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Opiate vs non-opiate eootshock induced analgesia (FSIA): Descending and intraspinal components
Brain Research, 245 (1982) 97-106 Elsevier Biomedical Press
97
Opiate vs Non-Opiate Eootshock Induced Analgesia (FSIA): Descending and Intraspinal Components L. R. WATKINS, D. A. COBELLI and D. J. MAYER Department of Physiology, Medical College of Virginia/VCU, Richmond, VA 23298 (U.S.A.)
(Accepted December 3rd, 1981) Key words: footshock induced analgesia - - descending inhibition - - dorsolateral funiculus - - intraspinal inhibition opiate analgesia - - non-opiate analgesia
Opiate and non-opiate footshock induced analgesia (FSIA) can be differentially elicited dependent upon the body region shocked. As measured by the spinally-mediated tail flick test, hind paw shock produces non-opiate analgesia whereas front paw shock produces opiate analgesia. The present series of experiments utilized cord lesions and transections to identify descending and intraspinal pathways mediating front paw and hind paw FSIA. The results of these studies indicate that front paw shock leads to activation of supraspinal sites which mediate analgesia via descending pathways lying solely within the dorsolateral funiculus (DLF) of the spinal cord; direct intraspinal pathways are not involved. Hind paw FSIA is also mediated by a descending DLF pathway but is unlike front paw FSIA in that it involves intraspinal pathways as well. This work provides further parallels between the analgesias produced by morphine, electrical brain stimulation and environmental stimuli. INTRODUCTION Electrical footshock has been demonstrated to reliably produce potent analgesia in rats 1,4,11-14. The magnitude and duration of footshock induced analgesia (FSIA) is dependent upon the intensity and duration of shock 12 and can be elicited by very brief (15 s) exposure to the stimulus 7. The behavioral deficits produced by this manipulation appear to be specific to nociception insofar as normal motor behavior, righting and corneal reflexes, vocalization, startle responses and response to touch remain unimpaired12,14,17. The neural mechanisms involved in FSIA are complex in that both opiate and non-opiate systems can mediate this effect. Differential activation of these systems has been observed which is dependent upon the duration of shock: opiate pathways are activated following prolonged (30 min) footshock whereas non-opiate mediation is observed after footshock of shorter (up to 3 min) duration 1,11,12,14. Thus it appeared that opiate systems were only activated following extended exposure to painful 0006-8993/82/0000-0000/$02.75 © Elsevier Biomedical Press
shock; non-opiate mediation being observed to briefer stress. Recently, it has become evident that the body region shocked is also a critical determinant of the opiate/non-opiate nature of FSIA. I f brief (90 s) shock is restricted to the front paws, the resultant analgesia: (1) is significantly antagonized by 1 /zg intrathecal or 0.1 mg/kg systemic naloxone 9,17,22,28, and (2) demonstrates cross-tolerance to morphine 17,22. In contrast, the analgesia resulting from shock restricted to the hind paws is neither reversible by naloxone nor does it show cross-tolerance with morphine 9,17,22,2s. Since the tail flick test 2,10 was used to assay analgesia in both of these paradigms, it remains plausible that non-opiate analgesia results from shock delivered within or near the dermatome being tested, and opiate analgesia is produced by shock delivered to more distant body regions. Due to the striking similarities now recognized between front paw FSIA and morphine analgesia (MA), it is plausible that c o m m o n neural pathways may mediate both effects. Supraspinal loci mediate
98 MA via descending pathways within the dorsolateral funiculus (DLF) of the spinal cord 5,6,13,1s. This projection is critical to the production of MA since bilateral lesions of this axon tract abolish the analgesia produced by systemic morphine 5,6,13. The effect of D L F lesions on FSIA has previously been examined 13. Hayes et al. 13 reported that FSIA was not attenuated by D L F lesions. However, in that study: (1) non-opiate FSIA was elicited by shock delivered to all 4 paws, and (2) the magnitude of analgesia was only evaluated within 30 s post-shock. Since (1) FSIA is nowrec ognized to be divisible into opiate (front paw) and non-opiate (hind paw) forms and (2) we have previously demonstrated that the duration of analgesia is by far more malleable than the initial degree of analgesia 9,17,22, we have reevaluated the effect of D L F lesions on FSIA. In addition, the effect of spinal transection was examined to determine whether intraspinal mechanisms contribute to the analgesia elicited by front paw or hind paw shock. EXPERIMENT 1. EFFECT OF BILATERAL THORACIC DLF LESIONS ON FRONT PAW FSIA
Methods Nineteen adult male Sprague-Dawley rats (350500 g) were used in the present experiment. Prior to surgery, all rats were tested for baseline pain responsivity, using a modification 2 of the D'Amour-Smith tail flick test 11. Bulb voltage was adjusted to attain a 3.5-4.0 s baseline latency (BL). Three tail flick trials, with a 2 min intertrial interval (ITI), were averaged to yield a mean baseline latency for each rat. Following baseline determinations, rats were anesthetized with Metofane (Pitman-Moore) and a laminectomy was performed at the second thoracic (T2) vertebral level. Following reflection of the dura, the D L F was located by identifying the dorsal root entry zone. Nine of the animals received bilateral D L F lesions using a carbon dioxide laser (Sharplan 733). The other 10 animals served as sham-operated controls; in these animals, the dura was reflected but no lesion was made. The exposed spinal cord was covered with Gel-foam (Upjohn) and the wound was then closed. All animals were treated with 0.3 ml distracillin immediately after sugery and 0.2 ml at one day following surgery.
One week after surgery, rats were again tested for baseline pain responsivity using the tail flick test (3 trials, 2 min ITI). Immediately following baseline measures, a soft nylon loop was gently placed around the hips of the animal and the rat placed on the non-electrified grid. The nylon cord was then elevated such that only the front paws were in contact with the grid. Each rat received one exposure to 90 s of constant current 60 Hz shock (1.6 mA rms). Upon termination of shock, tail flick trial, were used to assess pain responsivity through 14 mir post-shock. The radiant heat was automaticall3 terminated at 8 s if no tail flick occurred, in order tc avoid tissue damage. The post-shock tail flick latencies were expressec as a percent of maximal possible effect ( ~ M P E ) using the following equation: ~ M P E = [ ( T L - - B L ) / ( 8 . 0 - - BL)] × 100 where T L = post-shock test latency and BL = mean post-surgical baseline latency. Within eact shock paradigm (front paw and hind paw), meai ~ M P E for each post-shock test was compare~ between sham-operated and D L F lesioned group'. using the Student's t-test (I-tailed); Satterthwaite': approximation of the Student's t was used whel significant differences in sample variance occurred 2~ Paired t-tests comparing postshock and baselinq latencies were also calculated for each group. After behavioral testing was completed, rats wer~ overdosed with sodium pentobarbital and perfuse~ transcardially with 10 ~ formalin. The exposed tho racic cords of control animals were visually exam ined for any evidence of spinal damage. The cords o lesioned animals were removed and post-fixed iJ 10 ~ formalin. Following paraffin embedding, thes, cords were cutinto 15 # m sections, mounted on glas slides and stained with the Klfiver-Barrera method Drawings were then made of the maximal extent o the lesions, which were clearly identifiable by gliosi and myelin disruption. All lesion analyses were per formed blind with respect to the behavioral results
Results The bilateral D L F lesions are illustrated in Fig 1A. In agreement with previous reports 13, bilatera D L F lesions failed to alter baseline pain responsivit:
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Fig. 1. Composite drawing of the dorsolateral funiculus (DLF) lesions of rats used in Experiments 1, 2 and 4. A: bilateral DLF lesions at the second thoracic (T2) vertebral level of rats tested for front paw FSIA (Expt. 1). B: bilateral DLF lesions at the third cervical (C3) vertebral level of rats tested for front paw FSIA 1 week after surgery (Expt. 2). C: bilateral DLF lesions at C3 of rats tested for front paw FSIA 3 days after surgery (Expt. 2). D: bilateral DLF lesions at T2 of rats tested for hind paw FSIA (Expt. 4). After behavior testing was completed, rats were perfused with 10 ~ formalin. The lesion sites were cut at 15 #m and stained using the Kliiver-Barrera method. Drawings were made of the maximal extent of the lesions, which were clearly identifiable by gliosis and myelin disruption. (P >0.59). In addition, no significant differences (P >0.50) were observed in postsurgical baseline pain responsivity of lesioned and sham animals. Analgesia induced by front paw shock was profoundly attenuated in rats with bilateral T2 D L F lesions (Fig. 2). A highly significant (P <0.00005) difference was observed between sham-operated and D L F lesioned rats immediately post-shock. Whereas sham-operated rats remained significantly analgesic throughout testing, DLF-lesioned rats had returned to baseline latencies by 2 min after shock.
in the present experiment lesions were made above the pain input, at the third cervical (C3) vertebral level. In addition, the DLF-lesioned rats were subdivided into two groups: 7 rats were tested 3 days and 9 rats were tested 1 week after surgery in order to observe whether differential effects occur at varying times after D L F section. With the exception of level of surgical intervention and post-surgical times of testing, all methods were identical to those in Experiment 1.
