The neural basis of footshock analgesia: The effect of periaqueductal gray lesions and decerebration

Brain Research, 276 (1983) 317-324 Elsevier

317

The Neural Basis of Footshock Analgesia: The Effect of Periaqueductal Gray Lesions and Decerebration L. R. WATKINS, I. B. KINSCHECK and D. J. MAYER

Department of Physiology and Biophysics, Medical College of Virginia, Richmond, VA 23298 (U.S.A.) (Accepted February 8th, 1983)

Key words: footshock induced analgesia - - opiate analgesia - - non-opiate analgesia - - periaqueductal gray - - decerebration

Previous studies have demonstrated that brief front paw shock and brief hind paw shock produce potent opiate and non-opiate analgesia, respectively. Front paw footshock-induced analgesia (FSIA) and hind paw FSIA are similar in that each is mediated by a medullospinal pathway. A question which arises is whether these opiate and non-opiate descending pathways are activated in direct response to afferent information from the spinal cord or whether indirect activation via more rostral centers is required. The first experiment examined the effect of lesions of the rostral periaqueductal gray (PAG) and caudal PAG on front paw (opiate) FSIA and hind paw (nonopiate) FSIA. In no case did PAG lesions markedly reduce the magnitude of these pain inhibitory states. Since this result raised the possibility that rostral centers may not have any major involvement in the production of these phenomena, the second experiment examined the effect of decerebration on front paw FSIA and hind paw FSIA. Decerebration had no effect on hind paw FSIA and, at most, produced only a very modest decrease in front paw FSIA. The fact that potent and prolonged analgesia can still be elicited after decerebration clearly demonstrates that limbic, cortical, thalamic, and rostral midbrain structures are not critical to the production of these pain inhibitory effects. Thus this work provides the first demonstration of opiate and non-opiate analgesia systems within the • caudal brainstem and spinal cord which can be activated by environmental stimuli. INTRODUCTION Brief transcutaneous shock delivered to either the front paws or hind paws of rats has been repeatedly demonstrated to produce analgesia31, 37,3s. Behaviorally, front paw and hind paw shock appear to produce an identical phenomenon, in that the shock-induced inhibitory effects appear to be specific to nociception 20, and each reliably elicits potent analgesia which long outlasts the period of stimulationS1,33,34,37. However, recent investigations have revealed that the underlying mechanisms mediating front paw footshock-induced analgesia (FSIA) and hind paw F S I A are markedly different. Pharmacological studies have shown that front paw shock produces opiate analgesia and hind paw shock produces non-opiate analgesia31,37, 3s. Front paw F S I A appears to be mediated at the level of the spinal cord by endogenous opioids37.,3s, serotonin20,36 and, to a lesser extent, norepinephrine20, 36 since antagonists delivered directly to the cord attenuate the pain inhibition. In contrast, the neurotransmitters involved in hind paw F S I A have not yet been identified; all that is known is that 0006-8993/83/$03.00 ~) 1983 Elsevier Science Publishers B.V.

opiate antagonists31,37, as and spinal monoamine depletion20, 36 fail to attenuate this non-opiate analgesic state. Front paw F S I A and hind paw F S I A also differ with regard to their neuroanatomical substrates. Front paw F S I A is mediated via a centrifugal pathway which projects through the dorsolateral funiculus (DLF) of the spinal cord 33 and originates within the medullary nucleus raphe alatus (NRA)38-40. In contrast, hind paw F S I A is dependent upon both an intraspinal pathway and a centrifugal D L F pathway33,3s. The latter originates, in part, in the N R A ; another supraspinal area(s) must also be involved but, to date, has not been identified 38-40. Thus all available evidence indicates that the underlying bases of front paw F S I A and hind paw FSIA are distinct. However, one fundamental similarity between front paw F S I A and hind paw F S I A is that each is mediated by a medullospinal pain inhibitory pathway. A question which arises is whether these opiate and non-opiate descending pathways are activated in direct response to afferent information from the spinal cord or whether indirect activation via more rostral

