Psychopharmacology (1992) 108:123-130
Psychopharmacology © Springer-Verlag 1992
Role of spinal serotoninl receptor subtypes in thermally and mechanically elicited nociceptive reflexes Anne Z. Murphy ~, R. Maureen Murphy 2, and Frank P. Zemlan 2 Alzheimer's Research Center, 1Department of Physiology, 2Department of Psychiatry, University of Cincinnati College of Medicine, 231 Bethesda Avenue, Cincinnati, OH 45267-0559, USA Received April 23, 1991 / Final version December 10, 1991
Abstract. The ability of 5-HT1A and 5-HT1B agonists to alter a spinal animal's nociceptive threshold was examined using two analgesiometric tests. In the spinal withdrawal reflex test, administration of the selective 5-HT1A agonists ipsapirone, gepirone and PAPP resulted in significant dose-dependent increases in receptive field (RF) area for withdrawal reflexes when compared to predrug baseline values, indicating an increase in nociceptive sensitivity. The average overall percent maximal increase in RF area following administration of 5-HT1A selective compounds was: 80+ 16% for the ventroflexion reflex, 904-6% for the dorsiflexion reflex and 874-8% for the lateral flexion reflex. Similar to the effects noted with 5-HT1A agonists, administration of 5-HTtB agonists RU24969, mCPP and TFMPP resulted in a hyperalgesic response with an overall percent maximal increase of 434-6% for the ventroflexion reflex, 514-6% for the dorsiflexion reflex and 384-9% for the lateral flexion reflex. In the tail-flick analgesiometric test, administration of the 5-HTzA agonists 8-OH-DPAT and ipsapirone and the 5-HT~B agonists RU24969 and mCPP resulted in a significant dose-dependent increase in tailflick latencies when compared to predrug baseline values, indicating a decrease in nociceptive sensitivity to noxious thermal stimuli. No differences in magnitude of the effect of the two receptor subtypes were found, indicating that stimulation of either 5-HT1A or 5-HT~B receptors was equipotent in producing the antinociceptive tail-flick response. Administration of the 5-HT1A antagonist metergoline completely reversed the increase in TFL produced by RU24969, providing further evidence that the effects seen here are mediated by spinal 5-HT receptors. The results of this study indicate that spinal 5-HT~ receptor subtypes may either facilitate or inhibit nociceptive input depending upon the type of nociceptor that is activated, as opposed to the type of receptor subtype that is stimulated. Key words: Serotonin- Spinal reflexes - N o c i c e p t i o n 5-HT1A - 5-HTtB - Spinal cord Offprint requests to: A.Z. Murphy
Several lines of evidence have shown that spinally projecting serotonergic neurons play an important role in the modulation of nociception. Serotonin intrathecally applied produces behaviorally defined analgesia in the rat, rabbit and cat, and is reversed by administration of the 5-HT antagonist methysergide (Yaksh and Wilson 1979). Iontophoretic application of 5-HT on the spinal cord inhibits dorsal horn neuronal responses to pinch (Randic and Yu 1976), and electrical stimulation of the NRM, which results in an increase in spinal cord 5-HT release, produces an inhibition of dorsal horn units responsive to noxious stimulation (Light et al. 1986). While the results of these studies appear to clearly implicate 5-HT as a major neurotransmitter involved in the descending analgesia system, it has been shown that depending upon the experimental procedures used, 5-HT may facilitate, have no effect or inhibit pain transmission. Several researchers have reported that changes in nociceptive thresholds following either 5-HT administration or lesioning of specific pathways are dependent upon the type of analgesiometric test being employed. York and Maynert (1978) found that following raphe lesions in rats, squeal thresholds, but not reflex responses to noxious stimulation, were increased. Fasmer et al. (1985) found that lesions of the descending 5-HT pathways produced the expected results of hyperalgesia in the tail flick test but produced the opposite effect of hypoalgesia in the formalin test. Eide et al. (1987) have also reported test-dependent changes in nociceptive threshold. They found that IP administration of metitepin, a 5-HT antagonist, resulted in hyperalgesia in the tail-flick test as expected, but induced dose-dependent increases in nociceptive threshold in the hot plate and formalin test. Several researchers have also shown that changes in nociceptive threshold are dependent upon the route of drug administration. Fasmer et al. (1984) found reduced response latencies following intrathecal injection of metergoline, a 5-HT antagonist, in both the tail flick and hot plate tests. However, increased response latencies for the hot plate were observed following intraperitoneal (IP) and intracerebroventricular (ICV) injections of the same drug.
