THE JOURNAL OF COMPARATIVE NEUROLOGY 325:435-445 (1992)

Noradrenergic Modulation of Noxious Heat-Evoked Fos-Like Immunoreactivity in the Dorsal Horn of the Rat Sacral Spinal Cord S.L. JONES Department of Pharmacology, College of Medicine, University of Oklahoma, Oklahoma City, Oklahoma 73190

ABSTRACT The tail-flick withdrawal reflex commonly is used to study spinal nociceptive mechanisms; noradrenergic agonists administered intrathecally inhibit the tail-flick reflex in a dosedependent manner. The objectives of the present study were: (1)to use fos-like immunoreactivity as a marker for neuronal activity to examine the population of neurons in the spinal cord dorsal horn that are engaged by activation of nociceptive tail afferents, and (2) to determine whether fos-like immunoreactivity can be modulated by intrathecally administered alpha adrenoceptor agonists. Neurons demonstrating heat-evoked fos-like immunoreactivity were identified bilaterally in the sacral spinal cord in superficial and deep dorsal horn laminae. Heat-evoked fos-like immunoreactivity was inhibited dose-dependently by intrathecal norepinephrine (NE). The inhibition was attenuated significantly by: (1)phentolamine (PHEN), a nonselective alpha adrenoceptor antagonist; (2) yohimbine (YOH), an alpha-2 adrenoceptor antagonist; and ( 3 ) prazosin (PRAZ), an alpha-1 adrenoceptor antagonist. Thus, both spinal alpha-1 and alpha-2 adrenoceptors mediate the inhibition of heat-evoked fos-like immunoreactivity produced by intrathecal NE. ST-91, an alpha-2 adrenoceptor agonist, also inhibited significantly the expression of fos-like immunoreactivity; the inhibition was antagonized by YOH. In the absence of noxious heat, intrathecal NE dose-dependently evoked the expression of fos-like immunoreactivity in the superficial dorsal horn, which was antagonized by PHEN and PRAZ, but not by YOH, suggesting that the effect is mediated by spinal alpha-1 adrenoceptors. t ) 1992 Wiley-Liss, Inc. Key words: norepinephrine, descending inhibition, antinociception, immunohistochemistry, intrathecal

Intrathecally administered norepinephrine (NE) and selective alpha-2 adrenoceptor agonists produce analgesia in humans (e.g., Eisenach et al., '891, and an antinociception in both visceral and cutaneous experimental models of pain (e.g., Yaksh, '85; Solomon et al., '89; Danzebrink and Gebhart, '90; Nagasaka and Yaksh, '90; Fisher et al., '91; Jones, '91a). Recently, considerable attention has been focused on understanding the mechanisms and sites of action of nonopioid systems of analgesia. It has been established that: (I)spinal sites of action contribute to the antinociceptive effects produced by monoamines, (2) spinal alpha-2 adrenoceptors predominantly mediate the antinociceptive effects produced by monoamines, and (3) intrathecally administered NE and alpha adrenoceptor agonists modulate selectively nociceptive transmission (e.g., Reddy et al., '80; Howe et al., '83; Yaksh, '85). O

1992 WILEY-LISS, INC.

Electrophysiological data do support behavioral data that suggest that NE modulates sensory transmission in the spinal cord dorsal horn. However, electrophysiological evidence does not entirely support the notion that NE inhibits selectively spinal nociceptive transmission; indeed, NE has both excitatory (e.g., Todd and Millar, '83; Howe and Zieglgansberger, '87; Millar and Williams, '89) and inhibitory (e.g., Belcher et al., '78; Headley et al., '78; North and Yoshimura, '84; Willcockson et al., '84; Fleetwood-Walker et al., '85) effects on dorsal horn neuronal activity. Interestingly, the excitatory effects of NE appear to occur predomiAccepted July 31, 1992. Address reprint requests to Dr. S.L. Jones, Department of Pharmacology, 764 BMSB, University of Oklahoma Health Sciences Center, 940 Stanton L. Young Blvd., Oklahoma City, OK 73190.

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nantly in superficial dorsal horn laminae. Thus, superficial dorsal horn neurons may be modulated by NE in a manner different from deep dorsal horn neurons, and consequently may subserve different functions. In the spinal cord dorsal horn, primary afferent fibers conveying specific nociceptive information from the periphery terminate in superficial laminae; thus, dorsal horn neurons located in laminae I and I1 of the spinal cord are important participants in spinal nociceptive processing (e.g., Light and Perl, '79). In the rat, dense distributions of alpha-2 adrenoceptor binding sites have been identified in lamina 11, adjacent to the central canal, and in the intermediolateral cell column; these distributions parallel the distributions of catecholamine-containing varicosities in the spinal cord dorsal horn (Young and Kuhar, '79; Seybold and Elde, '84; see Seybold, '86 for review). As a result, catecholamine-containing terminations and alpha-2 adrenoceptors anatomically are ideally situated to be involved in the modulation of spinal nociceptive transmission. Prior to the development of fos immunohistochemistry, it was not possible to examine the neurons in the spinal cord that, as a population, functionally are involved in the transmission of nociceptive information. Thus, the objectives of the present study were: (1)to utilize fos immunohistochemistry as a marker for neuronal activity to examine the population of neurons in the spinal cord dorsal horn that are engaged by activation of nociceptive tail afferents, and (2) to examine the ability of intrathecally administered alpha adrenoceptor agonists to modulate fos-like immunoreactivity. Some of these data have been presented in abstract form (Jones, '9 lb).

