Brain Research, 594 (1992) 99-108 © 1992 Elsevier Science Publishers B.V. All rights reserved 0006-8993/92/$05.00

99

BRES 18196

Parallel activation of multiple spinal opiate systems appears to mediate 'non-opiate' stress-induced analgesias L.R. Watkins, E.P. Wiertelak, J.E. Grisel, L.H. Silbert and S.F. Maier Department of Psychology, University of Colorado at Boulder, Boulder, CO 80309 (USA) (Accepted 26 May 1992)

Key words: Opiate; Non-opiate; Analgesia; Spinal cord;/~ Opiate receptor; 8 Opiate receptor; K Opiate receptor

Pain is powerfully modulated by circuitries within the CNS. Two major types of pain inhibitory systems are commonly believed to exist: opiate (those that are blocked by systemic opiate antagonists and by systemic morphine tolerance) and non-opiate (those that are not). We used intrathecal delivery of/~, 8, and K opiate receptor antagonists to exam;no 3 well-accepted non-opiate stress-induced analgesias. Combined blockade of all 3 classes of opiate receptors antagonized all of the 'non-opiate' analgesias. Further experiments demonstrated that blocking and 8 or IL and K was sufficient to abolish 'non-opiate' analgesias. Combined blockade of K and ~ receptors was without effect. The clear conclusion is that all endogerLous analgesia systems may in fact be opiate at the level of the spinal cord. Phenomena previously thought to be non-opiate appear to involve parallel activation of multiple spinal opiate processes. These findings suggest the need for a fundamental shift in conceptualizations regarding the orgat~ization and function of pain modulatory systems in particular, and opiate systems in general.

INTRODUCTION The CNS contains circuitry capable of powerfully and selectively suppressing pain x22's2'ss. These pain inhibitory systems can be activated by either focal electrical stimulation of, or morphine microinjection into, specific CNS sites. The discovery of brain stimulation produced analgesia (SPA) and morphine microinjection analgesia (MA) corresponded closely in time with the identification of both opiate receptors and endogenous opiates within the brain and spinal cord. Subsequent work confirmed that multiple opiate receptor subtypes (termed /~, 8 and K) and multiple forms of endogenous opiates (enkephalins, endorphins and dynorphins) are involved in pain modulation 15'22'27'4°'45'47's5.The striking anatomical overlap of CNS sites containing opiates and their receptors with sites from which SPA and MA can be produced strongly suggested that both SPA and MA may be mediated by activation of intrinsic opiate-like systemst5'27'4°'45'47'5~'55. The ability of opiate receptor antagonists to abolish both SPA and MA provided pharmacological support for such a notion 27.

The fact that pain inhibition systems can be activated by either electrical stimulation or exogenous opiates does not provide insight into the functions that these systems might serve. There should be environmental situations which trigger their activation if they are physiologically relevant. Indeed, profound analgesic states can be produced by a wide variety of laboratory procedures, including transcutaneous shock, cold water swims, food deprivation, centrifugal rotation and defeat in conspecific encounters, amongst others 22'44'52. Since situations which activate endogenous analgesia systems tend to be stressful, the resultant phenomena have been collectively termed 'stressinduced analgesias' (SIA). The existence of SIA has led to a variety of proposals concerning the functional significance of pain inhibitory systems22'44'52. Although the study of analgesia systems was originally guided by the premise that endogenous opiates would naturally be key mediators, a steady stream of observations challenged this assumption. The earliest such observations arose from the study of SPA. Numerous reports now exist of SPAs which are neither blocked by systemically administered opiate antago-

Correspondence: L.R. Watkins, Department of Psychology, University of Colorado at Boulder, Boulder, CO 80309, USA. Fax: (1) (303) 492-2967.

