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 degrees C and cold exposed (4 degrees 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.

Differences in pattern of release of triiodothyronine (T3) and tetraiodothyronine (T4) associated receptors from chromatin by micrococcal nuclease.

Our study aimed to determine whether tetraiodothyronine (T4) binds to chromatin associated receptors and to compare such binding with that of triiodothyronine (T3). We found that the in vitro binding profile of both hormones to receptor-containing fragments released from chromatin by micrococcal nuclease was similar, with the exception of the well known lower T4 affinity. In contrast, the in vivo experiments revealed differences in the sedimentation profile of T3 and T4 bound receptors. More specifically, while T3-bound receptors are excised by micrococcal nuclease as an abundant approximately 6.2 S chromatin-receptor complex, T4-bound receptors are released as fragments of lower sedimentation coefficient (approximately 5.4 S) and as free receptors (not bound to DNA) (approximately 3.9 S). These data indicate that, in vivo, T4 binds with receptors which are differently organized in chromatin than those of T3 and could have a special, as yet undefined, intrinsic nuclear activity.

Interaction between [3H]flunitrazepam and [3H]GABA binding in the cerebellum of reeler mice.

It has been shown that in the cerebellum of reeler mutant mice GABA levels and GABA uptake increase while GABA binding decreases. This study shows that in the cerebellum of these mutants there is also an increase of benzodiazepine receptors. This increase is observed in cerebellar homogenates, in nuclei and in membranes. The increase in the density of central (i.e. clonazepam displacable) benzodiazepine receptors is primarily reflected in binding sites located in the GABA-receptor complex. In comparison to wild-type, GABA-modulin extracted from reeler cerebellum inhibits with a greater potency [3H]GABA binding. The increase in the central-type of benzodiazepine binding and its interaction with GABA binding, observed in cerebellar membranes, is interpreted as a functional response to the decrease in GABA binding and may reflect benzodiazepine receptor condensation and/or changes of subunit composition of the GABA/benzodiazepine receptor complex. The enhanced activity of reeler GABA-modulin reflects a functional response to the increased GABA levels in reeler cerebellum. The increase of the peripheral-type (i.e. PK 11195 displacable) of benzodiazepine receptors is probably due to metabolic changes that may accompany reeler cerebellar mutation. Differences in nuclear benzodiazepine binding between reeler and wild-type mice add a physiological importance to the nuclear binding of this drug.

Effects of LiCl on triiodothyronine (T3) binding to nuclei from rat cerebral hemispheres.

The therapeutic effects of lithium in mania and depression are thought to be mediated by its effects on plasma thyroid hormone (T4, T3) levels. Inasmuch as T3 affects transcription by binding with its nuclear receptors, in this study we examined whether Li+ alters T3 nuclear binding. Although plasma T3 and T4 levels were not affected, 125I-T3 uptake was decreased, and both in vivo and in vitro studies showed a significant increase in nuclear T3 binding in brain and liver tissue. This increase was shown to reflect an increase in maximal binding density. On the basis of these findings, it is proposed that Li+ exerts its action by inducing "cellular hypothyroidism." Integrating existing information on thyroid hormones and affective diseases and the effects of hypothyroidism on neurotransmitters thought to be altered in mania and depression, this hypothesis is supported and contributes to understanding of the effects of LiCl and thyroid hormones in affective diseases.

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.

Cerebellar and striatal dopamine receptors: effects of reeler and weaver murine mutations.

The presence and the binding characteristics of D1 and D2 receptors were investigated in normal-reeler and normal-weaver mutant mice utilizing [3H]spiperone (D2 antagonist), [3H]SKF 38393 (D1 agonist), and [3H]DA as ligands. Analysis of the binding data showed that in the cerebellum there are two binding components for all [3H]ligands. Comparison of the binding constants from cerebellum and striatum showed that in cerebellum the high affinity-low capacity component has similar affinity with that of striatum. The reeler and weaver mutations affected the binding of all ligands: In reeler, total cerebellar specific binding sites for [3H]spiperone and [3H]SKF 38393 decrease significantly (approximately 50% and approximately 70%, respectively), while those for [3H]DA show a small (approximately 10-15%) but not significant decrease. In weaver, total cerebellar specific binding sites for [3H]spiperone, [3H]SKF 38393, and [3H]DA also decrease significantly (approximately 60%, approximately 70%, and approximately 50%, respectively). In reeler striatum [3H]SKF 38393 binding (Bmax) is significantly decreased (approximately 24%), while [3H]spiperone and [3H]DA binding (Bmax) is not affected. In weaver striatum, [3H]SKF 38393 binding is significantly increased (approximately 40%), while [3H]DA binding (Bmax) decreases significantly (approximately 70%). On the basis of the cytoarchitectural aberrations that characterize the cerebellum of these mutants and some well-established information regarding the dopaminergic system of the cerebellum, the above results indicate that in this region a) D1 receptors are mainly localized on granule cells and b) D2 receptors are localized postsynaptically on granule cells and presynaptically on the DA fibers innervating the cerebellum.

Kinetics of dopamine and noradrenaline transport in synaptosomes from cerebellum, striatum and frontal cortex of normal and reeler mice.

