Brain Research, 88 (1975) 181-185
181
© Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands
Differential effect of morphine on central
versus
peripheral nociception
J. PETER ROSENFELD AND ROBERT KOWATCH D~Tartment of Psychology, Cresap Laboratory of Neuroscience and Behavior, Northwestern University, Evanston, Ill. 60201 (U.S.A.)
(Accepted January 14th, 1975)
Reported below is the fact that morphine, given in doses producing clear analgesia to peripheral pain, fails to alter the nociceptive reaction threshold to aversive brain stimulation in rats. This fact bears on the question of whether opiate analgesia is mediated by (1) inhibitory effects of opiates on pain perception loci in brain, versus (2a) the activation by morphine of central structures having a downward inhibitory effect on the transmission of nociceptive impulses through the spinal cord 1z,19, or (2b) direct inhibitory action of morphine on cord transmission v. Stimulation of brain loci in the spino-reticular pain system extensively studied by Mehler 13 has highly aversive behavioral consequencesS, 1°,14,17,Is,z°. It is known that many of the structures in this system respond electrophysiologically to nociceptire peripheral stimulationt,4-6, 9,11. It is therefore not unreasonable to assume that brain stimulation along this 'pain path' activates at least some of the same higher pain perception centers normally activated by natural (peripheral) aversive events. The higher activation, however, bypasses spinal cord transmission, which, according to hypotheses 2a and 2b given above, is a site of crucial inhibitory action during opiate analgesia. Thus a lack of effect of morphine on centrally evoked pain may aid in validation of the second set of hypotheses given above. Ten male albino rats (400-500 g) were used in the study. These were implanted with standard bipolar stimulating electrodes as described in Rosenfeld et al. 17. The electrode tip diameters were 76 # m in the present report and the intertip distance was 0.5 ram. The electrodes were insulated to the tips. The aversive brain stimulation sites used in the study, as confirmed by later histological examination, were in the nucleus subcoeruleus of the pontine reticular formation (n = 4), the intercollicular nucleus of the midbrain (n 4), and the dorsal-lateral central gray substance of the midbrain (n = 2). (The terminology is based on Mehler's 13 usages.) The noxiousness of electrical stimulation at these central sites was confirmed prior to (n = 7) or after (n --- 3) the main experiments by data on performance in a 2-way avoidance box 17,22. In this situation, the rats were given a brain stimulation every 5 sec unless they moved to the 'safe' side of the running box. The safe side was redefined every minute to control for positional preferences. If the tested brain stimulation has nociceptive consequences, the
182 rats will learn to avoid :~. 50'j!.~ of the shocks, typically during the first of the 20 rain daily test sessions. The 2-way avoidance box involves active escape and passive avoidance. It might be objected that central stimulation could produce stereotyped running behavior in the absence of pain, thus rendering dubious any interpretations with the present paradigm. In the present situation, however, the rats" escape beha~ ior involves not only running but goal-directed running, e.g., often they have to turn themselves about prior to running so that the run will be in the proper direction. (Details of the procedure are given in refs. 17 and 22.) In the present study, only placements which at reasonable current levels mediated ::~ 80 ~o avoidance behavior were considered aversive and used in the study. Other indicators of nociception such as running, excreting, vocalizing and attempts to climb out of the situation were also noted during avoidance testing. In 7 rats the avoidance was learned before other treatments. It was reinstated under morphine in 3 of these 7 rats as described below; in the remaining 3 (of the initial 10) rats the avoidance was learned for the first time under morphine. All aversive brain stimulation consisted of a 500 msec train of biphasic balanced pulse pairs of 200 #sec pulse width (per phase) at 300 Hz. Currents used were 50-200 #A, peak-to-peak. It was confirmed that the current level which led to an immediate behavioral response that a naive observer and the experimenter classified as aversive in the main experiments, always mediated > 8 0 ~o avoidance in the confirmatory avoidance tests. The objective criterion used to score a reaction as aversive involved a burst of running from one place to another in the test box and other distress signs as described below for peripheral nociception. The test of peripheral aversion used to confirm the state of morphine analgesia employed a 30 sq. cm box whose grid floor was electrifiable with 500 msec bursts of 'scrambled' 60 Hz, sinusoidal current, variable in 30/~A steps from 50 to 400 #A. All currents in the study were checked by monitoring voltage across a known resistor in series with the rat. In each rat, the procedure was as follows: after 30 min of habituation to the peripheral test box, 2 ascending series of footshock trains were delivered at the approximate rate of one train/5 sec in 30 #A steps beginning at a value of 50 #A. Threshold for peripheral nociception was considered to be the lowest current level (mean of 2) at which a clear aversive reaction was detected by two independent observers, e.g., the reaction had to involve motion of the animal from one place to another. Typically, it was accompanied by squeaking, excreting, and/or jumping. In some rats, apparently aversive ractions, without motion from one place to another, were seen at currents below those defined as threshold. Thus the threshold values actually used here were conservative. Further confirmation of the aversive quality of the footshock used is provided by a published report 3 of passive avoidance learning using the exact same equipment with similar current levels. Following determination of baseline peripheral threshold, determination of central aversive stimulation threshold was accomplished. The rat was placed in his home cage (a 20 cm ;/. 50 cm transparent plastic box; 20 cm deep), brain stimulation electrodes attached, and central shocks delivered every 5 sec at increasing current amplitudes (starting from 10/~A) until an aversion reaction in-
