unit recording techniques we further studied effects of. s.c. formalin on response ... Formalin injection into the RF of LTM units also result- ed in an increase in ...
Exp Brain Res (1998) 118:14–18
© Springer-Verlag 1998
R E S E A R C H A RT I C L E
&roles:Jun Chen · Natsu Koyama
Differential activation of spinal dorsal horn units by subcutaneous formalin injection in the cat: an electrophysiological study
&misc:Received: 28 August 1996 / Accepted: 2 January 1997
&p.1:Abstract In our previous report we found that subcutaneous (s.c.) formalin injection into the cutaneous receptive field (RF) of dorsal horn wide-dynamic-range (WDR) units and nociceptive primary afferent units resulted in a tonic, long-lasting increase in firing. However, s.c. formalin injection only resulted in a short-lasting increase in firing of non-nociceptive primary afferent units. In the present study, by using extracellular singleunit recording techniques we further studied effects of s.c. formalin on response properties of identified superficial-layer nociceptive-specific (NS) units and deeper-layer, low-threshold mechanoreceptive (LTM) units of L7 dorsal horn in urethane-chloralose-anesthetized cats. s.c. formalin injection into the RF of NS units resulted in a tonic, long-lasting increase in firing (7.08 ± 0.42 spikes/s, n = 5), for more than 1 h, compared with the spontaneous background (1.42 ± 0.03 spikes/s, n = 5). Formalin injection into the RF of LTM units also resulted in an increase in firing; however, the duration was short-lasting, for 25–520 s (152.92 ± 46.73 s, n = 12). The present study demonstrated that s.c. injection of dilute formalin solution resulted in activation of not only nociceptive but also non-nociceptive dorsal horn units, suggesting that tissue injury caused by s.c. formalin results in vigorous injury discharges of peripheral nerve terminals, which subsequently leads to activation of primary afferent neurons and secondary dorsal horn neurons. &kwd:Key words Subcutaneous formalin · Dorsal horn · Nociception · Central neuronal changes · Cat&bdy:
Introduction Subcutaneous (s.c.) injection of dilute formalin solution has been widely used in rodents as an animal model to J. Chen (✉) · N. Koyama Department of Physiology, Shiga University of Medical Science, Seta, Tsukinowa-Cho, Otsu 520–21, Japan Tel. +81-775-48-2147, Fax: +81-775-48-2048,&/fn-block:
study the mechanisms of sustained and prolonged pain or to evaluate the effects of a variety of endogenous and exogenous substances on the nociceptive responses in behavioral studies (for review, see Tjølsen et al. 1992). It has been well established that s.c. formalin injection to rodents (rats and mice) results in biphasic nociceptive response, including an acute phasic response lasting for about 3–5 min followed 15–20 min later by a tonic prolonged response lasting for 20–40 min in behavioral studies (Dubuisson and Dennis 1977; Hunskaar and Hole 1987; Tjølsen et al. 1992; Abbott et al. 1995). Some electrophysiological studies demonstrated that the response of dorsal horn convergent neurons to s.c. formalin correlated well with biphasic behavioral response in rats (Dickenson and Sullivan 1987a, b; Haley et al. 1990). Recently, we have demonstrated that s.c. formalin injection into the low-threshold center of the cutaneous receptive field (RF-ltc) of dorsal horn wide-dynamic-range (WDR) neurons in cats resulted in tonic, long-lasting increase in spike discharges and an expansion of the high-threshold surround of RF (RF-hts) in a monophasic manner (Chen et al. 1996). Our results also showed that activation of the long-lasting increase in spike discharges and expansion of the RF-hts of WDR neurons were induced and maintained by activation of an N-methyl-Daspartate (NMDA) receptor and were primarily dependent upon the ongoing peripheral afferents (Chen et al. 1996). In electrophysiological studies of rat (Dickenson and Sullivan 1987b) and cat (Banna et al. 1986), it has been reported that dorsal horn WDR units were activated by s.c. formalin injection, whereas few or none of the low-threshold mechanoreceptive (LTM) and dorsal column (DC) units were activated by s.c. formalin. In more recent experiments, however, it was reported that s.c. formalin could activate not only Aδ- and C-fibers but also Aβ-fibers in a single-fiber recording study in rats (Puig and Sorkin 1996) and our single primary afferent cell recording from cat L7 dorsal root ganglia (DRG; Chen et al. 1996). Moreover, so far the effects of s.c. formalin on dorsal horn nociceptive-specific (NS) neurons have never been reported.
