Nutrient Requirements and Interactions Analgesic Response to Stress Is Reduced in Perinatally Undernourished Rats1,2 A. C. Gutiérrez and E. A. Keller Departamento de Farmacologı́a, Facultad de Ciencias Quı́micas, Universidad Nacional de Córdoba, Agencia Postal 4, C.C. 61, 5000 Córdoba, Argentina KEY WORDS: • perinatal undernutrition • opiate agent • rats • stress • analgesia • protein undernutrition Early-age undernutrition induces, even after a nutritional recovery period, alterations in different behavioral paradigms, such as exaggerated behavioral responses to stress (Barnes et al. 1968, Levitsky and Barnes 1970, Smart 1971). We previously suggested that this insult during early life probably affects the ability of undernourished rats to cope with environmental demands in adulthood (Keller et al. 1994). This point of view is based on much evidence suggesting that the adaptive changes in monoaminergic receptors may play a role in the maintenance of normal behaviors after exposure to stressful or aversive events (Cancela and Molina 1987, Cancela et al. 1988, Kennett et al. 1985, Stone 1987) and on the fact that rats undernourished during early life are unable to develop these adaptive changes in monoaminergic receptors following successive stress exposures (Keller et al. 1990 and 1994). On the other hand, a large number of findings show that exposure to a variety of stressors leads to a pain suppression effect, termed stress-induced analgesia (SIA),3 which may be mediated by either opiate or nonopiate mechanisms (Akil et al. 1978, Amir et al. 1980, Grisel et al. 1993, Lewis et al. 1980, Olson et al. 1994). For example, SIA induced by restraint and long forced swims has been reported to have an opiate origin, since this analgesic response is blocked by naloxone and potentiated when enzymatic degradation is prevented (Amir and Amit 1978, Bodnar et al. 1980, Christie et al. 1981, Greenberg and O’Keefe, 1982, Kelly and Franklin 1987) and, conversely, short swims induced nonopiate analgesia (Galea et al. 1993, Tierney et al. 1991). Several pieces of evidence suggest that, apart from inducing a pain-suppressing effect, SIA would play an adaptive role in aversive situations, necessary for coping with stressful conditions (Amit and Galina 1986, Sumová and Jakoubek 1989). In accordance with our hypothesis that early undernutrition probably affects the adult’s ability to cope with aversive situations and that SIA could be regarded as an adaptation of the organism to aversive situations, we have evaluated the painsuppression effects induced by two aversive situations, namely forced swimming (FS, short and long) and acute restraint, in rats undernourished in early life. In addition, the analgesic responses induced by different morphine (MOR) doses were evaluated in rats given the same dietary treatment. MATERIAL AND METHODS Animals and diets. A previously described protein deprivation regimen (Marichich et al. 1979) was used. Briefly, Wistar strain female rats from our colony were divided into two groups at d 14 of pregnancy and fed isocaloric diets containing 240 and 80 g casein/ kg, respectively, for control and undernourished rats. Diets also contained 4 g D,L-methionine/kg. After weaning (30 d), pups continued to receive the same diet as their dams until 50 d of age. Both groups were thereafter fed a balanced nonpurified diet (g/kg diet: protein, 180; fiber, 55; fat, 40; energy, 11.6 MJ/kg diet; Cargill, Buenos Aires, Argentina) for at least 90 d before the experiment (i.e., rats were at least 140 d old). Undernourished and control male rats used in these experiments came from different litters, so that sibling replication was consistently avoided. When the experiments were performed, 1 Supported by grants from the Consejo Nacional de Investigaciones Cientı́ficas y Técnicas (CONICET) and from CONICOR, Córdoba, Argentina. 2 The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked ‘‘advertisement’’ in accordance with 18 USC section 1734 solely to indicate this fact. 3 Abbreviations used: FS, forced swimming; MOR, morphine; % MPE, percent maximum possible effect; SAL, saline; s.c., subcutaneous; SIA, stress induced analgesia. 0022-3166/97 $3.00 q 1997 American Society for Nutritional Sciences. Manuscript received 10 May, 1996. Initial review completed 6 June 1996. Revision accepted 9 December 1996. 