Brazilian Journal of Medical and Biological Research (2005) 38: 73-80 abbreviatus as a model for nociceptive experiments 73 ISSN 0100-879X

The terrestrial Megalobulimus abbreviatus as a useful model for nociceptive experiments. Effects of morphine and naloxone on thermal avoidance behavior

M. Achaval1, M.A.P. Penha1, 1Laboratório de Histofisiologia Comparada, Departamento de Ciências Morfológicas, A. Swarowsky1, P. Rigon1, and 2Laboratório de Neurobiologia, Departamento de Fisiologia, L.L. Xavier1, G.G. Viola1 and Instituto de Ciências Básicas da Saúde, Universidade Federal do Rio Grande do Sul, D.M. Zancan2 Porto Alegre, RS, Brasil

Abstract

Correspondence We describe the behavior of the snail Megalobulimus abbreviatus Key words M. Achaval upon receiving thermal stimuli and the effects of pretreatment with • Snail Laboratório de Histofisiologia morphine and naloxone on behavior after a thermal stimulus, in order • Thermal avoidance Comparada to establish a useful model for nociceptive experiments. Snails sub- • Nociception Departamento de Ciências mitted to non-functional (22ºC) and non-thermal hot-plate stress • Morphine Morfológicas, ICBS, UFRGS • Naloxone R. Sarmento Leite, 500 (30ºC) only displayed exploratory behavior. However, the 90050-070 Porto Alegre, RS submitted to a thermal stimulus (50ºC) displayed biphasic avoidance Brasil behavior. Latency was measured from the time the was placed Fax: +55-51-3316-3092 on the hot plate to the time when the animal lifted the head-foot E-mail: [email protected] complex 1 cm from the substrate, indicating aversive thermal behav- ior. Other animals were pretreated with morphine (5, 10, 20 mg/kg) or Research supported by naloxone (2.5, 5.0, 7.5 mg/kg) 15 min prior to receiving a thermal UFRGS/FINEP (No. 66.91.0509.00) and CNPq. stimulus (50ºC; N = 9 in each group). The results (means ± SD) showed an extremely significant difference in response latency be- tween the group treated with 20 mg/kg morphine (63.18 ± 14.47 s) and the other experimental groups (P < 0.001). With 2.5 mg/kg (16.26 ± Received January 30, 2004 3.19 s), 5.0 mg/kg (11.53 ± 1.64 s) and 7.5 mg/kg naloxone (7.38 ± 1.6 Accepted October 13, 2004 s), there was a significant, not dose-dependent decrease in latency compared to the control (33.44 ± 8.53 s) and saline groups (29.1 ± 9.91 s). No statistically significant difference was found between the naloxone-treated groups. With naloxone plus morphine, there was a significant decrease in latency when compared to all other groups (minimum 64% in the saline group and maximum 83.2% decrease in the morphine group). These results provide evidence of the involve- ment of endogenous opioid peptides in the control of thermal with- drawal behavior in this snail, and reveal a stereotyped and reproduc- ible avoidance behavior for this snail , which could be studied in other pharmacological and neurophysiological studies.