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severalfold: (1)because the distal end of the catheter was placed rostral to the region of interest, potential mechanical damage to the sacral spinal cord by the catheter was minimized, (2) placement of the catheter at the rostral extent of the sacral spinal cord allowed the volume of injectate to flow over the sacral spinal cord, and (3) one control indicated that insertion of the intrathecal catheter resulted in no significant expression of fos-like immunoreactivity in the spinal cord in the region of interest. Injections were initiated immediately following insertion of the catheter. The location of the distal end of the catheter was verified at the conclusion of the experiment when the spinal cord was removed; results obtained from two animals in which the catheter placement was not at the level of the sacral spinal cord, or in which the spinal cord was grossly damaged, were not included in the data analysis. Pharmacologic agonists and antagonists were administered intrathecally in volumes of 10 pl, followed by 10 ~1 flushes of normal saline. All intrathecal injections were made slowly over a period of about 1 minute. The drugs used were: norepinephrine HC1 (NE; 15, 30 and 60 nmoles; pH 5.43, 5.38 and 5.20, respectively; Sigma, St. Louis, MO), phentolamine HCl (PHEN; 60 nmoles; pH 4.38; Sigma), prazosin HCl (PRAZ; 60 nmoles; pH 5.65; Sigma), yohimbine HCl (YOH; 60 nmoles; pH 5.30; Sigma) and ST-91 (80 nmoles; pH 6.4; supplied by Boehringer Ingelheim, Ridgefield, CT). The doses of agonists (NE and ST-91) used in these experiments were chosen on the basis of reports that they produce antinociceptive effects in a variety of nociceptive tests (hot plate, tail-flick, and colorectal distention) when administered intrathecally; similarly, the doses of antagonists (PHEN, PRAZ, YOH) used were doses that MATERIALS AND METHODS have been reported to block those antinociceptive effects Experiments were performed on 112 adult male Sprague- (e.g., Reddy et al., '80; Howe et al., '83; Danzebrink and Dawley rats (Sasco Labs, Omaha, NE) weighing between Gebhart, '90; Nagasaka and Yaksh, '90). All drugs were prepared freshly for each experiment and, 300 and 350 g. Rats were deeply anesthetized with sodium pentobarbital (45 mgikg, i.p.), and were maintained in a with the exception of prazosin, were dissolved in sterile surgical plane of anesthesia (i.e., withdrawal reflexes were physiological saline (0.9% NaC1). Prazosin was dissolved in absent) throughout the duration of the experiments with distilled water. Heating and stirring were required to supplemental doses of sodium pentobarbital (3-6 mgikgi dissolve both the prazosin and yohimbine. hr, i.p.). Body temperature was maintained at 36-38°C with Experimental protocol an electric heating pad. All drugs were administered intrathecally in the absence Nociceptive stimulus and in the presence of noxious heating of the tail to The tail was immersed in 55-60°C water for a duration of determine their effects on foa-like immunoreactivity in the 10 seconds for 30 consecutive trials at 2 minute intervals. spinal cord dorsal horn and their effects on noxious heatWith the exception of a slight reddening of the skin, the evoked fos-like immunoreactivity, respectively. protocol used resulted in no blister formation or other Following insertion of the intrathecal catheter, four visible damage to the skin of the tail. Preliminary experi- injections of agonist (10 pl each) were administered intraments indicated that this protocol reliably and reproducibly thecally at 30 minute intervals. Each injection of agonist evoked the expression of fos-like immunoreactivity in the was followed by a 10 ~1 flush of normal saline to clear the sacral spinal cord. catheter. In animals exposed to noxious heat, the tail was immersed in 55-60°C water for 10 second durations immeIntrathecal injections diately following the first intrathecal injection of agonist, Intrathecal injections of pharmacologic agonists and and was continued for 30 consecutive trials at 2 minute antagonists were made at the level of the sacral spinal cord. intervals. Intrathecal injections of agonists were continued A horizontal slit was made in the atlanto-occipital mem- following completion of noxious heating of the tail to brane and an 8.5 cm length of PE-10 tubing measured to minimize possible confounding influences of continued extend to the rostral level of the sacral spinal cord was stimulation of nociceptive afferents due to secondary effects inserted into the subarachnoid space (Yaksh and Rudy, produced by the noxious heat (e.g., inflammation, edema, '76). The catheter was sutured to muscle at its rostral end. etc.). NE and ST-91, administered intrathecally, have been Afferent terminations from the tail are localized in the demonstrated to: (1)produce maximal antinociceptive efsacral spinal cord. Thus, the reasons for placement of the fects within 20-30 minutes of administration, and (2) have catheter at the rostral extent of the sacral spinal cord were a duration of action of approximately 60-120 minutes (e.g.,

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NA MODULATION OF HEAT-EVOKED FOS PROTEINS Reddy et al., '80; Howe et al., '83; Aran and Proudfit, '90; Nagasaka and Yaksh, '90). Thus, by administering the agonists at 30 minute intervals, maximal antinociceptive effects were maintained for the duration of the experiment. In experiments in which antagonists were administered in combination with agonists, four intrathecal injections of antagonist (10 p1 each) also were administered at 30 minute intervals. Injections of antagonist were initiated 10 minutes prior to the first injection of agonist; each injection of antagonist was followed by a 10 pI flush of normal saline to clear the catheter. Agonists and noxious heat then were administered according to the protocol described above. Intrathecally administered antagonists have been reported to produce their effects within 5-10 minutes following administration (e.g., Howe et al., '83; Sagen and Proudfit, '84).

Time course studies revealed that a 2 hour survival time (following the first noxious stimulus) resulted in optimal expression of heat-evoked fos-like immunoreactivity in the sacral spinal cord (see Results). Therefore, for all subsequent experiments, a 2 hour survival time was used.

Histology At the conclusion of the experiments, the rats were euthanized with an overdose of sodium pentobarbital (75100 mgikg, i.p.1, and perfused transcardially with 100-150 ml of heparinized phosphate buffered saline (PBS, 0.01 M, pH 7.5, 37'0, followed by 500 ml of 4% paraformaldehyde (4°C). A laminectomy was done to expose the spinal cord, and placement of the intrathecal catheter was confirmed. Spinal cord segments L5-L6 and the remaining caudal extent of the sacral spinal cord were blocked in situ, removed and postfixed overnight in 4% paraformaldehyde. The spinal cord tissue was sectioned on a vibratome (50 km coronal sections); all sections were saved and processed for fos-like immunoreactivity according to the following protocol.