765 / 4w16$$0018 04-02-97 14:08:46 nutra LP: J Nut April Downloaded from jn.nutrition.org by guest on January 18, 2016 ABSTRACT Stress-induced analgesia was evaluated in adult rats submitted early in life to a protein deprivation schedule. Rats were undernourished with a hypoproteic diet containing 80 g casein/kg diet from d 14 of gestation until 50 days of age. Rats were thereafter fed a balanced nonpurified diet until 140 days of age, when they were exposed to two stressors: forced swimming and acute restraint, after which the analgesic response was evaluated. In addition, the analgesic response induced by different morphine doses was determined in another group of rats. Basal latency was not different in deprived and control rats. Undernourished rats presented a significantly lower analgesic response in both stress situations. However, when the analgesic response induced by different morphine doses (1, 2, 4 and 8 mg/kg, s.c.) was assessed, a significantly higher response occurred in undernourished rats compared to control rats. This lower stress-induced analgesia in undernourished rats may account for the behavioral alterations attributed to early undernutrition. J. Nutr. 127: 765–769, 1997. GUTIERREZ AND KELLER 766 / 4w16$$0018 04-02-97 14:08:46 FIGURE 1 Effect of acute restraint on the analgesic response in control and undernourished rats. On d 6, following a 5-d habituation period, all rats were immobilized for 2 h, and the tail flick-latencies were determined 30, 60 and 120 min after the start of the restraint session. Data are expressed as the mean percent maximum possible effect (% MPE) computed from the tail-flick response latencies using the unrestrained baseline latencies. Each point represents the mean { SEM of 25 rats, SEM not shown if less than point radius. *Significantly different than control rats (Fisher’s test, P õ 0.05). stylet wires of the appropriate size. Rats were allowed to recover from surgery for at least 10 d before testing. Rats were habituated to handling and the tail-flick apparatus for 5 d before the experiment. Intraventricular injections of MOR were administered using a 10-mL Hamilton syringe inserted in the guide cannula by means of a length of sterile polyethylene tubing (PE-10). Injections were administered over 20-30 s periods, in a volume of 4 mL MOR dissolved in a physiologically balanced solution consisting of NaCl (7.46 g), KCl (0.20 g), MgCl2 (0.19 g) and CaCl2 (0.14 g) per L of distilled water (Yaksh and Rudy 1977). On completion of the experiment the cannula position was checked by injecting a 10 g/L solution of methylene blue in the same manner as for the drug microinjection. The rats were thereafter decapitated and the brains were removed and preserved in formaldehyde solution, 100 mL/L. A stereotaxic atlas (Koning and Kipllel 1963) was used as a guide for the identification of anatomic structures. Only the data from rats whose catheter placement was confirmed was taken into account. Statistical analysis. Stress-response data was analyzed by a twoway ANOVA for repeated measures followed by Fisher’s least significant difference test. In order to draw a morphine dose-response curve for each group, the areas below the %MPE curves were computed in terms of time using a statistical and pharmacokinetical data analysis program (PKCALC, Shumaker 1986). Morphine dose-response data was analyzed by three-way ANOVA for repeated measures and by Fisher’s least significant differences test (Steel and Torrie, 1980) for post-hoc comparisons of groups. A difference of P õ 0.05 was considered significant. A software package (STATISTICA Version 4.3, Statsoft, Inc. 1993) was used to calculate statistics. RESULTS Effect of acute restraint on the analgesic response. Acute immobilization induced a significant increase in tail-flick latency by control rats (Fig. 1). However, acute stress-induced analgesia was significantly lower in undernourished rats. Diet (P õ 0.04) and time (P õ 0.00001), but not their interaction, affected %MPE. The increase in tail-flick latency in control rats was significantly greater than that observed in undernourished rats at 30 min (P õ 0.05). nutra LP: J Nut April Downloaded from jn.nutrition.org by guest on January 18, 