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Introduction thermal stimulus can be suppressed by the opiate antagonist, naloxone (3,4,7). Thus, in According to Sherrington, the capacity these gastropods the ability of the nonselec- of animals to detect and react to stimuli that tive opiate antagonist naloxone to produce a may compromise their integrity is embodied complete blockade of morphine analgesia in the term “nociception” cited in Ref. 1. The indicates that a µ receptor is related to this ability to respond to aversive environmental behavior (4,8). Naloxone alone decreases stimuli is a basic characteristic of animals the latency of the thermal stress (2). It was and this adaptive animal behavior implies also demonstrated that thermal stimulus-in- the presence of nociceptors to detect the duced analgesia was blocked by a δ antago- aversive nature of the stimuli and effectors nist (9). In addition, enkephalin immunore- which respond to the sensory input with activity was identified in the central nervous reflex and/or non-reflex behavior. Descrip- system of different snails such as A. califor- tions of postures and movements associated nica, Lymnaea stagnalis, Helix pomatia, and with nociception and avoidance behavior M. abbreviatus (10,11). can be quantitative and objective if we re- The present study reports the avoidance frain from interpreting them with connota- behavior of M. abbreviatus, as well as the tions of human experience. The avoidance effects of morphine and its antagonist nalox- behavior of snails, such as Cepaea nemoralis, one on this snail after a thermal stimulus. Megalobulimus sanctipauli and Aplysia This study represents a contribution as a californica has been described (1-4). In avoid- starting point for the functional detection of ance behavior, the snails presented a with- the nociceptive circuit (12,13) present in this drawal reaction accompanied by mucus se- snail. cretion to all kinds of noxious stimuli. In response to moderately intense tactile stimu- Material and Methods lation (15-40 g/mm2) the snail remained out- side the shell, locomotion ceased, and the Animals animal showed pneumostome closure, ten- tacle contraction, head withdrawal, and a Adult land snails, Megalobulimus abbre- latency of about 0.2 s. In response to a strong viatus (Becquaert, 1948), previously called noxious stimulus, the snails showed all the M. oblongus (Müller, 1774), with shell aforementioned reactions and retraction of lengths of 65-80 mm and weighing 50-79 g the whole body into the shell (5). In response were collected in Charqueadas county, State to thermal stimuli, within a few seconds C. of Rio Grande do Sul, Brazil. The animals nemoralis (40ºC) and M. sanctipauli (52ºC) were kept in a screened terrarium, subjected showed aversive behavior characterized by to normal annual light and temperature var- lifting the anterior regions of the head-foot iations, and fed lettuce and water ad libitum. (H-F) complex. This behavior is not detected To minimize the novelty stress (4,9,14), the in snails at room temperature or at lower satiated snails were allowed to rehydrate in temperatures such as 30-33ºC (4,6). individual humid chambers 2 h prior to the Moreover, the administration of morphine experiments. Pharmacological agents were sulfate, methionine-enkephalin or ß-endor- dissolved in saline and injected into the phin resulted in a significant increase in the intrahemocelic cavity through the anterior latency of thermal avoidance behavior (4,6). ventral zone of the foot. Fifteen minutes Also, the increased latency resulting from after the injection, the snails were submitted morphine administration to C. nemoralis, to a thermal stimulus. To avoid sensitization Limax maximus and M. sanctipauli after a or habituation, each snail was used only

Braz J Med Biol Res 38(1) 2005 Megalobulimus abbreviatus as a model for nociceptive experiments 75 once. Additionally, to avoid the probable naloxone. Four groups (N = 9 in each) were effect of the lack of humidity on the sub- injected with naloxone hydrochloride (Cris- strate during exposure of the snails to the hot tália) at concentrations of 2.5, 5.0 or 7.5 mg/ plate, a plastic film covered with 10 ml of kg body weight, or with saline (1.0 ml) be- distilled water was placed over the plate. It fore being placed on a heated surface (50ºC). was then necessary to wait for the plate to Another group of untreated hydrated snails reacquire the predetermined temperature. All (control, N = 9) was also placed on a heated the experimental procedures were carried surface. out during the mid-light period (14-17 h). To reverse the action of morphine 9 snails were pretreated with naloxone hydrochlo- Experimental procedures ride (5.0 mg/kg body weight) and after 5 min were injected with morphine sulfate (20.0 The nociceptive thresholds of individual mg/kg body weight); after another 15 min, snails were determined by measuring the the snails were placed on a heated surface latency of their avoidance or withdrawal (50ºC). Another group was injected with 1.0 behaviors (15) in response to the thermal ml saline (N = 9). stimulus. The measurement of latency started at the time when the snail was placed on the Statistical analysis hot plate and ended as soon as the animal lifted the H-F complex to a minimum of 1 cm Data were analyzed statistically by anal- (4). Individual hydrated snails were placed ysis of variance (ANOVA) followed by the on a hot plate (model-DS37, Socrel, Comerio, Tukey test, with the level of significance set VA, Italy) at 22ºC ambient temperature (non- at P < 0.05. The analyses were carried out functional hot plate), at a non-stressful ther- using the SPSS 7.0 software. mal condition of 30ºC or at a stressful ther- mal condition of 50ºC (N = 9 in each group) Results and the response latency of their avoidance behavior was determined. Nociceptive response In order to determine the variation of the thermal stress (50ºC) response latency to The snails placed on the nonfunctional morphine and naloxone, individual hydrated hot plate (22ºC) displayed only exploratory snails were injected with pharmacological behavior, with their H-F complex remaining drugs, as described below, 15 min prior to extended, touching the substrate and show- thermal stimuli. After their response laten- ing slow undulatory waves in the parapodial cies were determined, the snails were quickly region. In the anterior region of the H-F removed from the hot plate. complex, the parapodium was lifted. Pro- Four groups of hydrated snails (N = 9 in tracted labial palps and oral tentacles were in each) were injected with morphine sulfate constant movement and when they touched (Cristália do Brasil S/A, São Paulo, SP, Bra- the substrate they displayed a quick retrac- zil) at concentrations of 5.0, 10.0, and 20.0 tion. Immediately, they again performed ten- mg/kg body weight or saline (1.0 ml) before tacular palpation of the substrate, exploring being placed on a heated surface (50ºC). the environment. The snails exhibited a slight Another group (control; N = 9) of untreated elevation of the anterior aspect of the head hydrated snails was also placed on a heated and then turning movements of the body surface (16). without turning movements of the shell, but Latencies were also determined for ani- did not show any locomotory activity. Thus, mals treated with the morphine antagonist the snails exposed to 22ºC displayed neither

