ẟ-Opioid Receptor Systems in Rodent Brain: Effects of Manipulation by Developmental and Gene Knockout Approaches

Abstract

1. Weaning rat pups at day 21 stimulates a μ- to d-opioid receptor transition in the mediation of swim-stress-induced analgesia (swim SIA) by 25 days of age. Maintaining pups with their mother until 25 days of age delays this weaning-induced receptor transition with pups retaining only μ-opioid receptor mediated swim SIA. We have addressed the possibility that removal of maternal milk is the stimulus for the weaning-induced μ- to d-receptor transition, by studying the effect of lactating and non-lactating surrogate mothers and two milk substitutes (casein-rich and casein-free) on opioid receptor control of swim SIA. Litters were either left with their mother throughout studies, weaned at day 21, switched to a surrogate or provided with a milk-substitute at day 21. SIA was induced by warm water (20°C) swimming for 3 min periods and analgesia assessed by the tail immersion test (50°C).

2. Naltrindole (1mg kg^-1) significantly antagonised swim SIA in 25 day old weaned rats, in rats provided with a non-lactating surrogate and those provided with casein-free milk substitute. Naltrindole had no effect in non-weaned pups, pups given a casein-rich milk substitute or in pups from litters provided with a lactating surrogate from day 21 to day 25.

3. In 40 day old pups, swim SIA responses were attenuated by naltrindole (1 mg kg^-1) pretreatment after extended housing with the birth mother, provision of a lactating surrogate mother from weaning until this age and following weaning at the normal age of postnatal day 21. In contrast, swim SIA responses in 40 day old pups provided with a casein-rich milk substitute (after removal of the birth mother at 21 days of age) were not affected by pretreatment with naltrindole (1 mg kg^-1). The findings indicate that loss of maternal milk and thus removal of casein from the neonatal rat diet at the time of weaning is the stimulus for a p- to 5-receptor transition in the mediation of swim SIA responses. This transition can be delayed by provision of a casein-rich milk formula to pups beyond that achieved by extended housing with the birth mother or a lactating surrogate mother.

4. To study the effect of chronic blockade of the 5-opioid receptor during the neonatal period rats were chronically treated with naltrindole (1 mg kg^-1, s. c. ) from the day of birth until postnatal day 19 in the laboratory of Paz Viveros, Universidad Complutense, Madrid, Spain. Brains and spinal cords were removed and frozen at 20 or 25 days of age and autoradiographic mapping performed on sections from these tissues for the analysis of binding to μ-, d-, and κ-opioid receptors. Chronic naltrindole treatment did not produce major changes in the level or distribution of [³H]deltorphin I, [³H]DAMGO or [³H]CI-977 binding to d-, μ- , or κ-receptors respectively. Small, regional differences in binding were observed, particularly in 20 day old females where [³H]DAMGO was slightly elevated throughout the brain although this effect was not observed in the spinal cord and was absent in 25 day old animals suggesting age and gender both influence p-5-receptor inter-activity during development.

5. Brains from 5-receptor gene (DOR) knockout mice were provided by the laboratory of Professor Brigitte Kieffer, Universite Louis Pasteur, Strasbourg, France. Opioid receptors were mapped autoradiographically in these animals using [³H]deltorphin I, [³H]DAMGO, [³H]CI-977 and [³H]bremazocine to label d-, μ- , κ₁-and total κ-receptors respectively. Following short term film exposure no [³H]deltorphin I binding could be detected in homozygous DOR-deficient mice at any level throughout the brain suggesting successful deletion of the 5-receptor gene. However, following long periods of exposure residual [³H]deltorphin I binding was detected throughout the brain, particularly in regions commonly associated with high u-receptor expression. [³H]Deltorphin I binding in the DOR-deficient mice significantly correlated with regions demonstrating moderate to high levels of [³H]DAMGO binding to μ-receptors suggesting that [³H]deltorphin I is not as selective a 5-receptor ligand as some groups have previously described and demonstrates cross-over labelling with μ-sites in the absence of a 5-receptor population.

6. Autoradiographic mapping showed significant overall changes in μ- and κ-receptor binding in brains from mice lacking both copies of the 6-receptor (DOR) gene. Both p- and κ₁-receptor populations demonstrated an overall decrease in the level of binding in homozygous 5-receptor gene knockout mice, compared to wild-type animals. In contrast, [³H]bremazocine labelling of a total κ-receptor population was elevated in homozygous animals suggesting that this ligand and the κ₁-receptor selective [³H]CI-977 recognise different κ-receptor populations. The findings also emphasise the co-operativity that exists between the opioid receptor types and suggests developmental changes occur in the absence of the DOR gene in order to maintain homeostasis within the endogenous opioid system.