318 centers is required. Direct medullary activation may underlie these phenomena since certain neurons within NRA have been found which are excited by noxious peripheral stimuli 3A3. Yet only the lateral extent of NRA (the nucleus reticularis paragigantocellularis, PGC) receives monosynaptic input from the spinal cordl. The medial portion of NRA (the nucleus raphe magnus, NRM) does not appear to receive afferents from the cord1, yet discrete lesions of this area reduce front paw FSIA and hind paw FSIA 38-40. Furthermore, Abols and Basbaum 1 reported that the afferent projection from the cord to the N R A area arises predominantly from cervical levels: almost no cells in lumbosacral cord were found to terminate in NRA. Thus activation of NRA in response to hind. paw or front paw shock may well be indirect, being mediated through more rostral sites. If these opiate and non-opiate medullospinal pathways are indeed activated by rostral centers, then it would be important to identify which areas are involved. One area which has long been implicated as having a critical role in centrifugal pain inhibitory systems is the periaqueductal gray (PAG) 4,19. The observation that pain could be inhibited by electrical stimulation of the PAG provided the first behavioral evidence for the existence of endogenous analgesia systems 29. Since that time, numerous studies have shown that powerful analgesia can be elicited by stimulation of, or morphine microinjection into, sites throughout the rostrocaudal extent of the P A G 43,44. A role for the PAG in FSIA is supported by several lines of evidence. First, the demonstration that the PAG receives input from ascending pain pathways21 and, in turn, sends direct projections to the N R A 1.8 provides anatomical support for the idea that the PAG may act as a pivotal point between centripetal pain transmission and centrifugal pain inhibition. Second, electrophysiological studies have demonstrated that activation of the P A G leads to activation of the NRA 11.18,22,23,25,28,30. Third, several important similarities have been recognized between the analgesias produced by footshock and by PAG activation in that, for both: (1) either opiate or nonopiate analgesia can be elicited7,38.44, (2) pain inhibition is mediated via the D L F 5,24,38, (3) this descending spinal pathway originates, at least in part, in the N R A 6.35,38,45,46, and (4) opiate analgesia is mediated by monoaminergic, and non-opiate analgesia by non-

monoaminergic mechanisms at the level of the spinal cord14,20,36,42. Thus, presently available evidence suggests that the PAG may be involved in the production of FSIA. The purpose of the present study was to directly test this hypothesis by examining the effect of P A G lesions on front paw FSIA and hind paw FSIA.

EXPERIMENT 1. EFFECT OF ROSTRAL AND C A U D A L PAG LESIONS ON FRONT PAW FSIA AND HIND PAW FSIA

Methods A total of 56 adult male Sprague-Dawley rats (350--500 g) were used in the present experiment. Rats were anesthetized with 50 mg/kg sodium pentobarbital, supplemented with Metofane (PitmanMoore) as needed. For each rat, a lesioning electrode was stereotaxically21 lowered into either the rostral P A G (AP = +4.4, L = 0, D = +3.75) or caudal PAG (AP = +1.9, L = _+ 0.6, D = +2.95). The electrode was constructed of a stainless steel wire (0.2 mm diameter, Medwire) which was insulated with Teflon except for the cross section of the tip. Rostral PAG lesions (n = 16) and caudal PAG lesions (n = 20) were made by delivering 1.3 mA DC current for 15 s; sham (control) lesions (n = 20) consisted of lowering the electrode within 2.5 mm of the caudal P A G site with no current being passed. Upon completion of the surgical procedure, the skull opening was covered with gel foam powder (Up John) and the wound closed; rats were treated with penicillin as needed. Twelve days after surgery, rats were tested to assess their baseline pain responsivity using a modification2 of the D'Amour-Smith tail flick test 10. Three tail-flick trials, with a 2 min inter-trial interval (ITI), were averaged to attain a mean baseline latency for each rat. Immediately following this baseline measure, rats were exposed to 90 s of constant current 60 Hz shock delivered differentially to either the front paws (1.6 m A rms) or to the hind paws (1.2 m A r m s ) . Details of this methodology have been presented previously3X. Upon termination of shock, tail flick trials were used to assess pain responsivity at 0, 1, 2 rain, and every second minute through 14 min after shock. The radiant heat was automatically termi-