100 nists such as naloxone or naltrexone, nor show cross tolerance with morphine 4''x23'33'5'. Likewise, a variety of environmental stressors have been identified which produce SIA unaffected by these same procedures :-''~4's:. The concept of multiple analgesia systems gradually evolved in order to account for such disparate observations. Bo~'~,ic,piate and non-opiate analgesias were proposed to exist under this new formulation, and to be subserved by separate mechanisms. By definition, opiate analgesias included phenomena which could be attenuated by systemically administered opiate antagonists (naloxone or naltrexone) and/or showed cross-tolerance with morphine. Non-opiate analgesias encompassed everything else. This opiate/ non-opiate dichotomy has become widely accepted as a fundamental tenet for pain modulation systems 22'27'44's2. We recently made a surprising observation while investigating whether ~, ~ or x opiate receptor subtypes mediate opiate forms of SIA in rats "~3's4.We used transcutaneous tail shock to produce analgesia in these initial experiments. This procedure was chosen because it sequentially elicits three distinct analgesic states as the shock session progresses s. Based on the effect of high doses (14 mg/kg s.c.) of systemic opiate antagonists '''~'~ and on the etf,,ct of prior morphine tolerance 7, this procedure had been found to reliably produce opiate analgesia after I-2 tail shocks, non-opiate anal. gesia after 5-4{) tail shocks, and a second opiate anal. gesia after 80-100 tail shocks. Furthermore, all 3 of these analgesic states were known to be produced entirely through shock-induced activation of supraspim:i si~¢s~°,~,~.These brain sites, in turn, activate descending projections, with the end result being sup. pression of pain transmission at the level of the spinal cord. Thus, these 3 analgesic states are similar to SPA and MA in their dependence on centrifugal (e.g. de, scending from brain to spinal cord) pain suppression pathwaysXZ'-.'-7.sz. Given that all 3 opiate receptor subtypes are found within pain modulatory areas of the spinal cord t'~''2'2~'4°'4s'4~,we s'~ initially tested whether tail shock induced attalgcsias could be blocked by intrathecal (injection into the cerebrospinal fluid surrounding the cord) delivery of either naltrexone (the relatively nonselective opiate antagonist used systemically to define the opiate/non-opiate nature of the 3 tail shock induced analgesias) or highly selective/z, 6 and K opiate receptor antagonists. Similar to the effects observed following systemically administered naltrexone, the opiate analgesias elicited by 2 shocks and 80-100 shocks were blocked by 18.5 nM (7/J.g), but not 9.25 nM (3.5 /zg), naltrexone -s-~. In this study s3 we also found that, following 18.5 nM microinjection of the selective antag-

onists, these opiate analgesias were blocked only by the x antagonist. Thus~ at the level of the spinal cord, x opiate receptors appear to mediate both opiate analgesias. Surprisingly, in this same study 53, we observed that the 'non-opiate' analgesia elicited by 5-40 tail shocks was also markedly attenuated by 7/~g (but not 3.5/zg) naltrexone. Three alternative explanations readily come to mind to explain this finding. First, perhaps the biodistribution of systemic naltrexone is such that relatively little actually reaches the lumbosacral cord. If this were so, perhaps the effects seen with 7 #g (18.5 nM) i.t. naltrexone reflect mediation of 'non-opiate' analgesia by receptors that do not readily bind naltrexone (and thus might not be exposed to sufficiently high concentrations of naltrexone following systemic administration). Indeed naltrexone has low affinity for ~: receptors. Certainly, reports do exist of a 'non-opiate' stress-induced analgesia actually being found to be mediated by x opiate receptors 32. Unfortunately, this explanation for 'non-opiate' tail shock analgesia is contradicted by the finding that 18.5 nM i.t. binaltorphimine, a highly specific x receptor antagonist 4z, was without effect s'~. A second alternative explanation is that perhaps 7 ~g (18.5 nM) naltrexone is a sufficiently high dose that the drug is non-specifically binding to non-opiate receptors ~''~, This does not appear to be the case since this dose of i.t. naltrexone has no effect on baseline tail flick latencies s.~ and does not affect the non-opiate analgesia induced by i.t. norepinephrine (Wiertelak, Maier and Watkins, unpublished observations). Furthermore, none of the highly selective ~, 8, or ~¢opiate antagonists, delivered at the same molar concentration as 7/zg naitrexone, had any effect on 'non-opiate' tail shock analgesia s3. The third alternative led to the present series of experiments. Perhaps the fact that 7/~g naltrexone does block 'non-opiate' tail shock analgesia, yet equimolar/z, ,$, or ~ antagonists do not block 'nonopiate' tail shock analgesia indicates that multiple opiate receptor subtypes are being activated in parallel. By such a mechanism, analgesia would not be bIocked unless more than one opiate receptor subtype were simultaneously bound by antagonist. The data to be presented here fully support this third possibility. The present series of experiments indicate that 'non-opiate' endogenous analgesia systems may not be non-opiate after all. Instead, the procedures that in the past have yielded 'non-opiate' endogenous analgesia may actually produce analgesia by activating multiple spinal opiate systems in parallel. As such, these results indicate the need for a shift in conceptualizations

101 regarding the organization and function of endogenous pain modulatory systems in particular, and perhaps opiate systems in general. MATERIALS AND METHODS

Subjects Adult male Sprague-Dawley rats (450-600 g; Holtzman Labs.) were used in all experiments. The animals were singly housed and maintained on a 12 h/12 h light/dark cycle with food and water available ad libitum. Care and use 'ff the animals in all described procedures were in accordance w|th protocols approved by the University of Colorado Laboratory Animal Research Committee.

lntrathecal (i.t.) catheterization l.t catheters were implanted 1-2 weeks prior to testing to allow for drug delivery into the cerebrospinal fluid surrounding the lumbosacral spinal cord 56. I.t. catheterizations were performed under sodium pentobarbital anesthesia (Nembutal; Abbott Laboratories, 55 mg/kg, intraperitoneally), supplemented with methox3,flurane (Metofane; Pitman-Moore) as needed. All animals were treated with penicillin-streptomycin post-operatively to prevent infection. Catheter construction and implantation have been described previously 51.

