- Email: [email protected]
The existence of a monosynaptic reflex arc in the spinal cord of the frog
EXPERIMENTAL
The
NEUROLOGY
Existence the
14, 175-186
of a Monosynaptic Spinal
K. C. HOLEMANS, Department
(1966)
of Physiology,
Cord
of the
H. S. MEIJ, of
University Received
August
Reflex
Arc
in
Frog
AND B. J. MEYERS Pretoria,
16,
Pretoria,
South
Africa
1965
Many observations on the spinal cord of the frog, reported in the literature, could satisfactorily be explained by assuming the operation of a two-neuron reflex arc, but no direct physiological evidence for the existence of such a simple nervous pathway in this species has yet been put forward. The present investigation represents a successful attempt to establish experimentally that such a reflex arc does operate in the frog. The basis for these experiments was the fact that spinal reflexes in this species can be facilitated by applying a previous conditioning stimulus to the relevant afferent nerve. Conduction time, central reflex time and synaptic delay were estimated by classical methods, and it was concluded that the response to a second stimulus following a suitable conditioning volley had the properties of a monosynaptic reflex. The points in favor of this view were: (i) A highly synchronized, high-potential discharge, such as that recorded from the ventral roots, requires the simultaneous activation of many motoneurons. This could hardly occur if the excitatory process had to pass through more than one synaptic connection. (ii) The central reflex time was very similar (difference less than 2%) to that recorded for synaptic delay. (iii) The reflex was elicited by activation of the fastest conducting, lowestthreshold afferent fibers.
Introduction The motoneuron reflex recorded from a ventral root of the spinal cord of the frog following stimulation of the corresponding dorsal root showsa prolonged (15-20 msec) asynchronous pattern of electrical activity (5, 17; see also Fig. 1.4, first response). In the cat a similar asynchronous discharge is preceded by an initial prominent peak which is transmitted through arcs of two neurons (13, 18). The existence of a two-neuron reflex arc in the frog had not been establishedeither through physiological experimentation or through histological observation. Histological studies indicate that the frog apparently for
1 We wish to express his helpful criticism
our gratitude to Professor of the manuscript. 175
F. Bremer,
University
of Brussels,
176
HOLEMANS,
MEIJ,
AND
MEYER
lacks the sensory-motor collaterals that are so prominent in the cat, for no degenerating fibers are seen in the ventral horn after section of the dorsal roots ( 12). However, numerous connections between dorsal-root terminals and dendritic expansions of motoneurons have been demonstrated in the frog (19). Bremer (3) suggested in 1942 that some of his findings on the frog’s spinal cord could be explained by assuming the operation of a two-neuron reflex arc. More recently Brookhart and Fadiga (5) mentioned several experiments (1, 9, 17) where the operation of a monosynaptic reflex arc would provide a satisfactory explanation of the results. Observations were made on the isolated spinal cord of the frog by Brookhart and Fadiga, but these furnished only indirect evidence for the existence of such a simple nervous pathway. In their studies it was not possible to elicit a typical synchronized, short-latency reflex spike in a ventral root after stimulation of a dorsal root. The present study represents a successful attempt to demonstrate the existence of a monosynaptic reflex arc in the spinal cord of the frog. Advantage was taken of the fact, discovered by Bremer (4) and applied by several investigators (10, 17), that the reflex response of the spinal cord of the frog can be facilitated by applying a suitable conditioning volley. It will be shown that the reaction to the test stimulus following the conditioning volley had the characteristics of a monosynaptic reflex discharge. Methods
After a number of preliminary experiments, the present study was undertaken with South African frogs (Xenopus 2eavis), their weights ranging from 7.5 to 125 g. Room temperature varied from 18 to 22 C in different experiments. The frogs were made spinal by transection of the cord at the level of the fourth ventricle. Laminectomy was performed on the five lower vertebrae (IV-VIII), thus exposing the well-developed three inferior dorsal and ventral nerve roots (VIII-X) together with the cord segments of entry or exit of these roots. The right dorsal roots VIII and IX, as well as ventral roots VIII, 1,X and X were distally severed. The ipsilateral sciatic nerve was exposed and severed at the distal end of the femur. Thus stimuli applied to the sciatic nerve could reach the spinal cord only through dorsal root X which was left intact. (In the frog, fibers from spinal nerves VIII, IX and X constitute the sciatic nerve.) Reflex activity was elicited by means of square-wave pulses applied
MONOSYNAPTIC
REFLEX
ARC
177
to the sciatic nerve (cathode proximal) and recorded from the ipsilateral distally severed ventral root X by a single hooked platinum electrode, its potential being pitted against ground. Intervals between reflexes (elicited by single or double stimuli) were kept constant at 3 sec. The height of the reflex spike was always expressed as the mean value of ten consecutive measurements. Synaptic delay was estimated by a method analogous to that employed by Lorente de No (14, 15) and by Renshaw (18), a brief stimulus (0.05 msec) being applied to the lateral surface of the spinal cord. Afferent conduction time was measured (18) by applying the stimulus to the sciatic nerve and recording the potential change of a cord dorsum electrode placed at the point of entry of dorsal root X. Efferent conduction time was measured from the latency of the m spike recorded for the estimation of the synaptic delay (18). An electronic stimulator (DISA Multistim) equipped with a stimulus isolation transformer was used and potential differences were displayed and eventually photographed on the screen of a Tektronix 502 double beam oscilloscope. Under our recording conditions (single electrode, recording from nerve at point of exit from volume, pitted against ground) a predominantly positive deflection could be expected (16). Nevertheless, negativity was recorded upwards. For ordinary operation the amplifier was a-c coupled, but slower waves were checked by using the same instrument in d-c coupled operation. Results