183 volving motion from one place to another occurred. Also noted were other behavioral distress signals such as vocalizing, excreting, attempts to climb out of the situations, etc. A 20-turn potentiometer was employed to increase current; each quarter turn yielded an increase of about 5 #A. The aversion reaction was confirmed by an independent observer and by comparison with previously determined levels yielding > 8 0 ~ avoidance as described above. It was typically noted that apparently aversive reactions such as vocalizing, jerking, orienting, attempts to climb the wall, etc. preceded the running response, so that the thresholds for central pain reported are probably conservative. Following determination of baseline central and peripheral nociceptive thresholds, the rats were injected with morphine sulfate solution, 6 mg/kg, subcutaneously (in the distensible skin on the dorsal neck region). Forty-five minutes later, thresholds were taken again in the manner just described. Peripheral threshold was always determined before central shock threshold since central shock could produce analgesia possibly potentiating the opiate lz. In all rats, the 2-way avoidance procedure was employed (as was described above) immediately after central threshold was determined in the drug state to confirm that the threshold determined would mediate > 80 },~ avoidance in addition to producing an aversive reaction as scored by visual observation. Five rats were additionally tested 24 h following morphine treatment to confirm the return of peripheral shock threshold to the baseline level. The results are summarized in Table I. The control threshold values for each rat were defined as 100 ~ values; the values from the drug conditions were divided by the control values and multiplied by 100 to yield treatment data expressed as percentages of control measures. Table I shows that the peripheral nociceptive threshold following morphine was typically increased to at least 3 0 0 ~ while the central threshold remained near 100 ~o of the control value. A correlated, 2-tailed t-test performed to determine the significance of the difference between percentage change means for the peripheral v e r s u s the central shock treatments yielded a significant value of t, P
181
© Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands
Differential effect of morphine on central
versus
peripheral nociception
J. PETER ROSENFELD AND ROBERT KOWATCH D~Tartment of Psychology, Cresap Laboratory of Neuroscience and Behavior, Northwestern University, Evanston, Ill. 60201 (U.S.A.)
(Accepted January 14th, 1975)
Reported below is the fact that morphine, given in doses producing clear analgesia to peripheral pain, fails to alter the nociceptive reaction threshold to aversive brain stimulation in rats. This fact bears on the question of whether opiate analgesia is mediated by (1) inhibitory effects of opiates on pain perception loci in brain, versus (2a) the activation by morphine of central structures having a downward inhibitory effect on the transmission of nociceptive impulses through the spinal cord 1z,19, or (2b) direct inhibitory action of morphine on cord transmission v. Stimulation of brain loci in the spino-reticular pain system extensively studied by Mehler 13 has highly aversive behavioral consequencesS, 1°,14,17,Is,z°. It is known that many of the structures in this system respond electrophysiologically to nociceptire peripheral stimulationt,4-6, 9,11. It is therefore not unreasonable to assume that brain stimulation along this 'pain path' activates at least some of the same higher pain perception centers normally activated by natural (peripheral) aversive events. The higher activation, however, bypasses spinal cord transmission, which, according to hypotheses 2a and 2b given above, is a site of crucial inhibitory action during opiate analgesia. Thus a lack of effect of morphine on centrally evoked pain may aid in validation of the second set of hypotheses given above. Ten male albino rats (400-500 g) were used in the study. These were implanted with standard bipolar stimulating electrodes as described in Rosenfeld et al. 17. The electrode tip diameters were 76 # m in the present report and the intertip distance was 0.5 ram. The electrodes were insulated to the tips. The aversive brain stimulation sites used in the study, as confirmed by later histological examination, were in the nucleus subcoeruleus of the pontine reticular formation (n = 4), the intercollicular nucleus of the midbrain (n 4), and the dorsal-lateral central gray substance of the midbrain (n = 2). (The terminology is based on Mehler's 13 usages.) The noxiousness of electrical stimulation at these central sites was confirmed prior to (n = 7) or after (n --- 3) the main experiments by data on performance in a 2-way avoidance box 17,22. In this situation, the rats were given a brain stimulation every 5 sec unless they moved to the 'safe' side of the running box. The safe side was redefined every minute to control for positional preferences. If the tested brain stimulation has nociceptive consequences, the
