Iodothyronines were extracted and purified from the hypothalamus and T4 and T3 were measured by RIA, as described previously [25,26]. In brief, the entire hypothalamic block was homogenized directly in methanol, and 2000 cpm of outer ring labeled [131I]T4 and [125I]T3 were added to each sample as internal tracers for recovery. Appropriate volumes of chloroform were added to extract with chloroform/methanol (2:1) twice. The iodothyronines were then back-extracted into an aqueous phase and purified by passing this aqueous phase through Bio-Rad AG 1 �� 2 resin columns (Bio-Rad, Veenendaal, The Netherlands). After a pH gradient, the iodothyronines were eluted with 70% acetic acid, evaporated to dryness, and resuspended in RIA buffer. The extracts were counted to determine the recovery of [131I]T4 and [125I]T3 added to each sample. For the present experiment the average recovery was 50.5% for [131I]T4, and 74.5% for [125I]T3. Concentrations were calculated using the amounts of T4 and T3 found in the respective RIAs, the individual recovery of [131I]T4 and [125I]T3 added to each sample, and the weight of the tissue sample submitted for extraction.Cloning of rabbit genesTotal RNA was isolated from rabbit hypothalamic tissue using Qiazol lysis reagent (Qiagen, Venlo, The Netherlands) and subsequently purified using the RNeasy mini RNA isolation kit (Qiagen, Venlo, The Netherlands). cDNA was obtained by reverse transcription of 2 ��g total RNA using random hexamer primers. Oligonucleotides homologous to sequences surrounding the start or stop codons of human, mouse and rat proTRH, D2, MCT8, MCT10, OATP1C1, thyroid hormone receptor (TR) ��1, TR��2, TR��1 and TR��2 were designed and used for PCR. The amplified fragments were cloned into the pGEM-T vector followed by sequence analysis.