylvania School, Philadelphia, Pennsylvania Received
November
Diseases and of Anatomy Medical
10, 1958
The pcssibility of an interneuron in the so-called “direct” inhibitory pathway has been examined by studying postsynaptic potentials of motor horn cells in the SZ-S3 segments of cat’s spinal ccrd following excitatory and inhibitory volleys in ipsilateral and contralateral dorsal roots. Monosynaptic connections were demonstrated histologically from ipsilateral dorsal roots to motor horn cell somata and dendrites and from contralateral dorsal roots to motor horn cell dendrites alone. However, direct comparison of ipsilateral EPSP and contralateral IPSP latencies in the same unit showed an IPSP latency longer than the EPSP latency by 0.3 to 0.7 msec. Since no adequate explanation could be found to account for this extra latency on the basis of monosynaptic connections, the presence of an interneuron in the direct inhibitory pathway remains a definite possibility. Introduction
The first definition and measurement of the shortest latency reflex discharge and its “direct” inhibition in the spinal cord were obtained from recordings on ventral root and peripheral nerve (14, 19). The minimal latency of this excitation was such that it was thought to be monosynaptic and to be mediated by the reflex fibers shown by Cajal (5) to extend directly from dorsal funiculus to motoneurons. The striking similarities between short latency excitation and inhibition suggested rather strongly that “direct” inhibition was also monosynaptic. These similarities are seen in (a) the exponential decay of the two processes acting on a test reflex (16) ;” (b) the almost linear relationship between the 1 Supported in part by a research grant (B-461) from the National Institute Neurological Diseases and Blindness of the National Institutes of Health, U. Public Health Service. 2 However, see the conclusions of Bradley, Easton, and Ecdes (2). 28
of S.
DIRECT
INHIBITION
29
degree of inhibition or facilitation and the amplitude of the responsible dorsal root spike, beginning with the smallest measurable input (13, 1.5) ; and (c) the demonstration that the fibers in the dorsal root responsible for both actions have the same threshold (1.5). Precise measurement of the latency of “direct” inhibition of a test monosynaptic ventral root discharge is difficult because even the most synchronous discharge has a duration of about 1 msec. Microelectrode studies (10) show that the individual action potentials of the single ventral root fibers making up this discharge have a duration of 0.3 to 0.5 msec. Thus, the ventral root discharge reflects the time of initiation of only its earliest components, while the late units alone are inhibited when excitatory and inhibitory volleys approach synchrony (Fig. 1). Brock, Coombs, and Eccles (3) found by use of intracellular microelectrodes that the firing of a motoneuron is preceded by a depolarization of the cell membrane, the excitatory postsynaptic potential or EPSP, and is prevented by hyperpolarization, the inhibitory postsynaptic potential or IPSP. This technique permits a comparison of inhibitory and excitatory latencies, each measured between the time of the initiation of the afferent volley and the first evidence of its postsynaptic effect in the motoneuron. This comparison deals only with “direct” inhibition and does not apply to “remote” inhibition (12)) which acts without producing postsynaptic hyperpolarization. Eccles, Fatt, and Landgren (8) have challenged the conclusion that “direct” inhibition is monosynaptic. They found, using nerves innervating a variety of synergistic and antagonistic muscles of the hind leg, that IPSP trailed EPSP by minimal intervals of 0.5 to 1.0 msec. This difference in latency was believed by them to implicate an interneuron in the inhibitory pathway, an interpretation expanded into a general hypothesis that all central inhibitory pathways act through small interneurons which produce a chemical inhibitory transmitter. Following Cajal’s (5, Fig. 113) demonstration that monosynaptic fibers passing from the dorsal funiculus to the motoneurons give off collaterals to a nearby interneuron nucleus, Eccles, Fatt, and Landgren (8) studied the activity of these or adjacent interneurons to stimulation of muscle nerves. They reported that many of these cells are activated specifically by group IA fibers from muscle, and in their opinion, their firing to this activation is such that most of the previously described characteristics of direct inhibition can be accounted for by such a disynaptic mechanism. One of the difficulties in interpreting the differences in latency between
30
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AND
SPRAGUE
excitatory (EPSP) and inhibitory postsynaptic potentials (IPSP) in the lumbosacral segments, is the fact that pathways of different lengths are usually involved. More satisfactory, in this respect, are the lower sacral segments (SZ-S3) in which ipsilateral monosynaptic reflexes (serving the
FIG. 1. Direct inhibition of S3 ventral root spike evoked by ipsilateral dorsal root 53, E, and inhibited by contralateral dorsal root S3, I. A: Mean of 10 traces stimulating ipsilateral dorsal root S3 twice at F and E. B: Mean of 10 traces stimulating ipsilateral dorsal root S3 at F and E and contralateral dorsal roct S3 at I. C: Difference between A and B. A large stable monosynaptic test reflex frequently requires two shccks to dorsal root S3. Note that even for this stimulus interval (0.4 msec) the late components of the spike are those which are selectively inhibited.
