Mu and kappa opioid receptor expression in the mediobasal hypothalamus and effectiveness of selective antagonists on prolactin release during lactation

Abstract

Endogenous opioid peptides are involved in prolactin release during lactation, in part by decreasing tuberoinfundibular dopaminergic (TIDA) neuronal activity. Both mu (mu) and kappa (kappa) opioid receptors have a role in the suckling-induced prolactin rise after 4-5 h up deprivation. The aim of this study was to investigate effects of mu opioid receptor antagonist, beta-funaltrexamine (beta-FNA), and kappa opioid receptor antagonist, nor-binaltorphimine (nor-BNI), on prolactin secretion and TIDA neuronal activity in lactating rats after 18 h pup deprivation. After 4 h separation from pups, the suckling-induced prolactin rise was abolished by 16 microg nor-BNI and 5 microg beta-FNA, coincident with increased dihydroxyphenylacetic acid (DOPAC):dopamine ratio in the stalk-median eminence (SME). However, after 18 h pups separation, these same doses of nor-BNI and beta-FNA did not alter the prolactin surge or DOPAC:dopamine ratios in the SME. Higher doses of nor-BNI (32 microg) and beta-FNA (10 microg) were required to inhibit suckling-induced prolactin secretion. beta-FNA (10 microg) increased the DOPAC:dopamine ratio in the SME, whereas nor-BNI (32 microg) treatment had no effect. The mu and kappa opioid receptor mRNA levels in the mediobasal hypothalamus were similar to suckled control rats after 4 h pup deprivation, but increased 1.4-fold after 18 h pup deprivation. These data support involvement of endogenous opioidergic systems in the suckling-induced prolactin rise after a prolonged (18 h) period of pup deprivation, as well as the shorter (4 h) pup deprivation period previously reported. Suppression of TIDA neuronal activity likely played a part in mu opioid receptor input to the suckling-induced prolactin rise after both 4 h and 18 h separation, whereas non-dopaminergic input was implicated with kappa opioid receptors after 18 h pup deprivation. Increased mu and kappa opioid receptors gene expression in the mediobasal hypothalamus may contribute to reduced effectiveness of opioid receptor antagonists to block suckling-induced prolactin release after 18 h pup deprivation.

Figure 1

Plasma prolactin (ng/ml) profiles from lactating dams deprived of pups for 4h (A, B) or 18h (C, D, E, F) and injected i.c.v. at 15 min before pup return with β-funaltrexamine (β-FNA) at a dose of 5 μg/5μl (A, C) or 10 μg/5μl (E) or with nor-binaltorphimine (nor-BNI) at a dose of 16 μl/5μl (B, D) or 32 μg/5μl (F). Plasma prolactin profiles (open square) of dams deprived of pups for 4h and injected i.c.v. with saline (5 μl, n=5) are repeated in panels A and B. Dams deprived of pups for 4h (at −240 min) were injected with (A) β–FNA (5 μg/5 μl, n=3) or (B) nor-BNI (16 μg/5 μl, n=4) at 15 min before time 0 min and allowed to suckle for 60 min. Plasma prolactin profiles (open square) of dams deprived of pups for 18h and injected i.c.v. with saline (5 μl, n=9) are repeated in panels C, D, E and F. Dams deprived of pups for 18h (at −1080 min) were injected with (C) β -FNA (5 μg/5 μl, n=5), (D) nor-BNI (16 μg/5 μl, n=6), (E) β -FNA (10 μg/5 μl, n=6), or (F) nor-BNI (32 μg/5 μl, n=6) at 15 min before time 0 min and allowed to suckle for 60 min. Each value represents a mean ± SEM. Significance level compared to saline-treated controls (* p < 0.05, **p<0.01, ***p<0.001).

Figure 2

3,4-dihydroxyphenylacetic acid (DOPAC) content (A, E, I), dopamine content (B, F, J), DOPAC:dopamine ratio (C, G, K) in the SME and serum prolactin concentration (D, H, L) on day 7 of lactation after i.c.v. injection of nor-BNI, β-FNA or saline. Lactating dams deprived of pups for 4h were injected with nor-BNI (16 μg/5 μl, n=6), β -FNA (5 μg/5 μl, n=6) or saline (5 μl, n=13) at 15 min before pup return and sacrificed at 60 min after suckling onset (A, B, C, D). Lactating dams deprived of pups for 18h were injected with nor-BNI (16 μg/5 μl, n=6), β-FNA (5 μg/5 μl, n=6) or saline (5 μl, n=11) at 15 min before pup return and sacrificed at 60 min after suckling onset (E, F, G, H). Lactating dams deprived of pups for 18h were injected with nor-BNI (32 μg/5 μl, n=14), β -FNA (10 μg/5 μl, n=14) or saline (5 μl, n=15) at 15 min before pup return and sacrificed at 60 min after suckling onset (I, J, K, L). Each value represents a mean ± SEM. Significance level compared to saline-treated controls (*p< 0.05, **p<0.01, ***p<0.001).

Figure 3

(A) μ opioid receptor and (B) κ opioid receptor mRNA expression (normalized to GAPDH) levels in the mediobasal hypothalamus of suckled lactating dams, 4h pup-deprived dams, 18h pup-deprived dams and diestrous rats. Data show fold differences, relative to suckled lactating rats. Each value represents a mean ± SEM of determinations from six to nine rats for pup-deprived and diestrous rats and 18 rats for suckled lactating dams used as control. Significance level compared to suckled dams (*p< 0.05, **p<0.01).