A Neuronal Correlate of Secondary Hyperalgesia in the Rat Spinal Dorsal Horn Is Submodality Selective and Facilitated by Supraspinal Influence

EXPERIMENTAL NEUROLOGY ARTICLE NO.

149, 193–202 (1998)

EN976688

A Neuronal Correlate of Secondary Hyperalgesia in the Rat Spinal Dorsal Horn Is Submodality Selective and Facilitated by Supraspinal Influence Antti Pertovaara Department of Physiology, Institute of Biomedicine, University of Helsinki, Helsinki; and Department of Physiology, Institute of Biomedicine, University of Turku, Turku, Finland Received June 10, 1997; accepted September 10, 1997

Tissue injury produces hyperalgesia not only in the injured area (primary hyperalgesia) but also outside of it (secondary hyperalgesia). In the present investigation, the submodality selectivity and the contribution of supraspinal influence to a neural correlate of the secondary hyperalgesia induced by neurogenic inflammation was studied in the presumed pain relay neurons of the rat spinal dorsal horn. Mechanically and thermally evoked responses to wide-dynamic range (WDR) neurons of the spinal dorsal horn were recorded under sodium pentobarbital anesthesia in rats. Neurogenic inflammation was induced by application of mustard oil outside of the receptive fields of WDR neurons. To study the contribution of supraspinal influence to mustard oil-induced changes in neuronal responses, the spinal cord was transected at a midthoracic level or lidocaine was microinjected into the rostroventromedial medulla (RVM). Furthermore, the antidromically evoked compound volley in the sural nerve was determined to reveal excitability changes in the central terminals of primary afferent A-fibers induced by mustard oil. The results indicate that mustard oil adjacent to the receptive fields of spinal WDR neurons significantly enhanced their responses to mechanical but not to noxious heat stimuli, without a significant influence on their spontaneous activity. Both high- and low-threshold mechanoreceptive input to WDR neurons was equally facilitated, whereas mechanoreceptive input to spinal dorsal horn neurons mediating innocuous messages (low-threshold mechanoreceptive neurons) was not changed. Mustard oil in a remote site (forepaw) did not produce any hyperexcitability to responses evoked by hindpaw stimulation. Spinal transection or lidocaine block of the RVM significantly attenuated the mustard oil-induced mechanical hyperexcitability in spinal dorsal horn neurons. Mustard oil had no significant effect on a compound Avolley in the sural nerve induced by intraspinal stimulation of sural nerve terminals at a submaximal intensity. The selective mechanical hyperexcitability in spinal WDR neurons, without a change in their spontaneous activity, can be explained by a heterosyn-

aptic facilitatory action on presynaptic terminals mediating mechanical signals to these nociceptive spinal neurons. These findings indicate that brain stem– spinal pathways, involving the RVM, do not only suppress nociception but under some pathophysiological conditions concurrent facilitatory influence may predominate and lead to enhancement of mechanical hyperexcitability. The descending facilitatory feedback loop to nociceptive spinal neurons may help to protect the wounded tissue and thus promote healing. r 1998 Academic Press Key Words: secondary hyperalgesia; spinal dorsal horn neuron; plasticity in nociception; mustard oil; descending facilitation; presynaptic facilitation.

INTRODUCTION

A tissue injury may lead to enhanced pain responses (hyperalgesia) both in the injured area and in its neighborhood. It is generally considered that the hyperalgesia outside of the injured area, also called secondary hyperalgesia, is predominantly due to central mechanisms (46). In humans, secondary hyperalgesia is selective to mechanical stimulation (46), whereas in animal studies the submodality selectivity of adjacent hyperalgesia is still a controversial issue. Under physiological conditions, brain stem–spinal pathways predominantly exert a strong inhibitory influence on pain relay neurons at the spinal dorsal horn level (3, 52), although also descending facilitatory influence has been demonstrated following stimulation of some brainstem sites (2, 6, 8, 14, 43, 56). Under pathophysiological conditions, various pain regulatory systems, including brain stem–spinal pathways, may be subject to dramatic plastic changes that may contribute to pathophysiological pain (9, 51). For example, it has been shown that dorsal rhizotomy may reverse the raphe magnus or locus coeruleus-induced inhibition of spinal dorsal horn neurons to descending facilitation (16). On the other hand, following experimental arthritis or carrageenan-induced inflammation

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0014-4886/98 $25.00 Copyright r 1998 by Academic Press All rights of reproduction in any form reserved.