Results EXPERIMENT 2. EFFECT OF BILATERAL CERVICAL DLF LESIONS ON FRONT PAW FSIA
Methods Since the front paw shock stimulus enters the neuraxis above T2, the results of Experiment 1 cannot exclude the possibility that intraspinal pathways as well as pain modulatory pathways of supraspinal origin may have been interrupted. Therefore,
No significant differences were observed in the baseline pain responsivity of sham- ( n = 10) or DLFlesioned rats tested 3 days ( n = 6 ) or 1 week ( n = 9 ) after surgery. Histologies of the C3 D L F lesions are presented in Fig. 1B and C. Analgesia was significantly attenuated in C3 DLF-lesioned rats at every time tested following shock; no differences were observed in the time-
I00 All rats were treated with 0.3 ml distracillin immediately after surgery. Brisk tail flick response was observed in spinalized rats within 5-30 min after spinal transection. Behavioral testing began 8 h after surgery. Foliowing assessment of baseline tail flick latencies (3 trials, 2 min ITI), rats were shocked on their front paws for 90 sec, as described in Experiment 1. U p o n termination of shock, tail flick trials were used to assess pain responsivity through 4 rain post-shock. Statistical analyses were as outlined in Experiment
course of analgesia produced in groups tested 3 days or 1 week after surgery (Fig. 2). Significant analgesia was only observed immediately after shock termination (0 rain) in both C3 DLF-lesioned groups. In contrast, sham-operated controls demonstrated pronotmced analgesia throughout the 14 min test.
EXPERIMENT 3. EFFECT OF THORACIC SPINAL TRANSECTIONOF FRONT PAW FSIA Methods
1.
Sixteen adult male Sprague-Dawley rats (350-500 g) were used in this experiment. Following assessment of baseline tail flick latencies (3 trials, 2 min ITI), all rats were anesthetized with Metofane (Pitm a n - M o o r e ) , a laminectomy was performed at T2 and the dura was reflected. The spinal cords of 9 rats were transected with a heated cauterizing electrode; the other 7 rats served as sham operated controls. The exposed spinal cord was covered with GelF o a m (Upjohn) and the wound was then closed. Each surgery required approximately 5 min to complete.
After behavioral testing was completed, rats were overdosed with pentobarbital and perfused transcardially with 10 ~ formalin. The thoracic cords of sham operated and lesioned rats were visually examined and manually probed for any evidence of spinal damage or incomplete transection, respectively.
Results Spinalization failed to significantly decrease tail
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TIME POST-SHOCK (min) Fig. 2. Effect of bilateral dorsolateral funiculus (DLF) lesions and spinalization on front paw FSIA. Bilateral DLF lesions at either the second thoracic (T2) or third cervical (C3) vertebral levels virtually abolished front paw FSIA (left, center). Since DLF lesions at C3 leave all potential intraspinal connections intact between the level of nociceptive input (front paws) and the lumbosacral cord (controlling the spinally mediated tail flick response), direct intraspinal pathways cannot be involved in the analgesia induced by front paw shock; pain inhibition must be mediated by supraspinal sites which inhibit pain via descending pathways within the DLF. Transient, although significant, analgesia is observed following spinalization below the level of nociceptive input (right), indicating that humoral factors may mediate this brief effect. *--P<0.05, **=P<0.01, ***=P<0.005, ****=P<0.001, ***** =P<0.0005, ****** =P<0.0001.
101 HIND PAW FSIA IO0
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Fig. 3. Effect of bilateral dorsolateral funiculus (DLF) lesions and spinalization on hind paw FSIA. Bilateral DLF lesions at the second thoracic (T2) vertebral levels greatly attenuated, but did not abolish, hind paw FSIA. Immediately after shock termination (0 min), profound analgesia was observed, which then slowly dissipated. No further significant reduction in analgesia was observed following T2 spinalization; spinalized rats remained analgesic through 12 min after shock. These results imply that descending pathways involved in hind paw FSIA only exist within the DLF and that intraspinal pathways account for the remaining potent analgesia. *--P<0.05, **--P<0.01, *** ~P<0.005, ***** =P<0.0005, ****** =P<0.0001. flick latencies (P >0.05). This result is in agreement with previous reports 18. At all postshock times tested (Fig. 2), analgesia induced by front paw shock was greatly attenuated in spinalized rats, as compared to shams (P <0.001). However, despite total cord transection below the level of shock input, spinalized rats exhibited significant analgesia immediately post-shock (P <0.05). As in Experiment 2, this was a very transient effect since latencies were not significantly different than baseline values by 1 min. EXPERIMENT 4. EFFECT OF BILATERAL THORACIC DLF LESIONS ON HIND PAW FSIA
No significant difference was observed in baseline (BL) pain responsivity of DLF-lesioned ( n = 8 ) or sham ( n = 10) animals. Histological reconstructions of the lesions are shown in Fig. 1D. Immediately following hind paw shock, profound analgesia was observed in both DLF-lesioned and sham groups; however, only the sham surgery group remained analgesic throughout the 14 min postshock test (Fig. 3). In contrast, the DLF-lesioned animals remained significantly analgesic only until 4 min after shock. EXPERIMENT 5. EFFECT OF THORACIC SPINAL TRANSECTION ON HIND PAW FSIA Methods
Seventeen adult male Sprague-Dawley rats (350500 g) received either T2 spinal transections or sham surgery as outlined in Experiment 3. Behavioral testing of hind paw FSIA was delayed until 8 h after surgery. Following assessment of baseline tail flick latencies (3 trials, 2 min ITI), the animals received hind paw shock as described in Experiment 4. Shock parameters, post-shock testing, statistical analyses and histological verification were as outlined in Experiment 3. Results
Methods
Eighteen adult male Sprague-Dawley rats (350490 g) received either T2 D L F lesions or sham surgery as outlined in Experiment 1. Behavioral testing of hind paw FSIA was delayed until one week after surgery. Following assessment of baseline tail flick latencies (3 trials, 2 min ITI), a soft nylon cord was gently placed around the chest of the rat, and the animal
Significant differences were observed in the postsurgical baseline (BL) pain responsivity of sham and spinalized rats (P <0.05). Sample size (n) and average tail flick latencies (mean 4- S.E.) for each group were as follows. Sham: n = 7 , BL=3.85 40.12 s; T2 spinal transection: n----10, BL=3.01 0.12 s. Throughout the 14 rain post-shock test, sham operated animals remained significantly more anal-
102 gesic than the spinalized group (Fig. 3); the sham group remained analgesic throughout the 14 min test (P < 0.00005 at 14 min). Importantly, rats with total spinal transections were significantly analgesic through 12 min post-shock. Except for 0 min postshock (P <0.05), no significant differences were observed in the analgesia induced in T2 D L F or T2 spinalized rats.
