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J. Phyriol. (1978), 279, pp. 153- 166 With 10 text-figure8 Printed in Great Britain
SENSITIZATION AND HABITUATION OF DORSAL HORN CELLS IN CATS
BY M. DAVID EGGER From the Department of Anatomy, CMDNJ-Rutgers Medical School, Piscataway, New Jersey 08854, U.S.A.
(Received 30 November 1977) SUMMARY
1. Extracellular recordings were obtained from spinal dorsal horn cells in acutely spinalized cats anaesthetized with sodium pentobarbitone. The dorsal horn cells studied responded to ipsilateral tactile stimulation of the central pad of the hind foot. Eleven short latency dorsal horn cells driven by electrical stimulation of the foot pad were studied intensively; these short latency dorsal horn cells all discharged within 1-5 msec of the arrival of an afferent volley at the dorsal root entry zone. Electrode tip sites were histologically verified to lie near the medial border of the dorsal horn in the seventh lumbar segment, in Rexed's laminae III and IV. 2. Electrical stimulation of the foot pad not only activated the dorsal horn cells studied, but also produced a reflex discharge which was monitored by recording from the ipsilateral first sacral ventral root, which had been sectioned intradurally and mounted on bipolar recording electrodes. Repeated stimulation of the foot pad at moderate intensities and frequencies (e.g. three times threshold for the ventral root response at 5 Hz) typically produced a transitory increase in the magnitude of the reflex discharge (sensitization) followed by a marked waning of the reflex magnitude (habituation). Within a few minutes following cessation of stimulation, the reflex magnitude returned to its prestimulation value. During repeated bouts of five hundred stimuli each, at frequencies from 1.0 to 10-0 Hz, and intensities 1-5-10-0 times reflex threshold, the firing pattern of a short latency dorsal horn cell was monitored along with the magnitude of the ventral root response. Changes in response patterns of the dorsal horn cells were compared to those of the reflex
discharges. 3. The short latency dorsal horn cells fell into two distinct patterns of response. The firing pattern of six dorsal horn cells paralleled the response pattern of the reflex discharge; when the reflex increased in magnitude, each of these dorsal horn cells increased in number of responses per stimulus; when the reflex discharge decreased in magnitude, each of these dorsal horn cells decreased the number of responses per stimulus. These dorsal horn cells were characterized by the following: intermediate thresholds to tactile stimulation, comparable to that of the reflex discharge itself; relatively low numbers of responses per stimulus (mean: 183/stimulus); low spontaneous activity rates (once per 10 see or less). 4. The firing patterns of the other class of short latency dorsal horn cells did not parallel the response pattern of the reflex discharge; these showed only a rather rapid,
M. D. EGGER though moderate, decrease in responses per stimulus over the entire range of intensities and frequencies tested. These five dorsal horn cells were characterized by the following: thresholds of tactile stimulation considerably below that of the reflex discharge itself; bursts of responses following each stimulation (mean: 7-1/stimulus); high spontaneous activity rates (mean: 2.4/sec). 5. It is suggested that the intermediate threshold short latency dorsal horn cells are capable of mediating the reflex discharge elicited by central pad stimulation, and that the changes in firing patterns ofthese dorsal horn cells with repeated stimulation may contribute appreciably to both the sensitization and the habituation ofthe reflex discharge. 154
INTRODUCTION
In a variety of reflex preparations, repeatedly applying the same stimulus leads first to an enhanced response (sensitization), then to a diminution in response magnitude (habituation) (e.g. Egger, Bishop & Cone, 1976; Groves, & Thompson, 1970; Thompson & Spencer, 1966). Problems of major concern for neurophysiologists studying the changes in transmission represented by sensitization and habituation are, first, the sites, and secondly, the mechanisms of these observed changes. In invertebrate preparations in which mechanisms of habituation have been studied in detail, a major site of changes in transmission has been identified as the region of the primary afferent synapse on the first central neurone in the transmission chain (Callec, Guillet, Pichon & Boistel, 1971; Kandel, Brunelli, Byrne & Castellucci, 1976; Zucker, 1972). In vertebrates, among the best-studied preparations are the isolated frog spinal cord and hind-limb flexion reflex in the acute spinal cat. In the frog, Farel, Glanzman & Thompson (1973) demonstrated monosynaptic habituation in the descending lateral column pathway to the spinal moteneurones; subsequently Farel (1974) has shown that this synaptic connexion can also show posttetanic potentiation. However, the preparation studied by Farel and his colleagues is not, strictly speaking, a reflex pathway, but rather the monosynaptic activation of motoneurones by descending lateral column fibres arising from neurones within the central nervous system. In the cat, dorsal horn neurones which show patterns of response relating to sensitization or to habituation of the hind-limb flexion reflex have been observed, but it is not clear, what, if any, relationship these interneurones might have to the transmission of the reflex. Groves and colleagues (Groves, DeMarco & Thompson, 1969; Groves & Thompson, 1970, 1973) found that interneurones displaying only habituation patterns (found in laminae I-V) were anatomically distinct from and generally of shorter latencies than those showing a sensitization-habituation pattern (found in laminae V-VII). On the basis of these related observations, Groves & Thompson (1970, 1973) proposed a 'dual-proces' model involving separate neuronal elements to explain observed changes in interneuronal and reflex firing patterns during habituation and sensitization. A major point of their model is that habituation and sensitization are independently mediated by different neuronal circuits. However, Farel's work on the frog spinal cord (Farel, 1974; Farel & Thompson, 1976) and Castellucci & Kandel's work on Aplysia (1974, 1976) have indicated that both decreases and increases in
155 SENSITIZATION A ND HABITUATION reflex transmission may be mediated by differing mechanisms operating within or upon the same synapses. Analysis of a cutaneous reflex in the cat, the plantar cushion (PC) reflex, has revealed the location of dorsal horn cells that presumably mediate this reflex (Egger & Wall, 1971): In an extensive search of the lumbosacral spinal cord, we found a distinctive population of dorsal horn cells with intermediate thresholds very similar to those of the PC reflex itself. Subsequent work demonstrated that the PC reflex is mediated by group II cutaneous afferents; that it shows classical properties of sensitization and habituation; that sensitization and habituation do not occur peripherally, but rather within the spinal cord; and that these changes are not mediated in a simple way by mechanisms related to presynaptic inhibition (Egger et al. 1976). METHODS were from dorsal horn cells in twenty adult cats, male recorded Extracellular action potentials and female, weighing 2 1-5 1 kg. The cats were anaesthetized with sodium pentobarbitone (Nembutal), 40 mg/kg i.P., supplemented when necessary. Atropine sulphate was administered routinely, 0.15 mg I.M. Before recording, the cats were immobilized by i.v. infusion of gallamine
triethiode (Flaxedil), and artificially respirated. The lumbosacral spinal cord was exposed and immersed under mineral oil maintained near 36 0C. Body temperature was maintained with a circulating hot water bath. The first sacral ventral root (VRS1) on the left was identified, cut intradurally near its exit, and mounted on bipolar platinum hook electrodes. The spinal cord was severed in the lower thoracic region. The left hind limb was mounted rigidly with a pin driven through the ankle joint. Electrical stimuli were applied to the plantar cushion (PC) through 27-gauge hypodermic needles inserted into the septa separating medial and lateral lobes from the central lobe. The electrical stimuli delivered to the PC were monophasic, rectangular pulses of 0 05 msec, delivered through an isolation transformer. To ensure that physiological conditions were maintained, the blood pressure of the right femoral artery was monitored continuously, and repeated small samples of arterial blood were analyzed using an IL Model 113 blood gas analysis system. Arterial blood pH, Pco0, and %02 saturation were monitored. All data presented in this paper were taken when these parameters were within normal physiological ranges. Especially for studies of sensitization and habituation, it is important that these parameters be held constant. The magnitude of the PC reflex response is exquisitely sensitive to changes in pH and/or Pco. of the arterial blood (Egger et al. 1976). The magnitude of the PC reflex was determined by monitoring the electrical response in VRS1. These electrical responses, amplified by a Tektronix FM 122 