Hypothalamic dopaminergic neurons in transgenic dwarf mice: histofluorescence, immunocytochemical, and in situ hybridization studies.
Spontaneous dwarf mice, in which both growth hormone (GH) and prolactin (PRL) are undetectable, are severely deficient in the PRL-inhibiting catecholamine dopamine (DA), as well as its synthetic enzyme, tyrosine hydroxylase (TH), in the basal hypothalamus (Phelps et al., Cell Tissue Res., 240:19-25, 1985; Phelps, Brain Res., 416:354-358, 1987). In contrast, transgenically constructed dwarf mice (Behringer et al., Genes Dev., 2:453-461, 1988) show complete ablation of pituitary GH cells, but PRL cells are retained at a level of approximately 10% of normal. In order to determine the feedback effect of this reduced, rather than absent, PRL on hypothalamic DA neurons, brains of transgenic dwarf mice were examined for catecholamine transmitters by histofluorescence, for the synthetic enzyme TH by immunocytochemistry, and for TH mRNA expression by in situ hybridization. DA histofluorescence in transgenic dwarfs was comparable to that of normal littermate mice in nonpituitary regulating areas (perikarya of zona incerta [A13] of hypothalamus and in midbrain substantia nigra area [A9]). Arcuate nucleus (A12) DA neurons that inhibit PRL secretion, however, showed dim to absent fluorescence in perikarya and in external median eminence terminals in dwarfs. There were reduced (P less than 0.05) numbers of A12 TH-immunoreactive neurons in transgenic dwarfs, to approximately 60% of those in normal mice. In contrast, TH-positive neurons in other hypothalamic areas (A13, A14) had average populations equivalent to those in normal mice. Quantification of TH mRNA abundance by in situ hybridization using both image analysis of hybridization over the arcuate nucleus, and grain counts per individual A12 cell in this nucleus, indicated that relative mRNA levels were the same in normal and transgenic dwarfs. The observations indicate that reduction in pituitary PRL is accompanied by defective expression in hypothalamic tuberoinfundibular neurons, which is severe at the DA neurotransmitter level, significant regarding observable TH immunoreactivity, and undetectable with regard to TH mRNA expression. Collectively, the findings suggest that posttranscriptional processes are involved with the mediation of PRL feedback upon hypothalamic neurons. Technically and quantitatively, the report presents the feasibility of simultaneous evaluation of transmitter histofluorescence, synthetic enzyme immunocytochemistry, and mRNA expression in individual animals.
Phelps
C J
CJ
Department of Neurobiology and Anatomy, University of Rochester School of Medicine, New York.
Carlson
S
S
Hurley
D L
DL
D000818:Animals; D001111:Arcuate Nucleus of Hypothalamus / Q000737:chemistry / Q000166:cytology / Q000201:enzymology; D004298:Dopamine / Q000032:analysis* / Q000172:deficiency / Q000235:genetics; D004392:Dwarfism / Q000235:genetics*; D005260:Female; D013006:Growth Hormone / Q000032:analysis; D007031:Hypothalamus / Q000737:chemistry* / Q000166:cytology* / Q000201:enzymology; D007150:Immunohistochemistry; D051379:Mice; D008822:Mice, Transgenic / Q000235:genetics*; D008856:Microscopy, Fluorescence; D009474:Neurons / Q000737:chemistry* / Q000166:cytology* / Q000201:enzymology; D009693:Nucleic Acid Hybridization; D011388:Prolactin / Q000032:analysis / Q000502:physiology; D012333:RNA, Messenger / Q000032:analysis / Q000235:genetics; D013004:Somatostatin / Q000032:analysis; D014446:Tyrosine 3-Monooxygenase / Q000032:analysis / Q000172:deficiency / Q000235:genetics
D012333:RNA, Messenger; D013004:Somatostatin; D011388:Prolactin; D013006:Growth Hormone; D014446:Tyrosine 3-Monooxygenase; D004298:Dopamine