SUN-094 Elevated opioid signaling in the arcuate nucleus dopaminergic (A12) neurons in lactating rats

Abstract Disclosure: P. Cromb: None. D.R. Grattan: None. S. Yip: None. The arcuate nucleus (ARN) dopaminergic (A12) neurons release dopamine to inhibit prolactin secretion. During lactation, these neurons cease dopamine production, allowing prolactin levels to rise, but begin synthesizing the opioid peptide enkephalin. However, the functional significance of enkephalin remains unknown. The reciprocal innervation of A12 neurons and the alteration of DA release by opioid receptor (OPR) antagonists have led to the hypothesis that there is an increase in opioid signaling in A12 neurons to reduce their activity during lactation, thereby decreasing dopamine output. To address this, we determined the expression of opioid receptor mRNAs in A12 neurons and evaluated the functional response of the neurons to enkephalin.RNAscope and immunohistochemical labeling of pro-enkephalin (Penk), opioid receptor (OPR) mRNAs, and tyrosine hydroxylase (TH; a marker for dopamine) were performed on the ARN of diestrous (D; n=4) and lactating (L; n=4) rats. Co-expression of Penk or OPR mRNAs in TH neurons was quantified using QuPath and compared using Student’s t-test (mean ± SEM). For functional analysis, diestrous and lactating TH-Cre female rats (n=4) were injected with adeno-associated virus (AAV)-GCaMP6s into the ARN. Four weeks later, the basal calcium activity (rhythmicity index; RI) of the TH neurons was monitored and compared following treatment with met-enkephalin (Enk; 3uM) with or without naloxone (nal; 1M). The A12 neurons co-express mu- and kappa-OPR subtypes but not the delta-OPR subtype. A significant increase in the percentage of TH neurons co-expressing Penk (D: 19.13% ± 1.45 vs. L: 57.24% ± 1.45, p < 0.002) and kappa-OPR (D: 45.11% ± 1.85 vs. L: 67.18% ± 3.26, p < 0.01), but not mu-OPR, was observed in A12 neurons of lactating rats compared to diestrous rats. Calcium imaging revealed that Enk treatment abolished the relative intensity (RI) of all A12 neurons in both diestrous (baseline [BL]: 0.712 ± 0.014 vs. Enk: 0.086 ± 0.006, p < 0.0001) and lactating rats (BL: 0.155 ± 0.018 vs. Enk: 0.035 ± 0.004, p < 0.0001); however, a subset of neurons remained unaffected during lactation. This effect was blocked by naloxone pre-treatment in both groups. Collectively, our findings demonstrate that the increased percentage of A12 neurons producing enkephalin, along with the elevated expression of kappa-OPR in these neurons, may suppress their own activity, thereby reducing dopamine output. This elevated opioid signaling in A12 neurons allows prolactin levels to rise, supporting lactation. Presentation: Sunday, July 13, 2025