124 While 5 - H T agonist administration generally results in behaviorally-defined analgesia in the intact animal, the opposite effect occurs in a spinal animal ( Z e m l a n et aI. 1983). Similar differences in spinal versus intact animals have also been reported. Berge et al. (1985) showed that 8 - O H - D P A T , a 5-HT~A agonist, had no effect on an intact animal's nociceptive threshold to radiant heat, while M u r p h y and Z e m l a n (1990) f o u n d t h a t this same agonist decreased a spinally transected animal's nociceptive threshold to noxious mechanical stimuli. Thus, 5 - H T m a y have a differential effect on c o m p l e x (supraspinal) versus reflex (spinal) responses to noxious stimulation. Several other researchers have confirmed the a b o v e findings that u n d e r varying experimental conditions, i.e. route o f administration and type o f noxious stimulus used, 5 - H T m a y facilitate, p r o d u c e no effect, or inhibit behaviorally-defined analgesia (Dennis and Melzack 1980; Berge 1982). Electrophysiological studies have also reported multiple effects o f 5 - H T on spinal processing o f nociceptive information. Anatomically, b o t h excitatory and inhibitory 5 - H T synapses have been identified in the dorsal h o r n ( R u d a and G o b e l 1980). While the action o f 5 - H T on dorsal h o r n wide d y n a m i c range neurons is primarily inhibitory, it was recently s h o w n that a p p r o x i m a t e l y 20-30% o f dorsal h o r n nociceptive n e u r o n s d e m o n s t r a t e an increase in unit activity following N R M stimulation or 5 - H T iontophoresis (Zemlan et al. 1988). Based u p o n receptor binding studies three subtypes o f 5 - H T receptors have been identified in the C N S : 5-HT~, 5 - H T 2 and 5-HT3. R e c e p t o r characterization studies have d e m o n s t r a t e d that the p r e d o m i n a n t class o f 5 - H T receptors in the spinal c o r d are o f the heterogeneous 5-HT~ type and it was recently d e m o n s t r a t e d that 5 - H T t A and 5-HT1B receptor subtypes a c c o u n t for approximately 50% o f the total [ a H ] 5 - H T binding sites in the rat spinal c o r d (Zemlan and Schwab 1991). W i t h the recent discovery o f multiple 5 - H T t receptor subtypes in the spinal cord, it is possible that the heterogeneous effects o f 5 - H T at the spinal level (hyperalgesia versus analgesia) m a y be mediated by particular 5-HT~ receptor subtype(s). The p u r p o s e o f the present study was to determine if 5 - H T ~ receptor subtypes differentially mediate spinal (reflex) responses to noxious stimulation. Specifically, we examined whether 5-HT1A and 5-HT~B agonists selectively alter a spinal animal's nociceptive threshold using two analgesiometric tests. The p u r p o s e o f spinal transection was t w o f o l d ; first, spinal transection frees dorsal h o r n neurons f r o m any descending inhibitory control. Second, spinal transection also causes retrograde degeneration o f terminals caudal to the lesion, thus removing the effect o f e n d o g e n o u s l y released 5 - H T on behavior. Therefore, only 5 - H T p h a r m a c o l o g i c a l l y introduced into the system in functionally relevant.
Materials and methods Animalpreparation. Male Sprague-Dawley rats, weighing between
250 and 300 g, were spinally transected at the tenth thoracic verte-
DORSIFLE×ION
I
Fig. 1. Receptive field areas of the three spinal cord nociceptive withdrawal reflexes elicited by noxious levels (> 400 mm Hg pressure) of mechanical stimulation
bra according to a procedure described by Zemlan et al. (1978). Briefly, animals were anesthetized with Equithesan (42.5 mg/ml choral hydrate and 9.72 mg/ml sodium pentobarbital dissolved in physiological saline) administered IP in a volume of 2.5 cc/kg body weight. The dorsal body surface of the rat was shaved and an incision made exposing the vertebral column. A laminectomy was performed at the tenth thoracic vertebra, exposing the dura mater and the spinal cord, and a 1-2 mm portion of the spinal cord was removed. The completeness of the transection was verified visually by cleaning the area between the two severed ends of the spinal cord and observing the tissue free vertebra. Animals were then housed in conventional rat cages, given free access to food and water, and maintained on a 12 : 12 h light/dark cycle. Because spinal transection disrupts normal bladder function, all animals were voided and bathed twice daily until normal urinary function returned. All animals were given a minimum of 2 days post-operative recovery time; animals that appeared unhealthy were sacrificed immediately. Animals were tested in either the flexor measurement or the tail-flick test, and each animal was tested a maximum of three times. Nocieeptive tests." flexor measurement. The behavioral endpoints
