- Email: [email protected]
Analgesia induced by brief footshock is inhibited by 5-hydroxytryptamine but unaffected by antagonists of 5-hydroxytryptamine or by naloxone
Neuropharmacoh~#.' Vol. 21, pp. 51 to 56, 1982
0028-3908,82'010051-06503.00,0 Pergamon Press Ltd
Printed in Great Britain
ANALGESIA INDUCED BY BRIEF FOOTSHOCK IS INHIBITED BY 5-HYDROXYTRYPTAMINE BUT UNAFFECTED BY ANTAGONISTS OF 5-HYDROXYTRYPTAMINE OR BY NALOXONE M. D. T R I C K L E B A N K , P. H. HUTSONand G. CURZON Department of Neurochemistry, Institute of Neurology, 33 John's Mews, London WCI N 2NS, U.K. (Accepted 1 duh' 1981)
Summary Exposureto footshock (1 mA) for 30see induced a marked analgesia that was enhanced by pretreatment with the 5HT synthesis inhibitor, p-chlorophenylalanine, and attenuated by the 5HT releasing drugs p-chloroamphetamine and fenfluramine, by the 5HT re-uptake inhibitor, ftuoxetine and by the 5HT agonists, 5-methoxy-N,N-dimethyltryptamine and MK212. However, agonists, quipazine and triftuoromethylphenylpiperazine, with greater reported affinities for 5HT binding sites on rat brain membranes than MK212 were without effect as were the antagonists metergoline, methysergide, cyproheptadine, mianserine and methiothepin. The specific opioid antagonist naloxone was also without effect. The results in general indicate that analgesia induced by brief footshock (l mA, 30 sec) is inversely related to 5HT availability but there is little evidence of involvement of known 5HT receptors.
Responsiveness to noxious stimulation is decreased by enhancing the availability of central 5HT (Messing, Phebus, Fisher and Lytle, 1975; Yaksh and Wilson, 1979; Ogren and Holm, 1980) and by stimulating 5HT receptors (Samanin, Bernasconi and Quattrone, 1976) or areas of brain rich in 5HT neuronal cell bodies (Oliveras, Guilband and Besson, 1979; Satoh, Akinori, Takahiro and Takagi, 1980). Conversely, responsiveness is increased when the availability of 5HT is decreased by inhibiting 5HT synthesis (Tenen, 1967; Fibiger, Mertz and Campbell, 1972; Messing, Fisher, Phebus and Lytle, 1976) or lesioning 5HT tracts (Lints and Harvey, 1969). Responsiveness is decreased not only by increasing 5HT availability but also by prior exposure to stress such as that induced by electroshock, rotation, forced swimming, or injections of hypotonic saline or 2-deoxy-D-glucose (Madden, Akil, Patrick and Barchas, 1977; Hayes, Bennett, Newlon and Mayer, 1978; Bodnar, Kelly, Brutus and Glusman, 1980; Chance, 1980). Many investigators have studied the role of endogenous opioids in this behaviour but while some have found it to be attenuated by small doses of the specific opiate antagonist naloxone (Chesher and Chan, 1977; Amir and Amit, 1978; Willer and AlbeFessard, 1980) others have found this drug to be without effect or effective only with very large doses (Chance and Rosecrans, 1979; Hayes et al., 1978; Bodnar, Kelly, Spiaggia, Ehrenberg and Glusman, 1978; Millan, Przewlocki, Jerlicz, Gramsh, Hollt and Herz, 1981) or only against analgesia provoked by prolonged stress (Lewis, Cannon and Liebeskind, 1980). The term "analgesia" is used here in a corn-
monly accepted sense i.e. to indicate a decreased response to a noxious stimulus. In view of the above evidence, the effects of altered 5HT availability and naloxone on the analgesia induced by brief exposure to footshock were examined. The results show that even with large doses, naloxone did not diminish the analgesic response to footshock and that (in contrast to 5HT-dependent nociceptive mechanisms operating in the absence of prior stressful stimulation) the analgesia was enhanced by drugs which inhibit 5HT synthesis and attenuated by drugs which increase extraneuronal concentrations of 5HT. However, the evidence is largely against the involvement of 5HT receptors. METHODS
Animals Male Sprague-Dawley rats (mean body weight _+ SD 234 _+ 12 g) were obtained from Charles River Ltd, Margate, Kent, U.K. and fed ALGH standard rodent diet. They were housed singly for 8 9 days under a 12hr light dark (white red) cycle with white light commencing at 06.00 hr. Experiments were carried out between 14.00 and 17,00hr. Footshock Animals were placed in an aluminium box (240 × 210 × 90ram high) with a front wall of clear perspex. The roof was low enough to prevent the animals from minimising shock by rearing. The floor was a grid of 4 m m diameter stainless steel bars spaced 14mm between centres, through which shock of 1 mA intensity was delivered for 30sec via a constant current shock source with scrambler (6 periods of 5 sec, less than 1 sec intervals between shocks).
Key words: footshock, analgesia, naloxone, 5 hydroxytryptamine. 51
M.D. TRICKLEBANKet al.
52
Nociceptive testing
Assay of 5HT
Responses to noxious heat were monitored using a paw lick method. Animals were placed in a 51 glass beaker heated from below by water at 5 6 C and the time taken to lick the paws noted. In the experiment with p-chlorophenylalanine, a tail-flick procedure was also used in which the caudal 2 c m of the tail was immersed in a water bath at 51°C and the time taken to withdraw the tail noted. Tail-flick latencies were determined immediately before the paw-lick test. In both tests, latencies to respond were measured immediately before (L1) and immediately after (L2) 30sec exposure to footshock. Non-shocked controls were returned to the home cage for 30sec between determination of L1 and L2. Analgesic responses were expressed as the percentage analgesia score calculated from: (L2/L1) x 100. L1 values for control animals were usually about 20 sec. In determining the dose-response relationship for naloxone, the procedure was modified so that 3 pawlick determinations were made on each rat. After taking the initial latency (L1) the second (L2) was determined after returning the animal to its home cage for 30see. It was then exposed to footshock and the latency determined again (L3) immediately after termination of shock. The percentage analgesia score after footshock was then expressed as: L3/L2 × 100. The ratio: L2/LI × 100 provided a non-shocked control value. Preliminary experiments (to be reported in detail elsewhere) showed that L~, L2 and L3 did not differ significantly in the absence of footshock and that the analgesic effect of footshock (1 mA, 30 sec) was still significant (though smaller) 10 min after shock. Analgesic effects of exposure times from 10 to 120 sec were comparable but increasing the current to 2 mA caused increased analgesia. The effect was considerably greater between 14.00 and 16.00hr than between 8.00hr and 10.00hr and became less marked on repeated daily shock exposure.
In some experiments the animals were killed irnmediately after nociceptive testing and the brain, minus cerebellum, pons + medulla oblongata and olfactory lobes, taken for the determination of 5HT by the method of Curzon and Green (1970).
Statistics Statistical comparisons were made using Student's t-test.