2016 body weights for control and undernourished rats were 312 { 8.7 g and 239 { 6.1 g, respectively. Rats were kept at 22 { 27C with a 12-h light:dark cycle, lights on at 0700 h, with free access to food and water. These conditions meet the standards for the care of laboratory animals as outlined in the Guide for the Care and Use of Laboratory Animals (NRC 1985). Drugs. Morphine-HCl (Lab Verardo, Buenos Aires, Argentina) was dissolved in saline, 9 g NaCl/L (SAL). Injections were administered at 1 mL/kg body weight for all doses (1, 2, 4, and 8 mg/kg body weight). Analgesic response to acute restraint. Male adult rats from control and undernourished groups were habituated to manipulation by the experimenter, to the tail-flick apparatus (handling) and to the plexiglas restraining device daily, starting 5 d before immobilization. This handling procedure was carried out so that the novelty of the experimental situation would not influence the effect of restraint. On the sixth day, each rat was placed for 2 h in a plexiglas device capped with a wooden plug, the whole device fitting the rat’s body for complete immobilization. The analgesic response was evaluated 30, 60 and 120 min after the restraint session started. Measurement of analgesic response. Analgesia was measured by the tail-flick procedure (D’Amour and Smith 1941), with modifications. Each rat was held loosely wrapped in a towel or placed in its restraint device and then taken to the tail-flick apparatus. The ventral side of the tail (about 4 cm from the tip) was exposed to a beam of radiant heat. An automated counter recorded the latency in seconds between the onset of the beam and the movement of the tail (tailflick), which cut the beam off. The stimulus intensity was adjusted so that baseline tail-flick latency for the non-stressed control rats was in the 1.40-3.70 s range. To prevent tissue damage, a 12-s cut-off time was used. Tail-flick latency data are expressed as the mean percent maximum possible effect (%MPE) according to the formula (Dewey and Harris 1975): %MPE Å [(TL 0 BL)/(12 0 BL)] 1 100; where TL is observed latency after stressor application, 12 is cut-off time in seconds and BL is baseline latency determined for unstressed rats (control and undernourished) in three trials conducted on different places on the tail at 5-s intervals. The average of the last two trials was taken as the baseline measure for each rat. Analgesic response to forced swimming. Male adult rats, both control and undernourished, were habituated to manipulation by the experimenter, to the tail-flick apparatus and to the experimental conditions of forced swimming (FS). This habituation process lasted for 5 d and was conducted between 0900 and 1200 h. It consisted of carrying the rats in individual cages from the animal room to the stressor room, placing them in a drying cage and then taking them to the analgesia-recording room. On the sixth day, the basal analgesic response (baseline latency) was determined for each rat as described above. Five minutes later, rats were segregated into two groups, each comprising both control and undernourished rats, and groups were subjected to the FS task for 5 or 15 min. Swimming was forced by placing the rats individually in acrylic cylinders (40 cm high, 18 cm wide) filled with 257C water to 17 cm deep. Thereafter each rat was placed in a cage in front of a fan delivering hot air for 10 min, put back in the home cage and taken to the analgesia-recording room. Tail-flick response was recorded 15, 30, 45, 60, 120 and 180 min after stressor. Analgesic response induced by different morphine doses. Male adult rats from control and undernourished groups were subjected to the handling process for 5 d and also were habituated to being placed on the scales then returned to their home cages. On the sixth day the basal analgesic (predrug) latency was determined for all animals. The control and undernourished rats were divided into five subgroups, and these were injected with SAL (1 mL/kg) or MOR (1, 2, 4 and 8 mg/kg). Postdrug latency was determined for all rats 30, 60, 90, 120 and 180 min after injection. For intraventricular injections rats were anesthetized with chloral hydrate, 160 g/L (400 mg/kg body weight, i.p.), and 22-gauge stainless steel guide cannulae