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Environmental modulation of behavioral tolerance in spinal rats
46
Brain Research, 561 (1992) 40-52 © 1992 Elsevier Science Publishers B.V. All rights reserved. 0006-8993/92/$05.00
BRES 17751
Environmental modulation of behavioral tolerance in spinal rats Claire Advokat and Carla Mclnnis Department of Psychology, Louisiana State University, Baton Rouge, LA 70803 (USA) (Accepted 7 January 1992) Key words: Behavioral tolerance; Opiate antinociception; Spinal rat
We previously reported that the antinociceptive effect of morphine on the thermal tail withdrawal reflex (tail flick, TF) was reduced in rats that had practiced the TF response prior to morphine administration. This phenomenon, termed behavioral tolerance, was observed even when rats were spinally transected following the TF tests. The present experiments were conducted to determine whether such retention of behavioral tolerance after spinal transection was dependent on prior experience with the TF procedure, or whether other nociceptive pretreatments would also reduce opiate antinoeiception on the TF test in spinal rats. Separate groups of rats were pretreated with either nociceptive thermal TF or Hot Plate (HP), or mechanical Tail Pinch (TP) stimuli, as well as non-nociceptive exposure to the experimental context (Habituation) or No Pretreatment. Approximately half of the rats were spinally transected after their respective pretreatment, while the other half were left intact. When Intact rats were subsequently tested on the TF at 30, 60, 120 and 180 min after morphine administration. only the TF pretreated group was tolerant, relative to the Non-pretreated control group. However, this was a transient effect, and was only significant at the first, 30-rain test; there was no difference among the groups in the total duration of antinoeiception across all the time points. When spinal rats were tested after morphine administration, there was also an overall significant difference among the groups at 30 min. In this case, the differences were maintained across all time points. All three nociceptive pretreatments (TF, HP and TP) significantly reduced the total duration of opiate antinociception in acute spinal rats, relative to the Non-pretreatment condition, whereas the Habituation treatment did not. These data reveal a differential effect of behavioral pretreatment on morphine-induced antinociception in Intact vs. Spinal rats. In Intact rats, behavioral tolerance on the TF test was transient and only observed in animals that had prior experience with the TF procedure. In contrast, when rats were spinally transected after noeiceptive pretreatment, behavioral tolerance on the TF test was maintained throughout the test session and was observed in all of the nociceptive pretreatment groups. These data indicate first that noeiceptive stimulation can produce a persistent change in the spinal cord which significantly influences spinal reflex function and second that when the neuraxis is intact, the effect of such stimulation may be modulated by descending supraspinal input. INTRODUCTION N u m e r o u s investigations have shown that the effect o f drugs on a variety of behavioral responses can be reduced if subjects are allowed to practice the response before the drug is administered. This p h e n o m e n o n , t e r m e d behavioral tolerance (BT), has b e e n d e m o n strated with a variety of drugs and behaviors in intact animals 15. In contrast, little is known a b o u t the neural basis of this a d a p t a t i o n , p r e s u m a b l y because most behavioral p r e p a r a t i o n s are too complex for such analyses. In previous research, we e x a m i n e d B T in a response system that m a y ultimately be a m e n a b l e to neural analysis; the involuntary, defensive tail withdrawal reflex (tail flick, TF) elicited by noxious thermal stimulation. This reflex is used extensively as a nociceptive index to assess the analgesic (antinociceptive) effects of opiates. Most i m p o r t a n t in this context is the fact that the T F reflex can be elicited in the spinally transected rat and, that o p i a t e - i n d u c e d antinociception 2'13, tolerance L5 and dep e n d e n c e 6 can also be o b s e r v e d with this response after
spinalization. Because a vast a m o u n t of information exists concerning neurophysiological mechanisms of o p i a t e action at the spinal level, the possibility arises that behavioral adaptations o b s e r v e d within this reflex system might ultimately be related to neural mechanism. Previous results from o u r l a b o r a t o r y showed that after r e p e a t e d exposure to the T F test p r o c e d u r e o v e r several days, the analgesic effect of systemic m o r p h i n e in intact rats is significantly reduced, relative to non-TFexperienced rats, indicating the d e v e l o p m e n t o f BT. The d a t a also showed that such tolerance was r e t a i n e d for 24 h after spinalization: the antinociceptive response to m o r p h i n e in T F p r e t r e a t e d rats that were spinally transected was significantly less than that of non-pretreated, spinal rats 1'1s. The fact that B T could be m a i n t a i n e d for at least 24 h after a spinal transection suggested that some type of neural m o d u l a t i o n had occurred within the spinal cord as a result of prior experience with the T F procedure. F u r t h e r examination indicated that the d e v e l o p m e n t of B T required an intact neuraxis. If rats were spinally
Correspondence: C. Advokat, Department of Psychology, Louisiana State University, Baton Rouge, LA 70803, USA.
47 t r a n s e c t e d before t h e y w e r e t e s t e d o n t h e TF, b e h a v i o r a l t o l e r a n c e t o s y s t e m i c m o r p h i n e a d m i n i s t r a t i o n did n o t occur. That result indicated that supraspinal input was n e c e s s a r y f o r t h e a c q u i s i t i o n o f b e h a v i o r a l t o l e r a n c e 1. The main p u r p o s e of the p r e s e n t study was to determ i n e w h e t h e r t h e d e v e l o p m e n t o f B T o f t h e T F reflex required prior TF exposure or, w h e t h e r the p h e n o m e n o n c o u l d also b e p r o d u c e d b y e x p o s u r e t o o t h e r n o c i c e p t i v e p r o c e d u r e s . I n a d d i t i o n , t h e p r e s e n t s t u d i e s also determined whether BT would develop when TF pret r e a t m e n t w a s a d m i n i s t e r e d w i t h i n a single e x p e r i m e n t a l s e s s i o n , a n d w h e t h e r this p h e n o m e n o n
w o u l d b e ex-
p r e s s e d in a d o s e - d e p e n d e n t m a n n e r .
MATERIALS AND METHODS
Subjects A total of 101 male albino Sprague-Dawley derived rats, of the Holtzman strain, weighing 250-350 g were used as subjects. They were bred and raised at the animal facility of the Baton Rouge campus of Louisiana State University. All animals were housed individually in suspended stainless steel cages in a colony room maintained on a 12 h:12 h light-dark cycle, with dark onset at 17.00 h. Food and water were available ad libitum.
Surgical procedures Spinal transections were performed under ether anesthesia. A laminectomy was made between thoracic vertebrae 6 and 9 and a 1-2 mm portion of the spinal cord was removed by excavation and replaced with gelfoam to reduce bleeding, after which the wound was sutured and the cages placed on heating pads to maintain body temperature. On the morning after surgery, the hindquarters of each rat were washed with warm water and their urine was expressed manually by the application of pressure to their bladders (see ref. 1 for details),
Nociceptive assessment For all rats, the final nociceptive response was assessed with the tail flick (TF) test7. Noxious stimulation was produced by a beam of high intensity light, focused on the tail. The response time was measured automatically and was defined as the interval between the onset of the thermal stimulus and the abrupt flick of the tail. Each determination consisted of three trials; the mean score was taken as the response latency. In order to minimize tissue damage to the tail, animals not responding within 14 s were removed from the apparatus and assigned a response latency of 14 s.
Drug administration Morphine sulfate (Penick Corp., Lyndhurst, N J) was dissolved in 0.9% saline and administered subcutaneously such that the appropriate dose was injected in a volume of 1 ml/kg.
Statistical analyses In the first experiment, the data consisted of the TF latency scores, in seconds, and are presented as the group mean + S.E.M. In subsequent studies, the duration of opiate antinociception was determined by testing the animals at 30, 60, 120 and 180 min after morphine administration and then obtaining the Area Under the Curve (AUC) with the aid of a computer program (PHARM/ PCS) 2°. For each animal the A_UC was determined by entering the four xy data pairs, in which x = tail flick latency and y = 30, 60, 120 or 180 min. The computer program calculated the total area (i.e. the integral) based on an approximation using the trapezoidal rule. Statistical tests were then performed on the AUC values that comprised each experimental group. These results are expressed in
the figures as the group mean + S.E.M. AUC scores. In each case, statistical calculations, performed with a computer program (CRUNCH Interactive Statistical Program) consisted of analyses of variance, for either repeated measures within groups or for differences among several groups, with the appropriate post hoc comparisons and Student's t-test. Results were considered significant at P = 0.05 or less.