Drugs The following drugs were kindly donated by their manufacturers: p-chloroamphetamine (Regis), fenfluramine (Servier), fluoxetine (Lilly), quipazine (Miles), methysergide (Sandoz), mianserine (Organon), methiothepin (Roche), metergoline (Farmitalia), naloxone (Endo), 6-chloro-2-11-piperazinyl]-pyrazine (MK2121 and cyproheptadine (Merck, Sharpe and Dohme). The remainder were purchased from the indicated companies: p-chlorophenylalanine methyl ester and 5-methoxy-N,N-dimethyhryptamine (Sigmal, trifluoromethylphenylpiperazine (Aldrich). Metergoline was dissolved in 1'~,, ascorbic acid. All other drugs were dissolved or suspended in 0.9'!, saline. Drugs were given intraperitoneally at l ml/kg body weight. RESt I~I'S
E~'ct qf footshock on po'centaqe analgesia .score Of non-drug treated rats In the absence of footshock, mean percentage analgesia scores of non-drug treated rats werc between 97 and 128"ii (paw-lick method: Tables 1, 2, 3t. The average of these mean values was 107. Thus, in the absence of intervening footshock, there was virtually no analgesic response to serial analgesia tests. Foolshock invariably significantly increased analgesia scores to mean values between 194 and 291'~.. The average of these means was 219. When the tail-flick
Table 1. Effect of p-chlorophenylalanine (PCPA) on shock-induced analgesia and brain 5HT concentration Percentage analgesia score No shock Shock
Analgesia test
A
5HT concentration I/~g,g)~
Saline
101.8 + 44.2
211.1 ± 54.9**
109
0.699 + 0.051
PCPA
97.3 + 23.8
292.8 +_ 95.4**+
195
0.132 + 0.021+
Saline
112.8 _+ 22.6
156.0+ 22.6*
43
PCPA
123.1 + 22.1
214.9 + 33.7**+t
92
Paw-lick
Tail-flick Values are mean + SD N = 9 10per group. PCPA (150 mg/kg) was given daily for 3 days and animals were tested 24 hi" after the last iniection. A = difference between shock and non-shock values. Significance of differences from non-shocked animals: *P < 0.01: **P < 0.001. Significance of differences from shocked animals given saline: +P < 0.05: q+P < 0.001. §5HT concentrations were determined after serial tail-flick and paw-lick determination.
5HT and footshock induced analgesia
53
Table 2. Effects of p-chloroamphetamine, fenfluramine and fluoxetine on shockinduced analgesia Percentage analgesia score No shock Shock
A
Saline
113.9 ± 27.0 (10)
204.8 _+ 52.4** (8)
91
p-chloroamphetamine ( 1.5 mg/kg, 30 min before test)
122.0 ± 36.3 (17)
155.3 ± 41.7"++ (17)
33
Saline
103.7 _+ 33.9 (10)
206.4 ±- 48.2** {9)
103
Fenfluramine (5 mg/kg, 30 min before test)
I15.1 ± 32.3 (9)
147.4 + 23.9++ (91
32
Saline
115.5 ± 51.0 (24)
194.7 _+ 71.5"* 1231
79
Fluoxetine ( 10 mg/kg, 120 min before test)
120.9 _+ 43.7 (14)
152.1 ± 57.7+ (15)
31
Values are means ± SD. N u m b e r of animals in parentheses. A = Difference between shock and non-shock values. Significance of differences fron non-shocked animals: *P < 0.02: **P < 0.001. Significance of differences from saline treated shocked animals: +P = 0.05: ++P < 0.01.
Table 3, Effects of 5-methoxy-N,N-dimethyltryptamine, MK212, trifluoromethylphenylpiperazine and quipazine on shock-induced analgesia Percentage analgesia score No shock Shock Saline
103.0 + 27.8 (10)
216.8 ± 44.3* (10)
114
5-Methoxy-N,N-dimethyltryptamine
116.2 ± 393 110)
150.8 ± 49.5+ (91
35
Saline
104.5 ± 33.4 (9)
209.5 _+ 73.8* ll0)
105
MK212 (2.5 mg/kg, 30 rain before test)
1I 1.0 +_ 35.3 (8)
130.3 -L-_38.2+ (11)
19
Saline
128.1 + 38.3 (10)
291.4 ± 88.0* (10)
163
Trifluoromethylphenylpiperazine ( 10 mg/kg, 120 mi n before test )
113.2 _+ 33.7 [9)
267.0 _+ 79.2* ( 101
I54
97.3 + 19.7 (101
219.0 + 49.0* (10)
i22
117.4 ± 55.1 (I 0j
225.9 -+__72.7* (10)
109
(2 mg/kg, 20 min before test)
Saline
Quipazine (2.5 mg/kg, 30 min before test)
Values are means ± SD N u m b e r of animals in parentheses. A = Difference between shock and non-shock values. Significance of differences from non-shocked animals: *P < 0.001. Significance of differences from saline treated shocked animals: +P < 0.01.
~'. 21 I
i~
A
54
M.D. TRICKLEBANKet al.
method was used (Table 1) the results were qualitatively similar though the increase of the analgesia score after footshock was smaller.
given at doses and times shown in Table 4, were without effect on the rise of percentage analgesia score after footshock.
E[]ect of inhibition of 5HT synthesis on percentage analgesia score after footshock
E~Ibct ~[ naloxone on percentaqe analgesia score ~!fier [botshock
Inhibition of 5HT synthesis with p-chlorophenylalanine (150mg/kg, 72, 48 and 24 hr before testing) decreased brain 5HT by 81"~,. The percentage analgesia score after footshock was significantly increased above its value for saline-treated rats whether the paw-lick or tail-flick methods were used (Table 1). In both cases the increases in scores after footshock were approximately doubled by p-chlorophenylalanine.
Naloxone (1, 5, 10, 20 mg/kg, 15 rain before testing} did not attenuate the rise in the percentage analgesia score after footshock (Table 5). On the contrary, the rises tended to be somewhat greater than those found for a concurrently studied control group though thc differences were not significant.
Ef[ect of increased extraneuronal al,ailahility oJ 5 H T on percentage analgesia score after Jbotshoek Thirty minutes after giving either p-chloroamphetamine (1.5mg/kg) or fenfluramine (5.0mg/kg) to enhance 5HT release (Trulson and Jacobs, 1976), the effect of footsh0ck on percentage analgesia score was significantly reduced (Table 2). Similar results were obtained with fluoxetine (10mg/kg), a specific 5HT re-uptake inhibitor (Fuller and Wong, 1977).
Effect of drugs on initial paw-lick or tail flick latencies None of the drugs significantly altered latenc~ to lick the paws on initial exposure to heat before footshock, except for p-chloroamphetamine which gave a latency of 27.7 _+ 11.2 sec (n = 35), which was significantly greater (p < 0.001) than the value of 21.4_+ 6.Ssec (n = 18) obtained for saline-trcatcd controls (values + SD). None of the drugs caused an obvious behavioural disturbance that may have intcrfered with the determination of paw-lick latencies.