were permanently implanted in the third ventricle (anteroposterior: 01.8, from the bregma; lateral: 0.0; Ventral: 04.5, from the skull top) according to the procedure described by Tseng and Fujimoto (1984). The guide cannulae were fitted with UNDERNUTRITION AND STRESS-INDUCED ANALGESIA 767 doses in undernourished rats was significantly displaced to the left compared to that of the controls (P õ 0.001). In addition, intracerebroventricular administration of MOR also produced a significantly greater tail-flick latency in undernourished rats compared to controls. Drug (P õ 0.007), time (P õ 0.0001) and the interactions diet 1 time (P õ 0.003), drug 1 time (P õ 0.00001) and diet 1 drug 1 time (P õ 0.03) affected %MPE. The Fisher’s test for the posterior individual comparisons revealed that a 5 mg per rat MOR injection provoked an increase in tail-flick latency in undernourished rats expressed as %MPE, significantly higher than that of control rats at 15 and 30 min. Percent MPE at 15 min was 23.53 { 4.56 for control rats and 36.56 { 4.41 for undernourished rats; at 30 min, values were 27.91 { 4.98 for control rats and 48.49 { 10.44 for undernourished rats (P õ 0.001). DISCUSSION Effect of forced swimming on the analgesic response. Control rats subjected to the 15 min FS test had an increase in tail-flick latency, which reached its peak between 15 and 30 min after test (Fig. 2). This increase was significantly lower in undernourished rats. Diet (P õ 0.05), time (P õ 0.00001) and their interaction (P õ 0.01) affected %MPE. The increase in the tail-flick latency in control rats was significantly greater than that observed in undernourished rats at 15, 30 and 45 min (P õ 0.05). When the FS test lasted for 5 min, the analgesic response of undernourished rats did not differ from controls (data not shown). Effect of different MOR doses on the analgesic response. The subcutaneous administration of different MOR doses was effective in inducing analgesia, as evidenced by the increased tail-flick latency for control and undernourished rats (Fig. 3). However, this analgesic effect was significantly stronger in undernourished rats than in controls. On the other hand, as expected, SAL administration (vehicle) produced no significant difference in analgesic response between the two groups (Fig. 3). Diet (P õ 0.0007), MOR dose (P õ 0.00001), time (P õ 0.00001) and the three-way interaction (P õ 0.02) affected % MPE. The increase in tail-flick latency in undernourished rats administered the 1 and 2 mg/kg MOR injection, expressed as % MPE, was significantly higher than that observed in control rats at 30 and 60 min (P õ 0.05). The 4 mg/ kg MOR injection produced an increase in post-drug latency in undernourished rats that was significantly greater than that of control rats at 90, 120 and 180 min (P õ 0.02). To compare the effects of different MOR doses in the control and undernourished groups, tail-flick latency expressed as %MPE was transformed to the area under the dose-effect curve for the 30-180 min period, for each group and for each dose (Fig. 4). In undernourished rats different MOR doses (1, 2, 4 and 8 mg/kg) provoked a left-hand displacement of the doseresponse curve compared to that for similarly treated control rats (Fig. 4). The dose-response curve for the different MOR / 4w16$$0018 04-02-97 14:08:46 nutra LP: J Nut April Downloaded from jn.nutrition.org by guest on January 18, 2016 FIGURE 2 Effect of the 15-min forced swimming test (FS) on the analgesic response in control and undernourished rats. All rats were subjected to handling for 5 d and habituated to the experimental conditions under which the stressor was to be applied. On d 6, rats were subjected to FS for 15 min. The tail-flick latency was recorded 15, 30, 45, 60, 120 and 180 min after stressor. Each point represents the mean { SEM of 17-19 rats. *Significantly different from control rats (Fisher’s test, P õ 0.05). As previously reported, after acute immobilization the analgesic response of control animals increases over time (Amir and Amit 1978, Bodnar et al. 1980). However, in the stressed undernourished rats, this analgesic response was lower than that of controls. Similar results were found after a 15 min FS session; that is, stressed controls displayed a higher response than