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descending inhibition of spinal dorsal horn neurons receiving input from the inflammed area has been shown to be enhanced (35, 37). However, hyperreflexia to A-fiber stimulation in carrageenan-treated rats was dependent on supraspinal facilitation (15). Also opioid abstinence-induced hyperreflexia was dependent on brain stem–spinal facilitatory influence (18). Concerning secondary hyperalgesia, there is recent behavioral evidence indicating that brain stem–spinal pathways may actually enhance spinal segmental mechanisms underlying hyperalgesia (25, 47, 50). However, behavioral studies do not indicate whether the facilitation of spinal pain reflexes is due to increased responsiveness of sensory or motor neurons. Thus, it remains to be studied whether the recently demonstrated brain stem– spinal facilitatory effects are ‘‘truly’’ hyperalgesic (action on sensory neurons) or only hyperreflexive (action on motoneurons). Furthermore, behavioral studies do not reveal the exact spinal target of hyperalgesic action (e.g., presynaptic action on selective peripheral input or nonselective postsynaptic action on spinal pain-relay neurons). The present study addressed the questions whether a presumed spinal neuronal correlate of secondary hyperalgesia induced by neurogenic inflammation is submodality selective and whether it is dependent on supraspinal influence. Mustard oil was applied outside the receptive fields of the wide-dynamic range (WDR) or low-threshold mechanoreceptive (LTM) neurons of the rat spinal spinal dorsal horn to produce neurogenic inflammation and secondary hyperalgesia. Mustard oil produces a tonic activation of nociceptive primary afferent fibers (34) and behaviorally it produces a secondary hyperalgesia of rapid onset as well in humans as in rats (7, 19, 20, 26, 47). Neuronal responses were determined before and after mustard oil application to natural mechanical and thermal stimuli. To study the contribution of supraspinal influence to mustard oil-induced changes in spinal neuronal responses, the spinal cord was transected at a midthoracic level or lidocaine was microinjected into the rostroventromedial medulla. Finally, to reveal a possible presynaptic excitability change in central terminals of primary afferent A-fibers, the effect of mustard oil on intraspinally induced antidromic compound A-volley in the sural nerve was determined. METHODS

Adult Hannover–Wistar rats (The Finnish National Laboratory Animal Center; weight 350–450 g) were used in the experiments. The experiment was approved by the Institutional Ethics Committee of the University of Helsinki and the ethical guidelines of the IASP were strictly followed.

Experimental Surgery The rats were anesthetized with sodium pentobarbital (50 mg/kg ip). The level of anesthesia was frequently determined by observing the size of the pupils, the general mucle tone, and behavioral responses to noxious pinching. Supplemental doses of sodium pentobarbital (20 mg/kg) were administered as required. In general, a supplemental dose of pentobarbitone was administered before testing a neuron and no additional doses were given before the neuron was completely tested (testing took no more than 30 min). A homeothermic blanket was used to keep the body temperature at 36.6°C. The rats were spontaneously breathing. The peripheral vascularization was checked by considering the color of the ears and the extremities. Animals were placed in a standard stereotaxic frame according to the atlas of Paxinos and Watson (31). A laminectomy was performed at the level of vertebrae T12–L2, the dura were removed, and a pool of skin was formed and filled with warm mineral oil. Two spinal clamps, one rostral and one caudal to the laminectomy, were used to stabilize the preparation. In a group of rats, an additional laminectomy was performed at the midthoracic level and through it the spinal cord was surgically transected under visual control. Spinalization was performed 2 to 3 h prior to the start of electrophysiological recordings. For microinjections of lidocaine/saline in the rostroventromedial medulla (AP 22.6, ML 0.0, and DV 0.5 mm), the skull was exposed and a hole was drilled for placement of a guide cannula (26 gauge) 2 mm above the target. In experiments on the effect of mustard oil on primary afferent terminal excitability, the sural nerve was dissected free in a pool of mineral oil. Electrophysiological Recording Spinal unit activity was recorded extracellularly with lacquer-coated tungsten electrodes (tip impedance 5–10 MV at 1 kHz) and then amplified and filtered using standard techniques. The signal was fed through a window discriminator (F. Haer, ME) to a rate monitor (F. Haer) and to a timed counter (F. Haer). The peristimulus time histograms and integrated spike counts were observed on a digital storage oscilloscope (Tektronix DS420, Portland, OR) and hard copies of the data in the oscilloscope screen were made with a laser printer (HP LaserJet III) for off-line analysis. Primary afferent terminal excitability was recorded as originally described by Wall (49). Briefly, the sural nerve was placed on a pair of platinum–iridium wires for recording antidromic compound potentials evoked by stimulating the cord with a lacquer-coated tungsten microelectrode at the site of sural nerve terminals. The exact stimulus site in the lumbar spinal cord was selected by observing the submaximal electrically