measured were the expansion or reduction of the receptive field (RF) areas of three spinal nociceptive withdrawal reflexes: (1) the vertebral column ventroflexion reflex, RF area located on tail base and immediately adjacent portion of the dorsal body surface; (2) the dorsiflexion reflex, RF area located on the dorsal body surface along the midline at the level of the iliac crest; and (3) the lateral flexion/tail deviation reflex, RF located on the entire lumbar dotsum off the midline (Zemlan et al. 1983). RF area locations are illustrated in Fig. 1. The size of the RF area was determined by repeated mechanical stimulation of the dorsal body surface with calibrated forceps. Noxious mechanical stimulation (noxious being defined as > 400 mm of mercury of pressure) of the same area had to elicit a withdrawal response three times in order to be included as part of the RF area. This procedure was continued until the perimeters of all three reflexes were determined. The sensitivity of the reflexes were then quantified as the size of the RF area in cmz. In order to ensure that changes in RF size were directly related to changes in nociceptive sensitivity, threshold measurements for the amount of pressure necessary to elicit the flexor reflexes were determined. In agreement with previous reports (Zemlan et al. 1983), we found that the size of the RF area was inversely related to the flexor reflex threshold. Thus, animals with a lower flexor reflex threshold have a concomitant increase in the size of the RF area. Based on preliminary experiments, quantifying the size of the RF area was found to be a more reliable, quicker measurement of
125 nociceptive sensitivity than threshold measurement, and was thus employed in these studies. Reflex measurement was conducted on days 3, 6, and 9 posttransection; no significant changes in recovery of function over this time period were noted. In order to generate dose-response curves, each animal was injected at 20 min intervals with either saline or increasing equimolar doses ofa 5-HT1A or 5-HTtB agonist, and the size of the RF area for all three spinal reflexes was quantified. Each test session consisted of one baseline measurement and four doses of drug or saline. Preliminary studies indicated that the 5-HT compounds used in this study affected RF areas for up to 2 h post-injection; all drug effects were totally absent 24 h post-injection. Animals were randomly assigned to one of the drug conditions (5-HT~A or 5-HT~B agonist, or saline) at the beginning of each test session. While some animals were tested on the same drug twice, no effects of tolerance were noted.
Tail-flick test. Animals were spinally transected as previously mentioned. Following a minimum often days post-transection, tail-flick latencies (TFL) were determined using a standard procedure initially described by D'Amour and Smith (1941). Briefly, the test is conducted by positioning the rat's tail over a slit in a metal plate and a beam of light is focused on a spot 1-2 cm from the caudal tip of the tail. The time elapsed from the onset of the thermal stimulus to reflex removal of the tail from the light source is defined as the tail-flick latency. Because the effect of these compounds on TFL was unknown, a low beam intensity was used which produced control response latencies between 4.5 and 5.0 s. To prevent excessive tissue damage, a maximum TFL of 10 s was employed. To increase the stability of the response, animals were handled in the test situation daily 1 week prior to actual testing and all animals were tested in the same room in which they were housed. Due to the nature of the test and the length of the intertrial interval, cumulative doses were not given. In order to conserve animals, several IP doses of the same drug were given to each animal. A minimum of 3 days was allowed between administration of different doses, and the doses were given in random order to control for interference between possible sequential effects and dose response. For the 5-HT~ agonist studies, animals were tested at 15 rain intervals and each session consisted of six trials. A baseline TFL was established for each rat as the mean of trials 1 through 3 ; all drugs were injected immediately following trial 3. Preliminary time-course studies for the compounds used indicated no significant mean differences in TFL between trials 4, 5 and 6; thus the mean latency for these trials was used as the TFL for that session. For the antagonist studies, the same procedure was used except that the test session consisted of a total of nine trials. The antagonist was injected immediately following trial 6 and the mean of trials 7, 8 and 9 was used as the TFL. Drugs and solutions. The 5-HT~A agonists