Experimental procedures Behavioral tolerance to morphine in intact rats. The first experiment was designed to determine whether a series of TF trials, administered within a single experimental session, would reduce the subsequent analgesic effect of morphine on the TF. The rats in this condition (Tail Flick Pretreatment) were brought to the experimental room, wrapped in a cloth towel and given three TF trials in succession, five different times, at 1-h intervals. This resulted in a total of 15 trials, administered between 09.00-10.00 h and 13.0014.00 h. Two days later these subjects were brought back to the experimental room, and randomly divided into two groups, one injected with 0.75 mg/kg of morphine and the other injected with 1.5 mg/kg of morphine. The group injected with 0.75 mg/kg was tested once on the TF, 30 rain after injection (n = 5). All animals injected with 1.5 mg/kg were also tested on the TF at 30 min (n = 13) and some of these subjects (n = 5) were tested again at 60, 120 and 180 min after injection. Two additional groups, which were not previously exposed to the experimental context or the TF apparatus, were injected with the same doses of morphine (No Pretreatment Groups: 0.75 mg/kg, n = 5; 1.5 mg/kg, n = 10). Again, all of the rats were tested 30 rain postinjection, whereas half of the animals injected with 1.5 mg/kg were also tested at 60, 120 and 180 min after injection. This procedure resulted in 4 experimental groups, consisting of rats that were either Pretreated or Not Pretreated with the TF procedure prior to systemic injection of either 0.75 or 1.5 mg/kg of morphine. All groups were tested 30 rain after injection, and those rats injected with 1.5 mg/kg of morphine were then tested repeatedly, to determine whether or not the duration of analgesia would be altered by prior exposure to the TF test. Several additional groups were preexposed to treatments which did not involve the TF test: a fifth group of rats was brought into the experimental room and received a nociceptive hot plate (HP) test once every hour for 5 h, for a total of five trials. For this test, a Plexiglas cylinder (28.5 cm in height and 20.5 cm in diameter), which restrained the rat, was placed on top of a metal plate. The temperature of the plate was maintained at 54 +_ 1.0°C by a heated water bath. Animals were placed within the cylinder and the latency until either the animal licked its hindpaws or jumped out of the cylinder was manually recorded. Animals not responding within 40 s were removed and assigned a 40-s response latency (Hot Plate Group, n = 5). A sixth group of rats was brought to the experimental room and received three nociceptive tail pinch (TP) trials, five times, at onehour intervals, for a total of 15 trials. In this case, the rats were wrapped in a cloth towel and placed on the TF apparatus. Pressure was applied to the rat's tails with a hemostat, whose teeth were covered with clear polyethylene tubing. The hemostat was closed at a constant setting for 5 s on each of the three trials. This elicited vigorous movements of the tail and vocalizations in all subjects. A different patch of skin was pinched on each trial (Tail Pinch Group, n = 6). A seventh group of rats was exposed to the experimental room during a 'pretreatment' session but was not placed in the TF apparatus and did not receive TF tests (Habituation Group, n = 9). Two days later, the subjects in the Hot Plate, Tail Pinch and Habituation groups were injected with 1.5 mg/kg of morphine and tested on the tail flick 30 rain later. All of the animals in the Hot Plate and Tail Pinch groups, and five of the animals in the Habituation group were tested again at 60, 120 and 180 min after the injection. Behavioral tolerance to morphine in spinal rats. It is well-known
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Fig. 1. Mean Tail Flick latency + S.E.M. of intact rats (n --- 30) on each of five pretest trials, administered at one-hour intervals within a single session. Each trial consisted of three tail flick tests.
that the antinociceptive effect of systemic morphine on the TF test is significantly reduced within 24 h after spinal transection. Therefore, in order to obtain an equivalent response on the TF the dose of morphine administered to spinal rats must be greater than (approximately double) the dose given to intact rats3. To confirm that observation in the context of these experiments, a dose-response function to morphine was first determined in rats that were spinally transected 24 h previously. Separate groups of spinal rats were injected with either 0.75 (n = 3), 1.5 (n = 4), 2.25 (n = 6) or 3.0 (n = 10) mg/kg of morphine and tested on the TF at 30, 60, 120 and 180 min after the injection. In the second experiment, separate groups of intact rats received each of the four different pretreatments. One group was exposed to the experimental room, but was not placed on the TF apparatus (Habituation Group, n = 5). The second, third and fourth groups received the same HP (n = 6), TP (n = 6) and TF (n = 8) pretreatments as described above. On the day after each of these respective treatments, all rats were spinally transected. On the next day, all rats were injected with 3.0 mg/kg of morphine and tested on the TF at 30, 60, 120 and 180 min after the injection. RESULTS
Effect of tail flick pretreatment in intact rats There was a slight, but statistically significant decline in baseline latency during the T F p r e t r e a t m e n t trials. A summary of this decrease is shown in Fig. 1, which presents the data for all rats that received the 15 T F pretreatment trials prior to their respective (Intact or Spinal) experimental m a n i p u l a t i o n (n = 30, F29' 4 = 8 . 9 8 , P < 0.0001). The baseline latency was reduced from 4.7 s on the first trial to 3.4 s on the fifth, demonstrating the development of within session facilitation, or sensitization, of the T F response in intact rats.
Behavioral tolerance in intact rats All rats, both T F pretreated (n = 18) and Non-pretreated (n = 15) were given a preliminary T F trial prior to their respective m o r p h i n e injection. The m e a n + S.E.M. T F latency for Pretreated rats was 4.9 + 0.32 s and for Non-pretreated rats, 4.5 ___ 0.32 s (t = 0.83, n.s.). This result indicates that, although reflex latency was significantly reduced during pretest trials this effect had dissipated within 48 h and would not account for any
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1.5
Morphine Dose (mg/kg)
Fig. 2. Mean Tail Flick latency + S.E.M., 30 min after a s.c. morphine injection in four groups of rats. Two groups had no prior exposure to the experimental context or tail flick procedure (No Pretreatment; 0.75 mg/kg, n = 5; 1.5 mg/kg, n = 10). Two groups received a series of 15 tail flick trials administered 48 h previously (Tail Flick; 0.75 mg/kg, n = 5; 1.5 mg/kg, n = 13).
differences among the groups observed after m o r p h i n e administration. In addition, the latencies of the HP, TP and H a b i t u a t i o n groups were 4.5 + 0,25 s, 4.1 + 0.34 s and 4.4 + 0.22 s respectively, suggesting that these pretreatments also did not modify baseline T F latency. The effect of T F p r e t r e a t m e n t in intact rats o n the subsequent analgesic response to morphine two days later, is summarized in Fig. 2. This figure shows the response of the Non-pretreated and T F Pretreated rats, 30 min after m o r p h i n e administration for each of the two doses. As indicated, T F p r e t r e a t m e n t reduced opiate analgesia in a dose-related m a n n e r . A two-way A N O V A showed a significant effect of p r e t r e a t m e n t (n = 33, F32,1 = 19.5, P = 0.0001) and of dose (F32a = 34.3, P < 0.0001), with no interaction. These data demonstrate that a series of T F tests, administered within a single session, produced behavioral tolerance to the analgesic effect of morphine which was retained for at least two days. Additional analyses were performed on the scores of the five groups that were injected with the higher, 1.5 mg/kg, dose of morphine. These groups were first compared at the 30-rain time point. The results showed an overall significant effect of p r e t r e a t m e n t (n = 43, F42.4 = 4.21, P = 0.0064). However, post hoc tests indicated that only the scores of the T F Pretreated group were significantly different (i.e. lower) than that of the Nonpretreated group ( D u n n e t t ' s test, P = < 0.05). The m e a n T F latencies + S.E.M. for all the groups at 30 min after m o r p h i n e were: No pretreatment, 12.6 + 0.63 s; Habituation, 11.06 + 0.87 s; HP, 9.76 + 1.15 s; TP, 10.82 + 0.82 s and TF, 8.77 + 0.65 s. However, a second analysis of the complete time effect curve ( A U C ) showed no difference among the groups. These data, summarized in Fig. 3 show that over the complete 180-min course of the test session, there was no effect of the respective pretreatment conditions
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0 0 Pretreotment Fig. 3. Effect of pretreatment condition on morphine-induced antinociception in intact rats. Separate groups of intact rats received either No Pretreatment (n = 5), Habituation to the experimental context (n = 5) or nociceptive Hot Plate (n = 5), Tail Pinch (n = 6) or Tail Flick trials (n = 5). All rats were injected with 1.5 mg/kg s.c. morphine and tested at 30, 60, 120 and 180 min postinjection. The data are presented as the mean + S.E.M. area under the time effect curve (AUC).
on m o r p h i n e - i n d u c e d a n t i n o c i c e p t i o n in intact rats.
