Brain Research, 103 (1976) 215-228 ,t;3 Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands
SENSITIZATION AND R E F L E X IN CATS
HABITUATION
OF
THE
PLANTAR
215
CUSHION
M. DAVID EGGER, JOHN W. BISHOP AND CONSTANCE H. CONE
Department O] Anatomy, Rutgers Medical School, Piscataway, N.J. 08854 (U.S.A.) (Accepted July 16th, 1975)
SUMMARY
The plantar cushion reflex in cats was examined as a model system in a mammal for the study of the effects of repeated stimulation on neural transmission. Effects of various frequencies and intensities of stimulation were similar to those seen in other reflex systems. For instance, for a fixed number of stimuli, habituation of the plantar cushion reflex was more marked at 10 Hz than at 2.0 Hz, and with 1.0 × threshold stimulation than with 5.0 × threshold stimulation. Sensitization occurred at intermediate intensities and frequencies of stimulation. Dorsal root potentials were studied; changes in dorsal root potentials during iterated stimulation did not correlate with the changes in the plantar cushion reflex. These changes in the plantar cushion reflex were also unrelated to variations in afferent transmission peripheral to the spinal cord. Sensitization and habituation in the plantar cushion reflex occurred during iterated stimulation, were produced centrally, and were unrelated to mechanisms of presynaptic inhibition.
l NTRODUCTION
Identical stimuli presented repeatedly may elicit varying reflex responses. The effects of repeated stimulation have been studied recently in a variety of invertebrate and vertebrate preparations14,17. The mammalian preparation most thoroughly studied is the hind-limb flexion reflex in the acute spinal cat12, 23 25. Detailed neuronal analysis of the flexion reflex has not proved feasible, because the locations of interneurons mediating this reflex are unknown. The plantar cushion (PC) reflex, on the other hand, is a comparatively well-analyzed reflex: it is basically trisynaptic; the locations of the first-order interneurons are known; the locations of the second-order interneurons can be inferred 6. The PC reflex can be easily elicited, with stimulation intensities low enough to excite only the largest cutaneous afferents (group II), an
216 order of magnitude or more below threshold for the hind-limb flexion reflex in anesthetized preparations. If the PC reflex does show 'plastic' changes in transmission with repeated stim u lation, it ought to be possible to analyze the firing patterns of neuronal elements responsible for changes in reflex transmission. This paper is concerned with the phenomenology of changes in PC reflex magnitudes. A preliminary report has appeared L A later paper will present a neuronal analysis of these changes ~. METHODS
The PC reflex was studied in 41 adult cats, male and female, weighing 2.4-5.9 kg, anesthetized with Nembutal, 40 mg/kg i.p. Atropine sulfate was routinely administered, 0.15 mg i.m. The cats were immobilized with Flaxedil and artificially respirated. Conventional neurophysiological methods of spinal cord exposure, electrical stimulation and recording were used. The lumbosacral spinal cord was exposed and immersed under mineral oil maintained near 36 °C with thermoservocontrolled heat lamps and a hot water bath. The first sacral ventral root (VRS1) on the left was identified, cut intraduralty near its exit, and mounted on bipolar platinum hook electrodes. In some preparations the first sacral dorsal root was also cut. In most preparations the spinal cord was severed in the lower thoracic region. When dorsal root potentials were recorded, appropriate rootlets were severed. A hind-limb was mounted rigidly with a pin driven through the ankle joint. Electrical stimuli were applied to PC through 27gauge hypodermic needles inserted into the septa separating medial and lateral lobes from the central lobe. The magnitude of the PC reflex was determined by monitoring the electrical response in VRS1. These electrical responses were recorded, both photographically and on FM tape, for later analysis. In some cases, the areas of the monophasic responses were measured planimetrically; in other cases, response magnitudes were estimated by measuring the maximum voltage of the VRS1 response. In general, the electrical stimuli delivered to PC were monophasic, rectangular pulses of 0.05 msec or 0.5 msec duration. To ensure that physiological conditions were maintained, the blood pressure of the right femoral artery was continuously monitored, and repeated small samples of arterial blood were analyzed using an IL model 113 blood gas analysis system. Arterial blood pH, Pco~ and O/o02saturation were monitored. All data presented in this paper were taken when these parameters were within normal physiological ranges 10,2'),'~. Especially for studies of sensitization and habituation, it is important that these parameters be held constant. During control studies, it was found that the magnitude of the PC response is exquisitely sensitive to changes in p H and/or Pco2 of the arterial blood. Figs. 1 and 2 illustrate the changes in reflex magnitudes of the PC reflex occurring during forced hyperventilation. Kuno and Per116 demonstrated a linear increase in the size of a monosynaptic response evoked in the cat as Pcoz - measured in expired air - - decreased from around 30 to about 10 torr; similar data
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Fig. 1. Magnitude of plantar cushion (PC) reflex in one cat as a function of arterial pH during forced hyperventilation. The magnitude of the PC reflex was arbitrarily defined as 100 corresponding to pH = 7.44 (near the upper limit of the normal range TM) on the experimentally determined linear regression line (r = 0.90).
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for the PC reflex are illustrated in Fig. 2. Kitahata et al. 15 studied dorsal-to-ventral root responses in L7 segment in the cat, finding that with hyperventilation both the monosynaptic and polysynaptic components of the reflex increased, the monosynaptic component increasing maximally to about 300 ~ of control, whereas the polysynaptic
218 component increased maximally to about 150 °/o of control. This was about the magmtude of increase shown maximally by the oligosynaptic PC reflex (Fig. 2), thot_igh u n occasion increases to 200'!i, of control were observed (Fig. 1). RESU LTS
(1) Effects of variousfrequencies of stimulation For most habituating reflex systems, for a given intensity of stimulation, habituation occurs more rapidly, i.e., with a fewer number of stimuli, the higher the frequency of stimulation 25. This was found true of the PC reflex. The typical habituation sequence was carried out as follows: the electrical threshold for the reflex was determined by gradually increasing the voltage of the stimulation pulse at l Hz. Once this threshold had been determined, the voltage was increased about three-fold. Fig. 3 shows 5 consecutive responses at 1 Hz and 3.3 < threshold. To determine the effects of iterated stimulation, the PC was stimulated for at least 3 rain at 0.1 Hz, to establish a control level. The stimulation frequency was then increased for a fixed number of stimuli, usually about 500. After the 500th stimulus, the frequency of stimulation was reduced to 0.1 Hz again, for at least 3 rain. This sequence was repeated. Each PC reflex, recorded from an S I ventral root, was photographed, and the size of the response measured. Fig. 4 illustrates changes in response magnitude in an acutely spinalized cat, lor stimuli delivered at 3.0 ~ threshold, and 2, 5, and 10 Hz. Each point corresponds to the average of 5 consecutive responses. For each sequence, the control magnitude is defined as 100, which corresponds to the average of the last 10 stimuli at 0.1 Hz preceding the higher frequency stimuli. Following the higher frequency sequence, the stimulation frequency was again reduced to 0. l Hz. Each point during the recovery portion of the graph in Fig. 4 is the average of 5 consecutive responses recorded at 0.1 Hz. Note that for each of the 3 test frequencies - - 2, 5, and 10 Hz ........ there was an initial tendency for the response magnitude to increase. This is reflex sensitization, which was most marked at 2 Hz. Both 5 and 10 Hz produced a rather rapid drop in response magnitude by 100 stimuli, though at 5 Hz there was a tendency for the response magnitude to recover, while at 10 Hz, the habituation continued to decline to below 50 ~J~of control by 500 stimuli. At 2 Hz, habituation was marginal. At 5 Hz, the response magnitude was intermediate, at about 75 ~o of control value after 520 stimuli. Complete recovery occurred in all 3 cases by 100 sec during 0. I Hz stimulation.