stressed undernourished rats. It should be noted that no differences were observed in the latency of analgesic response between nonstressed controls and nonstressed undernourished rats. The stress-induced analgesic response is considered to be an adaptive response necessary for coping with stressful situations (Amit and Galina 1986, Sumová and Jakoubek 1989). Because our present results show that undernourished rats have a lower analgesic response after enduring the stress of restraint and 15 min of forced swimming, these data confirm our hypothesis that early-life undernutrition affects the ability to cope with aversive situations. It has been shown that opioid mechanisms mediate analgesic responses induced by restraint and long-duration swims (Amir and Amit 1978, Bodnar et al. 1980, Christie et al. 1981, Greenberg and O’Keefe, 1982, Kelly and Franklin 1987); therefore, the lower analgesic response observed in undernourished rats may be mediated by opioid mechanisms. In support of this, the same analgesic response was observed in control and undernourished rats after a short swim that induces nonopiate analgesia (Galea et al. 1993, Tierney et al. 1991). On the other hand, when the analgesic response elicited by s.c. administration of different morphine doses was assessed in both groups, the response was significantly higher in undernourished than in control rats. Also, since both intracerebroventricular and s.c. MOR administration in undernourished rats induced the same analgesic response, the possibility of a pharmacokinetic alteration may be discarded. Thus, the analgesic response observed after the administration of the opiate agonist may indicate supersensitivity of the opioid brain receptors in undernourished rats. Since MOR induces its analgesic effect by selectively interacting with the m-type opioid receptor (Olson et al. 1994), it follows that undernourished rats present supersensitive m-type opiate brain receptors. In turn, this higher reactivity to MOR indicates that the lower analgesic response observed in the undernourished rats subjected to stressful situations may be the consequence of reduced release of opiates caused by such situations, and that the development of supersensitivity of the opiate receptors was not sufficient to compensate for the deficit in opiate release. In support of this, a reduced release of endorphin following stressful situations has been demonstrated in undernourished rats (Perry and Izquierdo 1989, Vendite et al. 1985). In addition, recent evidence from GUTIERREZ AND KELLER 768 our laboratory has demonstrated that the combined treatment with MOR or b-endorphin and stress in undernourished rats causes adaptive changes in their brain monoaminergic receptors (Keller et al. 1994, and in press), suggesting that the absence of this adaptive change in response to stress following early undernutrition was due, at least in part, to a functional deficiency in the activation of an endogenous opiate mechanism, triggered by repeated aversive experiences (Keller et al. 1994, and in press). Since several reports demonstrated alterations in different brain receptors as a consequence of early life undernutrition (Keller et al. 1982, Wiggins et al. 1984), the supersensitivity observed in undernourished rats may be due to a higher density of m-type brain receptors; however, signal transduction alterations (G protein and/or effectors) cannot be discarded. Even though further investigation is necessary to clarify this topic, it should be kept in mind that present as well as previous evidence from our laboratory strongly suggest that perinatal undernourishment may result in a permanent functional deficit in the opiate process involved in behavioral responses to stress. Recent evidence has shown that the modification taking place in the endogenous opiate system during development induces in adult rats behavioral alterations similar to those reported for undernourished rats (De Cabo et al. 1995). Furthermore, several reports indicate that the endogenous opiate system participates directly in the elaboration of behavioral adaptations to stressful stimuli, and that the analgesia induced by opiate release when animals are confronted with a certain stressful situation would allow the animal to adequately interact with