Drugs Naloxone hydrochloride (Sigma Chemical), a relatively nonspecific opiate antagonist, was used systemically in Expt. 2 to verify that hind paw footshock induced analgesia was, using our laboratory conditions, indeed 'non-opiate' according to published criteria. The dosage and injection procedure (see below) were according to our previously published protocol 45'52. Highly specific opiate antagonists were used to further define the receptor subtypes involved (Expts. 1-5). The following drugs were administered intrathecally: vehicle (dimethylsulfoxide (DMSO), pH 7.0; Sigma), the ~¢-receptor antagonist Nor-binaltorphimine dihydrochloride (Research Biochemicals; MW 734.78)42, the 8-receptor antagonist naltrindole hydrochloride (Research Biochemicals; MW 450,6) 35, and the tt-receptor antagonist C'ys -Tyr' .Orn'-Pen -amide ( c r o P ; Penninsula Laboratories; MW 1062.24)24. All of these specific antagonists were delivered in a volume of I ~tl. Dosages were delivered so that the total drug delivered was equimolar to 7.0 ~tg (18,5 nM) of naltrexone hydrochloride (MW 377.9). This molar dose was chosen based on the observed antagonism of 'non-opiate' tail shock analgesia by 7 ~tg naltrexone i.t. 53. Since the specific antagonists used are chemically unstable and/or only slightly soluble in saline, the following procedures were strictly followed. Naltrindole and binaltorphimine were dissolved in 100% DMSO (pH 7.0; Sigma Chemical), aliquoted into 15 ~tl units, and frozen in O-ring sealed vials at -70°C until used. On the days that these aliquots were to be used, a single vial of the test drug (used that day only) was slowly thawed, kept cold on ice, and used within 4 h. Lyophilized c r o p in pre-weighed vials was maintained at - 20°C until used. On the days that these aliquots were to be used, a single vial was brought to room temperature and dissolved in DMSO pH 7.0. The drug was then kept cold on ice and used within 4 h.

ter, Each animal's tail extended through a hole in the rear of its tube, allowing for either tail-flick testing or the delivery of tail shock without removing the animal from the tube. Animals were placed in the tubes in the dimly lit, warm experimental room approximately 60 rain prior to the onset of testing. Tail]lick (TF) test. Animals were tested for their threshold responsivity to radiant heat pain using a modification 1 of the TF test 5. The radiant heat source was adjusted by a variable AC transformer so that baseline latencies were typically 2.5-3.0 s. In the event that no TF occurred, the lamp was automatically terminated at 10 s to prevent tissue damage. Baseline TF latencies were recorded prior to any injection procedures (see above). Four TF trials were recorded at 1 rain iatervals, the last 3 being averaged to attain a mean pre-drug baseline latency (labelled B1 in all figures). A post-drug baseline latency was determined in the same manner, beginning 10 rain after intrathecal microinjection of drugs (labelled B2 in all figures). TF latencies were also recorded following varying numbers of tail shocks (see below), or at specific times following either hind paw shock or cold water swim (see below). In Expts. 1 and 4, rats were tested after 2, 5, 10, 20, 40, 60, 80 and 100 tail shocks. For each animal, 3 TF trials were recorded at 1 rain intervals after each of these shock termination points; these 3 latencies were averaged to attain a single mean test trial latency following 2, 20, 40 and 80 tail shocks. In Expts. 2 and 5, rats were tested at 0, 2, 4, 6, 8, 10, 12 and 14 rain after completion of the 90 s hind paw shock exposure (see below). At each of these timepoints, a single TF latency was recorded for every animal. In Expt. 3, rats were tested at 60 rain after completion of the cold water swim. For each animal, two TF trials were recorded and averaged to form a single mean test trial latency. Testing 60 rain after cold water swim was chosen since this allowed the animals' core body temperature to have returned to normal ~1. Tail shock procedure. Computer-controlled inescapable 5 s tail ~hocks (1.0 mA) were delivered through fuse clip electrodes taped to the rat's tail and augmented by electrode paste. Shock was delivered on a 1 rain variable interval schedule. The shock source was designed after the Orason-Stadler Model 700. For details, see ref. 8. Hind paw shock procedure. Transcutaneous hind paw shock (00 s, 1.6 rms, Coulbourn Electronics) was delivered using the same apparatus and technique as described previously 45''~2. Cold water swim procedure. Under our experimental conditions, potent non-opiate analgesia is induced by 4 rain of continuous swimming in 14°C water. For details, see refs. I I and 12.