Fig. 3. Recording from VRS1 of 5 successive traces following stimulation of the PC at 1.0 Hz and 3.3 × threshold. Small deflections at extreme left are stimulus artifacts. Horizontal calibration is 2.0 msec. Vertical calibration is 200/iV.
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Fig. 4. PC reflex m a g n i t u d e s as percentages of control values at 3.0 x threshold during 2 ((D), 5 ( ~ ) , a n d 10 Hz ( U ) stimulation, followed by s t i m u l a t i o n at 0.1 Hz. Each point is the average of 5 consecutive responses. For further explanation, see text.
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Fig. 5. C o n d i t i o n i n g curve. T h e abscissa indicates the d u r a t i o n between two stimuli to the P C ; the ordinate, the second response percentage o f control responses. R e s p o n s e m a g n i t u d e s were d e t e r m i n e d by planimetric analysis of p h o t o g r a p h e d oscilloscope traces. E a c h point is the average of 10 traces obtained at 1.0 Hz, 3.3 x threshold. Bars indicate the range of the m e a n s of 5 s t i m u l u s pairs each, at each of the indicated intervals.
220 There was a slight tendency for the response magnitudes to increase above comrol values. These curves are typical for the PC response at these parameters, both in preparations with low thoracic spinal sections and in those with intact spinal cords. These data show the expected course of sensitization-habituation for a wellbehaved' reflex; there does not seem to be any obvious, straightforward way to explain them on the basis of a classical neurophysiological 'conditioning curve' (Fig. 5).
(2) Effects of various intensities of stimulation As the intensity of" stimulation increases, habituation becomes less marked 2:,. Fig. 6 illustrates the results from an experiment conducted similarly to that of Fig. 4, except that in this case, intensity of stimulation was varied, rather than frequency. During 2 Hz stimulation, 4 intensities were tested: 1.0, 1.5, 2.0, and 5.0 >.; threshold. For 5.0 :,~ threshold, only sensitization occurred, whereas at 1.0 ':. threshold there was no sensitization, only habituation, reducing the magnitude of the reflex to less than 40~o of control value in 500 stimuli. In another experiment, similar data were obtained, but in this case (Fig. 7), absolute values of the peak magnitudes of the responses (in /,V) are plotted. At 1.0 Hz and 5.0 >: threshold, the magnitude of the response was over 750 #V, and little or no habituation occurred. Note that at 1.5 ~: threshold, under similar conditions, the reflex declined to about 50 jg~i of its control value, but that the absolute value of the control level was only about 400 #V. In several preparations, attempts were made to compare the threshold o1' the PC reflex with that of the hind-limb flexion reflex. In all cases, the flexion reflex threshold was at least an order of magnitude greater than that of the PC reflex. For instance, in the acutely spinalized cat from which the data of Fig. 6 were taken, no flexion reflex
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Fig. 6. PC reflex magnitudes as percentages of control values c]icited at 2 Hz duringstimu]ation at 1.0 ( 0 ) , 1.5 (©), 2.0 ( × ), and 5.0 (•) × threshold, followed by stimulation at 0.1 Hz. Each point is the average of 5 consecutive responses. For further explanation, see text.
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Fig. 7. Absolute values of peak responses recorded from VRS1 following stimulation at 1.0 Hz, and 1.5 (HI), 2.0 (~') and 5.0 (O) × threshold stimulation. Each point is the average of 5 consecutive responses. Data for stimuli 51-220 omitted. could be elicited even though the stimulation voltage was increased to 33 × threshold voltage for elicitation of the PC reflex. This indicates that the intensity effects could not have been due to elicitation of a flexion reflex competing with the PC reflex at higher stimulation intensities. In preparations in which the PC reflex had been markedly habituated by repetitive stimulation, squeezing of the skin of the foot proximal to PC during continued stimulation returned the magnitude of the PC reflex toward control values (dishabituation).
(3) Location of changes in magnitude of PC reflex during repeated stimulation: peripheral versus central (a) The plantar cushion. Did any of the observed changes in effectiveness of stimulation during repeated stimulation occur at the PC itself, perhaps related to polarization of the stimulating electrodes? To test for possible effects of polarization, the general course of sensitization, habituation, and recovery was determined for bouts of stimulation with balanced, biphasic constant-current pulses of 2.4 mA, 0.5 msec duration, separated by 0.1 msec. (The constant-current pulses were obtained from ELS CCS-1A stimulators). N o r m a l sensitization-habituation patterns were observed in these cases - - as they were with monophasic constant-current pulses - -
222 indicating that polarization of electrodes during stimulation of P ( could nol h~t\e caused the observed changes in reflex magnitude. (b) Peripheral m'rl'e~. Afferent impulses resulting from stimulation of PC ~erc recorded in the lateral plantar nerve of the foot, and from the tibial nerve ill the popliteal fossa. No marked changes in the magnitudes of afferent volleys occurred at these recording sites during bouts of stimulation that produced normal sensitizationhabituation in the PC reflex. (c) Dorsal root entrl' zone. The magnitude of the afferent w)lley entering the spinal cord was monitored by mounting the intact dorsal root at the seventh lumbar (L7) level on 30-gauge platinum bipolar electrodes just proximal to the dorsal root entry zone. The graph in Fig. 8 compares magnitudes of the afferent volleys entering the spinal cord with those of the PC reflex during more than 800 stimuli at 5 Hz, 5 threshold. Note that although the PC reflex first showed marked sensitization, then marked habituation, these changes were not paralleled by changes in the magnitude of the afferent volley. The observed large-scale changes in PC reflex magnitude must have occurred centrally.