preamplifier, frequency range 0.8 Hz-1*0 kHz, were recorded both photographically with a Grass C4 kymograph camera and on FM tape (HP 3960 instrumentation tape recorder) for later analysis. After an experiment was completed, the response magnitudes were determined by measuring the maximum voltage of each VRS1 response from the photographic record, or by averaging several responses with a Nicolet Model 1072 signal averaging system, then reading the maximum voltage of the averaged responses digitally from the computer. Comparing maximum voltages of the monophasic responses recorded from the ventral roots led to essentially the same results as comparing response areas measured planimetrically (Egger et al. 1976; Oapek & Esplin, 1977). Recordings in the dorsal horn were made through etched tungsten metal microelectrodes, insulated with glass, according to a method modified from Merrill & Ainsworth (1972). The electrodes, with 1.0 kHz impedances of 0 45-0 MQ, were lowered into the spinal cord with a Transvertex stepping micromanipulator locked to a Transvertex spinal-cord frame. The electrodes were connected to a WP DAM-5 preamplifier, frequency range 10 Hz-10 kHz, the output of which was displayed on a Tektronix 565 oscilloscope. Cells responding to tactile stimulation of the PC were located in the previously mapped medial portion of the seventh lumbar (L7) dorsal horn (Egger & Wall, 1971). The action potentials of the dorsal horn cells studied were negative, then positive, spikes of peak-to-peak magnitudes from 0 23 to 1.6 mV. The PC was stimulated at 1.0 Hz during searching, so that neurones with
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M. D. EGGER
no resting discharge could be detected. Dorsal horn cells were distinguished from primary afferent fibres on the basis of wave forms and patterns of response. None of the dorsal horn cells studied followed stimulation frequencies above 20-40 Hz. Furthermore, although dorsal horn cells were recorded very near the medial dorsal horn-dorsal colunm border, all recording sites were found to have been in the grey matter of the dorsal horn. The following characteristics were determined for the dorsal horn cells: (1) spontaneous frequency; (2) tactile threshold and receptive field; (3) electrical threshold; (4) the number of responses per electrical stimulus to repetitive stimulation at various intensities and frequencies; (5) minimum latency. The tactile thresholds of many dorsal horn cells were semi-quantitatively measured with calibrated hairs (Model PR-11 Pressure Aesthesiometer, Research Media, Inc.). In some experiments, the firing frequencies per stimulus of the dorsal horn cells were determined by direct measurements from photographs of oscilloscope traces. In other experiments, the frequencies were determined with the output of a WP Model 120 window discriminator, displayed on an HP 1201 storage oscilloscope. A typical iterated stimulation sequence was carried out as follows: the electrical stimulation threshold for the PC reflex was first determined by gradually increasing the voltage of the pulse at 1-0 Hz. Then, iterated test sequences were conducted at 1-5, 3 0, 5-0 and 10 0 times threshold. These intensities of stimulation evoked a volley in the group II cutaneous afferents innervating the PC; they are well below thresholds that will evoke a hind-limb flexion reflex (Egger et al. 1976). At each of these intensities, the PC was stimulated for at least 3 min at 01 Hz, to establish a control level. The stimulation frequency was then increased to 1-0, 2-0, 5 0 or 10-0 Hz for 500 stimuli. After the 500th stimulus, the frequency of stimulation was again reduced to 0-1 Hz for at least 3 min. The effects of this range of intensities and frequencies of stimulation on the PC reflex under these conditions has been described (Egger et al. 1976). At the end of the recording session, a cathodal current of 20 /,A for 30 sec was passed through the electrode tip; the electrodes were withdrawn; and the spinal cord immersed in 10 % formalsaline. After soaking for at least 15 min, the lumbosacral portion of the spinal cord was excised and placed in 10% formal-saline for several days. The fixed spinal cords were then cut into 25 jsm sections, and stained with cresyl violet. The positions of the recording electrode tips were determined by measurements of the locations of the lesions from the stained sections. A total of eleven short latency dorsal horn cells, one each from eleven cats, were studied for a sufficient length of time to make possible detailed analyses of their responses to iterated stimulation. Short latency cells were defined as those with a central latency of 1-5 msec or less. The central latency was the interval between the arrival of the earliest afferent impulses at the dorsal root entry zone following electrical stimulation of the PC, and the occurrence of an action potential in the dorsal horn cell (Egger & Wall, 1971). RESULTS
In characterizing responses of short latency dorsal horn cells in the L7 segment to tactile or electrical stimulation of the ipsilateral central foot pad or plantar cushion (PC), two populations were found. One group, referred to as LT (low threshold) dorsal horn cells, responded to light touch or brushing the PC. In contrast, the IT (intermediate threshold) group typically required a tap on the PC with a probe before a response was elicited. The use of calibrated hairs to compare roughly the tactile thresholds between the two groups indicated that at least two orders of magnitude in mg-force applied to the PC separated the thresholds of the two groups of dorsal horn cells. Table 1 lists additional characteristics by which these two groups differed, based on a sample of 5 LT and 6 IT dorsal horn cells. Each of these differences is statistically significant (P < 0- 05): (1) the LT group had a higher spontaneous frequency, with a mean of 2-4 action potentials per sec, versus 0-02 per see for the IT group; (2) the threshold for responses to electrical pulses differed between the two groups. Defining as 1-0 the threshold intensity for eliciting a PC reflex monitored in VRS1, the LT dorsal horn cells had a mean electrical threshold of 0-84, versus
157 SENSITIZATION AND HABITUATION 1 45 for the IT group. Finally, (3) stimulating the PC once per see at 3 0 times threshold for the elicitation of the PC reflex evoked, on the average, 741 firings per stimulus for the LT group, vs. 1*3 for the IT group. This is a particular instance of the more general observation that, over a wide range of stimulus intensities, there was a marked tendency for the LT dorsal horn cells to fire multiply to each stimulus, TABLE 1. Characteristics of short latency dorsal horn cells driven from the plantar cushion Firings per Spontaneous stimulus Threshold Units frequency 11*0 1*0 LT-1 04 2*2 LT-2 0*8 04 7.7 1*0 1*2 LT-3 7-6 07 LT-4 5*6 7'2 07 4.5 LT-5 1.0 1.5 0.0 IT-1 1.2 0.0 2-0 IT-2 1-3 2*0 IT-3 0.0 2*5 1.0 0.0 IT-4 09 0-1 IT-5 1-4 1*1 08 0.0 IT-6
LT refers to the low threshold, IT to the intermediate threshold group of dorsal horn cells. LT dorsal horn cells responded to light brushing of the plantar cushion (PC). IT dorsal horn cells did not respond to brushing, but did respond to tap on the PC with a stiff hair or probe. Spontaneous frequency measured during repeated 10 sec intervals of recording, indicated by mean action potentials/sec. Threshold is the multiple of the PC reflex threshold to electrical stimulation; both unit and PC reflex threshold determined at 1 0 Hz stimulation frequency. Firings per stimulus is the mean number of action potentials per stimulus for ten consecutive stimuli at a frequency of 0 1 Hz. All stimulation intensities were 3 0 times threshold for the PC reflex.
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Fig. 1. The top trace is a recording from an intermediate threshold dorsal horn cell (unit IT-2); the bottom trace, the plantar cushion (PC) reflex recorded from VRS1. The PC was stimulated at 3-0 times threshold for eliciting the PC reflex. The arrow at lower left indicates the stimulus artifact. Calibration: horizontal, 2-5 msec; vertical, upper trace (negative polarity upwards) 160 ,uV, lower trace 350 REV.
M. D. EGGER while the IT dorsal horn cells characteristically fired only once or twice per stimulus. These contrasting firing patterns are illustrated in Figs. 1 and 2. On the other hand, the two groups did not differ significantly in their minimum latencies: 1 2 msec for the short latency LT dorsal horn cells and 1P0 msec for the IT group. The two groups also did not differ in the size or location of their receptive fields, which included all or part of the PC. Nor did the LT and IT cells differ in their location within the doisal horn (Fig. 3). Both groups clustered tightly at the extreme medial edge of the L7 dorsal horn, in laminae III and IV of Rexed (1954). 158
9
Fig. 2. The top trace is a recording from a low threshold dorsal horn cell (unit LT-5); the bottom trace, the PC reflex recorded from VRS1. The PC was stimulated at 3-0 times threshold for eliciting the PC reflex. The arrow at lower left indicates the stimulus artifact. Calibration: horizontal, 2-5 msec; vertical, upper trace (negative polarity upwards) 500 ,uV, lower trace 130 ,uV.