the stressful stimulus, and therefore reach an appropriate balance with the environment (Amir et al. 1980, Amit and Galina 1986). That is to say, opiates also participate in the elaboration and expression of the emotional response to stress (Amir et al. 1980). The preceding results strongly indicate that further research on the functioning of the endogenous opiate system in undernourished animals may elucidate the neural basis of behavioral alterations observed in these animals. ACKNOWLEDGMENTS We are grateful to Gabriela Bazán for her English technical assistance and to Elsa R. Pereyra for her laboratory technical assistance. LITERATURE CITED FIGURE 4 Dose-response curves comparing the analgesic effect of different morphine (MOR) doses in control and undernourished rats. Each point was derived from the area under the corresponding 180min time course curve shown in Fig. 3. The dose-response curves for different MOR doses in undernourished rats showed a significant displacement to the left compared with the controls (P õ 0.001). / 4w16$$0018 04-02-97 14:08:46 Akil, H., Madden, J., Patrick, R. L. & Barchas, J. D. (1978) Stress-induced increase in endogenous opiate peptide: Concurrent analgesia and its partial reversal by naloxone. In: Opiate and Endogenous Peptides (W. Kosterlitz, ed.), pp. 63–70. North Holland, Amsterdam. Amir, S. & Amit, Z. (1978) Endogenous opioid ligands may mediate stressinduced changes in the affective properties of pain related behavior in rats. Life Sci. 23: 1143–1152. nutra LP: J Nut April Downloaded from jn.nutrition.org by guest on January 18, 2016 FIGURE 3 Effect of the administration of different morphine (MOR) doses on the analgesic response in control and undernourished rats. All rats were subjected to handling for 5 d. On d 6, the predrug latency was determined in all rats. Rats were subsequently injected with SAL or one of four doses (1, 2, 4 or 8 mg/kg body weight) of MOR. Postdrug latency was determined in all rats 30, 60, 90, 120 and 180 min after drug administration. Each point represents the mean { SEM of 5-14 rats. *Significantly different from control rats injected with 1 mg MOR/kg body weight (Fisher’s test, P õ 0.05). /Significantly different from control rats injected with 2 mg MOR/kg body weight (Fisher’s test, P õ 0.05). **Significantly different from control rats injected with 4 mg MOR/kg body weight (Fisher’s test, P õ 0.05). UNDERNUTRITION AND STRESS-INDUCED ANALGESIA / 4w16$$0018 04-02-97 14:08:46 Kennett, G. A., Dickinson, S. L. & Curzon, G. (1985) Enhancement of some 5HT-dependent behavioral responses following repeated immobilization in rats. Brain Res. 330: 253–263. Koning, J.F.B. & Kipllel, R. A. (1963) The Rat Brain. A Stereotoxic Atlas of the Forebrain and Lower Parts of the Brain Stem. Robert F. Krieger Publishing Co., Huntington, NY. Levitsky, D. A. & Barnes, R. H. (1970) Effects of early malnutrition on the reaction in adult rats to aversive stimuli. Nature 225: 468–469. Lewis, J. W., Cannon, J. T. & Liebeskind, J. C. (1980) Opioid and non-opioid mechanisms of stress analgesia. Science 208: 623–625. Marichich, E. S., Molina, V. A. & Orsingher, O. A. (1979) Persistent changes in central catecholaminergic system after recovery of perinatally undernourished rats. J. Nutr. 109: 1045–1050. National Research Council (1985) Guide for the use and care of laboratory animals. Publicaiton no. 85–23(rev.), National Institutes of Health, Bethesda, MD. Olson, G. A., Olson, R. D. & Rastin, A. J. (1994) Endogenous opiates: 1993 (Review). Peptides 15: 1513–1556. Perry, M.L.S. & Izquierdo, I. (1989) Effects of posttraining and pretest endorphin and ACTH administration in normal and protein malnourished rats. Peptides 10: 1117–1120. Shumaker, R. C. (1986) PKCALC: A basis interactive computer program for statistical and pharmacokinetic analysis of data. Drug Metab. Rev. 17: 331– 348. Smart, J. L. (1971) Long-lasting effects of early nutritional deprivation on the behavior of rodents. Psychiatr. Neurol. Neurochi. 