(Cambridge Research Biomedical) and used in the protocol described above. Incubating fos antibody overnight with 7 mM synthetic fos peptide resulted in no staining indicative of fos-like immunoreactivity, thus demonstrating that the primary antibody and resultant neuronal labeling are specific for the fos oncoprotein. Omitting the c-fos antiserum from the protocol described above also produced no neuronal labeling in the sacral spinal cord, thus indicating that the observed labeling is not nonspecific labeling produced by the secondary antibody.

Data analysis Every fourth tissue section (i.e.,approximately every 200 pm of tissue; 20-30 sections per animal) was scanned by brightfield microscopy; labeled neurons were plotted with the aid of a camera lucida drawing tube. It should be noted that neurons demonstrating fos-like immunoreactivity exhibited varying intensities of staining (see Fig. 1).However, neurons were not classified on the basis of their intensity of staining; all neurons demonstrating staining were considered to be positively labeled. For each animal, the numbers of labeled neurons in laminae 1/11 and in deep laminae (laminae 111-VI) were totaled and the average numbers of neurons demonstrating fos-like immunoreactivity per section were calculated. Individual means within each experimental group then were averaged; results are expressed as s.d. All statistics were done with the CSS the mean Statistica program; comparisons were made by a one-way analyses of variance with Newman-Keuls post-hoc tests. P < .05 was considered significant.

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RESULTS Distribution of heat-evoked fos-like immunoreactivity

Neurons demonstrating fos-like immunoreactivity evoked by noxious heating of the tail were identified bilaterally in Immunohistochemistry the dorsal horn in all sacral spinal cord segments (Fig. 1A). The avidin-biotin-peroxidase complex (ABC) technique No labeled cells were identified in the lumbar spinal cord. was used for immunohistochemical localization of fos-like That the distribution of fos-like immunoreactivity was immunoreactivity, and diaminobenzidine (DAB) served as topographically organized in a manner consistent with the the chromagen. The reaction was carried out at room termination sites of tail afferents suggests that: (1) the temperature unless otherwise indicated. Free floating tis- expression of heat-evoked fos-like immunoreactivity is spesue sections were incubated sequentially in the following cific for the activation of tail afferents, and ( 2 ) mechanical solutions: 3 rinses with 0.01 M PBS, 10 minutes each; 0.3% perturbation of the spinal cord tissue produced by insertion triton X-100/4% normal rabbit serum (NRS)/0.01M PBS, of the intrathecal catheter rostra1 to S1 was not sufficient to 1 hour; sheep anti-c-fos oncoprotein (Cambridge Research nonspecificallyevoke the expression of fos-like immunoreacBiomedical) 1:6,000 diluted in 4%NRSiO.O1 M PBS, over- tivity. Neurons demonstrating heat-evoked fos-like immunorenight at 4°C; 2 rinses with 4% NRSi0.01 M PBS, 10 activity predominantly were located in the superficial lamiminutes each; biotinylated anti-goat IgG (Vector Labs, 47 p1110 ml of 4% NRS/0.01 M PBS), 1hour; 2 rinses with 4% nae (laminae 1/11]of the spinal cord dorsal horn; however, NRSi0.01 M PBS, 10 minutes each; Vectastain ABC solu- cells also were identified in deep dorsal horn laminae. tion (Vector Labs, 2 drops A and 2 drops B in 10 mlO.01 M Labeled neurons were identified throughout the mediolatPBS), 45 minutes; 2 rinses with 4% NRSi0.01 M PBS, 10 era1 extent of the spinal cord. minutes each; biotinylated anti-goat IgG, 30 minutes; 2 Time course rinses with 0.01 M PBS, 10 minutes each; Vectastain ABC solution, 30 minutes; 3 rinses with 0.01 M PBS, 10 minutes The expression of fos-like immunoreactivity in the spinal each; filtered solution of 0.05% DAB and 0.004% HzOz in cord evoked by nociceptive stimuli (e.g., the formalin test 0.01 M PBS, 20-30 minutes; 5 rinses with 0.01 M PBS, 5 and thermal cutaneous stimuli) has been reported to vary minutes each. Tissue sections were mounted onto gelatin- with time (e.g., Presley et al., '90; Williams et al., '90). coated slides, air-dried, dehydrated in xylene for 5 minutes Thus, initial experiments determined systematically the optimal survival time following initiation of noxious heatand coverslipped. To establish the specificity of the primary antibody, fos ing of the tail for the expression of fos-like immunoreactivantiserum was preabsorbed with synthetic fos peptide ity in the spinal cord dorsal horn using the following

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Fig. 1. Example photomicrographs of fos-like immunoreactivity in 50 +m transverse sections of the spinal cord dorsal horn. A Vehicle control (intrathecal normal saline, NS) for heat-evoked fos-like immunoreactivity. B. Inhibition of heat-evoked fos-like immunoreactivity by intrathecal norepinephrine WE, 60 nmoles). C:Fos-like immunoreac-

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tivity evoked by NE (60 nmoles) in the absence of noxious heat. D: Attenuation of the inhibition of heat-evoked fos-like immunoreactivity produced by NE (60 nmoles) by the alpha-2 antagonist yohimbine (YOH, 60 nmoles).

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Considering the neuronal population as a whole, there were no significant differences in the average numbers of cells per section expressing fos-like immunoreactivity at the 1,2, and 2.5 hour survival times (Fig. 2A). However, after 3 hours, there were significantly fewer neurons expressing fos-like immunoreactivity than at any of the shorter survival times. If the neurons are segregated according to their location in laminae 1/11 versus deep laminae (Fig. 2B), a slightly different pattern of significance appears. In laminae 1/11, there were no significant differences in the average numbers of labeled neurons per section at the 1, 2, and 2.5 hour survival times; however, the average number of labeled neurons per section at 3 hours was significantly less than at the 1or 2 hour survival times. In deep laminae, at 2 hours, the average number of labeled neurons per section was significantly greater than at any of the other survival times. At 3 hours, the average number of fos-positive cells per section was significantly less than the average number at 2 and 2.5 hours, but not at 1 hour. Thus, the maximal number of neurons expressing fos-like immunoreactivity occurred following a 2 hour survival time; the labeling was significantly diminished after 3 hours. As a result, a 2 hour survival time was used in all subsequent experiments.