Facilitatiolt of the Spinal IZeftex Response. If a single stimulus, several times maximal for the lowest threshold fibers of the dorsal roots, was applied to the sciatic nerve or the tenth dorsal root, the reaction recorded from ventral root X was only exceptionally a synchronous spike. Usually a prolonged ( 15-25 msec) asynchronous discharge such as the first response in Fig. 1A was recorded from ventral root X. If, however, the same stimulus was preceded 5 to 250 msec by an identical stimulus, the reflex response changed to a high intensity, perfectly synchronized spike such as the second response in Fig. 1A. By varying the interval between the two stimuli, a facilitation curve such as presented in Fig. 2 could be constructed. Two peaks such as shown in Fig. 2 were observed in nineteen out of twenty facilitation curves. These peaks were observed when the intervals between the two stimuli were 5 and 25 msec, respectively, and the notch
178
HOLEMANS,
MEIJ,
AND
MEYER
between the two peaks was situated at an interval of 10 msec. These values for the twenty facilitation curves are presented in Table 1. The mean value for the interval at which the first peak occurred was 5.6 msec (5.0-7.0) and the second peak was observed at an interval of
FIG. 1. A. Upper beam: Response to two successive stimuli applied to the sciatic nerve and recorded from the ipsilateral ventral root X. Stimuli were 20 msec apart. The strength of both stimuli was maximal (1.0 v, duration 0.3 msec). The intensity of the second response is more than ten times that of the first. Time scale: 10 msec. Potential scale: 10 mv. Lower beam: Marking of the stimuli. B. Upper beam: Electrical activity recorded from ventral root X after stimulation (square wave, 0.05 msec, 4 v) of the ipsilateral side of segment X. The first spike (m spike of Lorente de No) begins with the stimulus; the interval between stimulus and second spike (S scale: 20 mv. spike of Lorente de No) is 3.9 msec. Time scale: 2 msec. Potential Lower beam: Marking of the stimulus. C. Upper beam: Potential changes of a cord dorsum electrode, above the point of entry of dorsal root X after stimulation of the sciatic nerve. Seven superimposed traces. Afferent conduction time is measured from such records. Time scale: 2 msec. Potential scale: 2 mv. Lower beam: Marking of the stimulus. D. Upper beam: Neurogram of dorsal root X with maximal (7.0 v, 0.3 msec) stimulus applied to the sciatic nerve. The alpha, beta, gamma and delta elevations described by Erlanger and Gasser can easily be recognized. Time scale: 2 msec. Potential scale: 10 mv. Lower beam: Marking of the stimulus.
MONOSYNAPTIC
REFLEX
ARC
179
24.4 msec (13-40). The mean value for the interval at which the notch between the two peaks occurred was 9.6 msec (7-15). In seventeen out of nineteen curves the second peak was higher than the first. The intensity of the recorded reflex reaction at the peaks of the facilitation curve was usually several millivolts (Table 1) , and in some cases spikes of 30 mv were recorded (Fig. 1A). The total duration of the facilitation curves varied between 80 and more than 300 msec. Not only could the reflex response in ventral root X be facilitated by applying a conditioning stimu-
INTERVAL
50 SEPARATING
FIRST AN0
100 SECOND
STIMULI
1 150 IN MSEC
c 200
FIG. 2. A typical facilitation curve of a ventral-root response elicited by a stimulus on the sciatic nerve, preceded by a conditioning stimulus of equal strength. The interval separating the conditioning and test stimulus varied between 5 and 200msec and at the highest peak of the facilitation curve the intensity of the reflex response was 6.7 mv.
lus to the same nerve as the test stimulus (sciatic nerve or dorsal root X) but facilitation was also observed although less pronounced if the conditioning stimulus was applied to ipsilateral dorsal roots VIII and IX, contralateral dorsal roots IX and X and in some cases to the brachial plexus. Although characteristics and duration of the facilitation curves differed considerably, it was always possible to elicit strong synchronized activity in a ventral root by means of this double stimulus technique. Synaptic DeZay. Figure 1B represents the electrical activity recorded in ventral root X after stimulation of the ipsilateral side of segment X. The foot of the spike (m spike of Lorente de N6; 14, 15) was synchronous with the stimulus artifact. In this particular case (Fig. IB) a second peak was registered 3.9 msec after the stimulus had been applied (s spike). The interval between the foot of the m and that of the s spike was ascribed by Lorente de N6 to the synaptic delay. The mean interval
180
HOLEMANS,
MEIJ,
AND
MEYER
in ten preparations (Table 2) was 3.03 msec. In ten additional preparations the side of segment IX was stimulated and activity recorded at a distance of 25 to 30 mm from the point of stimulation in spinal nerve IX after section of the ipsilateral dorsal roots VIII, IX and X. The m spike was thus separated from the stimulus artifact by about 0.2 msec, but the mean interval m to s remained the same. TABLE MEAN
AND
TEST STIMULI
INDIVIDUAL
VALUES
AT WHICH
No.
Interval (msec)
peak
Second
Intensity (mv)
1 14/5
5.0
2.1
2 14/5
5.0 7.0 -
3.6 0.9 -
1 20/s
5.5
1.6
2 20/5
6.0 6.0
3.2 4.5
5 .o 7.0
3.5 2.8
1 18/5 2 18/5
2b20/5 la21/5 lb21/5 2 21/j
5.5
0.4
2 24/4 la25/5
6.0 5.0
1.9 1.6
lb25/5
5.0 5.0
1 .o 0.3
lc25/5 2a25/5
Interval (msec)
CONDITIONING THEM
peak
A~-;D
OCCURRED
Notch
Intensity (mv)
Interval (msec)
Intensity
1.1
10
(mv)
2.9 6.6
30 20
1.2 2.9
15 -
2.6 0.6 -
30
1.8
10
0.5
20 13 25
3.0 3.1 6.0
10
0.6 2.0
30
2.1
40 46
1.3 2.3
10 10
40 30
3.4 1.8
25
2.6
20 15
3 .2 3.8
2.1
7.0
3.2
2c25/5 la26/5
5.0 5.0 5.0
1.7 0.7 1.6
15 30
5 .o
2.4
5.6
THE
25 25
7.0
Mean
BETWEEN
10
2b25/5
lb26/5 lc26/5
1
INTERVAL
THE Two PEAKS AND THE NOTCH BETWEEN IN TWENTY FACILITATION CURVES First
Exp.