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DISCUSSION The present series of experiments provide the first systematic examination of the complex neural pathways involved in opiate and non-opiate FSIA. Previous work has demonstrated that the body region shocked is a determinant of the pain inhibitory system activated. Based on studies of the effects of systemic naloxone 9,22, intrathecal naloxone 27 and morphine tolerance 22, front paw and hind paw shock were dearly shown to produce opiate and non-opiate analgesia, respectively. Both of these analgesias appear to be neurally, rather than hormonally, mediated. Specifically, the production of opiate analgesia by front paw shock does not depend on hypophyseal, sympathetic or adrenal medullary opioids (for discussion, see ref. 23). Thus it is now reasonable to examine the neural pathways activated by footshock. The fact that footshock results in the inhibition of the spinally mediated tail flick reflex requires that the pain inhibitory circuitry either exists within the spinal cord or consists of a descending pathway with supraspinal origin. The neurally mediated analgesia produced by morphine and electrical brain stimulation are both mediated by descending pathways within the dorsolateral funiculus (DLF) of the cord 5,6,18. Identification in the present study of a critical descending D L F pathway for both front paw FSIA and hind paw FSIA draws a further parallel between these environmentally induced analgesias, classically conditioned analgesia 24, morphine analgesia (MA) and stimulation produced analgesia (SPA). Thus it appears plausible that the D L F may form a final common pathway for all neural endogenous pain inhibitory systems. Although front paw FSIA and hind paw FSIA are both dependent upon the functional integrity of the DLF, they differ with regard to the involvement of
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Fig. 4. The neural circuitry of front paw FSIA (left) and hin~ paw FSIA (right). The demonstration that both T2 and C DLF lesions virtually abolish front paw FSIA implies tha direct intraspinal pathways cannot be involved in this pai~ inhibition since any potential neural pathway between th front paws (which receive the shock) and the tail (which i tested for analgesia) remains intact following C3 lesions, Therefore, front paw shock activates supraspinal structure which mediate analgesia via a descending pathway lyin within the DLF. Hind paw FSIA is also mediated, in part, b an ascending-descending loop since T2 DLF lesions signif candy attenuate, but do not abolish, hind paw FSIA. Unlik front paw FSIA, hind paw FSIA is also mediated by intrasp: nal pathways since significant and prolonged analgesia i observed following T2 spioalization. Antagonism of fror paw FSIA by spinal naloxone indicates the involvement of a endogenous opioid-like peptide (EOLP). intraspinal components. Front paw FSIA is virtu ally abolished following bilateral D L F lesions at th second thoracic (T2) vertebral level. As seen i: Fig. 4, interruption of intraspinal pathways can not account for the effects observed following DL] lesions, since D L F lesions below the spinal level c sensory input (T2) and those above the spinal lev¢ of sensory input (C3) result in equivalent atten uation of front paw FSIA. Therefore, like MA, fron paw FSIA is mediated by descending pathway which originate from supraspinal sites. Interestingl5 no further reduction of front paw FSIA occurre following T2 spinal transection. Two conclusion
103 can be drawn from this observation: (1) the DLF is the only spinal pathway involved in the opiate analgesia induced by front paw shock, and (2) since all neural connections were severed below the level of pain input, a non-neural component appears to be responsible for the transient analgesia observed immediately post-shock (cf. Fig. 2). Although at present, the identity of this non-neural factor remains elusive, preliminary data gathered in our laboratory indicate that this small, transient effect may be opiate mediated since systemic naloxone prevents the analgesic state observed immediately after front paw shock in thoracic spinal transected rats 8. As previously stated, non-opiate analgesia induced by hind paw shock is also mediated by descending pathways within the DLF (Fig. 4). Although Hayes et al. 13 concluded that the DLF does not mediate non-opiate FSIA, careful examination of their work reveals that their data are not inconsistent with the results of the present experiments. As reported by Hayes et al. 13, potent analgesia is observed imediately following shock. However, it is now known that the duration, rather than the initial magnitude, of non-opiate FSIA is markedly attenuated following T2 DLF lesions. Since Hayes et al. 13 only examined the magnitude of analgesia within 30 s after shock, they were unable to observe the effect of DLF lesions on the duration of non-opiate FSIA. It is important to note that intraspinal, rather than descending, pain modulatory pathways are responsible for the analgesia observed following T2 DLF lesions (Fig. 4). Since the time course of analgesia observed following T2 spinal transections and T2 DLF lesions do not differ significantly, it is concluded that segmental circuitry and descending pathways within the DLF account for the entire analgesic response to hind paw shock. Since, in spinalized animals, significant analgesia was observed through 12 min after shock, intraspinal pathways are capable of producing potent and prolonged pain inhibition. Thus, this series of papers on footshock induced analgesia (see refs. 22, 23, 24 and 27) provide strong evidence for the existence of multiple endogenous pain inhibitory systems within the central nervous system. Studies of front paw and hind paw FSIA have identified at least three neural pain inhibitory pathways. The first produces an opiate analgesia, is
observed following front paw shock or classical conditioning to either front paw or hind paw shock, is mediated by descending pathways within the DLF, and is dependent upon an opiate synapse within the spinal cord. The other two systems produce the non-opiate analgesia observed following hind paw shock. One consists of a descending pathway which exists entirely within the DLF and the second involves intraspinal pathways. However, it is evident from a review of the literature that these three pathways cannot account for all of the presently available data on endogenous control of pain. As seen in Table I, four major classes of endogenous pain inhibitory systems are readily evident: neural/opiate, hormonal/opiate, neural/nonopiate and hormonal/non-opiate. Front paw FSIA serves as the prototype of the neural/opiate pain inhibitory class (Table I). Members of this class are characterized as being antagonized by naloxone, showing cross-tolerance to morphine and not being dependent upon peripheral opioids (i.e. not attenuated by adrenalectomy or hypophysectomy). Included as neural/opiate analgesias are front paw FSIA, analgesia classically conditioned to either front paw or hind paw shock, stimulation produced analgesia and morphine analgesia following systemic, intracerebral, or intrathecal administration. As can be seen in Table I, presently available data are consistent with the idea that these analgesias are mediated by common neural pathways. Although controversy presently exists regarding the involvement of the nucleus raphe alatus 26 in SPA and systemic morphine analgesia (for discussion, see ref. 26), recent work has demonstrated that analgesia produced by morphine microinjection into the PAG is abolished following nucleus raphe alatus lesions 2s. Furthermore, a significant correlation is observed after lesioning this region between reduction in the degree of analgesia produced by systemic morphine and by front paw shock 2s. This result again strongly argues in favor of a common neural basis for these phenomena. Analgesia produced by electrical brain stimulation is a special case in that it belongs to two classes: neural/opiate and neural/non-opiate (Table I). Although striking similarities exist between morphine analgesia and stimulation produced analgesia (SPA), brain stimulation appears to activate both
104 TABLE I
Summary of currently available data on variousforms of analgesia
As seen in the table, the analgesias produced by various manipulations fall into 4 distinguishable classes • neural/opiate, hormonal/ opiate, neural/non-opiate, and hormonal/non-opiate. Analgesia is categorized as hormonal if it is reduced or abolished by removal of the pituitary or adrenal glands. The criteria for opiate analgesia include antagonism by naloxone and morphine tolerance. ~, = analgesia is reduced or abolished, ~ = analgesia is potentiated, 0 = analgesia is unaffected, ? = disparate evidence exists whether analgesia is reduced or unaffected, • = no data are available. A blank space indicates a category which is inappropriate to the analgesia. For literature citations, see ref. 14. Similarity to opiates Systemic naloxone
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Neural/opiate Brief front paw shock Conditioning to footshock Systemic morphine Intracerebral morphine Intrathecal morphine Brain stimulation Hormonal/opiate Acupuncture Prolonged 4 paw shock Immobilization Neural/non-opiate Brief hind paw shock Brief 4 paw shock 2-deoxy-D-glucose Brain stimulation Hormonal/non-opiate Cold water swims Insulin Unknown/opiate TNS (lo freq/hi int) Food deprivation Unknown/non-opiate TNS (hi freq/lo int) Hypnosis
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opiate and non-opiate pain inhibitory systems. Naloxone has a variable effect on SPA ranging f r o m complete reversal to n o antagonism at allZ,19, z0. Part o f this variability is accounted for by the site o f stimulation. Prieto et al. 21 have reported that stimulation sites ventral to the dorsal raphe support a naloxone-reversible analgesia, whereas SPA elicited f r o m more dorsal areas fails to be reversed by this opiate antagonist. Furthermore, the fact that crosstolerance between morphine analgesia and SPA is incomplete implies that a non-opiate c o m p o n e n t exists for SPA 16.