Statistics Statistical comparisons were accomplished using analysis of variance (ANOVA) to determine main effects. The Scheffe F-test was used for post hoc specific comparisons ( P - 0.05). in cases of multiple comparisons, alpha levels were adjusted using the Bonferroni method.

EXPERIMENT 1. EFFECT OF COMBINED ~, 8, x OPIATE ANTAGONIST ADMINISTRATION ON 'NONOPIATE' TAIL SHOCK ANALGESIA Rationale and Methods

Injection procedures In all experiments involving intrathecal drug &livery, baseline pain responses (see below) were measured prior to a~,d again 10 min after completion of intrathecal microinjection. For ea~:h animal, one of these drugs tested (see above) was injected into the i.t. catheter, followed by a 10/zl sterile saline flush over 30 s (for details, see ref.

51). Behavioral testing procedures All behavioral testing occurred during the animals' light cycle and was performed in a small, dimly lit room mai.ntained at a constant 27.5:1:0.5°C. Testing was performed while the rats were loosely restrained in Plexiglas tubes 17.5 cm in length and 7.0 cm in diame-

As described in the Introduction, it was hypothesized that there was a logical reason that 7 ~g i.t. naltrexone does block 'non-opiate' tail shock analgesia, yet equimolar (18.5 nM)/~, 8, or K antagonists i.t. do not block 'non-opiate' tail shock analgesia 53. That is, it was hypothesized that these data indicate that multiple opiate receptor subtypes are being simultaneously activated in parallel. By such a mechanism, analgesia would not be blocked unless more than one specific opiate receptor antagonist were simultaneously administered.

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Fig. 1. The effect of combined intrathecal (i.t.) delivery of 6.17 nM CTOP (# antagonist), 6.17 nM binaltorphimine (e antagonist), and 6.17 nM naltrindole (6 antagonist)on tail shock induced analgesia. Combin,~g these 3 antagonists virtually abolished the 'non-opiate' analgesia induced by 5-40 tail shocks,whereas 18.5nM of any one of these drugs alone had no effect on this analgesicstate5"~.II. vehicle controls; [3. combined deliveryof/a.. 3, and ,c antagonists. In order to test this hypothesi,~, rats were injected i.t. with either a total of 18.5 nM opiate antagonists (6.17 nM CTOP + 6.17 nM binaltorphimine + 6.17 nM naltrindole; n - 8) or equivolume (1 ~1) vehicle (n -- 7). Surgical preparation, i.t. microinjection, and behavioral testing were all as described in Materials and Methods (see above).

Restdts and Discussio~ The vehicle and ~ + B + ~¢ groups did not differ in their baseline TF latencies, either before (BI) or after (B2) drug administration. In response to tail shock, however, marked differences in their analgesic response were observed, in striking contrast to the data we reported previously where 18.5 nM of neither CI'OP, binaltorphimine, nor naltrindole had any effect on 'non-opiate' tail shock analgesia s3' combined administration of all 3 antagonists (combining 6.17 nM el! each drug; herein referred to as '~t + 8 + K antagonists') blocked 'non-opiate' analgesia induced by 5-40 tail shocks (Fig. 1) (Fi,ta = 65.084, P < 0,001), These results strongly suggest that analgesia induced by 5-40 tail shocks is mediated by parallel activation of multipie opiate receptors, rather than by 'non-opiate' mechanic;ms as previously believed. In agreement with our previous finding that t;~¢ 2 shock and 80-100 shock opiate analgesia are mediated by ~¢ receptors s'~, both of these opiate analgesias were also significantly reduced in the group receiving # + 6 + K antagonists,

EXPERIMENT 2. COMBINED /z, 6 and K ANTAGONISTS BLOCK 'NON-OPIATE' HIND PAW FOOTSHOCK INDUCED ANALGESIA

Rationale and Method In order to test the generality of this finding, the # + ~ + K antagonists were tested against two other