Discussion Effect of 5HT agonists on percentage analgesia score after footshock
Analgesia induced by 30see footshock and measured by a paw-lick test was attenuated by drugs The percentage analgesia score after footshock was which increased extraneuronal 5HT i.e. the 5HT also significantly attenuated by the 5HT agonists releasing agents, p-chloroamphetamine and fenflur5-methoxy-N,N-dimethyltryptamine (2 mg/kg, 20 rain amine, and the 5HT reuptake inhibitor fluoxetine. before testing) and MK212 (2.5 mg/kg, 30min before Conversely, analgesia was enhanced when brain 5HT testing) (Table 3). However, two other agonists, quipa- was depleted by p-chlorophenylalanine. The effect of zine (2.5 mg/kg, 30rain before testing) and trifluoro- the latter drug was manifest not only when analgesia methylphenylpiperazine (10 mg/kg, 2 hr before testing) was measured by the paw-lick but also when the tailhad negligible effects on the rise of percentage analge- rick test was used. This suggests that 5HT is involved sia score after footshock. in the refex response to noxious stimuli and not merely in the locomotor component of the paw-lick E~bct oJ" 5HT antagonists on percentage analgesia test. score after jbotshock The results strongly indicate that analgesia induced A series of 5HT antagonists (methysergide, meter- by brief footshock is inversely related to 5HT awfilgoline, cyproheptadine, methiothepin, mianserine), ability or release. They contrast with the positive relationship found by others between 5HT and nociceptive thresholds determined without prior stressful Table 4. Antagonists of 5HT without significant effect on stimulation (see introduction for references). They also contrast with findings on the role of 5HT in analshock-induced analgesia gesia resulting from prolonged electroshock or immoDose Time bilization, which was attenuated by 5HT antagonists (mg/kg) before testing P* or synthesis inhibitors, or enhanced by administration of the 5HT precursor 5-hydroxytryptophan (BhattaMethysergide 2.0 2 hr > 0.2 charya, Keshary and Sanyal, 1978: Kulkarni, 1980: Metergoline 1.0 2 hr > 0.2 Shimizu, Koja, Fujisaki and Fukuda, 1981). Another Metergoline 5.0 2 hr > 0.1 difference between the present results and those of Metergoline 5.0 30 min > 0.5 Kulkarni (1980) and of Shimizu et al. (1981) is that the analgesic effect of brief footshock was not opposed by Cyproheptadine 10.0 20 min > 0.2 naloxone (1 20mg/kg, Table 5) but these authors Methiothepin 2.5 30 rain > 0.5 found that the drug was effective at 1 mg/kg against analgesia induced by more prolonged stresses. Mianserine 2.5 30 min > 0.5 The above differences, and the report by Lewis et * V. analgesia scores of vehicle-treated animals exposed ul. (1980) that naloxone antagonised thc analgesic to footshock. effect of prolonged intermittent footshock but not
5HT and footshock induced analgesia Table 5. Effect of naloxone on shock induced analgesia Percentage analgesia score Non-shock Shock
A
Saline
102.3 + 36.4
219.6 + 67.7
117
Naloxone 1 mg/kg 5 mg/kg 10 mg/kg 20mg/kg
77.7 102.8 93.1 98.1
244.9 228.1 246.7 284.5
167 126 154 187
+ + + +
20.9 28.6 29.6 17.5
± + + +
81.4 33.9 62.5 78.3
Values are mean ± SD N = 6 animals per group. A = Difference between shock and non-shock values.
55
is poor in view of the lack of effect of trifluoromethylphenylpiperazine, which is a powerful agonist (Fuller and Clemens, 1979; Fuller et al., 1980) and the uncertain interpretation of the effects of 5-methoxy-N,Ndimethyltryptamine and MK212. Furthermore, metergoline (inactive at both doses and times used) is equipotent at both receptor types (Peroutka, Lebovitz and Snyder, 1981). These findings thus raise the possibility of mechanisms dependent on a third type of central 5HT receptor or one not involving 5HT receptors at all. Acknowled,qements We thank the MRC for financial support and Mr R. Scraggs for technical assistance.
that of brief continuous footshock, suggests two distinct mechanisms for stress-induced analgesia i.e. an acute non-opioid mechanism depending inversely on 5HT availability, and a chronic opioid mechanism depending directly on 5HT availability. Opposing 5HT-dependent analgesic mechanisms are also indicated by the finding by Berge, Hole and Dahle (1980) that the 5HT agonist 5-methoxy-N,N-dimethyltryptamine caused increased and decreased nociception at low and high dose levels respectively. Interaction between such mechanisms and various reported effects of stress on brain 5HT (Curzon and Green, 1971; Palkovits, Brownstein, Kizer, Saavedra and Kopin, 1976; Kennett and Joseph, 1981) implies that relationships between brain 5HT and stress-provoked analgesia may vary considerably with conditions. A striking feature of the present results is that shock-provoked analgesia was affected by drugs which alter 5HT availability (Tables 1, 2) and by lesioning of spinal 5HT neurones (Hutson, Tricklebank and Curzon, 1982) but not by 5HT antagonists (Table 4) or by the 5HT agonists, trifluoromethylphenylpiperazine and quipazine (Table 3). Although the response was attenuated by the agonist, 5-methoxyN,N-dimethyltryptamine, this drug is structurally similar to 5HT and hence may well have a similar spectrum of biological activity. The response was also decreased by the agonist MK212 but this drug may also act on other transmitter systems such as dopamine (Clineschmidt, 1979) and we have recently shown that dopamine (like 5HT) opposes footshockinduced analgesia (Curzon, Hutson and Tricklebank, 1982). Furthermore, MK212 is probably a weaker 5HT agonist than the two structurally related agonists which did not affect the analgesic response (see above) as it is less potent as a displacer of 3H-5HT from rat brain membranes in vitro (Fuller, Mason and Molloy, 1980). Therefore, the drug experiments as a whole suggest that the action of 5HT on shock-provoked analgesi a does not involve known 5HT receptors. Evidence against 5HTz receptors is particularly strong in view of the lack of effect of all five antagonists tested as these receptors are usually more sensitive to antagonists than to agonists (Peroutka and Snyder, 1979) while evidence for the involvement of 5HT1 receptors
REFERENCES
Amir, S. and Amit, Z. (1978). Endogenous opioid ligands may mediate stress-induced changes in the affective properties of pain related behaviour in rats. Life Sci. 23: 1143 1152. Berge, O. G., Hole, K. and Dahle, H. (1980). Nociception is enhanced after low doses and reduced after high doses of the receptor agonist 5-methoxy-N,N-dimethyltryptamine. Neurosci. Lett. 19: 219-223. Bhattacharya, S. K., Keshary, P. R. and Sanyal, A. K. (19781. Immobilization stress induced antinociception in rats: possible role of serotonin and prostaglandins Eur. J. Pharmacol. 50: 83-85. Bodnar, R. J., Kelly, D. D., Spiaggia, A., Ehrenberg, C. and Glusman, M. (19781. Dose-dependent reductions by naloxone of analgesia induced by cold-water stress. Pharmac. Biochem. Behae. 8:667 672. Bodnar, R. J., Kelly, D. D., Brutus, M. and Glusman, M. (1980). Stress-induced analgesia: neural and hormonal determinants. Neurosci. Biobehav. Rev. 4: 87-100. Chance, W. T. (1980). Autoanalgesia: opiate and nonopiate mechanisms. Neurosci. Biobehav. Rev. 4:55 68. Chance, W. T. and Rosecrans, J. A. (1979). Lack of effect of naloxone on autoanalgesia. Pharmac. Biochem. Behav. 11 : 643 646. Chesher, G. B. and Chan, B. (1977). Footshock induced analgesia in mice: its reversal by naloxone and cross tolerance with morphine. Life Sci. 21" 1569-1574. Clineschmidt, B. V. (1979). MK212: A serotonin-like agonist in the CNS. Gen. Pharmac. 10:287 290. Curzon, G. and Green, W. R. (1970). Rapid method for the determination of 5-hydroxytryptamine and 5-hydroxyindoleacetic acid in small regions of rat brain. Br. J. Pharmac. 39:653 655. Curzon, G. and Green, A. R. (1971). Regional and subcellular changes in the concentration of 5-hydroxytryptamine and 5-hydroxyindoleacetic acid in the rat brain caused by hydrocortisone, DL-~-methyl-tryptophan, L-kynurenine and immobilisation. Br. J. Pharmac. 43:39 52. Curzon, G., Hutson, P. H. and Tricklebank, M. D. (1982). Involvement of dopamine in the analgesic response induced by footshock. Br. J. Pharmac. In press. Fibiger, H. C., Mertz, P. H. and Campbell, B. A. (1972). The effect of para-chlorophenylalanine on aversion thresholds and reactivity to foot shock. Physiol. Behav. 8:259 263. Fuller, R. L. and Wong, D. T. (1977). Inhibition of serotonin reuptake. Fedn Proc. Fedn Am. Socs exp. Biol. 36: 2154-2158. Fuller, R. W. and Clemens, J. A. (1979). 1-(m-Trifluoromethylphenyl)-piperazine, a serotonin agonist, increases serum corticosterone and prolactin concentration in rats. I R C S Med. Sci. 7: 106.