Non-opiate mechanisms are involved in other pain inhibitory pathways as well. The analgesias p r o d u c e d by 2-deoxy-D-glucose (2-DG), brief hind paw shock and brief shock of all four paws are included in the neural/non-opiate class (Table I). Like systemic morphine analgesia, 2 - D G analgesia is potentiated after hypophysectomy. T h o u g h this m a y indicate that a h o r m o n a l antagonist to 2 - D G analgesia exists, it seems more plausible that, like the potentiation observed for morphine, this is due to peripheral effects secondary to hypophysectomy, including alterations in b l o o d - b r a i n barrier, degrad-
105 ation rates and bound/free ratios in the blood (for disc. see ref. 23). On the other hand, the endocrine system does appear to be involved in other forms of pain inhibition. Analgesia produced by prolonged footshock, acupuncture and immobilization apparently depend upon peripheral opioids. However, it remains equivocal whether the critical source of peripheral opioids is the pituitary gland or the adrenal medulla. As discussed in detail elsewhere (for disc. see ref. 23), hypophysectomy can indirectly lead to pronounced alterations in adrenal medullary function. Whether synthesis of adrenal enkephalin is affected as well is presently unknown. The work of Lewis et al. 15 suggests that this may indeed be the case. They demonstrated that adrenal demedullation significantly attenuated analgesia following prolonged footshock. The attenuation was, in fact, greater than they observed after hypophysectomy. The effect of adrenal demedullation on acupuncture and immobilization induced analgesia, the other two members of this class, has not yet been investigated. The hormones apparently involved in hormonal/ non-opiate analgesia (i.e., insulin and cold water swim induced analgesias) have also not been identified. Since hypophysectomy has been the only manipulation tested for this class, involvement of adrenal cortical or adrenal medullary substances cannot be ruled out. For example, epinephrine has previously been demonstrated to be capable of producing prolonged analgesia. Therefore, it remains plausible that non-hypophyseal factors may be the
REFERENCES 1 Akil, H., Madden, J., Patrick, R. L. and Barchas, J. D., Stress-induced increase in endogenous opiate peptides" concurrent analgesia and its partial reversal by naloxone. In H. W. Kosterlitz (Ed.), Opiates and Endogenous Opioid Peptides, North Holland, Amsterdam, 1976, pp. 63-70. 2 Akil, H. and Mayer, D. J., Antagonism of stimulationproduced analgesia by p-CPA, a serotonin synthesis inhibitor, Brain Research, 44 (1972) 692-697. 3 Akil, I-I., Mayer, D. J. and Liebeskind, J. C., Antagonism of stimulation-produced analgesia by naloxone, a narcotic antagonist, Science, 191 (1976) 961-962. 4 Amir, A. and Amit, Z., Enhanced analgesic effects of stress following administration of naltrexone in rats, Europ. J. Pharmacol., 59 (1979) 137-140. 5 Barton, C., Basbaum, A. I. and Fields, H. L., Dissociation of supraspinal and spinal actions of morphine: a
actual basis of hormonal/non-opiate analgesia. Thus, it is clear that multiple endogenous systems exist which cart act to inhibit the perception of pain. These systems are highly complex; analgesia can be produced via opiate/ or non-opiate/hormonal, as well as opiate/or non-opiate/neural, pathways. It is not yet clear whether the hormonal systems act directly at the level of the spinal cord or whether they may act indirectly by activating descending pain inhibitory pathways with supraspinal origin. It is clear that pain inhibitory pathways within the D L F are critically involved in every descending neurally mediated analgesia studied to date. However, beyond this point, little is known regarding the pharmacology or anatomy of the various systems. Identification and characterization of these multiple pain inhibitory pathways has direct significance for the control of pain in man since an understanding of these circuits will potentially provide a battery of methods with which to treat clinical pain. Sequential activation of the various pathways may allow effective treatment of pain in man while obviating the problems of tolerance and addiction. ACKNOWLEDGEMENTS We would like to thank Ms. Ingrid Kinscheck for her invaluable technical assistance, and Dr. D. D. Becker for allowing us to use Sharplan carbon dioxide laser. This research was supported by PHS Grant DA 00576 to D . J . M . L . R . W . was supported by a NSF Graduate Fellowship.
quantitative evaluation, Brain Research, 188 (1980) 487-498. 6 Basbaum, A., Marley, N. J. E., O'Keefe, J. and Clanton, C. H., Reversal of morphine and stimulation-produced analgesia by subtotal spinal cord lesions, Pain, 3 (1977) 43-56. 7 Chance, W. T., Autoanalgesia: opiate and non-opiate mechanisms, Neurosci. Biobehav. Rev., 4 (1979) 55-67. 8 Cobelli,D. A.,lnvestigationsoftheSympatheticandNeural Components of Front Paw Footshock Induced Analgesia,
unpublished Master's Thesis, Medical College of Virginia/Virginia Commonwealth University, 1981. 9 Cobelli, D. A., Watkins, L. R and Mayer, D. J., Dissociation of opiate and non-opiate footshock produced analgesia (FSA), Neurosci. Abstr., 6 (1980) 247. 10 D'Amour, F. E. and Smith, D. L., A method for determining loss of pain sensation, J. PharmacoL exp. Ther., 72 (1941) 74-79.
106 11 Hayes, R. L., Bennet, G. J., Newlon, P. and Mayer, D. J., Analgesic effects of certain noxious and stressful manipulations in the rat, Neurosci. Abstr., 2 (1976) 939. 12 Hayes, R. L., Bennett, G. J., Newlon, P. and Mayer, D. J., Behavioral and physiological studies of non-narcotic analgesia in the rat elicited by certain environmental stimuli, Brain Research, 155 (1978) 69-90. 13 Hayes, R. L., Price, D. D., Bennet, G. J., Wilcox, G. L. and Mayer, D. J., Differential effects of spinal cord lesions on narcotic and non-narcotic suppression of nociceptive reflexes: further evidence for the physiologic multiplicity of pain modulation, Brain Research, 155 (1978) 91-101. 14 Lewis, J. W., Cannon, J. T. and Liebeskind, J. C., Opioid and nonopioid mechanisms of stress analgesia, Science, 208 (1980) 623-625. 15 Lewis, J. W., Tordoff, M. G., Sherman, J. E. and Liebeskind, J. C., Role of the adrenal medulla in opioid stress analgesia, Neurosci. Abstr., 7 (1981) 735. 16 Mayer, D. J. and Hayes, R. L., Stimulation-produced analgesia: development of tolerance and cross-tolerance to morphine, Science, 188 (1975) 941-943. 17 Mayer, D. J. and Watkins, L. R., The role of endorphins in endogenous pain control systems. In H. M. Emrich (Ed.), Modern Problems of Pharmacopsychiatry: The Role ofEndorphins inNeuropsychiatry, Karger Basel, 1981, 68-96. 18 Murfin, R., Bennett, G. J. and Mayer, D. J., The effects of dorsolateral spinal cord (DLF) lesions on analgesia from morphine microinjected into the periaqueductal grey matter (PAG) of the rat, Neurosci. Abstr., 2 (1976) 946. 19 Oliveras, J. L., Hosobuchi, Y., Redjemi, F., Guilbaud, F. and Besson, J. M., Opiate antagonist, naloxone, strongly reduces analgesia induced by stimulation of a raphe nucleus (centralis inferior), Brain Research, 120 (1977) 221-229. 20 Pert, A. and Walter, M., Comparison between naloxone reversal of morphine and electrical stimulation induced analgesia in the rat mesencephalon, Life Sci., 19 (1976) 1023-1032. 21 Prieto, G. J., Giesler, G. J. and Cannon, J. T., Evidence for
22
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25
26
27
28
29
30
site specificity in naloxone's antagonism of stimulation produced analgesia in the rat, Neurosci. Abstr., 4 (1979) 460. Watkins, L. R., Cobelli, D. A., Faris, P., Aceto, M. D. and Mayer, D. J., Opiate vs. non-opiate footshock induced analgesia: the body region shocked is a critical factor, Brain Research, in press. Watkins, L. R., Cobelli, D. A., Newsome, H. H. and Mayer, D. J., Footshock induced analgesia is dependent neither on pituitary nor sympathetic activation, Brain Research, 245 (1982) 81-96. Watkins, L. R., Cobelli, D.A. and Mayer, D. J., Classical conditioning of front paw and hind paw footshock induced analgesia (FSIA): naloxone reversibility and descending pathways, Brain Research, in press. Watkins, L. R., Griffin, G., Leichnetz, G. R. and Mayer, D. J., Identification and somatotopic organization of nuclei projecting via the dorsolateral funiculus in rats: a retrograde tracing study using HRP slow-release gels, Brain Research, 223 (1981) 237-255. Watkins, L. R., Griffin, G., Leichnetz, G. R. and Mayer, D. J., The somatotopic organization of the nucleus raphe magnus and surrounding brain stem structures as revealed by HRP slow-release gels, Brain Research, 181 (1980) 1-15. Watkins, L. R. and Mayer, D. J., Involvement of spinal opioid systems in footshock induced analgesia (FSIA): antagonism by naloxone is possible only before induction of analgesia, Brain Research, in press. Watkins, L. R. and Mayer, D. J., The neural organization of endogenous opiate and non-opiate pain control systems, Science, in press. Watkins, L. R., Young, E., Kinscheck, I. and Mayer, D. J., Effects of nucleus raphe magnus lesions and periaqueductal gray lesions on footshock induced analgesia and morphine analgesia: evidence for common neural pathways, Neurosci. Abstr., 7 (1981) 340. Winer, B. J., Statistical Principles in Experimental Design, McGraw-Hill, New York, 1962, p. 37.
97
Opiate vs Non-Opiate Eootshock Induced Analgesia (FSIA): Descending and Intraspinal Components L. R. WATKINS, D. A. COBELLI and D. J. MAYER Department of Physiology, Medical College of Virginia/VCU, Richmond, VA 23298 (U.S.A.)