widely accepted forms of non-opiate stress-induced analgesia. In Expt. 3, this drug combination was tested against cold water swim analgesia i1'~2'22. In the present experiment, it was tested against hind paw footshock induced analgesia (FSIA) 4s'52. Prior to testing the effect of the i.t. ~ + 8 + K antagonists, it was necessary to verify that hind paw footshock would indeed produce non-opiate analgesia under our present laboratory conditions. In accordance with the published criterion used to establish hind paw FSIA as a non-opiate analgesia4s'52, rats were exposed to hind paw shock after receiving either i.p. saline or i.p. naloxone. Each rat received a series of two i.p. injections delivered 10 rain apart. Each injection consisted of either 10 m g / k g naloxone or equivolume vehicle (1 m i / k g sterile physiological saline). The first injection was delivered after assessment of pre-drug baseline TF latencies (B1). The second injection was delivered immediately after post-drug baseline TF testing (B2) and immediately prior to hind paw shock. This injection protocol is identical to that previously used to investigate hind paw FSIA4s's2. Hind paw shock and behavioral testing were as described in Materials and Methods (see above). Two groups of rats were then tested to determine the effect of i.t. tt + 8 + K antagonists on non-opiate hind paw FSIA. One group received 18.5 nM ~t + B + ~¢ antagonists (n m 8); the other received equivolume vehicle (1 ~1 DMSO; n "! 7). Surgical preparation, i.t. microinjection, and behavioral testing were as described in Materials and Methods (see above).

Restdts and Discussion Systemic administration of 20 m g / k g naloxone failed to attenuate hind paw FSIA (Fig. 2A)(Ft,t4 = 2.431, A. Systemic

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Time After Hind Paw Shock (rain.) Fig, 2. A: the effect of systemic naloxone (20 m g / ~ i.p., total dose) on hind paw footshock induced analgesia(FSIA). Under our laboratory conditions, hind paw FSIA was again found to be non-opiate, in that naloxone (13) failed to attenuate hind paw FSIA, compared to saline controls (i). B: the effect of combined intrathecai (i.t.) delivery of 6.17 nM CTOP (~ antagonist),6.17 nM binaltorphimine Oc antagonist), and 6.17 nM naltrindole (8 antagonist)on hind paw FSIA. Like the effect observed for 'non-opiate' tail shock analgesia, this combination of drugs markedly attenuated this 'non-opiate' analgesic state, i , vehicle controls; 13, combined delivery of/a., 8, and ~cantagonists.

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T i m e A f t e r Cold W a t e r S w i m ( r a i n . ) Fig. 3, Effect of combined intrathecal (i.t.) delivery of 6.17 nM CTOP (# antagonist), 6.17 nM binaltorphimine (t< antagonist), and 6.17 nM naltrindole (6 antagonist) on cold water swim analgesia. Like the effect observed for both 'non-opiate' tail shock analgesia and 'nonopiate' hind paw FSIA, this combination of drugs markedly attenuated this 'non-opiate' analgesic state.

P > 0.05), thus verifying the apparent non-opiate nature of this phenomenon 4s'52. In contrast, i.t. tz + 6 + ~< antagonists markedly attenuated hind paw FSIA relative to vehicle controls (Ft,~3 - 22.516, P < 0.001). While hind paw FSIA was not affected immediately after shock termination ('0 min', Fig. 2B) (FI,L~ = 1.704, P > 0.05), analgesia was abolished from 4 to 14 min after shock (F~,~.~- 21.459, P < 0.001). This pattern is strikingly similar to that observed following spinal transection. Hind paw FSIA is not attenuated in spinally transected rats immediately after shock termination,

E X P E R I M E N T 3. C O M B I N E D #, 6 and K A N T A G O NISTS B L O C K ' N O N - O P I A T E ' C O L D W A T E R SWIM INDUCED ANALGESIA

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Fig. 4. Effect of specific opiate antagonist pairs on tail shock induced analgesia. Opiate antagonist mixtures were delivered as # + x (A), # + 6 (B), or/z + K (C) pairs. Each antagonist was delivered at 9.25 nM so to maintain a total delivered dose of 18.5 nM. The 8-K pair had no effect on 'non-opiate' tail shock analgesia. In contrast, both the/z + 6 and # + K pairs blocked 'non-opiate' tail shock analgesia. This antagonism cannot be accounted for by the presence of the tz antagonist alone, because delivery of this 9.25 nM dose of the/z antagonist alone had no effect on analgesia (D).

104 behavioral testing were as described in Materials and Methods (see above).

