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 pain-suppression 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

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).

Tail-flick latency data are expressed as the mean percent maximum possible effect (%MPE) according to the formula (Dewey and Harris 1975): %MPE = [(TL  BL)/(12  BL)] × 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.

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: 1.8, from the bregma; lateral: 0.0; Ventral: 4.5, from the skull top) according to the procedure described by Tseng and Fujimoto (1984). The guide cannulae were fitted with 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-µL 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 µL 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.

RESULTS

When the FS test lasted for 5 min, the analgesic response of undernourished rats did not differ from controls (data not shown).

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 dose-response curve compared to that for similarly treated control rats (Fig. 4). The dose-response curve for the different MOR doses in undernourished rats was significantly displaced to the left compared to that of the controls (P < 0.001). In addition, intracerebroventricular administration of MOR also produced a significantly greater tail-flick latency in undernourished rats compared to controls. Drug (P < 0.007), time (P < 0.0001) and the interactions diet × time (P < 0.003), drug × time (P < 0.00001) and diet × drug × time (P < 0.03) affected %MPE. The Fisher's test for the posterior individual comparisons revealed that a 5 µg 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 < 0.001).

DISCUSSION

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 µ-type opioid receptor (Olson et al. 1994), it follows that undernourished rats present supersensitive µ-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 our laboratory has demonstrated that the combined treatment with MOR or -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 µ-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.