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Naloxone-reversible stress-induced feeding and analgesia in the slug Limax maximus
Life Sciences, Vol. 38, pp. 203-209 Printed in the U.S.A.
Pergamon Press
NALOXONE-REVERSIBLE STRESS-INDUCED FEEDING AND ANALGESIA IN THE SLUG LIM_AXMAXIM~JS Martin K a v a l i e r s 1 and M a u r t c e H t r s t 2 Department of Psychology I , University of Alberta, Edmonton, Alberta Canada T6G 2E9 2 and Department of Pharmacology and Toxicology University of Western Ontario, London, Ontario Canada N6A 5B7 (Received in final form October 29, 1985) SUMMARY Exposure to tail-pinch stress increased the thermal nociceptive thresholds and food intakes of the slug, Limax maximus. These stressinduced "analgesic" and feeding responses, which were similar to the behaviors observed after treatment with exogenous opiates, were blocked by the opiate antagonist, naloxone. These results indicate that exposure to stress increases endogenous opioid activity in slugs and induces various behavioral and physiological responses in a manner analogous to that reported in mammals. There is substantial evidence that exposure to either physical or psychological stress can increase endogenous opioid activity in mammals (1-4) and induce a variety of behavioral and physiological responses, including analgesia (1,2,5,6) and increased feeding (6-8). These stress-induced responses are suppressed by the opiate antagonist, naloxone, and are similar to the effects observed after administration of exogenous opiate agonists such as morphine (1,2,6,9,10). These observations suggest that endogenous opioid systems are activated during stress and may form part of an intrinsic mechanism of basic survival value (6,9,10). It has become evident, recently, that invertebrates also possess regulatory opioid systems that resemble those found in mammals (11-13). In molluscs, administration of morphine produces naloxone reversible analgesic (14-16) and ingestive (17-19) responses that are analogous to those obtained in mammals (1,2,6,9). In this study we present evidence to show that exposure to stress also induces fundamental opioid mediated responses in invertebrates. We report that "tail-pinch" stress induces naloxone-reversible analgesic and ingestive responses in the terrestrial slug, Limax maximus. METHODS Animals. Slugs (10-15 g) were field collected (Victoria, British Columbia) and maintained in the laboratory under 12 hours of light and 12 hours of darkness (light, 25 ~w/cm =) at 22 i °i. They were communally housed in a glass aquarium (95 x 51 x 46 cm) that was provided with a soil suhstrate. Animals were kept moist and had continuous access to water, lettuce and other leafy vegetation.
0024-3205/86 $3.00 + .00 Copyright (c) 1986 Pergamon Press Ltd.
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Experimental Procedures Nociceptive Responses. Nociceptive thresholds of slugs were determined by measuring the latency of their thermal detection and avoidance behaviors. Individual slugs were placed on a heated surface (hot-plate, Technilab, N.J.) at 40 ± 0.5 ° and the latency of their foot or anterior body-lifting response to the thermal stimulus was recorded, after which the animals were removed from the aversive surface. Ingestive responses. Ingestive responses were determined by the amounts of food consumed by individual slugs over a three hour period. Seventy-two hours prior to testing, slugs were placed individually in i0 cm diameter petri dishes to which they were confined by 5 cm high plastic mesh sides and a plastic cover. They were acclimated to the chambers with both lettuce and water present. In the feeding determinations a substrate of pre-weighted lettuce was placed in the bottom of the dish. Romaine lettuce from the same portions of the head was used for each determination. To ensure full hydration of the slugs, a thin film of water was also maintained in each dish. At the end of each hour of the measurement period, slugs were removed from the dishes and the remaining lettuce was blotted dry and weighed. Any food adhering to the slugs was removed and corrected for in the hourly food intake determinations. Stress Responses. Slugs were stressed by gently pinching with blunt plastic tipped forceps the posterior or "tail" portion of the body using a pressure of 0.10-0.15 dynes/cm 2 (Bellco Strain Gauge, N.Y.). This "tail-pinch" stress was applied for two minutes, after which the thermal response latencies and ingestive responses of individual animals over three hours were determined. Individuals receiving only handling served as controls for the nociception determinations. Ingestive responses of unstressed animals, and of individuals exposed to the tail-pinch stress without intervening determinations of thermal response latencies, were also carried out. Ten minutes