(4) Possible role of presynaptic inhibition: dorsal root potentials Stimulation of PC elicits dorsal root potentials (DRPs) that can be recorded
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Fig. 8. PC reflex magnitudes as percentages of control values during more than 800 stimuli at 5 Hz and 5 × threshold (O), compared with simultaneously recorded magnitudes of the afferent volley (Q) reaching the spinal cord at the dorsal root entry zone of L7. Each point is the average of 5 consecutive responses. The inset illustrates sample responses recorded during recovery period at 0.1 Hz. Top trace was recorded from dorsal root L7; bottom trace was recorded from ventral root SI. Small deflection at the extreme left of bottom trace is stimulus artifact. Horizontal calibration is 2.0 msec. Vertical calibration is 15/zV (top trace) and 375 FV (bottom trace).
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Fig, 9. Eight consecutive DRPs recorded from a strand of Sl dorsal root during 0.1 Hz stimulation of PC at 5 > threshold for the PC reflex. Sharp, upward deflections at left are stimulus artifacts. The large, late downward deflections correspond to the negative DR V of Lloyd and MclntyrO s. Horizontal calibration is 20 msec.]Vertical calibration is 20 HV.
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Fig. 10. Solid lines correspond to PC reflex; dotted lines to DR V of DRP recorded from a strand of SI dorsal root. Stimuli delivered at 5 Hz to PC at 3 × threshold ( 0 ) and 10 x threshold (A). Each point corresponds to the average of 5 responses, taken in alternation of recording from VRSI (PC reflex) and SI dorsal root (DRP). Ordinate indicates percentage of control values. Data for stimuli 101 400 omitted. For further explanation, see text. from dorsal rootlets n e i g h b o r i n g L7. Fig. 9 illustrates 8 consecutive D R P s recorded from a s t r a n d of dorsal root S1. PC was stimulated at 0.1 Hz, 5 x threshold for the PC reflex. D u r i n g repeated stimulation of PC at frequencies that p r o d u c e d sensitization a n d h a b i t u a t i o n of the PC reflex, the D R P s typically d e m o n s t r a t e d the following behavior : the m a g n i t u d e o f the D R P ( c o m p o n e n t V) 18 d r o p p e d very rapidly to a b o u t 40-60 ~ of its c o n t r o l value, a n d r e m a i n e d at a b o u t this level until stimulation ceased, recovering very rapidly d u r i n g 0.1 Hz stimulation. The form of the h a b i t u a t i o n curve did n o t parallel that of the PC reflex, n o r did the D R P s ever show any signs o f sensitiz-
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ation. Fig. 10 illustrates data from an acutely spinalized cat in which 5 Hz stimulation at 3 ~: threshold produced sensitization-habituation of the PC reflex, while 5 Hz stimulation at l0 threshold, unexpectedly produced habituation without preceding sensitization. However, in neither case did changes in magnitude of tile DRPs parallel, or correlate well with, the PC curves. Thus, to the extent that the DRP can be taken as a sign of presynaptic inhibition19, 20, this process does not seem to underlie, in any straightforward way, the changes seen in sensitization-habituation of the PC reflex. DISCUSSION
(1) The P C reflex Engberg 7 showed that gentle pressure on PC evokes plantar flexion of the toes, activating all the intrinsic plantar muscles as well as the flexor digitorum longus and the plantaris muscle. The motoneurons of these muscles were mainly inhibited by volleys in flexor reflex afferents, as ascertained by intracellular recording from the motoneurons. Engberg therefore suggested that the plantar muscles should be classified as physiological extensors of the limb, but Clare and Landau 2 found that some of these muscles showed features of reflex behavior that were neither consistently flexor nor extensor. One peculiarity of the muscles activated in the PC reflex is the lack of reciprocal inhibition with motoneurons of the antagonists, the toe dorsiflexors. Egger and Wall 6 showed, by intracellular recording from motoneurons participating in the PC reflex, extraceIlular recording from interneurons, as well as recording from peripheral nerves of the hind-limb, that the PC reflex is mediated by group II afferents from the PC, with a minimum of two serially connected populations of interneurons between the afferents and the motoneurons. (2) Effects o f iterated stimulation Modern neurophysiological investigation of the mechanisms of habituation and sensitization o f mammalian spinal reflexes began with the study of Spencer et al. °"'~of the flexion reflex in the acute spinal cat. At the same time, Thompson and Spencer '-'5 published a theoretical paper, reviewing the neurophysiological literature and suggesting nine parametric relations of stimulus presentation and response characteristic of habituation. These criteria have become the focus of many neurophysiological analyses of habituation since 1966, including, for example, studies of the isolated frog spinal cordS, 9. Later work has suggested that some of the 9 characteristics may be redundant 13. But these criteria, with their reformulations, have guided the work presented in this paper. The principal criteria relate to the effects of varying frequency and intensity of stimulation, viz. habituation occurs more rapidly as the frequency of stimulation increases, and less rapidly as the intensity of stimulation is increased. These criteria were fulfilled by the PC reflex. Furthermore, dishabituation or recovery toward, or beyond, control levels, occurs with a strong stimulus added to the habituating train. Spencer et al. 23 noted that dishabituation seems to be simply a superimposed facilitation, which is consistent with our observations that pinching the skin proximal to PC dishabituates the PC reflex.
225 Engberg 7 noted, and we confirmed, that the PC reflex can be elicited from stimulation of the skin proximal to PC, but at a higher threshold than PC stimulation. Groves et al. 12 analyzed parametrically the effects of stimulus frequency and intensity on habituation and sensitization of the flexor withdrawal reflex in the acute spinal cat. They observed that sensitization occurred with two distinct time courses, with 'long term' sensitization seen only at higher stimulation intensities. This is consistent with our observations on the PC reflex. Furthermore, we observed that 'long term' sensitization occurred at intermediate habituating frequencies: in Fig. 4 'short term' sensitization is seen at all 3 frequencies - - 2, 5, and 10 Hz - - but only the 5 Hz trace shows 'long term' sensitization. Especially with the frequency effects of habituation, it is necessary to compare the behavior of the reflex at high frequency with the interactions produced by two eliciting stimuli separated by various intervals. For the monosynaptic responses of biceps-semitendinosus motoneurons in the cat, Curtis and Eccles 3 were able to explain most of the behavior at higher frequencies from an analysis of a physiological 'conditioning curve'. That this is not possible in our preparation can be observed from Fig. 5, which would lead one to predict, incorrectly, that habituation of the PC reflex would proceed only slowly at 10 Hz, and probably not at all above about 5 Hz.
