BDNF increases the early expression of TRH mRNA in fetal TrkB+ hypothalamic neurons in primary culture.
Known effects of neurotrophins in the developing central nervous system include induction or regulation of peptide expression. Hypothalamic postmitotic thyrotropin-releasing hormone (TRH)-producing neurons may require neurotrophins for survival and/or differentiation. This issue was investigated using primary cell cultures derived from 17-day-old fetal rat hypothalamus seeded in serum-free medium and analysed up to 4 days in vitro culture. Neurotrophin receptor (TrkB and TrkC) mRNA expression was detected by RT-PCR in fetal hypothalamus and throughout the culture period. Western blots confirmed the expression of the full-length proteins in vitro. Semi-quantitative RT-PCR showed that the addition of brain-derived neurotrophic factor (BDNF) increases TRH mRNA levels while the addition of neurotrophin-3 does not. TRH cell content was not modified. Studies on the effect of cell density or homologous conditioned medium demonstrated that endogenous factors probably contribute to determine TRH mRNA levels. One of these factors was BDNF because basal TRH mRNA levels were reduced by the addition of a Trk inhibitor or anti-BDNF. TrkB mRNA was expressed in 27% of cells and TRH mRNA in 2% of cells. The number of TRH+ cells was not affected by BDNF treatment. Forty-eight per cent of TRH neurons contained TrkB mRNA; these neurons had higher amounts of TRH mRNA than TrkB- neurons. Only TrkB+ cells responded to BDNF by increasing their TRH mRNA levels suggesting that BDNF may directly affect TRH biosynthesis. In conclusion, fetal hypothalamic TRH neurons are probably heterogeneous in regard to the neurotrophic factors enhancing peptide and mRNA levels. BDNF enhances TRH mRNA levels in a population of TrkB+ fetal hypothalamic TRHergic neurons in primary culture. However, additional influences may be necessary for the establishment of peptide phenotype in the TrkB+ neurons.
Guerra-Crespo
M
M
Departamento de Genética y Fisiología Molecular, Instituto de Biotecnología, Universidad Nacional Autónoma de México, A.P. 510-3, Cuernavaca, Mor., 62271, México.
Ubieta
R
R
Joseph-Bravo
P
P
Charli
J L
JL
Pérez-Martínez
L
L
D000818:Animals; D015153:Blotting, Western; D019208:Brain-Derived Neurotrophic Factor / Q000494:pharmacology*; D002452:Cell Count; D002478:Cells, Cultured; D017077:Culture Media, Conditioned; D004076:Digoxigenin; D007031:Hypothalamus / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D009474:Neurons / Q000187:drug effects / Q000378:metabolism*; D011233:Precipitin Tests; D012333:RNA, Messenger / Q000096:biosynthesis*; D011863:Radioimmunoassay; D051381:Rats; D017208:Rats, Wistar; D020813:Receptor, trkB / Q000235:genetics / Q000378:metabolism*; D020812:Receptor, trkC / Q000096:biosynthesis; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013973:Thyrotropin-Releasing Hormone / Q000096:biosynthesis*
D019208:Brain-Derived Neurotrophic Factor; D017077:Culture Media, Conditioned; D012333:RNA, Messenger; D013973:Thyrotropin-Releasing Hormone; D020813:Receptor, trkB; D020812:Receptor, trkC; D004076:Digoxigenin