musclesof the tail) are inhibited “directly” by volleys in the contralateral dorsal roots ( 17, 22, and unpublished results of Lloyd, Sprague, and Edisen, 1955). After unilateral section of these dorsal roots, terminal degeneration was found on cell bodies and dendrites of ipsilateral motoneurons and on dendrites only of contralateral motoneurons. Thus, mono-
DIRECT
31
INHIBITION
synaptic connections exist between each S2-S3 dorsal root and the motoneurons of both sides of the cord, pathways of approximately equal length (Fig. 10). Wilson and Lloyd (22) recorded monosynaptic reflex discharges concurrently in the two S3 ventral roots, while changing the timing of one S3 dorsal root volley with respect to the other. With this arrangement, each dorsal root volley sets up a monosynaptic reflex on the ipsilateral side, and inhibits the reflex on the opposite side. They found that both reflexes may be inhibited for as much as 0.1 msec on either side of synchrony of dorsal root shocks, and concluded that no time is present for an interneuron in the inhibitory pathway. Because of the reservations about ventral root recording discussed earlier in this paper, and because of the above anatomical evidence for contralateral monosynaptic connections, it was felt that “direct” inhibition in the S2-S3 spinal segments should be investigated using intracellular microelectrodes. Methods
Ten cats were prepared by high spinal section, intercollicular decerebration, or by use of pentobarbital sodium (Nembutal), d-tubocurarine being used for immobilization in all animals. Dorsal and ventral roots of the second and third sacral segments, were exposed and cut bilaterally at equal distances from the cord for stimulation and recording. Ventral root reflexes were recorded following dorsal root shocks (up to 10X threshold) throughout the experiment to ascertain the presence of ipsilateral, monosynaptic responses, and their “direct” inhibition by near simultaneous stimulation of the contralateral dorsal roots. The crossed inhibition was not always found, and when present, it was usually in S3 segment. In those cases, where the lumbosacral plexus was subsequently dissected, the absence of crossed inhibition was correlated with a postfixed plexus (unpublished results of Lloyd, Sprague, and Edisen, 1955) in which these segments contributed to the innervation of the hind leg, and thus were not associated solely with crossed innervation of the tail muscles. Search was made in the S2-S3 area of the cord with a microelectrode for motoneurons which showed responses to ipsilateral and/or contralateral dorsal root stimulation. Excitatory and inhibitory postsynaptic potentials of the lower sacral 3 During the course of this investigation it was learned being conducted by Curtis, Kmjevic, and Miledi (7).