FOR THE
7 10
3.6 1.4 0.2
8 15
1.5 0
10
0 0
10 10
1.7
10
1.3
3 .o 3.7
7 7
20
4.0
7
0.7 0.1 0.5
20
3.6
7
1.6
24.4
9.6
ABerent and Efferent Conduction Time. The afferent conduction time could be measuredby means of a cord dorsum electrode placed at the point of entry of dorsal root X (18). This electrode recorded a potential change, such as that illustrated in Fig. 1C. The latter is identical in shape and time course with the potential change recorded by a microelectrode in the dorsal horn of the frog’s spinal cord by Brookhart and Fadiga (5) and from the cord dorsum of the cat by Renshaw ( 18). Like
MONOSYNAPTIC
REFLEX
181
ARC
Renshaw we defined as afferent conduction time the interval between the stimulus and the foot of the first positive deflection. The mean and individual values for afferent conduction time obtained with ten preparations are presented in Table 2. Efferent conduction time could be estimated from the latency of the m spike in the synaptic delay records (Fig. 1B). In these records, however, the foot of the positive deflection, that is, the source of current preceding the impulse proper (16), was synchronous with the stimulus. TABLE 2 MEAN AND INDIVIIJUAL VALUES FOR TOTAL REFLEX LATENCY, CENTRAL REFLEX TIME (TOTAL REFLEX LATENCY MINUS AFFERENT CONDUCTION TEME) AND SYNAPTIC DELAY. THE FIGURES WERE RECORDED FROM TEN DIFFERENT PREPARATIONS 2
3 Latency
First reaction (msec)
Second reaction (msec)
9 10
4.5 5.5 4.0 3.2 5.0 4.0 4.2 4.4 3.8 4.0
4.2 5.5 4.0 3.2 5.0 4.0 4.2 4.4 3.8 4.2
Mean
4.26
4.25
1 Exp. No.
8
4 Afferent conduction time (msec)
S(3-4) Central reflex (msec)
6 Synaptic delay (msec)
1.4 1.7 1.2 1.1 1 .a 1 .o 1.2 1 .o 1.2 1.2
2.8 3.8 2.8 2.1 3.2 3.0 3 .o 3.4 2.6 3.0
3.2 3.9 3.0 2.0 3.5 2.6 3.0 3.4 2.7 3.0
1.28
2.97
3.03
The initiation of this positive deflection was taken as the reference point for all the measurements and the efferent conduction time, according to this technique, was therefore zero. In other words, a ventral-root electrode 6 to 8 mm from the point of exit of this root recorded positivity as soon as the corresponding motoneurons were activated. Fibers Responsible for Reflex Activity. If dorsal root X was severed near its entry into the cord and charged onto a recording electrode, the different fiber groups described by Erlanger, Bishop and Gasser (7) could easily be distinguished (Fig. 1D) and the strength-response curve for each group could be constructed (2). The strength-response curve of the fastest conducting fiber group has been compared to the strength-
182
HOLEMANS,
MEIJ,
AND
MEYFaR
response curve of the reflex response in ventral root X. The results are plotted in Fig. 3. It was found that the reflex response in ventral root X reached its maximum when the strength of the second stimulus was just maximal for the fastest conducting fibers of dorsal root X; these constitute the alpha group of Erlanger, Bishop and Gasser. In Fig. 3 a slight decrease in growth of the alpha elevation with increasing stimulus strength is seen after the stimulus reaches the value
0.1 ST%lL”S
FIG.
root
3.
Comparison
X neurogram
between (interrupted
0.4 0.3 STRENGTH
the line)
0.5
0.6
0.7
o-3
0.0
IN VOLTS
response of the alpha and the reflex response
elevation in ventral
of the dorsal root X (con-
tinuous line). Stimulus strength pIotted against response. of 0.15 v. This pattern of increase in the alpha elevation of the neurogram could be found in many of the preparations, and was ascribed by Erlanger, Bishop and Gasser (7) to the presence of a slightly faster conducting group of fibers within the alpha group. The term “pre-alpha group” was applied by these authors to the fibers in question, The stepwise increase in the reflex activity with increasing stimulus strength, seen in Fig. 3, was not always so conspicuous but could be found in most stimulus strength-reflex response curves. Fibers Responsible jar Facilitation. The fibers responsible for facilitation were also of the lowest-threshold fiber group of the dorsal roots. The relationship between the strength of the first stimulus and the reflex response to a second stimulus could be investigated when the interval between the successivestimuli was kept constant and when the second stimulus was supramaximal for reflex activity. With this arrangement it
MONOSYNAPTIC
REFLEX
ARC
183
was found that a weak first stimulus, exciting the fastest fiber group of the dorsal root only, had a maximal facilitory influence. Discussion The main object of the present study was to establish whether or not two-neuron reflex arcs exist in the spinal cord of the frog. The data obtained seem to prove the existence of such a simple nervous pathway for the reasons discussed below. Facilitation. The possibility to facilitate spinal reflexes in the frog by means of a conditioning stimulus preceding a test stimulus by 5 to 300 msec has been thoroughly studied by Bremer (4) and Kleyntjens (10). Although the technical arrangement in these early studies (stimulation of a peripheral nerve, recording of a reflex muscle contraction) was considerably different from that employed in the present study, there are no major contradictions in the findings. In Bremer’s facilitation curves the second peak of facilitation was observed when the interval between the first and second stimulus was 30 to 60 msec, and in many cases 40 to 50 msec. In the facilitation curves obtained in the present study the second peak occurred at a mean interval of 24.4 msec, which is considerably shorter than in Bremer’s experiments. It will be shown in a separate paper that this difference can be accounted for by the fact that in the present study antidromic stimulation of the ventral-root fibers has been excluded. Brevity and Intensity of the Reflex ilctivity. The highly synchronized short spike potential recorded from ventral root X after the second stimulus can only be explained by a massive, synchronous activation of the corresponding motoneurons. The spike was sustained for 2 to 3 msec and reached a potential of 30 mv in certain preparations (Fig. 1.4). Such synchrony would scarcely be possible if the excitatory process had to travel through more than one synaptic connection. Synchronism of SynaPtic Delay and Central Reflex Time. Central reflex time is equal to total reflex latency minus afferent and efferent conduction times. The total latency of the facilitated reflex responsewas identical to the latency of the unfacilitated response.The initial deflection of the unfacilitated response,therefore, could not have passedthrough more synaptic connections than the second responsebut was too small to result in a prominent spike such as that seenin the cat (13) or in the facilitated responsein the frog. Kleyntjens (10) reported a considerable reduction in the latency of the facilitated response (muscle contraction) but his