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Spinal dorsal horn WDR and NS neurons have been demonstrated to be very important in mediation of nociceptive transmission (Christensen and Perl 1970; Light et al. 1979; Willis and Coggeshall 1991). It has been reported that nociceptive and thermoreceptive Aδ-primary afferent fibers project mostly to laminae I, IIo, and V, where NS and/or WDR neurons are located; whereas Aδ LTM fibers project mostly to laminae IIi and III, where most of LTM neurons are located (Christensen and Perl 1970; Light and Perl 1979a, b; Brown 1981; Traub and Mendell 1988; Koerber et al. 1991). It has also been reported that C-fibers supplying LTMs project only in the DC, whereas those innervating high-threshold mechanoreceptors project either through DC or Lissauer’s tract (Chung and Coggeshall 1979; Light and Perl 1979a; Traub and Mendell 1988). In order to elucidate the mechanisms underlying formalin-induced nociception, systematic investigation of the effects of s.c. formalin on dorsal horn neurons is necessary. In the present study, by using an extracellular single-unit recording technique, we further identified NS units in the superficial layer and LTM units in deeper layers of dorsal horn and studied the effects of s.c. formalin on them.
Materials and methods Anesthesia and physiological controls The experiments were performed on either male or female cats weighing from 2.0 to 4.0 kg. The animals were provided by the Laboratory Animal Center of Shiga University of Medical Science (SUMS), and use of the animals was reviewed and approved by the SUMS Animal Care and Use Committee; Principles of laboratory animal care (NIH publication No. 86-23, revised 1985) were followed. The cats were initially anesthetized by intramuscular injection of ketamine HCl (25 mg/kg). Anesthesia was maintained with an intravenous dose of 3.5 ml/kg urethane-chloralose solution
Fig. 1A–D An example of effects of s.c. formalin on nociceptive-specific (NS) units. A Receptive field (RF) of the NS unit. B The unit only responded to noxious pinch but not to brush and pressure applied to the RF, indicated by an arrow in A. Bin width 1 s. C Effects of s.c. formalin on the NS unit. The first arrow indicates insertion, and formalin injection followed 2 min later. Bin width 5 s. D Effect of s.c. formalin on arterial blood pressure. E Locations of seven NS units; one symbol represents one unit. Star, the unit shown in A–D; diamonds, the units recorded from the contralateral side. F Pooled curve from five NS units. Vertical bar, mean ± SEM&ig.c:/f
(urethane 125 mg/ml; chloralose 10 mg/ml) supplemented as required. A tracheal cannula was inserted and the animal was placed in a stereotaxic frame. The animal was then paralyzed by an intravenous injection of pancuronium bromide (2–4 mg/kg per hour) and artificially ventilated with room air. The end-tidal CO 2 was monitored and maintained at 3.5–4.5%. Arterial blood pressure was monitored through a cannula of the carotid artery. Adequate anesthesia was confirmed intermittently by examining whether the animal had spontaneous movements or arousal responses to noxious pinch applied to the skin, especially at the time when the muscle relaxant wore off. Core body temperature was also monitored through a thermister probe inserted into the esophagus and maintained at 37.5 ± 0.5°C by means of a feedback-controlled heating pad under the ventral surface of the abdomen. Surgical preparation and recording procedure Both the left and the right superficial peroneal (SP) and