(3) Reflex habituation and the D R P s
A classical description of the dorsal root potential (DRP) in cats was provided by Lloyd and McIntyre is who termed the prolonged negative component of the DRP, D R V. They showed that DRP components I-III, and part of IV, were reflections of afferent activity entering the spinal cord. The remainder of IV was due to activity in interneurons. D R V appeared to result from the polarization of primary afferent fibers by current flows associated with interneuronal activation. These interpretations were confirmed and further clarified by Wall 27,zs. Subsequent work has been reviewed by Schmidtlg, 20. There is now agreement that D R V is due to the electrotonic spread of depolarization of the terminal arborizations of afferent fibers. Thus, DR V is a sign of primary afferent depolarization, presumably due to presynaptic inhibition of these afferents. Presynaptic inhibition is thought to be mediated by interneurons in the dorsal horn. Spencer et al. ~4 noted that, during habituation of the flexion reflex, the cord dorsum potential also was altered. In particular, the P wave, which corresponds to D R V of the DRP, decreased during habituating stimulation. However, they were unable to determine the time course of these changes. Thus, there was no straightforward way that the changes in the P wave could be compared to the changes in reflex magnitude. Wickelgren 29 observed that D R V of the D R P was markedly decreased following 8 min of 2 Hz stimulation. Recovery to control levels occurred within 10 sec when the DRP was recorded from a distally severed dorsal rootlet. Somjen and Lothman zl illustrate (in their Fig. 1) the rapid waning of the DRP with 3, i0 and 100 Hz stimulation. Their records are similar to ours, in that the DRPs
226
drop rapidly to a lower wilue, then maintain that reduced level more or less steadily during iterated stimulation, Thus, the rates of decline as well as the rates of reaching asymptotic levels. ~rc much more rapid lbr DR V of the DRP than for either the flexion reflex or the P ( reflex. We are unaware of any previously published detailed comparison between D R V and reflex changes, as in our Fig. 10. We could also find no prior mention in the literature relating to our observation of the failure of DRPs to demonstrate sensitiz~tion. In any case, it is abundantly clear from our data that the neuronal mechanisms responsible for changes in magnitude of the DRPs cannot be identical to those producing changes in the magnitudes of the PC reflex. Interestingly, Abrahams Lhas noted that prolonged inhibition of the PC reflex resulting from stimulation in the maj~r descending fiber systems also turned out to be unrelated to the elicitation of DRPs. Furthermore, Groves et al. ~, in a direct investigation of the excitability ol" afferent terminals during habituation and sensitization of the flexion reflex, failed to find any evidence at all of tonic presynaptic inhibition during habituation, or of tonic presynaptic facilitation during sensitization.
(4) Conclusions The PC reflex has been shown to be well-behaved according to the principal criteria of Thompson and Spencer '~5. Over a range of approximately 1.0-10 Hz, the PC reflex shows patterns of sensitization-habituation that are frequency and intensity specific. Sensitization seems to be a normal concomitant of the changing transmission process with iterated stimulation, over a wide range of stimulation parameters, excepting those at the lowest threshold and the highest frequencies. Dishabituation of the PC reflex occurs, though this is probably not different from facilitation of the reflex. By recording along the afferent pathway from the PC to the spinal cord, we demonstrated that the changes in the PC reflex magnitude during iterated stimulation do not occur in the periphery, but rather within the spinal cord itself. The sensitization-habituation changes cannot be simply explained on the basis of an inhibition-facilitation curve determined at low frequency, in contrast to the situation for a monosynaptic response 3. Furthermore, changes in DRPs with iterated stimulation appear unrelated to the changes in PC reflex transmission, strongly suggesting that presynaptic inhibition is not a basic mechanism for the changes observed. Because its neuronal reflex circuitry is fairly well established 6, the PC reflex provides an excellent model for analysis of sensitization-habituation in a mammalian preparation. ACKNOWLEDGEMENTS
Work reported in this paper was supported by the following grants to M.D.E. : N I N D S NS06297, NSF BMS 75-02312, and N1MH Research Scientist Development Award M H 11952.
227 REFERENCES 1 ABRAHAMS,V. C., On the induction of prolonged change in the functional state of the spinal cord. In J. J. BONICA (Ed.), International Synlposium on Pain, Advances in Neurology, Vol. 4, Raven Press, New York, 1974, pp. 243-251. 2 CLARE, M. H., AND LANDAU, W. M., Distal hind-limb reflex responses in cats with largely intact spinal reflex circuits, Exp. Neurol., 46 (1975) 470-495. 3 CURTIS, D. R., AND ECCLES, J. C., Synaptic action during and after repetitive stimulation, J. Physiol. (Lon¢L), 150 (1960) 374-398. 4 EGGER, m. D., ADAMS, N. R., BISHOP, J. W., AND CONE, C. H., Sensitization and habituation of the plantar cushion reflex in cats, Program and Abstracts, Societ), /or Neuroscience, 3rd A/mual Meeting, 1973, p. 256. 5 EGGER, M. D., AND CONE, C. 1-t., First-order interneurons: sensitization and habituation, Program and Abstracts, Society Jbr Neuroscience, 4th Annual Meeting, 1974, p. 199. 6 EGGER, M. D., AND WALL, P. D., The plantar cushion reflex circuit: an oligosynaptic cutaneous reflex, J. Physiol, (Lond.), 216 (1971) 483-501. 7 ENGBERG, !., Reflexes to foot muscles in the cat, Acta physiol, scand., 62, Suppl. 235 (1964) 1-64. 8 FAREL, P. B., GLANZMAN, D. L., AND THOMPSON, R. F., Habituation of a monosynaptic response in vertebrate central nervous system: lateral column-motoneuron pathway in an isolated frog spinal cord, J. Neurophysiol., 36 (1973) 1117-1130. 9 FAREL, P. B., ANt) THOMPSON, R. F., Habituation and dishabituation to dorsal root stimulation in the isolated frog spinal cord, Behav. Biol., 7 (1972) 37-45. 10 FINK, B. R., AND SCHOOt,MAY, M., Arterial blood acid-base balance in unrestrained waking cats, Fed. Proc., 21 (1962) 440. 11 GROVES, P, M., GLANZMAN, D. L., PATTERSON, M. M., AND THOMPSON, R. F., Excitability of cutaneuns afferent terminals during habituation and sensitization in acute spinal cat, Brain Research, 18 (1970) 388 392. 12 GROVES, P, M., LEE, D., AND THOMPSON, R. F., Effects of stimulus frequency and intensity on habituation and sensitization in acute spinal cat, Physiol. Behav., 4 (1969) 383 388. 13 HINDE, R. A., Behavioral habituation. In G. HORN AND R. A. HINDE (Eds.), Short-term Changes in Neural Activity and Behaviour, Cambridge University Press, Cambridge, 1970, pp. 3-40. 14 KANDEL, E. R., An invertebrate system for the cellular analysis of simple behaviors and their modifications. In F. O. SCHMITT AND F. G. WORDEN (Eds.), The Neurosciences: Third Study Program, M.I.T, Press, Cambridge, Mass., 1974, pp. 347 370. 15 KITAHATA, L. M., TAUB, A., AND SATO, I., Hyperventilation and spinal reflexes, Anesthesiology, 31 (1969) 321 326. 16 KUNO, M., AND PERL, E. R., Alteration of spinal reflexes by interaction with suprasegmental and dorsal root activity, J. Physiol. (Lond.), 151 (1960) 103-122. 17 KUPFERMANN, I,, Neurophysiology of learning, Ann. Rev. Psychol., 26 (1975) 367-391. 