that
a similar
study
was
32
FRANK
AND
SPRAGUE
motoneurons were recorded with 3 M KCl-filled micropipettes according to the technique of Brock, Coombs, and Eccles (3) and Frank and Fuortes (10). It was commonly found that hyperpolarizing synaptic potentials reversed their sign soon after penetration of the motoneuron, presumably due to leakage of Cl- from the micropipette (6)) which shifts the ionic balance and thus raises the IPSP equilibrium potential of the membrane. Identification as IPSPs of such reversed potentials was accomplishedby passing depolarizing current through the electrode using the bridge technique of Araki and Otani (1) and Frank and Fuortes (10). Application of the depolarizing current changes the resting membrane potential, so it is again above the shifted IPSP equilibrium potential, and the IPSP reappears as a membrane hyperpolarization. The fact that the EPSP depolarization is not so reversed by either ion leakage or moderate depolarization makes this a convenient method of identification. Figure 2 shows the difference in effect of applied currents on EPSP (+) and IPSP (0) in sacral motoneurons. Note that the IPSP is depolarizing at zero current, but is correctly identified by the effect of the applied current.
+ + +
+ FIRING
“0: z i;j E I: Q: p:: t! %
0 ,-2
?
0 0
HYPERPOLARIZING
CURRENT
0 0
-4
0
CEPOLARIZING
CURRENT
FIG. 2. Effects of polarizing currents on postsynaptic potentials (PSP) of two S3 motoneurons. EPSP’s (+) are unaffected by positive and negative currents which alter and reverse IPSP’s (0). Ordinate: amplitude of PSP; reversal occurs at 0. Abscissa: amplitude of applied current; firing for EPSP occurs at arrow.
DIRECT
33
INHIBITION
Latencies for PSP’s were measured from the shock artifact rather than from the arrival of the volley at the cord surface. This is justified by measurements showing that within the range of stimuli used in these experiments (up to 10X threshold), the arrival time of the volley at the cord varied by not more than 0.06 msec. The relatively small number of cells recorded is explained by the diffrculty in penetrating motoneurons in the small part of the spinal cord. Results
Ipsilaterd PSP’s. Many motor horn cells showed EPSP’s to ipsilateral S2-S3 dorsal root stimulation which were typically monosynaptic in time course (11). The minimum latencies of 11 such units ranged from 0.65
FIG. 3. Variation of latency of EPSP with subthreshold single S3 motoneuron. A-C: increasing stimulus strength S3; D shows beginning of antidromic firing of same unit.
stimulus strength to ipsilateral dorsal Time: msec.
in a root
to 1.2 msec. For any one of these units, EPSP latency was distinctly a function of stimulus strength. Figure 3 shows that in a single unit the EPSP latency can be varied by as much as 0.6 msec. The maximum latency is difficult to determine because with weak shocks the PSP is lost in the noise level, but 3 cells had maximum latencies which were at least 1.0, 1.4, and 1.6 msec. The latter is illustrated in Fig. 3A. This EPSP has a time course which appears monosynaptic but could have a plurisynaptic contribution. The unit of Fig. 3 could not be fired within the range of stimuli used. On other units suprathreshold stimuli further reduced the EPSP latency as seen in Fig. 4. Threshold monosynaptic EPSP’s in lower sacral motoneurons usually rise to maximum in about 0.9 to 1.0 msec. Firing in
34
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these units for threshold excitation shows a total latency equal to the sum of this EPSP rise time plus the time required for the presynaptic volley to initiate the EPSP. As illustrated in Fig. 4, a strong afferent volley can produce maximal reduction of both of these times: the EPSP rise time from 1 msec down to 0.45 msec, and EPSP latency from 0.95 down to 0.65 msec, so that total firing latency is reduced from 2.0 to 1.1 msec. Thus, in the spectrum of action potentials contributing to ventral root discharge, there will be a range of latencies equal to the sum of at least these two effects.
ABc-
II FIG. 4. Variation of latency and rise time of EPSP ipsilateral dorsal root S3 in a single S3 motoneuron. strength. Trace discontinued after beginning of spike in cate range of latency of IPSP in same cell (see Fig. 5).
with stimulus strength to A-D: decreasing stimulus A-C. Vertical lines indiTime: msec.