56
M . D . TRICKLI!BANK t'g ol.
Fuller, R. W.. Mason, N. R. and Molloy, B. B. (1980). Structural relationships in the inhibition of ['~H] serotonin binding to rat brain membranes in citro by 1-phenyl-piperazines. Biochem. Pharnlac. 29:833 835. Hayes, R. L., Bennett, G. J., Newlon, P. G. and Mayer, D. J. (1978}. Behavionral and physiological studies of non-narcotic analgesia in the rat elicited by certain environmental stimuli. Brain Re~. 155:69 90. Hutson, P. H., Tricklebank, M. D. and Curzon, G. (19821. Enhancement of footshock-induced analgesia by spinal 5,7-dihydroxytryptamine lesions. Brain Res. In press. Kennett, G. A. and Joseph, M. H. (1981). The functional importance of increased brain tryptophan in the serotonergic response to restraint stress. Neurc~pharmac. 20: 39 43. Kulkarni, S. K. (1980). Heat and other physiological stressinduced analgesia: catccholamine mediated and naloxone reversible response. L(I~" Sci. 27:185 188. Lewis, J. W.. Cannon, J. T. and Licbeskind, J. C. (1980). Opioid and non-opioid mechanisms of stress analgesia. Science 208:623 625. Lints, C. E. and Harvey, J. A. (1969). Drug induced reversal of brain damage in the rat. Physiol. Behac. 4:29 31. Madden, J., Akil, H., Patrick, R. L. and Barchas, J. D. (1977). Stress-induced parallel changes in central opioid levels and pain responsiveness in the rat. Nature, Lond 265:358 360. Messing, R. B., Fisher, L. A., Phebus, L. and Lytle, L. D. (1976). Interaction of diet and drugs in the regulation of brain 5-hydroxyindoles and the response to painful electric shock. Lili, Sci. 18:707 714. Messing, R. B., Phebus, L., Fisher, L. A. and Lytle, L. D. (1975). Analgesic effect of fluoxetine hydrochloride (Lilly 1101401 a specific inhibitor of serotonin uptake. Psychophurmac. Comm. 1:511 521. Millan, M. J., Przewlocki, R., Jerlicz, M., Gramsch, C. H., Hollt, V. and Herz, A. (1981). Stress-induced release of brain and pituitary-endorphin: major role of endorphins in generation of hyperthermia, not analgesia. Bruin Res. 208:325 338. Ogren, S. O. and Holm. A.-C. (1980). Test-specific effects of the 5HT rcuptake inhibitors alaproclate and zimelidine on pain sensitivity and morphine analgesia. J. Nem'al Trans. 47:253 271.
Oliveras, J. L., Guilband, G. and Besson, J. M. 11979). A map of serotoninergic structures involved in stimulation producing analgesia in unrestrained freely moving cats. Brain Res. 164:317 322. Palkovits. M., Brownstein, M., Kizer, J. S., Saavedra, J. M. and Kopin, h J. (1976). Effects of stress on serotonin and tryptophan hydroxylasc activity of brain nuclei. In: Catecholammes aml Stre.~s (Usdin, E.. Kvctnansky, R. and Kopin, I. J., Eds). pp. 51 88. Pergamon Press, Oxford, Peroutka, S. J., Lebovitz, R. M. and Snyder, S. H. (1981). Two distinct central serotonin receptors with different physiological functions. Sciem'e 212:827 828. Peroutka. S. J. and Snyder. S. H. 11979}. Multiple serotonm receptors: differential binding of [3H] 5-hydroxytryptamine, [~H] Lysergic acid diethylamide and [~H] spiroperidol, Moh'c. Phurmac. 16:687 699. Samanin, R.. Bernasconi. S, and Quattrone, A. (1976). Antinociceptive action of quipazine: relation to central serotonergic receptor stimulation. Psychopharmuc. 46: 219 222. Satoh. M., Akinori, A., Takahirn, N. and Takagi, H. 11980). Evidence for involvement of separate mechanisms in the production of analgesia by electrical stimulation of the nncleus paragigantocellularis and nucleus raphe magnus in the rat. Bruin Res. 194:525 529. Shimizu. T., Koja, T.. Fnjisaki, T. and Fukuda, T. (1981). Effects of methysergide and naloxone on analgesia induced by the peripheral electrical stimulation in mice. Brain Re.~. 208:463 467. Tenen, S. S. 11967). The effects of/>chlorophenylalanine, a scrotonin depleter, on avoidance acquisition, pain sensitivity and behaviour in the rat. Ps.vchophurmucoh~fia, Berlin 10:204 219. Trulson, M. E. and Jacobs, B. L. (1976). Behavioural evidence for the rapid release of CNS serotonin by PCA and fenfluramine. Era'. J. Pharnluc. 36:149 154. Wilier, J. C, and Albe-Fessard, D. {19801. Electrophysiological evidence for a release of endogenous opiates in stress-induced "analgesia' in man. Bruill Res. 198: 419 426. Yaksh. T. L. and Wilson, P. R. (19791. Spinal serotonm terminal system mediates antmociception. J. Pharmac. exp. Ther. 208:446 453.