Intrathecal vehicle controls

To determine the effects of intrathecally administered alpha adrenoceptor agonists on fos-like immunoreactivity, + $ 20it was essential to establish that the intrathecal injection + +O a (i.e., the volume of the injectate and/or the drug vehicle) itself did not nonspecifically evoke fos-like immunoreactivB I .o 2 .O 2.5 3.0 ity. Injections of vehicle (normal saline, NS; four injections; Survival Time (hrs) 10 ~1 each) were administered intrathecally at 30 minute intervals in the absence of noxious heating of the tail. Fig. 2 . Average numbers of cells per section, demonstrating fos-like Intrathecal injections of NS evoked no significant expresimmunoreactivity in the spinal cord dorsal horn (A), and in superficial (laminae 1/11) and deep laminae fB)a t varying survival times following sion of fos-like immunoreactivity in the spinal cord dorsal horn, in laminae 1/11 or in deep dorsal horn laminae (mean initiation of noxious heating of the tail. A *, indicates a significant difference compared to the 1hour, 2 hour and 2.5 hour survival times, 2.2 ? 3.2,2.2 2 3.1,O.l 5 0.1 cellsper section, respectively; (P < .05). B: *, +, or 0 indicates a significant difference compared to n = 2). Thus, the possible mechanical perturbation of the 1 hour, 2 hour or 2.5 hour survival times, respectively (P < ,051. spinal cord tissue produced by the volume of the injectate Statistical comparisons were made between the various survival times and/or nonspecific effects produced by NS are not sufficient within either laminae 1/11or deep laminae. to evoke the expression of fos-like immunoreactivity in the spinal cord dorsal horn. To establish control values for the expression of fos-like experimental paradigm. The tail was immersed in 55-60°C water for 10 seconds for 30 consecutive trials at 2 minute immunoreactivity evoked by noxious heat, injections of NS intervals; the animals were allowed to survive for 1, 2, 2.5, were administered intrathecally at 30 minute intervals in or 3 hours following initiation of the noxious stimulus. the presence of noxious heating of the tail. In the spinal Following a 1, 2, 2.5, or 3 hour survival time, the average cord dorsal horn, the average number of cells per section numbers of cells per section expressing heat-evoked fos-like demonstrating heat-evoked fos-like immunoreactivity was immunoreactivity in the spinal cord dorsal horn were 84.7 5 10.6 (range 101.8-71.6, n = 6). Specifically, in 80.4 ? 9.0 (range 71.4-92.6, n = 41, 108.5 5 25.4 (range laminae 1/11 and in deep dorsal horn laminae, the average 67.5-135.3, n = 51, 92.3 ? 10.1 (range 85.8-104.0, n = 3) numbers of cells per section demonstrating heat-evoked 9.0 (range 63.2and 55.2 f 5.7 (range 49.4-62.7, n = 4), respectively (Fig. fos-like immunoreactivity were 74.4 86.6) and 9.0 2.8 (range 6.1-13.6), respectively (see also 2A). Cell counts specifically in laminae 1/11 and in deep dorsal horn laminae revealed a similar pattern of heat- Fig. 1A). These values were used as controls against which to evoked fos-like immunoreactivity (Fig. 2B). In laminae 1/11 following a 1, 2, 2.5, or 3 hour survival time, the average compare other intrathecal drug effects on fos-likeimmunorenumbers of cells per section demonstrating fos-like immu- activity both in the absence and in the presence of noxious noreactivity were 69.2 5 6.6 (range 64.1-78.9), 85.2 i- 20.6 heating of the tail (seebelow and Figs. 3 and 4). (range 51.7-108.01, 74.8 ? 10.6 (range 68.3-87.0) and 48.5 Intrathecal norepinephrine dose response 2 5.9 (range 44.1-56.8), respectively. In deep dorsal horn relationships laminae the average numbers of cells per section demonstrating fos-like immunoreactivity were 8.6 ? 2.2 (range Dose response relationships were established for intra5.8-11.0), 20.3 k 5.4 (range 13.4-28.11, 13.6 ? 0.9 (range thecal NE in the absence and presence of noxious heat (Fig. 12.7-14.5) and 4.6 ? 1.2 (range 3.4-6.2) following a 1, 2, 3). Four injections of 15430, or 60 nmoles of NE (10 p1 each) were administered intrathecally at 30 minute intervals and 2.5, or 3 hour survival time, respectively. 0