In one cat two cells showed hyperpolarizing PSP following ipsilateral stimulation, but their latencies were 3 and 4 msec respectively-clearly plurisynaptic. Contralateral PSP’s. The PSP’s which were recorded from motor horn cells following contralateral dorsal root volleys were usually initially hyperpolarizing potentials which were often followed by depolarization. Minimum latencies of these IPSP’s ranged from 1.1 to 1.6 msec in 6 units. Due to the difficulty of finding and holding these units (see Methods) the effect of stimulus strength on the latency was not carefully studied, but data on hand suggest that the variation of latency with stimulus strength was much less (ca. 0.2 msec) than that of the ipsilateral EPSP (ca. 0.6 msec). Figure 5 shows an example. The depolarization which often followed hyperpolarization in response to contralateral stimulation appeared to rise out of the IPSP more or less abruptly as seen in Fig. 6.
DIRECT
INHIBITION
35
The range of latencies for this contralateral EPSP was 1.8 to 2.3 msec in 7 cells (range 0.3 msec for 6 of the 7 cells). Polarizing currents applied through the microelectrode, as described in the section on Methods, served to distinguish between EPSP’s and IPSP’s which had been reversed pre-
FIG. 5. Variation of latency of IPSP with decreasing stimulus strength, A-E, to contralateral dorsal root S3 in the same S3 motoneuron as in Fig. 4. Note that stronger stimulus strengths in A-B bring in late depolarization, curtailing full development of early IPSP. Time: msec.
A-A FIG. 6. IPSP interrupted by EPSP in an S3 motoneuron following stimulation of contralateral dorsal root S3 (A). EPSP for the same motoneuron following stimulation of ipsilateral dorsal root S3 (B). Time: msec.
sumably due to leak of Cl- ions. Figure 7 shows an example. It may be seen that such applied currents do not affect the latencies of the PSP’s. Three cells in this study showed only brief latency EPSP’s to contralateral stimulation. Their latencies were 0.9, 1.0, and 1.4 msec-comparable to ipsilateral EPSP latencies. Comparison of EPSP and IPSP L&en&es. While the ranges of minimal latencies for ipsilateral EPSP’s and contralateral IPSP’s in different cells are broad enough to actually overlap, the more significant comparisons are obtained when both types of PSP’s are seen in the same cell. Five cells satisfied this condition and showed a longer IPSP than EPSP
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latency. Differences in the two latencies were: 0.3, 0.5, 0.6, 0.65, 0.7 msec, mean 0.55 msec. Figure 8 shows tracings of the extremes of this range.
FIG. 7. Left. Reversal of IPSP with applied currents in a single S3 motoneuron stimulated by contralateral volley in dorsal root S3 (A-B) ; hyperpolarizing current EPSP in same cell following ipsilateral volley in (A), depolarizing current (B). dorsal root S3 (C-E) ; depolarizing current (C), hyperpolarizing current (D), no current (E). Cell apparently failing between C and D. Latencies of PSP’s unaffected by applied currents or cell deterioration. Time: msec. FIG. 8. Right. Latency differences for EPSP and IPSP in S3 motoneurons illustrating minimum (A, B) and maximum (C, D) differences. Stimulus to ipsilateral (A, C) and contralateral (B, D) dorsal root S3. Time: msec.