Repeated electrical stimulation of the PC at 1.0-10.0 Hz led to reversible changes in the magnitude of the PC response. An example of these changes is shown in Fig. 4, based on recordings from VRS 1. The PC was stimulated at 3 0 times threshold for eliciting a response in this ventral root. Trace 4A, which serves as a control, is the average of sixteen consecutive responses elicited at 0-1 Hz. Trace 4B is the average of the next sixteen consecutive responses, with the frequency of stimulation increased to 5.0 Hz, indicating that an increase in response magnitude, or 'sensitization', occurred early in the 5 0 Hz sequence. Trace 4C is the average of responses 81-96 elicited at 5 0 Hz, showing a decrease in response magnitude, or 'habituation', which is much more marked by responses 449-464 in the 5 0 Hz sequence (trace 4D). After 500 stimuli at 5 0 Hz, the stimulation frequency was returned to 0.1 Hz. Complete recovery (including some 'rebound' above control levels) is indicated by trace 4E, which is the average of responses 9-16 of the 0 1 Hz sequence which followed the 5.0 Hz bout of stimulation. Some combinations of stimulation intensities and frequencies produced patterns of response increases and decreases, similar to that shown in Fig. 4; others produced only habituation (Egger et al. 1976). During various combinations of intensities and frequencies of stimulation, it was possible to record the number of responses per
159 SENSITIZATION AND HABITUATION stimulus for both LT and IT dorsal horn cells. The response patterns of the two groups differed markedly. Regardless of the pattern of changes in response shown by the PC reflex as a whole, LT dorsal horn cells showed only habituation (Figs. 5, 7 and 8). On the other hand, IT dorsal horn cells exhibited response patterns that tended to
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B
Fig. 3. A, cresyl-violet stained section through middle portion of L7 segment of spinal cord. Arrow indicates site of lesion produced by electrode from which an intermediate threshold dorsal horn cell (unit IT-1) was recorded. Calibration: 300 ,um. B, drawing of dorsal horn, with lamninae II-IV indicated (after Rexed (1954), Fig. 24). The shaded area at the medial edge of laminae III and IV indicates the common locus of the eleven short latency dorsal horn cells activated by electrical stimulation of the PC.
160 M. D. EGGER follow those of the PC response as a whole (Figs. 9 and 10). The early sensitization of an IT dorsal horn cell is indicated in Fig. 6. Habituation of this neurone occurred later in the 2-0 Hz bout of stimulation. No sensitization of an LT dorsal horn cell was ever observed.
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Fig. 4. Averaged responses to PC stimulation recorded from VRS 1. The PC was stimulated at 3 0 times threshold for eliciting the PC reflex. A, responses to 0.1 Hz stimulation (n = 16). B, first sixteen responses at 5 0 Hz, immediately following responses shown in A. C, responses 81-96 of the 5-0 Hz series. D, responses 449-464 of the 5 0 Hz series. E, responses 9-16 of 0.1 Hz series immediately following the 500th stimulus of the 5.0 Hz series. Arrow at lower left indicates stimulus artifact. Calibration: horizontal, 5 0 msec; vertical, 165 iV.
For the IT dorsal horn cells, the close relationship between firings/stimulus and magnitudes of the PC reflex as a whole occurred early in the bout of 1.0-10.0 Hz stimulation, before the response magnitudes approached asymptotic values. For example, for the data shown in Fig. 9, the coefficient of determination, r2, is 0 71 during the first 96 stimuli in the two bouts of 5*0 Hz stimulation. Similarly, for the first 96 stimuli in the two bouts of stimulation shown in Fig. 10, r2 = 068.
SENSITIZATION AND HABITUATION 0
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Fig. 5. Sample sequence showing habituation in a low threshold dorsal horn cell (LT-5). Each dot represents one action potential of this unit. Each horizontal line of dots indicates the number and latency of action potentials per stimulus to the PC at 5-0 times threshold for the reflex. Each time the stimulus recurred, the response line moved upward. This Figure shows the total number of responses to the first 48 stimuli in a 5 0 Hz series. The arrow at the lower left indicates the time of occurrence of the stimuli. Calibration: 2-0 msec. 11
0 011
00 0 11
Fig. 6. Sample sequence of sensitization in an intermediate threshold dorsal horn cell (IT- 1). Representation similar to that of Fig. 5, of action potentials per stimulus to the PC at 3 0 times threshold for the reflex. The lower part of the Figure indicates responses to fourteen occurrences of the stimulus delivered at 0-1 Hz. Note that on several occasions, no responses occurred to the stimulation. The horizontal arrow indicates the beginning of a series at 2-0 Hz which produced sensitization. The 2-0 Hz stimulation occurred immediately following the 0-1 Hz series shown below the arrow. Responses to fifty-seven occurrences of the 2-0 Hz stimulation are shown. Vertical arrow at lower left indicates the time of occurrence of the stimuli. Calibration: 2-0 msec. 6
PRY 279
M. D. EGGER 162 In contrast, for the corresponding data for the LT dorsal horn cell shown in Fig. 7, r2 = 0*001; and 0 19 for the data for the LT dorsal horn cell shown in Fig. 8. DISCUSSION
Two populations of short latency dorsal horn cells were found which respond to stimulation of the PC. In general the short latency LT group was similar to low threshold tactile dorsal horn cells studied by other investigators (e.g. Armett, Gray & Palmer, 1961; Wall, 1973). The short latency IT group was qualitatively character150
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10 160 240 0 80 100 400 450 500 Time (sec) Stimuli Fig. 7. Graph indicates lack of correspondence between response patterns of a low threshold dorsal horn cell (LT-5, continuous lines) and reflex simultaneously recorded from VRS1 (interrupted lines) to stimulation of the PC. The portion of the abscissa labelled 'Stimuli' refers to responses during bouts of stimulation at 5 0 Hz. The portion of the abscissa labelled 'sec' refers to responses during recovery periods of 0 1 Hz stimulation immediately following the 5 0 Hz stimulation. The circles (0) indicate responses during stimulation at 5 0 times threshold for the PC reflex; the triangles (V), responses to stimulation at 1.5 times threshold for the reflex. The ordinate gives the percentage of firings per stimulus for the dorsal horn cell, or of the response magnitude for the PC reflex, with 100 % defined as the average observed during series of 16 stimuli delivered at 0 I Hz immediately prior to the 5.0 Hz series. Each of the points of the 'Stimuli' portion of the graph is the average of sixteen responses; each point in the 'sec' portion is the average of eight responses. 0
50
ized by Egger & Wall (1971). Both of these short latency groups appear to be members of larger groups of dorsal horn cells with central latency distributions that extend for many msec; members of both of these larger groups of dorsal horn cells were typically found during each experiment (Egger & Wall, 1971) The differential tactile sensitivities shown by these two groups may be due, at least in part, to activation by different mechanoreceptors which innervate the PC (Iggo & Ogawa, 1977; Janig, Schmidt & Zimmerman, 1968; Lynn, 1969). The LT dorsal horn cells may be activated by Pacinian corpuscles, while the IT dorsal horn cells appear to have response characteristics related to the rapidly adapting afferents.
163 SENSSITIZATION AND HABITUATION An unusual characteristic of the dorsal horn cells excited by mechanical stimulation of the PC is that none appear to belong to the spinocervical tract (Brown, 1973, p. 324).
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80 160 0 100 400 450 500 Time (sec) Stimuli Fig. 8. Response patterns of another low threshold dorsal horn cell (LT.3), illustrating lack of correspondence between response patterns of this unit and that of the PC reflex. Graph similar to Fig. 7. Continuous lines refer to the responses of the dorsal horn cell. Interrupted lines refer to responses recorded from VRS1. PC stimulated at 3 0 times threshold for the reflex bout of 500 stimuli at 5 0 Hz indicated by circles (@), or at 10 0 Hz indicated by triangles (v). 0
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Fig. 9. Graph, similar to that of Fig. 7, but illustrating the correspondence between response patterns of an intermediate threshold dorsal horn cell (IT-3, continuous lines) and reflex recorded simultaneously from VRS1 (interrupted lines) to stimulation of the PC. PC stimulated 500 times at 5 0 Hz, at 3 0 times threshold for the reflex indicated by circles (@), or at 10 0 times threshold indicated by triangles (v). 6-2
164 ,11. D. EGGER LT short latency dorsal horn cells showed only habituation following iterated stimulation, but these neurones were activated at thresholds considerably below those of the PC reflex itself, responding, for instance, to light brushing of the PC, a stimulus which never elicited the PC reflex. These LT dorsal horn cells which showed only a habituation pattern were most likely not part of the PC reflex circuit. Short latency IT units had threshold levels similar to those of the PC reflex. These IT units showed sensitization and habituation, resembling the
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Fig. 10. Response patterns of another intermediate threshold dorsal horn cell (IT-6), illustrating the correspondence between response patterns of this unit and that of the PC reflex. Graph similar to Fig. 7. Continuous lines refer to responses of the dorsal horn cell. Interrupted lines refer to responses recorded from VRS1. PC stimulated at 3-0 times threshold for the reflex, bout of 500 stimuli at 1 0 Hz indicated by circles (0), or at 5.0 Hz indicated by triangles (V).