posterior tibial (PT) nerves were dissected free from the surrounding tissues at the level of the ankle joint, and a pair of bipolar platinum hookelectrodes was applied to each of them for electrical stimulation of primary afferent volleys. A laminectomy was performed from the 4th to the 6th vertebrae to expose the lumbar enlargement of the spinal cord. The vertebral column was suspended in a horizontal position by means of two vertebral clamps fixing the 3rd and 7th lumbar vertebrae to the stereotaxic frame. The dura mater was longitudinally opened and the exposed cord was covered with warm paraffin oil (37°C) to prevent it from drying. Extracellular single-unit recordings were made with glass capillary microelectrodes (10–20 MΩ) filled with 2% pontamine sky blue in 0.5 M sodium acetate. Explorations with microelectrodes were made into the L7 spinal dorsal horn in steps of 5 µm with an electronically controlled microstepping micromanipulator. Light stroking and probing of the skin at the hindpaw or electrical stimulation of the SP or PT nerve ipsilateral to the recording side was used as a search stimulus to identify a dorsal horn neuron to be studied. The strength of electrical stimulation was supramaximal for A-fibers but subthreshold for C-fibers (less than 2 V at 0.1 ms, 1 Hz). Single-unit activities of the dorsal horn neuron were amplified and displayed on an oscilloscope, and its output was also fed to a window discriminator. The window discriminator was connected to a spike counter to allow real-time recording of peristimulus time histogram (PSTH) and spikes. Digital data of the firing
16 rates per 1 s or 5 s were also simultaneously recorded by a built-in printer in the spike counter for off-line and statistical analysis. A dorsal horn unit was classified as a NS unit on the basis of its characteristic response only to a noxious mechanical stimulus such as a pinch with small serrated forceps or an alligator clip applied to the whole RF (Fig. 1A, B). They also responded to electrical stimulation of PT or SP nerve with latencies corresponding to conduction velocities of probably Aδ- and C-fiber afferents. However, LTM units responded best to innocuous, low-intensity stimuli such as air-puff, brushing, or indentation of the skin by a glass rod applied to the RF (Fig. 2A), and they responded to electrical stimulation of PT or SP nerve only with latencies corresponding to conduction velocities of Aβ-fiber afferents. Subcutaneous injection with formalin and vehicle Throughout the experiments, 0.1 ml of 5% formalin (1.85% formaldehyde diluted in normal saline) was used. Sterile 0.9% saline was injected into the RF of NS or LTM units, respectively, as a control. In this case, if no further increase in firing was observed, s.c. formalin injection was administered at 32 min or 35 min after vehicle injection. The second NS unit was never studied on the same side; however, the same side could be used for the study of further LTM units, but the distance between the two injection sites had to be more than 3–4 cm. Histological localization of the recording site Recording sites were marked by an electrophoretic deposition of pontamine sky blue from the electrode tip, passing a negative current of 5 µA for 15–20 min. At the termination of each experiment, the animals were deeply anesthetized, flushed with 1000 ml of normal saline, and then perfused with 3000 ml of 10% formalin solution through the ascending aorta. Locations of the dye marks were identified and drawn with camera lucida in cresyl violetstained sections.