Discussion Intracellular potentials from motoneurons show that excitation is preceded by a depolarizing synaptic potential (EPSP) and the beginning of the potential is the first indication of the arrival of the afferent volley at the postsynaptic cell. Similarly, direct inhibition in this cell is indicated by the generation of a hyperpolarizing synaptic potential. There seems to be no reasonable doubt that these changes in membrane polarization are causally related to the reflex excitation and inhibition of the cell (see, however, 22). The measurements made in this study clearly demonstrate that the time required for a contralateral dorsal root volley
DIRECT
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INHIBITION
to produce an IPSP in a motor horn cell in the lower sacral cord of the cat is 0.3 to 0.7 msec longer than the time for an ipsilateral dorsal root volley to produce an EPSP in the same cell. RANGE
OF EFFECTIVE
EXCITATORY
AND
INHIBITORY
LATENCIES
An excitatory afferent volley produces different degrees and latencies of depolarization (EPSP) in different motor cells. Many are depolarized subliminally, others reach threshold after maximum build-up of 1 msec, and some are so strongly depolarized that they reach threshold in less than 0.5 msec. The latencies of these EPSP’s vary directly (not necessarily linearly) with their build-up times (Figs. 3 and 4). The resulting mofrosynaptic reflex volley will be made up of action potentials in the ventral root fibers showing this same total temporal dispersion. The motoneuron of Fig. 4 shows a range of firing latency with varying stimulus strength similar to the temporal dispersion found among different cells contributing to a monosynaptic discharge elicited by a single stimulus. If an inhibitory volley to the same motoneurons is to be effective it must arrive in time to prevent some of the cells from being depolarized to their threshold levels. The tracings of Fig. 4 at different stimulus strengths are taken to represent the range in EPSP’s which occurs in different cells for a single afferent volley. It will be seen that an inhibitory volley, to be effective, can arrive as late as 1 msec after the beginning of a just threshold EPSP, or as late as 0.35 msec after the beginning of the strongest EPSP’s. The latencies for contralateral IPSP’s in the same cell are shown in Fig. 5 and the limits are indicated in Fig. 4 by the two vertical lines for synchronous delivery of ipsilateral and contralateral shocks. For this unit under these conditions, the inhibitory IPSP would arrive in time to prevent excitation by all but the strongest excitatory volleys. Thus, the contralateral inhibitory volley could have been delivered 0.6 msec later than synchrony and would still have been effective in blocking firing of the cell by weak excitatory volleys. THE
FALLACY
OF RENSHAW’S
CENTRAL
LATENCY
MEASUREMENTS
Renshaw (19) attempted to measure the central latencies for excitation and inhibition of motoneurons in the sixth and seventh lumbar segments. Figure 9 is a diagram showing the temporal relations of Renshaw’s findings to the potentials recorded with an intracellular microelectrode. He first measured the time between arrival of the excitatory volley at the
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L6 segment of the cord (Fig. 9, E) and the beginning of the reflex response on a branch of the crural nerve. From this time he subtracted the conduction time between direct excitation of these quadriceps motoneurons (by stimulation through a 50 u electrode in their midst) to the beginning of the action potential on the crural nerve. Since both of these measurements sampled the most rapidly conducting axons of the lowest threshold cells, his difference gives the central time (from arrival at the cord to beginning of spike) for the first cells to be fired by the excitatory volley
L
FIG. 9. Diagram
cf intracellular postsynaptic potentials in different cells to illustrate the fallacy in Renshaw’s measurements of central inhibitory latencies. Responses in 5 cells (I-5) to an excitatory volley arriving at the L6 segment at E ; (l-3) fire at inflection points, the earliest (1) at F. Responses in 5 cells (6-10, not necessarily l-5) to an inhibitory volley arriving at the L6 segment at I, 0.2 msec before E. Dashed line indicates the inhibition of firing in cell 3 resulting from the earliest IPSP (10).