were: 8-OH-DPAT [8-hydroxy-2-(di-n-propylamino)tetralin hydrobromide], Research Biochemicals; gepirone [(4,4-dimethyl-l-4-4-(2-pyrimidinyl)-lpiperazinyl butyl-2,6-piperidinedione hydrochloride], BristolMeyers; ipsapirone [(2-(4-(4-(2-pyrinidinyl)-l-piperazinyl) butyl1,2-benzisothiazol-3-(2H)one-1,1-dioxidehydrochloride], Troponwerke; PAPP [pNHz-PE-TFMPP], Dept of Human Resources. The 5-HTIB agonists were: mCPP [1-(3-chlorophenyl)piperazine HC1] and TFMPP [1-(m-triflouro-methylphenyl)piperazine], Aldrich Chemicals; RU24969 [5-methoxy 3(1,2,3,6-tetrahydro 4-pyridinyl)lH indole succinate neutre], Roussel-UCLAF. The 5-HT I antagonist was metergoline from Farmitalia. All drugs were dissolved in 0.9 % NaC1 (physiological saline) and were injected in volumes of 1 ml/kg. The metergoline solution contained 5.0 mg/ml ascorbic acid. All drugs had pH values between 3.5 and 7.0, and were made up fresh for each test session. All drugs were administered intraperitoneally (IP). In the flexor withdrawal studies, the 5-HTIA agonists used were gepirone, ipsapirone and PAPP. The 5-HT1B agonists used were
mCPP, TFMPP and RU24969. In the tail-flick studies, the 5-HTIA agonists used were 8-OH-DPAT and ipsapirone; mCPP and RU24969 were the 5-HT1B agonists used. The rationale for using 8-OH-DPAT in the tail-flick studies is that this agonist is one of the most selective 5-HT~A agonist available (Zemlan et al. 1990a, b). However, the results of this agonist on nociceptive sensitivity in the flexor withdrawal test have already been published by this laboratory (Murphy and Zemlan 1990), and it was thought to be more useful to examine other available 5-HT~A agonists. In preliminary studies, 8-OH-DPAT was tested in the flexor withdrawal test using the doses 0.0, 0.05, 0.20, 0.50 and 2.0 mg/kg, and the same pattern of results reported by Murphy and Zemlan were found (results not included).
Statistics. As previously stated, all RF areas were quantified in terms of cmz. Because baseline RF areas for all animals were variable, these values were normalized by converting them to percentage of maximal expansion using the formula: I postdrug R F - predrug RF ] x ~ ~ ~ ~ j 100 where maximum RF area is defined as those areas seen in a nondrug-treated animal 1 month after spinal transection. The maximum RF areas (cm 2) were: 7.065 for ventroflexion, 2.648 for dorsiflexion, and 18.135 for lateral flexion. To allow for direct comparisons between animals with different baseline TFLs, these values were transformed into percentage of basal TFL. This allowed for the construction of dose response curves which were normalized for differing predrug values. All statistical comparisons between baseline and dose were conducted using two-tailed t-tests for correlated measures. Due to the large number of a priori specified comparisons, all significance tests were analyzed using the Bonferroni adjusted degrees of freedom.
Results Flexor reflex studies A d m i n i s t r a t i o n o f the selective 5-HT1A a g o n i s t s g e p i r o n e , i p s a p i r o n e a n d P A P P (0.0, 0.05, 0.20, 0.50, 2.0 m g / k g ) resulted in significant d o s e - d e p e n d e n t increases in R F a r e a for all three spinal w i t h d r a w a l reflexes w h e n c o m p a r e d to p r e d r u g b a s e l i n e values (see Fig. 2). F o r e x a m p l e , as s h o w n in Fig. 2, a d m i n i s t r a t i o n o f g e p i r o n e resulted in a n e x p a n s i o n o f the v e n t r o f l e x i o n R F a r e a f r o m 36 4- 10% o f the m a x i m a l r e s p o n s e following the 0.05 m g / k g dose, t o 100 4- 0% after the 2.0 m g / k g dose. S i m i l a r effects were seen for the d o r s i f l e x i o n a n d the l a t e r a l flexion reflexes. F o r b o t h these reflexes, R F a r e a s were i n c r e a s e d to 31 4- 15% a n d 464- 10%, respectively, f o l l o w i n g the 0.05 m g / k g d o s e to a n overall e x p a n sion for b o t h reflexes o f 984- 2% o f t h e m a x i m u m a r e a f o l l o w i n g a d m i n i s t r a t i o n o f the 2.0 m g / k g dose. A d m i n i s t r a t i o n o f i p s a p i r o n e a n d P A P P also resulted in significant R F a r e a e x p a n s i o n s a c r o s s all t h r e e reflexes, similar to t h e effects o b t a i n e d w i t h g e p i r o n e . F o l l o w i n g i p s a p i r o n e a d m i n i s t r a t i o n , the overall p e r c e n t m a x i m a l e x p a n s i o n o f R F a r e a s for e a c h reflex w a s : v e n t r o f l e x i o n 91 4- 6%, d o r s i f l e x i o n 95 4- 2%, a n d l a t e r a l flexion 92 4- 6 %. T h e effect o f P A P P o n the overall p e r c e n t m a x i m a l e x p a n s i o n o f R F a r e a s for each reflex w a s : v e n t r o flexion 48 4- 5 %, d o r s i f l e x i o n 78 4- 5 % a n d l a t e r a l flexion 72 4- 5%.