prior to handling or tail-pinch additional groups of slugs received intramuscular (i.m) injections of either naloxone hydrochloride (I.0 mg/Kg) or the saline (20) vehicle (i0 ml/Kg). The effects of i.m morphine sulfate (0.i0, 1.0, i0 mg/Kg, B.D.H. Toronto) or saline control (I0 ml/kg) on the thermal response latencies of slugs at time periods of 15, 30, 60 and 90 minutes after injection were also examined. As well, the effects of pre-treatment with naloxone hydrochloride (i.0 mg/Kg, Sigma St. Louis) or saline (I0 ml/Kg) control on the responses to morphine (i0 mg/Kg) or saline (i0 ml/Kg), as well as the effects of naloxone (I0 mg/Kg) by itself, were determined. Morphine-induced feeding responses in slugs are described elsewhere (17-19). All determinations were carried out in the early to mid-light periods, with only one treatment per animal. In the stress determinations, each treatment was replicated with ten animals. Five different slugs were used for each of the dose and time determinations with morphine. Data were analysed by two-way analysis of variance and Students t-tests. RESULTS Stress-Induced Analgesia. A sterotypical elevation of the extended portion of the body was observed in slugs that were placed on the 40 ° surface. This behavior was not observed in slugs that were exposed to ambient or non-aversive thermal conditions (32°). Tail-pinch stressed slugs were "analgesic", displaying significantly (p< 0.01) greater latencies to respond to the heated
Vol. 38, No. 3, 1986
40'
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A STRESS
L)
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NON-STRESS
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.
FIGURE i Effects of tail pinch (stress) and control handling procedures (non-stress) on the A. thermal (40°C) response latencies and B. ingestive responses over three hours of individual slugs, Limax maximus. C. represents animals whose measurements of ingestive responses were preceded by thermal response latency determinations, while N represents animals who only had ingestive responses determined). All animals received either naloxone hydrochloride (nx., 1.0 mg/Kg), saline vehicle (sal., i0 ml/Kg) or no injection (cntrl., control) i0 min before exposure to the tail-pinch stress or control handling non-stress procedures, n=10 in all cases. Vertical lines denote two standard errors of the mean.
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FIGURE 2 A. Time course of the efforts of morphine (0.i0, 1.0, i0 mg/Kg) and saline (I0 ml/Kg) on the thermal response latencies of individual slugs, Limax maximus. B. Effects of naloxone (nx., 1.0 mg/Kg) pre-treatment on the thermal response latencies of morphine (morph., i0 mg/Kg) and saline (sal., i0 ml/Kg) injected slugs. Response latencies of non-injected control (cntr.) and morphine (i0 mg/Kg) treated slugs with and without saline (S morph; C morph) pre-treatments are also provided. N=5 in all cases. Vertical lines denote two standard errors of the mean.
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surface than control, handled animals (Fig. IA). Pre-treatment with naloxone blocked this stress-induced analgesia, with saline injections having no evident effects on the thermal response latencies (Fig. IA). Morphine also caused a significant (p < 0.01, for 1.0 mg/Kg), dose-dependent increase in the thermal response latencles of slugs, with maximal responses occurring 15-30 min after injection (Fig. 2A). Pre-treatment with naloxone blocked this morphine-induced analgesia (Fig. 2B), with naloxone by itself having no evident effects on basal (40 °) nociceptive responses. Stress-Induced Feeding. Tail-plnch stress caused a significant (p< 0.01) increase over three-hours in the ingestive responses and food intakes of slugs (Fig. 3). Maximal increases in ingestion occurred during the first one-two hours, with a decline to basal levels by the third hour. These feeding responses were not affected by intervening nociceptive determinations. As well, control handling procedures had no significant stimulatory effects on the food intake of slugs. Naloxone suppressed the tail-pinch stress-induced feeding (Fig. IB), but had no significant effects on the basal food intakes of free-feeding slugs (17). DISCUSSION The present results demonstrate the presence of naloxone-reversible, stress-induced feeding and analgesia in the slug. Application of a tail-plnch stress resulted in a significant increase in the latency of response to an aversive thermal stimulus. This increase in nociceptive thresholds is analogous to the a qlgesic responses observed in rats and mice after exposure to a variety of stressurs, including body pinch (1,2,6,9). Pre-treatment with naloxone blocked the stress-induced analgesia in slugs, with saline injections having no evident effects on response