Triiodothyronine Receptors in Developing Mouse Neuronal and Glial Cell Cultures and in Chick-Cultured Neurones and Astrocytes

Abstract

The evolution of L-triiodothyronine (T₃) receptors was studied in developing cultures of cells dissociated from cerebral hemispheres of 14-day-old mouse embryos, which present successive distinct periods of cell proliferation and/or maturation. These periods are characterized essentially as neuronal from 1 to 12 days in vitro (DIV) and glial between 12 and 60 DIV. Furthermore myelin-related membranes are produced in this culture system. Binding capacities of the T₃ nuclear receptors increased from 1 to 6 DIV, when it reached a maximum (16 fmol/100 µg DNA). A similar increase of the DNA content of the cell was observed until 8 DIV. Thereafter a sharp fall of receptor concentration leading to a 5-fold decrease in the binding capacity occurred until day 15, a period at which neurones disappeared from the cultures. From 25 to 50 DIV (coinciding with the glial period), the concentration of receptor remained more or less constant (1–2 fmol/100 µg DNA). In parallel, the DNA content did not vary greatly between 30 and 50 DIV. Scatchard analysis revealed the presence of a single class of receptors at 6 and 20 DIV, representative of ''neuronal'' and ''glial'' periods, respectively. The equilibrium dissociation constant (Kd) of the nuclear receptor from cells at 6 DIV (2 x 10^–10M) was similar to that found at 20 DIV. These results were confirmed using pure cultured neurones and astrocytes prepared from embryonic chick brain. The effect of T₃ on the cellular gangliosides used as an index of neuronal cell maturation, and on cerebroside sulfotransferase (CST), an enzyme involved in the production of myelin sulfatides, was studied to determine a possible correlation between the binding capacity of the T₃ nuclear receptor and the response of the cultured cells to thyroid hormone. Our data demonstrate that T₃ had no significant effect either on the content of gangliosides or on their developmental pattern, while it increased the level of CST activity by 75% between 18 and 25 DIV. These results show that, although the concentration of T₃ receptors per 100 µg DNA in glial cells was lower than that in neurones, it was nevertheless sufficient to elicit a response in oligodendrocytes.

Regulation of Neuronal Thyroid Hormone Receptor α₁ mRNA by Hydrocortisone, Thyroid Hormone and Retinoic Acid

“…A number of studies show the presence of thyroid hormone receptors in mammalian brain [14,[24][25][26][27][28][29][30]. Evidence indicates that T3 nuclear receptor in brain is not regulated by T3 [14][15][16][17].…”

“…Evidence indicates that T3 nuclear receptor in brain is not regulated by T3 [14][15][16][17]. In other tissues, T3 regulates its receptor niRNA levels but in an inverse manner [16].…”

“…Second, in mixed-cell cultures the stimulation by HC reaches a peak at 13 days and attains about the same degree of stimula tion (5-fold) as that which was attained in the neuronenriched culture. In the mixed-cell cultures of brain cells used in these studies, the period of neuronal maturation is between 1 and about 13 days and the period of glial maturation usually reaches its maximum between 20 and 35 days [14], Also during the latter period neurons no longer exist in the culture. The HC stimulation therefore coincides with the period of neuronal maturation.…”

“…15, 19 and 24 days (mixed cells) and 7, 9, 11 and 13 days (neurons). Studies with neuronalenriched cultures were purposely limited to 13 days, since mainte nance of nontransformed neurons is difficult beyond 2 weeks [14].…”

“…T3 a r and a 2-receptors are identical in the first 370 amino acids, but differ in the C'terminal region, which contains the hormone-binding domain in the a,-receptor. The a 2, which can bind the DNA-responsive element but not the ligand, may play an inhibitory role in T3 action [11][12][13], Existing evidence indicates that the T3 nuclear recep tor in brain is not regulated by T3 [14][15][16][17]. The specific DNA sequence to which the TRoq binds with highest affinity is the octamer 5'-TAAGGTCA.…”