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evoked volley in the sural nerve and then finding a stimulus location in which a conditioning intraspinal stimulus (stimulus duration, 0.1 ms; interstimulus interval, 10 ms) produced a clear enhancement in the test volley. Characterization of Neurons During the search for spinal neurons the glabrous skin of the ipsilateral hindpaw was stimulated repeatedly with a brush. After a neuron responding to a mechanical stimulus was found, its receptive field characteristics were determined using mechanical stimuli (calibrated monofilaments producing forces of 4, 12, and 46 g; Stoelting, Wood Dale, IL) and a series of thermal stimuli of 5 s duration from the adaptation temperature of 35°C: 1, 44, 46, 48, 50, 52, and 54°C. Thermal stimulation was performed with a feedbackcontrolled contact thermostimulator (stimulus surface 82.8 mm2, LTS3-Stimulator, Thermal Devices Inc., Golden Valley, MN). Each mechanical stimulus force was consecutively presented three times for the duration of 1 s at 1-s intervals to reveal a possible wind-up phenomenon. Only neurons that were considered to be in the spinal dorsal horn according recording depth from the cord surface (,1000 µm) were included (52). The neurons activated by brush were classified into wide-dynamic range neurons and low-threshold mechanoreceptive neurons according to the criteria described earlier (52). Briefly, neurons that gave differential responses to mechanical stimulation of high ($46 g) and low (#2 g) intensity were classified as WDR neurons, whereas the responses of LTM neurons were not increased with an increase in stimulus force from 4 to 46 g. It should be also noted that all WDR neurons of the present study also gave differential responses to increases in the intensity of the heat stimulus within noxious range (46 to 52°C), whereas none of the LTM neurons of the present study was activated by noxious heat. Other types of spinal dorsal horn neurons, such as nociceptive specific ones with high thresholds, were not considered in this study. Drug Administrations Mustard oil (50% in ethanol; Merck, Darmstadt, Germany) was applied for 2 min on a piece of filter paper (2 cm2 ) to a site in the hindpaw glabrous skin that was about 1–2 cm outside the borders of the receptive field of studied neuron, except in one group of rats mustard oil was applied in the glabrous skin of the forepaw (remote site). The microinjection of lidocaine (40 µg in 1 µl; Astra, So¨derta¨lje, Sweden) or physiological saline (1 µl) was made as described in detail elsewhere (32). Lidocaine (4%) at the volume of 1.0 µl should effectively suppress neuronal activity within a radius of 1.4–1.7 mm (30, 36). At the completion of the

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experiment, the microinjection sites were histologically verified. Course of the Study Under all conditions, the responses to test stimuli of submaximal intensity were determined before mustard oil (reference response; 100%), 5 min after the application of mustard oil, and 5 min after the microinjection of lidocaine/saline. The statistical comparison of mustard oil-induced effects on mechanically evoked responses were performed using the phasic responses evoked by 46g force, unless otherwise specified. In statistical evaluation paired t test was used, unless otherwise specified. P , 0.05 was considered to represent a significant difference. RESULTS

Submodality Dependence of Hyperexcitability Mustard oil applied outside of the receptive field of spinal WDR neurons produced a significant enhancement of their responses to mechanical but not to noxious heat stimuli (n 5 16; Figs. 1 and 2A). Spontaneous activity of spinal dorsal horn WDR neurons was not significantly increased by mustard oil applied outside of their receptive fields. Before mustard oil the average spontaneous activity of spinal dorsal horn WDR neurons was 1.9 6 0.6 Hz (n 5 10) and following mustard oil it was 2.4 6 0.7 Hz (ns, paired t test). When mustard oil was occasionally applied to the center of the receptive field, WDR neurons strongly increased their spontaneous activity for several minutes (n 5 4, data not shown). Mustard oil applied in a remote site (forepaw skin) did not produce any hyperexcitability in the spinal dorsal horn WDR neurons receiving their input from the hindpaw (n 5 5; Fig. 2A). The selective mechanical hyperexcitability induced by mustard oil in spinal WDR neurons raised the question whether the hyperalgesic action was selective with respect to innocuous versus noxious mechanical signals. This question was addressed in two experiments. First, mustard oil-induced facilitation of mechanical input to spinal WDR neurons was independent of the stimulus force (Fig. 2B) indicating that both noxious and innocuous mechanical input to spinal WDR neurons was equally facilitated. Second, mustard oil did not facilitate mechanically evoked input to those spinal dorsal horn neurons that mediate innocuous mechanical signals (so called low-threshold mechanoreceptive or LTM neurons; n 5 6; Figs. 2B and 3). Effect of Mustard Oil on Central Terminal Excitability of Primary Afferent A-Fibers Effect of mustard oil on a compound A-volley in the sural nerve evoked by intraspinal stimulation of the

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FIG. 1. Mustard oil-induced enhancement of responses of one spinal dorsal horn WDR neuron to mechanical (A–C) stimulation and lack of effect on responses to thermal (D, E) stimulation. In A–C three consecutive mechanical stimuli applied at three different forces (4, 12, and 46g) were applied at about 1-s intervals, and this was followed by continuous pressure at the force of 46g (46 g cont.). Notice change of scale bar from A to B. In D and E a noxious heat stimulus (iii, from 35 to 52°C) was applied to the receptive field. F shows the receptive field (RF) of the neuron and the site of mustard oil application. i, ratemeter record (bin width 0.2 s; scale bar, 10 Hz); ii, integral of impulse count (scale bar, 100 imp.). B, C, and E, the time elapsed from the application of mustard oil is shown above each graph.

sural nerve terminals was determined to elucidate the possible role of presynaptic mechanisms to the mustard oil-induced facilitatory action (Fig. 4). Before mustard oil, intraspinal conditioning stimulus produced a significant increase in the submaximal antidromic A-volley in

the sural nerve (P , 0.05, paired t test). Mustard oil had no effect either on an unconditioned or on a conditioned compound A-volley in the sural nerve induced by intraspinal stimulation of sural nerve terminals at a submaximal intensity (n 5 7; Fig. 4).