18 LLOYD, D. P., AND MCINTYRE, A. K., On the origins of dorsal root potentials, J. gen. Physiol., 32 (I 949) 409-443. 19 SCHMIDT, R. F., Presynaptic inhibition in the vertebrate central nervous system, Ergebn. Physiol., 63 (1971) 20-101. 20 SCHMIDT, R. F., Control of the access of afferent activity to somatosensory pathways. In A. IGGO (Ed.), Somatosensory System, Handbook of Sensory Physiology, Vol. 2, Springer, New York, 1973, pp. 151 206. 21 SOMJEN, G. G., AND LOTHMAN, E. W., Potassium, sustained focal potential shifts, and dorsal root potentials of the mammalian spinal cord, Brain Research, 69 (1974) 153-157. 22 SORENSEN, S. C., Arterial Pco2 in awake cats calculated from gas tensions in subcutaneous pockets, Respirat. Physiol., 3 (1967) 261-265. 23 SPENCER, W. A., THOMPSON, R. F., AND NE1LSON, D. R., Response decrement of the flexion reflex in the acute spinal cat and transient restoration by strong stimuli, J. Neurophysiol., 29 (1966) 221 239. 24 SPENCER, W. A., THOMPSON, R. F., AND NEILSON, D. R., Alterations in responsiveness of ascending and reflex pathways activated by iterated cutaneous afferent volleys, J. Neurophysiol., 29 (1966) 240-252. 25 THOMPSON, R. F., AND SPENCER, W. a . , Habituation: a model phenomenon for the study of neuronal substrates of behavior, Ps.vchol. Rev., 73 (1966) 16 43.
228 26 VON KOEPPEN, F., D1ECKHOI.I, I)., KANZOW, E., UND KOTSERONIS,J., Uber die CO2-Spannung im arteriellen Blur nicht-narkotisierter Katzen, Pfliigers Arck. ges. Pkysiol., 297 (1967) R34-R35. 27 WALL, P. D., Excitability changes in afferent fibre terminations and their relation to sims. pc, tentials, J. Pk)'siol. (LoAd.), 142 (1958) 1-21. 28 WALL, P. D., The origin of a spinal-cord slow potential, J. Pkysiol. (LoAd.), 164 (1962) 508 526. 29 WICKELC,REN, B. G., Habituation of spinal motoneurons, J. Neuropkysiol., 30 (1967) 1404-1423.
SENSITIZATION AND R E F L E X IN CATS
HABITUATION
OF
THE
PLANTAR
215
CUSHION
M. DAVID EGGER, JOHN W. BISHOP AND CONSTANCE H. CONE
Department O] Anatomy, Rutgers Medical School, Piscataway, N.J. 08854 (U.S.A.) (Accepted July 16th, 1975)
SUMMARY
The plantar cushion reflex in cats was examined as a model system in a mammal for the study of the effects of repeated stimulation on neural transmission. Effects of various frequencies and intensities of stimulation were similar to those seen in other reflex systems. For instance, for a fixed number of stimuli, habituation of the plantar cushion reflex was more marked at 10 Hz than at 2.0 Hz, and with 1.0 × threshold stimulation than with 5.0 × threshold stimulation. Sensitization occurred at intermediate intensities and frequencies of stimulation. Dorsal root potentials were studied; changes in dorsal root potentials during iterated stimulation did not correlate with the changes in the plantar cushion reflex. These changes in the plantar cushion reflex were also unrelated to variations in afferent transmission peripheral to the spinal cord. Sensitization and habituation in the plantar cushion reflex occurred during iterated stimulation, were produced centrally, and were unrelated to mechanisms of presynaptic inhibition.
l NTRODUCTION
Identical stimuli presented repeatedly may elicit varying reflex responses. The effects of repeated stimulation have been studied recently in a variety of invertebrate and vertebrate preparations14,17. The mammalian preparation most thoroughly studied is the hind-limb flexion reflex in the acute spinal cat12, 23 25. Detailed neuronal analysis of the flexion reflex has not proved feasible, because the locations of interneurons mediating this reflex are unknown. The plantar cushion (PC) reflex, on the other hand, is a comparatively well-analyzed reflex: it is basically trisynaptic; the locations of the first-order interneurons are known; the locations of the second-order interneurons can be inferred 6. The PC reflex can be easily elicited, with stimulation intensities low enough to excite only the largest cutaneous afferents (group II), an
216 order of magnitude or more below threshold for the hind-limb flexion reflex in anesthetized preparations. If the PC reflex does show 'plastic' changes in transmission with repeated stim u lation, it ought to be possible to analyze the firing patterns of neuronal elements responsible for changes in reflex transmission. This paper is concerned with the phenomenology of changes in PC reflex magnitudes. A preliminary report has appeared L A later paper will present a neuronal analysis of these changes ~. METHODS
The PC reflex was studied in 41 adult cats, male and female, weighing 2.4-5.9 kg, anesthetized with Nembutal, 40 mg/kg i.p. Atropine sulfate was routinely administered, 0.15 mg i.m. The cats were immobilized with Flaxedil and artificially respirated. Conventional neurophysiological methods of spinal cord exposure, electrical stimulation and recording were used. The lumbosacral spinal cord was exposed and immersed under mineral oil maintained near 36 °C with thermoservocontrolled heat lamps and a hot water bath. The first sacral ventral root (VRS1) on the left was identified, cut intraduralty near its exit, and mounted on bipolar platinum hook electrodes. In some preparations the first sacral dorsal root was also cut. In most preparations the spinal cord was severed in the lower thoracic region. When dorsal root potentials were recorded, appropriate rootlets were severed. A hind-limb was mounted rigidly with a pin driven through the ankle joint. Electrical stimuli were applied to PC through 27gauge hypodermic needles inserted into the septa separating medial and lateral lobes from the central lobe. The magnitude of the PC reflex was determined by monitoring the electrical response in VRS1. These electrical responses were recorded, both photographically and on FM tape, for later analysis. In some cases, the areas of the monophasic responses were measured planimetrically; in other cases, response magnitudes were estimated by measuring the maximum voltage of the VRS1 response. In general, the electrical stimuli delivered to PC were monophasic, rectangular pulses of 0.05 msec or 0.5 msec duration. To ensure that physiological conditions were maintained, the blood pressure of the right femoral artery was continuously monitored, and repeated small samples of arterial blood were analyzed using an IL model 113 blood gas analysis system. Arterial blood pH, Pco~ and O/o02saturation were monitored. All data presented in this paper were taken when these parameters were within normal physiological ranges 10,2'),'~. Especially for studies of sensitization and habituation, it is important that these parameters be held constant. During control studies, it was found that the magnitude of the PC response is exquisitely sensitive to changes in p H and/or Pco2 of the arterial blood. Figs. 1 and 2 illustrate the changes in reflex magnitudes of the PC reflex occurring during forced hyperventilation. Kuno and Per116 demonstrated a linear increase in the size of a monosynaptic response evoked in the cat as Pcoz - measured in expired air - - decreased from around 30 to about 10 torr; similar data
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Fig. 1. Magnitude of plantar cushion (PC) reflex in one cat as a function of arterial pH during forced hyperventilation. The magnitude of the PC reflex was arbitrarily defined as 100 corresponding to pH = 7.44 (near the upper limit of the normal range TM) on the experimentally determined linear regression line (r = 0.90).