0028-3908,82'010051-06503.00,0 Pergamon Press Ltd
Printed in Great Britain
ANALGESIA INDUCED BY BRIEF FOOTSHOCK IS INHIBITED BY 5-HYDROXYTRYPTAMINE BUT UNAFFECTED BY ANTAGONISTS OF 5-HYDROXYTRYPTAMINE OR BY NALOXONE M. D. T R I C K L E B A N K , P. H. HUTSONand G. CURZON Department of Neurochemistry, Institute of Neurology, 33 John's Mews, London WCI N 2NS, U.K. (Accepted 1 duh' 1981)
Summary Exposureto footshock (1 mA) for 30see induced a marked analgesia that was enhanced by pretreatment with the 5HT synthesis inhibitor, p-chlorophenylalanine, and attenuated by the 5HT releasing drugs p-chloroamphetamine and fenfluramine, by the 5HT re-uptake inhibitor, ftuoxetine and by the 5HT agonists, 5-methoxy-N,N-dimethyltryptamine and MK212. However, agonists, quipazine and triftuoromethylphenylpiperazine, with greater reported affinities for 5HT binding sites on rat brain membranes than MK212 were without effect as were the antagonists metergoline, methysergide, cyproheptadine, mianserine and methiothepin. The specific opioid antagonist naloxone was also without effect. The results in general indicate that analgesia induced by brief footshock (l mA, 30 sec) is inversely related to 5HT availability but there is little evidence of involvement of known 5HT receptors.
Responsiveness to noxious stimulation is decreased by enhancing the availability of central 5HT (Messing, Phebus, Fisher and Lytle, 1975; Yaksh and Wilson, 1979; Ogren and Holm, 1980) and by stimulating 5HT receptors (Samanin, Bernasconi and Quattrone, 1976) or areas of brain rich in 5HT neuronal cell bodies (Oliveras, Guilband and Besson, 1979; Satoh, Akinori, Takahiro and Takagi, 1980). Conversely, responsiveness is increased when the availability of 5HT is decreased by inhibiting 5HT synthesis (Tenen, 1967; Fibiger, Mertz and Campbell, 1972; Messing, Fisher, Phebus and Lytle, 1976) or lesioning 5HT tracts (Lints and Harvey, 1969). Responsiveness is decreased not only by increasing 5HT availability but also by prior exposure to stress such as that induced by electroshock, rotation, forced swimming, or injections of hypotonic saline or 2-deoxy-D-glucose (Madden, Akil, Patrick and Barchas, 1977; Hayes, Bennett, Newlon and Mayer, 1978; Bodnar, Kelly, Brutus and Glusman, 1980; Chance, 1980). Many investigators have studied the role of endogenous opioids in this behaviour but while some have found it to be attenuated by small doses of the specific opiate antagonist naloxone (Chesher and Chan, 1977; Amir and Amit, 1978; Willer and AlbeFessard, 1980) others have found this drug to be without effect or effective only with very large doses (Chance and Rosecrans, 1979; Hayes et al., 1978; Bodnar, Kelly, Spiaggia, Ehrenberg and Glusman, 1978; Millan, Przewlocki, Jerlicz, Gramsh, Hollt and Herz, 1981) or only against analgesia provoked by prolonged stress (Lewis, Cannon and Liebeskind, 1980). The term "analgesia" is used here in a corn-
monly accepted sense i.e. to indicate a decreased response to a noxious stimulus. In view of the above evidence, the effects of altered 5HT availability and naloxone on the analgesia induced by brief exposure to footshock were examined. The results show that even with large doses, naloxone did not diminish the analgesic response to footshock and that (in contrast to 5HT-dependent nociceptive mechanisms operating in the absence of prior stressful stimulation) the analgesia was enhanced by drugs which inhibit 5HT synthesis and attenuated by drugs which increase extraneuronal concentrations of 5HT. However, the evidence is largely against the involvement of 5HT receptors. METHODS
Animals Male Sprague-Dawley rats (mean body weight _+ SD 234 _+ 12 g) were obtained from Charles River Ltd, Margate, Kent, U.K. and fed ALGH standard rodent diet. They were housed singly for 8 9 days under a 12hr light dark (white red) cycle with white light commencing at 06.00 hr. Experiments were carried out between 14.00 and 17,00hr. Footshock Animals were placed in an aluminium box (240 × 210 × 90ram high) with a front wall of clear perspex. The roof was low enough to prevent the animals from minimising shock by rearing. The floor was a grid of 4 m m diameter stainless steel bars spaced 14mm between centres, through which shock of 1 mA intensity was delivered for 30sec via a constant current shock source with scrambler (6 periods of 5 sec, less than 1 sec intervals between shocks).
Key words: footshock, analgesia, naloxone, 5 hydroxytryptamine. 51
M.D. TRICKLEBANKet al.
52
Nociceptive testing
Assay of 5HT
Responses to noxious heat were monitored using a paw lick method. Animals were placed in a 51 glass beaker heated from below by water at 5 6 C and the time taken to lick the paws noted. In the experiment with p-chlorophenylalanine, a tail-flick procedure was also used in which the caudal 2 c m of the tail was immersed in a water bath at 51°C and the time taken to withdraw the tail noted. Tail-flick latencies were determined immediately before the paw-lick test. In both tests, latencies to respond were measured immediately before (L1) and immediately after (L2) 30sec exposure to footshock. Non-shocked controls were returned to the home cage for 30sec between determination of L1 and L2. Analgesic responses were expressed as the percentage analgesia score calculated from: (L2/L1) x 100. L1 values for control animals were usually about 20 sec. In determining the dose-response relationship for naloxone, the procedure was modified so that 3 pawlick determinations were made on each rat. After taking the initial latency (L1) the second (L2) was determined after returning the animal to its home cage for 30see. It was then exposed to footshock and the latency determined again (L3) immediately after termination of shock. The percentage analgesia score after footshock was then expressed as: L3/L2 × 100. The ratio: L2/LI × 100 provided a non-shocked control value. Preliminary experiments (to be reported in detail elsewhere) showed that L~, L2 and L3 did not differ significantly in the absence of footshock and that the analgesic effect of footshock (1 mA, 30 sec) was still significant (though smaller) 10 min after shock. Analgesic effects of exposure times from 10 to 120 sec were comparable but increasing the current to 2 mA caused increased analgesia. The effect was considerably greater between 14.00 and 16.00hr than between 8.00hr and 10.00hr and became less marked on repeated daily shock exposure.
In some experiments the animals were killed irnmediately after nociceptive testing and the brain, minus cerebellum, pons + medulla oblongata and olfactory lobes, taken for the determination of 5HT by the method of Curzon and Green (1970).
Statistics Statistical comparisons were made using Student's t-test.