(Fig. 9, E to F). He then determined the time of arrival at the samesegmentof the cord of the latest inhibitory volley in dorsal root L7 (Fig. 9, I) which produced a barely discernible reduction in the reflex. The time between arrival of this latest effective inhibitory volley and the production of a spike in the earliest firing motoneurons (I to F) was taken as the central inhibitory time. Actually this inhibitory volley blocked not the earliest but only the latest firing motoneurons (Fig. 9, 3). His figure of 0 to 0.2 msec for the difference in central times for excitation and inhibition is thus in error by the temporal spread in firing time described in the previous section. In addition, his difference takes no
DIRECT
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INHIBITION
account of the fact that even the most strongly excited motoneurons require nearly 0.5 msec from the beginning of the EPSP to initiation of the action potential. It should be pointed out, of course, that at the time Renshaw’s paper was published it was not known that motoneuron excitation is the result of synaptic depolarization to a threshold level. ORIGIN
OF LONGER
INHIBITORY
LATENCY
Differences in Conduction Time. The present experiments, as well as those of Wilson and Lloyd (22) and Curtis, Krnjevic, and Miledi (7), were designed specifically to use excitatory and inhibitory pathways which were as similar as possible in order to minimize the uncertainty introduced by difference in conduction paths. The extramedullary pathways in the dorsal roots of the same segment are of identical length and the cord is so small in the S2-S3 region that intramedullary paths could scarcely differ by as much as 1 mm. It is considered unlikely that any marked difference exists in conduction velocity between the excitatory and inhibitory fibers since a corresponding difference in threshold would then be expected and no such difference was found. However, the possibility remains that dorsal root fibers with low thresholds and high conduction velocities were excitatory to ipsilateral motoneurons while those with higher thresholds and lower conduction velocities were inhibitory across the cord. Monitoring the arrival of the inhibitory volley at the cord does not reveal difference in coduction velocity, since a slightly later arrival of inhibitory impulses would be masked by the earlier arriving excitatory impulses. A comparison of latency differences between afferent volleys initiated at different distances from the cord would permit the measurement of conduction velocities of excitatory and inhibitory fibers; this was not considered practical in the present study. Where this type of experiment has been done (15)) no such conduction velocity differences were found. It is therefore assumed that similar fibers are responsible for “direct” inhibition in the lower sacral cord and that volleys arrive at the terminals of both types of afferents with substantially the same latencies. Differences in Synaptic Times. All measurements in this study are made to the beginning of the postsynaptic potential so that the buildup times for these potentials are not involved, at least for the motoneurons. But, it has been suggested that the longer time from the arrival of the inhibitory volley at the afferent terminals until the beginning of the IPSP in the motor horn cell might be due to a slower synaptic process than that
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involved in excitation. No evidence bearing on this point is available from the present experiments, but Eccles, Fatt, and Landgren (8) have recorded, in the biceps-semitendinosus nucleus (L7), a presynaptic spike evoked by stimulation of the quadriceps nerve about 1.1 msec after its cord entry at dorsal root L6. Subtracting this figure from the mean IPSP delay (1.57 msec) recorded from 25 biceps-semitendinosus motoneurons, leaves a figure of 0.47 msec for the mean inhibitory synaptic delay. This is reasonably comparable to the 0.3 msec synaptic delay for excitation reported by Brock, Coombs, and Eccles (3) and the 0.3 to 0.45 msec of Brooks and Eccles (4). Although the identification of the presynaptic spike as inhibitory would have been more satisfying had it and the postsynaptic IPSP been measured simultaneously in the same preparation, there is no contrary evidence that the excitatory and inhibitory synaptic delays are not approximately equal. Dendritic Conduction Time. The anatomical findings of Sprague (20, 2 1) demonstrate monosynaptic endings on the cell bodies and dendrites of ipsilateral motoneurons and on the dendrites only of contralateral motoneurons in the S2-S3 region of the cat’s cord (Fig. 10). Either of two PSP responses in the motoneurons