184
HOLEMANS,
MEIJ,
AND
MEYER
technical arrangement was different from that employed in the present study. The mean central reflex time of the ten preparations presented in Table 1 was 2.97 msec. Kleyntjens found a minimum delay of 3 msec between the application of a stimulus to the dorsal roots and activation of the motoneurons. Synaptic delay was found to correspond closely to central reflex time, the difference being in the order of 2%. The synaptic delay of about 3 msec is considerably longer than that found in cats (0.7-0.8 msec) with a similar technique (18). However, the total reflex latency is also considerably shorter in the cat ( 1.0-1.4 msec) than in the frog (4.25 msec in Table 1). According to Kubota and Brookhart ( 11) the most reliable estimates indicate that synaptic delay in the isolated spinal cord of frogs exceeds 1 msec. The actual times reported by these authors were 1.88 msec (5) and 1.5 to 2 msec (8). The first of these values represents the interval from the moment that the stimulus was applied to the moment of onset of the ventral-horn focal potential, and the second set of values the interval from the application of the stimulus to the onset of the EPSP recorded within the cell. The initiation of the spike was, however, delayed in Fadiga and Brookhart’s recordings (8) by about 2 msecwith regard to the onset of the EPSP. In the present study the interval measuredwas that between the moment of application of the stimulus and onset of the spike and the value of 3 msec obtained, therefore, does not contradict the findings of Brookhart and Fadiga. The possibility of studying synaptic delay by stimulating synaptic connections through an electrode in contact with the lateral surface of the cord rather than through needle electrodes inserted into the motor nucleus (14, 15, 18) deserves some comment. In the spinal cord of the frog, dendrites of the motoneurons extend into the white matter and the finer branchesof the dendrites terminate in the subpial zone of the lateral funiculus (19). Silver (19) found a lower threshold for motoneuron stimulation on the lateral surface of the cord than on either the dorsal or the ventral funiculi. He stated that the synaptic junctions of the lateral neuropil region were less than 20 lo away from the intact surface. Threshold of Responsible Fibers. The reflex responsewas elicited by activation of the lowest threshold, fastest conducting afferent fibers. This may constitute an additional argument for the presence of only one synaptic connection in the reflex arc, since in mammalsonly large afferents establish monosynaptic connections with motoneurons (6). Conclusions. The present study demonstratesthe occurrence of a monosynaptic reflex in the spinal cord of the frog. The existence of a two-
MONOSYNAPTIC
REFLEX
ARC
185
neuron reflex arc in this species had been suggested by Bremer (3) in 1942 and postulated by Fadiga and Brookhart (8) in 1960. However, the fact that it was possible to elicit strong synchronized activity in the motoneurons by means of afferent impulses travelling through the dorsal roots is at variance with the views of Brookhart and Fadiga (5). These authors ascribed the lack of synchronized activity in a ventral root following stimulation of a dorsal root to the localization of dorsal-root terminals on the dendritic expansions of the motoneurons rather than on the motoneuron soma. The data obtained in this study show that activation and synchronous spike generation in the frog’s spinal motoneuron nucleus can be induced by axodendritic synaptic connections. References 1. 2. 3.
4. 5.
6.
7.
8. 9. 10.
11. 12.
C. MATTIIEWS. 1938. The interpretation of potential changes in the spinal cord. J. Pkysiol. London 92: 276-321. BRADLEY, K., and J. C. ECCLES. 1953. Analysis of the fast afferent impulses from the thigh muscles. J. Pkysiol. London 133: 462-473. BREMER, F., and V. BONNET. 1942. Contributions a l’etude de la physiologie g&kale des centres nerveux. II. L’inhibition reflexe. Arch. Intern. Pkysiol. 52: 153-194. BREMER, F., and F. KLEYNTJENS. 1937. Nouvelles recherches sur le phenomene de la sommation centrale d’influx nerveux. Arch. Intern. Pkysiol. 45: 382-413. BROOKHART, J. M., and E. FADICA. 1960. Potential fields initiated during monosynaptic activation of frog motoneurons. J. Pkysiol. London 150: 633655. ECCLES, J. C., R. M. ECCLES, and A. LUNDBERG. 1957. Synaptic actions on motoneurons in relation to the two components of the group I muscle afferent volley. J. Pkysiol. London 136: 527-546. ERLANGER, J., G. BISHOP, and H. S. GASSER. 1926. The action potential waves transmitted between the sciatic nerve and its spinal roots. Am. J. Pkysiol. 78: 575-589. FADIGA, E., and J. M. BROOKHART. 1960. Monosynaptic activation of different portions of motor neuron membrane. Am. J. Pkysiol. 195: 693-703. FUORTES, M. G. F. 1951. Potential changes of the spinal cord following different types of afferent excitation. J. Pkysiol. London 113: 372-386. KLEYNTJENS, F. 1937. Contribution a l’etude des effets d’excitations antidromiques sur I’activitC retlexe de la grenouille spinale. I. Action d’une vollee d’influx antidromiques sur le Processus de sommation centrale. Arch. Intern. Pkysiol. 45: 415-441. KUBOTA, K., and J. M. BROOKHART. 1963. Recurrent facilitation of frog motoneurons. J. Neuvopkysiol. 26: 877-893. LIU, C. N., and W. W. CHAMBERS. 1957. Experimental study of the anatomical organization of the frog’s spinal cord. Amt. Record 127: 326. BARRON,
D. H.,
and
B. H.
186 13. 14. 15. 16. 17. 18. 19.
HOLEMANS,
MEIJ,
AND
MEYER
LLOYD, D. P. 1943. Reflex action in relation to pattern and peripheral source of afferent stimulation. J. Xeurophysiol. 6: 111-119. LORENTE DE No, R. 1939. Transmission of impulses through cranial motor nuclei. J. LVezuophysiol. 2: 402-464. LORENTE DE No, R. 1935. The electrical excitability of the motoneurons. J. Cell. Comp. Physiol. 7: 47-71. LORENTE DE No, R. 1947. A study of nerve physiology. St&es Rockefeller Inst. Med. Res. 131~132(2): 465-466. MARX, C. 1950. Mise en evidence de l’action centrale des propriocepteurs musculaires de la grenouille. Arch. Intern. Physiol. 57: 447-451. RENSHAW, B. 1940. Activity in the simplest spinal reflex pathways. J. Neurophysiol. 3: 373-387. SILVER, M. L. 1942. Motoneurons of the spinal cord of the frog. J. Camp. Neural. 77: l-39.