Pretreatment Fig. 5. Effect of pretreatment condition on morphine-induced antinociception in acute spinal rats. Separate groups of intact rats received either No Pretreatment (n = 10), Habituation to the experimental context (n = 5), or nociceptive Hot Plate (n = 6), Tail Pinch (n = 6) or Tail Flick (n = 8) trials. On the next day all rats were spinally transected. They were injected 24 h later with 3.0 mg/kg, s.c., morphine and tested at 30, 60, 120 and 180 min postinjection. The data are presented as the mean + S.E.M. total area under the time effect curve (AUC).
t r a n s e c t e d on the n e x t day, i n j e c t e d on the f o l l o w i n g day with 3.0 m g / k g of m o r p h i n e and tested o n the T F 30,
Behavioral tolerance in spinal rats
60, 120 and 180 min later. All spinally t r a n s e c t e d rats,
T h e results of the d o s e - r e s p o n s e
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like intact rats, w e r e t e s t e d on the T F p r i o r to m o r p h i n e
p h i n e in spinal rats is s u m m a r i z e d in Fig. 4, which shows
a d m i n i s t r a t i o n . A s e x p e c t e d f r o m p r e v i o u s r e p o r t s h2`Sn3
the A U C
T F l a t e n c i e s w e r e r e d u c e d after t r a n s e c t i o n . T h e m e a n
+ S . E . M . of the f o u r g r o u p s of rats i n j e c t e d
with 0.75, 1.5, 2.25 and 3.0 m g / k g of m o r p h i n e . A n a l y -
_+ S . E . M . latency of p r e t e s t e d spinal rats (n = 8) was
sis of t h e s e d a t a i n d i c a t e d a significant o v e r a l l effect of
2.6 _+ 0.13 s and of n o n - p r e t e s t e d spinal rats (n = 23),
dose (n = 23, F22 ' 3 = 5.3, P = 0.0076). H o w e v e r , post
3.0 + 0.16 s (t = 1.6, n.s.). T h e latencies of the HP, T P
h o c c o m p a r i s o n s s h o w e d that the o n l y significant differ-
and H a b i t u a t i o n g r o u p s w e r e 3.3 +_ 0.57 s, 3.2 _+ 0.15 s
e n c e was b e t w e e n the lowest and highest d o s e ( T u k e y - A ,
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P < 0.01). T h e s e d a t a are consistent with p r e v i o u s re-
T h e results of the v a r i o u s p r e t e s t t r e a t m e n t s are s h o w n
suits and c o n f i r m that a d o s e of 3.0 m g / k g was r e q u i r e d
in Figs. 5 and 6, which also include the d a t a f r o m the
in spinal rats to p r o d u c e
an a n t i n o c i c e p t i v e r e s p o n s e
N o n - p r e t r e a t e d g r o u p of spinal rats. A s i n d i c a t e d in Fig.
c o m p a r a b l e to the 1.5 m g / k g d o s e in intact rats. T h e r e -
5, t h e r e was a significant overall d i f f e r e n c e in A U C
fore, in the n e x t e x p e r i m e n t the 3.0 m g / k g dose was u s e d
a m o n g the five g r o u p s (n = 35, F34 ' 4 = 7.84, P =
to assess the effect of the v a r i o u s p r e t r e a t m e n t s on m o r p h i n e a n t i n o c i c e p t i o n in spinal rats. In this study, s e p a r a t e g r o u p s of rats r e c e i v e d o n e of the f o u r p r e t r e a t m e n t
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Log Morphine Dose ( m g / k g ) Fig. 4. Dose-response function to the antinociceptive effect of s.c. morphine in rats that were spinally transected 24 h previously. The data are presented as the mean + S.E.M. total area under the time-effect curve (AUC) for separate groups that were injected with either 0.75 mg/kg (n = 3), 1.5 mg/kg (n = 4), 2.25 mg/kg (n = 6) or 3.0 mg/kg (n = 10) and tested on the TF at 30, 60, 120 and 180 min postinjection.
60
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Fig, 6. Mean Tail Flick latencies of spinally transected rats as a function of time after a s.c. injection of 3.0 mg/kg of morphine. The open circles represent the latencies of Non-pretreated rats; filled circles indicate the latencies of TF pretreated rats; filled squares indicate the latencies of HP pretreated rats; filled triangles indicate the latencies of TP pretreated rats; filled diamonds indicate the latencies of Habituated rats (n = 5). The mean +_ S.E.M. AUC values for these groups are summarized in Fig, 5.
50 0.0002). Post hoc comparisons (Dunnett's test) showed that the Hot Plate (n = 6, P < 0.01), Tail Pinch (n = 6, P < 0.05) and Tail Flick (n = 8, P < 0.01) pretreatment groups differed from the control (No Pretreatment Group, n = 10), whereas the Habituation (n = 5) group did not. Fig. 6 shows the tail flick scores of each of the five groups of spinally transected rats for each of the four time points after morphine administration. It can be seen that the latencies of the control (Non-pretreated) group are substantially greater than those of the TF, TP and HP groups, whereas the scores of the Habituation group fall in between the control and nociceptive pretreatment values. DISCUSSION The results of the pretreatment session show that when a standard test procedure of three successive tail flick trials is administered to intact rats at hourly intervals, performance of the withdrawal reflex is facilitated (i.e. latencies decline significantly). This represents shortterm sensitization of the TF response. When morphine was administered 48 h later to irltact rats, antinoeiception was reduced in a dose-related manner, at the 30-min time point, indicating that prior elicitation of the reflex produced behavioral tolerance. This result is consistent with an earlier report, in which behavioral tolerance was observed 40 min after administration of a 3.0 mg/kg dose of morphine in intact rats that were pretreated with the TF procedure 3 times per week, for 7 sessions 1. The present data show that repeated TF tests within a single session will also produce behavioral tolerance to morphine and that the expression of BT is dose dependent. These data also show that none of the other pretreatments were effective in reducing the analgesic response to morphine on the T E Moreover, analysis of the time-effect curves indicated that even this expression of behavioral tolerance was short-lived. When the total duration of antinoeiception was taken into account, there was no difference among the groups. It is possible that the parameters chosen for the pretreatment conditions were not optimal and that behavioral tolerance in intact rats would have been maintained (in the TF pretreated group) or acquired (in the HP and TP groups) if the pretreatments were prolonged or if the interval between pretest and tolerance tests was greater or less than 48 h. Nevertheless, when these same manipulations were performed prior to spinal transection, the outcome was different. In contrast to the transient influence of TF pretreatment in intact rats, this manipulation produced behavioral tolerance in spinal rats that was still signifi-
cant when the total duration of antinociception was taken into account. It should be emphasized that the effect of the behavioral pretreatments in intact animals was not compared directly with the effect of the same manipulations in spinal animals. Rather, the effects of the various pretreatments were compared within each of these two conditions, between animals who were exposed to nociceptive and non-nociceptive stimulation and those who were not. This pattern of results indicates first, that in the intact rat, nociceptive stimulation can modify spinal reflex responsivity. The data support prior results showing that TF elicitation produces enduring changes within the spinal cord 1 and extend that observation to include other nociceptive manipulations. Further, by examining the time course of behavioral tolerance, the present results show that systemically administered morphine may inhibit, suppress or override the intrinsic changes in spinal responsivity that were produced by prior nociceptive experience. Presumably, morphine prevented behavioral tolerance from being maintained in intact rats by acting on descending supraspinal pathways which modulate reflex circuits. As a result, the phenomenon was only expressed when the drug and the test procedure were paired for the first time. Second, even in the intact rat, the short-lived expression of BT was very specific. Only those animals which practiced the TF response were subsequently tolerant to the analgesic effect of morphine on the TF. Apparently, this specificity is mediated supraspinally because all of the nociceptive pretreatments, hot plate and tail pinch, as well as the tail flick, significantly reduced the antinociceptive effect of morphine in acute spinal rats. Because there was no obvious way of matching the intensity of the three nociceptive treatments, the parameters of each manipulation were arbitrarily chosen and it is not possible to directly compare their relative effectiveness. Nevertheless~ all of these modest nociceptive procedures produced a significant change in spinal reflex function which was sustained after spinalization. Third, the fact that exposure to the hot plate and the tail pinch were also effective, indicates that activation of the same pattern of neuronal activity produced by the TF procedure was not necessary for the development of BT. Furthermore, the fact that the mechanical tail pinch stimulus was also effective suggests that the nociceptive stimulus need not be restricted to the thermal modality. Presumably, the primary requirement for retention of BT after spinalization is the application of an appropriate noxious stimulus and other aversive stimuli, such as tail shock, should also be effective. The difference in retention of BT between the Intact and Spinal condition suggests that prior exposure to nox-
