The Temporal Pattern of Mating‐Induced Immediate‐Early Gene Product Immunoreactivity in LHRH and Non‐LHRH Neurons of the Estrous Ferret Forebrain

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Abstract
The mating-induced preovulatory surge of luteinizing hormone (LH) lasts for at least 12 h in the female ferret. This prolonged increase in circulating LH is presumably accompanied by a corresponding elevation in the activity and output of luteinizing hormone-releasing hormone (LHRH) neurons projecting to the hypothalamic-hypophyseal portal blood vessels and adenohypophysis. We used the protein products of the immediate early genes (IEGs) c-fos, and c-jun as markers of neural activation in order to determine whether a sub-population of LHRH neurons is differentially activated by mating and whether non-LHRH neurons in specific forebrain regions are selectively activated at different times during the mating-induced preovulatory LH surge. In Experiment 1, estrous female ferrets were perfused 0.5, 1.5, 3.0, 6.0 or 12.0 h after receiving one 5-min intromission from a male or after being placed alone in a testing cage for 20 min. Fos-like immunoreactivity (Fos-IR; Oncogene Ab-2 antiserum) and LHRH-like immunoreactivity (LHRH-IR; LR-1 antiserum) were visualized. The percentage of Fos-IR LHRH neurons was significantly augmented 1.5 h after mating but had returned to basal levels by 3.0 h. The double-labeled LHRH neurons were concentrated in the caudal medio-basal hypothalamus. In non-LHRH neurons the number of Fos-IR neural nuclei was significantly increased by mating in the medial preoptic area (MPOA), bed nucleus of the stria terminalis (BNST), medial amygdala (MA), ventrolateral hypothalamus (VLH), and midbrain central tegmental field (CTF) 1.5 h after mating but, as in LHRH neurons, had returned to basal levels by 3.0 h. In Experiment 2, estrous females were perfused 1.5 h or 8.0 h after either receiving one 5-min intromission or being placed alone in a testing cage, and the brains were processed for LHRH and c-Fos-like (DCH-1, Dr Gerard Evan), c-Jun-like (Jun-IR; Oncogene Ab-2) or Egr-1-like (Egr-IR; Santa Cruz) immunoreactivity. The percentage of LHRH neurons colabeled with both Fos-IR and Jun-IR was significantly greater in the 1.5 h group than in the unpaired group. Again, the induction of these IEG products occurred in LHRH neurons in the caudal medio-basal hypothalamus. Mating significantly increased the number of Fos-IR non-LHRH neural nuclei in the MPOA, BNST, MA, VMH and CTF, as well as the number of Egr-IR nuclei in the MPOA, BNST and MA in the 1.5 h group. By contrast, the number of Jun-IR non-LHRH neurons was unaffected by mating. In these Experiments we have identified a sub-population of LHRH neurons which, using Fos and Jun as markers of neural activation, is activated by mating and may be differentially involved in the generation of the preovulatory LH surge. Although the LHRH system is presumably activated throughout the duration of the 12 h preovulatory LH surge, c-Fos and c-Jun immunoreactivity in LHRH neurons is augmented only transiently. Fos-IR and Egr-IR in non-LHRH neurons show a similar time-course. Together, these results suggest that the presence of augmented levels of these proteins is not required for the maintenance or termination of the preovulatory output of LHRH.

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