Braz J Med Biol Res 38(1) 2005 76 M. Achaval et al.

foot lifting behavior nor locomotion. No tion and searching movements in the second modification was detected in mucus secre- (17). tion. The snails were observed for 300 s on First phase. When placed on the hot the hot plate, always displaying the same plate, M. abbreviatus exhibited an extended behavior, which was also observed in snails foot which was immediately retracted later- submitted to a non-thermal stress of 30ºC. ally. At the same time, the oral tentacles and The snails submitted to a thermal stimu- labial palps retracted and this movement was lus of 50ºC displayed biphasic avoidance followed by a medial-ventral retraction of behavior, exhibiting a retraction of the body the H-F complex, beginning at the rostral into the shell in the first phase and protrac- region and extending to the caudal region. Thus, only the lateral portion of the foot Figure 1. Schematic representa- remained in contact with the substrate, with tion of the snail Megalobulimus the occurrence of a partial retraction of the abbreviatus in the first phase of body into the shell. When the snail was ob- aversive behavior. Note the re- tracted oral tentacles and labial served from the anterior region, the H-F com- palps and the triangular aspect plex showed a triangular aspect (Figure 1). of the head-foot complex when Second phase. After 25 s, on average, the the snail was placed on the hot plate. retracted oral tentacles and labial palps be- gan to protract and the snail displayed search- ing movements followed by lateral parapo- dial waves characterizing exploratory be- havior. Subsequently, the animals exhibited anterior lifting of the H-F complex 1 cm or more from the substrate. The average la- tency of the avoidance behavior response or of the H-F complex elevation to a height of 1 cm was 33 s. After the lifting of the H-F complex, the rostral portion of the head turned (Figure 2). All the animals in this group exhibited foot swelling and intense secretion of yellowish mucus.

Nociceptive responses after morphine and naloxone administration

Administration of different concentra- Figure 2. Schematic representa- tions of morphine before the thermal stress tion of the second phase of the (50ºC) promoted an increased latency of aversive behavior of the snail Megalobulimus abbreviatus. withdrawal behavior in the snails, with the Note that, after protraction, the oral tentacles and optic tentacles remaining snail exhibited anterior lifting of partially retracted and flaccid. the head-foot complex 1 cm from the substrate, characteriz- The animals treated with 5.0, 10.0 or ing aversive behavior. 20.0 mg/kg body weight morphine displayed a partial retraction of the H-F complex, less intense than that observed in control snails. In the second phase of avoidance behavior, the snails showed a partial protraction of

Braz J Med Biol Res 38(1) 2005 Megalobulimus abbreviatus as a model for nociceptive experiments 77 flaccid palps and oral tentacles. morphine. The snails injected with a higher dose of The snails pre-treated with naloxone (5.0 morphine (20.0 mg/kg body weight) showed mg/kg body weight) and injected with mor- pedal muscle rigidity before being placed on phine (20.0 mg/kg body weight) 5 min later the hot plate. The optic and oral tentacles of displayed both phases of the avoidance be- these snails remained protracted and flaccid havior, with an average response latency of throughout all phases of the test. All animals 10.6 s. The response latency of this group in this group that received morphine before the thermal aversive stimulus presented a 90 thick yellowish mucus secretion. With this 80 * higher morphine dose the response latency 70 was significantly longer (means ± SD, 63.18 60 ± 14.47 s) than those recorded with 5.0 mg/ 50 kg (34.22 ± 12.7 s) or 10.0 mg/kg (34.8 ± 40 7.78 s) of morphine. Nevertheless, there was 30 no significant difference in the response la-