51 ious stimulation can significantly influence the responsivity of spinal reflex circuits but that such intrinsic changes are modulated by supraspinal input. While nociceptive stimulation produced a general decrease in the antinociceptive action of morphine within the spinal cord, the behavioral expression of this effect was limited, by a supraspinal mechanism, to the particular response system that was used to assess morphine-induced antinociception in intact animals. The present studies represent one of several paradigms which have been used to investigate the adaptive capacities of the isolated mammalian spinal cord. A nonassociative form of spinal plasticity, known as 'spinal fixation '8 has been extensively studied by several investigators, most recently Patterson et al. 16. In this procedure, a postural asymmetry of the hindlimb is induced in decerebrate or anesthetized dogs, rabbits or rats by lesions in the cerebellum. Typically, these asymmetries involve flexing of one limb and extension of the opposite limb. If at least 45 min is allowed between the brain lesion and a subsequent spinal transection, the asymmetry will outlast the spinal transection for at least two hours. In fact, Patterson et al. have shown that spinal fixation can also be produced by 45 min of direct hindlimb stimulation even when it is administered immediately after a spinal transection~9. Although associative, Pavlovian, conditioning of a spinal flexion reflex in the acute spinal dog was reported as early as 1940, subsequent attempts to verify this phenomenon were unsuccessful (see ref. 16 for a review of spinal conditioning). Nevertheless, a number of more recent studies have demonstrated conditioning of the flexor nerve 4 and the flexion reflex 9'1° in acute spinal cats. Most recently, Grau et al. 11 have reported that a conditioned antinociceptive response, assessed with the tail flick reflex, could be established in the acute spinal rat. Within several hours after a spinal transection rats received differential conditioning trials in which a mild shock to one leg (CS+) was paired with a strong tail shock, while the same mild shock to the other leg (CS-) was not followed by tail shock. After training, the conditioned stimuli were presented in conjunction with no-
REFERENCES 1 Advokat, C., Tolerance to the antinociceptive effect of morphine in spinally transected rats, Behav. Neurosci., 103 (1989) 1091-1098. 2 Advokat, C. and Burton, P., Antinociceptive effect of systemic and intrathecal morphine in spinally transected rats, Eur. J. PharmacoL, 139 (1987) 335-343. 3 Advokat, C. and Gulati, A., Spinal transection reduces both spinal antinociception and CNS concentration of systemically administered morphine in rats, Brain Res., 555 (1991) 251-258. 4 Beggs, A.L., Steinmetz, J.E., Romano, A.G. and Patterson,
ciceptive thermal TF tests. There was a significant difference between the response to the C S + and C S - during the first four test trials, i.e. the TF reflex latency was elevated when preceeded by the C S + but not the C S - , indicating the acquisition of a conditioned stress-induced antinociceptive response. Furthermore, an elegant series of experiments by Wolpaw et al. has provided evidence for operant conditioning of the monosynaptic spinal stretch reflex in monkeys (see ref. 21 for review and references). Although the conditioning task is acquired by monkeys with an intact neuraxis, the effects of conditioning are still present three days after a spinal transection. While these studies provide evidence for the acquisition of both associative and non-associative changes in reflex activity in the acute spinal preparation, it remains to be seen whether or not comparable plasticity can be sustained in the chronic preparation. Attempts to condition the hindlimb withdrawal reflex in chronic spinal dogs were unsuccessful 14. The chronic spinal rat also showed no evidence of behavioral tolerance 1. However, Buerger et al. reported successful instrumental avoidance conditioning of both leg withdrawal and leg lengthening in chronic spinal rats (1-3 weeks after transection) 17. A classical conditioning paradigm has also been reported to produce a 'reliable conditioned response of urination' in a man with a complete spinal transection 12. While it appears that the spinal cord is capable of significant functional adaptation it is clear that much still needs to be learned about the neural mechanisms responsible, particularly in regard to long-term retention of spinal reflex modulation. Because of the extensive amount of information available concerning the neurophysiological, neuropharmacological and behavioral characteristics of the spinally mediated withdrawal reflex, this preparation may prove useful for investigations relating behavioral function to neural substrate.
Acknowledgements. This research was supported by US Public Health Service Grant DA 02845 from the National Institute on Drug Abuse.
M.M., Extinction and retention of a classically conditioned flexor nerve response in acute spinal cat, Behav. Neurosci., 97 (1983) 530-540. 5 Berge, O.-G. and Hole, K., Tolerance to the antinociceptive effect of morphine in the spinal rat, Neuropharmacology, 20 (1981) 653-657. 6 Buccafusco, J.J. and Marshall, D.C., Dorsal root lesions block the expression of morphine withdrawal elicited from the rat spinal cord, Neurosci. Lett., 59 (1985) 319-324. 7 D'Amour, EE. and Smith, D.L., A method for determining loss of pain sensation, J. Pharmacol. Exp. Ther., 72 (1941) 7479.
52 8 DiGiorgio, A.M., Persistenza nell'animale spinale, di asimmetrie posturali e motorie di origine cerebellare. Nota I-III, Arch. FisioL, 27 (1929) 518-580. 9 Durkovic, R.G., Retention of a classically conditioned reflex response in spinal cat, Behav. Neural Biol., 43 (1985) 12-20. 10 Durkovic, R.G. and Damianopoulos, E.N., Forward and backward classical conditioning of the flexion reflex in the spinal cat, J. Neurosci., 6 (1986) 2921-2925. 11 Grau, J.W., Salinas, J.A., IUich, P.A. and Meagher, M.W., Associative learning and memory for an antinociceptive response in the spinalized rat, Behav. Neurosci., 104 (1990) 489494. 12 Ince, L.P., Brucker, B.S. and Alba, A., Reflex conditioning in a spinal man, J. Comp. Physiol. Psychol., 92 (1978) 796-802. 13 Irwin, S., Houde, R.W., Bennett, D.R., Hendershot, L.C. and Seevers, M.H., The effects of morphine, methadone and meperidine on some reflex responses of spinal animals to nociceptive stimulation, J. PharmacoL Exp. Ther., 101 (1951) 132143. 14 Lloyd, A.J., Wikler, A. and Whitehouse, J.M,, Nonconditionability of the flexor reflex in the chronic spinal dog, J. Comp. Physiol. Psychol., 68 (1969) 576-579.
15 MaCrae, J.R., Scoles, M.T. and Siegel, S., Fhe contribution of Pavlovian conditioning to drug tolerance and dependence, Br. J. Addict., 82 (1987) 371-380. 16 Patterson, M.M., Mechanisms of classical conditioning and fixation in spinal animals. In A.H. Riesen and R.F. Thompson (Eds.), Advances in Psychobiology, Vol. 3. Wiley. New York, 1976. pp. 381-436. 17 Sherman, B.S., Hoehler, EK. and Buerger, A.A., Instrumental avoidance conditioning of increased leg lowering in the spinal rat, Physiol. Behav., 25 (1982) 123-128. 18 Siuciak, J.A. and Advokat, C., Antinociceptive effect of intrathecal morphine in tolerant and nontolerant spinal rats, Pharmacol. Biochem. Behav., 34 (1989) 445-452. 19 Steinmetz, J.E., Cervenka, J., Robinson, C., Romano, A.G. and Patterson, M.M., Fixation of spinal reflexes in rats by central and peripheral sensory input, J. Comp. Physiol. Psychol., 95 (1981) 548-555. 20 Tallarida, R.J. and Murray, R.B., Manual of Pharmacologic Calculations with Computer Programs, Springer, New York, 1987. 21 Wolpaw, J.R. and Carp, J.S., Memory traces in spinal cord, Trends" Neurol. Sci., 13 (1990) 137-142.