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for the PC reflex are illustrated in Fig. 2. Kitahata et al. 15 studied dorsal-to-ventral root responses in L7 segment in the cat, finding that with hyperventilation both the monosynaptic and polysynaptic components of the reflex increased, the monosynaptic component increasing maximally to about 300 ~ of control, whereas the polysynaptic
218 component increased maximally to about 150 °/o of control. This was about the magmtude of increase shown maximally by the oligosynaptic PC reflex (Fig. 2), thot_igh u n occasion increases to 200'!i, of control were observed (Fig. 1). RESU LTS
(1) Effects of variousfrequencies of stimulation For most habituating reflex systems, for a given intensity of stimulation, habituation occurs more rapidly, i.e., with a fewer number of stimuli, the higher the frequency of stimulation 25. This was found true of the PC reflex. The typical habituation sequence was carried out as follows: the electrical threshold for the reflex was determined by gradually increasing the voltage of the stimulation pulse at l Hz. Once this threshold had been determined, the voltage was increased about three-fold. Fig. 3 shows 5 consecutive responses at 1 Hz and 3.3 < threshold. To determine the effects of iterated stimulation, the PC was stimulated for at least 3 rain at 0.1 Hz, to establish a control level. The stimulation frequency was then increased for a fixed number of stimuli, usually about 500. After the 500th stimulus, the frequency of stimulation was reduced to 0.1 Hz again, for at least 3 rain. This sequence was repeated. Each PC reflex, recorded from an S I ventral root, was photographed, and the size of the response measured. Fig. 4 illustrates changes in response magnitude in an acutely spinalized cat, lor stimuli delivered at 3.0 ~ threshold, and 2, 5, and 10 Hz. Each point corresponds to the average of 5 consecutive responses. For each sequence, the control magnitude is defined as 100, which corresponds to the average of the last 10 stimuli at 0.1 Hz preceding the higher frequency stimuli. Following the higher frequency sequence, the stimulation frequency was again reduced to 0. l Hz. Each point during the recovery portion of the graph in Fig. 4 is the average of 5 consecutive responses recorded at 0.1 Hz. Note that for each of the 3 test frequencies - - 2, 5, and 10 Hz ........ there was an initial tendency for the response magnitude to increase. This is reflex sensitization, which was most marked at 2 Hz. Both 5 and 10 Hz produced a rather rapid drop in response magnitude by 100 stimuli, though at 5 Hz there was a tendency for the response magnitude to recover, while at 10 Hz, the habituation continued to decline to below 50 ~J~of control by 500 stimuli. At 2 Hz, habituation was marginal. At 5 Hz, the response magnitude was intermediate, at about 75 ~o of control value after 520 stimuli. Complete recovery occurred in all 3 cases by 100 sec during 0. I Hz stimulation.
Fig. 3. Recording from VRS1 of 5 successive traces following stimulation of the PC at 1.0 Hz and 3.3 × threshold. Small deflections at extreme left are stimulus artifacts. Horizontal calibration is 2.0 msec. Vertical calibration is 200/iV.
219
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Fig. 4. PC reflex m a g n i t u d e s as percentages of control values at 3.0 x threshold during 2 ((D), 5 ( ~ ) , a n d 10 Hz ( U ) stimulation, followed by s t i m u l a t i o n at 0.1 Hz. Each point is the average of 5 consecutive responses. For further explanation, see text.
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Fig. 5. C o n d i t i o n i n g curve. T h e abscissa indicates the d u r a t i o n between two stimuli to the P C ; the ordinate, the second response percentage o f control responses. R e s p o n s e m a g n i t u d e s were d e t e r m i n e d by planimetric analysis of p h o t o g r a p h e d oscilloscope traces. E a c h point is the average of 10 traces obtained at 1.0 Hz, 3.3 x threshold. Bars indicate the range of the m e a n s of 5 s t i m u l u s pairs each, at each of the indicated intervals.
220 There was a slight tendency for the response magnitudes to increase above comrol values. These curves are typical for the PC response at these parameters, both in preparations with low thoracic spinal sections and in those with intact spinal cords. These data show the expected course of sensitization-habituation for a wellbehaved' reflex; there does not seem to be any obvious, straightforward way to explain them on the basis of a classical neurophysiological 'conditioning curve' (Fig. 5).
(2) Effects of various intensities of stimulation As the intensity of" stimulation increases, habituation becomes less marked 2:,. Fig. 6 illustrates the results from an experiment conducted similarly to that of Fig. 4, except that in this case, intensity of stimulation was varied, rather than frequency. During 2 Hz stimulation, 4 intensities were tested: 1.0, 1.5, 2.0, and 5.0 >.; threshold. For 5.0 :,~ threshold, only sensitization occurred, whereas at 1.0 ':. threshold there was no sensitization, only habituation, reducing the magnitude of the reflex to less than 40~o of control value in 500 stimuli. In another experiment, similar data were obtained, but in this case (Fig. 7), absolute values of the peak magnitudes of the responses (in /,V) are plotted. At 1.0 Hz and 5.0 >: threshold, the magnitude of the response was over 750 #V, and little or no habituation occurred. Note that at 1.5 ~: threshold, under similar conditions, the reflex declined to about 50 jg~i of its control value, but that the absolute value of the control level was only about 400 #V. In several preparations, attempts were made to compare the threshold o1' the PC reflex with that of the hind-limb flexion reflex. In all cases, the flexion reflex threshold was at least an order of magnitude greater than that of the PC reflex. For instance, in the acutely spinalized cat from which the data of Fig. 6 were taken, no flexion reflex
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Fig. 6. PC reflex magnitudes as percentages of control values c]icited at 2 Hz duringstimu]ation at 1.0 ( 0 ) , 1.5 (©), 2.0 ( × ), and 5.0 (•) × threshold, followed by stimulation at 0.1 Hz. Each point is the average of 5 consecutive responses. For further explanation, see text.