latency. This inhibitory effect of naloxone, which was similar to that observed in mammals (1-3), supports the hypothesis of endogenous opioid activation and involvement in the mediation of the stress-lnduced analgesic response of slugs. The dose of naloxone used (I.0 mg/kg) is primarily considered to affect opiate responses. This contention of opiate involvement is further supported by the observations that morphine has a similarly, naloxone reversible, analgesic effect in Limax and other molluscs (14-16). These dose- and time-dependent analagesic effects of morphine are analogous to the effects reported in laboratory rats and mice given opiates (1,2,6). At the test temperature of 40 ° , naloxone did not have any evident effects on the basal nociceptive responses of the slugs. In general, naloxone does not have consistent, significant effects on vertebrate behavioral responses to many aversive stimuli (2,10). It may be considered that the nociceptive or analgesic affects of morphine arise from inhibition of locomotor responses (13). However, control studies with the doses of morphine (i0 mg/kg) used in the present study revealed no adverse affects on locomotor behavior (19). In addition, if there had been either an impairment of locomotor activity or a non-speclfic body flexion, this would not have resulted in the initiation of a significant opioid-mediated ingestive response. The occurrence of both increased feeding and an elevated nociceptive response is similar to that observed in stressed mammals, and further supports the applicability of the term 'stress-induced analgesia' in the present study. Both high and low affinity opioid binding sites, as well as leuclne- and methionine-enkephalin have been demonstrated to be present in molluscan neural tissue (11-13). Immunocytochemical studies have confirmed the existence of
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$-endorphin and methionine-enkephalin in various ganglia of Limax (21). These findings provide additional support for the proposal that endogenous opioids are involved in the mediation of stress-induced analgesia in Limax. However, further investigations of possible non-specific effects of naloxone (22), and the roles of non-opioid mechanisms (5) in the mediation of stress-induced analgesia in Limax are needed. Tail-pinch stress also caused significant increases in the food intakes of free-feeding slugs. These ingestive responses were similar to the feeding behaviors obtained after administration of morphine to Limax. Equivalent feeding responses were obtained using either the lettuce diet described above (18,19), or a more refined uniform carrot puree diet (17). Naloxone blocked tail-pinch, as well as morphine-induced feeding (17), supporting the hypothesis of stress-induced, opioid mediated feeding in slugs. However, examinations of the possible roles of non-opioid mechanisms, as well as the relative contributions of food and water uptakes to the ingestive responses, are needed. As the animals in the present experiment were well hydrated, it is not known whether water uptake accompanied feeding, analogous to the situation in mammals (23,24). There is some evidence, however, to suggest that endogenous opiates may also have independent stimulatory effects on mammalian drinking (25,26). It remains to be determined whether or not opioid systems have a role in the control of fluid balance and water uptake by Limax and other molluscs. These ingestive reponses of Limax are similar to the stress-lnduced, naloxone-reversible, feeding and drinking behaviors observed in mammals (7,8). Naloxone had no significant effects on basal food intake of free-feeding slugs (17), but, as in mammals, could block the ingestive responses of food deprived animals (9). In mammals it has been suggested that stress-induced feeding may involve interactions between endogenous opioids, monoamines, serotonergic and other neurotransmitters (9). There is evidence for dopamine and opioid involvement in the control of Limax feeding (27), while serotonergic mechanisms have been suggested to be involved in the mediation of stress-induced decrease in the latency to the initiation of feeding by the marine mollusc, Aplysia (28). The present results indicate that opioid systems have a functional role in the mediation of stress-induced analgesic and feeding responses of slugs and suggest that this role is in many respects comparable to that reported for mammals. Furthermore, these observations suggest that there may have been an early evolutionary development and phylogenetic continuity of a fundamental opioid involvement in the mediation of basic stress induced behavioral and physiological responses. ACKNOWLEDGEMENTS Supported by a Natural Sciences and Engineering Research Council of Canada grant to M.K. 15222A8) REFERENCES i. 2. 3.