Brain Research, 561 (1992) 40-52 © 1992 Elsevier Science Publishers B.V. All rights reserved. 0006-8993/92/$05.00
BRES 17751
Environmental modulation of behavioral tolerance in spinal rats Claire Advokat and Carla Mclnnis Department of Psychology, Louisiana State University, Baton Rouge, LA 70803 (USA) (Accepted 7 January 1992) Key words: Behavioral tolerance; Opiate antinociception; Spinal rat
We previously reported that the antinociceptive effect of morphine on the thermal tail withdrawal reflex (tail flick, TF) was reduced in rats that had practiced the TF response prior to morphine administration. This phenomenon, termed behavioral tolerance, was observed even when rats were spinally transected following the TF tests. The present experiments were conducted to determine whether such retention of behavioral tolerance after spinal transection was dependent on prior experience with the TF procedure, or whether other nociceptive pretreatments would also reduce opiate antinoeiception on the TF test in spinal rats. Separate groups of rats were pretreated with either nociceptive thermal TF or Hot Plate (HP), or mechanical Tail Pinch (TP) stimuli, as well as non-nociceptive exposure to the experimental context (Habituation) or No Pretreatment. Approximately half of the rats were spinally transected after their respective pretreatment, while the other half were left intact. When Intact rats were subsequently tested on the TF at 30, 60, 120 and 180 min after morphine administration. only the TF pretreated group was tolerant, relative to the Non-pretreated control group. However, this was a transient effect, and was only significant at the first, 30-rain test; there was no difference among the groups in the total duration of antinoeiception across all the time points. When spinal rats were tested after morphine administration, there was also an overall significant difference among the groups at 30 min. In this case, the differences were maintained across all time points. All three nociceptive pretreatments (TF, HP and TP) significantly reduced the total duration of opiate antinociception in acute spinal rats, relative to the Non-pretreatment condition, whereas the Habituation treatment did not. These data reveal a differential effect of behavioral pretreatment on morphine-induced antinociception in Intact vs. Spinal rats. In Intact rats, behavioral tolerance on the TF test was transient and only observed in animals that had prior experience with the TF procedure. In contrast, when rats were spinally transected after noeiceptive pretreatment, behavioral tolerance on the TF test was maintained throughout the test session and was observed in all of the nociceptive pretreatment groups. These data indicate first that noeiceptive stimulation can produce a persistent change in the spinal cord which significantly influences spinal reflex function and second that when the neuraxis is intact, the effect of such stimulation may be modulated by descending supraspinal input. INTRODUCTION N u m e r o u s investigations have shown that the effect o f drugs on a variety of behavioral responses can be reduced if subjects are allowed to practice the response before the drug is administered. This p h e n o m e n o n , t e r m e d behavioral tolerance (BT), has b e e n d e m o n strated with a variety of drugs and behaviors in intact animals 15. In contrast, little is known a b o u t the neural basis of this a d a p t a t i o n , p r e s u m a b l y because most behavioral p r e p a r a t i o n s are too complex for such analyses. In previous research, we e x a m i n e d B T in a response system that m a y ultimately be a m e n a b l e to neural analysis; the involuntary, defensive tail withdrawal reflex (tail flick, TF) elicited by noxious thermal stimulation. This reflex is used extensively as a nociceptive index to assess the analgesic (antinociceptive) effects of opiates. Most i m p o r t a n t in this context is the fact that the T F reflex can be elicited in the spinally transected rat and, that o p i a t e - i n d u c e d antinociception 2'13, tolerance L5 and dep e n d e n c e 6 can also be o b s e r v e d with this response after
spinalization. Because a vast a m o u n t of information exists concerning neurophysiological mechanisms of o p i a t e action at the spinal level, the possibility arises that behavioral adaptations o b s e r v e d within this reflex system might ultimately be related to neural mechanism. Previous results from o u r l a b o r a t o r y showed that after r e p e a t e d exposure to the T F test p r o c e d u r e o v e r several days, the analgesic effect of systemic m o r p h i n e in intact rats is significantly reduced, relative to non-TFexperienced rats, indicating the d e v e l o p m e n t o f BT. The d a t a also showed that such tolerance was r e t a i n e d for 24 h after spinalization: the antinociceptive response to m o r p h i n e in T F p r e t r e a t e d rats that were spinally transected was significantly less than that of non-pretreated, spinal rats 1'1s. The fact that B T could be m a i n t a i n e d for at least 24 h after a spinal transection suggested that some type of neural m o d u l a t i o n had occurred within the spinal cord as a result of prior experience with the T F procedure. F u r t h e r examination indicated that the d e v e l o p m e n t of B T required an intact neuraxis. If rats were spinally
Correspondence: C. Advokat, Department of Psychology, Louisiana State University, Baton Rouge, LA 70803, USA.
47 t r a n s e c t e d before t h e y w e r e t e s t e d o n t h e TF, b e h a v i o r a l t o l e r a n c e t o s y s t e m i c m o r p h i n e a d m i n i s t r a t i o n did n o t occur. That result indicated that supraspinal input was n e c e s s a r y f o r t h e a c q u i s i t i o n o f b e h a v i o r a l t o l e r a n c e 1. The main p u r p o s e of the p r e s e n t study was to determ i n e w h e t h e r t h e d e v e l o p m e n t o f B T o f t h e T F reflex required prior TF exposure or, w h e t h e r the p h e n o m e n o n c o u l d also b e p r o d u c e d b y e x p o s u r e t o o t h e r n o c i c e p t i v e p r o c e d u r e s . I n a d d i t i o n , t h e p r e s e n t s t u d i e s also determined whether BT would develop when TF pret r e a t m e n t w a s a d m i n i s t e r e d w i t h i n a single e x p e r i m e n t a l s e s s i o n , a n d w h e t h e r this p h e n o m e n o n
w o u l d b e ex-
p r e s s e d in a d o s e - d e p e n d e n t m a n n e r .
MATERIALS AND METHODS
Subjects A total of 101 male albino Sprague-Dawley derived rats, of the Holtzman strain, weighing 250-350 g were used as subjects. They were bred and raised at the animal facility of the Baton Rouge campus of Louisiana State University. All animals were housed individually in suspended stainless steel cages in a colony room maintained on a 12 h:12 h light-dark cycle, with dark onset at 17.00 h. Food and water were available ad libitum.
Surgical procedures Spinal transections were performed under ether anesthesia. A laminectomy was made between thoracic vertebrae 6 and 9 and a 1-2 mm portion of the spinal cord was removed by excavation and replaced with gelfoam to reduce bleeding, after which the wound was sutured and the cages placed on heating pads to maintain body temperature. On the morning after surgery, the hindquarters of each rat were washed with warm water and their urine was expressed manually by the application of pressure to their bladders (see ref. 1 for details),
Nociceptive assessment For all rats, the final nociceptive response was assessed with the tail flick (TF) test7. Noxious stimulation was produced by a beam of high intensity light, focused on the tail. The response time was measured automatically and was defined as the interval between the onset of the thermal stimulus and the abrupt flick of the tail. Each determination consisted of three trials; the mean score was taken as the response latency. In order to minimize tissue damage to the tail, animals not responding within 14 s were removed from the apparatus and assigned a response latency of 14 s.
Drug administration Morphine sulfate (Penick Corp., Lyndhurst, N J) was dissolved in 0.9% saline and administered subcutaneously such that the appropriate dose was injected in a volume of 1 ml/kg.
Statistical analyses In the first experiment, the data consisted of the TF latency scores, in seconds, and are presented as the group mean + S.E.M. In subsequent studies, the duration of opiate antinociception was determined by testing the animals at 30, 60, 120 and 180 min after morphine administration and then obtaining the Area Under the Curve (AUC) with the aid of a computer program (PHARM/ PCS) 2°. For each animal the A_UC was determined by entering the four xy data pairs, in which x = tail flick latency and y = 30, 60, 120 or 180 min. The computer program calculated the total area (i.e. the integral) based on an approximation using the trapezoidal rule. Statistical tests were then performed on the AUC values that comprised each experimental group. These results are expressed in
the figures as the group mean + S.E.M. AUC scores. In each case, statistical calculations, performed with a computer program (CRUNCH Interactive Statistical Program) consisted of analyses of variance, for either repeated measures within groups or for differences among several groups, with the appropriate post hoc comparisons and Student's t-test. Results were considered significant at P = 0.05 or less.