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440 the effects on fos-like immunoreactivity determined. NE dose-dependently evoked the expression of fos-like immunoreactivity in the absence of noxious heat, and dosedependently inhibited the expression of heat-evoked foslike immunoreactivity in the spinal cord dorsal horn. Thus, NE had both excitatory and inhibitory effects on the expression of fos-like immunoreactivity. In the absence of noxious heat, NE dose-dependently evoked the expression of fos-like immunoreactivity in the spinal cord dorsal horn (Fig. 3A, see also Fig. 1C).Considering the neuronal population as a whole, the average numbers of cells per section expressing fos-like immunoreactivity following 15, 30, and 60 nmole doses of intrathecal NE were 23.4 f 5.1 (range 15.6-28.4, n = 5),24.0 t 5.1 (range 15.8-27.2, n = 5) and 37.6 t 7.2 (range 30.1-49.5, n = 5), respectively. At all doses of NE, the average numbers of cells per section expressing fos-like immunoreactivity were significantly greater compared to control (0 nmole NE). As illustrated in Figure 3C, intrathecal NE evoked fos-like immunoreactivity in neurons predominantly located in laminae 1/11.Following 15, 30, and 60 nmole doses of NE, the average numbers of fos-positive cells per section in laminae 1/11 were 21.1 2 4.9 (range 14.3-26.01, 21.6 t 5.7 (range 12.9-26.2) and 34.2 2 7.0 (range 28.4-45.9), respectively. At all doses of NE, the average numbers of cells per section expressing fos-like immunoreactivity were significantly greater compared to control (0 nmole NE). In deep dorsal horn laminae, the average numbers of fos-positive cells per section following 15,30, and 60 nmole doses of NE were 2.3 2 0.9 (range 1.2-3.8), 2.4 2 1.0 (range 1.5-3.9) and 3.3 t 1.5 (range 1.6-4.8), respectively. However, these were not significantly different from control. In the presence of noxious heating of the tail, NE dose-dependently inhibited heat-evoked fos-like immunoreactivity (Fig. 3). Considering the neuronal population as a whole, the average numbers of cells per section expressing heat-evoked fos-like immunoreactivity following 15, 30, and 60 nmole doses of intrathecal NE were 88.5 t 14.4 (range 65.4-101.2, n = 51, 60.0 k 14.6 (range 47.3-84.6, n = 6), and 47.4 +- 5.9 (range 37.1-56.5, n = 8 ) , respectively (Figs. 1 B and 3A). Following 30 and 60 nmoles of intrathecal NE, the average numbers of cells per section expressing heat-evoked fos-like immunoreactivity were significantly different compared to control (0 nmole NE) and to each other. The inhibition of heat-evoked fos-like immunoreactivity produced by 15 nmoles of intrathecal NE was not significant compared to control; however, as discussed above, 15 nmoles of NE did significantly evoke fos-like

Fig. 3. Intrathecal norepinephrine (NE) dose-dependently modulates the expression of fos-like immunoreactivity in the spinal cord dorsal horn. A: Average numbers of cells per section, demonstrating fos-like immunoreactivity in the spinal cord dorsal horn with varying doses of intrathecal NE in the presence ( 0 ,NE + Heat) and absence (A,NE) of noxious heat. NE's inhibitory effect on heat-evoked fos-like immunoreactivity is indicated by subtracting the average numbers of cells per section expressing NE evoked fos-like immunoreactivity from the average numbers of cells per section expressingheat-evoked fos-like immunoreactivity in the presence of NE (m, [NE + Heat] - NE). +, significantly different from 30 nmole NE. B: Dose-dependent inhibition of heat-evoked fos-like immunoreactivity in laminae 1/11 and deep dorsal horn laminae by NE. f, significantly different from 15 nmole NE. C: NE dose-dependently evoked fos-like imrnunoreactivity in laminae 1/11 and deep dorsal horn laminae in the absence of noxious heat. A, Band C: *, significantly different from control ( 0 nmole NE); 0, significant difference between NE + Heat and NE. All data are presented as means % s.d.; P < .05 was considered significant.

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+ Heat Fig. 4. Effects of intrathecal alpha adrenoceptor agonists and antagonists on fos-like immunoreactivity in laminae 1/11 and deep dorsal horn laminae in the presence (cross-hatched bars) and absence (open bars) of noxious heat. Note the different scales on the y-axes. All drugs were administered in volumes of 10 pl,followed by a 10 p1 flush of normal saline. PHEN, (nonselective alpha-lialpha-2antagonist), PRAZ (alpha-1 antagonist), and YOH (alpha-2 antagonist) blocked significantly the inhibition of heat-evoked fos-like immunoreactivity produced by NE, suggesting that both spinal alpha-1 and alpha-2 adrenoceptors mediate the inhibition produced by NE. ST-91 (alpha-2 agonist) also inhibited the expression of heat-evoked fos-like immunoreactivity and was blocked by YOH. In contrast, only PHEN and PRAZ blocked significantly fos-like immunoreactivity evoked by intratbecal NE in the

absence of noxious heat; ST-91 did not evoke significantly fos-like immunoreactivity compared to control (NS). Thus, fos-like immunoreactivity evoked by intrathecal NE is mediated by spinal alpha-1 adrenoceptors. *, significantly different compared to NS or NS + heat; +, significantly different compared to NE or NE + heat; 0, significantly different compared to ST-91 + heat; 0, significantly different from NE + YOH. Statistical comparisons were made between groups within laminae 1/11or deep laminae. All data are presented as means t s.d.; P < .05 was considered significant. Abbreviations: NS, 10 pl normal saline; NE, 60 nmoles norepinephrine; PHEN, 60 nmoles phentolamine; PRAZ, 60 nmoles prazosin; YOH, 60 nmoles yohimbine; ST-91, 80 nmoles 2-[2,6-dimethylphenylaminol2-imidazoline.

immunoreactivity in the absence of noxious heat. Following 15 and 30 nmoles of NE, significant differences were noted between the average numbers of cells per section expressing fos-like immunoreactivity in the absence and presence of noxious heat (Fig. 3A). However, following 60 nmoles of NE, the average number of fos-positive cells per section inhibited by NE was not significantly different from the average number of fos-positive cells per section evoked by NE in the absence of noxious heat.

If the average numbers of fos-positive neurons per section were examined according to their location in the spinal cord dorsal horn (i.e., laminae 1/11 vs. deep laminae), similar patterns of dose-dependent inhibition of heatevoked fos-like immunoreactivity were observed (Fig. 3B). In laminae 1/11, the average numbers of cells per section expressing fos-like immunoreactivity following 15, 30, and 60 nmole doses of NE were 83.5 13.4 (range 61.8-95.01, 57.9 f 14.4 (range 43.8-81.8), and 44.3 2 5.9 (range 34.2-

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50.0), respectively. The inhibition of heat-evoked fos-like immunoreactivity was not significantly different from control (0 nmoles NE) following 15 nmoles of intrathecal NE; however, the average numbers of cells per section demonstrating fos-like immunoreactivity were significantly fewer than control following 30 and 60 nmoles of NE. In deep dorsal horn laminae, the average numbers of fos-positive cells per section following 15,30, and 60 nmole doses of NE were 4.9 i 1.1 (range 3.6-6.11, 2.3 1.3 (range 0.6-3.5) and 3.2 2 0.9 (range 1.5-4.61, respectively. All three doses of NE significantly reduced labeling compared to control; however, there was no significant dose-related effect, probably due to the small number of fos-positive cells in deep laminae. That inhibition of heat-evoked fos-like immunoreactivity was also produced in deep dorsal horn laminae suggests that intrathecal NE did penetrate the spinal cord in concentrations sufficient to influence the expression of heat-evoked fos-like immunoreactivity.