S.Z. AMIR, Z.W. BROWN and Z.AMIT, Neurosci. Biobeha. Rev. 4: 77-86 (1980). R. BODNAR, D.D. KELLY, M. BRUTUS and M. GLUSMAN, Neurosci. Biobehav. Rev. 4: 87-96 (1980). W.T. CHANCE, A.C. WHITE, G.M. KRYNOCK and J.A. ROSECRANS, Brain Res. 141: 371-374 (1982).
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4. 5. 6. 7. 8. 9. i0. ii. 12. 13.
14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28.
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J.E. MORLEY, A.S. LEVINE, S.S. MURRAY, J. KNEIP and M. GRACE, Am. J. Physiol., (Regulatory Integrative Comp. Physiol.) 12:RI59-RI63 (1982). G.S. TERM_AN, K. SHAVIT, J.W. LEWIS, J.T. CANNON and J.C. LIEBESKIND, Science 226:1270-1277 (1984). G.C. TESKEY, M. KAVALIERS and M. HIRST, Life Sci. 35:303-315 (1984). J.E. MORLEY and A.S. LEVINE, Science 206:1259-1261 (1980). J.E. MORLEY, A.S. LEVINE and N.E. ROWLAND, Life Sci. 32:2169-2182 (1983). J.E. MORLEY, A.S. LEVlNE, G.K. YIM and M.T. LOWY, Neurosci. Biobehav. Rev. 7: 281-305 (1983). ~.L. HENRY, Neurosci. Biobehav. Rev. 6:229-245 (1982). R.M. KREAM, R.S. ZUKIN and G.B. STEFANO, J. Biol. Chem. 225:9218-9224 (1980). M.K. LEUNG and G.B. STEFANO, Proc. Natl. Acad. Sci. 81:955-958 (1984). G.B. STEFANO, B. HALL, M.H. MARKHAM and B. DVORKIN, Science, 213:928-930 (1980). M. KAVALIERS, M. HIRST and G.C. TESKEY, Science 2 2 0 : 9 9 - 1 0 1 (1983). M. KAVALIERS and M. HIRST, Neuropharmacol. 22:1321-1326 (1983). M. KAVALIERS, M. HIRST and G.C. TESKEY, Neuropharmacol. 24: 621-626. M. KAVALIERS, M. HIRST and G.C. TESKEY, Physiol. Behav. 33:765-767 (1984). M. KAVALIERS, K-P. OSSENKOPP, A. MATHERS, Pharmacol. Biochem. Behav. In press (1986). M. KAVALIERS, M. HIRST, G.C. TESKEY, Brain Res. Bull. In press (1985). J.J. CHANG an A. GELPERIN, Proc. Nat. Acad. Sci. 77:6204-6206 (1980). C.R. MARCHAND, P.G. SOKOLOVE and M.P. DUBOIS, Cell Tiss. Res. 238:349-353 (1984). J.C. SAWYNOK, J.C. PINSKY and F.S. LABELLA, Life Sci. 25:1621-1627 (1979). T.J. FITZSIMONS and J. LE MAGNEN, J. Comp. Physiol. Psychol. 67:273-283 (1969). H.R. KISSILEEF, J. Comp. Physiol. Psychol. 67:284-300 (1969). B. BRANDS, J.A. THORNHILL, M. HIRST and C.W. GOWDEY, Life Sci. 24:1773-1778 (1979). D.R. BROWN and S.G. HOLTZM~, Pharmacol. Biochem. Behav. ii: 507-573 (1979). S.J. WIELAND and A. GELPERIN, J. Neurosci. 2:1735-].745 (1983). I. KUPFERMAN and K.R. WEISS, Behav. Neurol. Biol. 32:126-132 (1981).