FIG. 2. (A) Mustard oil applied adjacent to the receptive fields of spinal dorsal horn WDR neurons did not enhance their responses to noxious heat stimuli (52°C; n 5 16), whereas responses to mechanical stimuli (46g; n 5 16) were significantly enhanced. Mustard oil in a remote site (forepaw) did not enhance mechanically evoked responses (46g; n 5 5). (B) Mustard oil adjacent to the receptive field of spinal pain neurons (n 5 13) produced an equal enhancement of mechanically evoked responses both to low (4g) and high (46g) intensity stimuli, whereas responses to spinal dorsal horn LTM neurons were not influenced (n 5 6).

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relate of secondary hyperalgesia) in the WDR neurons of the spinal dorsal horn is predominantly to mechanically evoked responses. Furthermore, a spinal–brain stem–spinal feedback loop, involving the RVM, facilitates the spinal segmental mechanisms underlying the secondary hyperexcitability. At the spinal dorsal horn level, the mustard oil-induced facilitatory action may be explained by a predominantly presynaptic facilitatory action on all mechanical input to wide-dynamic range neurons. Alternatively, mustard oil application had different actions on interneuronal pathways conveying mechanical vs heat inputs to WDR neurons. Descending Facilitatory Action

FIG. 3. Responses of one spinal dorsal horn LTM neuron with and without mustard oil. (A) The stimulus–response function for mechanical stimulation shows that the response was saturated already at low stimulus forces (2g) indicating that this neuron was a LTM neuron. (B) Mechanical responses evoked by a 2g stimulus before mustard oil. (C) Ten minutes following mustard oil application the mechanical responses were identical to that before mustard oil. (D) The receptive field (RF) of the neuron and the site of mustard oil application. The scale bar for ratemeter recors represents 10 Hz.

Supraspinal Contribution to the Mustard Oil-Induced Facilitatory Action The possibility that supraspinal influence contributed to hyperexcitability in the spinal dorsal horn WDR neurons was tested in two ways. First, the spinal cord was transected at a midthoracic level to find out the importance of descending input to hyperexcitability. The results indicated that following spinal transection mustard oil applied adjacent to the receptive fields of WDR neurons still caused hyperexcitability; it was, however, markedly weaker than in animals with an intact spinal cord (Fig. 5A). According to ratemeter recordings, the mustard oil-induced absolute increase of responses evoked by a 46g force was 6.2 6 1.6 Hz (n 5 6) in spinalized animals and 29.5 6 6 Hz (n 5 12) in animals with an intact spinal cord (P , 0.03). Second, lidocaine, a local anesthetic, was microinjected into the rostroventromedial medulla (RVM) in an attempt to modulate hyperexcitability in spinal WDR neurons. Lidocaine (n 5 7), unlike saline (n 5 5), in the RVM produced a significant attenuation of hyperexcitability (Figs. 5B and 6). Supraspinally administered lidocaine did not attenuate the mustard oil-induced hyperexcitability in the lumbar spinal dorsal horn of rats with a transected cord (n 5 5; Fig. 5A). DISCUSSION

According to the present results mustard oil-induced secondary hyperexcitability (a presumed neuronal cor-

The results of this study are in line with the previous evidence indicating that a chemical irritant, mustard oil, applied outside of the receptive fields of nociceptive spinal dorsal horn neurons can induce mechanical hyperexcitability due to spinal segmental mechanisms (54, 55) as shown in spinally transected animals of the present study. Importantly, adjacent hyperexcitability in nociceptive spinal neurons was markedly attenuated by spinal transection and by a lidocaine block of the RVM. This observation is in line with recent behavioral evidence indicating that mustard oil-induced adjacent hyperreflexia is facilitated by descending input (25, 47). Consistent with the present findings is the recent observation that the descending inhibition of spinal dorsal horn neurons driven from the periaqueductal gray was attenuated following intracutaneous capsaicin treatment (24) or chronic spinal nerve ligation (33). Also in some other hyperalgesia or under tonic pain

FIG. 4. (A) Average amplitudes of antidromic compound volleys in the sural nerve (n 5 7) evoked by the first (uncond.) and the latter (conditioned) stimulus of the pair before (open bars) and 5 min after application of mustard oil to the distal innervation area (hatched bars). 100%, response to the first stimulus of the pair before mustard oil. The enhancements of responses to the latter stimulus of the pair indicate increased primary afferent excitability caused by the preceding stimulus. Importantly, mustard oil did not induce significant changes. (B) An original recording of a compound A-volley induced by an intraspinally applied electrical twin stimulus (arrows) before (i) and 5 min after (ii) mustard oil application (each trace is an average of 15 consecutive responses).

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FIG. 5. (A) Following spinal transection mustard oil applied adjacent to the receptive field produced a significant hyperexcitability to mechanical stimuli in WDR neurons (n 5 5) that was significantly weaker than that in rats with an intact spinal cord (P , 0.01, compare with B or Fig. 1A) and not attenuated by lidocaine (40 µg) in the RVM. (B) Lidocaine (LIDO; 40 µg in 1 µl) in the RVM significantly attenuated mustard oil-induced hyperexcitability in WDR neurons (n 5 7). (C) Saline in the rostroventromedial medulla (RVM) did not attenuate mustard oil-induced hyperexcitability (n 5 5). (D) Centers of the injection sites in the RVM. Rmg, raphe magnus nucleus; GiA, gigantocellularis pars alpha nucleus; Gi, gigantocellular nucleus; DPGi, dorsal paragigantocellular nucleus; g7, genu of the facial nerve. For further details, see the legend to Fig. 1.