Experimental procedures Behavioral tolerance to morphine in intact rats. The first experiment was designed to determine whether a series of TF trials, administered within a single experimental session, would reduce the subsequent analgesic effect of morphine on the TF. The rats in this condition (Tail Flick Pretreatment) were brought to the experimental room, wrapped in a cloth towel and given three TF trials in succession, five different times, at 1-h intervals. This resulted in a total of 15 trials, administered between 09.00-10.00 h and 13.0014.00 h. Two days later these subjects were brought back to the experimental room, and randomly divided into two groups, one injected with 0.75 mg/kg of morphine and the other injected with 1.5 mg/kg of morphine. The group injected with 0.75 mg/kg was tested once on the TF, 30 rain after injection (n = 5). All animals injected with 1.5 mg/kg were also tested on the TF at 30 min (n = 13) and some of these subjects (n = 5) were tested again at 60, 120 and 180 min after injection. Two additional groups, which were not previously exposed to the experimental context or the TF apparatus, were injected with the same doses of morphine (No Pretreatment Groups: 0.75 mg/kg, n = 5; 1.5 mg/kg, n = 10). Again, all of the rats were tested 30 rain postinjection, whereas half of the animals injected with 1.5 mg/kg were also tested at 60, 120 and 180 min after injection. This procedure resulted in 4 experimental groups, consisting of rats that were either Pretreated or Not Pretreated with the TF procedure prior to systemic injection of either 0.75 or 1.5 mg/kg of morphine. All groups were tested 30 rain after injection, and those rats injected with 1.5 mg/kg of morphine were then tested repeatedly, to determine whether or not the duration of analgesia would be altered by prior exposure to the TF test. Several additional groups were preexposed to treatments which did not involve the TF test: a fifth group of rats was brought into the experimental room and received a nociceptive hot plate (HP) test once every hour for 5 h, for a total of five trials. For this test, a Plexiglas cylinder (28.5 cm in height and 20.5 cm in diameter), which restrained the rat, was placed on top of a metal plate. The temperature of the plate was maintained at 54 +_ 1.0°C by a heated water bath. Animals were placed within the cylinder and the latency until either the animal licked its hindpaws or jumped out of the cylinder was manually recorded. Animals not responding within 40 s were removed and assigned a 40-s response latency (Hot Plate Group, n = 5). A sixth group of rats was brought to the experimental room and received three nociceptive tail pinch (TP) trials, five times, at onehour intervals, for a total of 15 trials. In this case, the rats were wrapped in a cloth towel and placed on the TF apparatus. Pressure was applied to the rat's tails with a hemostat, whose teeth were covered with clear polyethylene tubing. The hemostat was closed at a constant setting for 5 s on each of the three trials. This elicited vigorous movements of the tail and vocalizations in all subjects. A different patch of skin was pinched on each trial (Tail Pinch Group, n = 6). A seventh group of rats was exposed to the experimental room during a 'pretreatment' session but was not placed in the TF apparatus and did not receive TF tests (Habituation Group, n = 9). Two days later, the subjects in the Hot Plate, Tail Pinch and Habituation groups were injected with 1.5 mg/kg of morphine and tested on the tail flick 30 rain later. All of the animals in the Hot Plate and Tail Pinch groups, and five of the animals in the Habituation group were tested again at 60, 120 and 180 min after the injection. Behavioral tolerance to morphine in spinal rats. It is well-known
48 6i
~
14.
v
0~0~
•
3
t
E
o
7¸
i--O
No
A--A
Toil
Pretreetment Flick
IT
o 1
2
3
4
5
Pretest Triat
Fig. 1. Mean Tail Flick latency + S.E.M. of intact rats (n --- 30) on each of five pretest trials, administered at one-hour intervals within a single session. Each trial consisted of three tail flick tests.
that the antinociceptive effect of systemic morphine on the TF test is significantly reduced within 24 h after spinal transection. Therefore, in order to obtain an equivalent response on the TF the dose of morphine administered to spinal rats must be greater than (approximately double) the dose given to intact rats3. To confirm that observation in the context of these experiments, a dose-response function to morphine was first determined in rats that were spinally transected 24 h previously. Separate groups of spinal rats were injected with either 0.75 (n = 3), 1.5 (n = 4), 2.25 (n = 6) or 3.0 (n = 10) mg/kg of morphine and tested on the TF at 30, 60, 120 and 180 min after the injection. In the second experiment, separate groups of intact rats received each of the four different pretreatments. One group was exposed to the experimental room, but was not placed on the TF apparatus (Habituation Group, n = 5). The second, third and fourth groups received the same HP (n = 6), TP (n = 6) and TF (n = 8) pretreatments as described above. On the day after each of these respective treatments, all rats were spinally transected. On the next day, all rats were injected with 3.0 mg/kg of morphine and tested on the TF at 30, 60, 120 and 180 min after the injection. RESULTS
Effect of tail flick pretreatment in intact rats There was a slight, but statistically significant decline in baseline latency during the T F p r e t r e a t m e n t trials. A summary of this decrease is shown in Fig. 1, which presents the data for all rats that received the 15 T F pretreatment trials prior to their respective (Intact or Spinal) experimental m a n i p u l a t i o n (n = 30, F29' 4 = 8 . 9 8 , P < 0.0001). The baseline latency was reduced from 4.7 s on the first trial to 3.4 s on the fifth, demonstrating the development of within session facilitation, or sensitization, of the T F response in intact rats.
Behavioral tolerance in intact rats All rats, both T F pretreated (n = 18) and Non-pretreated (n = 15) were given a preliminary T F trial prior to their respective m o r p h i n e injection. The m e a n + S.E.M. T F latency for Pretreated rats was 4.9 + 0.32 s and for Non-pretreated rats, 4.5 ___ 0.32 s (t = 0.83, n.s.). This result indicates that, although reflex latency was significantly reduced during pretest trials this effect had dissipated within 48 h and would not account for any
o
75
1.5
Morphine Dose (mg/kg)
Fig. 2. Mean Tail Flick latency + S.E.M., 30 min after a s.c. morphine injection in four groups of rats. Two groups had no prior exposure to the experimental context or tail flick procedure (No Pretreatment; 0.75 mg/kg, n = 5; 1.5 mg/kg, n = 10). Two groups received a series of 15 tail flick trials administered 48 h previously (Tail Flick; 0.75 mg/kg, n = 5; 1.5 mg/kg, n = 13).
differences among the groups observed after m o r p h i n e administration. In addition, the latencies of the HP, TP and H a b i t u a t i o n groups were 4.5 + 0,25 s, 4.1 + 0.34 s and 4.4 + 0.22 s respectively, suggesting that these pretreatments also did not modify baseline T F latency. The effect of T F p r e t r e a t m e n t in intact rats o n the subsequent analgesic response to morphine two days later, is summarized in Fig. 2. This figure shows the response of the Non-pretreated and T F Pretreated rats, 30 min after m o r p h i n e administration for each of the two doses. As indicated, T F p r e t r e a t m e n t reduced opiate analgesia in a dose-related m a n n e r . A two-way A N O V A showed a significant effect of p r e t r e a t m e n t (n = 33, F32,1 = 19.5, P = 0.0001) and of dose (F32a = 34.3, P < 0.0001), with no interaction. These data demonstrate that a series of T F tests, administered within a single session, produced behavioral tolerance to the analgesic effect of morphine which was retained for at least two days. Additional analyses were performed on the scores of the five groups that were injected with the higher, 1.5 mg/kg, dose of morphine. These groups were first compared at the 30-rain time point. The results showed an overall significant effect of p r e t r e a t m e n t (n = 43, F42.4 = 4.21, P = 0.0064). However, post hoc tests indicated that only the scores of the T F Pretreated group were significantly different (i.e. lower) than that of the Nonpretreated group ( D u n n e t t ' s test, P = < 0.05). The m e a n T F latencies + S.E.M. for all the groups at 30 min after m o r p h i n e were: No pretreatment, 12.6 + 0.63 s; Habituation, 11.06 + 0.87 s; HP, 9.76 + 1.15 s; TP, 10.82 + 0.82 s and TF, 8.77 + 0.65 s. However, a second analysis of the complete time effect curve ( A U C ) showed no difference among the groups. These data, summarized in Fig. 3 show that over the complete 180-min course of the test session, there was no effect of the respective pretreatment conditions
49
1500
No PretreQtment
1500
l ~ No Pretreotment Habituated
Habituated Hot Plate
Toil Pinch 0
IT'A Toil Pinch
~
Toil Flick
750
Tail Flick
750
0 0 Pretreotment Fig. 3. Effect of pretreatment condition on morphine-induced antinociception in intact rats. Separate groups of intact rats received either No Pretreatment (n = 5), Habituation to the experimental context (n = 5) or nociceptive Hot Plate (n = 5), Tail Pinch (n = 6) or Tail Flick trials (n = 5). All rats were injected with 1.5 mg/kg s.c. morphine and tested at 30, 60, 120 and 180 min postinjection. The data are presented as the mean + S.E.M. area under the time effect curve (AUC).
on m o r p h i n e - i n d u c e d a n t i n o c i c e p t i o n in intact rats.