Drugs The following drugs were kindly donated by their manufacturers: p-chloroamphetamine (Regis), fenfluramine (Servier), fluoxetine (Lilly), quipazine (Miles), methysergide (Sandoz), mianserine (Organon), methiothepin (Roche), metergoline (Farmitalia), naloxone (Endo), 6-chloro-2-11-piperazinyl]-pyrazine (MK2121 and cyproheptadine (Merck, Sharpe and Dohme). The remainder were purchased from the indicated companies: p-chlorophenylalanine methyl ester and 5-methoxy-N,N-dimethyhryptamine (Sigmal, trifluoromethylphenylpiperazine (Aldrich). Metergoline was dissolved in 1'~,, ascorbic acid. All other drugs were dissolved or suspended in 0.9'!, saline. Drugs were given intraperitoneally at l ml/kg body weight. RESt I~I'S
E~'ct qf footshock on po'centaqe analgesia .score Of non-drug treated rats In the absence of footshock, mean percentage analgesia scores of non-drug treated rats werc between 97 and 128"ii (paw-lick method: Tables 1, 2, 3t. The average of these mean values was 107. Thus, in the absence of intervening footshock, there was virtually no analgesic response to serial analgesia tests. Foolshock invariably significantly increased analgesia scores to mean values between 194 and 291'~.. The average of these means was 219. When the tail-flick
Table 1. Effect of p-chlorophenylalanine (PCPA) on shock-induced analgesia and brain 5HT concentration Percentage analgesia score No shock Shock
Analgesia test
A
5HT concentration I/~g,g)~
Saline
101.8 + 44.2
211.1 ± 54.9**
109
0.699 + 0.051
PCPA
97.3 + 23.8
292.8 +_ 95.4**+
195
0.132 + 0.021+
Saline
112.8 _+ 22.6
156.0+ 22.6*
43
PCPA
123.1 + 22.1
214.9 + 33.7**+t
92
Paw-lick
Tail-flick Values are mean + SD N = 9 10per group. PCPA (150 mg/kg) was given daily for 3 days and animals were tested 24 hi" after the last iniection. A = difference between shock and non-shock values. Significance of differences from non-shocked animals: *P < 0.01: **P < 0.001. Significance of differences from shocked animals given saline: +P < 0.05: q+P < 0.001. §5HT concentrations were determined after serial tail-flick and paw-lick determination.
5HT and footshock induced analgesia
53
Table 2. Effects of p-chloroamphetamine, fenfluramine and fluoxetine on shockinduced analgesia Percentage analgesia score No shock Shock
A
Saline
113.9 ± 27.0 (10)
204.8 _+ 52.4** (8)
91
p-chloroamphetamine ( 1.5 mg/kg, 30 min before test)
122.0 ± 36.3 (17)
155.3 ± 41.7"++ (17)
33
Saline
103.7 _+ 33.9 (10)
206.4 ±- 48.2** {9)
103
Fenfluramine (5 mg/kg, 30 min before test)
I15.1 ± 32.3 (9)
147.4 + 23.9++ (91
32
Saline
115.5 ± 51.0 (24)
194.7 _+ 71.5"* 1231
79
Fluoxetine ( 10 mg/kg, 120 min before test)
120.9 _+ 43.7 (14)
152.1 ± 57.7+ (15)
31
Values are means ± SD. N u m b e r of animals in parentheses. A = Difference between shock and non-shock values. Significance of differences fron non-shocked animals: *P < 0.02: **P < 0.001. Significance of differences from saline treated shocked animals: +P = 0.05: ++P < 0.01.
Table 3, Effects of 5-methoxy-N,N-dimethyltryptamine, MK212, trifluoromethylphenylpiperazine and quipazine on shock-induced analgesia Percentage analgesia score No shock Shock Saline
103.0 + 27.8 (10)
216.8 ± 44.3* (10)
114
5-Methoxy-N,N-dimethyltryptamine
116.2 ± 393 110)
150.8 ± 49.5+ (91
35
Saline
104.5 ± 33.4 (9)
209.5 _+ 73.8* ll0)
105
MK212 (2.5 mg/kg, 30 rain before test)
1I 1.0 +_ 35.3 (8)
130.3 -L-_38.2+ (11)
19
Saline
128.1 + 38.3 (10)
291.4 ± 88.0* (10)
163
Trifluoromethylphenylpiperazine ( 10 mg/kg, 120 mi n before test )
113.2 _+ 33.7 [9)
267.0 _+ 79.2* ( 101
I54
97.3 + 19.7 (101
219.0 + 49.0* (10)
i22
117.4 ± 55.1 (I 0j
225.9 -+__72.7* (10)
109
(2 mg/kg, 20 min before test)
Saline
Quipazine (2.5 mg/kg, 30 min before test)
Values are means ± SD N u m b e r of animals in parentheses. A = Difference between shock and non-shock values. Significance of differences from non-shocked animals: *P < 0.001. Significance of differences from saline treated shocked animals: +P < 0.01.
~'. 21 I
i~
A
54
M.D. TRICKLEBANKet al.
method was used (Table 1) the results were qualitatively similar though the increase of the analgesia score after footshock was smaller.
given at doses and times shown in Table 4, were without effect on the rise of percentage analgesia score after footshock.
E[]ect of inhibition of 5HT synthesis on percentage analgesia score after footshock
E~Ibct ~[ naloxone on percentaqe analgesia score ~!fier [botshock
Inhibition of 5HT synthesis with p-chlorophenylalanine (150mg/kg, 72, 48 and 24 hr before testing) decreased brain 5HT by 81"~,. The percentage analgesia score after footshock was significantly increased above its value for saline-treated rats whether the paw-lick or tail-flick methods were used (Table 1). In both cases the increases in scores after footshock were approximately doubled by p-chlorophenylalanine.
Naloxone (1, 5, 10, 20 mg/kg, 15 rain before testing} did not attenuate the rise in the percentage analgesia score after footshock (Table 5). On the contrary, the rises tended to be somewhat greater than those found for a concurrently studied control group though thc differences were not significant.
Ef[ect of increased extraneuronal al,ailahility oJ 5 H T on percentage analgesia score after Jbotshoek Thirty minutes after giving either p-chloroamphetamine (1.5mg/kg) or fenfluramine (5.0mg/kg) to enhance 5HT release (Trulson and Jacobs, 1976), the effect of footsh0ck on percentage analgesia score was significantly reduced (Table 2). Similar results were obtained with fluoxetine (10mg/kg), a specific 5HT re-uptake inhibitor (Fuller and Wong, 1977).
Effect of drugs on initial paw-lick or tail flick latencies None of the drugs significantly altered latenc~ to lick the paws on initial exposure to heat before footshock, except for p-chloroamphetamine which gave a latency of 27.7 _+ 11.2 sec (n = 35), which was significantly greater (p < 0.001) than the value of 21.4_+ 6.Ssec (n = 18) obtained for saline-trcatcd controls (values + SD). None of the drugs caused an obvious behavioural disturbance that may have intcrfered with the determination of paw-lick latencies.