conditions participation of descending facilitation has been demonstrated (32, 48, 50). Furthermore, the present findings extend these behavioral findings by demonstrating that the hyperreflexic effect of brain stem– spinal pathways (28) may be explained by an increased responsiveness of sensory neurons of the spinal dorsal horn, i.e., by a ‘‘true’’ hyperalgesic action. The present neurophysiological results, together with recent behavioral studies, indicate that under neurogenic inflammation the neurons originating in or passing through the rostroventromedial medulla may tonically enhance spinal pain signals from the neighbourhood of an injured area. Following experimental arthritis or carrageenaninduced inflammation, spinal responses from the area of primary hyperalgesia have been shown to be under an enhanced supraspinal inhibitory influence (35, 37) or under a supraspinal facilitatory influence (15) depending on the type of response studied. Thus, whether a descending inhibitory or facilitatory control predomi-

nates may markedly vary depending on experimental conditions and the underlying pathophysiology. In the present study, pentobarbitone may have unmasked descending inhibition (10) and this may have contributed to a predominance of descending facilitation. Moreover, pentobarbitone may have attenuated the spinal segmental mechanisms underlying adjacent hyperexcitability (7). Hyperexcitability was spatially restricted to the neighborhood of the mustard oil application site as in previous mustard oil studies (54, 55) and it was submodality selective as in a previous capsaicin study (41). These observations indicate that the hyperexcitability was not due to a general change in neuronal excitability caused by the painful conditioning stimulus. The lack of effect by mustard oil in a remote site also excludes the possibility that changes in anesthesia level during testing could explain the hyperexcitability induced by mustard oil in an adjacent site, since anesthesia was identically induced and maintained under these two

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FIG. 6. Responses of one spinal WDR neuron demonstrating mustard oil-induced hyperexcitability that is selective to mechanical stimuli and attenuated by lidocaine (LIDO) in the rostroventromedial medulla (RVM). A–C, three repetitive mechanical stimuli (4g, 12g, and 46g) were presented to the receptive field in each condition followed by a continuous pressure at the force of 46g (horizontal bar). Concomitantly, response to noxious heat in the same neuron was not enhanced by mustard oil (D and E, respectively). (F) The receptive field (RF) of the neuron and the site of application of mustard oil. i, rate meter recording (bin width 0.2 s; scale bar, 10 Hz); ii, integral of impulse count (scale bar, 100 imp.); iii, heat stimulus from 35 to 52°C and back.

experimental conditions. It should be noted, however, that under other experimental conditions a painful conditioning stimulus may produce remote facilitatory (53) or even inhibitory effects (22). Possible Neurochemical Changes Underlying Spinal Facilitation Mustard oil as capsaicin activates predominantly nociceptive C-fibers at the peripheral level (34, 39) and this causes various changes in central neurotransmission that may underlie the secondary hyperalgesia and allodynia (41). It has been shown that neurokinin A, acting through NK2 receptors, and glutamate, acting through metabotropic (mGlu) receptors, play an important role in mediating sustained responses to neurogenic inflammation induced by mustard oil (29). Intradermal capsaicin is known to produce a release of excitatory amino acids and substance P in the spinal dorsal horn (11, 42). Additionally, capsaicin in the skin has been shown to cause an activation of proteinkinase C in the spinal cord, which is likely to contribute to the development of allodynia and secondary hyperalgesia by enhancing the responses of excitatory amino acid receptors and by desensitizing glycine and GABA receptors (23, 24). Also, there is recent evidence indicating that proteinkinase C plays a significant role in the attenuation of the descending inhibition from the peri-

aqueductal gray in rats treated with intracutaneous capsaicin injection (24). On the other hand, the antiallodynic potency of a selective a2-adrenoceptor agonist was enhanced due to spinal mechanisms in mustard oil-treated rats (26). Thus, neurogenic inflammation may induce complex changes in pain regulatory systems. Some of these changes may be beneficial and potentiate the antihyperalgesic effects of exogenous compounds whereas some are harmful and tend to reduce the effect of pain therapy. Submodality Selectivity of Adjacent Hyperalgesia The selective secondary hyperexcitability to mechanical stimulation but not to heat in spinal dorsal horn WDR neurons is in line with previous electrophysiological results in primates (41) and with previous human psychophysical findings following capsaicin or mustard oil treatment of the skin (1, 12, 13, 19–21, 41, 44). Furthermore, mustard oil-induced an equal facilitation of mechanically evoked responses to spinal WDR neurons following both low- and high-intensity stimulation. This finding is consistent with psychophysical results demonstrating that capsaicin or mustard oil induce both mechanical allodynia (touch-evoked pain) and mechanical hyperalgesia (enhanced pain evoked by stimulating at a noxious intensity) (5). Lack of mustard oil-induced effect on mechanical responses to LTM

