Conversion and binding of tetraiodothyronine in developing rat brain

In this study we have examined whether rat brain nuclear thyroid hormone receptors bind T₄ or metabolites of T₄ and whether there is a developmental change in brain T₄ metabolism and binding. Developing animals were injected with trace [¹²⁵I]3,5-tetraiodothyronine ([¹²⁵I]T₄) and after sacrifice brain nuclear and cytoplasmic fractions were examined to determine whether their radioactivity was represented by the injected [¹²⁵I]T₄ or any of its metabolites. Of the radiothyronines specifically bound to the nucleus, 90% was found to be triiodothyronine ([¹²⁵I] T₃) and 10% was [¹²⁵I]T₄. Of the cytoplasmic, protamine sulfate-precipitable fraction, 40% was [¹²⁵I]T₄ and 60% [¹²⁵I]T₃. Inasmuch as the percentage of [¹²⁵I] T₃ found in plasma during the same postinjection interval was similar to that present as contaminant of the injected material, it was concluded that brain [¹²⁵I] T₃ derives from local monodeiodination of T₄ to T₃. The main developmental change observed was a marked decline in the total cytoplasmic and nuclear [¹²⁵I] T₄ uptake. However, with development, the T₃/T₄ ratio remained constant in the nuclear fraction while it decreased in the cytoplasmic fraction. It is concluded that although T₃, deriving from monodeiodianation of T₄, is the main form of thyroid hormone that regulates brain development by its binding to brain nuclear receptors, the fact that T₄ is the most available from during the critical period makes it, indirectly, very important to brain development. Further, the decline observed with development in T₄ uptake and monodeiodination to T₃, may contribute to the concomitantly declining role of thyroid hormones on brain tissue.     

Effect of Cold Exposure on Thyroid Hormone Metabolism and Nuclear Binding in Rat Brain

In this study we examined whether adult rat brain tissue (cerebral hemispheres) would under cold exposure respond with changes in the local metabolism and nuclear binding of thyroid hormones (T₃, T₄). Adult, control rats kept at 22°C and cold exposed (4°C, 20 h) rats were injected with trace of ¹²⁵I-T₄ or ¹²⁵I-T₃ returned to their respective environment and sacrificed four hours later. The radioactive hormonal forms were identified and quantified in the cytoplasmic and nuclear fractions. It was found that in cold exposed rats injected with ¹²⁵I-T₄, the total cytoplasmic radioactivity was higher than that of controls. This increase was not associated with ¹²⁵I-T₄ but it reflected an increase (88 %) in its deiodination product ¹²⁵I-T₃ (¹²⁵I-T₃ (T₄)). Although total cytoplasmic ¹²⁵I-T₄ did not change, there was a decrease (28%) in its protein free cytoplasmic fraction. ¹²⁵I-T₃ (T₄) and ¹²⁵I-T₄ bound to the nuclear fraction were found to decrease by 58 and 46% respectively. Cold exposed animals injected with ¹²⁵I-T₃ also showed an increase in cytoplasmic ¹²⁵I-T₃ (81%) and a decrease in ¹²⁵I^– (40%) whereas ¹²⁵I-T₃ bound to the nuclear fraction decreased by 64%. These results indicate that cold exposure of rats decreases brain local T₃ metabolism and nuclear binding while it does not effect local T₄ metabolism.     

Effects of hypothyroidism on RNA synthesis in the adult rat brain

In this study we investigated the effects of hypothyroidism on adult brain RNA synthesis. Our data show that in the cerebral hemispheres of hypothyroid rats there is a decrease in microsomal RNA content and microsomal [³H]uridine incorporation. Sucrose gradient analysis revealed that these changes are mainly associated with free ribosomes and subunits and reflect changes in rRNA. The above changes are accompanied by a decrease in RNA polymerase I activity. All of the above mentioned changes returned to normal after thyroxine (T₄) treatment. In contrast to RNA polymerase I, RNA polymerase II activity was not affected. However, electrophoretic analysis of the in vitro poly(A)⁺RNA translation products revealed that hypothyroidism affects a few mRNAs. These results indicate that thyroid hormones have a role in adult brain tissue metabolism.     

[3H]GABA binding in the cerebellum of the reeler murine mutant

In the cerebellum of the reeler mutant mouse, characterized morphologically by depletion of the granule cell population and abnormal synapse formation, increased GABA concentration and alterations in [³H]GABA binding have been observed. This study shows decreased affinity of the Na⁺-independent, high affinity GABA binding component of synaptosomal membranes and an increased affinity of the Na⁺-dependent, high affinity GABA binding component in reeler cerebellar homogenate and synaptic membranes. In contrast to the changes in affinity, the number of both Na⁺-dependent and Na⁺-independent binding sites was not significantly altered. The decreased affinity of the Na⁺-independent GABA binding and the increased affinity of the Na⁺-dependent binding, evidenced only in cerebellar tissue, were interpreted to indicate, respectively, hypo- and hypersensitivity of the postsynaptic and presynaptic elements of cerebellar GABAergic synapses, induced by the depressed excitatory granule cell input and/or the increased mossy fiber contact with the ectopic Purkinje cells.     