126 VENTROFLEXION
o ~% z
Psychopharmacology © Springer-Verlag 1992
Role of spinal serotoninl receptor subtypes in thermally and mechanically elicited nociceptive reflexes Anne Z. Murphy ~, R. Maureen Murphy 2, and Frank P. Zemlan 2 Alzheimer's Research Center, 1Department of Physiology, 2Department of Psychiatry, University of Cincinnati College of Medicine, 231 Bethesda Avenue, Cincinnati, OH 45267-0559, USA Received April 23, 1991 / Final version December 10, 1991
Abstract. The ability of 5-HT1A and 5-HT1B agonists to alter a spinal animal's nociceptive threshold was examined using two analgesiometric tests. In the spinal withdrawal reflex test, administration of the selective 5-HT1A agonists ipsapirone, gepirone and PAPP resulted in significant dose-dependent increases in receptive field (RF) area for withdrawal reflexes when compared to predrug baseline values, indicating an increase in nociceptive sensitivity. The average overall percent maximal increase in RF area following administration of 5-HT1A selective compounds was: 80+ 16% for the ventroflexion reflex, 904-6% for the dorsiflexion reflex and 874-8% for the lateral flexion reflex. Similar to the effects noted with 5-HT1A agonists, administration of 5-HTtB agonists RU24969, mCPP and TFMPP resulted in a hyperalgesic response with an overall percent maximal increase of 434-6% for the ventroflexion reflex, 514-6% for the dorsiflexion reflex and 384-9% for the lateral flexion reflex. In the tail-flick analgesiometric test, administration of the 5-HTzA agonists 8-OH-DPAT and ipsapirone and the 5-HT~B agonists RU24969 and mCPP resulted in a significant dose-dependent increase in tailflick latencies when compared to predrug baseline values, indicating a decrease in nociceptive sensitivity to noxious thermal stimuli. No differences in magnitude of the effect of the two receptor subtypes were found, indicating that stimulation of either 5-HT1A or 5-HT~B receptors was equipotent in producing the antinociceptive tail-flick response. Administration of the 5-HT1A antagonist metergoline completely reversed the increase in TFL produced by RU24969, providing further evidence that the effects seen here are mediated by spinal 5-HT receptors. The results of this study indicate that spinal 5-HT~ receptor subtypes may either facilitate or inhibit nociceptive input depending upon the type of nociceptor that is activated, as opposed to the type of receptor subtype that is stimulated. Key words: Serotonin- Spinal reflexes - N o c i c e p t i o n 5-HT1A - 5-HTtB - Spinal cord Offprint requests to: A.Z. Murphy
Several lines of evidence have shown that spinally projecting serotonergic neurons play an important role in the modulation of nociception. Serotonin intrathecally applied produces behaviorally defined analgesia in the rat, rabbit and cat, and is reversed by administration of the 5-HT antagonist methysergide (Yaksh and Wilson 1979). Iontophoretic application of 5-HT on the spinal cord inhibits dorsal horn neuronal responses to pinch (Randic and Yu 1976), and electrical stimulation of the NRM, which results in an increase in spinal cord 5-HT release, produces an inhibition of dorsal horn units responsive to noxious stimulation (Light et al. 1986). While the results of these studies appear to clearly implicate 5-HT as a major neurotransmitter involved in the descending analgesia system, it has been shown that depending upon the experimental procedures used, 5-HT may facilitate, have no effect or inhibit pain transmission. Several researchers have reported that changes in nociceptive thresholds following either 5-HT administration or lesioning of specific pathways are dependent upon the type of analgesiometric test being employed. York and Maynert (1978) found that following raphe lesions in rats, squeal thresholds, but not reflex responses to noxious stimulation, were increased. Fasmer et al. (1985) found that lesions of the descending 5-HT pathways produced the expected results of hyperalgesia in the tail flick test but produced the opposite effect of hypoalgesia in the formalin test. Eide et al. (1987) have also reported test-dependent changes in nociceptive threshold. They found that IP administration of metitepin, a 5-HT antagonist, resulted in hyperalgesia in the tail-flick test as expected, but induced dose-dependent increases in nociceptive threshold in the hot plate and formalin test. Several researchers have also shown that changes in nociceptive threshold are dependent upon the route of drug administration. Fasmer et al. (1984) found reduced response latencies following intrathecal injection of metergoline, a 5-HT antagonist, in both the tail flick and hot plate tests. However, increased response latencies for the hot plate were observed following intraperitoneal (IP) and intracerebroventricular (ICV) injections of the same drug.