Pergamon Press
NALOXONE-REVERSIBLE STRESS-INDUCED FEEDING AND ANALGESIA IN THE SLUG LIM_AXMAXIM~JS Martin K a v a l i e r s 1 and M a u r t c e H t r s t 2 Department of Psychology I , University of Alberta, Edmonton, Alberta Canada T6G 2E9 2 and Department of Pharmacology and Toxicology University of Western Ontario, London, Ontario Canada N6A 5B7 (Received in final form October 29, 1985) SUMMARY Exposure to tail-pinch stress increased the thermal nociceptive thresholds and food intakes of the slug, Limax maximus. These stressinduced "analgesic" and feeding responses, which were similar to the behaviors observed after treatment with exogenous opiates, were blocked by the opiate antagonist, naloxone. These results indicate that exposure to stress increases endogenous opioid activity in slugs and induces various behavioral and physiological responses in a manner analogous to that reported in mammals. There is substantial evidence that exposure to either physical or psychological stress can increase endogenous opioid activity in mammals (1-4) and induce a variety of behavioral and physiological responses, including analgesia (1,2,5,6) and increased feeding (6-8). These stress-induced responses are suppressed by the opiate antagonist, naloxone, and are similar to the effects observed after administration of exogenous opiate agonists such as morphine (1,2,6,9,10). These observations suggest that endogenous opioid systems are activated during stress and may form part of an intrinsic mechanism of basic survival value (6,9,10). It has become evident, recently, that invertebrates also possess regulatory opioid systems that resemble those found in mammals (11-13). In molluscs, administration of morphine produces naloxone reversible analgesic (14-16) and ingestive (17-19) responses that are analogous to those obtained in mammals (1,2,6,9). In this study we present evidence to show that exposure to stress also induces fundamental opioid mediated responses in invertebrates. We report that "tail-pinch" stress induces naloxone-reversible analgesic and ingestive responses in the terrestrial slug, Limax maximus. METHODS Animals. Slugs (10-15 g) were field collected (Victoria, British Columbia) and maintained in the laboratory under 12 hours of light and 12 hours of darkness (light, 25 ~w/cm =) at 22 i °i. They were communally housed in a glass aquarium (95 x 51 x 46 cm) that was provided with a soil suhstrate. Animals were kept moist and had continuous access to water, lettuce and other leafy vegetation.
0024-3205/86 $3.00 + .00 Copyright (c) 1986 Pergamon Press Ltd.
204
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Experimental Procedures Nociceptive Responses. Nociceptive thresholds of slugs were determined by measuring the latency of their thermal detection and avoidance behaviors. Individual slugs were placed on a heated surface (hot-plate, Technilab, N.J.) at 40 ± 0.5 ° and the latency of their foot or anterior body-lifting response to the thermal stimulus was recorded, after which the animals were removed from the aversive surface. Ingestive responses. Ingestive responses were determined by the amounts of food consumed by individual slugs over a three hour period. Seventy-two hours prior to testing, slugs were placed individually in i0 cm diameter petri dishes to which they were confined by 5 cm high plastic mesh sides and a plastic cover. They were acclimated to the chambers with both lettuce and water present. In the feeding determinations a substrate of pre-weighted lettuce was placed in the bottom of the dish. Romaine lettuce from the same portions of the head was used for each determination. To ensure full hydration of the slugs, a thin film of water was also maintained in each dish. At the end of each hour of the measurement period, slugs were removed from the dishes and the remaining lettuce was blotted dry and weighed. Any food adhering to the slugs was removed and corrected for in the hourly food intake determinations. Stress Responses. Slugs were stressed by gently pinching with blunt plastic tipped forceps the posterior or "tail" portion of the body using a pressure of 0.10-0.15 dynes/cm 2 (Bellco Strain Gauge, N.Y.). This "tail-pinch" stress was applied for two minutes, after which the thermal response latencies and ingestive responses of individual animals over three hours were determined. Individuals receiving only handling served as controls for the nociception determinations. Ingestive responses of unstressed animals, and of individuals exposed to the tail-pinch stress without intervening determinations of thermal response latencies, were also carried out. Ten minutes prior to handling or tail-pinch additional groups of slugs received intramuscular (i.m) injections of either naloxone hydrochloride (I.0 mg/Kg) or the saline (20) vehicle (i0 ml/Kg). The effects of i.m morphine sulfate (0.i0, 1.0, i0 mg/Kg, B.D.H. Toronto) or saline control (I0 ml/kg) on the thermal response latencies of slugs at time periods of 15, 30, 60 and 90 minutes after injection were also examined. As well, the effects of pre-treatment with naloxone hydrochloride (i.0 mg/Kg, Sigma St. Louis) or saline (I0 ml/Kg) control on the responses to morphine (i0 mg/Kg) or saline (i0 ml/Kg), as well as the effects of naloxone (I0 mg/Kg) by itself, were determined. Morphine-induced feeding responses in slugs are described elsewhere (17-19). All determinations were carried out in the early to mid-light periods, with only one treatment per animal. In the stress determinations, each treatment was replicated with ten animals. Five different slugs were used for each of the dose and time determinations with morphine. Data were analysed by two-way analysis of variance and Students t-tests. RESULTS Stress-Induced Analgesia. A sterotypical elevation of the extended portion of the body was observed in slugs that were placed on the 40 ° surface. This behavior was not observed in slugs that were exposed to ambient or non-aversive thermal conditions (32°). Tail-pinch stressed slugs were "analgesic", displaying significantly (p< 0.01) greater latencies to respond to the heated
Vol. 38, No. 3, 1986
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A STRESS
L)
3O
NON-STRESS
e.
