Introduction

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Stress-induced analgesia (SIA) can be divided into opioid and nonopioid types (on the basis of naloxone reversibility) and is dependent on the nature of the stressful stimulus (Bodnar, 1990). SIA has been observed in young rats in response to warm water (20°C) swimming. Swimming periods of 3 min produce an opioid form of SIA that is dependent on the hypothalamic-pituitary-adrenal axis (Jackson and Kitchen, 1989). Studies with naloxone and the -selective antagonists ICI 174864 and naltrindole have shown that the opioid form of SIA is mediated by µ-receptors in 20-day-old rat pups, but by day 25 it is predominantly -sites that operate this behavior (Jackson and Kitchen, 1989; Kitchen and Pinker, 1990). We have shown that weaning plays a critical role in this transition. Swim SIA responses in pups weaned at day 21 are partially reversed following pretreatment with the -selective antagonist naltrindole, indicating an active -opioid receptor component. However, pups housed with their mother until 25 days beyond the normal day of weaning continue to exhibit µ-receptor-mediated swim SIA, insensitive to naltrindole (Muhammad and Kitchen, 1993). Delay of weaning was able to halt the µ- to -opioid receptor transition in the mediation of swim SIA by 5 to 10 days (Muhammad and Kitchen, 1993). The molecular mechanisms underlying the effect of weaning have been addressed by homogenate binding and autoradiography and these have suggested weaning activates a subpopulation of -receptors predominantly located in cortical structures (Kitchen et al., 1995). The precise physiological mechanisms underlying this receptor transition remain to be elucidated and we have now examined the behavioral trigger for this weaning-induced effect. We report here that provision of casein-rich milk for pups is the critical component in maintaining µ-receptor-mediated swim SIA up to day 25.

	Materials and Methods

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Animals and Experimental Conditions. Wistar albino rats (University of Surrey bred) of either sex were used and maintained in litters of 10 pups. Animals were cross-fostered at birth and allowed free access to water and rat chow. Other conditions and assignment to experimental groups were as previously described (Muhammad and Kitchen, 1993). Weaned, nonweaned, lactating surrogate mother, nonlactating surrogate mother, casein-rich, casein-free milk substitute, and casein-spiked, casein-free milk substitute experimental groups were studied for SIA responses at 25 days of age. Weaned, nonweaned, lactating surrogate mother, and casein-rich milk substitute groups were also tested for SIA responses at 40 days of age.

Swim-Stress Procedures and Analgesic Testing. Animals were divided into treatment groups so that analgesic testing took place for saline and drug-treated rats on separate days to minimize interday and interlitter variation. Analgesic responses were recorded immediately before drug administration by use of the tail immersion test at 50°C as described previously (Kitchen et al., 1984). Naltrindole (1 mg/kg) was administered in 0.9% saline i.p. in dose volumes no greater than 0.2 ml, 10 min before swimming stress. Animals were stressed by placing them individually in a plastic water tank (29 × 22 × 28 cm deep) containing water at 20 ± 1°C for a 3-min period as previously described for young rats (Jackson and Kitchen, 1989). At the end of the swimming period, rats were removed from the water, dried, and returned to the home cage before subsequent analgesic testing. Tail immersion responses were measured at 1, 5, 10, 15, and 30 min following swimming stress.

Drugs and Statistical Procedures. Naltrindole was purchased from Sigma and 0.9% w/v saline was obtained from Phoenix Pharmaceuticals (Gloucester, UK). Nociceptive treatment groups were compared using repeated measures ANOVA and post hoc comparisons made by Duncan's multiple range test using the Superanova software package for the Macintosh. Repeated measures ANOVA was carried out to determine an overall effect between groups. Where significant differences were observed by repeated measures ANOVA individual post hoc comparison were then carried out across treatment groups at individual time points.

	Results

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Naltrindole (1 mg/kg) had no significant effect on nociceptive latencies in unstressed 25-day-old animals from any experimental groups studied (Figs. 1 and 2). The magnitude of SIA in response to swimming was similar in all groups (latency 5-7 s; Figs. 1 and 2). Naltrindole (1 mg/kg) did not significantly alter swim SIA in 25-day-old nonweaned animals or in pups from litters provided with a lactating surrogate mother (Fig. 1, b and d). In weaned pups and those housed with a nonlactating surrogate, naltrindole significantly reduced swim SIA (Fig. 1, a and c).

View larger version (31K): [in this window] [in a new window]   Fig. 1.   Effect of naltrindole (1 mg/kg) on analgesia induced by a 3-min swim in 25-day-old weaned rats (a), 25-day-old nonweaned rats (b), 25-day-old pups transferred to a nonlactating surrogate at day 21 (c), and 25-day-old pups transferred to a lactating surrogate at day 21 (d). Values represent mean ± S.E.M. for 8 to 10 animals. Saline-injected, unstressed (); saline-injected, swim-stressed (); naltrindole (1 mg/kg), unstressed (); naltrindole (1 mg/kg), swim-stressed (). Pre, pretest response 10 min before swim stress. *P < 0.05 swim-stressed versus appropriate unstressed control; +P < 0.05 saline-treated, swim-stressed versus naltrindole-treated, swim-stressed.