Free amino acids in synaptic vesicles isolated from the cerebellum and cerebral hemispheres of control and neonatally X-irradiated rats

X-irradiation of the rat brain (1000R, at two days of age), suppresses the normal age-related increase in the weight of the cerebellum and cerebral hemispheres and influences amino acid levels. The decrease in glutamic acid concentration, particularly in the cerebellum, supports the previously advanced proposition that this amino acid may be associated with, or may be the transmitter of, the rat cerebellar granule cells. Subfractionation of the cerebellar tissue reveals that the decrease in the glutamic acid level consequent to the loss of granule cells, is reflected in the cytoplasmic fraction but not in the synaptic vesicle subfraction, where glutamic acid was increased. The reduced weight gain in the cerebral hemispheres after irradiation, is accompanied by a significant decrease of aspartate in the cytoplasmic fraction, changes which suggest that a specific cell type, with aspartic acid as its neurotransmitter (possibly in the hippocampus), may also be radiosensitive in the early postnatal period. In contrast, in the synaptic vesicle fraction from cerebral hemispheres, all free amino acids, with the exception of glutamine, increased significantly. Overall, the changes in free amino acid concentration induced by X-irradiation in the cytoplasmic fraction in both brain regions studied are opposite to those found in the synaptic vesicle fraction and although they may indicate changes in specific cell populations, as proposed above, they could also reflect changes in cellular compartmentalization and metabolism or changes in the relative axonal arborization of the affected regions.Special Issue dedicated to Dr. Elizabeth Roboz-Einstein.     

EFFECT OF HYPOTHYROIDISM ON IONIC METABOLISM AND Na-K ACTIVATED ATP PHOSPHOHYDROLASE ACTIVITY IN THE DEVELOPING RAT BRAIN1

The effects of neonatal hypothyroidism on electrolyte contents and the Na⁺ and K⁺ activated ATPase system was studied in the cerebral cortex and cerebellum of the developing rat. Neonatal hypothyroidism increased Na⁺ and CI^? contents and decreased K⁺ and Mg²⁺ contents in both brain areas. Hypothyroidism also resulted in a decrease in the specific activity of the Na-K ATPase extracted by deoxycholate treatment from brain homogenate as well as in the specific activity of this enzyme in the heavy microsomal fraction. The decrease in Mg²⁺ content and ATPase activity is discussed in relation to the changes occurring in Na⁺ and K⁺. Both enzymic and ionic changes may underlie the biochemical and physiological abnormalities observed when the brain is deprived of thyroxine at critical stages of its development.     

Glutamic acid: A strong candidate as the neurotransmitter of the cerebellar granule cell

Free amino acids and cholinergic enzymes were investigated in the cerebellum of reeler and weaver mice in an attempt to identify the neurotransmitter characteristic of the granule cell population and to clarify any neurotransmitter abnormalities of their pre- and postsynaptic neurons induced by their depletion. The data indicate that glutamic acid may be the neurotransmitter of the granule cells. Pre- and postsynaptic neurotransmitter activity seemed not to be markedly altered in cerebellar granule cell dysgenesis.     

The tyrosine phosphorylation site of the acetylcholine receptor beta subunit is located in a highly immunogenic epitope implicated in channel function: antibody probes for beta subunit phosphorylation and function.

Tyrosine phosphorylation of the nicotinic acetylcholine receptor (AChR) seems to be involved in AChR desensitization and localization on the postsynaptic membrane. This study reveals a probable function of the single known beta subunit phosphorylation site (beta Tyr355) and provides suitable tools for its study. The epitopes for 15 monoclonal antibodies (mAbs) against the cytoplasmic side of the AChR beta subunit were precisely mapped using > 100 synthetic peptides attached on polyethylene rods. Eleven mAbs bound to a very immunogenic cytoplasmic epitope (VICE-beta) on Torpedo beta 352-359, which contains the beta Tyr355, and to the corresponding sequence of human AChR. The contribution of each VICE-beta residue to mAb binding was then studied by peptide analogues having single residue substitutions. Overall, each of the residues beta 354-359, including beta Tyr355, proved critical for mAb binding. Two of our four mAbs known to block the ion channel were found to bind at (mAb148) or close (mAb10) to VICE-beta. Tyrosine phosphorylation of Torpedo AChR by endogenous kinase(s) selectively reduced binding of some VICE-beta mAbs, including the channel blocking mAb148. We conclude that VICE-beta probably plays a key role in AChR function. Elucidation of this role should be facilitated by the identified mAb tools.