182 rats will learn to avoid :~. 50'j!.~ of the shocks, typically during the first of the 20 rain daily test sessions. The 2-way avoidance box involves active escape and passive avoidance. It might be objected that central stimulation could produce stereotyped running behavior in the absence of pain, thus rendering dubious any interpretations with the present paradigm. In the present situation, however, the rats" escape beha~ ior involves not only running but goal-directed running, e.g., often they have to turn themselves about prior to running so that the run will be in the proper direction. (Details of the procedure are given in refs. 17 and 22.) In the present study, only placements which at reasonable current levels mediated ::~ 80 ~o avoidance behavior were considered aversive and used in the study. Other indicators of nociception such as running, excreting, vocalizing and attempts to climb out of the situation were also noted during avoidance testing. In 7 rats the avoidance was learned before other treatments. It was reinstated under morphine in 3 of these 7 rats as described below; in the remaining 3 (of the initial 10) rats the avoidance was learned for the first time under morphine. All aversive brain stimulation consisted of a 500 msec train of biphasic balanced pulse pairs of 200 #sec pulse width (per phase) at 300 Hz. Currents used were 50-200 #A, peak-to-peak. It was confirmed that the current level which led to an immediate behavioral response that a naive observer and the experimenter classified as aversive in the main experiments, always mediated > 8 0 ~o avoidance in the confirmatory avoidance tests. The objective criterion used to score a reaction as aversive involved a burst of running from one place to another in the test box and other distress signs as described below for peripheral nociception. The test of peripheral aversion used to confirm the state of morphine analgesia employed a 30 sq. cm box whose grid floor was electrifiable with 500 msec bursts of 'scrambled' 60 Hz, sinusoidal current, variable in 30/~A steps from 50 to 400 #A. All currents in the study were checked by monitoring voltage across a known resistor in series with the rat. In each rat, the procedure was as follows: after 30 min of habituation to the peripheral test box, 2 ascending series of footshock trains were delivered at the approximate rate of one train/5 sec in 30 #A steps beginning at a value of 50 #A. Threshold for peripheral nociception was considered to be the lowest current level (mean of 2) at which a clear aversive reaction was detected by two independent observers, e.g., the reaction had to involve motion of the animal from one place to another. Typically, it was accompanied by squeaking, excreting, and/or jumping. In some rats, apparently aversive ractions, without motion from one place to another, were seen at currents below those defined as threshold. Thus the threshold values actually used here were conservative. Further confirmation of the aversive quality of the footshock used is provided by a published report 3 of passive avoidance learning using the exact same equipment with similar current levels. Following determination of baseline peripheral threshold, determination of central aversive stimulation threshold was accomplished. The rat was placed in his home cage (a 20 cm ;/. 50 cm transparent plastic box; 20 cm deep), brain stimulation electrodes attached, and central shocks delivered every 5 sec at increasing current amplitudes (starting from 10/~A) until an aversion reaction in-
183 volving motion from one place to another occurred. Also noted were other behavioral distress signals such as vocalizing, excreting, attempts to climb out of the situations, etc. A 20-turn potentiometer was employed to increase current; each quarter turn yielded an increase of about 5 #A. The aversion reaction was confirmed by an independent observer and by comparison with previously determined levels yielding > 8 0 ~ avoidance as described above. It was typically noted that apparently aversive reactions such as vocalizing, jerking, orienting, attempts to climb the wall, etc. preceded the running response, so that the thresholds for central pain reported are probably conservative. Following determination of baseline central and peripheral nociceptive thresholds, the rats were injected with morphine sulfate solution, 6 mg/kg, subcutaneously (in the distensible skin on the dorsal neck region). Forty-five minutes later, thresholds were taken again in the manner just described. Peripheral threshold was always determined before central shock threshold since central shock could produce analgesia possibly potentiating the opiate lz. In all rats, the 2-way avoidance procedure was employed (as was described above) immediately after central threshold was determined in the drug state to confirm that the threshold determined would mediate > 80 },~ avoidance in addition to producing an aversive reaction as scored by visual observation. Five rats were additionally tested 24 h following morphine treatment to confirm the return of peripheral shock threshold to the baseline level. The results are summarized in Table I. The control threshold values for each rat were defined as 100 ~ values; the values from the drug conditions were divided by the control values and multiplied by 100 to yield treatment data expressed as percentages of control measures. Table I shows that the peripheral nociceptive threshold following morphine was typically increased to at least 3 0 0 ~ while the central threshold remained near 100 ~o of the control value. A correlated, 2-tailed t-test performed to determine the significance of the difference between percentage change means for the peripheral v e r s u s the central shock treatments yielded a significant value of t, P