Discussion Effect of 5HT agonists on percentage analgesia score after footshock
Analgesia induced by 30see footshock and measured by a paw-lick test was attenuated by drugs The percentage analgesia score after footshock was which increased extraneuronal 5HT i.e. the 5HT also significantly attenuated by the 5HT agonists releasing agents, p-chloroamphetamine and fenflur5-methoxy-N,N-dimethyltryptamine (2 mg/kg, 20 rain amine, and the 5HT reuptake inhibitor fluoxetine. before testing) and MK212 (2.5 mg/kg, 30min before Conversely, analgesia was enhanced when brain 5HT testing) (Table 3). However, two other agonists, quipa- was depleted by p-chlorophenylalanine. The effect of zine (2.5 mg/kg, 30rain before testing) and trifluoro- the latter drug was manifest not only when analgesia methylphenylpiperazine (10 mg/kg, 2 hr before testing) was measured by the paw-lick but also when the tailhad negligible effects on the rise of percentage analge- rick test was used. This suggests that 5HT is involved sia score after footshock. in the refex response to noxious stimuli and not merely in the locomotor component of the paw-lick E~bct oJ" 5HT antagonists on percentage analgesia test. score after jbotshock The results strongly indicate that analgesia induced A series of 5HT antagonists (methysergide, meter- by brief footshock is inversely related to 5HT awfilgoline, cyproheptadine, methiothepin, mianserine), ability or release. They contrast with the positive relationship found by others between 5HT and nociceptive thresholds determined without prior stressful Table 4. Antagonists of 5HT without significant effect on stimulation (see introduction for references). They also contrast with findings on the role of 5HT in analshock-induced analgesia gesia resulting from prolonged electroshock or immoDose Time bilization, which was attenuated by 5HT antagonists (mg/kg) before testing P* or synthesis inhibitors, or enhanced by administration of the 5HT precursor 5-hydroxytryptophan (BhattaMethysergide 2.0 2 hr > 0.2 charya, Keshary and Sanyal, 1978: Kulkarni, 1980: Metergoline 1.0 2 hr > 0.2 Shimizu, Koja, Fujisaki and Fukuda, 1981). Another Metergoline 5.0 2 hr > 0.1 difference between the present results and those of Metergoline 5.0 30 min > 0.5 Kulkarni (1980) and of Shimizu et al. (1981) is that the analgesic effect of brief footshock was not opposed by Cyproheptadine 10.0 20 min > 0.2 naloxone (1 20mg/kg, Table 5) but these authors Methiothepin 2.5 30 rain > 0.5 found that the drug was effective at 1 mg/kg against analgesia induced by more prolonged stresses. Mianserine 2.5 30 min > 0.5 The above differences, and the report by Lewis et * V. analgesia scores of vehicle-treated animals exposed ul. (1980) that naloxone antagonised thc analgesic to footshock. effect of prolonged intermittent footshock but not
5HT and footshock induced analgesia Table 5. Effect of naloxone on shock induced analgesia Percentage analgesia score Non-shock Shock
A
Saline
102.3 + 36.4
219.6 + 67.7
117
Naloxone 1 mg/kg 5 mg/kg 10 mg/kg 20mg/kg
77.7 102.8 93.1 98.1
244.9 228.1 246.7 284.5
167 126 154 187
+ + + +
20.9 28.6 29.6 17.5
± + + +
81.4 33.9 62.5 78.3
Values are mean ± SD N = 6 animals per group. A = Difference between shock and non-shock values.
55
is poor in view of the lack of effect of trifluoromethylphenylpiperazine, which is a powerful agonist (Fuller and Clemens, 1979; Fuller et al., 1980) and the uncertain interpretation of the effects of 5-methoxy-N,Ndimethyltryptamine and MK212. Furthermore, metergoline (inactive at both doses and times used) is equipotent at both receptor types (Peroutka, Lebovitz and Snyder, 1981). These findings thus raise the possibility of mechanisms dependent on a third type of central 5HT receptor or one not involving 5HT receptors at all. Acknowled,qements We thank the MRC for financial support and Mr R. Scraggs for technical assistance.
that of brief continuous footshock, suggests two distinct mechanisms for stress-induced analgesia i.e. an acute non-opioid mechanism depending inversely on 5HT availability, and a chronic opioid mechanism depending directly on 5HT availability. Opposing 5HT-dependent analgesic mechanisms are also indicated by the finding by Berge, Hole and Dahle (1980) that the 5HT agonist 5-methoxy-N,N-dimethyltryptamine caused increased and decreased nociception at low and high dose levels respectively. Interaction between such mechanisms and various reported effects of stress on brain 5HT (Curzon and Green, 1971; Palkovits, Brownstein, Kizer, Saavedra and Kopin, 1976; Kennett and Joseph, 1981) implies that relationships between brain 5HT and stress-provoked analgesia may vary considerably with conditions. A striking feature of the present results is that shock-provoked analgesia was affected by drugs which alter 5HT availability (Tables 1, 2) and by lesioning of spinal 5HT neurones (Hutson, Tricklebank and Curzon, 1982) but not by 5HT antagonists (Table 4) or by the 5HT agonists, trifluoromethylphenylpiperazine and quipazine (Table 3). Although the response was attenuated by the agonist, 5-methoxyN,N-dimethyltryptamine, this drug is structurally similar to 5HT and hence may well have a similar spectrum of biological activity. The response was also decreased by the agonist MK212 but this drug may also act on other transmitter systems such as dopamine (Clineschmidt, 1979) and we have recently shown that dopamine (like 5HT) opposes footshockinduced analgesia (Curzon, Hutson and Tricklebank, 1982). Furthermore, MK212 is probably a weaker 5HT agonist than the two structurally related agonists which did not affect the analgesic response (see above) as it is less potent as a displacer of 3H-5HT from rat brain membranes in vitro (Fuller, Mason and Molloy, 1980). Therefore, the drug experiments as a whole suggest that the action of 5HT on shock-provoked analgesi a does not involve known 5HT receptors. Evidence against 5HTz receptors is particularly strong in view of the lack of effect of all five antagonists tested as these receptors are usually more sensitive to antagonists than to agonists (Peroutka and Snyder, 1979) while evidence for the involvement of 5HT1 receptors
REFERENCES
Amir, S. and Amit, Z. (1978). Endogenous opioid ligands may mediate stress-induced changes in the affective properties of pain related behaviour in rats. Life Sci. 23: 1143 1152. Berge, O. G., Hole, K. and Dahle, H. (1980). Nociception is enhanced after low doses and reduced after high doses of the receptor agonist 5-methoxy-N,N-dimethyltryptamine. Neurosci. Lett. 19: 219-223. Bhattacharya, S. K., Keshary, P. R. and Sanyal, A. K. (19781. Immobilization stress induced antinociception in rats: possible role of serotonin and prostaglandins Eur. J. Pharmacol. 50: 83-85. Bodnar, R. J., Kelly, D. D., Spiaggia, A., Ehrenberg, C. and Glusman, M. (19781. Dose-dependent reductions by naloxone of analgesia induced by cold-water stress. Pharmac. Biochem. Behae. 8:667 672. Bodnar, R. J., Kelly, D. D., Brutus, M. and Glusman, M. (1980). Stress-induced analgesia: neural and hormonal determinants. Neurosci. Biobehav. Rev. 4: 87-100. Chance, W. T. (1980). Autoanalgesia: opiate and nonopiate mechanisms. Neurosci. Biobehav. Rev. 4:55 68. Chance, W. T. and Rosecrans, J. A. (1979). Lack of effect of naloxone on autoanalgesia. Pharmac. Biochem. Behav. 11 : 643 646. Chesher, G. B. and Chan, B. (1977). Footshock induced analgesia in mice: its reversal by naloxone and cross tolerance with morphine. Life Sci. 21" 1569-1574. Clineschmidt, B. V. (1979). MK212: A serotonin-like agonist in the CNS. Gen. Pharmac. 10:287 290. Curzon, G. and Green, W. R. (1970). Rapid method for the determination of 5-hydroxytryptamine and 5-hydroxyindoleacetic acid in small regions of rat brain. Br. J. Pharmac. 39:653 655. Curzon, G. and Green, A. R. (1971). Regional and subcellular changes in the concentration of 5-hydroxytryptamine and 5-hydroxyindoleacetic acid in the rat brain caused by hydrocortisone, DL-~-methyl-tryptophan, L-kynurenine and immobilisation. Br. J. Pharmac. 43:39 52. Curzon, G., Hutson, P. H. and Tricklebank, M. D. (1982). Involvement of dopamine in the analgesic response induced by footshock. Br. J. Pharmac. In press. Fibiger, H. C., Mertz, P. H. and Campbell, B. A. (1972). The effect of para-chlorophenylalanine on aversion thresholds and reactivity to foot shock. Physiol. Behav. 8:259 263. Fuller, R. L. and Wong, D. T. (1977). Inhibition of serotonin reuptake. Fedn Proc. Fedn Am. Socs exp. Biol. 36: 2154-2158. Fuller, R. W. and Clemens, J. A. (1979). 1-(m-Trifluoromethylphenyl)-piperazine, a serotonin agonist, increases serum corticosterone and prolactin concentration in rats. I R C S Med. Sci. 7: 106.