ST-91 effects To confirm that spinal alpha-2 adrenoceptors mediate the inhibition of heat-evoked fos-like immunoreactivity, the selective alpha-2 adrenoceptor agonist ST-91 (2-[2,6diethylphenylaminol-2-imidazoline, 80 nmoles) was administered intrathecally in the presence and absence of noxious heat (Fig. 4). In laminae 1/11,ST-91 inhibited significantly noxious heat-evoked fos-like immunoreactivity (mean 30.8 ? 6.7 cells per section, range 21.1-39.8, n = 5) compared to control (74.4 t 9.0). The inhibition was significantly greater than that produced by NE alone, and was abolished by intrathecal YOH (64.5 f 9.2 cells per section, range 53.4-74.8, n = 5; P < .05). In contrast to NE, ST-91 administered in combination with YOH did not increase significantly the average number of cells per section expressing heat-evoked fos-like immunoreactivity compared to control. Similarly, in contrast to NE, when ST-91 was administered intrathecally in the absence of noxious heating of the tail, ST-91 did not evoke significantly the expression of fos-like immunoreactivity in laminae 1/11 compared to control (mean 10.4 3.5 cells per section, range 5.6-14.2, n = 5 vs. 2.2 2 3.1 cells per section; P = .61). In deep dorsal horn laminae, ST-91also inhibited significantly heat-evoked fos-like immunoreactivity compared to control (mean 1.3 t_ 0.7 cells per section, range 0.6-2.2, n = 5 vs. 9.0 & 2.8). However, the inhibition was not attenuated significantly by intrathecal YOH.

*

Antagonism of NE effects Selective alpha adrenoceptor antagonists were administered intrathecally to elucidate which alpha adrenoceptor subtypes mediate the effects on fos-like immunoreactivity produced by NE. As described previously (see Materials and Methods), intrathecal NE (60 nmoles) was administered in combination with the nonselective alpha adrenoceptor antagonist phentolamine (PHEN, 60 nmoles), the selective alpha-1 adrenoceptor antagonist prazosin (PRAZ, 60 nmoles), or the selective alpha-2 adrenoceptor antagonist yohimbine (YOH, 60 nmoles) (Fig. 4). Results indicate that fos-like immunoreactivity in laminae 1/11 evoked by intrathecal NE in the absence of noxious heat is mediated by spinal alpha-1 adrenoceptors. The expression of fos-like immunoreactivity evoked by 60 nmoles of NE in the absence of noxious heat (mean 34.2 -+ 7.0 cells per section, range 28.4-45.9, n = 5)was abolished (P < .05) by PHEN (mean 9.8 r 7.0 cells per section, range 2.4-18.7, n = 5) and PRAZ (mean 12.7 t 6.4 cells per section, range 8.2-22.8, n = 5), but not by YOH (mean 31.9 12.2 cells per section, range 19.2-46.9, n = 5) (Fig. 4). In laminae 1/11, the inhibition of heat-evoked fos-like immunoreactivity produced by 60 nmoles of NE (mean 44.3 f 5.9 cells per section, range 34.2-52.6, n = 8 ) was abolished (P < ,051 by PHEN (mean 66.8 2 4.1 cells per section, range 59.9-70.8, n = 51, PRAZ (mean 65.7 2 13.4 cells per section, range 49.4-85.3, n = 5) and YOH (mean 88.5 ? 10.2 cells per section, range 75.6-102.2, n = 5 , see also Fig. 1D). Thus, both spinal alpha-1 and alpha-2 adrenoceptors mediate the inhibition of heat-evoked foslike immunoreactivity produced by intrathecal NE. Furthermore, YOH elevated fos labeling above control (74.4 ? 9.0 vs. 88.5 c 10.2; P < .05), suggesting that YOH antagonized the inhibitory alpha-2-mediated effects produced by NE, but not the alpha-1-mediated excitatory effects produced by NE (Fig. 4). In deep dorsal horn laminae, the average numbers of cells per section demonstrating fos-like immunoreactivity were few; thus, statistical significance was not achieved in the antagonist studies (Fig. 4). As a result, it was not possible to make conclusions regarding the role of specific alpha adrenoceptors in the modulation of fos-like immunoreactivity in deep dorsal horn laminae.

*

DISCUSSION Results of this study indicate that intrathecally administered NE has both excitatory and inhibitory effects on the expression of fos-like immunoreactivity in the spinal cord dorsal horn; the overall effect of NE on heat-evoked fos-like immunoreactivity represents a complicated interaction of alpha-1 and alpha-2-mediated effects. The rationale for this interpretation is as follows. In the absence of noxious heat, NE produced an increase in fos labeling. The effect was abolished by PHEN and PRAZ, but was unaffected by YOH, suggesting that the increase in fos-like immunoreactivity produced by intrathecal NE is mediated by spinal alpha-1 adrenoceptors. In contrast, NE inhibited heat-evoked foslike immunoreactivity; the inhibition was abolished by PHEN, PRAZ, and YOH. Similarly, administration of ST-91 inhibited heat-evoked fos-like immunoreactivity, and the inhibition was abolished by YOH. Thus, both spinal alpha-1 and alpha-2 adrenoceptors mediate the inhibition of heatevoked fos-like immunoreactivity produced by NE. That the increase in fos labeling evoked by the stimulation of alpha-1 adrenoceptors is superimposed on the overall inhibitory effect of NE on heat-evoked fos labeling is suggested by the following lines of evidence. First, ST-91 inhibited fos-like immunoreactivity to a greater extent than did NE, suggesting that fos-like immunoreactivity expressed following intrathecal NE represents a combination of both excitatory and inhibitory effects. Second, YOH not only abolished the inhibition of fos-like immunoreactivity produced by NE, but also unmasked NE’s ability to significantly enhance heat-evoked fos-like immunoreactivity, suggesting that excitation predominantly is an alpha-1-mediated effect. In summary, the increase in fos-like immunoreactivity produced by intrathecal NE in the absence of noxious heat is an alpha-1-mediated effect. The inhibition of heat-evoked fos-like immunoreactivity produced by intrathecal NE rep-