Results and Discussion l.t.p. + 6 + r antagonists again did not affect baseline TF latencies. However, 'non-opiate' cold water swim analgesia was markedly attenuated by i.t. /.~ + + r antagonists (Fig. 3) (Ft,~t = 7.479, P < 0.05). These data, combined with the results of Expt. 2 using hind paw FSIA and Expt. l using tail shock analgesia, provide strong evidence that i.t. /.t + ($ + x antagonists can block a variety of analgesic states that have been considered to be non-opiate analgesias, according to classical criteria. E X P E R I M E N T 4. WHICH PAIRS OF A N T A G O N I S T S BLOCK ' N O N - O P I A T E ' TAIL SHOCK INDUCED ANAL-

Therefore, intrathecal delivery of antagonist pairs (($ + K, /~ + ($, and /z + K) was examined using tail shock :,nduced analgesia. Each of the 3 antagonists was delivered as 9.25 nM so that a total of 18.5 nM was again delivered in a total volume of 1 #1. The following groups of rats were tested: vehicle control (n = 6), ~$+ K (n = 7), g, + ~ (n = 7), a n d / z + I< (n = 8). Based on pilot experiments, one additional group was tested which received only 9.25 nM of the/~ antagonist (n = 8) to verify that this dose of CTOP had no affect on analgesia induced by 5-40 tail shocks. Due to the very specific question being addressed., the animals were not subjected to shock beyond the 40 required to determine if CTOP would affect analgesia within this 'nonopiate' shock rar, ge. Surgical preparation, i.t. microinjection, and be~avioral testing were as described in Materials and Methods (see above).

GESIA?

Rationale and Method Antagonism of all forms of 'non-opiate' endogenous analgesia systems by the # + ,$ + ~ antagonist mixture suggests activation of multiple opiate receptor subtypes, but does not clarify which are critically involved.

Results and Discussion A very clear pattern of results emerged from this study. The 8 + K antagonist pair had no effect on the 'non-opiate' analgesia induced by 5-40 tail shocks (Fig. 4A), The opiate analgesias produced by 2 shocks and by 80-100 shocks were attenuated due to the presence

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Fig. 5. Effect of .~pecific opiate antagonist pairs on hind paw f~tshock induced analgesia (FSIA). Opiate antagonist mixtures were delivered as ,i + ~ (At. # + ,S (B), or # + ~ (C) pairs. Each antagonist was delivered at 9.25 nM so to maintain a total delivered dose of 18.5 nM. The 8 - K pair had no effect on aralgcsia. In contrast, both the ~ + 8 and p. + ~< pairs blocked *non-opiate' hind paw FS]A. This antagonism cannot be accouatcd for by (he presence of the p. antagonist alone, because delivery of this 9.25 nM dose of the p. antagonist alone had no effect on

analgesia (D).

105 of the r antagonist, as predicted from previous studies -~3. In contrast with the lack of effect of the + K antagonist pair on 'non-opiate' tail shock analgesia, this analgesia was abolished whenever the p, a:~}.agonist was present as one of the antagonist pair. That is, both the # + ~ and the p, + ~: mixtures blocked 'nonopiate' analgesia (Fig. 4B, C). This antagonism was caused by the concomitant application of the antagonist pairs, rather than to the # antagonist alone since 9.25 nM of the /~ antagonist applied singly had no effect on this 'non-opiate' analgesia (Fig. 4D). These observations were supported by the analysis of variance (ANOVA) which revealed a reliable effect of group assignment in both opiate (F3.24 = 8.166, P < 0.001) and 'non-opiate' (F4.3~ = 12.777, P < 0.001) analgesias. Subsequent post hoc Scheffe F-tests revealed significant differences between the vehicle controls and both the # + ~: and 6 + K antagonist pair groups in r-mediated opiate analgesia (produced by 2 shocks and by 80-100 shocks) 53. In contrast, in 'non-opiate' analgesia (produced by 5-40 shocks), significant differences existed between the vehicle controls and both the # + and p. + ~: antagonist pair groups. Moreover, both the + 8 and ~ + ~: antagonist pair were significantly different from either the ~ + K antagonist pair or p.antagonist groups. Neither the #-antagonist nor the 8 + ~: antagonist pair groups differed from the vehicle controls. EXPERIMENT 5. WltlCH PAIRS OF ANTAGONISTS BLOCK 'NON-OPIATE' HIND PAW SHOCK INDUCED ANALGESIA?

Rationale and Method T o test the generality of these findings, Expt. 4 was repeated using hind paw shock as the method of inducing 'non.opiate' analgesia. Five groups of rats were tested: vehicle control (n = 7), 8 + K (n - 7), /z + 8 (n = 7), ~ +K (n = 7) and 9.25 nM tz alone (n = 6). Surgical preparation, i.t. microinjection, and behavioral testing were as described in Materials and Methods (see above).