56
M . D . TRICKLI!BANK t'g ol.
Fuller, R. W.. Mason, N. R. and Molloy, B. B. (1980). Structural relationships in the inhibition of ['~H] serotonin binding to rat brain membranes in citro by 1-phenyl-piperazines. Biochem. Pharnlac. 29:833 835. Hayes, R. L., Bennett, G. J., Newlon, P. G. and Mayer, D. J. (1978}. Behavionral and physiological studies of non-narcotic analgesia in the rat elicited by certain environmental stimuli. Brain Re~. 155:69 90. Hutson, P. H., Tricklebank, M. D. and Curzon, G. (19821. Enhancement of footshock-induced analgesia by spinal 5,7-dihydroxytryptamine lesions. Brain Res. In press. Kennett, G. A. and Joseph, M. H. (1981). The functional importance of increased brain tryptophan in the serotonergic response to restraint stress. Neurc~pharmac. 20: 39 43. Kulkarni, S. K. (1980). Heat and other physiological stressinduced analgesia: catccholamine mediated and naloxone reversible response. L(I~" Sci. 27:185 188. Lewis, J. W.. Cannon, J. T. and Licbeskind, J. C. (1980). Opioid and non-opioid mechanisms of stress analgesia. Science 208:623 625. Lints, C. E. and Harvey, J. A. (1969). Drug induced reversal of brain damage in the rat. Physiol. Behac. 4:29 31. Madden, J., Akil, H., Patrick, R. L. and Barchas, J. D. (1977). Stress-induced parallel changes in central opioid levels and pain responsiveness in the rat. Nature, Lond 265:358 360. Messing, R. B., Fisher, L. A., Phebus, L. and Lytle, L. D. (1976). Interaction of diet and drugs in the regulation of brain 5-hydroxyindoles and the response to painful electric shock. Lili, Sci. 18:707 714. Messing, R. B., Phebus, L., Fisher, L. A. and Lytle, L. D. (1975). Analgesic effect of fluoxetine hydrochloride (Lilly 1101401 a specific inhibitor of serotonin uptake. Psychophurmac. Comm. 1:511 521. Millan, M. J., Przewlocki, R., Jerlicz, M., Gramsch, C. H., Hollt, V. and Herz, A. (1981). Stress-induced release of brain and pituitary-endorphin: major role of endorphins in generation of hyperthermia, not analgesia. Bruin Res. 208:325 338. Ogren, S. O. and Holm. A.-C. (1980). Test-specific effects of the 5HT rcuptake inhibitors alaproclate and zimelidine on pain sensitivity and morphine analgesia. J. Nem'al Trans. 47:253 271.
Oliveras, J. L., Guilband, G. and Besson, J. M. 11979). A map of serotoninergic structures involved in stimulation producing analgesia in unrestrained freely moving cats. Brain Res. 164:317 322. Palkovits. M., Brownstein, M., Kizer, J. S., Saavedra, J. M. and Kopin, h J. (1976). Effects of stress on serotonin and tryptophan hydroxylasc activity of brain nuclei. In: Catecholammes aml Stre.~s (Usdin, E.. Kvctnansky, R. and Kopin, I. J., Eds). pp. 51 88. Pergamon Press, Oxford, Peroutka, S. J., Lebovitz, R. M. and Snyder, S. H. (1981). Two distinct central serotonin receptors with different physiological functions. Sciem'e 212:827 828. Peroutka. S. J. and Snyder. S. H. 11979}. Multiple serotonm receptors: differential binding of [3H] 5-hydroxytryptamine, [~H] Lysergic acid diethylamide and [~H] spiroperidol, Moh'c. Phurmac. 16:687 699. Samanin, R.. Bernasconi. S, and Quattrone, A. (1976). Antinociceptive action of quipazine: relation to central serotonergic receptor stimulation. Psychopharmuc. 46: 219 222. Satoh. M., Akinori, A., Takahirn, N. and Takagi, H. 11980). Evidence for involvement of separate mechanisms in the production of analgesia by electrical stimulation of the nncleus paragigantocellularis and nucleus raphe magnus in the rat. Bruin Res. 194:525 529. Shimizu. T., Koja, T.. Fnjisaki, T. and Fukuda, T. (1981). Effects of methysergide and naloxone on analgesia induced by the peripheral electrical stimulation in mice. Brain Re.~. 208:463 467. Tenen, S. S. 11967). The effects of/>chlorophenylalanine, a scrotonin depleter, on avoidance acquisition, pain sensitivity and behaviour in the rat. Ps.vchophurmucoh~fia, Berlin 10:204 219. Trulson, M. E. and Jacobs, B. L. (1976). Behavioural evidence for the rapid release of CNS serotonin by PCA and fenfluramine. Era'. J. Pharnluc. 36:149 154. Wilier, J. C, and Albe-Fessard, D. {19801. Electrophysiological evidence for a release of endogenous opiates in stress-induced "analgesia' in man. Bruill Res. 198: 419 426. Yaksh. T. L. and Wilson, P. R. (19791. Spinal serotonm terminal system mediates antmociception. J. Pharmac. exp. Ther. 208:446 453.