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NA MODULATION OF HEAT-EVOKED FOS PROTEINS resents an interaction of inhibitory effects mediated by spinal alpha-2 adrenoceptors and mixed excitatoryiinhibitory effects mediated by spinal alpha-1adrenoceptors. Based on the results discussed above, it might be predicted that intrathecal NE given in combination with PRAZ should produce an inhibitory effect equivalent to that produced by ST-91 (i.e., a pure alpha-2 effect). It is unclear why this apparent inconsistency in the results was obtained. Further investigation will be required to adequately explain this phenomenon.

Inhibitory effects of norepinephrine Intrathecal studies. Intrathecally administered adrenoceptor agonists produce dose-dependent antinociceptive effects in both rats and cats. Systematic studies examining structure activity relationships have determined that spinal alpha adrenoceptors mediate the elevated nociceptive thresholds produced by intrathecally administered adrenoceptor agonists, including NE (e.g., Reddy and Yaksh, '80; Reddy et al., '80; Howe et al., '83; Fisher et al., '91). The relative roles of alpha-1 and alpha-2 adrenoceptors have been assessed by examining: (1)the ability of intrathecally administered selective adrenoceptor agonists to produce antinociceptive effects, and (2) the ability of selective adrenoceptor antagonists to alter the antinociceptive effects produced by the intrathecal administration of alpha adrenoceptor agonists. Both alpha-1 (e.g., methoxamine) and alpha-2 (e.g., ST-91) adrenoceptor agonists elevate thermally evoked nociceptive thresholds when administered intrathecally in the rat; the antinociceptive effects produced by methoxamine and ST-91 are antagonized by PRAZ and YOH, respectively (Howe et al., '83). Similarly, intrathecally administered NE produces dose-dependent antinociceptive effects that are antagonized by both PRAZ and YOH (e.g., Howe et al., '83). That spinal adrenoceptors are postsynaptically located is suggested by experiments in which intrathecally administered 6-hydroxydopamine fails to block the antinociceptive effects of intrathecal methoxamine and ST-91 (e.g., Howe et al., '83). Thus, both postsynaptic spinal alpha-1 and alpha-2 adrenoceptors appear to contribute to the antinociceptive effects produced by adrenoceptor agonists administered intrathecally. The results reported in the present study are consistent with these observations. Intrathecally administered NE inhibited significantly heat-evoked fos-like immunoreactivity; the inhibition was blocked by both PRAZ and YOH, suggesting that both alpha-1 and alpha-2 spinal adrenoceptors contribute to the inhibitory effects produced by NE. Similarly, intrathecally administered ST-91 inhibited heat-evoked fos-like immunoreactivity, an effect which was blocked by intrathecal YOH. Interestingly, antinociceptive effects produced by NE released via activation of endogenous descending systems have been demonstrated to be mediated predominantly by spinal alpha-2 adrenoceptors. Inhibition of the tail-flick reflex produced by electrical and/or chemical stimulation in the nucleus locus coeruleus (e.g., Jones and Gebhart, '86), the nucleus raphe magnus (e.g., Aimone et al., '87), the A5 cell group (e.g., Burnett and Gebhart, '91), or the lateral reticular nucleus (e.g., Gebhart and Ossipov, '86; Janss and Gebhart, '87) is blocked significantly by YOH, but not by PRAZ. Thus, it is possible that different spinal mechanisms are engaged by endogenously released NE compared to intrathecally administered NE and alpha adrenoceptor agonists.

Microiontophoretic studies. A role for the noradrenergic system in the modulation of sensory transmission in the dorsal horn has been demonstrated by microiontophoretic studies (see Jones, '91a for review). Belcher et al. ('78) reported that iontophoretically applied NE inhibited selectively unit activity of nociceptive-specific neurons in deep laminae of the cat dorsal horn. Headley et al. ('78) also reported that the iontophoresis of NE onto cat dorsal horn neurons in deep laminae produced a selective reduction of activity of neurons responding to noxious, but not to non-noxious stimuli. Fleetwood-Walker et al. ('85) further established that postsynaptic alpha-2 adrenoceptors mediate the inhibition produced by the iontophoresis of NE onto spinocervical tract or dorsal column neurons in the cat. Alpha-2 adrenoceptor agonists mimicked the selective inhibition of nociceptive-evoked unit activity produced by NE; alpha-1 and beta adrenoceptor agonists did not. Additionally, alpha-2 adrenoceptor antagonists either reversed or reduced the potency of the inhibition of nociceptive-evoked unit activity produced by NE; selective alpha-1 adrenoceptor antagonists failed to produce any reversal of the NE effect. In the primate, iontophoretically applied NE inhibited glutamate-evoked neuronal activity of spinothalamic tract neurons; however, no correlation was noted between the inhibition of unit activity and the modality of the neuron (Willcockson et al., '84). Thus, electrophysiological evidence supports behavioral data that suggest that NE modulates sensory transmission in the spinal cord dorsal horn. However, from the data currently available, it is difficult to conclude that NE has selective inhibitory effects on spinal nociceptive transmission. Additionally, few studies have examined: (1) the effects of NE on neurons specificallyin laminae 1/11, a region that receives nociceptivespecific primary afferent input and has dense concentrations of alpha-2 binding sites, and (2)the specific adrenoceptor subtypes that mediate the different effects on dorsal horn neuronal activity observed. In the present study it was not possible to determine if cells expressing fos-like immunoreactivity in response to noxious heat are nociceptive-specificneurons; however, fos immunohistochemistry does reveal the population of neurons in the spinal cord dorsal horn that are involved in the host of responses that occur following noxious heat stimulation. Dorsal horn neurons located in laminae 1/11comprised the majority of neurons demonstrating fos-like immunoreactivity in response to noxious heat stimulation. NE inhibited significantly fos expression in laminae 1/11dorsal horn neurons, and the inhibition was abolished by both alpha-1 and alpha-2 adrenoceptor antagonists. Thus, neurons in the superficial laminae of the spinal cord dorsal horn appear to be important in the modulation of spinal nociceptive transmission by NE.