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neurons of the spinal dorsal horn is in line with the results of an earlier cat study (40) and this finding indicates that the mustard oil-induced facilitatory action was selective not only concerning the submodality of the peripheral input but also concerning the type of central neuron receiving the input. Only responses to the spinal dorsal horn WDR neurons that are presumably important for mediating painful messages (52) were facilitated. Together the facilitation of low- and high-intensity mechanical input but not heat-evoked input to WDR neurons, lack of facilitatory effect on mechanically evoked responses to LTM neurons and lack of a significant change in spontaneous activity of WDR neurons could be explained by a presynaptic facilitation of all mechanical input to spinal WDR neurons. This hypothesis was further elucidated by determining the effect of mustard oil on excitability of central terminals of a cutanous nerve, which was expected to be changed if presynaptic facilitation occurred (38). Mustard oil had no effect on the excitability of central terminals of cutaneous A-fibers that mediate low-intensity mechanical input. This lack of mustard oil-induced effect could be explained by the assumption that only a minority of the fastly conducting A-fibers studied are connected to WDR neurons. The present methodology of recording compound A-volleys should reveal an excitability change in central terminals only if it occurred in a majority of the fiber population studied. Therefore, the lack of excitability change in cutaneous nerve terminals was not in discrepancy with the hypothesis that presynaptic facilitation occurred only in a subpopulation of mechanoreceptive fibers mediating mechanical signals to WDR neurons. However, it should be noted that according to studies performed in invertebrates, presynaptic inhibition or facilitation may take place without a change in excitability of primary afferent terminals (17). Moreover, it should be noted that the absence of presynaptic excitability changes of large myelinated fibers does not rule out possible changes in terminal excitability of small afferent fibers, some of which respond to innocuous mechanical stimuli. Since mustard oil was applied outside of the receptive fields of the WDR neurons, this would be a case of heterosynaptic presynaptic facilitation. The present study focused on changes in response characteristics of spinal dorsal horn WDR neurons as a cause of secondary hyperalgesia. Spinal dorsal horn WDR neurons are generally considered important for the mediation of painful (52) and hyperalgesic (45) responses and the present findings further support this line of evidence. However, there is also previous evidence indicating that nociceptive specific neurons or so called high-threshold neurons of the spinal dorsal horn, that were not studied in this experiment, may significantly contribute to secondary hyperalgesia and allo-

dynia (4). It should be noted that these alternative mechanisms need not be mutually exclusive. Furthermore, one more mechanism not studied here is the expansion of receptive fields of spinal dorsal horn neurons. An expansion of receptive fields has been previously described following administration of mustard oil (54, 55) or capsaicin (11) and it may also contribute to hyperalgesia and allodynia observed behaviorally. Conclusions Chemical activation of cutaneous nociceptors by mustard oil leads to a selective mechanical hyperexcitability outside the receptive fields of nociceptive spinal dorsal horn neurons. The spinal segmental mechanisms underlying this presumed neuronal correlate of secondary hyperalgesia are facilitated by a spinal– brain stem–spinal feedback loop involving the RVM. From the evolutional point of view, descending facilitation of pain signals originating from the neighborhood of injured tissue may have been beneficial for tissue healing, since it helps to protect and immobilize the injured region. However, under human clinical conditions this type of pain-enhancing loop is likely to be harmful. Further understanding of the neurochemical basis of this facilitatory loop may help to find completely new therapeutic tools to alleviate clinical hyperalgesia and allodynia. ACKNOWLEDGEMENTS The contribution of Dr. E. Mecke to histology is gratefully acknowledged. This study was financially supported by the MRC, Academy of Finland.

REFERENCES 1.

2.

3.

4. 5.

6.

7.

Ali, Z., R. A. Meyer, and J. N. Campbell. 1996. Secondary hyperalgesia to mechanical but not heat stimuli following a capsaicin injection in hairy skin. Pain 68: 401–411. Almeida, A., A. Tjolsen, D. Lima, A. Coimbra, and K. Hole. 1996. The medullary dorsal reticular nucleus facilitates acute nociception in the rat. Brain Res. Bull. 39: 7–15. Basbaum, A. I., and H. L. Fields. 1984. Endogenous pain control systems: Brainstem spinal pathways and endorphin circuitry. Annu. Rev. Neurosci. 7: 309–338. Cervero, F., and J. M. A. Laird. 1996. Mechanisms of touchevoked pain (allodynia): A new model. Pain 68: 13–23. Cervero, F., R. A. Meyer, and J. N. Campbell. 1994. A psychophysical study of secondary hyperalgesia: Evidence for increased pain to input from nociceptors. Pain 58: 21–28. Cervero, F., and J. H. Wolstencroft. 1984. A positive feedback loop between spinal cord nociceptive pathways and antinociceptive areas of the cat’s brain stem. Pain 20: 125–138. Cleland, C. L., F.-Y. Lim, and G. F. Gebhart. 1994. Pentobarbital prevents the development of C-fiber-induced hyperalgesia in the rat. Pain 57: 31–43.

SECONDARY HYPEREXCITABILITY IN SPINAL NEURONS 8.

9.

10.

11.

12.

13.

14.

15.

16.

17. 18.

19.

20.

21.

22.

23.

24.

25.

26.