'type S' neurones of Groves & Thompson (1970, 1973), except that the IT units were short latency neurones in laminae III and IV, rather than long latency neurones in laminae V-VII. Because the IT dorsal horn cells showed either sensitization-habituation, or habituation alone, depending on the stimulus parameters (Figs. 9 and 10), it appears that these neurones, which are thought to mediate the PC reflex, could mediate both habituation and sensitization-habituation; A 'dualprocess' model involving independent neuronal circuits is unnecessary to explain sensitization-habituation of the PC reflex. On the basis of known values for synaptic delays and e.p.s.p. rise-times, it is possible to argue that the short latency dorsal horn cells in this study were monosynaptically activated by primary afferents. In the mammalian central nervous system, synaptic delay for e.p.s.p.s is about 0 3 msec, and a delay of about 0*5 msec occurs between the onset of the e.p.s.p. and the production of an action potential (Berry & Pentreath, 1976). Thus, for the spinal cord, one would expect that the shortest latencies between
SENSITIZATION AND HABITUATION 165 arrival of an afferent volley at the dorsal root entry zone and the occurrence of an action potential from a dorsal horn cell would be about 0*8 msec. Making no additional allowance for axonal conduction times, an action potential produced by a disynaptic linkage should not occur in less than 1P6 msec following the arrival of a volley at the dorsal root entry zone. The criterion selected in this paper, of a central latency of 1-5 msec or less, should include only dorsal horn cells driven monosynaptically by primary afferents. However, the possible existence of very short-latency disynaptic responses has recently been discussed by Watt, Stauffer, Taylor, Reinking, & Stuart, (1976). It is also possible, of course, that dorsal horn cells with central delays exceeding 1.5 msec may have been monosynaptically driven. In summary, during iterated stimulation that leads to sensitization and habituation of the PC reflex, important reflex changes may occur between the arrival of the afferent volley at the dorsal root entry zone and the firing of first-order neurones. Because individual short latency IT dorsal horn cells can display both sensitization and habituation, no 'dual-process' theory involving anatomically distinct pathways is needed to explain PC reflex sensitization and habituation. Similar changes occurring between afferents and first-order neurones have been demonstrated in the marine mollusc Aplysia (Kandel et al. (1976), although these authors use 'sensitization' to refer to a slightly different phenomenon), and, for habituation alone, in the cockroach (Callec et al. 1971) and in the crayfish (Zucker, 1972). I thank John Bishop, Constance Cone, Peter Kahrilas, Irmenia Meadows, Eric Proshansky and Susan Sachs for advice and assistance. A preliminary report has been published which included some of the data presented here (Egger & Cone, 1974). This research was supported by Public Health Service Grants NS-06297 and NS-13456, National Science Foundation Grant BMS 75-02312, and General Research Support Grant 27-1917 from CMDNJ-Rutgers Medical School. REFERENCES
ARMETT, C. J., GRAY, J. A. B. & PALMER, J. F. (1961). A group of neurones in the dorsal horn associated with cutaneous mechanoreceptors. J. Physiol. 156, 611-622. BERRY, M. S. & PENTREATH, V. W. (1976). Criteria for distinguishing between monosynaptic and polysynaptic transmission. Brain Rea. 105, 1-20. BROWN, A. G. (1973). Ascending and long spinal pathways: Dorsal columns, spinocervical tract and spinothalamic tract. Handbook of Sensory Physiology, vol. ii. Somatosensory System, ed. IGGO, A., pp. 315-338. New York: Springer. CALLEc, J. J., GUILLET, J. C., PICHON, Y. & BOISTEL, J. (1971). Further studies on synaptic transmission in insects. II. Relations between sensory information and its synaptic integration at the level of a single giant axon in the cockroach. J. exp. Biol. 55, 123-149. CAPEK, R. & ESPLIN, B. (1977). Homosynaptic depression and transmitter turnover in spinal monosynaptic pathway. J. Neurophysiol. 40, 95-105. CASTELLUCCI, V. F. & KANDEL, E. R. (1974). A quantal analysis of the synaptic depression underlying habituation of the gill-withdrawal reflex in Aplysia. Proc. natn. Acad. Sci. U.S.A. 71, 5004-5008. CASTELLUCCI, V. & KANDEL, E. R. (1976). Presynaptic facilitation as a mechanism for behavioral sensitization in Aplysia. Science, N.Y. 194, 1176-1178. EGGER, M. D., BISHOP, J. W. & CONE, C. H. (1976). Sensitization and habituation of the plantar cushion reflex in cats. Brain Re8. 103, 215-228. EGGER, M. D. & CONE, C. H. (1974). First-order interneurons: sensitization and habituation. Proceedings of Fourth Annual Meeting, Society for Neuroscience, p. 199.
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EGGER, M. D. & WALL, P. D. (1971). The plantar cushion reflex circuit: an oligosynaptic cutaneous reflex. J. Phy8iol. 216, 483-501. FAREL, P. B. (1974). Dual processes control response habituation across a single synapse. Brain Re8. 72, 323-327. FAREL, P. B., GLANZMAN, D. L. & THOMPSON, R. F. (1973). Habituation of a monosynaptic response in vertebrate central nervous system: lateral column-motoneuron pathway in isolated frog spinal cord. J. Neurophy8iol. 36, 1117-1130. FAREL, P. B. & THOMPSON, R. F. (1976). Habituation of a monosynaptic response in frog spinal cord: evidence for a presynaptic mechanism. J. Neurophy8iol. 39, 661-666. GROVES, P. M., DEMARCO, R. & THOMPSON, R. F. (1969). Habituation and sensitization of spinal interneuron activity in acute spinal cat. Brain Res. 14, 521- 525. GROVES, P. M. & THOMPSON, R. F. (1970). Habituation: a dual-process theory. P8ychol. Rev. 77, 419-450. GROvES, P. M. & THOMPSON, R. F. (1973). A dual-process theory of habituation: neural mechanisms. Habituation, vol. II, Phy8iological Substrate8, ed. PEEKE, H. V. S. & HERZ, M. J., pp. 175-205. New York: Academic. IGGo, A. & OGAWA, H. (1977). Correlative physiological and morphological studies of rapidly adapting mechanoreceptors in cat's glabrous skin. J. Physiol. 266, 275-296. JANIG, W., SCHMIDT, R. F. & ZIMMERMAN, M. (1968). Single unit responses and the total afferent outflow from the cat's foot pad upon mechanical stimulation. Expl Brain Re8. 6, 100-115. KANDEL, E. R., BRUNELLI, M., BYRNE, J. & CASTELLUCCI, V. (1975). A common presynaptic locus for the synaptic changes underlying short-term habituation and sensitization of the gillwithdrawal reflex in Aply8ia. Cold Spring Harb. Symp. quant. Biol. XL, 465-482. LYNN, B. (1969). The nature and location of certain phasic mechanoreceptors in the cat's foot. J. Physiol. 201, 765-773. MERRILL, E. G. & AINSWORTH, A. (1972). Glass-coated platinum-plated tungsten microelectrodes. Med. biol. Engng 10, 662-672. REXED, B. (1954). A cytoarchitectonic atlas of the spinal cord in the cat. J. comp. Neurol. 100, 297-379. THOMPSON, R. F. & SPENCER, W. A. (1966). Habituation: a model phenomenon for the study of neuronal substrates of behavior. P8ychol. Rev. 73, 16-43. WALL, P. D. (1973). Dorsal horn electrophysiology. Handbook of Sen8ory Phy8iology, vol. II. Somato8en8ory Sy8tem, ed. Ioo, A., pp. 253-270. New York: Springer. WATT, D. G. D., STAUFFER, E. K., TAYLOR, A., REINKING, R. M. & STUART, D. G. (1976). Analysis of muscle receptor connections of spike-triggered averaging. 1. Spindle primary and tendon organ afferents. J. Neurophysiol. 39, 1375-1392. ZUCKER, R- S. (1972). Crayfish escape behavior and central synapses. 11. Physiological mechanisms underlying behavioral habituation. J. Neurophysiol. 35, 621-637.