Pretreatment Fig. 5. Effect of pretreatment condition on morphine-induced antinociception in acute spinal rats. Separate groups of intact rats received either No Pretreatment (n = 10), Habituation to the experimental context (n = 5), or nociceptive Hot Plate (n = 6), Tail Pinch (n = 6) or Tail Flick (n = 8) trials. On the next day all rats were spinally transected. They were injected 24 h later with 3.0 mg/kg, s.c., morphine and tested at 30, 60, 120 and 180 min postinjection. The data are presented as the mean + S.E.M. total area under the time effect curve (AUC).
t r a n s e c t e d on the n e x t day, i n j e c t e d on the f o l l o w i n g day with 3.0 m g / k g of m o r p h i n e and tested o n the T F 30,
Behavioral tolerance in spinal rats
60, 120 and 180 min later. All spinally t r a n s e c t e d rats,
T h e results of the d o s e - r e s p o n s e
f u n c t i o n to m o r -
like intact rats, w e r e t e s t e d on the T F p r i o r to m o r p h i n e
p h i n e in spinal rats is s u m m a r i z e d in Fig. 4, which shows
a d m i n i s t r a t i o n . A s e x p e c t e d f r o m p r e v i o u s r e p o r t s h2`Sn3
the A U C
T F l a t e n c i e s w e r e r e d u c e d after t r a n s e c t i o n . T h e m e a n
+ S . E . M . of the f o u r g r o u p s of rats i n j e c t e d
with 0.75, 1.5, 2.25 and 3.0 m g / k g of m o r p h i n e . A n a l y -
_+ S . E . M . latency of p r e t e s t e d spinal rats (n = 8) was
sis of t h e s e d a t a i n d i c a t e d a significant o v e r a l l effect of
2.6 _+ 0.13 s and of n o n - p r e t e s t e d spinal rats (n = 23),
dose (n = 23, F22 ' 3 = 5.3, P = 0.0076). H o w e v e r , post
3.0 + 0.16 s (t = 1.6, n.s.). T h e latencies of the HP, T P
h o c c o m p a r i s o n s s h o w e d that the o n l y significant differ-
and H a b i t u a t i o n g r o u p s w e r e 3.3 +_ 0.57 s, 3.2 _+ 0.15 s
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c o m p a r a b l e to the 1.5 m g / k g d o s e in intact rats. T h e r e -
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to assess the effect of the v a r i o u s p r e t r e a t m e n t s on m o r p h i n e a n t i n o c i c e p t i o n in spinal rats. In this study, s e p a r a t e g r o u p s of rats r e c e i v e d o n e of the f o u r p r e t r e a t m e n t
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Log Morphine Dose ( m g / k g ) Fig. 4. Dose-response function to the antinociceptive effect of s.c. morphine in rats that were spinally transected 24 h previously. The data are presented as the mean + S.E.M. total area under the time-effect curve (AUC) for separate groups that were injected with either 0.75 mg/kg (n = 3), 1.5 mg/kg (n = 4), 2.25 mg/kg (n = 6) or 3.0 mg/kg (n = 10) and tested on the TF at 30, 60, 120 and 180 min postinjection.
60
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Postinjection Time
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Fig, 6. Mean Tail Flick latencies of spinally transected rats as a function of time after a s.c. injection of 3.0 mg/kg of morphine. The open circles represent the latencies of Non-pretreated rats; filled circles indicate the latencies of TF pretreated rats; filled squares indicate the latencies of HP pretreated rats; filled triangles indicate the latencies of TP pretreated rats; filled diamonds indicate the latencies of Habituated rats (n = 5). The mean +_ S.E.M. AUC values for these groups are summarized in Fig, 5.
50 0.0002). Post hoc comparisons (Dunnett's test) showed that the Hot Plate (n = 6, P < 0.01), Tail Pinch (n = 6, P < 0.05) and Tail Flick (n = 8, P < 0.01) pretreatment groups differed from the control (No Pretreatment Group, n = 10), whereas the Habituation (n = 5) group did not. Fig. 6 shows the tail flick scores of each of the five groups of spinally transected rats for each of the four time points after morphine administration. It can be seen that the latencies of the control (Non-pretreated) group are substantially greater than those of the TF, TP and HP groups, whereas the scores of the Habituation group fall in between the control and nociceptive pretreatment values. DISCUSSION The results of the pretreatment session show that when a standard test procedure of three successive tail flick trials is administered to intact rats at hourly intervals, performance of the withdrawal reflex is facilitated (i.e. latencies decline significantly). This represents shortterm sensitization of the TF response. When morphine was administered 48 h later to irltact rats, antinoeiception was reduced in a dose-related manner, at the 30-min time point, indicating that prior elicitation of the reflex produced behavioral tolerance. This result is consistent with an earlier report, in which behavioral tolerance was observed 40 min after administration of a 3.0 mg/kg dose of morphine in intact rats that were pretreated with the TF procedure 3 times per week, for 7 sessions 1. The present data show that repeated TF tests within a single session will also produce behavioral tolerance to morphine and that the expression of BT is dose dependent. These data also show that none of the other pretreatments were effective in reducing the analgesic response to morphine on the T E Moreover, analysis of the time-effect curves indicated that even this expression of behavioral tolerance was short-lived. When the total duration of antinoeiception was taken into account, there was no difference among the groups. It is possible that the parameters chosen for the pretreatment conditions were not optimal and that behavioral tolerance in intact rats would have been maintained (in the TF pretreated group) or acquired (in the HP and TP groups) if the pretreatments were prolonged or if the interval between pretest and tolerance tests was greater or less than 48 h. Nevertheless, when these same manipulations were performed prior to spinal transection, the outcome was different. In contrast to the transient influence of TF pretreatment in intact rats, this manipulation produced behavioral tolerance in spinal rats that was still signifi-
cant when the total duration of antinociception was taken into account. It should be emphasized that the effect of the behavioral pretreatments in intact animals was not compared directly with the effect of the same manipulations in spinal animals. Rather, the effects of the various pretreatments were compared within each of these two conditions, between animals who were exposed to nociceptive and non-nociceptive stimulation and those who were not. This pattern of results indicates first, that in the intact rat, nociceptive stimulation can modify spinal reflex responsivity. The data support prior results showing that TF elicitation produces enduring changes within the spinal cord 1 and extend that observation to include other nociceptive manipulations. Further, by examining the time course of behavioral tolerance, the present results show that systemically administered morphine may inhibit, suppress or override the intrinsic changes in spinal responsivity that were produced by prior nociceptive experience. Presumably, morphine prevented behavioral tolerance from being maintained in intact rats by acting on descending supraspinal pathways which modulate reflex circuits. As a result, the phenomenon was only expressed when the drug and the test procedure were paired for the first time. Second, even in the intact rat, the short-lived expression of BT was very specific. Only those animals which practiced the TF response were subsequently tolerant to the analgesic effect of morphine on the TF. Apparently, this specificity is mediated supraspinally because all of the nociceptive pretreatments, hot plate and tail pinch, as well as the tail flick, significantly reduced the antinociceptive effect of morphine in acute spinal rats. Because there was no obvious way of matching the intensity of the three nociceptive treatments, the parameters of each manipulation were arbitrarily chosen and it is not possible to directly compare their relative effectiveness. Nevertheless~ all of these modest nociceptive procedures produced a significant change in spinal reflex function which was sustained after spinalization. Third, the fact that exposure to the hot plate and the tail pinch were also effective, indicates that activation of the same pattern of neuronal activity produced by the TF procedure was not necessary for the development of BT. Furthermore, the fact that the mechanical tail pinch stimulus was also effective suggests that the nociceptive stimulus need not be restricted to the thermal modality. Presumably, the primary requirement for retention of BT after spinalization is the application of an appropriate noxious stimulus and other aversive stimuli, such as tail shock, should also be effective. The difference in retention of BT between the Intact and Spinal condition suggests that prior exposure to nox-