Clineschmidt, B. V., and E. G. Anderson. 1970. The blockade of bulbospinal inhibition by 5-hydroxytryptamine antagonists. Exp. Brain Res. 11: 175–186. Coderre, T. J., J. Katz, and R. Melzack. 1993. Contribution of central neuroplasticity to pathological pain: Review of clinical and experimental evidence. Pain 52: 259–285. Collins, J. G., and K. Ren. 1987. WDR response profiles of spinal dorsal horn neurons may be unmasked by barbiturate anesthesia. Pain 28: 369–378. Dougherty, P. M., and W. D. Willis. 1992. Enhanced responses of spinothalamic tract neurons to excitatory amino acids accompany capsaicin-induced sensitization in the monkey. J. Neurosci. 12: 883–894. Gro¨nroos, M., and A. Pertovaara. 1993. Capsaicin-induced central facilitation of a nociceptive flexion reflex in humans. Neurosci. Lett. 159: 215–218. Gro¨nroos, M., H. Naukkarinen, and A. Pertovaara. 1994. Capsaicin-induced central facilitation of a sympathetic vasoconstrictor response to painful stimulation in humans. Neurosci. Lett. 182: 163–166. Haber, L. H., R. F. Martin, J. M. Chung, and W. D. Willis. 1980. Inhibition and excitation of primate spinothalamic tract neurons by stimulation in region of nucleus reticularis gigantocellularis. J. Neurophysiol. 43: 1578–1593. Herrero, J. F., and F. Cervero. 1996. Supraspinal influence on the facilitation of rat nociceptive reflexes induced by carrageenan monoarthritis. Neurosci. Lett. 209: 21–24. Hodge, C. J., Jr., A. V. Apkarian, M. P. Owen, and B. S. Hanson. 1983. Changes in the effects of stimulation of locus coeruleus and nucleus raphe magnus following dorsal rhizotomy. Brain Res. 288: 325–329. Kandel, E. R., J. H. Schwartz, and T. M. Jessell. 1991. Principles of Neural Science, 3rd ed. Elsevier, New York. Kaplan, H., and H. L. Fields. 1991. Hyperalgesia during acute opioid abstinence: Evidence for a nociceptive facilitating function of the rostroventromedial medulla. J. Neurosci. 11: 1433– 1439. Koltzenburg, M., L. E. R. Lundberg, and H. E. Torebjo¨rk. 1992. Dynamic and static components of mechanical hyperalgesia in human hairy skin. Pain 51: 207–219. Koltzenburg, M., H. E. Torebjo¨rk, and L. K. Wahren. 1994. Nociceptor modulated central sensitization causes mechanical hyperalgesia in acute chemogenic and chronic neuropathic pain. Brain 117: 579–591. Lamotte, R. H., C. A. Shain, D. A. Simone, and E. F. Tsai. 1991. Neurogenic hyperalgesia: Psychophysical studies of underlying mechanisms. J. Neurosci. 66: 190–211. Lebars, D., A. H. Dickenson, and J.-M. Besson. 1979. Diffuse noxious inhibitory controls (DNIC). I. Effects on dorsal horn convergent neurones in the rat. Pain 6: 283–304. Lin, Q., Y. B. Peng, and W. D. Willis. 1996. Inhibition of primate spinothalamic tract neurons by spinal glycine and GABA is reduced during central sensitization. J. Neurophysiol. 76: 1005– 1014. Lin, Q., Y. B. Peng, and W. D. Willis. 1996. Possible role of protein kinase C in the sensitization of primate spinothalamic tract neurons. J. Neurosci. 16: 3026–3034. Mansikka, H., and A. Pertovaara. 1997. Supraspinal influence on hindlimb withdrawal thresholds and mustard oil-induced secondary allodynia in rats. Brain Res. Bull. 42: 359–365. Mansikka, H., J.-J. Ida¨npa¨a¨n-Heikkila¨, and A. Pertovaara. 1996. Different roles of a2-adrenoceptors of the medulla versus the spinal cord in modulation of mustard oil-induced central hyperalgesia in rats. Eur. J. Pharmacol. 297: 19–26.

27.

28.

29.

30.

31. 32.

33.

34.

35.

36.

37. 38.

39.

40.

41.

42.

43.

44.