J. Phyriol. (1978), 279, pp. 153- 166 With 10 text-figure8 Printed in Great Britain
SENSITIZATION AND HABITUATION OF DORSAL HORN CELLS IN CATS
BY M. DAVID EGGER From the Department of Anatomy, CMDNJ-Rutgers Medical School, Piscataway, New Jersey 08854, U.S.A.
(Received 30 November 1977) SUMMARY
1. Extracellular recordings were obtained from spinal dorsal horn cells in acutely spinalized cats anaesthetized with sodium pentobarbitone. The dorsal horn cells studied responded to ipsilateral tactile stimulation of the central pad of the hind foot. Eleven short latency dorsal horn cells driven by electrical stimulation of the foot pad were studied intensively; these short latency dorsal horn cells all discharged within 1-5 msec of the arrival of an afferent volley at the dorsal root entry zone. Electrode tip sites were histologically verified to lie near the medial border of the dorsal horn in the seventh lumbar segment, in Rexed's laminae III and IV. 2. Electrical stimulation of the foot pad not only activated the dorsal horn cells studied, but also produced a reflex discharge which was monitored by recording from the ipsilateral first sacral ventral root, which had been sectioned intradurally and mounted on bipolar recording electrodes. Repeated stimulation of the foot pad at moderate intensities and frequencies (e.g. three times threshold for the ventral root response at 5 Hz) typically produced a transitory increase in the magnitude of the reflex discharge (sensitization) followed by a marked waning of the reflex magnitude (habituation). Within a few minutes following cessation of stimulation, the reflex magnitude returned to its prestimulation value. During repeated bouts of five hundred stimuli each, at frequencies from 1.0 to 10-0 Hz, and intensities 1-5-10-0 times reflex threshold, the firing pattern of a short latency dorsal horn cell was monitored along with the magnitude of the ventral root response. Changes in response patterns of the dorsal horn cells were compared to those of the reflex
discharges. 3. The short latency dorsal horn cells fell into two distinct patterns of response. The firing pattern of six dorsal horn cells paralleled the response pattern of the reflex discharge; when the reflex increased in magnitude, each of these dorsal horn cells increased in number of responses per stimulus; when the reflex discharge decreased in magnitude, each of these dorsal horn cells decreased the number of responses per stimulus. These dorsal horn cells were characterized by the following: intermediate thresholds to tactile stimulation, comparable to that of the reflex discharge itself; relatively low numbers of responses per stimulus (mean: 183/stimulus); low spontaneous activity rates (once per 10 see or less). 4. The firing patterns of the other class of short latency dorsal horn cells did not parallel the response pattern of the reflex discharge; these showed only a rather rapid,
M. D. EGGER though moderate, decrease in responses per stimulus over the entire range of intensities and frequencies tested. These five dorsal horn cells were characterized by the following: thresholds of tactile stimulation considerably below that of the reflex discharge itself; bursts of responses following each stimulation (mean: 7-1/stimulus); high spontaneous activity rates (mean: 2.4/sec). 5. It is suggested that the intermediate threshold short latency dorsal horn cells are capable of mediating the reflex discharge elicited by central pad stimulation, and that the changes in firing patterns ofthese dorsal horn cells with repeated stimulation may contribute appreciably to both the sensitization and the habituation ofthe reflex discharge. 154
INTRODUCTION
In a variety of reflex preparations, repeatedly applying the same stimulus leads first to an enhanced response (sensitization), then to a diminution in response magnitude (habituation) (e.g. Egger, Bishop & Cone, 1976; Groves, & Thompson, 1970; Thompson & Spencer, 1966). Problems of major concern for neurophysiologists studying the changes in transmission represented by sensitization and habituation are, first, the sites, and secondly, the mechanisms of these observed changes. In invertebrate preparations in which mechanisms of habituation have been studied in detail, a major site of changes in transmission has been identified as the region of the primary afferent synapse on the first central neurone in the transmission chain (Callec, Guillet, Pichon & Boistel, 1971; Kandel, Brunelli, Byrne & Castellucci, 1976; Zucker, 1972). In vertebrates, among the best-studied preparations are the isolated frog spinal cord and hind-limb flexion reflex in the acute spinal cat. In the frog, Farel, Glanzman & Thompson (1973) demonstrated monosynaptic habituation in the descending lateral column pathway to the spinal moteneurones; subsequently Farel (1974) has shown that this synaptic connexion can also show posttetanic potentiation. However, the preparation studied by Farel and his colleagues is not, strictly speaking, a reflex pathway, but rather the monosynaptic activation of motoneurones by descending lateral column fibres arising from neurones within the central nervous system. In the cat, dorsal horn neurones which show patterns of response relating to sensitization or to habituation of the hind-limb flexion reflex have been observed, but it is not clear, what, if any, relationship these interneurones might have to the transmission of the reflex. Groves and colleagues (Groves, DeMarco & Thompson, 1969; Groves & Thompson, 1970, 1973) found that interneurones displaying only habituation patterns (found in laminae I-V) were anatomically distinct from and generally of shorter latencies than those showing a sensitization-habituation pattern (found in laminae V-VII). On the basis of these related observations, Groves & Thompson (1970, 1973) proposed a 'dual-proces' model involving separate neuronal elements to explain observed changes in interneuronal and reflex firing patterns during habituation and sensitization. A major point of their model is that habituation and sensitization are independently mediated by different neuronal circuits. However, Farel's work on the frog spinal cord (Farel, 1974; Farel & Thompson, 1976) and Castellucci & Kandel's work on Aplysia (1974, 1976) have indicated that both decreases and increases in
155 SENSITIZATION A ND HABITUATION reflex transmission may be mediated by differing mechanisms operating within or upon the same synapses. Analysis of a cutaneous reflex in the cat, the plantar cushion (PC) reflex, has revealed the location of dorsal horn cells that presumably mediate this reflex (Egger & Wall, 1971): In an extensive search of the lumbosacral spinal cord, we found a distinctive population of dorsal horn cells with intermediate thresholds very similar to those of the PC reflex itself. Subsequent work demonstrated that the PC reflex is mediated by group II cutaneous afferents; that it shows classical properties of sensitization and habituation; that sensitization and habituation do not occur peripherally, but rather within the spinal cord; and that these changes are not mediated in a simple way by mechanisms related to presynaptic inhibition (Egger et al. 1976). METHODS were from dorsal horn cells in twenty adult cats, male recorded Extracellular action potentials and female, weighing 2 1-5 1 kg. The cats were anaesthetized with sodium pentobarbitone (Nembutal), 40 mg/kg i.P., supplemented when necessary. Atropine sulphate was administered routinely, 0.15 mg I.M. Before recording, the cats were immobilized by i.v. infusion of gallamine
triethiode (Flaxedil), and artificially respirated. The lumbosacral spinal cord was exposed and immersed under mineral oil maintained near 36 0C. Body temperature was maintained with a circulating hot water bath. The first sacral ventral root (VRS1) on the left was identified, cut intradurally near its exit, and mounted on bipolar platinum hook electrodes. The spinal cord was severed in the lower thoracic region. The left hind limb was mounted rigidly with a pin driven through the ankle joint. Electrical stimuli were applied to the plantar cushion (PC) through 27-gauge hypodermic needles inserted into the septa separating medial and lateral lobes from the central lobe. The electrical stimuli delivered to the PC were monophasic, rectangular pulses of 0 05 msec, delivered through an isolation transformer. To ensure that physiological conditions were maintained, the blood pressure of the right femoral artery was monitored continuously, and repeated small samples of arterial blood were analyzed using an IL Model 113 blood gas analysis system. Arterial blood pH, Pco0, and %02 saturation were monitored. All data presented in this paper were taken when these parameters were within normal physiological ranges. Especially for studies of sensitization and habituation, it is important that these parameters be held constant. The magnitude of the PC reflex response is exquisitely sensitive to changes in pH and/or Pco. of the arterial blood (Egger et al. 1976). The magnitude of the PC reflex was determined by monitoring the electrical response in VRS1. These electrical responses, amplified by