Excitatory effects of norepinephrine The bulk of evidence suggests that iontophoretically applied NE has predominantly inhibitory effects on dorsal horn neuronal activity; however, excitatory effects also have been reported. In the cat, Todd and Millar ('83) reported that NE excited approximately 50%of the neurons examined in laminae 1/11, but had no effect on most units examined in lamina 111; no correlation was noted between NE's effect on unit activity and neuronal modality. Howe and Zieglgansberger ('87) also reported both inhibitory and excitatory effects on dorsal horn neuronal activity in anesthetized rats; the effects were not modality-specific. NE

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inhibited low threshold and multireceptive neurons in laminae 1/11 and in lamina 111, respectively, and excited some multireceptive and high threshold neurons in laminae 1/11 and lamina 111, respectively. In contrast, Millar and Williams ('89) reported that, in the rat, the effects of iontophoretically applied NE on superficial dorsal horn neuronal activity were modality specific; high threshold and wide dynamic range neurons were inhibited by NE, and low threshold (69%) neurons predominantly were excited by NE. To date, the specific adrenergic receptor systems mediating the excitatory effects on dorsal horn neuronal activity have not been examined. Interestingly, excitatory effects produced by iontophoretically applied NE predominantly have been observed in the superficial laminae of the spinal cord dorsal horn (Todd and Millar, '83; Howe and Zieglgansberger, '87; Millar and Williams, '89). Similarly, facilitation of superficial dorsal horn neuronal activity has been reported following activation of descending systems, some of which likely involve the release of endogenous NE (e.g., Price et al., '79; Dubuisson and Wall, '80; Light et al., '86; McMahon and Wall, '88). The present results also suggest that, on a population basis, NE excites laminae 1/11neurons, but not neurons in deeper dorsal horn laminae. Thus, superficial dorsal horn neurons may be influenced by NE in a manner different from deep dorsal horn neurons.

dorsal horn neurons; the later distribution of fos-immunoreactive neurons in deep dorsal horn laminae was hypothesized to represent polysynaptic mechanisms that are induced in the spinal cord by noxious heat stimulation. In the present study, survival times longer than 3 hours were not investigated; however, as discussed previously (see Materials and Methods), following a 3 hour survival time, the average numbers of neurons per section demonstrating fos-like immunoreactivity already were decreased significantly in both the superficial and deep dorsal horn laminae. It is possible that had a longer survival time been employed, the population of fos positive dorsal horn neurons in deep laminae would have been larger.

Summary The results reported in the present study confirm that NE has both excitatory and inhibitory effects on spinal cord dorsal horn neurons. Fos-like immunoreactivity evoked by NE is mediated by spinal alpha-1 adrenoceptors. The inhibition of fos-like immunoreactivity produced by intrathecal NE is, in contrast, mediated by both spinal alpha-1 and alpha-2 adrenoceptors. In summary, fos expression in laminae 1/11was influenced significantly by intrathecal NE, suggesting that superficial dorsal horn neurons likely play an important role in the noradrenergic modulation of spinal nociceptive transmission.

Methodological considerations Fos immunohistochemistry. Classical neuroanatomical tracing techniques have advanced tremendously our understanding of the organization of the central nervous system. Likewise, the functional implications of anatomical connections and pathways within the central nervous system have been examined, on a single unit basis, by using electrophysiological techniques. However, until recently, it was not possible to examine the population of neurons as a whole that functionally are important in the processing of information within the central nervous system. The expression of fos-like immunoreactivity in neuronal nuclei is a sensitive marker for cellular activation evoked by a variety of stimuli, including nociceptive peripheral stimuli (e.g., Morgan and Curran, '86; Hunt et al., '87; Morgan et al., '87; White and Gall, '87; Dragunow and Robertson, '88; Menetrey et al., '89; Bullitt, '89, '90; Presley et al., '90). Thus, the immunohistochemical localization of fos proteins can be used as a powerful technique to examine pathways and populations of neurons in the central nervous system that are involved functionally in the processing of peripheral information. Distribution o f fos neurons. The expression of fos-like immunoreactivity in the spinal cord evoked by nociceptive stimuli has been reported to vary with time (e.g., Presley et al., '90; Williams et al., '90). Hunt and colleagues (Williams et al., '90) recently have reported that the distribution of fos-like immunoreactivity in the spinal cord dorsal horn evoked by brief noxious heat stimulation of the hindpaw changes with varying survival times. Following a 2 hour survival time, fos-immunoreactive neurons were observed primarily in superficial dorsal horn laminae; in contrast, following an 8 hour survival time, fos-immunoreactive neurons were distributed primarily in deep dorsal horn laminae and a marked contralateral component was noted. The initial superficial distribution of fos-immunoreactive neurons was hypothesized to represent primary afferent terminal depolarization and monosynaptic activation of

ACKNOWLEDGMENTS The author thanks Rebecca Powers for her technical assistance, R.W. Blair, Ph.D. for his editorial comments, and Boehringer Ingelheim, Ltd. for graciously supplying ST-91. This research was supported by the Oklahoma Center for the Advancement of Science and Technology, the Presbyterian Health Foundation, and the Pharmaceutical Manufacturer's Association Foundation.

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