51 ious stimulation can significantly influence the responsivity of spinal reflex circuits but that such intrinsic changes are modulated by supraspinal input. While nociceptive stimulation produced a general decrease in the antinociceptive action of morphine within the spinal cord, the behavioral expression of this effect was limited, by a supraspinal mechanism, to the particular response system that was used to assess morphine-induced antinociception in intact animals. The present studies represent one of several paradigms which have been used to investigate the adaptive capacities of the isolated mammalian spinal cord. A nonassociative form of spinal plasticity, known as 'spinal fixation '8 has been extensively studied by several investigators, most recently Patterson et al. 16. In this procedure, a postural asymmetry of the hindlimb is induced in decerebrate or anesthetized dogs, rabbits or rats by lesions in the cerebellum. Typically, these asymmetries involve flexing of one limb and extension of the opposite limb. If at least 45 min is allowed between the brain lesion and a subsequent spinal transection, the asymmetry will outlast the spinal transection for at least two hours. In fact, Patterson et al. have shown that spinal fixation can also be produced by 45 min of direct hindlimb stimulation even when it is administered immediately after a spinal transection~9. Although associative, Pavlovian, conditioning of a spinal flexion reflex in the acute spinal dog was reported as early as 1940, subsequent attempts to verify this phenomenon were unsuccessful (see ref. 16 for a review of spinal conditioning). Nevertheless, a number of more recent studies have demonstrated conditioning of the flexor nerve 4 and the flexion reflex 9'1° in acute spinal cats. Most recently, Grau et al. 11 have reported that a conditioned antinociceptive response, assessed with the tail flick reflex, could be established in the acute spinal rat. Within several hours after a spinal transection rats received differential conditioning trials in which a mild shock to one leg (CS+) was paired with a strong tail shock, while the same mild shock to the other leg (CS-) was not followed by tail shock. After training, the conditioned stimuli were presented in conjunction with no-
REFERENCES 1 Advokat, C., Tolerance to the antinociceptive effect of morphine in spinally transected rats, Behav. Neurosci., 103 (1989) 1091-1098. 2 Advokat, C. and Burton, P., Antinociceptive effect of systemic and intrathecal morphine in spinally transected rats, Eur. J. PharmacoL, 139 (1987) 335-343. 3 Advokat, C. and Gulati, A., Spinal transection reduces both spinal antinociception and CNS concentration of systemically administered morphine in rats, Brain Res., 555 (1991) 251-258. 4 Beggs, A.L., Steinmetz, J.E., Romano, A.G. and Patterson,
ciceptive thermal TF tests. There was a significant difference between the response to the C S + and C S - during the first four test trials, i.e. the TF reflex latency was elevated when preceeded by the C S + but not the C S - , indicating the acquisition of a conditioned stress-induced antinociceptive response. Furthermore, an elegant series of experiments by Wolpaw et al. has provided evidence for operant conditioning of the monosynaptic spinal stretch reflex in monkeys (see ref. 21 for review and references). Although the conditioning task is acquired by monkeys with an intact neuraxis, the effects of conditioning are still present three days after a spinal transection. While these studies provide evidence for the acquisition of both associative and non-associative changes in reflex activity in the acute spinal preparation, it remains to be seen whether or not comparable plasticity can be sustained in the chronic preparation. Attempts to condition the hindlimb withdrawal reflex in chronic spinal dogs were unsuccessful 14. The chronic spinal rat also showed no evidence of behavioral tolerance 1. However, Buerger et al. reported successful instrumental avoidance conditioning of both leg withdrawal and leg lengthening in chronic spinal rats (1-3 weeks after transection) 17. A classical conditioning paradigm has also been reported to produce a 'reliable conditioned response of urination' in a man with a complete spinal transection 12. While it appears that the spinal cord is capable of significant functional adaptation it is clear that much still needs to be learned about the neural mechanisms responsible, particularly in regard to long-term retention of spinal reflex modulation. Because of the extensive amount of information available concerning the neurophysiological, neuropharmacological and behavioral characteristics of the spinally mediated withdrawal reflex, this preparation may prove useful for investigations relating behavioral function to neural substrate.
Acknowledgements. This research was supported by US Public Health Service Grant DA 02845 from the National Institute on Drug Abuse.
M.M., Extinction and retention of a classically conditioned flexor nerve response in acute spinal cat, Behav. Neurosci., 97 (1983) 530-540. 5 Berge, O.-G. and Hole, K., Tolerance to the antinociceptive effect of morphine in the spinal rat, Neuropharmacology, 20 (1981) 653-657. 6 Buccafusco, J.J. and Marshall, D.C., Dorsal root lesions block the expression of morphine withdrawal elicited from the rat spinal cord, Neurosci. Lett., 59 (1985) 319-324. 7 D'Amour, EE. and Smith, D.L., A method for determining loss of pain sensation, J. Pharmacol. Exp. Ther., 72 (1941) 7479.
52 8 DiGiorgio, A.M., Persistenza nell'animale spinale, di asimmetrie posturali e motorie di origine cerebellare. Nota I-III, Arch. FisioL, 27 (1929) 518-580. 9 Durkovic, R.G., Retention of a classically conditioned reflex response in spinal cat, Behav. Neural Biol., 43 (1985) 12-20. 10 Durkovic, R.G. and Damianopoulos, E.N., Forward and backward classical conditioning of the flexion reflex in the spinal cat, J. Neurosci., 6 (1986) 2921-2925. 11 Grau, J.W., Salinas, J.A., IUich, P.A. and Meagher, M.W., Associative learning and memory for an antinociceptive response in the spinalized rat, Behav. Neurosci., 104 (1990) 489494. 12 Ince, L.P., Brucker, B.S. and Alba, A., Reflex conditioning in a spinal man, J. Comp. Physiol. Psychol., 92 (1978) 796-802. 13 Irwin, S., Houde, R.W., Bennett, D.R., Hendershot, L.C. and Seevers, M.H., The effects of morphine, methadone and meperidine on some reflex responses of spinal animals to nociceptive stimulation, J. PharmacoL Exp. Ther., 101 (1951) 132143. 14 Lloyd, A.J., Wikler, A. and Whitehouse, J.M,, Nonconditionability of the flexor reflex in the chronic spinal dog, J. Comp. Physiol. Psychol., 68 (1969) 576-579.
15 MaCrae, J.R., Scoles, M.T. and Siegel, S., Fhe contribution of Pavlovian conditioning to drug tolerance and dependence, Br. J. Addict., 82 (1987) 371-380. 16 Patterson, M.M., Mechanisms of classical conditioning and fixation in spinal animals. In A.H. Riesen and R.F. Thompson (Eds.), Advances in Psychobiology, Vol. 3. Wiley. New York, 1976. pp. 381-436. 17 Sherman, B.S., Hoehler, EK. and Buerger, A.A., Instrumental avoidance conditioning of increased leg lowering in the spinal rat, Physiol. Behav., 25 (1982) 123-128. 18 Siuciak, J.A. and Advokat, C., Antinociceptive effect of intrathecal morphine in tolerant and nontolerant spinal rats, Pharmacol. Biochem. Behav., 34 (1989) 445-452. 19 Steinmetz, J.E., Cervenka, J., Robinson, C., Romano, A.G. and Patterson, M.M., Fixation of spinal reflexes in rats by central and peripheral sensory input, J. Comp. Physiol. Psychol., 95 (1981) 548-555. 20 Tallarida, R.J. and Murray, R.B., Manual of Pharmacologic Calculations with Computer Programs, Springer, New York, 1987. 21 Wolpaw, J.R. and Carp, J.S., Memory traces in spinal cord, Trends" Neurol. Sci., 13 (1990) 137-142.