(Accepted December 3rd, 1981) Key words: footshock induced analgesia - - descending inhibition - - dorsolateral funiculus - - intraspinal inhibition opiate analgesia - - non-opiate analgesia
Opiate and non-opiate footshock induced analgesia (FSIA) can be differentially elicited dependent upon the body region shocked. As measured by the spinally-mediated tail flick test, hind paw shock produces non-opiate analgesia whereas front paw shock produces opiate analgesia. The present series of experiments utilized cord lesions and transections to identify descending and intraspinal pathways mediating front paw and hind paw FSIA. The results of these studies indicate that front paw shock leads to activation of supraspinal sites which mediate analgesia via descending pathways lying solely within the dorsolateral funiculus (DLF) of the spinal cord; direct intraspinal pathways are not involved. Hind paw FSIA is also mediated by a descending DLF pathway but is unlike front paw FSIA in that it involves intraspinal pathways as well. This work provides further parallels between the analgesias produced by morphine, electrical brain stimulation and environmental stimuli. INTRODUCTION Electrical footshock has been demonstrated to reliably produce potent analgesia in rats 1,4,11-14. The magnitude and duration of footshock induced analgesia (FSIA) is dependent upon the intensity and duration of shock 12 and can be elicited by very brief (15 s) exposure to the stimulus 7. The behavioral deficits produced by this manipulation appear to be specific to nociception insofar as normal motor behavior, righting and corneal reflexes, vocalization, startle responses and response to touch remain unimpaired12,14,17. The neural mechanisms involved in FSIA are complex in that both opiate and non-opiate systems can mediate this effect. Differential activation of these systems has been observed which is dependent upon the duration of shock: opiate pathways are activated following prolonged (30 min) footshock whereas non-opiate mediation is observed after footshock of shorter (up to 3 min) duration 1,11,12,14. Thus it appeared that opiate systems were only activated following extended exposure to painful 0006-8993/82/0000-0000/$02.75 © Elsevier Biomedical Press
shock; non-opiate mediation being observed to briefer stress. Recently, it has become evident that the body region shocked is also a critical determinant of the opiate/non-opiate nature of FSIA. I f brief (90 s) shock is restricted to the front paws, the resultant analgesia: (1) is significantly antagonized by 1 /zg intrathecal or 0.1 mg/kg systemic naloxone 9,17,22,28, and (2) demonstrates cross-tolerance to morphine 17,22. In contrast, the analgesia resulting from shock restricted to the hind paws is neither reversible by naloxone nor does it show cross-tolerance with morphine 9,17,22,2s. Since the tail flick test 2,10 was used to assay analgesia in both of these paradigms, it remains plausible that non-opiate analgesia results from shock delivered within or near the dermatome being tested, and opiate analgesia is produced by shock delivered to more distant body regions. Due to the striking similarities now recognized between front paw FSIA and morphine analgesia (MA), it is plausible that c o m m o n neural pathways may mediate both effects. Supraspinal loci mediate
98 MA via descending pathways within the dorsolateral funiculus (DLF) of the spinal cord 5,6,13,1s. This projection is critical to the production of MA since bilateral lesions of this axon tract abolish the analgesia produced by systemic morphine 5,6,13. The effect of D L F lesions on FSIA has previously been examined 13. Hayes et al. 13 reported that FSIA was not attenuated by D L F lesions. However, in that study: (1) non-opiate FSIA was elicited by shock delivered to all 4 paws, and (2) the magnitude of analgesia was only evaluated within 30 s post-shock. Since (1) FSIA is nowrec ognized to be divisible into opiate (front paw) and non-opiate (hind paw) forms and (2) we have previously demonstrated that the duration of analgesia is by far more malleable than the initial degree of analgesia 9,17,22, we have reevaluated the effect of D L F lesions on FSIA. In addition, the effect of spinal transection was examined to determine whether intraspinal mechanisms contribute to the analgesia elicited by front paw or hind paw shock. EXPERIMENT 1. EFFECT OF BILATERAL THORACIC DLF LESIONS ON FRONT PAW FSIA
Methods Nineteen adult male Sprague-Dawley rats (350500 g) were used in the present experiment. Prior to surgery, all rats were tested for baseline pain responsivity, using a modification 2 of the D'Amour-Smith tail flick test 11. Bulb voltage was adjusted to attain a 3.5-4.0 s baseline latency (BL). Three tail flick trials, with a 2 min intertrial interval (ITI), were averaged to yield a mean baseline latency for each rat. Following baseline determinations, rats were anesthetized with Metofane (Pitman-Moore) and a laminectomy was performed at the second thoracic (T2) vertebral level. Following reflection of the dura, the D L F was located by identifying the dorsal root entry zone. Nine of the animals received bilateral D L F lesions using a carbon dioxide laser (Sharplan 733). The other 10 animals served as sham-operated controls; in these animals, the dura was reflected but no lesion was made. The exposed spinal cord was covered with Gel-foam (Upjohn) and the wound was then closed. All animals were treated with 0.3 ml distracillin immediately after sugery and 0.2 ml at one day following surgery.
One week after surgery, rats were again tested for baseline pain responsivity using the tail flick test (3 trials, 2 min ITI). Immediately following baseline measures, a soft nylon loop was gently placed around the hips of the animal and the rat placed on the non-electrified grid. The nylon cord was then elevated such that only the front paws were in contact with the grid. Each rat received one exposure to 90 s of constant current 60 Hz shock (1.6 mA rms). Upon termination of shock, tail flick trial, were used to assess pain responsivity through 14 mir post-shock. The radiant heat was automaticall3 terminated at 8 s if no tail flick occurred, in order tc avoid tissue damage. The post-shock tail flick latencies were expressec as a percent of maximal possible effect ( ~ M P E ) using the following equation: ~ M P E = [ ( T L - - B L ) / ( 8 . 0 - - BL)] × 100 where T L = post-shock test latency and BL = mean post-surgical baseline latency. Within eact shock paradigm (front paw and hind paw), meai ~ M P E for each post-shock test was compare~ between sham-operated and D L F lesioned group'. using the Student's t-test (I-tailed); Satterthwaite': approximation of the Student's t was used whel significant differences in sample variance occurred 2~ Paired t-tests comparing postshock and baselinq latencies were also calculated for each group. After behavioral testing was completed, rats wer~ overdosed with sodium pentobarbital and perfuse~ transcardially with 10 ~ formalin. The exposed tho racic cords of control animals were visually exam ined for any evidence of spinal damage. The cords o lesioned animals were removed and post-fixed iJ 10 ~ formalin. Following paraffin embedding, thes, cords were cutinto 15 # m sections, mounted on glas slides and stained with the Klfiver-Barrera method Drawings were then made of the maximal extent o the lesions, which were clearly identifiable by gliosi and myelin disruption. All lesion analyses were per formed blind with respect to the behavioral results
Results The bilateral D L F lesions are illustrated in Fig 1A. In agreement with previous reports 13, bilatera D L F lesions failed to alter baseline pain responsivit:
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Fig. 1. Composite drawing of the dorsolateral funiculus (DLF) lesions of rats used in Experiments 1, 2 and 4. A: bilateral DLF lesions at the second thoracic (T2) vertebral level of rats tested for front paw FSIA (Expt. 1). B: bilateral DLF lesions at the third cervical (C3) vertebral level of rats tested for front paw FSIA 1 week after surgery (Expt. 2). C: bilateral DLF lesions at C3 of rats tested for front paw FSIA 3 days after surgery (Expt. 2). D: bilateral DLF lesions at T2 of rats tested for hind paw FSIA (Expt. 4). After behavior testing was completed, rats were perfused with 10 ~ formalin. The lesion sites were cut at 15 #m and stained using the Kliiver-Barrera method. Drawings were made of the maximal extent of the lesions, which were clearly identifiable by gliosis and myelin disruption. (P >0.59). In addition, no significant differences (P >0.50) were observed in postsurgical baseline pain responsivity of lesioned and sham animals. Analgesia induced by front paw shock was profoundly attenuated in rats with bilateral T2 D L F lesions (Fig. 2). A highly significant (P <0.00005) difference was observed between sham-operated and D L F lesioned rats immediately post-shock. Whereas sham-operated rats remained significantly analgesic throughout testing, DLF-lesioned rats had returned to baseline latencies by 2 min after shock.
in the present experiment lesions were made above the pain input, at the third cervical (C3) vertebral level. In addition, the DLF-lesioned rats were subdivided into two groups: 7 rats were tested 3 days and 9 rats were tested 1 week after surgery in order to observe whether differential effects occur at varying times after D L F section. With the exception of level of surgical intervention and post-surgical times of testing, all methods were identical to those in Experiment 1.