a Tektronix FM 122 preamplifier, frequency range 0.8 Hz-1*0 kHz, were recorded both photographically with a Grass C4 kymograph camera and on FM tape (HP 3960 instrumentation tape recorder) for later analysis. After an experiment was completed, the response magnitudes were determined by measuring the maximum voltage of each VRS1 response from the photographic record, or by averaging several responses with a Nicolet Model 1072 signal averaging system, then reading the maximum voltage of the averaged responses digitally from the computer. Comparing maximum voltages of the monophasic responses recorded from the ventral roots led to essentially the same results as comparing response areas measured planimetrically (Egger et al. 1976; Oapek & Esplin, 1977). Recordings in the dorsal horn were made through etched tungsten metal microelectrodes, insulated with glass, according to a method modified from Merrill & Ainsworth (1972). The electrodes, with 1.0 kHz impedances of 0 45-0 MQ, were lowered into the spinal cord with a Transvertex stepping micromanipulator locked to a Transvertex spinal-cord frame. The electrodes were connected to a WP DAM-5 preamplifier, frequency range 10 Hz-10 kHz, the output of which was displayed on a Tektronix 565 oscilloscope. Cells responding to tactile stimulation of the PC were located in the previously mapped medial portion of the seventh lumbar (L7) dorsal horn (Egger & Wall, 1971). The action potentials of the dorsal horn cells studied were negative, then positive, spikes of peak-to-peak magnitudes from 0 23 to 1.6 mV. The PC was stimulated at 1.0 Hz during searching, so that neurones with
156
M. D. EGGER
no resting discharge could be detected. Dorsal horn cells were distinguished from primary afferent fibres on the basis of wave forms and patterns of response. None of the dorsal horn cells studied followed stimulation frequencies above 20-40 Hz. Furthermore, although dorsal horn cells were recorded very near the medial dorsal horn-dorsal colunm border, all recording sites were found to have been in the grey matter of the dorsal horn. The following characteristics were determined for the dorsal horn cells: (1) spontaneous frequency; (2) tactile threshold and receptive field; (3) electrical threshold; (4) the number of responses per electrical stimulus to repetitive stimulation at various intensities and frequencies; (5) minimum latency. The tactile thresholds of many dorsal horn cells were semi-quantitatively measured with calibrated hairs (Model PR-11 Pressure Aesthesiometer, Research Media, Inc.). In some experiments, the firing frequencies per stimulus of the dorsal horn cells were determined by direct measurements from photographs of oscilloscope traces. In other experiments, the frequencies were determined with the output of a WP Model 120 window discriminator, displayed on an HP 1201 storage oscilloscope. A typical iterated stimulation sequence was carried out as follows: the electrical stimulation threshold for the PC reflex was first determined by gradually increasing the voltage of the pulse at 1-0 Hz. Then, iterated test sequences were conducted at 1-5, 3 0, 5-0 and 10 0 times threshold. These intensities of stimulation evoked a volley in the group II cutaneous afferents innervating the PC; they are well below thresholds that will evoke a hind-limb flexion reflex (Egger et al. 1976). At each of these intensities, the PC was stimulated for at least 3 min at 01 Hz, to establish a control level. The stimulation frequency was then increased to 1-0, 2-0, 5 0 or 10-0 Hz for 500 stimuli. After the 500th stimulus, the frequency of stimulation was again reduced to 0-1 Hz for at least 3 min. The effects of this range of intensities and frequencies of stimulation on the PC reflex under these conditions has been described (Egger et al. 1976). At the end of the recording session, a cathodal current of 20 /,A for 30 sec was passed through the electrode tip; the electrodes were withdrawn; and the spinal cord immersed in 10 % formalsaline. After soaking for at least 15 min, the lumbosacral portion of the spinal cord was excised and placed in 10% formal-saline for several days. The fixed spinal cords were then cut into 25 jsm sections, and stained with cresyl violet. The positions of the recording electrode tips were determined by measurements of the locations of the lesions from the stained sections. A total of eleven short latency dorsal horn cells, one each from eleven cats, were studied for a sufficient length of time to make possible detailed analyses of their responses to iterated stimulation. Short latency cells were defined as those with a central latency of 1-5 msec or less. The central latency was the interval between the arrival of the earliest afferent impulses at the dorsal root entry zone following electrical stimulation of the PC, and the occurrence of an action potential in the dorsal horn cell (Egger & Wall, 1971). RESULTS
In characterizing responses of short latency dorsal horn cells in the L7 segment to tactile or electrical stimulation of the ipsilateral central foot pad or plantar cushion (PC), two populations were found. One group, referred to as LT (low threshold) dorsal horn cells, responded to light touch or brushing the PC. In contrast, the IT (intermediate threshold) group typically required a tap on the PC with a probe before a response was elicited. The use of calibrated hairs to compare roughly the tactile thresholds between the two groups indicated that at least two orders of magnitude in mg-force applied to the PC separated the thresholds of the two groups of dorsal horn cells. Table 1 lists additional characteristics by which these two groups differed, based on a sample of 5 LT and 6 IT dorsal horn cells. Each of these differences is statistically significant (P < 0- 05): (1) the LT group had a higher spontaneous frequency, with a mean of 2-4 action potentials per sec, versus 0-02 per see for the IT group; (2) the threshold for responses to electrical pulses differed between the two groups. Defining as 1-0 the threshold intensity for eliciting a PC reflex monitored in VRS1, the LT dorsal horn cells had a mean electrical threshold of 0-84, versus
157 SENSITIZATION AND HABITUATION 1 45 for the IT group. Finally, (3) stimulating the PC once per see at 3 0 times threshold for the elicitation of the PC reflex evoked, on the average, 741 firings per stimulus for the LT group, vs. 1*3 for the IT group. This is a particular instance of the more general observation that, over a wide range of stimulus intensities, there was a marked tendency for the LT dorsal horn cells to fire multiply to each stimulus, TABLE 1. Characteristics of short latency dorsal horn cells driven from the plantar cushion Firings per Spontaneous stimulus Threshold Units frequency 11*0 1*0 LT-1 04 2*2 LT-2 0*8 04 7.7 1*0 1*2 LT-3 7-6 07 LT-4 5*6 7'2 07 4.5 LT-5 1.0 1.5 0.0 IT-1 1.2 0.0 2-0 IT-2 1-3 2*0 IT-3 0.0 2*5 1.0 0.0 IT-4 09 0-1 IT-5 1-4 1*1 08 0.0 IT-6
LT refers to the low threshold, IT to the intermediate threshold group of dorsal horn cells. LT dorsal horn cells responded to light brushing of the plantar cushion (PC). IT dorsal horn cells did not respond to brushing, but did respond to tap on the PC with a stiff hair or probe. Spontaneous frequency measured during repeated 10 sec intervals of recording, indicated by mean action potentials/sec. Threshold is the multiple of the PC reflex threshold to electrical stimulation; both unit and PC reflex threshold determined at 1 0 Hz stimulation frequency. Firings per stimulus is the mean number of action potentials per stimulus for ten consecutive stimuli at a frequency of 0 1 Hz. All stimulation intensities were 3 0 times threshold for the PC reflex.
4>'
s
Fig. 1. The top trace is a recording from an intermediate threshold dorsal horn cell (unit IT-2); the bottom trace, the plantar cushion (PC) reflex recorded from VRS1. The PC was stimulated at 3-0 times threshold for eliciting the PC reflex. The arrow at lower left indicates the stimulus artifact. Calibration: horizontal, 2-5 msec; vertical, upper trace (negative polarity upwards) 160 ,uV, lower trace 350 REV.
M. D. EGGER while the IT dorsal horn cells characteristically fired only once or twice per stimulus. These contrasting firing patterns are illustrated in Figs. 1 and 2. On the other hand, the two groups did not differ significantly in their minimum latencies: 1 2 msec for the short latency LT dorsal horn cells and 1P0 msec for the IT group. The two groups also did not differ in the size or location of their receptive fields, which included all or part of the PC. Nor did the LT and IT cells differ in their location within the doisal horn (Fig. 3). Both groups clustered tightly at the extreme medial edge of the L7 dorsal horn, in laminae III and IV of Rexed (1954). 158
9
Fig. 2. The top trace is a recording from a low threshold dorsal horn cell (unit LT-5); the bottom trace, the PC reflex recorded from VRS1. The PC was stimulated at 3-0 times threshold for eliciting the PC reflex. The arrow at lower left indicates the stimulus artifact. Calibration: horizontal, 2-5 msec; vertical, upper trace (negative polarity upwards) 500 ,uV, lower trace 130 ,uV.