to contralateral stimulation could be related to these monosynaptic dendritic endings. These endings might be producing the short latency contralateral EPSP’s seen in three motor horn cells in this study and also observed by Curtis, Krnjevic, and Miledi (7). The latencies for these EPSP’s are comparable to those of ipsilateral EPSP’s and support the view that conduction times for excitatory and inhibitory afferent volleys are equally comparable. Alternatively, the contralateral, monosynaptic, dendritic endings might be responsible for the contralaterally evoked IPSP. In this case, it would be necessary to postulate a delayed electrotonic conduction of IPSP down the dendrites to the microelectrode in the soma. Three facts argue against this explanation: (a) The IPSP’s recorded in this study have an abrupt beginning and at least as rapid an initial slope as do the EPSP’s. This is qualitatively the kind of behavior to be expected of local synaptic activity rather than that conducted electrotonically over a distance sufficient to delay its beginning by 0.3 to 0.7 msec (18). (b) Contralateral IPSP’s are very sensitive to polarizing currents applied through the microelectrode as would be expected were they originating close to the microelectrode. (c) Insofar as the “direct inhibition” of the lumbar cord is similar to that in the lower sacral segments, the observations of Eccles, Fatt, and Landgren (8, see above) suggest that “extra time” has not
DIRECT
INHIBITION
41
been spent between the arrival of the presynaptic spike at the motoneuron and the appearance of an IPSP at the microelectrode. A third possibility is that the contralateral monosynaptic endings are not active under the conditions of these experiments and so do not contribute to the potentials recorded by intracellular microelectrodes. This would not be in accord with the fact that low threshold afferent fibers are thought to be those with monosynaptic connections ( 15).
FIG. 10. Diagrammatic cross section of third sacral segment of the spinal cord of the cat showing distribution of degenerating moncsynaptic dcrsal root collaterals (broken line) to both sides of the cord. A, B and C indicate dorsal, lateral and medial dendrites of the motoneurons. Note pathway of collaterals through intermediate nucleus of Cajal (crosshatched area).
In view of the foregoing arguments, the first of these three alternatives is the most attractive, i.e., the brief latency contralaterally evoked EPSP’s are the result of excitation arriving monosynaptically over the dendritic synapsesobserved by Sprague (20, 21). The Inhibitory Interneuron. Three possible mechanisms accounting for the extra latency of the inhibitory pathway have been discussedand tentatively discarded. A fourth possibility is the existence of an inter-
42
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neuron in the inhibitory pathway. This possibility was proposed by Eccles, Fatt, and Landgren (S), opposed by Wilson and Lloyd (22), and supported by Curtis, Krnjevic, and Miledi (7). The present study shows that in the cat’s lower sacral cord the latency for the beginning of the IPSP is 0.3 to 0.7 msec longer than that for the EPSP. It is the view of the present authors that the lower limit of 0.3 msec is probably too short for the introduction of an interneuron but that the mean of 0.55 msec, or certainly Eccles’ value of 0.7 msec, does not preclude such a possibility. Eccles, Fatt, and Landgren (8) recognized that such a difference in latency does not in itself establish the presence of an interneuron nucleus in the inhibitory pathway. The cells of such a nucleus must have the anatomical connections and the physiological properties which satisfy the well known characteristics of “direct” inhibition, Both Eccles, Fatt, and Landgren (8) and Curtis, Krnjevic, and Miledi (7) have studied spinal interneurons whose behavior was consistent in certain respects with these characteristics.4 One of the difficulties in accepting the interneuron in the direct inhibitory pathway is the demonstration (13, 15) of the nearly identical and linear development of facilitation and inhibition of a test reflex with stimuli of increasing strength beginning with the smallest measurable volleys. Both Eccles, Fatt, and Landgren (8) and R. Eccles and Lundberg (9) have found evidence of a subliminal fringe in the relevant interneuron pool. This evidence of spatial summation in the development of the IPSP supports the interneuron hypothesis but does not agree with the observations on direct inhibition. This discrepancy has not yet been resolved. The suggestion of Eccles, Fatt, and Landgren (8) and Eccles and Lundberg (9) that depth of anesthesia might be responsible should be tested. 1. 2.
3.