124 While 5 - H T agonist administration generally results in behaviorally-defined analgesia in the intact animal, the opposite effect occurs in a spinal animal ( Z e m l a n et aI. 1983). Similar differences in spinal versus intact animals have also been reported. Berge et al. (1985) showed that 8 - O H - D P A T , a 5-HT~A agonist, had no effect on an intact animal's nociceptive threshold to radiant heat, while M u r p h y and Z e m l a n (1990) f o u n d t h a t this same agonist decreased a spinally transected animal's nociceptive threshold to noxious mechanical stimuli. Thus, 5 - H T m a y have a differential effect on c o m p l e x (supraspinal) versus reflex (spinal) responses to noxious stimulation. Several other researchers have confirmed the a b o v e findings that u n d e r varying experimental conditions, i.e. route o f administration and type o f noxious stimulus used, 5 - H T m a y facilitate, p r o d u c e no effect, or inhibit behaviorally-defined analgesia (Dennis and Melzack 1980; Berge 1982). Electrophysiological studies have also reported multiple effects o f 5 - H T on spinal processing o f nociceptive information. Anatomically, b o t h excitatory and inhibitory 5 - H T synapses have been identified in the dorsal h o r n ( R u d a and G o b e l 1980). While the action o f 5 - H T on dorsal h o r n wide d y n a m i c range neurons is primarily inhibitory, it was recently s h o w n that a p p r o x i m a t e l y 20-30% o f dorsal h o r n nociceptive n e u r o n s d e m o n s t r a t e an increase in unit activity following N R M stimulation or 5 - H T iontophoresis (Zemlan et al. 1988). Based u p o n receptor binding studies three subtypes o f 5 - H T receptors have been identified in the C N S : 5-HT~, 5 - H T 2 and 5-HT3. R e c e p t o r characterization studies have d e m o n s t r a t e d that the p r e d o m i n a n t class o f 5 - H T receptors in the spinal c o r d are o f the heterogeneous 5-HT~ type and it was recently d e m o n s t r a t e d that 5 - H T t A and 5-HT1B receptor subtypes a c c o u n t for approximately 50% o f the total [ a H ] 5 - H T binding sites in the rat spinal c o r d (Zemlan and Schwab 1991). W i t h the recent discovery o f multiple 5 - H T t receptor subtypes in the spinal cord, it is possible that the heterogeneous effects o f 5 - H T at the spinal level (hyperalgesia versus analgesia) m a y be mediated by particular 5-HT~ receptor subtype(s). The p u r p o s e o f the present study was to determine if 5 - H T ~ receptor subtypes differentially mediate spinal (reflex) responses to noxious stimulation. Specifically, we examined whether 5-HT1A and 5-HT~B agonists selectively alter a spinal animal's nociceptive threshold using two analgesiometric tests. The p u r p o s e o f spinal transection was t w o f o l d ; first, spinal transection frees dorsal h o r n neurons f r o m any descending inhibitory control. Second, spinal transection also causes retrograde degeneration o f terminals caudal to the lesion, thus removing the effect o f e n d o g e n o u s l y released 5 - H T on behavior. Therefore, only 5 - H T p h a r m a c o l o g i c a l l y introduced into the system in functionally relevant.
Materials and methods Animalpreparation. Male Sprague-Dawley rats, weighing between
250 and 300 g, were spinally transected at the tenth thoracic verte-
DORSIFLE×ION
I
Fig. 1. Receptive field areas of the three spinal cord nociceptive withdrawal reflexes elicited by noxious levels (> 400 mm Hg pressure) of mechanical stimulation
bra according to a procedure described by Zemlan et al. (1978). Briefly, animals were anesthetized with Equithesan (42.5 mg/ml choral hydrate and 9.72 mg/ml sodium pentobarbital dissolved in physiological saline) administered IP in a volume of 2.5 cc/kg body weight. The dorsal body surface of the rat was shaved and an incision made exposing the vertebral column. A laminectomy was performed at the tenth thoracic vertebra, exposing the dura mater and the spinal cord, and a 1-2 mm portion of the spinal cord was removed. The completeness of the transection was verified visually by cleaning the area between the two severed ends of the spinal cord and observing the tissue free vertebra. Animals were then housed in conventional rat cages, given free access to food and water, and maintained on a 12 : 12 h light/dark cycle. Because spinal transection disrupts normal bladder function, all animals were voided and bathed twice daily until normal urinary function returned. All animals were given a minimum of 2 days post-operative recovery time; animals that appeared unhealthy were sacrificed immediately. Animals were tested in either the flexor measurement or the tail-flick test, and each animal was tested a maximum of three times. Nocieeptive tests." flexor measurement. The behavioral endpoints