The
NEUROLOGY
Existence the
14, 175-186
of a Monosynaptic Spinal
K. C. HOLEMANS, Department
(1966)
of Physiology,
Cord
of the
H. S. MEIJ, of
University Received
August
Reflex
Arc
in
Frog
AND B. J. MEYERS Pretoria,
16,
Pretoria,
South
Africa
1965
Many observations on the spinal cord of the frog, reported in the literature, could satisfactorily be explained by assuming the operation of a two-neuron reflex arc, but no direct physiological evidence for the existence of such a simple nervous pathway in this species has yet been put forward. The present investigation represents a successful attempt to establish experimentally that such a reflex arc does operate in the frog. The basis for these experiments was the fact that spinal reflexes in this species can be facilitated by applying a previous conditioning stimulus to the relevant afferent nerve. Conduction time, central reflex time and synaptic delay were estimated by classical methods, and it was concluded that the response to a second stimulus following a suitable conditioning volley had the properties of a monosynaptic reflex. The points in favor of this view were: (i) A highly synchronized, high-potential discharge, such as that recorded from the ventral roots, requires the simultaneous activation of many motoneurons. This could hardly occur if the excitatory process had to pass through more than one synaptic connection. (ii) The central reflex time was very similar (difference less than 2%) to that recorded for synaptic delay. (iii) The reflex was elicited by activation of the fastest conducting, lowestthreshold afferent fibers.
Introduction The motoneuron reflex recorded from a ventral root of the spinal cord of the frog following stimulation of the corresponding dorsal root showsa prolonged (15-20 msec) asynchronous pattern of electrical activity (5, 17; see also Fig. 1.4, first response). In the cat a similar asynchronous discharge is preceded by an initial prominent peak which is transmitted through arcs of two neurons (13, 18). The existence of a two-neuron reflex arc in the frog had not been establishedeither through physiological experimentation or through histological observation. Histological studies indicate that the frog apparently for
1 We wish to express his helpful criticism
our gratitude to Professor of the manuscript. 175
F. Bremer,
University
of Brussels,
176
HOLEMANS,
MEIJ,
AND
MEYER
lacks the sensory-motor collaterals that are so prominent in the cat, for no degenerating fibers are seen in the ventral horn after section of the dorsal roots ( 12). However, numerous connections between dorsal-root terminals and dendritic expansions of motoneurons have been demonstrated in the frog (19). Bremer (3) suggested in 1942 that some of his findings on the frog’s spinal cord could be explained by assuming the operation of a two-neuron reflex arc. More recently Brookhart and Fadiga (5) mentioned several experiments (1, 9, 17) where the operation of a monosynaptic reflex arc would provide a satisfactory explanation of the results. Observations were made on the isolated spinal cord of the frog by Brookhart and Fadiga, but these furnished only indirect evidence for the existence of such a simple nervous pathway. In their studies it was not possible to elicit a typical synchronized, short-latency reflex spike in a ventral root after stimulation of a dorsal root. The present study represents a successful attempt to demonstrate the existence of a monosynaptic reflex arc in the spinal cord of the frog. Advantage was taken of the fact, discovered by Bremer (4) and applied by several investigators (10, 17), that the reflex response of the spinal cord of the frog can be facilitated by applying a suitable conditioning volley. It will be shown that the reaction to the test stimulus following the conditioning volley had the characteristics of a monosynaptic reflex discharge. Methods
After a number of preliminary experiments, the present study was undertaken with South African frogs (Xenopus 2eavis), their weights ranging from 7.5 to 125 g. Room temperature varied from 18 to 22 C in different experiments. The frogs were made spinal by transection of the cord at the level of the fourth ventricle. Laminectomy was performed on the five lower vertebrae (IV-VIII), thus exposing the well-developed three inferior dorsal and ventral nerve roots (VIII-X) together with the cord segments of entry or exit of these roots. The right dorsal roots VIII and IX, as well as ventral roots VIII, 1,X and X were distally severed. The ipsilateral sciatic nerve was exposed and severed at the distal end of the femur. Thus stimuli applied to the sciatic nerve could reach the spinal cord only through dorsal root X which was left intact. (In the frog, fibers from spinal nerves VIII, IX and X constitute the sciatic nerve.) Reflex activity was elicited by means of square-wave pulses applied
MONOSYNAPTIC
REFLEX
ARC
177
to the sciatic nerve (cathode proximal) and recorded from the ipsilateral distally severed ventral root X by a single hooked platinum electrode, its potential being pitted against ground. Intervals between reflexes (elicited by single or double stimuli) were kept constant at 3 sec. The height of the reflex spike was always expressed as the mean value of ten consecutive measurements. Synaptic delay was estimated by a method analogous to that employed by Lorente de No (14, 15) and by Renshaw (18), a brief stimulus (0.05 msec) being applied to the lateral surface of the spinal cord. Afferent conduction time was measured (18) by applying the stimulus to the sciatic nerve and recording the potential change of a cord dorsum electrode placed at the point of entry of dorsal root X. Efferent conduction time was measured from the latency of the m spike recorded for the estimation of the synaptic delay (18). An electronic stimulator (DISA Multistim) equipped with a stimulus isolation transformer was used and potential differences were displayed and eventually photographed on the screen of a Tektronix 502 double beam oscilloscope. Under our recording conditions (single electrode, recording from nerve at point of exit from volume, pitted against ground) a predominantly positive deflection could be expected (16). Nevertheless, negativity was recorded upwards. For ordinary operation the amplifier was a-c coupled, but slower waves were checked by using the same instrument in d-c coupled operation. Results
Facilitatiolt of the Spinal IZeftex Response. If a single stimulus, several times maximal for the lowest threshold fibers of the dorsal roots, was applied to the sciatic nerve or the tenth dorsal root, the reaction recorded from ventral root X was only exceptionally a synchronous spike. Usually a prolonged ( 15-25 msec) asynchronous discharge such as the first response in Fig. 1A was recorded from ventral root X. If, however, the same stimulus was preceded 5 to 250 msec by an identical stimulus, the reflex response changed to a high intensity, perfectly synchronized spike such as the second response in Fig. 1A. By varying the interval between the two stimuli, a facilitation curve such as presented in Fig. 2 could be constructed. Two peaks such as shown in Fig. 2 were observed in nineteen out of twenty facilitation curves. These peaks were observed when the intervals between the two stimuli were 5 and 25 msec, respectively, and the notch
178
HOLEMANS,
MEIJ,
AND
MEYER
between the two peaks was situated at an interval of 10 msec. These values for the twenty facilitation curves are presented in Table 1. The mean value for the interval at which the first peak occurred was 5.6 msec (5.0-7.0) and the second peak was observed at an interval of
FIG. 1. A. Upper beam: Response to two successive stimuli applied to the sciatic nerve and recorded from the ipsilateral ventral root X. Stimuli were 20 msec apart. The strength of both stimuli was maximal (1.0 v, duration 0.3 msec). The intensity of the second response is more than ten times that of the first. Time scale: 10 msec. Potential scale: 10 mv. Lower beam: Marking of the stimuli. B. Upper beam: Electrical activity recorded from ventral root X after stimulation (square wave, 0.05 msec, 4 v) of the ipsilateral side of segment X. The first spike (m spike of Lorente de No) begins with the stimulus; the interval between stimulus and second spike (S scale: 20 mv. spike of Lorente de No) is 3.9 msec. Time scale: 2 msec. Potential Lower beam: Marking of the stimulus. C. Upper beam: Potential changes of a cord dorsum electrode, above the point of entry of dorsal root X after stimulation of the sciatic nerve. Seven superimposed traces. Afferent conduction time is measured from such records. Time scale: 2 msec. Potential scale: 2 mv. Lower beam: Marking of the stimulus. D. Upper beam: Neurogram of dorsal root X with maximal (7.0 v, 0.3 msec) stimulus applied to the sciatic nerve. The alpha, beta, gamma and delta elevations described by Erlanger and Gasser can easily be recognized. Time scale: 2 msec. Potential scale: 10 mv. Lower beam: Marking of the stimulus.