Results and Discussion The results completely supported the findings of Expt. 4. As seen for 'non-opiate' tail shock analgesia, 'non-opiate' hind paw FSIA was not affected by the ~$+ t¢ antagonist pair (Fig. 5A) yet was markedly attenuated whenever the/x antagonist was included in the antagonist pair. That is, 'non-opiate' hind paw FSIA was attenuated by both the /z + (5 (Fig. 5B) and the /z + K (Fig. 5C) antagonists pairs. Again, these effects cannot be accounted for by the effects of 9.25 nM of

the p, antagonist alone, since this dose delivered singly had no effect on hind paw FSIA (Fig. 5D). These observations were supported by the analysis of variance (ANOVA) which revealed a reliable effect of group assignment, (F4,25 = 11.813, P < 0.001). Subsequent post hoc Scheffe F-tests revealed significant differences between the vehicle controls and both the /z + 6 and p. + K antagonist pair groups. Neither the 6 + K nor the /.t-antagonist groups differed from the vehicle controls. DISCUSSION The present series of experiments clearly demonstrate that combined intrathecal administration of #. (CTOP), 8 (naltrindole), and x (binaltorphimine) opiate receptor antagonists block eve:'y form of 'nonopiate' analgesia tested. Whereas 18.5 nM of any of these antagonists delivered singly had no effect on the analgesia induced by 5-40 tail shocks, combining 6.17 nM of each antagonist abolished 'non-opiate' tail shock analgesia, and markedly reduced both hind paw footshock induced analgesia and cold water swim analgesia. In order to determine which combinations of receptor blockade were critical, antagonists were tested in pairs against both tail shock analgesia and hind paw footshock induced analgesia. The identical pattern of results was observed for each of these 'non-opiate' states. Combined blockade of ~ and K receptors failed to have any effect on either phenomenon. In contrast, whenever a ~ antagonist was included in the antagonist pair, 'non.opiate' analgesia was blocked. That is, combined blockade of ~ and K receptors as well as combined blockade of # and 8 receptors blocked both 'non-opiate' analgesias. These effects could not be accounted for by the effect of the ~ antagonist alone, since this dose of the antagonist delivered singly had no effect on either analgesic state. While the combination of opiate antagonists blocked each of the classic 'non.opiate' SIAs, the nature of the effect was somewhat different in each case. The analgesia after 5-40 tail shocks was abolished, the analgesia after hind paw shock was unaffected immediately after the termination of the shock but abolished from 4 to 14 min after shock, and analgesia was only partially reversed at 60 rain after cold water swim. it is interesting to consider the relative importance of centrifugal and intraspinai pathways in the production of these analgesias, when interpreting this pattern. As used here, centrifugal pathways refer to neural pathways which have their cells of origin within the brain and send pain inhibitory projections down to the spinal cord dorsal horn. Correspondingly, intraspinal path-

106 ways refer to pain inhibitory circuitry which is contained entirely within the spinal cord, with no dependence upon the brain for its capability to suppress pain transmission. Spinal transection is used to distinguish whether an analgesia-inducing procedure relies on intraspinal or centrifugal pathways. Spinal transection completely prevents the analgesia produced by our tail shock procedure s3 and so the analgesia is entirely medi~,ted by centrifugal pathways and has no intraspinal component. In contrast, we have found that there is a strong intraspinal component to the analgesia observed 0-4 rain after hind paw footshock in that here the analgesia is not reduced by spinalization 49's2. However, the analgesia from 4 rain onward is eliminated by spinalization, suggesting centrifugal mediation at these timepoints. Cold water swim analgesia cannot be appropriately assessed in spinalized animals, and so it is not possible to determine the relative contribution of descending and intraspinai pathways for this analgesia. It would thus appear that the opiate antagonist combination failed to completely reverse analgesia only under circumstances that involve intraspinal induction mechanisms. The data are consistent with the possibility that all descending pain inhibition produced by stressors are mediated through endogenous opiates. These data are also entirely consistent with the hypothesis that 'non-opiate' analgesia results from parallel activation of multiple opiate receptor subtypes at the level of the spinal cord. In fact, the data clearly imply that all 3 spinal cord opiate receptor subtypes are simultaneously activated during 'non-opiate' stress-induced analgesias. The simplest circuit consistent with the pattern of results obtained would be that the ~ and x receptors occur in series, and that this pair exists in parallel with the/~ receptors (Fig. 6). 'Nonopiate' analgesias are only blocked when both of these parallel pathways are interrupted. Blocking only the p,, only the 8, or only the K sites is not sufficient to block analgesia since in each case one of the two parallel pathways is active. Blocking both ~ and ~ sites is not sufficient to block analgesia since, in this case, the/z pathway is still active, But blocking both /z and 6, or blocking both p, and K, sites is effective in blocking analgesia since this would effectively interrupt both of the parallel pathways. Taken together, these data call into question the fundamental belief that there exist both opiate and non-opiate endogenous pain inhibitory systems that are activated by stress 22,44,s:', in stress-induced analgesia research, each newly documented analgesia has been categorized as opiate or non-opiate based on the effect of systemic opiate antagonists (naloxone or naltrexone) and/or on the effect of prior morphine tolerance.