Results EXPERIMENT 2. EFFECT OF BILATERAL CERVICAL DLF LESIONS ON FRONT PAW FSIA
Methods Since the front paw shock stimulus enters the neuraxis above T2, the results of Experiment 1 cannot exclude the possibility that intraspinal pathways as well as pain modulatory pathways of supraspinal origin may have been interrupted. Therefore,
No significant differences were observed in the baseline pain responsivity of sham- ( n = 10) or DLFlesioned rats tested 3 days ( n = 6 ) or 1 week ( n = 9 ) after surgery. Histologies of the C3 D L F lesions are presented in Fig. 1B and C. Analgesia was significantly attenuated in C3 DLF-lesioned rats at every time tested following shock; no differences were observed in the time-
I00 All rats were treated with 0.3 ml distracillin immediately after surgery. Brisk tail flick response was observed in spinalized rats within 5-30 min after spinal transection. Behavioral testing began 8 h after surgery. Foliowing assessment of baseline tail flick latencies (3 trials, 2 min ITI), rats were shocked on their front paws for 90 sec, as described in Experiment 1. U p o n termination of shock, tail flick trials were used to assess pain responsivity through 4 rain post-shock. Statistical analyses were as outlined in Experiment
course of analgesia produced in groups tested 3 days or 1 week after surgery (Fig. 2). Significant analgesia was only observed immediately after shock termination (0 rain) in both C3 DLF-lesioned groups. In contrast, sham-operated controls demonstrated pronotmced analgesia throughout the 14 min test.
EXPERIMENT 3. EFFECT OF THORACIC SPINAL TRANSECTIONOF FRONT PAW FSIA Methods
1.
Sixteen adult male Sprague-Dawley rats (350-500 g) were used in this experiment. Following assessment of baseline tail flick latencies (3 trials, 2 min ITI), all rats were anesthetized with Metofane (Pitm a n - M o o r e ) , a laminectomy was performed at T2 and the dura was reflected. The spinal cords of 9 rats were transected with a heated cauterizing electrode; the other 7 rats served as sham operated controls. The exposed spinal cord was covered with GelF o a m (Upjohn) and the wound was then closed. Each surgery required approximately 5 min to complete.
After behavioral testing was completed, rats were overdosed with pentobarbital and perfused transcardially with 10 ~ formalin. The thoracic cords of sham operated and lesioned rats were visually examined and manually probed for any evidence of spinal damage or incomplete transection, respectively.
Results Spinalization failed to significantly decrease tail
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TIME POST-SHOCK (min) Fig. 2. Effect of bilateral dorsolateral funiculus (DLF) lesions and spinalization on front paw FSIA. Bilateral DLF lesions at either the second thoracic (T2) or third cervical (C3) vertebral levels virtually abolished front paw FSIA (left, center). Since DLF lesions at C3 leave all potential intraspinal connections intact between the level of nociceptive input (front paws) and the lumbosacral cord (controlling the spinally mediated tail flick response), direct intraspinal pathways cannot be involved in the analgesia induced by front paw shock; pain inhibition must be mediated by supraspinal sites which inhibit pain via descending pathways within the DLF. Transient, although significant, analgesia is observed following spinalization below the level of nociceptive input (right), indicating that humoral factors may mediate this brief effect. *--P<0.05, **=P<0.01, ***=P<0.005, ****=P<0.001, ***** =P<0.0005, ****** =P<0.0001.
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was placed on the non-electrified grid. The cord was then elevated such that only the hind paws were in contact with the grid. Shock parameters, post-shock testing, statistical analyses and histological verification of the lesions were as described in Experiment 1. Results
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Fig. 3. Effect of bilateral dorsolateral funiculus (DLF) lesions and spinalization on hind paw FSIA. Bilateral DLF lesions at the second thoracic (T2) vertebral levels greatly attenuated, but did not abolish, hind paw FSIA. Immediately after shock termination (0 min), profound analgesia was observed, which then slowly dissipated. No further significant reduction in analgesia was observed following T2 spinalization; spinalized rats remained analgesic through 12 min after shock. These results imply that descending pathways involved in hind paw FSIA only exist within the DLF and that intraspinal pathways account for the remaining potent analgesia. *--P<0.05, **--P<0.01, *** ~P<0.005, ***** =P<0.0005, ****** =P<0.0001. flick latencies (P >0.05). This result is in agreement with previous reports 18. At all postshock times tested (Fig. 2), analgesia induced by front paw shock was greatly attenuated in spinalized rats, as compared to shams (P <0.001). However, despite total cord transection below the level of shock input, spinalized rats exhibited significant analgesia immediately post-shock (P <0.05). As in Experiment 2, this was a very transient effect since latencies were not significantly different than baseline values by 1 min. EXPERIMENT 4. EFFECT OF BILATERAL THORACIC DLF LESIONS ON HIND PAW FSIA
No significant difference was observed in baseline (BL) pain responsivity of DLF-lesioned ( n = 8 ) or sham ( n = 10) animals. Histological reconstructions of the lesions are shown in Fig. 1D. Immediately following hind paw shock, profound analgesia was observed in both DLF-lesioned and sham groups; however, only the sham surgery group remained analgesic throughout the 14 min postshock test (Fig. 3). In contrast, the DLF-lesioned animals remained significantly analgesic only until 4 min after shock. EXPERIMENT 5. EFFECT OF THORACIC SPINAL TRANSECTION ON HIND PAW FSIA Methods
Seventeen adult male Sprague-Dawley rats (350500 g) received either T2 spinal transections or sham surgery as outlined in Experiment 3. Behavioral testing of hind paw FSIA was delayed until 8 h after surgery. Following assessment of baseline tail flick latencies (3 trials, 2 min ITI), the animals received hind paw shock as described in Experiment 4. Shock parameters, post-shock testing, statistical analyses and histological verification were as outlined in Experiment 3. Results
Methods
Eighteen adult male Sprague-Dawley rats (350490 g) received either T2 D L F lesions or sham surgery as outlined in Experiment 1. Behavioral testing of hind paw FSIA was delayed until one week after surgery. Following assessment of baseline tail flick latencies (3 trials, 2 min ITI), a soft nylon cord was gently placed around the chest of the rat, and the animal
Significant differences were observed in the postsurgical baseline (BL) pain responsivity of sham and spinalized rats (P <0.05). Sample size (n) and average tail flick latencies (mean 4- S.E.) for each group were as follows. Sham: n = 7 , BL=3.85 40.12 s; T2 spinal transection: n----10, BL=3.01 0.12 s. Throughout the 14 rain post-shock test, sham operated animals remained significantly more anal-
102 gesic than the spinalized group (Fig. 3); the sham group remained analgesic throughout the 14 min test (P < 0.00005 at 14 min). Importantly, rats with total spinal transections were significantly analgesic through 12 min post-shock. Except for 0 min postshock (P <0.05), no significant differences were observed in the analgesia induced in T2 D L F or T2 spinalized rats.