319 nated at 8 s if no tail flick occurred in order to avoid tissue damage. For all behavioral testing, the experimenter was blind as to the type of lesion that the animals had received. The tail flick latencies recorded after shock were expressed as a percentage of maximal possible effect (%MPE), using the following equationn: %MPE = [ ( T L - - B L ) / ( 8 . 0 - - B L ) ] x 100 where TL = the test latency recorded after shock and BL = the mean baseline latency. Within each shock paradigm (front paw and hind paw), analysis of variance was utilized to compare the effect of the lesions on the timecourse of footshock-induced analgesia. Student's t-tests were used to assess the effect of lesions on baseline pain responsivity and to make specific comparisons between groups of the degree of analgesia exhibited at various times after shock; Satterthwaite's approximation was utilized when significant differences in sample variance occurred41. Paired t-tests were used for each group to compare

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Fig. 1. Representative profiles of the rostral PAG and caudal PAG lesion groups used in Experiment 1. Numbers indicate the anterior-posterior plane of section according to the atlas of Palkovits and Jacobowitz 26. Abbreviations: CCGM, medial geniculate nucleus pars centralis; FR, fasciculus retroflexus; IP, interpeduncular nucleus; MCGM, medial geniculate nucleus pars marginalis; NCS, nucleus centralis superior; NMM, nucleus of the mammillary bodies; RN, red nucleus; SN, substantia nigra.

their average baseline tail flick latencies with those recorded after shock. After testing was completed, rats were overdosed with sodium pentobarbital and perfused transcardially with 10% formalin. Frozen serial sections (50/~m) were collected through the entire extent of the lesions and counterstained with Neutral Red. Lesion profiles (Fig. 1) were then compiled for all animals z6, with analyses being performed blind with respect to the behavioral results. Any animals with lesions which were not confined to the rostral or caudal PAG area were eliminated from analysis of behavioral data. Results Rostral and caudal PAG lesions failed to significantly alter baseline pain responsivity compared to controls. The average baseline latency (mean + S.E.) of each group was as follows: 3.86 ___0.1 s for the rostral PAG group, 3.81 + 0.1 s for the caudal PAG group, and 3.72 + 0.2 s for controls. Following front paw shock, the rostral PAG group (n = 9), caudal PAG group (n = 11), and control group (n = 12) each remained significantly analgesic throughout the 14-min test (P < 0.05 at 14 min, for each comparison) (Fig. 2, left). Rostral PAG lesions failed to reduce front paw FSIA, compared to controis. Student's t-tests comparing the tail flick latencies recorded for the caudal PAG group and controls failed to reveal that they differed significantly at any time after shock. However, despite the fact that no significant differences were observed at any single time tested, the degree of analgesia exhibited by the caudal PAG lesion group was consistently slightly lower than that of controls. This trend resulted in a significant overall effect using the ANOVA (P < 0.05). Similarly, hind paw shock produced analgesia in the rostral PAG group (n = 7), caudal PAG group (n = 8), and control group (n = 9) which persisted throughout the 14-rain test (P < 0.05 at 14 rain, for each group) (Fig. 2, right). Rostral PAG lesions failed to reduce analgesia, compared to controls; in fact, a potentiation was observed (P < 0.0005). Student's t-test comparisons between the caudal PAG lesion group and controls revealed that no reliable difference occurred through 12 min after shock;

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tail flick latencies of the caudal P A G lesion group were less than those of controls only at minute 14 (P < 0.01). EXPERIMENT 2: EFFECT OF DECEREBRATION ON FRONT PAW FSIA AND HIND PAW FSIA

Methods Since, unexpectedly, P A G lesions failed to have any marked antagonistic effect on either front paw FSIA or hind paw FSIA, the possibility arose that rostral centers may not have any major involvement in the production of these opiate and non-opiate mediated phenomena. To test this hypothesis, the effect of decerebration was examined. A total of 31 adult male Sprague-Dawley rats (350-500 g) were used in the present experiment. All surgery was performed using Metofane (Pitman-Moore), a short-acting inhalant anesthesia. Sham surgery was performed on 15 rats which consisted of removing a bone flap from the skull without lesioning the brain. Mid-collicular decerebrations were made in 16 rats using suction under visual guidance. Upon completion of brain transection, Oxycel (Parke-Davis) was placed in the wound to aid coagulation and the wound was closed. Behavioral testing was delayed until 8-10 h after surgery.