Swim SIA was unaffected by naltrindole (1 mg/kg) when the birth mother was replaced at day 21 by a bottle containing casein-rich milk substitute (Fig. 2a). However, swim SIA was significantly reduced following naltrindole (1 mg/kg) pretreatment in 25-day-old pups provided with a bottle of casein-free milk formula from day 21 to day 25 (Fig. 2b). Analgesic responses in pups provided with a bottle of casein-spiked milk formula were unaffected by pretreatment with naltrindole (1 mg/kg), corresponding to nonweaned, lactating surrogate mother, and casein-rich milk substitute-fed groups (Fig. 2c).

View larger version (24K): [in this window] [in a new window]   Fig. 2.   Effect of naltrindole (1 mg/kg) on analgesia induced by a 3-min swim in 25-day-old casein-rich milk-fed rats (a), 25-day-old casein-free milk-fed rats (b), and 25-day-old casein-spiked milk-fed rats (c). Values represent mean ± S.E.M. for 8 to 10 animals. Saline-injected, unstressed (); saline-injected, swim-stressed (); naltrindole (1 mg/kg), unstressed (); naltrindole (1 mg/kg), swim-stressed (). Pre, pretest response 10 min before swim stress. *P < 0.05 swim-stressed versus appropriate unstressed control; +P < 0.05 saline-treated, swim-stressed versus naltrindole-treated, swim-stressed.

Swim SIA was significantly antagonized in 40-day-old weaned and lactating surrogate-switched rats and completely blocked in nonweaned rats by naltrindole (1 mg/kg) pretreatment, whereas swim SIA in 40-day-old casein-rich milk-fed rats was unaffected by naltrindole (1 mg/kg) (Fig. 3).

View larger version (32K): [in this window] [in a new window]   Fig. 3.   Effect of naltrindole (1 mg/kg) on analgesia induced by a 3-min swim in 40-day-old weaned rats (a), 40-day-old nonweaned rats (b), 40-day-old rats transferred from mother to a lactating surrogate at 21 days (c), and 40-day-old casein-rich milk-fed rats (d). Values represent mean ± S.E.M. for 8 to 10 animals. Saline-injected, unstressed (); saline-injected, swim-stressed (); naltrindole (1 mg/kg), unstressed (); naltrindole (1 mg/kg), swim-stressed (). Pre, pretest response 10 min before swim stress. *P < 0.05 swim-stressed versus appropriate unstressed control; +P < 0.05 saline-treated, swim-stressed versus naltrindole-treated, swim-stressed.

At 25 days of age weaned animals and those provided with a nonlactating surrogate had significantly lower body weights than nonweaned groups, those provided with a lactating surrogate, and those fed milk substitutes. Weaned rats also had the lowest body weights at 40 days of age (Table 1).

	Discussion

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In common with our previous studies of delayed weaning, swim SIA was partially blocked by naltrindole (1 mg/kg) in 25-day-old weaned rats but was unaffected in nonweaned pups (Muhammad and Kitchen, 1993). This dose of naltrindole is selective for antagonism of -sites at this age (Kitchen and Pinker, 1990; Crook et al., 1992). Previous studies in this laboratory have demonstrated complete reversal of the swim SIA response in weaned animals by pretreatment with 5 mg/kg naltrindole, suggesting exclusive -mediation of this response. However, at this dose naltrindole may exert some µ-antagonist activity and therefore the lower concentration was used for the current experiments to address only the -receptor component (Muhammad and Kitchen, 1993).

Replacement of the mother with a lactating surrogate produced swim SIA responses similar to those of nonweaned pups, i.e., unaffected by naltrindole. In contrast, replacement with a nonlactating adult female resulted in responses comparable to those of weaned groups. Despite the likely absence of milk, pups are still able to suckle, although previous studies have demonstrated pups over 2 weeks of age choose to suckle, at a lactating nipple over nonlactating (Kenny et al., 1979; Blass, 1990). Our results therefore indicate that the opioid receptor switch in the mediation of swim SIA responses is not influenced by the psychological stimulus of maternal deprivation but is due to reduction in the suckling stimulus and/or dietary modification.