0
2G ,<,. Q 0¢,.,
-J
cntr. i
1.50
B
sal
STRESS
rC , co
E NON - STRESS
"0 0
o 0.50
LI.
c n
cntr.
sal.
.
FIGURE i Effects of tail pinch (stress) and control handling procedures (non-stress) on the A. thermal (40°C) response latencies and B. ingestive responses over three hours of individual slugs, Limax maximus. C. represents animals whose measurements of ingestive responses were preceded by thermal response latency determinations, while N represents animals who only had ingestive responses determined). All animals received either naloxone hydrochloride (nx., 1.0 mg/Kg), saline vehicle (sal., i0 ml/Kg) or no injection (cntrl., control) i0 min before exposure to the tail-pinch stress or control handling non-stress procedures, n=10 in all cases. Vertical lines denote two standard errors of the mean.
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A
1170 ~M°rphine _ -=
u., mg/k9 Satine
3c "b"
10 U~ fI
0
O
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0
50
,
L
*
30 Time
I
*
60
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90
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10 C
S
mOr.
FIGURE 2 A. Time course of the efforts of morphine (0.i0, 1.0, i0 mg/Kg) and saline (I0 ml/Kg) on the thermal response latencies of individual slugs, Limax maximus. B. Effects of naloxone (nx., 1.0 mg/Kg) pre-treatment on the thermal response latencies of morphine (morph., i0 mg/Kg) and saline (sal., i0 ml/Kg) injected slugs. Response latencies of non-injected control (cntr.) and morphine (i0 mg/Kg) treated slugs with and without saline (S morph; C morph) pre-treatments are also provided. N=5 in all cases. Vertical lines denote two standard errors of the mean.
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surface than control, handled animals (Fig. IA). Pre-treatment with naloxone blocked this stress-induced analgesia, with saline injections having no evident effects on the thermal response latencies (Fig. IA). Morphine also caused a significant (p < 0.01, for 1.0 mg/Kg), dose-dependent increase in the thermal response latencles of slugs, with maximal responses occurring 15-30 min after injection (Fig. 2A). Pre-treatment with naloxone blocked this morphine-induced analgesia (Fig. 2B), with naloxone by itself having no evident effects on basal (40 °) nociceptive responses. Stress-Induced Feeding. Tail-plnch stress caused a significant (p< 0.01) increase over three-hours in the ingestive responses and food intakes of slugs (Fig. 3). Maximal increases in ingestion occurred during the first one-two hours, with a decline to basal levels by the third hour. These feeding responses were not affected by intervening nociceptive determinations. As well, control handling procedures had no significant stimulatory effects on the food intake of slugs. Naloxone suppressed the tail-pinch stress-induced feeding (Fig. IB), but had no significant effects on the basal food intakes of free-feeding slugs (17). DISCUSSION The present results demonstrate the presence of naloxone-reversible, stress-induced feeding and analgesia in the slug. Application of a tail-plnch stress resulted in a significant increase in the latency of response to an aversive thermal stimulus. This increase in nociceptive thresholds is analogous to the a qlgesic responses observed in rats and mice after exposure to a variety of stressurs, including body pinch (1,2,6,9). Pre-treatment with naloxone blocked the stress-induced analgesia in slugs, with saline injections having no evident effects on response latency. This inhibitory effect of naloxone, which was similar to that observed in mammals (1-3), supports the hypothesis of endogenous opioid activation and involvement in the mediation of the stress-lnduced analgesic response of slugs. The dose of naloxone used (I.0 mg/kg) is primarily considered to affect opiate responses. This contention of opiate involvement is further supported by the observations that morphine has a similarly, naloxone reversible, analgesic effect in Limax and other molluscs (14-16). These dose- and time-dependent analagesic effects of morphine are analogous to the effects reported in laboratory rats and mice given opiates (1,2,6). At the test temperature of 40 ° , naloxone did not have any evident effects on the basal nociceptive responses of the slugs. In general, naloxone does not have consistent, significant effects on vertebrate behavioral responses to many aversive stimuli (2,10). It may be considered that the nociceptive or analgesic affects of morphine arise from inhibition of locomotor responses (13). However, control studies with the doses of morphine (i0 mg/kg) used in the present study revealed no adverse affects on locomotor behavior (19). In addition, if there had been either an impairment of locomotor activity or a non-speclfic body flexion, this would not have resulted in the initiation of a significant opioid-mediated ingestive response. The occurrence of both increased feeding and an elevated nociceptive response is similar to that observed in stressed mammals, and further supports the applicability of the term 'stress-induced analgesia' in the present study. Both high and low affinity opioid binding sites, as well as leuclne- and methionine-enkephalin have been demonstrated to be present in molluscan neural tissue (11-13). Immunocytochemical studies have confirmed the existence of
208
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$-endorphin and methionine-enkephalin in various ganglia of Limax (21). These findings provide additional support for the proposal that endogenous opioids are involved in the mediation of stress-induced analgesia in Limax. However, further investigations of possible non-specific effects of naloxone (22), and the roles of non-opioid mechanisms (5) in the mediation of stress-induced analgesia in Limax are needed. Tail-pinch stress also caused significant increases in the food intakes of free-feeding slugs. These ingestive responses were similar to the feeding behaviors obtained after administration of morphine to Limax. Equivalent feeding responses were obtained using either the lettuce diet described above (18,19), or a more refined uniform carrot puree diet (17). Naloxone blocked tail-pinch, as well as morphine-induced feeding (17), supporting the hypothesis of stress-induced, opioid mediated feeding in slugs. However, examinations of the possible roles of non-opioid mechanisms, as well as the relative contributions of food and water uptakes to the ingestive responses, are needed. As the animals in the present experiment were well hydrated, it is not known whether water uptake accompanied feeding, analogous to the situation in mammals (23,24). There is some evidence, however, to suggest that endogenous opiates may also have independent stimulatory effects on mammalian drinking (25,26). It remains to be determined whether or not opioid systems have a role in the control of fluid balance and water uptake by Limax and other molluscs. These ingestive reponses of Limax are similar to the stress-lnduced, naloxone-reversible, feeding and drinking behaviors observed in mammals (7,8). Naloxone had no significant effects on basal food intake of free-feeding slugs (17), but, as in mammals, could block the ingestive responses of food deprived animals (9). In mammals it has been suggested that stress-induced feeding may involve interactions between endogenous opioids, monoamines, serotonergic and other neurotransmitters (9). There is evidence for dopamine and opioid involvement in the control of Limax feeding (27), while serotonergic mechanisms have been suggested to be involved in the mediation of stress-induced decrease in the latency to the initiation of feeding by the marine mollusc, Aplysia (28). The present results indicate that opioid systems have a functional role in the mediation of stress-induced analgesic and feeding responses of slugs and suggest that this role is in many respects comparable to that reported for mammals. Furthermore, these observations suggest that there may have been an early evolutionary development and phylogenetic continuity of a fundamental opioid involvement in the mediation of basic stress induced behavioral and physiological responses. ACKNOWLEDGEMENTS Supported by a Natural Sciences and Engineering Research Council of Canada grant to M.K. 15222A8) REFERENCES i. 2. 3.
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