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Fig. 7. Absolute values of peak responses recorded from VRS1 following stimulation at 1.0 Hz, and 1.5 (HI), 2.0 (~') and 5.0 (O) × threshold stimulation. Each point is the average of 5 consecutive responses. Data for stimuli 51-220 omitted. could be elicited even though the stimulation voltage was increased to 33 × threshold voltage for elicitation of the PC reflex. This indicates that the intensity effects could not have been due to elicitation of a flexion reflex competing with the PC reflex at higher stimulation intensities. In preparations in which the PC reflex had been markedly habituated by repetitive stimulation, squeezing of the skin of the foot proximal to PC during continued stimulation returned the magnitude of the PC reflex toward control values (dishabituation).
(3) Location of changes in magnitude of PC reflex during repeated stimulation: peripheral versus central (a) The plantar cushion. Did any of the observed changes in effectiveness of stimulation during repeated stimulation occur at the PC itself, perhaps related to polarization of the stimulating electrodes? To test for possible effects of polarization, the general course of sensitization, habituation, and recovery was determined for bouts of stimulation with balanced, biphasic constant-current pulses of 2.4 mA, 0.5 msec duration, separated by 0.1 msec. (The constant-current pulses were obtained from ELS CCS-1A stimulators). N o r m a l sensitization-habituation patterns were observed in these cases - - as they were with monophasic constant-current pulses - -
222 indicating that polarization of electrodes during stimulation of P ( could nol h~t\e caused the observed changes in reflex magnitude. (b) Peripheral m'rl'e~. Afferent impulses resulting from stimulation of PC ~erc recorded in the lateral plantar nerve of the foot, and from the tibial nerve ill the popliteal fossa. No marked changes in the magnitudes of afferent volleys occurred at these recording sites during bouts of stimulation that produced normal sensitizationhabituation in the PC reflex. (c) Dorsal root entrl' zone. The magnitude of the afferent w)lley entering the spinal cord was monitored by mounting the intact dorsal root at the seventh lumbar (L7) level on 30-gauge platinum bipolar electrodes just proximal to the dorsal root entry zone. The graph in Fig. 8 compares magnitudes of the afferent volleys entering the spinal cord with those of the PC reflex during more than 800 stimuli at 5 Hz, 5 threshold. Note that although the PC reflex first showed marked sensitization, then marked habituation, these changes were not paralleled by changes in the magnitude of the afferent volley. The observed large-scale changes in PC reflex magnitude must have occurred centrally.
(4) Possible role of presynaptic inhibition: dorsal root potentials Stimulation of PC elicits dorsal root potentials (DRPs) that can be recorded
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Fig. 8. PC reflex magnitudes as percentages of control values during more than 800 stimuli at 5 Hz and 5 × threshold (O), compared with simultaneously recorded magnitudes of the afferent volley (Q) reaching the spinal cord at the dorsal root entry zone of L7. Each point is the average of 5 consecutive responses. The inset illustrates sample responses recorded during recovery period at 0.1 Hz. Top trace was recorded from dorsal root L7; bottom trace was recorded from ventral root SI. Small deflection at the extreme left of bottom trace is stimulus artifact. Horizontal calibration is 2.0 msec. Vertical calibration is 15/zV (top trace) and 375 FV (bottom trace).
223
Fig, 9. Eight consecutive DRPs recorded from a strand of Sl dorsal root during 0.1 Hz stimulation of PC at 5 > threshold for the PC reflex. Sharp, upward deflections at left are stimulus artifacts. The large, late downward deflections correspond to the negative DR V of Lloyd and MclntyrO s. Horizontal calibration is 20 msec.]Vertical calibration is 20 HV.
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Fig. 10. Solid lines correspond to PC reflex; dotted lines to DR V of DRP recorded from a strand of SI dorsal root. Stimuli delivered at 5 Hz to PC at 3 × threshold ( 0 ) and 10 x threshold (A). Each point corresponds to the average of 5 responses, taken in alternation of recording from VRSI (PC reflex) and SI dorsal root (DRP). Ordinate indicates percentage of control values. Data for stimuli 101 400 omitted. For further explanation, see text. from dorsal rootlets n e i g h b o r i n g L7. Fig. 9 illustrates 8 consecutive D R P s recorded from a s t r a n d of dorsal root S1. PC was stimulated at 0.1 Hz, 5 x threshold for the PC reflex. D u r i n g repeated stimulation of PC at frequencies that p r o d u c e d sensitization a n d h a b i t u a t i o n of the PC reflex, the D R P s typically d e m o n s t r a t e d the following behavior : the m a g n i t u d e o f the D R P ( c o m p o n e n t V) 18 d r o p p e d very rapidly to a b o u t 40-60 ~ of its c o n t r o l value, a n d r e m a i n e d at a b o u t this level until stimulation ceased, recovering very rapidly d u r i n g 0.1 Hz stimulation. The form of the h a b i t u a t i o n curve did n o t parallel that of the PC reflex, n o r did the D R P s ever show any signs o f sensitiz-
224
ation. Fig. 10 illustrates data from an acutely spinalized cat in which 5 Hz stimulation at 3 ~: threshold produced sensitization-habituation of the PC reflex, while 5 Hz stimulation at l0 threshold, unexpectedly produced habituation without preceding sensitization. However, in neither case did changes in magnitude of tile DRPs parallel, or correlate well with, the PC curves. Thus, to the extent that the DRP can be taken as a sign of presynaptic inhibition19, 20, this process does not seem to underlie, in any straightforward way, the changes seen in sensitization-habituation of the PC reflex. DISCUSSION
(1) The P C reflex Engberg 7 showed that gentle pressure on PC evokes plantar flexion of the toes, activating all the intrinsic plantar muscles as well as the flexor digitorum longus and the plantaris muscle. The motoneurons of these muscles were mainly inhibited by volleys in flexor reflex afferents, as ascertained by intracellular recording from the motoneurons. Engberg therefore suggested that the plantar muscles should be classified as physiological extensors of the limb, but Clare and Landau 2 found that some of these muscles showed features of reflex behavior that were neither consistently flexor nor extensor. One peculiarity of the muscles activated in the PC reflex is the lack of reciprocal inhibition with motoneurons of the antagonists, the toe dorsiflexors. Egger and Wall 6 showed, by intracellular recording from motoneurons participating in the PC reflex, extraceIlular recording from interneurons, as well as recording from peripheral nerves of the hind-limb, that the PC reflex is mediated by group II afferents from the PC, with a minimum of two serially connected populations of interneurons between the afferents and the motoneurons. (2) Effects o f iterated stimulation Modern neurophysiological investigation of the mechanisms of habituation and sensitization o f mammalian spinal reflexes began with the study of Spencer et al. °"'~of the flexion reflex in the acute spinal cat. At the same time, Thompson and Spencer '-'5 published a theoretical paper, reviewing the neurophysiological literature and suggesting nine parametric relations of stimulus presentation and response characteristic of habituation. These criteria have become the focus of many neurophysiological analyses of habituation since 1966, including, for example, studies of the isolated frog spinal cordS, 9. Later work has suggested that some of the 9 characteristics may be redundant 13. But these criteria, with their reformulations, have guided the work presented in this paper. The principal criteria relate to the effects of varying frequency and intensity of stimulation, viz. habituation occurs more rapidly as the frequency of stimulation increases, and less rapidly as the intensity of stimulation is increased. These criteria were fulfilled by the PC reflex. Furthermore, dishabituation or recovery toward, or beyond, control levels, occurs with a strong stimulus added to the habituating train. Spencer et al. 23 noted that dishabituation seems to be simply a superimposed facilitation, which is consistent with our observations that pinching the skin proximal to PC dishabituates the PC reflex.