Replacement of the mother at the time of weaning with a casein-rich milk formula prevents the µ- to -receptor switch in mediation of swim SIA at 25 days of age, whereas pups fed a similar composition casein-free formula exhibit -mediation. These findings suggest loss of suckling is not the stimulus for the µ- to -receptor transition observed in weaned and nonlactating surrogate groups. Furthermore, our results clearly show that the presence of casein in the milk substitute is the regulatory component for maintenance of µ-receptor mediation of swim SIA. Spiking of casein into the casein-free formula results in maintained µ-mediated swim SIA, confirming casein is the critical component. Because confirmation of µ-receptor mediation was not carried out in casein-treated groups it is not possible to be 100% sure that casein treatment does not impact on other receptor systems. Although there is the possibility that dietary alteration has a nonopioid receptor component we observed no difference in the magnitude of non--mediated analgesia nor in the duration of analgesia. It therefore seems likely that what is retained is indeed µ-receptor mediated.

Casein is a milk-derived protein that has been shown to contain peptide fragments that can exert opioid activity (Brantl et al., 1979; Brantl, 1984; Teschemacher et al., 1997). The most widely studied group of these peptides is the -casomorphins that have been identified in bovine, ovine, and human -casein (Teschemacher et al., 1997). Interestingly, -casomorphins had previously been undetected in -casein from rat and mouse (Teschemacher et al., 1990). Penetration of the blood-brain barrier is likely to be less restricted in 25-day-old rat pups (Ermisch et al., 1985; Reid and Hubbell, 1994; Teschemacher et al., 1997) and it is therefore possible that milk-derived -casomorphin-like peptides could elicit centrally mediated behavioral effects (Blass and Blom, 1996). The effect of casein upon opioid receptor development persists to day 40 when the blood-brain barrier should be fully developed in the rat and we must therefore entertain the possibility that the effect of these peptides may be exerted locally or peripherally rather than centrally. An alternative possibility is that casein contains a novel component distinct from the -casomorphins that is responsible for the regulation of opioid receptor development. Protein and carbohydrate levels in rat milk do not change significantly between days 5 and 20 after birth of the litter although fat levels decrease over this time (Godbole et al., 1981). However, by 25 days of age the time spent drinking and feeding by pups reaches adult levels and there is very little suckling (Thiels et al., 1990) evidenced by the low quantities of milk found in rat stomach at this age (Henning, 1981). The absolute amount of casein ingested from around the second week when they begin to eat solid food is thus likely to decrease and by 40 days of age rat pups are likely to be totally reliant upon rat chow diet and water as a possible food source. The critical component in casein is therefore diminishing after around 14 days of age.

The normal process of weaning takes pups from a high protein-high fat diet to a high carbohydrate-low protein diet (Jenness, 1974). The groups used in these experiments demonstrated significant differences in body weight at 25 days. Both weaned and nonlactating surrogate groups had lower body weights than nonweaned, lactating surrogate, and milk substitute-fed groups. There was no difference in body weight observed between animals fed on the three milk substitutes, which suggests the weaning-induced µ- to -transition is not the result of a gross nutritional change.

At the normal age of weaning (21 days) around 75% of the total adult levels of -receptors are developed in the rat brain, whereas µ- and -sites are fully mature (Spain et al., 1985; McDowell and Kitchen, 1986; Kitchen et al., 1992). Membrane homogenate binding and autoradiographic studies have demonstrated that weaning stimulates the development of a subpopulation of -receptors recognized by [³H]deltorphin I. These differences may indicate the expression of a -receptor subtype encoded for by a different gene, or that a subpopulation develops after weaning, which couples to a different G protein and that these coupled sites are recognized by [³H]deltorphin I (Kitchen et al., 1995; Kelly et al., 1998). This subpopulation of receptors may be responsible for the -receptor-mediated swim SIA observed in weaned, nonlactating surrogate mother, and casein-free milk substitute-fed groups.

By day 30 the effect of delayed weaning is lost because naltrindole is able to reverse swim SIA regardless of extended housing with their mother (Muhammad and Kitchen, 1993). Naltrindole partially reversed swim SIA in 40-day-old weaned and lactating surrogate-switched pups and completely reversed the response in 40-day-old nonweaned pups but swim SIA in animals provided with casein-rich milk substitute was unaffected by naltrindole. This suggests provision of dietary casein in a milk formula is able to delay the weaning transition beyond that achieved by extended housing with the mother. It therefore seems possible to provide prolonged alteration to brain opioid systems by dietary manipulation. Thiels et al. (1990) have demonstrated that suckling can continue up to day 34 although self-weaning can occur long before this and would suggest nonweaned pups would no longer be obtaining significant quantities of milk from their mother at day 40. The complete reversal of the swim SIA response in nonweaned 40-day-old rats by naltrindole compared with partial blockade in weaned and lactating surrogate groups may be linked to differences in body weight and therefore nutritional intake between these experimental groups. Nonweaned pups exhibit the highest body weights at 40 days of age and this may influence the complete transition from µ- to -receptor mediation of swim SIA responses observed in these animals.

In conclusion, removal of casein from the neonatal rat diet at the time of weaning results in a µ- to -transition in the mediation of swim SIA. This transition can be delayed up to at least 40 days of age by continued dietary provision of casein-rich milk substitute but not by extended housing with the mother.