At this time, rats were first tested for baseline pain responsivity (3 tail flick trials, 2 rain ITI), and then exposed to 90 s shock which was delivered either to the front paws or hind paws. Shock parameters, behavioral testing and statistical analyses were as in Experiment 1. Upon completion of testing, animals were perfused transcardially with 10% formalin. The brains of the control (sham) group were examined for any evidence of cortical damage. Frozen sections of the brains of the decerebrate group were collected in order to determine the level of transection. Control animals showing evidence of cortical damage and decerebrate animals with lesions rostral to the mid-coilicular level were excluded from statistical analyses.

Results Decerebration did not significantly alter baseline tail flick latencies from controls. The average baseline latency (mean + S.E.) was 3.74 + 0.09 s for the control group and 3.95 + 0.11 s for the decerebrate group. Following front paw shock, analgesia was observed throughout the 14-min test in both the control group and decerebrate group (p < 0.05 at 14 min for each comparison). Student's t-test comparisons of the degree of analgesia exhibited by the decerebrate and control groups revealed that no significant differences occurred at any time throughout the 14-min test. Although no significant differences were ob-

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321 served at any single time tested, the degree of analgesia exhibited by the decerebrate group was consistently slightly less than that of controls. This trend resulted in a significant overall effect using the ANOVA (P < 0.05). Similarly, hind paw shock produced potent analgesia in both the decerebrate and control groups throughout the 14 min test (P < 0.01, at 14 rain, for both groups). Decerebration failed to significantly reduce hind paw FSIA, compared to controls ( P > 0.1).

brainstem and spinal cord contain virtually all of the circuitry necessary to produce both opiate analgesia and non-opiate analgesia. These systems can be rapidly activated in direct response to environmental stimuli and result in potent analgesic states which long outlast the period of peripheral stimulation. Hind paw shock (Fig. 4) results in non-opiate analgesia by activation of both intraspinal and centrifugal

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DISCUSSION The present series of experiments demonstrate that PAG lesions and decerebration produce, at most, only a very modest decrease in either front paw FSIA or hind paw FSIA. Rostral PAG lesions fail to attenuate front paw FSIA or hind paw FSIA. Caudal PAG lesions and decerebration also failed to significantly reduce front paw FSIA at any single time tested, although a trend was observed in that these manipulations resulted in a consistent, though slight, decrease in the magnitude of analgesia (mean percent decrease compared to controls: 16% for caudal PAG lesions, 10.2% for decerebration). When tested for hind paw FSIA, caudal PAG lesions resulted in only a 7.6% decrease, and decerebration in a 6.4% decrease, in the magnitude of analgesia compared to controls. Thus these effects on front paw (opiate) FSIA and hind paw (non-opiate) FSIA appear to be quite minor, compared to manipulations such as DLF lesions which decrease front paw FSIA by 96.1% and hind paw FSIA by 61.8% 33. The fact that potent and prolonged analgesia can still be elicited after decerebration clearly demonstrates that limbic, cortical, thalamic, and rostral midbrain structures do not play an important role in the production of these pain inhibitory effects. Thus this work provides the first demonstration of opiate and nonopiate analgesia systems within the caudal brainstem and spinal cord which can be activated by environmental stimuli. Combining these data with the results of our previous studies of hind paw FSIA and front paw FSIA 31-34,36-40 allows the conceptualization of the neural circuitry shown in Fig. 4. As demonstrated by the present series of experiments, the caudal