MONOSYNAPTIC
REFLEX
ARC
179
24.4 msec (13-40). The mean value for the interval at which the notch between the two peaks occurred was 9.6 msec (7-15). In seventeen out of nineteen curves the second peak was higher than the first. The intensity of the recorded reflex reaction at the peaks of the facilitation curve was usually several millivolts (Table 1) , and in some cases spikes of 30 mv were recorded (Fig. 1A). The total duration of the facilitation curves varied between 80 and more than 300 msec. Not only could the reflex response in ventral root X be facilitated by applying a conditioning stimu-
INTERVAL
50 SEPARATING
FIRST AN0
100 SECOND
STIMULI
1 150 IN MSEC
c 200
FIG. 2. A typical facilitation curve of a ventral-root response elicited by a stimulus on the sciatic nerve, preceded by a conditioning stimulus of equal strength. The interval separating the conditioning and test stimulus varied between 5 and 200msec and at the highest peak of the facilitation curve the intensity of the reflex response was 6.7 mv.
lus to the same nerve as the test stimulus (sciatic nerve or dorsal root X) but facilitation was also observed although less pronounced if the conditioning stimulus was applied to ipsilateral dorsal roots VIII and IX, contralateral dorsal roots IX and X and in some cases to the brachial plexus. Although characteristics and duration of the facilitation curves differed considerably, it was always possible to elicit strong synchronized activity in a ventral root by means of this double stimulus technique. Synaptic DeZay. Figure 1B represents the electrical activity recorded in ventral root X after stimulation of the ipsilateral side of segment X. The foot of the spike (m spike of Lorente de N6; 14, 15) was synchronous with the stimulus artifact. In this particular case (Fig. IB) a second peak was registered 3.9 msec after the stimulus had been applied (s spike). The interval between the foot of the m and that of the s spike was ascribed by Lorente de N6 to the synaptic delay. The mean interval
180
HOLEMANS,
MEIJ,
AND
MEYER
in ten preparations (Table 2) was 3.03 msec. In ten additional preparations the side of segment IX was stimulated and activity recorded at a distance of 25 to 30 mm from the point of stimulation in spinal nerve IX after section of the ipsilateral dorsal roots VIII, IX and X. The m spike was thus separated from the stimulus artifact by about 0.2 msec, but the mean interval m to s remained the same. TABLE MEAN
AND
TEST STIMULI
INDIVIDUAL
VALUES
AT WHICH
No.
Interval (msec)
peak
Second
Intensity (mv)
1 14/5
5.0
2.1
2 14/5
5.0 7.0 -
3.6 0.9 -
1 20/s
5.5
1.6
2 20/5
6.0 6.0
3.2 4.5
5 .o 7.0
3.5 2.8
1 18/5 2 18/5
2b20/5 la21/5 lb21/5 2 21/j
5.5
0.4
2 24/4 la25/5
6.0 5.0
1.9 1.6
lb25/5
5.0 5.0
1 .o 0.3
lc25/5 2a25/5
Interval (msec)
CONDITIONING THEM
peak
A~-;D
OCCURRED
Notch
Intensity (mv)
Interval (msec)
Intensity
1.1
10
(mv)
2.9 6.6
30 20
1.2 2.9
15 -
2.6 0.6 -
30
1.8
10
0.5
20 13 25
3.0 3.1 6.0
10
0.6 2.0
30
2.1
40 46
1.3 2.3
10 10
40 30
3.4 1.8
25
2.6
20 15
3 .2 3.8
2.1
7.0
3.2
2c25/5 la26/5
5.0 5.0 5.0
1.7 0.7 1.6
15 30
5 .o
2.4
5.6
THE
25 25
7.0
Mean
BETWEEN
10
2b25/5
lb26/5 lc26/5
1
INTERVAL
THE Two PEAKS AND THE NOTCH BETWEEN IN TWENTY FACILITATION CURVES First
Exp.