Results A total of 31 single units were recorded from L7 dorsal horn of the spinal cord in 13 cats. Of 31 single units, 7 were NS units and 24 were LTM units. Locations of all NS units were in lamina I (Fig. 1E) and those of LTM units were mainly in lamina IV, with a few in laminar III (Fig. 3A, a) on basis of Rexed’s lamination scheme of spinal gray matter in cats (Rexed 1954). Subcutaneous formalin generally resulted in a temporary increase in arterial blood pressure for about 5–15 min in both NS and LTM units (Fig. 1D). Data in the present study were presented as mean ± SEM. Prolonged increase in firing of NS units induced by s.c. formalin Of seven NS units, two units were injected firstly with vehicle (0.1 ml of 0.9% saline), and it was found that vehicle injection into the RF only produced firing immediately after injection and the duration never exceeded over 5 min. However, s.c. formalin injection into a part of the RF of NS units (n = 7) resulted in a tonic, long-lasting firing for more than 1 h. Formalin injection produced a peak firing in the initial 1 min and then the firing rates reached a cer-
Fig. 2A–C An example of effects of s.c. vehicle and formalin on low-threshold mechanoreceptive (LTM) units. A The LTM unit responded to brush applied to the RF, indicated by an arrow above, but not to pressure and pinch. B Effects of s.c. vehicle injection on the LTM unit. Asterisk indicates response of the unit to brush applied to the RF prior to and 15 min and 20 min after injection. C Effects of s.c. formalin on the LTM unit. Asterisk, brush prior to and 20 min after injection. Bin width 1 s&ig.c:/f
tain level (7.08 ± 0.42 spikes/s, n = 5) above the control (1.42 ± 0.03 spikes/s, n = 5) at which it subsided gradually. Figure 1 A–D shows a typical example of the effects of s.c. formalin on NS units. Figure 1F shows a pooled curve representing the mean number of formalin-induced increases in spikes averaged from five NS units. The duration of formalin-induced prolonged firing of NS units was generally the same as that observed in WDR units, reported previously (Chen et al. 1996). Examination of the RF of all NS units 90 min after formalin showed that the injection site became unresponsive even though the units could still be evoked to discharge by noxious pinch applied to the remaining area of the RF. Short-lasting firing of LTM units induced by s.c. formalin Subcutaneous formalin injection into the RF of LTM units resulted in a short-lasting firing. Of a total of 24 LTM units tested with formalin, 20 were activated. Among the 20 units activated by s.c. formalin, most of them (n = 17) showed firing often with a robust delayed-
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Fig. 3A and the pooled curve from them is shown in Fig. 3B.
Discussion
Fig. 3 A Individual time courses of 12 LTM units in response to s.c. formalin injection. Inset a, locations of LTM units studied; one symbol represents one unit. Star indicates the unit shown in Fig. 2; reverse triangles, the units recorded from the contralateral side. B Pooled curve from 12 LTM units. Vertical bar, mean ± SEM&ig.c:/f
peak discharge lasting for 25–450 s (114.44 ± 43.65 s with 1786.22 ± 382.35 spikes averaged from 12 LTM units), the onset time of the delayed-peak discharge varied from 20 to 420 s (182.78 ± 51.56 s, n = 12; Fig. 3A), while the remaining three showed immediate firing (onset time from 0 to 5 s) lasting for 105–520 s (Fig. 3A). However, s.c. injection with vehicle (0.1 ml of sterile 0.9% saline) into the RF of LTM units (n = 5) produced only injecting action-evoked firing followed by none or sporadic discharge at much lower frequency (Fig. 2B). Figure 2 shows an example of the effects of vehicle and formalin on a LTM unit. As shown in Fig. 2B, C, vehicle injection only produced immediate peak firing during injection handling followed by sporadic discharge (less than 0.1 Hz) of less than 5 min, and no further discharge occurred in a 30-min time-course recording. When the RF was applied with brush stimulus at 15 min and 20 min after vehicle injection, the unit responded without any change compared with that prior to injection (Fig. 2B). However, formalin injection into the RF of the same unit 32 min after vehicle injection produced not only immediate firing but also a robust 420-s delayed-peak discharge lasting for 450 s with 3081 spikes (Fig. 2C). In the case of formalin injection, the peak discharge was often followed by a permanent loss of responsiveness of LTM units to any kind of mechanical stimuli applied to the RF (see Fig. 2C). The individual time courses of 12 LTM units are illustrated in