measured were the expansion or reduction of the receptive field (RF) areas of three spinal nociceptive withdrawal reflexes: (1) the vertebral column ventroflexion reflex, RF area located on tail base and immediately adjacent portion of the dorsal body surface; (2) the dorsiflexion reflex, RF area located on the dorsal body surface along the midline at the level of the iliac crest; and (3) the lateral flexion/tail deviation reflex, RF located on the entire lumbar dotsum off the midline (Zemlan et al. 1983). RF area locations are illustrated in Fig. 1. The size of the RF area was determined by repeated mechanical stimulation of the dorsal body surface with calibrated forceps. Noxious mechanical stimulation (noxious being defined as > 400 mm of mercury of pressure) of the same area had to elicit a withdrawal response three times in order to be included as part of the RF area. This procedure was continued until the perimeters of all three reflexes were determined. The sensitivity of the reflexes were then quantified as the size of the RF area in cmz. In order to ensure that changes in RF size were directly related to changes in nociceptive sensitivity, threshold measurements for the amount of pressure necessary to elicit the flexor reflexes were determined. In agreement with previous reports (Zemlan et al. 1983), we found that the size of the RF area was inversely related to the flexor reflex threshold. Thus, animals with a lower flexor reflex threshold have a concomitant increase in the size of the RF area. Based on preliminary experiments, quantifying the size of the RF area was found to be a more reliable, quicker measurement of
125 nociceptive sensitivity than threshold measurement, and was thus employed in these studies. Reflex measurement was conducted on days 3, 6, and 9 posttransection; no significant changes in recovery of function over this time period were noted. In order to generate dose-response curves, each animal was injected at 20 min intervals with either saline or increasing equimolar doses ofa 5-HT1A or 5-HTtB agonist, and the size of the RF area for all three spinal reflexes was quantified. Each test session consisted of one baseline measurement and four doses of drug or saline. Preliminary studies indicated that the 5-HT compounds used in this study affected RF areas for up to 2 h post-injection; all drug effects were totally absent 24 h post-injection. Animals were randomly assigned to one of the drug conditions (5-HT~A or 5-HT~B agonist, or saline) at the beginning of each test session. While some animals were tested on the same drug twice, no effects of tolerance were noted.
Tail-flick test. Animals were spinally transected as previously mentioned. Following a minimum often days post-transection, tail-flick latencies (TFL) were determined using a standard procedure initially described by D'Amour and Smith (1941). Briefly, the test is conducted by positioning the rat's tail over a slit in a metal plate and a beam of light is focused on a spot 1-2 cm from the caudal tip of the tail. The time elapsed from the onset of the thermal stimulus to reflex removal of the tail from the light source is defined as the tail-flick latency. Because the effect of these compounds on TFL was unknown, a low beam intensity was used which produced control response latencies between 4.5 and 5.0 s. To prevent excessive tissue damage, a maximum TFL of 10 s was employed. To increase the stability of the response, animals were handled in the test situation daily 1 week prior to actual testing and all animals were tested in the same room in which they were housed. Due to the nature of the test and the length of the intertrial interval, cumulative doses were not given. In order to conserve animals, several IP doses of the same drug were given to each animal. A minimum of 3 days was allowed between administration of different doses, and the doses were given in random order to control for interference between possible sequential effects and dose response. For the 5-HT~ agonist studies, animals were tested at 15 rain intervals and each session consisted of six trials. A baseline TFL was established for each rat as the mean of trials 1 through 3 ; all drugs were injected immediately following trial 3. Preliminary time-course studies for the compounds used indicated no significant mean differences in TFL between trials 4, 5 and 6; thus the mean latency for these trials was used as the TFL for that session. For the antagonist studies, the same procedure was used except that the test session consisted of a total of nine trials. The antagonist was injected immediately following trial 6 and the mean of trials 7, 8 and 9 was used as the TFL. Drugs and solutions. The 5-HT~A agonists were: 8-OH-DPAT [8-hydroxy-2-(di-n-propylamino)tetralin