FOR THE
7 10
3.6 1.4 0.2
8 15
1.5 0
10
0 0
10 10
1.7
10
1.3
3 .o 3.7
7 7
20
4.0
7
0.7 0.1 0.5
20
3.6
7
1.6
24.4
9.6
ABerent and Efferent Conduction Time. The afferent conduction time could be measuredby means of a cord dorsum electrode placed at the point of entry of dorsal root X (18). This electrode recorded a potential change, such as that illustrated in Fig. 1C. The latter is identical in shape and time course with the potential change recorded by a microelectrode in the dorsal horn of the frog’s spinal cord by Brookhart and Fadiga (5) and from the cord dorsum of the cat by Renshaw ( 18). Like
MONOSYNAPTIC
REFLEX
181
ARC
Renshaw we defined as afferent conduction time the interval between the stimulus and the foot of the first positive deflection. The mean and individual values for afferent conduction time obtained with ten preparations are presented in Table 2. Efferent conduction time could be estimated from the latency of the m spike in the synaptic delay records (Fig. 1B). In these records, however, the foot of the positive deflection, that is, the source of current preceding the impulse proper (16), was synchronous with the stimulus. TABLE 2 MEAN AND INDIVIIJUAL VALUES FOR TOTAL REFLEX LATENCY, CENTRAL REFLEX TIME (TOTAL REFLEX LATENCY MINUS AFFERENT CONDUCTION TEME) AND SYNAPTIC DELAY. THE FIGURES WERE RECORDED FROM TEN DIFFERENT PREPARATIONS 2
3 Latency
First reaction (msec)
Second reaction (msec)
9 10
4.5 5.5 4.0 3.2 5.0 4.0 4.2 4.4 3.8 4.0
4.2 5.5 4.0 3.2 5.0 4.0 4.2 4.4 3.8 4.2
Mean
4.26
4.25
1 Exp. No.
8
4 Afferent conduction time (msec)
S(3-4) Central reflex (msec)
6 Synaptic delay (msec)
1.4 1.7 1.2 1.1 1 .a 1 .o 1.2 1 .o 1.2 1.2
2.8 3.8 2.8 2.1 3.2 3.0 3 .o 3.4 2.6 3.0
3.2 3.9 3.0 2.0 3.5 2.6 3.0 3.4 2.7 3.0
1.28
2.97
3.03
The initiation of this positive deflection was taken as the reference point for all the measurements and the efferent conduction time, according to this technique, was therefore zero. In other words, a ventral-root electrode 6 to 8 mm from the point of exit of this root recorded positivity as soon as the corresponding motoneurons were activated. Fibers Responsible for Reflex Activity. If dorsal root X was severed near its entry into the cord and charged onto a recording electrode, the different fiber groups described by Erlanger, Bishop and Gasser (7) could easily be distinguished (Fig. 1D) and the strength-response curve for each group could be constructed (2). The strength-response curve of the fastest conducting fiber group has been compared to the strength-
182
HOLEMANS,
MEIJ,
AND
MEYFaR
response curve of the reflex response in ventral root X. The results are plotted in Fig. 3. It was found that the reflex response in ventral root X reached its maximum when the strength of the second stimulus was just maximal for the fastest conducting fibers of dorsal root X; these constitute the alpha group of Erlanger, Bishop and Gasser. In Fig. 3 a slight decrease in growth of the alpha elevation with increasing stimulus strength is seen after the stimulus reaches the value
0.1 ST%lL”S
FIG.
root
3.
Comparison
X neurogram
between (interrupted
0.4 0.3 STRENGTH
the line)
0.5
0.6
0.7
o-3
0.0
IN VOLTS
response of the alpha and the reflex response
elevation in ventral
of the dorsal root X (con-
tinuous line). Stimulus strength pIotted against response. of 0.15 v. This pattern of increase in the alpha elevation of the neurogram could be found in many of the preparations, and was ascribed by Erlanger, Bishop and Gasser (7) to the presence of a slightly faster conducting group of fibers within the alpha group. The term “pre-alpha group” was applied by these authors to the fibers in question, The stepwise increase in the reflex activity with increasing stimulus strength, seen in Fig. 3, was not always so conspicuous but could be found in most stimulus strength-reflex response curves. Fibers Responsible jar Facilitation. The fibers responsible for facilitation were also of the lowest-threshold fiber group of the dorsal roots. The relationship between the strength of the first stimulus and the reflex response to a second stimulus could be investigated when the interval between the successivestimuli was kept constant and when the second stimulus was supramaximal for reflex activity. With this arrangement it
MONOSYNAPTIC
REFLEX
ARC
183
was found that a weak first stimulus, exciting the fastest fiber group of the dorsal root only, had a maximal facilitory influence. Discussion The main object of the present study was to establish whether or not two-neuron reflex arcs exist in the spinal cord of the frog. The data obtained seem to prove the existence of such a simple nervous pathway for the reasons discussed below. Facilitation. The possibility to facilitate spinal reflexes in the frog by means of a conditioning stimulus preceding a test stimulus by 5 to 300 msec has been thoroughly studied by Bremer (4) and Kleyntjens (10). Although the technical arrangement in these early studies (stimulation of a peripheral nerve, recording of a reflex muscle contraction) was considerably different from that employed in the present study, there are no major contradictions in the findings. In Bremer’s facilitation curves the second peak of facilitation was observed when the interval between the first and second stimulus was 30 to 60 msec, and in many cases 40 to 50 msec. In the facilitation curves obtained in the present study the second peak occurred at a mean interval of 24.4 msec, which is considerably shorter than in Bremer’s experiments. It will be shown in a separate paper that this difference can be accounted for by the fact that in the present study antidromic stimulation of the ventral-root fibers has been excluded. Brevity and Intensity of the Reflex ilctivity. The highly synchronized short spike potential recorded from ventral root X after the second stimulus can only be explained by a massive, synchronous activation of the corresponding motoneurons. The spike was sustained for 2 to 3 msec and reached a potential of 30 mv in certain preparations (Fig. 1.4). Such synchrony would scarcely be possible if the excitatory process had to travel through more than one synaptic connection. Synchronism of SynaPtic Delay and Central Reflex Time. Central reflex time is equal to total reflex latency minus afferent and efferent conduction times. The total latency of the facilitated reflex responsewas identical to the latency of the unfacilitated response.The initial deflection of the unfacilitated response,therefore, could not have passedthrough more synaptic connections than the second responsebut was too small to result in a prominent spike such as that seenin the cat (13) or in the facilitated responsein the frog. Kleyntjens (10) reported a considerable reduction in the latency of the facilitated response (muscle contraction) but his