Repeated electrical stimulation of the PC at 1.0-10.0 Hz led to reversible changes in the magnitude of the PC response. An example of these changes is shown in Fig. 4, based on recordings from VRS 1. The PC was stimulated at 3 0 times threshold for eliciting a response in this ventral root. Trace 4A, which serves as a control, is the average of sixteen consecutive responses elicited at 0-1 Hz. Trace 4B is the average of the next sixteen consecutive responses, with the frequency of stimulation increased to 5.0 Hz, indicating that an increase in response magnitude, or 'sensitization', occurred early in the 5 0 Hz sequence. Trace 4C is the average of responses 81-96 elicited at 5 0 Hz, showing a decrease in response magnitude, or 'habituation', which is much more marked by responses 449-464 in the 5 0 Hz sequence (trace 4D). After 500 stimuli at 5 0 Hz, the stimulation frequency was returned to 0.1 Hz. Complete recovery (including some 'rebound' above control levels) is indicated by trace 4E, which is the average of responses 9-16 of the 0 1 Hz sequence which followed the 5.0 Hz bout of stimulation. Some combinations of stimulation intensities and frequencies produced patterns of response increases and decreases, similar to that shown in Fig. 4; others produced only habituation (Egger et al. 1976). During various combinations of intensities and frequencies of stimulation, it was possible to record the number of responses per
159 SENSITIZATION AND HABITUATION stimulus for both LT and IT dorsal horn cells. The response patterns of the two groups differed markedly. Regardless of the pattern of changes in response shown by the PC reflex as a whole, LT dorsal horn cells showed only habituation (Figs. 5, 7 and 8). On the other hand, IT dorsal horn cells exhibited response patterns that tended to
A
:q'' :....:
B
Fig. 3. A, cresyl-violet stained section through middle portion of L7 segment of spinal cord. Arrow indicates site of lesion produced by electrode from which an intermediate threshold dorsal horn cell (unit IT-1) was recorded. Calibration: 300 ,um. B, drawing of dorsal horn, with lamninae II-IV indicated (after Rexed (1954), Fig. 24). The shaded area at the medial edge of laminae III and IV indicates the common locus of the eleven short latency dorsal horn cells activated by electrical stimulation of the PC.
160 M. D. EGGER follow those of the PC response as a whole (Figs. 9 and 10). The early sensitization of an IT dorsal horn cell is indicated in Fig. 6. Habituation of this neurone occurred later in the 2-0 Hz bout of stimulation. No sensitization of an LT dorsal horn cell was ever observed.
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Fig. 4. Averaged responses to PC stimulation recorded from VRS 1. The PC was stimulated at 3 0 times threshold for eliciting the PC reflex. A, responses to 0.1 Hz stimulation (n = 16). B, first sixteen responses at 5 0 Hz, immediately following responses shown in A. C, responses 81-96 of the 5-0 Hz series. D, responses 449-464 of the 5 0 Hz series. E, responses 9-16 of 0.1 Hz series immediately following the 500th stimulus of the 5.0 Hz series. Arrow at lower left indicates stimulus artifact. Calibration: horizontal, 5 0 msec; vertical, 165 iV.
For the IT dorsal horn cells, the close relationship between firings/stimulus and magnitudes of the PC reflex as a whole occurred early in the bout of 1.0-10.0 Hz stimulation, before the response magnitudes approached asymptotic values. For example, for the data shown in Fig. 9, the coefficient of determination, r2, is 0 71 during the first 96 stimuli in the two bouts of 5*0 Hz stimulation. Similarly, for the first 96 stimuli in the two bouts of stimulation shown in Fig. 10, r2 = 068.
SENSITIZATION AND HABITUATION 0
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Fig. 5. Sample sequence showing habituation in a low threshold dorsal horn cell (LT-5). Each dot represents one action potential of this unit. Each horizontal line of dots indicates the number and latency of action potentials per stimulus to the PC at 5-0 times threshold for the reflex. Each time the stimulus recurred, the response line moved upward. This Figure shows the total number of responses to the first 48 stimuli in a 5 0 Hz series. The arrow at the lower left indicates the time of occurrence of the stimuli. Calibration: 2-0 msec. 11
0 011
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Fig. 6. Sample sequence of sensitization in an intermediate threshold dorsal horn cell (IT- 1). Representation similar to that of Fig. 5, of action potentials per stimulus to the PC at 3 0 times threshold for the reflex. The lower part of the Figure indicates responses to fourteen occurrences of the stimulus delivered at 0-1 Hz. Note that on several occasions, no responses occurred to the stimulation. The horizontal arrow indicates the beginning of a series at 2-0 Hz which produced sensitization. The 2-0 Hz stimulation occurred immediately following the 0-1 Hz series shown below the arrow. Responses to fifty-seven occurrences of the 2-0 Hz stimulation are shown. Vertical arrow at lower left indicates the time of occurrence of the stimuli. Calibration: 2-0 msec. 6
PRY 279
M. D. EGGER 162 In contrast, for the corresponding data for the LT dorsal horn cell shown in Fig. 7, r2 = 0*001; and 0 19 for the data for the LT dorsal horn cell shown in Fig. 8. DISCUSSION
Two populations of short latency dorsal horn cells were found which respond to stimulation of the PC. In general the short latency LT group was similar to low threshold tactile dorsal horn cells studied by other investigators (e.g. Armett, Gray & Palmer, 1961; Wall, 1973). The short latency IT group was qualitatively character150
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.
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10 160 240 0 80 100 400 450 500 Time (sec) Stimuli Fig. 7. Graph indicates lack of correspondence between response patterns of a low threshold dorsal horn cell (LT-5, continuous lines) and reflex simultaneously recorded from VRS1 (interrupted lines) to stimulation of the PC. The portion of the abscissa labelled 'Stimuli' refers to responses during bouts of stimulation at 5 0 Hz. The portion of the abscissa labelled 'sec' refers to responses during recovery periods of 0 1 Hz stimulation immediately following the 5 0 Hz stimulation. The circles (0) indicate responses during stimulation at 5 0 times threshold for the PC reflex; the triangles (V), responses to stimulation at 1.5 times threshold for the reflex. The ordinate gives the percentage of firings per stimulus for the dorsal horn cell, or of the response magnitude for the PC reflex, with 100 % defined as the average observed during series of 16 stimuli delivered at 0 I Hz immediately prior to the 5.0 Hz series. Each of the points of the 'Stimuli' portion of the graph is the average of sixteen responses; each point in the 'sec' portion is the average of eight responses. 0
50
ized by Egger & Wall (1971). Both of these short latency groups appear to be members of larger groups of dorsal horn cells with central latency distributions that extend for many msec; members of both of these larger groups of dorsal horn cells were typically found during each experiment (Egger & Wall, 1971) The differential tactile sensitivities shown by these two groups may be due, at least in part, to activation by different mechanoreceptors which innervate the PC (Iggo & Ogawa, 1977; Janig, Schmidt & Zimmerman, 1968; Lynn, 1969). The LT dorsal horn cells may be activated by Pacinian corpuscles, while the IT dorsal horn cells appear to have response characteristics related to the rapidly adapting afferents.
163 SENSSITIZATION AND HABITUATION An unusual characteristic of the dorsal horn cells excited by mechanical stimulation of the PC is that none appear to belong to the spinocervical tract (Brown, 1973, p. 324).
I
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80 160 0 100 400 450 500 Time (sec) Stimuli Fig. 8. Response patterns of another low threshold dorsal horn cell (LT.3), illustrating lack of correspondence between response patterns of this unit and that of the PC reflex. Graph similar to Fig. 7. Continuous lines refer to the responses of the dorsal horn cell. Interrupted lines refer to responses recorded from VRS1. PC stimulated at 3 0 times threshold for the reflex bout of 500 stimuli at 5 0 Hz indicated by circles (@), or at 10 0 Hz indicated by triangles (v). 0
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Fig. 9. Graph, similar to that of Fig. 7, but illustrating the correspondence between response patterns of an intermediate threshold dorsal horn cell (IT-3, continuous lines) and reflex recorded simultaneously from VRS1 (interrupted lines) to stimulation of the PC. PC stimulated 500 times at 5 0 Hz, at 3 0 times threshold for the reflex indicated by circles (@), or at 10 0 times threshold indicated by triangles (v). 6-2
164 ,11. D. EGGER LT short latency dorsal horn cells showed only habituation following iterated stimulation, but these neurones were activated at thresholds considerably below those of the PC reflex itself, responding, for instance, to light brushing of the PC, a stimulus which never elicited the PC reflex. These LT dorsal horn cells which showed only a habituation pattern were most likely not part of the PC reflex circuit. Short latency IT units had threshold levels similar to those of the PC reflex. These IT units showed sensitization and habituation, resembling the
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Fig. 10. Response patterns of another intermediate threshold dorsal horn cell (IT-6), illustrating the correspondence between response patterns of this unit and that of the PC reflex. Graph similar to Fig. 7. Continuous lines refer to responses of the dorsal horn cell. Interrupted lines refer to responses recorded from VRS1. PC stimulated at 3-0 times threshold for the reflex, bout of 500 stimuli at 1 0 Hz indicated by circles (0), or at 5.0 Hz indicated by triangles (V).