References ARAKI, T., and T. OTANI, Response of single motoneurone to direct stimulation in toad’s spinal cord. J. Neurophysiol. 18: 472-485, 1955. BRADLEY, K., D. M. EASTON, and J. C. ECCLES, An investigation of primary or direct inhibition. J. Physiol., Lond. 122: 474-488, 1953. BROCK, L. G., J. S. COOMBS, and J. C. ECCLES, The recording of potentials from motoneurones with an intracellular electrode. J. Physiol., Lond. 117: 431-460, 1952.
4 Such interneurons were not seen in the present study, but they were not specifically sought due to the difficuhy of identification described by Frank and Fuortes (11) and because it is not clear how such interneurons are to be distinguished from excitatory interneurons in the same nucleus.
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INHIBITION
4. BROOKS, C. McC., and J. C. ECCLES, Electrical investigation of the monosynaptic pathway through the spinal cord. J. Neurophysiol. 10: 251-273, 1947. 5. CAJAL, RAMON Y, “Histologie du Systeme Nerveux de 1’Homme et des Vertebres,” Paris, Maloine, 1911. 6. COOMBS,J. S., J. C. ECCLES,and P. FATT, The specific ionic conductances and the ionic movements acrcss the motoneuronal membrane that produce the inhibitory postsynaptic potential. J. Physiol., Lond. 130: 326-373, 1955. 7. CURTIS, D. R., K. KRNJEVIC, and R. MILEDI, Crossed inhibition of sacral motoneurones. J. Neurophysiol. 21: 319-326, 1958. 8. ECCLES, J. C., P. FATT, and S. LANDGREN, The central pathway for the direct inhibitory action of impulses in the largest afferent fibers of muscle. J. Neurophysiol.
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9. ECCLES, R. M., and A. LUNDBERG, Spatial facilitation in the direct inhibitory pathway. Nature, Lond. 179: 1305-1306, 19.57. 10. FRANK, K., and M. G. F. FUORTES, Potentials recorded from the spinal corl with microelectrodes. J. Physiol., Lond. 130: 625-654, 1955. 11. FRANK, K., and M. G. F. FUORTES, Stimulation of spinal motoneurones with intracellular electrodes. J. Physiol., Lond. 134: 451-470, 1956. 12. FRANK, K., and M. G. F. FUORTES, Presynaptic and postsynaptic inhibition of monosynaptic reflexes. Fed. PYOC., Bait. 16: 39-40, 19.57. 13. HUNT, C. C., Monosynaptic reflex response of spinal motoneurones to graded afferent stimulation. J. Gen. Physiol. 66: 813-852, 1955. 14. LLOYD, D. P. C., A direct central inhibitory action of dromically conducted impulses. J. Neurophysiol. 4: 184-190, 1941. 15. LLOYD, D. P. C., Reflex action in relation to pattern and peripheral source of afferent stimulation. J. Neuuophysiol. 6: 111-119, 1943. 16. LLOYD, D. P. C., Functional organization of the spinal cord. Physiol. Rev. 24: l-17, 1944. 17. LLOYD, D. P. C., Facilitation and inhibition of spinal motoneurons. J. Neurophysiol. 6: 421-438, 1946. 18. RALL, W., Membrane time constant of motoneurons. Science 125: 454, 1957. 19. RENSHAW, B., Reflex discharges in branches of the crural nerve. J. Neurophysiol.
6: 487-498,
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20. SPRAGUE, J. M., Site of spinal monosynaptic 15: 176-177,
inhibition.
Fed.
PYOC.,
Bolt.
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21. SPRAGUE,J. M., The distribution of dorsal root fibers on motor cells in the lumbosacral spinal cord of the cat, and the site of excitatory and inhibitory terminals of monosynaptic pathways. Proc. R. Sot., Ser. B, Biol. SC. Lond. 937: 534, 1958. 22. WILSON, V., and D. P. C. LLOYD, Bilateral spinal excitatory and inhibitory actions. Am. J. Physiol. 167: 641, 1956.