184
HOLEMANS,
MEIJ,
AND
MEYER
technical arrangement was different from that employed in the present study. The mean central reflex time of the ten preparations presented in Table 1 was 2.97 msec. Kleyntjens found a minimum delay of 3 msec between the application of a stimulus to the dorsal roots and activation of the motoneurons. Synaptic delay was found to correspond closely to central reflex time, the difference being in the order of 2%. The synaptic delay of about 3 msec is considerably longer than that found in cats (0.7-0.8 msec) with a similar technique (18). However, the total reflex latency is also considerably shorter in the cat ( 1.0-1.4 msec) than in the frog (4.25 msec in Table 1). According to Kubota and Brookhart ( 11) the most reliable estimates indicate that synaptic delay in the isolated spinal cord of frogs exceeds 1 msec. The actual times reported by these authors were 1.88 msec (5) and 1.5 to 2 msec (8). The first of these values represents the interval from the moment that the stimulus was applied to the moment of onset of the ventral-horn focal potential, and the second set of values the interval from the application of the stimulus to the onset of the EPSP recorded within the cell. The initiation of the spike was, however, delayed in Fadiga and Brookhart’s recordings (8) by about 2 msecwith regard to the onset of the EPSP. In the present study the interval measuredwas that between the moment of application of the stimulus and onset of the spike and the value of 3 msec obtained, therefore, does not contradict the findings of Brookhart and Fadiga. The possibility of studying synaptic delay by stimulating synaptic connections through an electrode in contact with the lateral surface of the cord rather than through needle electrodes inserted into the motor nucleus (14, 15, 18) deserves some comment. In the spinal cord of the frog, dendrites of the motoneurons extend into the white matter and the finer branchesof the dendrites terminate in the subpial zone of the lateral funiculus (19). Silver (19) found a lower threshold for motoneuron stimulation on the lateral surface of the cord than on either the dorsal or the ventral funiculi. He stated that the synaptic junctions of the lateral neuropil region were less than 20 lo away from the intact surface. Threshold of Responsible Fibers. The reflex responsewas elicited by activation of the lowest threshold, fastest conducting afferent fibers. This may constitute an additional argument for the presence of only one synaptic connection in the reflex arc, since in mammalsonly large afferents establish monosynaptic connections with motoneurons (6). Conclusions. The present study demonstratesthe occurrence of a monosynaptic reflex in the spinal cord of the frog. The existence of a two-
MONOSYNAPTIC
REFLEX
ARC
185
neuron reflex arc in this species had been suggested by Bremer (3) in 1942 and postulated by Fadiga and Brookhart (8) in 1960. However, the fact that it was possible to elicit strong synchronized activity in the motoneurons by means of afferent impulses travelling through the dorsal roots is at variance with the views of Brookhart and Fadiga (5). These authors ascribed the lack of synchronized activity in a ventral root following stimulation of a dorsal root to the localization of dorsal-root terminals on the dendritic expansions of the motoneurons rather than on the motoneuron soma. The data obtained in this study show that activation and synchronous spike generation in the frog’s spinal motoneuron nucleus can be induced by axodendritic synaptic connections. References 1. 2. 3.
4. 5.
6.
7.
8. 9. 10.
11. 12.
C. MATTIIEWS. 1938. The interpretation of potential changes in the spinal cord. J. Pkysiol. London 92: 276-321. BRADLEY, K., and J. C. ECCLES. 1953. Analysis of the fast afferent impulses from the thigh muscles. J. Pkysiol. London 133: 462-473. BREMER, F., and V. BONNET. 1942. Contributions a l’etude de la physiologie g&kale des centres nerveux. II. L’inhibition reflexe. Arch. Intern. Pkysiol. 52: 153-194. BREMER, F., and F. KLEYNTJENS. 1937. Nouvelles recherches sur le phenomene de la sommation centrale d’influx nerveux. Arch. Intern. Pkysiol. 45: 382-413. BROOKHART, J. M., and E. FADICA. 1960. Potential fields initiated during monosynaptic activation of frog motoneurons. J. Pkysiol. London 150: 633655. ECCLES, J. C., R. M. ECCLES, and A. LUNDBERG. 1957. Synaptic actions on motoneurons in relation to the two components of the group I muscle afferent volley. J. Pkysiol. London 136: 527-546. ERLANGER, J., G. BISHOP, and H. S. GASSER. 1926. The action potential waves transmitted between the sciatic nerve and its spinal roots. Am. J. Pkysiol. 78: 575-589. FADIGA, E., and J. M. BROOKHART. 1960. Monosynaptic activation of different portions of motor neuron membrane. Am. J. Pkysiol. 195: 693-703. FUORTES, M. G. F. 1951. Potential changes of the spinal cord following different types of afferent excitation. J. Pkysiol. London 113: 372-386. KLEYNTJENS, F. 1937. Contribution a l’etude des effets d’excitations antidromiques sur I’activitC retlexe de la grenouille spinale. I. Action d’une vollee d’influx antidromiques sur le Processus de sommation centrale. Arch. Intern. Pkysiol. 45: 415-441. KUBOTA, K., and J. M. BROOKHART. 1963. Recurrent facilitation of frog motoneurons. J. Neuvopkysiol. 26: 877-893. LIU, C. N., and W. W. CHAMBERS. 1957. Experimental study of the anatomical organization of the frog’s spinal cord. Amt. Record 127: 326. BARRON,
D. H.,
and
B. H.
186 13. 14. 15. 16. 17. 18. 19.
HOLEMANS,
MEIJ,
AND
MEYER
LLOYD, D. P. 1943. Reflex action in relation to pattern and peripheral source of afferent stimulation. J. Xeurophysiol. 6: 111-119. LORENTE DE No, R. 1939. Transmission of impulses through cranial motor nuclei. J. LVezuophysiol. 2: 402-464. LORENTE DE No, R. 1935. The electrical excitability of the motoneurons. J. Cell. Comp. Physiol. 7: 47-71. LORENTE DE No, R. 1947. A study of nerve physiology. St&es Rockefeller Inst. Med. Res. 131~132(2): 465-466. MARX, C. 1950. Mise en evidence de l’action centrale des propriocepteurs musculaires de la grenouille. Arch. Intern. Physiol. 57: 447-451. RENSHAW, B. 1940. Activity in the simplest spinal reflex pathways. J. Neurophysiol. 3: 373-387. SILVER, M. L. 1942. Motoneurons of the spinal cord of the frog. J. Camp. Neural. 77: l-39.