74: 443–452. Steel, R. G. & Torrie, J. M. (1980) Principles and Procedures of Statistics: A Biometrical Approach (Napier, C. & Maisel, J. W., eds.), pp.182–183. McGraw-Hill Book Co., New York, NY. Stone, E. A. (1987) Central cyclic-AMP-linked noradrenergic receptors: new findings on properties as related to the actions of stress. Neurosci. Biobehav. Rev. 11: 391–398. Sumová, A. & Jakoubek, B. (1989) Analgesia and impact induced by anticipation stress: involvement of the endogenous opioid system. Brain Res. 503: 273–280. Tierney, G., Carmody, J. & Jamison, D. (1991) Stress analgesia: the opioid analgesia of long swims suppresses the non-opioid analgesia induced by short swims in mice. Pain 46: 89–95. Tseng, L-F. & Fujimoto, J. M. (1984) Evidence that spinal endorphin mediates intraventricular b-endorphin-induced Tail Flick inhibition and catalepsy. Brain Res. 302: 231–237. Vendite, D., Wofchuk, S. & Souza, D. O. (1985) Effects of undernutrition during suckling on foot shock escape behavior and on related neurochemical parameters in rats. J. Nutr. 115: 1418–1424. Wiggins, R., Fuller, G. & Enna, S. (1984) Undernutrition and the development of brain neurotrasmitter systems. Life Sci. 35: 2085–2094. Yaksh, T. L. & Rudy, T. A. (1977) Studies of the direct spinal action of narcotics in the production of analgesia in the rat. J. Pharmacol. Exp. Ther. 202: 411– 428. nutra LP: J Nut April Downloaded from jn.nutrition.org by guest on January 18, 2016 Amir, S., Brown, Z. W. & Amit, Z. (1980) The role of endorphins in stress: Evidence and speculations. Neurosci. Biobehav. Rev. 4: 77–85. Amit, Z. & Galina, Z. H. (1986) Stress-induced analgesia: Adaptive pain suppression. Physiolog. Rev. 66: 1091–1114. Barnes, R. H., Neely, C. S., Kwong, E., Labadan, B. A. & Frankova, S. (1968) Postnatal nutritional deprivations as determinants of adult rat behavior toward food, its consumption and utilization. J. Nutr. 96: 467–476. Bodnar, R. J., Kelly, D. D., Brutus, M. & Glusman, M. (1980) Stress-induced analgesia: Neural and hormonal determinants. Neurosci. Biobehav. Rev. 4: 87–100. Cancela, L. M. & Molina, V. A. (1987) Reduced apomorphine-induced sedation following chronic stress. Europ. J. Pharmacol. 134: 117–119. Cancela, L. M., Volosin, M. & Molina, V. A. (1988) Chronic stress attenuation of alpha2-adrenoceptor reactivity is reversed by naltrexone. Pharmacol. Biochem. Behav. 31: 33–35. Christie, M. J., Chesher, G. B. & Bird, K. D. (1981) The correlation between swim-stress induced antinociception and [H3] Leu-enkephalin binding to brain homogenates in mice. Pharmacol. Biochem. Behav. 16: 853–857. D’Amour, F. E. & Smith, D. L. (1941) A method for determinig loss of pain sensation. J. Pharmacol. Exp. Ther. 72: 74–79. De Cabo, C., Pujol, A. & Viveros, M. P. (1995) Neonatally administered naltrexone affects several behavioral responses in adult rats of both genders. Pharmacol. Biochem. Behav. 50: 277–286. Dewey, W. L. & Harris, L. S. (1975) The tail flick test. In: Methods in Narcotic Research (Ehrenpreis, S & Neidle, A, eds.), pp. 101–109. Dekker, New York, NY. Galea, L.A.M., Kavaliers, M., Innes, D. L. & Ossenkopp, K-P. (1993) Sex differences in opioid and nonopioid mediated swim-stress induced analgesia in deer mice vary with the breeding season. Soc. Neurosci. Abstr. 19: 170. Greenberg, R. & O’Keefe, E. H. (1982) Thiorphan potentiation of stress-induced analgesia in the mouse. Life Sci. 31: 1185–1188. Grisel, J. E., Fleshner, M., Watkins, L. R. & Maier, S. F. (1993) Opioid and nonopioid interactions in two forms of stress-induced analgesia. Pharmacol. Biochem. Behav. 45: 161–172. Keller, E. A., Cancela, L. M., Molina, V. A. & Orsingher, O. A. (1994) Lack of adaptive changes in 5-HT sites in perinatally undernourished rats after chronic stress: Opioid influence. Pharmacol. Biochem. Behav. 47: 789–793. Keller, E. A., Molina, V. A. & Orsingher, O. A. (1990) Lack of neuronal adaptive changes following chronic treatment in perinatally undernourished rats. Pharmacol. Biochem. Behav. 37: 675–678. Keller, E. A., Munaro, N. I. & Orsingher, O. A. (1982) Perinatal undernutrition reduces alpha and beta adrenergic receptor binding in adult rat brain. Science 215: 1269–1270. Keller, E. A., Rey, A., Gutiérrez, A. C. & Cancela, L. M. (in press). Reversion by agonist opiate of the lack of adaptive changes on alpha2-adrenoceptor in perinatally undernourished rats after chronic stress. Pharmacol. Biochem. Behav. Kelly, S. & Franklin, K. (1987) Role of peripheral and central opioid activity in analgesia induced by restraint stress. Life Sci. 41: 789–794. 769