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Q2)_ Peripherol nerve DORSAL HORN ending Fig. 4. Neural circuitry of opiate and non-c fiate analgesia induced by front paw and hind paw shock. Front paw shock activates the nucleus raphe alatus (NRA) within the ventral medulla. This nucleus sends a descending projection through the DLF to the dorsal horn of the spinal cord. A serotonergicpathway lyingoutside of the DLF (non-DLF) is recruited as well. In turn, endogenous opiates are released, inhibiting pain transmission neurons (PTN). Hind paw shock inhibits PTN via two non-opiate pathways: an intraspinal pathway and a descending DLF pathway. The latter originates from the nucleus raphe alatus and from some other yet unidentified medullary area(s). Classically conditioned (opiate) analgesia seems to result from activation of the same DLF output pathway as front paw (opiate) FSIA. After conditioning trials in which the conditioned stimulus is paired with either front paw or hind paw shock (the unconditioned stimulus), the conditioned stimulus becomes capable of activating rostral centers in the brain, which, in turn, activate the periaqueductal gray (PAG) and subsequently the nucleus raphe alatus. This results, via a descending DLF pathway, in the release of endogenous opiates within the dorsal horn, producing analgesia.

322 pathways 33. The latter is apparently activated in response to an increase in activity within ascending pain pathways which terminate, in part, within the medulla 21. In turn, this leads, either directly or indirectly, to activation of the N R A and some other, yet unidentified, medullary area(s)3,13,39,40. These nuclei send medullospinal projections through the DLF33, 35, inhibiting pain at the level of the spinal cord through the release of non-opiate neurotransmitters37; the identity of these transmitters has yet to be determined39,40. In contrast, front paw (opiate) FSIA (Fig. 4) is mediated solely through activation of centrifugal pathways. Front paw shock leads, either directly or indirectly1, to activation of the NRA39, 40. In turn, this. area sends centrifugal projections to the cord, resulting in pain inhibition. Since bilateral DLF lesions abolish front paw FSIA 33, the D L F appears to be a critical pathway involved in the production of this opiate analgesia. In addition, serotonergic (5-HT) pathways, which originate within the N R A area 9 and project to the cord through pathways other than the DLF15A6, are critically involved in the production of front paw FSIA 20,36. Thus, it appears that, in this case, a non-additive interaction occurs between descending DLF and non-DLF pathways since blockade of either results in a profound loss of front paw FSIA20,33,36. Thus this series of investigations has led to the identification of at least 4 distinct pain inhibitory pathways which can be differentially activated by front paw and hind paw shock (Fig. 4). Although, as stated previously, shock-induced activation of these pathways is not mediated through rostral centers, previous investigations have demonstrated that, under certain circumstances, supramedullary areas may indeed profoundly modulate the activity of one of these pathways (Fig. 4). Using a classical conditioning paradigm, Watkins et al.32 showed that animals

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can learn to activate their endogenous opiate analgesia systems. The procedure followed in this paradigm consisted of pairing exposure to innocuous environmental cues (conditioned stimulus, CS) with either front paw or hind paw shock (unconditioned stimulus, UCS). Subsequently, exposure to the CS (in the absence of shock) reliably elicits analgesia (conditioned response, CR) through release of endogenous opiates within the spinal cord32. Since classical conditioning represents a learned phenomenon, this implies that information pertaining to the CS and UCS must be received by rostral centers in order for learning to occur (Fig. 4). After the association between the CS and UCS has formed, exposure solely to the CS apparently results in activation of these rostral areas that, in turn, lead to activation of the caudal dorsal PAG17, 39. This latter region, in turn, projects to the NRA1, 8 which is the origin of the descending DLF pathway critically involved in the production of this opiate analgesia 32. Thus it appears that animals can learn to activate rostral areas which powerfully modulate activity within the opiate centrifugal pathway previously described for front paw FSIA. In conclusion, these studies of front paw (opiate) FSIA, hind paw (non-opiate) FSIA and classically conditioned (opiate) analgesia provide strong evidence that endogenous neural systems exist which function as a negative feedback control system to dynamically modulate pain transmission. The existence of such centrifugal systems provides a parallel between pain sensation and other sensory modalities long known to be under negative feedback control. Importantly, these studies of FSIA and classically conditioned analgesia have provided evidence for the idea that invasive techniques, such as brain stimulation and morphine administration, may well be producing analgesia by artificially driving neural pathways which physiologically act to modulate pain.

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