hydrobromide], Research Biochemicals; gepirone [(4,4-dimethyl-l-4-4-(2-pyrimidinyl)-lpiperazinyl butyl-2,6-piperidinedione hydrochloride], BristolMeyers; ipsapirone [(2-(4-(4-(2-pyrinidinyl)-l-piperazinyl) butyl1,2-benzisothiazol-3-(2H)one-1,1-dioxidehydrochloride], Troponwerke; PAPP [pNHz-PE-TFMPP], Dept of Human Resources. The 5-HTIB agonists were: mCPP [1-(3-chlorophenyl)piperazine HC1] and TFMPP [1-(m-triflouro-methylphenyl)piperazine], Aldrich Chemicals; RU24969 [5-methoxy 3(1,2,3,6-tetrahydro 4-pyridinyl)lH indole succinate neutre], Roussel-UCLAF. The 5-HT I antagonist was metergoline from Farmitalia. All drugs were dissolved in 0.9 % NaC1 (physiological saline) and were injected in volumes of 1 ml/kg. The metergoline solution contained 5.0 mg/ml ascorbic acid. All drugs had pH values between 3.5 and 7.0, and were made up fresh for each test session. All drugs were administered intraperitoneally (IP). In the flexor withdrawal studies, the 5-HTIA agonists used were gepirone, ipsapirone and PAPP. The 5-HT1B agonists used were
mCPP, TFMPP and RU24969. In the tail-flick studies, the 5-HTIA agonists used were 8-OH-DPAT and ipsapirone; mCPP and RU24969 were the 5-HT1B agonists used. The rationale for using 8-OH-DPAT in the tail-flick studies is that this agonist is one of the most selective 5-HT~A agonist available (Zemlan et al. 1990a, b). However, the results of this agonist on nociceptive sensitivity in the flexor withdrawal test have already been published by this laboratory (Murphy and Zemlan 1990), and it was thought to be more useful to examine other available 5-HT~A agonists. In preliminary studies, 8-OH-DPAT was tested in the flexor withdrawal test using the doses 0.0, 0.05, 0.20, 0.50 and 2.0 mg/kg, and the same pattern of results reported by Murphy and Zemlan were found (results not included).
Statistics. As previously stated, all RF areas were quantified in terms of cmz. Because baseline RF areas for all animals were variable, these values were normalized by converting them to percentage of maximal expansion using the formula: I postdrug R F - predrug RF ] x ~ ~ ~ ~ j 100 where maximum RF area is defined as those areas seen in a nondrug-treated animal 1 month after spinal transection. The maximum RF areas (cm 2) were: 7.065 for ventroflexion, 2.648 for dorsiflexion, and 18.135 for lateral flexion. To allow for direct comparisons between animals with different baseline TFLs, these values were transformed into percentage of basal TFL. This allowed for the construction of dose response curves which were normalized for differing predrug values. All statistical comparisons between baseline and dose were conducted using two-tailed t-tests for correlated measures. Due to the large number of a priori specified comparisons, all significance tests were analyzed using the Bonferroni adjusted degrees of freedom.
Results Flexor reflex studies A d m i n i s t r a t i o n o f the selective 5-HT1A a g o n i s t s g e p i r o n e , i p s a p i r o n e a n d P A P P (0.0, 0.05, 0.20, 0.50, 2.0 m g / k g ) resulted in significant d o s e - d e p e n d e n t increases in R F a r e a for all three spinal w i t h d r a w a l reflexes w h e n c o m p a r e d to p r e d r u g b a s e l i n e values (see Fig. 2). F o r e x a m p l e , as s h o w n in Fig. 2, a d m i n i s t r a t i o n o f g e p i r o n e resulted in a n e x p a n s i o n o f the v e n t r o f l e x i o n R F a r e a f r o m 36 4- 10% o f the m a x i m a l r e s p o n s e following the 0.05 m g / k g dose, t o 100 4- 0% after the 2.0 m g / k g dose. S i m i l a r effects were seen for the d o r s i f l e x i o n a n d the l a t e r a l flexion reflexes. F o r b o t h these reflexes, R F a r e a s were i n c r e a s e d to 31 4- 15% a n d 464- 10%, respectively, f o l l o w i n g the 0.05 m g / k g d o s e to a n overall e x p a n sion for b o t h reflexes o f 984- 2% o f t h e m a x i m u m a r e a f o l l o w i n g a d m i n i s t r a t i o n o f the 2.0 m g / k g dose. A d m i n i s t r a t i o n o f i p s a p i r o n e a n d P A P P also resulted in significant R F a r e a e x p a n s i o n s a c r o s s all t h r e e reflexes, similar to t h e effects o b t a i n e d w i t h g e p i r o n e . F o l l o w i n g i p s a p i r o n e a d m i n i s t r a t i o n , the overall p e r c e n t m a x i m a l e x p a n s i o n o f R F a r e a s for e a c h reflex w a s : v e n t r o f l e x i o n 91 4- 6%, d o r s i f l e x i o n 95 4- 2%, a n d l a t e r a l flexion 92 4- 6 %. T h e effect o f P A P P o n the overall p e r c e n t m a x i m a l e x p a n s i o n o f R F a r e a s for each reflex w a s : v e n t r o flexion 48 4- 5 %, d o r s i f l e x i o n 78 4- 5 % a n d l a t e r a l flexion 72 4- 5%.
126 VENTROFLEXION
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