Our present results have demonstrated that s.c. formalin is able to produce a tonic, long-lasting increase in firing of all dorsal horn lamina I NS units tested in the current study; however, s.c. vehicle injection failed to do so, suggesting that dorsal horn NS units, like WDR units (Chen et al. 1996), are also likely to be responsible for formalin-induced nociceptive behavior in cats. The monophasic, long-lasting neuronal responses of dorsal horn nociceptive units to s.c. formalin injection in cats correlate very well to the behavioral responses reported by Dubuisson and Dennis (1977). In rodents, however, s.c. formalin injection results apparently in biphasic firing of dorsal horn WDR units (Dickenson and Sullivan 1987a, b; Haley et al. 1990), which also mirrors the behavioral responses showing flinching or licking of the injected hindpaw (Dubuisson and Dennis 1977; Hunskaar and Hole 1987; Tjølsen et al. 1992; Abbott et al. 1995). The species difference in behavioral responses between felines and rodents in the formalin test is most probably due to the species difference in dorsal horn neuronal responses. In previous behavioral and electrophysiological studies on rats, s.c. formalin-induced second tonic phase of response was considered to be peripheral-independent central sensitization, which might be initially evoked by the first acute phase (activation of C-fiber nociceptors; Dickenson and Sullivan 1987b; Coderre et al. 1990). However, more recently an increasing body of evidence from rodents has strongly demonstrated that biphasic formalin responses are primarily dependent upon peripheral afferents (Dickenson and Sullivan 1987a; Dallel et al. 1995; Taylor et al. 1995; Puig and Sorkin 1996). Similarly to the above results, we also demonstrated that s.c. formalin injection into the RF-ltc of dorsal horn WDR units of cats produces not only tonic, long-lasting firing but also an expansion of the RF-hts. These events are primarily dependent upon the ongoing peripheral afferent barrages, because blockade of the sciatic nerve results in a complete suppression of the formalin-induced responses. Moreover, by using an extracellular singleunit recording technique, we recorded all modalities of identified primary afferent units except for C-polymodal nociceptors from L7 DRG and found that s.c. formalin injection resulted in tonic, long-lasting firing of Aδ highthreshold mechanonociceptive units for 30–70 min and short-lasting firing of non-nociceptive Aβ-, Aδ-, and Cprimary afferent units for about 5–10 min. Our above results from cats are largely in consistence with those from rats reported more recently by Puig and Sorkin (1996). Taking all these results together, it is suggested that the tonic, long-lasting central neuronal changes induced by s.c. formalin injection are primarily dependent upon on-
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going peripheral afferent barrages mediated by nociceptive Aδ- and C-fibers in cats as well as in rats (Chen et al. 1996; Puig and Sorkin 1996). The present study also shows that s.c. formalin injection is able to activate dorsal horn LTM units with a short-lasting increase in firing for 25–520 s. Out of 24 LTM units tested with formalin, 20 were activated. The failure of firing induced by s.c. formalin in the remaining 4 LTM units was probably due to misplacing the injection outside the real RF. The formalin-induced increase in firing of most LTM units (85%, 17/20) shows firing often with a robust, delayed-peak discharge followed by a permanent loss of responsiveness of the unit to natural mechanical stimuli applied to the RF. This phenomenon was often observed in the Aβ primary afferent units recorded from L7 DRG of cats treated with s.c. formalin (Chen et al. 1996). It has been reported also, more recently, by Puig and Sorkin (1996), in a single-fiber recording study in rats. The loss of responsiveness of the injection site, caused by formalin, which has been commonly observed in both dorsal horn and primary afferent units, is probably due to fatal injury of the peripheral nerve terminals. Injured nerve terminals might give forth injury discharges that subsequently lead to activation of primary afferent neurons in the DRG and secondary neurons in the dorsal horn of the spinal cord (Chen et al. 1996). The dorsal horn neuronal activity induced by s.c. formalin correlates very well with that of primary afferent neurons recorded from the L7 DRG in cats (Chen et al. 1996) and that of peripheral afferent fibers recorded from sural nerve in rats (Puig and Sorkin 1996). Accordingly, it is suggested that the duration of formalin-induced firing of dorsal horn units is likely to be dependent primarily upon the input characteristics and number of peripheral afferent fibers innervated. The formalin-induced firing of WDR neurons is most likely to be mediated by both nociceptive and non-nociceptive peripheral afferent fibers and that of NS neurons mediated only by nociceptive ones, whereas that of LTM neurons is mediated only by non-nociceptive ones. &p.2:Acknowledgements J.C. is financially supported by a Japanese Government (Monbusho) Scholarship. The authors are grateful to Prof. K. Jinnai from the Department of Physiology, SUMS, for his encouragement.
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