Neural Circuitry Front Paw FSIA
Hind Paw FSIA
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DISCUSSION The present series of experiments provide the first systematic examination of the complex neural pathways involved in opiate and non-opiate FSIA. Previous work has demonstrated that the body region shocked is a determinant of the pain inhibitory system activated. Based on studies of the effects of systemic naloxone 9,22, intrathecal naloxone 27 and morphine tolerance 22, front paw and hind paw shock were dearly shown to produce opiate and non-opiate analgesia, respectively. Both of these analgesias appear to be neurally, rather than hormonally, mediated. Specifically, the production of opiate analgesia by front paw shock does not depend on hypophyseal, sympathetic or adrenal medullary opioids (for discussion, see ref. 23). Thus it is now reasonable to examine the neural pathways activated by footshock. The fact that footshock results in the inhibition of the spinally mediated tail flick reflex requires that the pain inhibitory circuitry either exists within the spinal cord or consists of a descending pathway with supraspinal origin. The neurally mediated analgesia produced by morphine and electrical brain stimulation are both mediated by descending pathways within the dorsolateral funiculus (DLF) of the cord 5,6,18. Identification in the present study of a critical descending D L F pathway for both front paw FSIA and hind paw FSIA draws a further parallel between these environmentally induced analgesias, classically conditioned analgesia 24, morphine analgesia (MA) and stimulation produced analgesia (SPA). Thus it appears plausible that the D L F may form a final common pathway for all neural endogenous pain inhibitory systems. Although front paw FSIA and hind paw FSIA are both dependent upon the functional integrity of the DLF, they differ with regard to the involvement of
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Fig. 4. The neural circuitry of front paw FSIA (left) and hin~ paw FSIA (right). The demonstration that both T2 and C DLF lesions virtually abolish front paw FSIA implies tha direct intraspinal pathways cannot be involved in this pai~ inhibition since any potential neural pathway between th front paws (which receive the shock) and the tail (which i tested for analgesia) remains intact following C3 lesions, Therefore, front paw shock activates supraspinal structure which mediate analgesia via a descending pathway lyin within the DLF. Hind paw FSIA is also mediated, in part, b an ascending-descending loop since T2 DLF lesions signif candy attenuate, but do not abolish, hind paw FSIA. Unlik front paw FSIA, hind paw FSIA is also mediated by intrasp: nal pathways since significant and prolonged analgesia i observed following T2 spioalization. Antagonism of fror paw FSIA by spinal naloxone indicates the involvement of a endogenous opioid-like peptide (EOLP). intraspinal components. Front paw FSIA is virtu ally abolished following bilateral D L F lesions at th second thoracic (T2) vertebral level. As seen i: Fig. 4, interruption of intraspinal pathways can not account for the effects observed following DL] lesions, since D L F lesions below the spinal level c sensory input (T2) and those above the spinal lev¢ of sensory input (C3) result in equivalent atten uation of front paw FSIA. Therefore, like MA, fron paw FSIA is mediated by descending pathway which originate from supraspinal sites. Interestingl5 no further reduction of front paw FSIA occurre following T2 spinal transection. Two conclusion
103 can be drawn from this observation: (1) the DLF is the only spinal pathway involved in the opiate analgesia induced by front paw shock, and (2) since all neural connections were severed below the level of pain input, a non-neural component appears to be responsible for the transient analgesia observed immediately post-shock (cf. Fig. 2). Although at present, the identity of this non-neural factor remains elusive, preliminary data gathered in our laboratory indicate that this small, transient effect may be opiate mediated since systemic naloxone prevents the analgesic state observed immediately after front paw shock in thoracic spinal transected rats 8. As previously stated, non-opiate analgesia induced by hind paw shock is also mediated by descending pathways within the DLF (Fig. 4). Although Hayes et al. 13 concluded that the DLF does not mediate non-opiate FSIA, careful examination of their work reveals that their data are not inconsistent with the results of the present experiments. As reported by Hayes et al. 13, potent analgesia is observed imediately following shock. However, it is now known that the duration, rather than the initial magnitude, of non-opiate FSIA is markedly attenuated following T2 DLF lesions. Since Hayes et al. 13 only examined the magnitude of analgesia within 30 s after shock, they were unable to observe the effect of DLF lesions on the duration of non-opiate FSIA. It is important to note that intraspinal, rather than descending, pain modulatory pathways are responsible for the analgesia observed following T2 DLF lesions (Fig. 4). Since the time course of analgesia observed following T2 spinal transections and T2 DLF lesions do not differ significantly, it is concluded that segmental circuitry and descending pathways within the DLF account for the entire analgesic response to hind paw shock. Since, in spinalized animals, significant analgesia was observed through 12 min after shock, intraspinal pathways are capable of producing potent and prolonged pain inhibition. Thus, this series of papers on footshock induced analgesia (see refs. 22, 23, 24 and 27) provide strong evidence for the existence of multiple endogenous pain inhibitory systems within the central nervous system. Studies of front paw and hind paw FSIA have identified at least three neural pain inhibitory pathways. The first produces an opiate analgesia, is
observed following front paw shock or classical conditioning to either front paw or hind paw shock, is mediated by descending pathways within the DLF, and is dependent upon an opiate synapse within the spinal cord. The other two systems produce the non-opiate analgesia observed following hind paw shock. One consists of a descending pathway which exists entirely within the DLF and the second involves intraspinal pathways. However, it is evident from a review of the literature that these three pathways cannot account for all of the presently available data on endogenous control of pain. As seen in Table I, four major classes of endogenous pain inhibitory systems are readily evident: neural/opiate, hormonal/opiate, neural/nonopiate and hormonal/non-opiate. Front paw FSIA serves as the prototype of the neural/opiate pain inhibitory class (Table I). Members of this class are characterized as being antagonized by naloxone, showing cross-tolerance to morphine and not being dependent upon peripheral opioids (i.e. not attenuated by adrenalectomy or hypophysectomy). Included as neural/opiate analgesias are front paw FSIA, analgesia classically conditioned to either front paw or hind paw shock, stimulation produced analgesia and morphine analgesia following systemic, intracerebral, or intrathecal administration. As can be seen in Table I, presently available data are consistent with the idea that these analgesias are mediated by common neural pathways. Although controversy presently exists regarding the involvement of the nucleus raphe alatus 26 in SPA and systemic morphine analgesia (for discussion, see ref. 26), recent work has demonstrated that analgesia produced by morphine microinjection into the PAG is abolished following nucleus raphe alatus lesions 2s. Furthermore, a significant correlation is observed after lesioning this region between reduction in the degree of analgesia produced by systemic morphine and by front paw shock 2s. This result again strongly argues in favor of a common neural basis for these phenomena. Analgesia produced by electrical brain stimulation is a special case in that it belongs to two classes: neural/opiate and neural/non-opiate (Table I). Although striking similarities exist between morphine analgesia and stimulation produced analgesia (SPA), brain stimulation appears to activate both
104 TABLE I
Summary of currently available data on variousforms of analgesia
As seen in the table, the analgesias produced by various manipulations fall into 4 distinguishable classes • neural/opiate, hormonal/ opiate, neural/non-opiate, and hormonal/non-opiate. Analgesia is categorized as hormonal if it is reduced or abolished by removal of the pituitary or adrenal glands. The criteria for opiate analgesia include antagonism by naloxone and morphine tolerance. ~, = analgesia is reduced or abolished, ~ = analgesia is potentiated, 0 = analgesia is unaffected, ? = disparate evidence exists whether analgesia is reduced or unaffected, • = no data are available. A blank space indicates a category which is inappropriate to the analgesia. For literature citations, see ref. 14. Similarity to opiates Systemic naloxone
Endocrine lesions
Neural lesions
lntrathecal Morphine D L F naloxone tolerance lesions (cross-
N. r. alatus PAG lesions lesions
Adrenalec- Adrenal de- Hypophystomy
medullation
ectomy
tolerance)
Neural/opiate Brief front paw shock Conditioning to footshock Systemic morphine Intracerebral morphine Intrathecal morphine Brain stimulation Hormonal/opiate Acupuncture Prolonged 4 paw shock Immobilization Neural/non-opiate Brief hind paw shock Brief 4 paw shock 2-deoxy-D-glucose Brain stimulation Hormonal/non-opiate Cold water swims Insulin Unknown/opiate TNS (lo freq/hi int) Food deprivation Unknown/non-opiate TNS (hi freq/lo int) Hypnosis
o
~,
~,
~,
~,
~,
0
~, ~
~ ~
~ ~
~, ~
~ ?
~, ?
f f
~ ~ ~
0 ~,
~
+
~
?
o
~
~
?
?
~
•
0
~,
~,
~,
~
0
?
0 ?
~, 0 0 ? 0 0
o
o f o
~, 0
0 0 ~, 0
~
0 0
0
0
~, 0 0
opiate and non-opiate pain inhibitory systems. Naloxone has a variable effect on SPA ranging f r o m complete reversal to n o antagonism at allZ,19, z0. Part o f this variability is accounted for by the site o f stimulation. Prieto et al. 21 have reported that stimulation sites ventral to the dorsal raphe support a naloxone-reversible analgesia, whereas SPA elicited f r o m more dorsal areas fails to be reversed by this opiate antagonist. Furthermore, the fact that crosstolerance between morphine analgesia and SPA is incomplete implies that a non-opiate c o m p o n e n t exists for SPA 16.