Fig. 6. One hypothetical neural circuit that could account for the data presented in Expts. I-5. The simplest circuit consistent with the pattern of results obtained would be that the 8 and K receptors occur in series, and that this pair exists in parallel with the /z receptors. 'Non-opiate' analgesias are only blocked when both of th~se parallel pathways are interrupted. Any manipulation which interrupts only one of these two parallel pathways do not interrupt analgesia. That is, blockade of only the p, 'arm' of the circuit still leaves the ~-K 'arm' intact, thereby allowing the expression of analge:,ia. Similarly, blockade of only the 8-g 'arm' (either by blocking 8 receptors, x receptors, or combined blockade of ~ + K receptors) still leaves the p, 'arm' intact, thereby allowing the expression of analgesia. However, simultaneous blockade of both 'arms' (by combined /~ + 8 blockade, or combined/~ + K blockade) would be p~edicted to disrupt analgesia. The inclusion of semtonin (SHT) and norepinephrine (NE) centrifugal pathways in the circuit derive from unpublished data from our lab which indicate that depletion of spinal cord 51tT abolishes the 2 shock and 80-100 shock opiate :malgcsias wh~r~ depletion of spinal cord NE abolishes the 5-40 shock 'non.opiate' lmalgesia. Ag;tin, for emphasis, this is a hyimtheticl~l circuit which may provide a working model for future experimentation in thi~ areal.

After being defined in this manner, 'non-opiate' analgesias have rarely been further challenged with opiate antagonists, The analgesias induced by 5-40 tail shocks, by hind paw shock, and by cold water swim were all documented to be non-opiate based on these classic criteria and using the same behavioral measure as used in the present study, The present series of experiments suggest that these classic criteria are inadequate and should not be used. Whether biodistributional factors account for the inability of high doses of systemic opiate antagonists to block non-opiate analgesia is unknown, Certainly, use of systemic doses greater than the 14-20 mg/kg already employed would not be reasonable given the probability that these relatively nonspecific drugs would bind to non-opiate receptors. Hence, any effects observed with higher systemic dosages would be suspect, regardless of the direction of the results. These data also suggest modification of current views concerning the operation of analgesia systems. Attempts at understanding what factors control whether

107 opiate or non-opiate systems are activated has been a major focus of pain research for over a decade 22'44's2. Currently, the 'severity hypothesis' is the most widely accepted view 44. The severity model proposes that opiate analgesia systems are the first line of defense when the organism encounters a stressful situation. According to this model, opiate systems shut down and non-opiate analgesia systems become recruited as the stressor becomes more intense or more prolonged 44. In contrast, our data imply that opiates are always involved. Instead of opiate systems shutting down as stressors become more intense or more prolonged, we propose instead that they become additive. That is, the organism is 'safeguarded' against debilitating pain by parallel activation of multiple opiate systems. The present data suggest opiate receptor interactions in the spinal cord. Previous investigations have clearly documented opiate receptor interactions at supraspinal sites. Whereas synergism in the behavioral effects of /z and 6 ligands has been observed, ~-8 antagonism has been reported as welil4'2°'21'34'36-39. Antagonism is also typically reported for K interactions with other opiate receptor subtypes in brain, x Agonists act to non-competitively inhibit/z and 8 binding, and to behaviorally antagonize the analgesic effects of morphine ~°a7'2°'2~. Our data suggest that opiate receptor interactions will be different in the spinal cord. Indeed, the little information that does exist supports this contention. Applied singly,/~, 8 and x agonists can all produce behavioral analgesia and inhibit the painevoked responses of spinal cord dorsal horn neurons ~'~s'2'~./~ and 8 agonists have recently been recognized to synergize at the level of the spinal cord dorsal horn, as they do in our behavioral data. Here, combined delivery of ~ and 8 agonists suppress the painevoked activity of pain transmission neurons to a far greater degree than delivery of either agonist alone 29. A complete understanding of these interactions will require a consideration of the centrifugal pathways that activate the spinal opiate mechanisms. As already noted, the opiate analgesias are initiated supraspinally, and different descending pathways activate different spinal opiate processes. For example, it is known that a descending serotonin pathway interacts with a spinal K system, while a noradrenergic pathway interacts with/.t sites 2'19'26'28'30'31'41A6.Indeed, this sort of arrangement makes it sensible that different environmental events and behavioral processes exert differential effects at the level of spinal opiate receptors. These data may also have implications regarding drug development for the control of pain in man. Part of the interest in non-opiate analgesias stems from the fact that they tend to be both very powerful analgesic

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