'type S' neurones of Groves & Thompson (1970, 1973), except that the IT units were short latency neurones in laminae III and IV, rather than long latency neurones in laminae V-VII. Because the IT dorsal horn cells showed either sensitization-habituation, or habituation alone, depending on the stimulus parameters (Figs. 9 and 10), it appears that these neurones, which are thought to mediate the PC reflex, could mediate both habituation and sensitization-habituation; A 'dualprocess' model involving independent neuronal circuits is unnecessary to explain sensitization-habituation of the PC reflex. On the basis of known values for synaptic delays and e.p.s.p. rise-times, it is possible to argue that the short latency dorsal horn cells in this study were monosynaptically activated by primary afferents. In the mammalian central nervous system, synaptic delay for e.p.s.p.s is about 0 3 msec, and a delay of about 0*5 msec occurs between the onset of the e.p.s.p. and the production of an action potential (Berry & Pentreath, 1976). Thus, for the spinal cord, one would expect that the shortest latencies between
SENSITIZATION AND HABITUATION 165 arrival of an afferent volley at the dorsal root entry zone and the occurrence of an action potential from a dorsal horn cell would be about 0*8 msec. Making no additional allowance for axonal conduction times, an action potential produced by a disynaptic linkage should not occur in less than 1P6 msec following the arrival of a volley at the dorsal root entry zone. The criterion selected in this paper, of a central latency of 1-5 msec or less, should include only dorsal horn cells driven monosynaptically by primary afferents. However, the possible existence of very short-latency disynaptic responses has recently been discussed by Watt, Stauffer, Taylor, Reinking, & Stuart, (1976). It is also possible, of course, that dorsal horn cells with central delays exceeding 1.5 msec may have been monosynaptically driven. In summary, during iterated stimulation that leads to sensitization and habituation of the PC reflex, important reflex changes may occur between the arrival of the afferent volley at the dorsal root entry zone and the firing of first-order neurones. Because individual short latency IT dorsal horn cells can display both sensitization and habituation, no 'dual-process' theory involving anatomically distinct pathways is needed to explain PC reflex sensitization and habituation. Similar changes occurring between afferents and first-order neurones have been demonstrated in the marine mollusc Aplysia (Kandel et al. (1976), although these authors use 'sensitization' to refer to a slightly different phenomenon), and, for habituation alone, in the cockroach (Callec et al. 1971) and in the crayfish (Zucker, 1972). I thank John Bishop, Constance Cone, Peter Kahrilas, Irmenia Meadows, Eric Proshansky and Susan Sachs for advice and assistance. A preliminary report has been published which included some of the data presented here (Egger & Cone, 1974). This research was supported by Public Health Service Grants NS-06297 and NS-13456, National Science Foundation Grant BMS 75-02312, and General Research Support Grant 27-1917 from CMDNJ-Rutgers Medical School. REFERENCES
ARMETT, C. J., GRAY, J. A. B. & PALMER, J. F. (1961). A group of neurones in the dorsal horn associated with cutaneous mechanoreceptors. J. Physiol. 156, 611-622. BERRY, M. S. & PENTREATH, V. W. (1976). Criteria for distinguishing between monosynaptic and polysynaptic transmission. Brain Rea. 105, 1-20. BROWN, A. G. (1973). Ascending and long spinal pathways: Dorsal columns, spinocervical tract and spinothalamic tract. Handbook of Sensory Physiology, vol. ii. Somatosensory System, ed. IGGO, A., pp. 315-338. New York: Springer. CALLEc, J. J., GUILLET, J. C., PICHON, Y. & BOISTEL, J. (1971). Further studies on synaptic transmission in insects. II. Relations between sensory information and its synaptic integration at the level of a single giant axon in the cockroach. J. exp. Biol. 55, 123-149. CAPEK, R. & ESPLIN, B. (1977). Homosynaptic depression and transmitter turnover in spinal monosynaptic pathway. J. Neurophysiol. 40, 95-105. CASTELLUCCI, V. F. & KANDEL, E. R. (1974). A quantal analysis of the synaptic depression underlying habituation of the gill-withdrawal reflex in Aplysia. Proc. natn. Acad. Sci. U.S.A. 71, 5004-5008. CASTELLUCCI, V. & KANDEL, E. R. (1976). Presynaptic facilitation as a mechanism for behavioral sensitization in Aplysia. Science, N.Y. 194, 1176-1178. EGGER, M. D., BISHOP, J. W. & CONE, C. H. (1976). Sensitization and habituation of the plantar cushion reflex in cats. Brain Re8. 103, 215-228. EGGER, M. D. & CONE, C. H. (1974). First-order interneurons: sensitization and habituation. Proceedings of Fourth Annual Meeting, Society for Neuroscience, p. 199.
166
M. D. EGGER
EGGER, M. D. & WALL, P. D. (1971). The plantar cushion reflex circuit: an oligosynaptic cutaneous reflex. J. Phy8iol. 216, 483-501. FAREL, P. B. (1974). Dual processes control response habituation across a single synapse. Brain Re8. 72, 323-327. FAREL, P. B., GLANZMAN, D. L. & THOMPSON, R. F. (1973). Habituation of a monosynaptic response in vertebrate central nervous system: lateral column-motoneuron pathway in isolated frog spinal cord. J. Neurophy8iol. 36, 1117-1130. FAREL, P. B. & THOMPSON, R. F. (1976). Habituation of a monosynaptic response in frog spinal cord: evidence for a presynaptic mechanism. J. Neurophy8iol. 39, 661-666. GROVES, P. M., DEMARCO, R. & THOMPSON, R. F. (1969). Habituation and sensitization of spinal interneuron activity in acute spinal cat. Brain Res. 14, 521- 525. GROVES, P. M. & THOMPSON, R. F. (1970). Habituation: a dual-process theory. P8ychol. Rev. 77, 419-450. GROvES, P. M. & THOMPSON, R. F. (1973). A dual-process theory of habituation: neural mechanisms. Habituation, vol. II, Phy8iological Substrate8, ed. PEEKE, H. V. S. & HERZ, M. J., pp. 175-205. New York: Academic. IGGo, A. & OGAWA, H. (1977). Correlative physiological and morphological studies of rapidly adapting mechanoreceptors in cat's glabrous skin. J. Physiol. 266, 275-296. JANIG, W., SCHMIDT, R. F. & ZIMMERMAN, M. (1968). Single unit responses and the total afferent outflow from the cat's foot pad upon mechanical stimulation. Expl Brain Re8. 6, 100-115. KANDEL, E. R., BRUNELLI, M., BYRNE, J. & CASTELLUCCI, V. (1975). A common presynaptic locus for the synaptic changes underlying short-term habituation and sensitization of the gillwithdrawal reflex in Aply8ia. Cold Spring Harb. Symp. quant. Biol. XL, 465-482. LYNN, B. (1969). The nature and location of certain phasic mechanoreceptors in the cat's foot. J. Physiol. 201, 765-773. MERRILL, E. G. & AINSWORTH, A. (1972). Glass-coated platinum-plated tungsten microelectrodes. Med. biol. Engng 10, 662-672. REXED, B. (1954). A cytoarchitectonic atlas of the spinal cord in the cat. J. comp. Neurol. 100, 297-379. THOMPSON, R. F. & SPENCER, W. A. (1966). Habituation: a model phenomenon for the study of neuronal substrates of behavior. P8ychol. Rev. 73, 16-43. WALL, P. D. (1973). Dorsal horn electrophysiology. Handbook of Sen8ory Phy8iology, vol. II. Somato8en8ory Sy8tem, ed. Ioo, A., pp. 253-270. New York: Springer. WATT, D. G. D., STAUFFER, E. K., TAYLOR, A., REINKING, R. M. & STUART, D. G. (1976). Analysis of muscle receptor connections of spike-triggered averaging. 1. Spindle primary and tendon organ afferents. J. Neurophysiol. 39, 1375-1392. ZUCKER, R- S. (1972). Crayfish escape behavior and central synapses. 11. Physiological mechanisms underlying behavioral habituation. J. Neurophysiol. 35, 621-637.