Non-opiate mechanisms are involved in other pain inhibitory pathways as well. The analgesias p r o d u c e d by 2-deoxy-D-glucose (2-DG), brief hind paw shock and brief shock of all four paws are included in the neural/non-opiate class (Table I). Like systemic morphine analgesia, 2 - D G analgesia is potentiated after hypophysectomy. T h o u g h this m a y indicate that a h o r m o n a l antagonist to 2 - D G analgesia exists, it seems more plausible that, like the potentiation observed for morphine, this is due to peripheral effects secondary to hypophysectomy, including alterations in b l o o d - b r a i n barrier, degrad-
105 ation rates and bound/free ratios in the blood (for disc. see ref. 23). On the other hand, the endocrine system does appear to be involved in other forms of pain inhibition. Analgesia produced by prolonged footshock, acupuncture and immobilization apparently depend upon peripheral opioids. However, it remains equivocal whether the critical source of peripheral opioids is the pituitary gland or the adrenal medulla. As discussed in detail elsewhere (for disc. see ref. 23), hypophysectomy can indirectly lead to pronounced alterations in adrenal medullary function. Whether synthesis of adrenal enkephalin is affected as well is presently unknown. The work of Lewis et al. 15 suggests that this may indeed be the case. They demonstrated that adrenal demedullation significantly attenuated analgesia following prolonged footshock. The attenuation was, in fact, greater than they observed after hypophysectomy. The effect of adrenal demedullation on acupuncture and immobilization induced analgesia, the other two members of this class, has not yet been investigated. The hormones apparently involved in hormonal/ non-opiate analgesia (i.e., insulin and cold water swim induced analgesias) have also not been identified. Since hypophysectomy has been the only manipulation tested for this class, involvement of adrenal cortical or adrenal medullary substances cannot be ruled out. For example, epinephrine has previously been demonstrated to be capable of producing prolonged analgesia. Therefore, it remains plausible that non-hypophyseal factors may be the
REFERENCES 1 Akil, H., Madden, J., Patrick, R. L. and Barchas, J. D., Stress-induced increase in endogenous opiate peptides" concurrent analgesia and its partial reversal by naloxone. In H. W. Kosterlitz (Ed.), Opiates and Endogenous Opioid Peptides, North Holland, Amsterdam, 1976, pp. 63-70. 2 Akil, H. and Mayer, D. J., Antagonism of stimulationproduced analgesia by p-CPA, a serotonin synthesis inhibitor, Brain Research, 44 (1972) 692-697. 3 Akil, I-I., Mayer, D. J. and Liebeskind, J. C., Antagonism of stimulation-produced analgesia by naloxone, a narcotic antagonist, Science, 191 (1976) 961-962. 4 Amir, A. and Amit, Z., Enhanced analgesic effects of stress following administration of naltrexone in rats, Europ. J. Pharmacol., 59 (1979) 137-140. 5 Barton, C., Basbaum, A. I. and Fields, H. L., Dissociation of supraspinal and spinal actions of morphine: a
actual basis of hormonal/non-opiate analgesia. Thus, it is clear that multiple endogenous systems exist which cart act to inhibit the perception of pain. These systems are highly complex; analgesia can be produced via opiate/ or non-opiate/hormonal, as well as opiate/or non-opiate/neural, pathways. It is not yet clear whether the hormonal systems act directly at the level of the spinal cord or whether they may act indirectly by activating descending pain inhibitory pathways with supraspinal origin. It is clear that pain inhibitory pathways within the D L F are critically involved in every descending neurally mediated analgesia studied to date. However, beyond this point, little is known regarding the pharmacology or anatomy of the various systems. Identification and characterization of these multiple pain inhibitory pathways has direct significance for the control of pain in man since an understanding of these circuits will potentially provide a battery of methods with which to treat clinical pain. Sequential activation of the various pathways may allow effective treatment of pain in man while obviating the problems of tolerance and addiction. ACKNOWLEDGEMENTS We would like to thank Ms. Ingrid Kinscheck for her invaluable technical assistance, and Dr. D. D. Becker for allowing us to use Sharplan carbon dioxide laser. This research was supported by PHS Grant DA 00576 to D . J . M . L . R . W . was supported by a NSF Graduate Fellowship.
quantitative evaluation, Brain Research, 188 (1980) 487-498. 6 Basbaum, A., Marley, N. J. E., O'Keefe, J. and Clanton, C. H., Reversal of morphine and stimulation-produced analgesia by subtotal spinal cord lesions, Pain, 3 (1977) 43-56. 7 Chance, W. T., Autoanalgesia: opiate and non-opiate mechanisms, Neurosci. Biobehav. Rev., 4 (1979) 55-67. 8 Cobelli,D. A.,lnvestigationsoftheSympatheticandNeural Components of Front Paw Footshock Induced Analgesia,
unpublished Master's Thesis, Medical College of Virginia/Virginia Commonwealth University, 1981. 9 Cobelli, D. A., Watkins, L. R and Mayer, D. J., Dissociation of opiate and non-opiate footshock produced analgesia (FSA), Neurosci. Abstr., 6 (1980) 247. 10 D'Amour, F. E. and Smith, D. L., A method for determining loss of pain sensation, J. PharmacoL exp. Ther., 72 (1941) 74-79.
106 11 Hayes, R. L., Bennet, G. J., Newlon, P. and Mayer, D. J., Analgesic effects of certain noxious and stressful manipulations in the rat, Neurosci. Abstr., 2 (1976) 939. 12 Hayes, R. L., Bennett, G. J., Newlon, P. and Mayer, D. J., Behavioral and physiological studies of non-narcotic analgesia in the rat elicited by certain environmental stimuli, Brain Research, 155 (1978) 69-90. 13 Hayes, R. L., Price, D. D., Bennet, G. J., Wilcox, G. L. and Mayer, D. J., Differential effects of spinal cord lesions on narcotic and non-narcotic suppression of nociceptive reflexes: further evidence for the physiologic multiplicity of pain modulation, Brain Research, 155 (1978) 91-101. 14 Lewis, J. W., Cannon, J. T. and Liebeskind, J. C., Opioid and nonopioid mechanisms of stress analgesia, Science, 208 (1980) 623-625. 15 Lewis, J. W., Tordoff, M. G., Sherman, J. E. and Liebeskind, J. C., Role of the adrenal medulla in opioid stress analgesia, Neurosci. Abstr., 7 (1981) 735. 16 Mayer, D. J. and Hayes, R. L., Stimulation-produced analgesia: development of tolerance and cross-tolerance to morphine, Science, 188 (1975) 941-943. 17 Mayer, D. J. and Watkins, L. R., The role of endorphins in endogenous pain control systems. In H. M. Emrich (Ed.), Modern Problems of Pharmacopsychiatry: The Role ofEndorphins inNeuropsychiatry, Karger Basel, 1981, 68-96. 18 Murfin, R., Bennett, G. J. and Mayer, D. J., The effects of dorsolateral spinal cord (DLF) lesions on analgesia from morphine microinjected into the periaqueductal grey matter (PAG) of the rat, Neurosci. Abstr., 2 (1976) 946. 19 Oliveras, J. L., Hosobuchi, Y., Redjemi, F., Guilbaud, F. and Besson, J. M., Opiate antagonist, naloxone, strongly reduces analgesia induced by stimulation of a raphe nucleus (centralis inferior), Brain Research, 120 (1977) 221-229. 20 Pert, A. and Walter, M., Comparison between naloxone reversal of morphine and electrical stimulation induced analgesia in the rat mesencephalon, Life Sci., 19 (1976) 1023-1032. 21 Prieto, G. J., Giesler, G. J. and Cannon, J. T., Evidence for
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24
25
26
27
28
29
30
site specificity in naloxone's antagonism of stimulation produced analgesia in the rat, Neurosci. Abstr., 4 (1979) 460. Watkins, L. R., Cobelli, D. A., Faris, P., Aceto, M. D. and Mayer, D. J., Opiate vs. non-opiate footshock induced analgesia: the body region shocked is a critical factor, Brain Research, in press. Watkins, L. R., Cobelli, D. A., Newsome, H. H. and Mayer, D. J., Footshock induced analgesia is dependent neither on pituitary nor sympathetic activation, Brain Research, 245 (1982) 81-96. Watkins, L. R., Cobelli, D.A. and Mayer, D. J., Classical conditioning of front paw and hind paw footshock induced analgesia (FSIA): naloxone reversibility and descending pathways, Brain Research, in press. Watkins, L. R., Griffin, G., Leichnetz, G. R. and Mayer, D. J., Identification and somatotopic organization of nuclei projecting via the dorsolateral funiculus in rats: a retrograde tracing study using HRP slow-release gels, Brain Research, 223 (1981) 237-255. Watkins, L. R., Griffin, G., Leichnetz, G. R. and Mayer, D. J., The somatotopic organization of the nucleus raphe magnus and surrounding brain stem structures as revealed by HRP slow-release gels, Brain Research, 181 (1980) 1-15. Watkins, L. R. and Mayer, D. J., Involvement of spinal opioid systems in footshock induced analgesia (FSIA): antagonism by naloxone is possible only before induction of analgesia, Brain Research, in press. Watkins, L. R. and Mayer, D. J., The neural organization of endogenous opiate and non-opiate pain control systems, Science, in press. Watkins, L. R., Young, E., Kinscheck, I. and Mayer, D. J., Effects of nucleus raphe magnus lesions and periaqueductal gray lesions on footshock induced analgesia and morphine analgesia: evidence for common neural pathways, Neurosci. Abstr., 7 (1981) 340. Winer, B. J., Statistical Principles in Experimental Design, McGraw-Hill, New York, 1962, p. 37.