Response latency (s) 20 * tency between snails that received 5.0 or 10 10.0 mg/kg body weight morphine (Figure 0 Control Saline Morphine Morphine Morphine Naloxone 3). The snails only injected with saline dis- 5 mg/kg 10 mg/kg 20 mg/kg 5 mg/kg + morphine played an response latency of 29.1 ± 9.91 s 20 mg/kg and exhibited a similar behavior to the con- trol group (33.44 ± 8.53 s). Furthermore, Figure 3. Effect of treatment with morphine, naloxone plus morphine, and saline (1.0 ml) on there was no significant difference between the response latency after thermal aversive stimuli (50ºC) 15 min after injection. Data are reported as means ± SEM for N = 9 animals in all groups. Note the significant difference the groups receiving saline or 5.0 or 10.0 mg/ between the group treated with 20.0 mg/kg body weight morphine and all other groups (P kg body weight morphine in terms of re- < 0.001), and the significant difference between the naloxone plus morphine-treated group sponse latency. and all other groups (P < 0.01). The vertical axis shows the response latency. *P < 0.01 (ANOVA followed by the Tukey test). The snails that received naloxone (2.5, 5.0, 7.5 mg/kg body weight) exhibited biphasic behavior, faster than that observed in control animals. However, lifting of the 90 H-F complex 1 cm above the substrate oc- 80 curred at 16.26 ± 3.19, 11.5 ± 1.64 and 7.38 70 ± 1.6 s, respectively, for each of the above 60 doses. The latency of the response obtained 50 with these naloxone doses was significantly 40 shorter than that of the control (33.44 ± 8.53 s; P < 0.01 for dose of 2.5 mg and P < 0.001 30 Response latency (s) for doses of 5.0 and 7.5 mg/kg) and saline 20 * * * groups (29.1 ± 9.91 s; P < 0.05 for dose of 10 * 2.5 mg/kg, P < 0.01 for dose of 5.0 mg/kg 0 Control Saline Naloxone Naloxone Naloxone Naloxone and P < 0.001 for dose of 7.5 mg/kg), but 2.5 mg/kg 5 mg/kg 7.5 mg/kg 5 mg/kg + morphine there were no significant differences between 20 mg/kg the three naloxone-treated groups (Figure 4). These naloxone doses did not interfere Figure 4. Effect of naloxone, naloxone plus morphine, and saline (1.0 ml) on the response with motor behavior. The snails exhibited latency after thermal aversive stimuli (50ºC) 15 min after injection. Data are reported as means ± SEM for N = 9 animals in all groups. *P < 0.05 compared to control and saline- retracted and flaccid oral and optic tentacles, treated groups (ANOVA followed by the Tukey test). No significant difference was ob- similar to those of the animals treated with served between the groups treated with different naloxone doses.

Braz J Med Biol Res 38(1) 2005 78 M. Achaval et al.