225 Engberg 7 noted, and we confirmed, that the PC reflex can be elicited from stimulation of the skin proximal to PC, but at a higher threshold than PC stimulation. Groves et al. 12 analyzed parametrically the effects of stimulus frequency and intensity on habituation and sensitization of the flexor withdrawal reflex in the acute spinal cat. They observed that sensitization occurred with two distinct time courses, with 'long term' sensitization seen only at higher stimulation intensities. This is consistent with our observations on the PC reflex. Furthermore, we observed that 'long term' sensitization occurred at intermediate habituating frequencies: in Fig. 4 'short term' sensitization is seen at all 3 frequencies - - 2, 5, and 10 Hz - - but only the 5 Hz trace shows 'long term' sensitization. Especially with the frequency effects of habituation, it is necessary to compare the behavior of the reflex at high frequency with the interactions produced by two eliciting stimuli separated by various intervals. For the monosynaptic responses of biceps-semitendinosus motoneurons in the cat, Curtis and Eccles 3 were able to explain most of the behavior at higher frequencies from an analysis of a physiological 'conditioning curve'. That this is not possible in our preparation can be observed from Fig. 5, which would lead one to predict, incorrectly, that habituation of the PC reflex would proceed only slowly at 10 Hz, and probably not at all above about 5 Hz.
(3) Reflex habituation and the D R P s
A classical description of the dorsal root potential (DRP) in cats was provided by Lloyd and McIntyre is who termed the prolonged negative component of the DRP, D R V. They showed that DRP components I-III, and part of IV, were reflections of afferent activity entering the spinal cord. The remainder of IV was due to activity in interneurons. D R V appeared to result from the polarization of primary afferent fibers by current flows associated with interneuronal activation. These interpretations were confirmed and further clarified by Wall 27,zs. Subsequent work has been reviewed by Schmidtlg, 20. There is now agreement that D R V is due to the electrotonic spread of depolarization of the terminal arborizations of afferent fibers. Thus, DR V is a sign of primary afferent depolarization, presumably due to presynaptic inhibition of these afferents. Presynaptic inhibition is thought to be mediated by interneurons in the dorsal horn. Spencer et al. ~4 noted that, during habituation of the flexion reflex, the cord dorsum potential also was altered. In particular, the P wave, which corresponds to D R V of the DRP, decreased during habituating stimulation. However, they were unable to determine the time course of these changes. Thus, there was no straightforward way that the changes in the P wave could be compared to the changes in reflex magnitude. Wickelgren 29 observed that D R V of the D R P was markedly decreased following 8 min of 2 Hz stimulation. Recovery to control levels occurred within 10 sec when the DRP was recorded from a distally severed dorsal rootlet. Somjen and Lothman zl illustrate (in their Fig. 1) the rapid waning of the DRP with 3, i0 and 100 Hz stimulation. Their records are similar to ours, in that the DRPs
226
drop rapidly to a lower wilue, then maintain that reduced level more or less steadily during iterated stimulation, Thus, the rates of decline as well as the rates of reaching asymptotic levels. ~rc much more rapid lbr DR V of the DRP than for either the flexion reflex or the P ( reflex. We are unaware of any previously published detailed comparison between D R V and reflex changes, as in our Fig. 10. We could also find no prior mention in the literature relating to our observation of the failure of DRPs to demonstrate sensitiz~tion. In any case, it is abundantly clear from our data that the neuronal mechanisms responsible for changes in magnitude of the DRPs cannot be identical to those producing changes in the magnitudes of the PC reflex. Interestingly, Abrahams Lhas noted that prolonged inhibition of the PC reflex resulting from stimulation in the maj~r descending fiber systems also turned out to be unrelated to the elicitation of DRPs. Furthermore, Groves et al. ~, in a direct investigation of the excitability ol" afferent terminals during habituation and sensitization of the flexion reflex, failed to find any evidence at all of tonic presynaptic inhibition during habituation, or of tonic presynaptic facilitation during sensitization.
(4) Conclusions The PC reflex has been shown to be well-behaved according to the principal criteria of Thompson and Spencer '~5. Over a range of approximately 1.0-10 Hz, the PC reflex shows patterns of sensitization-habituation that are frequency and intensity specific. Sensitization seems to be a normal concomitant of the changing transmission process with iterated stimulation, over a wide range of stimulation parameters, excepting those at the lowest threshold and the highest frequencies. Dishabituation of the PC reflex occurs, though this is probably not different from facilitation of the reflex. By recording along the afferent pathway from the PC to the spinal cord, we demonstrated that the changes in the PC reflex magnitude during iterated stimulation do not occur in the periphery, but rather within the spinal cord itself. The sensitization-habituation changes cannot be simply explained on the basis of an inhibition-facilitation curve determined at low frequency, in contrast to the situation for a monosynaptic response 3. Furthermore, changes in DRPs with iterated stimulation appear unrelated to the changes in PC reflex transmission, strongly suggesting that presynaptic inhibition is not a basic mechanism for the changes observed. Because its neuronal reflex circuitry is fairly well established 6, the PC reflex provides an excellent model for analysis of sensitization-habituation in a mammalian preparation. ACKNOWLEDGEMENTS
Work reported in this paper was supported by the following grants to M.D.E. : N I N D S NS06297, NSF BMS 75-02312, and N1MH Research Scientist Development Award M H 11952.
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