201

Martin, J. H. 1991. Autoradiographic estimation of the extent of reversible inactivation produced by microinjection of lidocaine and muscimol in the rat. Neurosci. Lett. 127: 160–164. Morgan, M. M., and H. L. Fields. 1994. Pronounced changes in the activity of nociceptive modulatory neurons in the rostral ventromedial medulla in response to prolonged thermal noxious stimuli. J. Neurosci. 72: 1161–1170. Munro, F. E., M. R. Young, S. M. Fleetwood-Walker, R. M. C. Parker, and R. Mitchell. 1994. Receptor and cellular mechanisms involved in mustard oil-induced activation of dorsal horn neurones. Prog. Pain Res. Manag. 2: 337–346. Myers, R. D. 1966. Injection of solutions into cerebral tissue: Relation between volume and diffusion. Physiol. Behav. 1: 171–174. Paxinos, G., and C. Watson. 1986. The Rat Brain in Stereotaxic Coordinates. Academic Press, Sydney. Pertovaara, A., H. Wei, and M. M. Ha¨ma¨la¨inen. 1996. Lidocaine in the rostroventromedial medulla and the periaqueductal gray attenuates allodynia in neuropathic rats. Neurosci. Lett. 218: 127–130. Pertovaara, A., V. K. Kontinen, and E. A. Kalso. 1997. Chronic spinal nerve ligation induces changes in response characteristics of nociceptive spinal dorsal horn neurons and in their descending regulation originating in the periaqueductal gray in the rat. Exp. Neurol., 147: 428–436. Reeh, P. W., L. Kocher, and S. Jung. 1986. Does neurogenic inflammation alter the sensitivity of unmyelinated nociceptors in the rat? Brain Res. 384: 42–50. Ren, K., and R. Dubner. 1996. Enhanced descending modulation of nociception in rats with persistent hindpaw inflammation. J. Neurophysiol. 76: 3025–3037. Sandku¨hler, J., B. Maisch, and M. Zimmermann. 1987. The use of local anesthetic microinjections to identify central neural pathways: A quantitative evaluation of the time course and extent of the neuronal block. Exp. Brain Res. 68: 168–178. Schaible, H.-G., and B. D. Grubb. 1993. Afferent and spinal mechanisms of joint pain. Pain 55: 5–54. Schmidt, R. F. 1973. Presynaptic inhibition in the vertebrate central nervous system. Rev. Physiol. Biochem. Pharmacol. 57: 21–101. Schmidt, R., M. Schmelz, R. Forster, M. Ringkamp, H. E. Torebjo¨rk, and H. O. Handwerker. 1995. Novel classes of responsive and unresponsive C nociceptors in human skin. J. Neurosci. 15: 333–341. Simone, D. A., T. K. Baumann, J. G. Collins, and R. H. Lamotte. 1989. Sensitization of cat dorsal horn neurons to innocuous mechanical stimulation after intradermal injection of capsaicin. Brain Res. 486: 185–189. Simone, D. A., L. S. Sorkin, U. Oh, J. M. Chung, C. Owens, R. H. Lamotte, and W. D. Willis. 1991. Neurogenic hyperalgesia: Central neural correlates in responses of spinothalamic tract neurons. J. Neurophysiol. 66: 228–246. Sorkin, L. S., and D. J. McAdoo. 1993. Amino acids and serotonin are released into the lumbar spinal cord of the anesthetized cat following intradermal capsaicin injection. Brain Res. 607: 89–98. Tattersal, J. E. H., F. Cervero, and B. M. Lumb. 1986. Viscerosomatic neurons in the lower thoracic spinal cord of the cat: Excitations and inhibitions evoked by splanchnic and somatic nerve volleys and by stimulation of brain stem nuclei. J. Neurophysiol. 56: 1411–1423. Torebjo¨rk, H. E., L. E. R. Lundberg, and R. H. Lamotte. 1992. Central changes in processing of mechanoreceptive input in capsaicin-induced secondary hyperalgesia in humans. J. Physiol. (Lond.) 448: 765–780.

202 45.

46.

47.

48.

49.

50.

ANTTI PERTOVAARA Treede, R.-D., and W. Magerl. 1995. Modern concepts of pain and hyperalgesia: Beyond the polymodal C-nociceptor. News Physiol. Sci. 10: 216–228. Treede, R.-D., R. A. Meyer, S. N. Raja, and J. N. Campbell. 1992. Peripheral and central mechanisms of cutaneous hyperalgesia. Prog. Neurobiol. 38: 397–421. Urban, M. O., M. C. Jiang, and G. F. Gebhart. 1996. Participation of central descending nociceptive facilitatory systems in secondary hyperalgesia. Brain Res. 737: 83–91. Vaccarino, A. L., and R. Melzack. 1992. Temporal processing of formalin pain: Differential role of the cingulum bundle, fornix pathway and medial bulboreticular formation. Pain 49: 257–271. Wall, P. D. 1958. Excitability changes in afferent fibre terminations and their relation to slow potentials. J. Physiol. (Lond.) 142: 1–21. Wiertelak, E. P., L. E. Furness, R. Horan, J. Martinez, S. F. Maier, and L. R. Watkins. 1994. Subcutaneous formalin produces centrifugal hyperalgesia at a noninjected site via the NMDA-nitric oxide cascade. Brain Res. 649: 19–26.

51.

Willis, W. D. 1994. Central plastic responses to pain. Prog. Pain Res. Manag. 2: 301–324.

52.

Willis, W. D., and R. E. Coggeshall. 1991. Sensory Mechanisms of the Spinal Cord, 2nd ed. Plenum, New York.

53.

Woolf, C. J. 1984. Long term alterations in the excitability of the flexion reflex produced by peripheral tissue injury in the chronic decerebrate rat. Pain 18: 325–343.

54.

Woolf, C. J., and A. E. King. 1990. Dynamic alterations in the cutaneous mechanoreceptive fields of dorsal horn neurons in the rat spinal cord. J. Neurosci. 10: 2717–2726.

55.

Woolf, C. J., P. Shortland, and L. G. Sivilotti. 1994. Sensitization of high mechanothreshold superficial dorsal horn and flexor motoneurones following chemosensitive primary afferent activation. Pain 58: 141–155. 56. Zhuo, M., and G. F. Gebhart. 1990. Characterization of descending inhibition and facilitation from the nuclei reticularis gigantocellularis and gigantocellularis pars alpha in the rat. Pain 42: 337–350.