differed significantly (P < 0.001) from that quite different from that observed at 22º and of the animals treated with morphine or sa- 30ºC. This withdrawal or avoidance behav- line and control, but not among the groups ior has also been identified in other gastro- injected with naloxone (Figure 3). pods, such as C. nemoralis, M. sanctipauli, The snails injected with saline alone dis- H. aspersa, and L. maximus (2,4,7,17,18), played an average response latency of 29.1 s although some differences were noted. Of and a behavior similar to that of the control particular note were the latency times for group. Also, there was no significant differ- snails to display the H-F lifting response (C. ence between the groups treated with saline nemoralis, 5-6 s; M. sanctipauli, 6-8 s, and or 5.0 and 10.0 mg/kg body weight morphine M. abbreviatus, 33 s). An explanation of the in terms of response latency. difference in response latency between the two species of Megalobulimus may be the Discussion time of day when the snails were stimulated. In the nociception of C. nemoralis, a light- In the present study, we examined the dark rhythm of thermal response latency was behavior of the snail M. abbreviatus after the found, with latency being longer during the application of thermal stimuli and the effects mid-dark period than during the mid-light of morphine and the antagonist naloxone on period (19). Nevertheless, the experiments thermal avoidance behavior. The snails on the two Megalobulimus species were car- showed exploratory behavior at ambient tem- ried out during the same mid-light period perature (22ºC) or non-thermal stress (30ºC), (4,20). but a stereotyped behavior at 50ºC. These During thermal stress, M. abbreviatus results were similar to those obtained with developed a complex behavior similar to other species of gastropods (4,7). that described for C. nemoralis (17). These M. abbreviatus displayed exploratory snails displayed biphasic avoidance behav- behavior without locomotion when placed ior including protraction and retraction of on the nonfunctional hot plate (22ºC) in the body. In the first phase of this behavior, relation to the animals in their habitat (cages). C. nemoralis displayed complete withdraw- The exploratory behavior consisted of move- al of the body into the shell, while M. ments of the H-F complex as well as of the abbreviatus displayed partial withdrawal oral tentacles and labial palps. The latter consisting of elevation of the ventral-medial structures showed retraction at first contact region of the anterior aspect of the body, with the substrate and were immediately supported only by the lateral parapodium. protracted, subsequently exhibiting a ten- These movements involve the columellar, tacular palpation of the substrate. This ex- tentacular retractor and pedal muscles (21), ploratory behavior was also described in the which are controlled by neurons located in Helix species, with satiated animals usually the pedal and cerebral ganglia (22,23). Thus, showing no locomotion (5). Also, the avoid- we can conclude that M. abbreviatus showed ance behavior was not observed at similar a sequence of escape behaviors similar to hot plate, (nonfunctional) temperatures in that shown by C. nemoralis (17). C. nemoralis, M. sanctipauli, H. aspersa or The color and consistency of the mucus L. maximus (2,4,7,9,18). secretion varied according to the thermal The pulmonate snail M. abbreviatus, stimulus. In M. abbreviatus stimulated at 22º when exposed to thermal stress (50ºC), pre- or 30ºC no modification was detected. How- sented withdrawal behavior characterized by ever, after thermal stress a copious, thick, lifting of the H-F complex, with an average yellowish mucus secretion, and swelling of response latency of 33 s. This behavior was the foot were observed, indicating a prob-

Braz J Med Biol Res 38(1) 2005 Megalobulimus abbreviatus as a model for nociceptive experiments 79 able inflammatory process in the foot. viatus. These results, together with the de- Our findings showed that morphine elic- tection of Met-enkephalin-like immunore- ited an increase in the latency of the aversive activity in the central nervous system of this behavior and that naloxone blocked this be- snail (11), provide evidence of the involve- havior causing an antiaversive effect in M. ment of an endogenous opioid system in the abbreviatus, as has been described in verte- control of thermal withdrawal behavior and brates and other invertebrates (1,2,4,7,9,18, nociception. On other hand, it is well known 20,24-27). The fact that naloxone suppressed that one of the most difficult problems in the analgesic response of morphine and re- neurobiology is functional identification of duced the nociceptive response of the en- neurons involved in a network underlying dogenous peptides suggests the existence of certain behaviors (31). Thus, this experi- an opiate mechanism in M. abbreviatus. mental model can be used for subsequent In addition, it is well known that mor- investigations about nociception to be con- phine produces behavioral inactivity and ducted in our laboratory, such as the detec- muscular rigidity in both humans and ani- tion of the nociceptive pathway and of the mals (26,28). At low doses it enhances loco- neurotransmitters and peptides involved in motion, while at higher doses it produces a this circuit. biphasic effect. In the first phase, spontane- ous activity is reduced and in the second Acknowledgments phase, locomotion increases. At the highest doses, morphine produces a state character- We are grateful to Dr. Alberto A. Rasia- ized by muscular rigidity and lack of sponta- Filho (Fundação Faculdade Federal de Ciên- neous activity (29,30). cias Médicas de Porto Alegre) for valuable Both morphine and naloxone affected comments on the manuscript and to Anto- the thermal avoidance behavior of M. abbre- nio. G. Severino for technical assistance.

References

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