Recent evidence indicates that the cerebellum has a dopaminergic system. In order to elucidate further the dopaminergic system in the cerebellum, we investigated the transport of dopamine (DA) in synaptosomal preparations of normal and reeler mice. For comparative purposes we also studied DA transport in synaptosomal preparations from striatum and frontal cortex and compared DA transport to noradrenaline (NA) transport. [3H]-DA transport into cerebellar synaptosomes was found to be a Na(+)-dependent, two component system--a high affinity, low capacity and a low affinity, high capacity. In striatum [3H]-DA is transported by a similar high but different low affinity component. Maximal velocities of both transport components in the striatum were higher than the corresponding ones in the cerebellum. In the frontal cortex we also observed two [3H]-DA transport components with affinities significantly lower than those in cerebellum and striatum. [3H]-NA transport into synaptosomes, prepared from the three brain regions studied, showed two transport components with similar Kt and Vmax values, except for the high affinity component in striatum whose affinity is lower. In reeler mice [3H]-DA transport was different from normal only in the cerebellum where the maximal velocity for both transport components was significantly higher (2x). In contrast, no significant difference was observed in the transport of [3H]-NA. The accumulated [3H]-DA from cerebellar slices was found to be releasable by K+ stimulation, in a Ca(++)-dependent manner, and most of the released radioactivity was in the form of [3H]-DA. These results indicate that in the cerebellum there is a low-density dopaminergic system which is distinct from the corresponding noradrenergic system.

Characterization of thyroid hormone transport in synaptosomes from rat brain.

In this study we examined the mode of triiodothyronine (T3) and tetraiodothyronine (T4) transport in synaptosomal preparations from cerebral hemispheres of adult rat brain. Our results show that these hormones are transported by different mechanisms: T3 uptake is a saturable process and Hofstee analysis of the data reveals two transport components--a high affinity (Kt approximately 50 pM), low density and a low affinity (Kt approximately 3.1 nM) high density system. The Vmax of both components is influenced by the extracellular/intracellular Na+ gradient. T3 uptake decreases in the presence of ouabain and gramicidin. T3 uptake also shows a temperature dependence and decreases in the presence of KCN. In contrast, T4 uptake is a nonsaturable process and is not influenced by metabolic inhibitors or Na ions. It is proposed that T3 entry into neurons is a carrier-mediated process and depends on Na ions. In contrast, T4 is transported by diffusion which is driven by high extracellular/intracellular differences in T4 concentration, maintained by the high rate of cellular deiodination of T4 to T3, characteristic of this tissue.

Kinetic and pharmacologic characterization of dopamine binding in the mouse cerebellum and the effects of the reeler mutation.

The purpose of this study was to characterize the dopaminergic system in the mouse cerebellum and to determine whether the dyskinesia of the reeler mutant is accompanied by alterations in cerebellar and/or striatal dopamine binding. From the analysis of (3H) dopamine ((3H)DA) and (3H)spiperone ((3H)Sp) binding, the study of the effects of several drugs on this binding, and the comparison of these parameters between the cerebellum and striatum, we conclude that a dopaminergic system exists in the cerebellum with properties common to the striatal system but also with some differences. That is, 1) with (3H)DA as ligand, we find two binding sites in cerebellum with similar Kd to those of striatum but of lower density, 2) with (3H)Sp as ligand we observe two binding sites in cerebellum and one in striatum, and 3) the competition of (3H)DA binding by various drugs shows that among the cerebellar sites, relative to striatum, there is a higher proportion that corresponds to high affinity D3 and D4 (D2 high) binding sites. In cerebellum and striatum of reeler mice, (3H)DA binding increases 125-174% and 14%, respectively.

Cholinergic neurotoxicity induced by ethylcholine aziridinium (AF64A) in neuron-enriched cultures.

The sequence of events in neuronal changes induced by the cholinotoxin ethylcholine aziridinium (AF64A) was studied. Neuron-enriched cultures derived from 8-day-embryonic chick cerebra were treated with AF64A at concentrations of 10(-5), 10(-4) and 10(-3) M. Choline acetyltransferase (ChAT) was used as an index of cholinergic neurons. Changes in cell morphology, the immunocytochemical and biochemical presence of ChAT, and DNA and protein content were assessed. Neuron-enriched cultures exposed to AF64A showed a dose-dependent response; after 24 h of exposure to 10(-3) M toxin all cells were dead, whereas a concentration of 10(-5) M did not alter culture morphology or DNA and protein contents. Despite the lack of cytological changes and the presence of ChAT immunoreactivity, biochemically assessed ChAT activity was reduced 36% in 10(-5) M treated cultures. Thus, the implicated decrease in acetylcholine synthesis in these cells cannot entirely account for the neuronal degeneration. Simultaneous exposure of cultures to both AF64A and 10 times higher concentrations of choline chloride delayed or diminished the neurotoxic changes. The protective effect of high choline concentrations was interpreted as evidence of competition between choline and AF64A for the high affinity choline transport system and as constituents in the cell membrane. Examination of the temporal sequence of cytotoxic changes in 10(-4) M exposed cultures revealed that disruption of neuronal aggregates and fragmentation of neurites occurred between 4 and 8 hours of exposure. After 24 h, some neurons survived but with attenuated arbors; in contrast, astrocytes appeared intact, suggesting that glial cells are more resistant than neurons to the toxic effects of AF64A. These findings suggest this culture model may be useful to further elucidate the mechanisms of AF64A drug action and study differentiation of cultured neuronal populations in the absence of cholinergic cells.

Thyroxine deiodination, cytoplasmic distribution and nuclear binding of thyroxine and triiodothyronine in liver and brain of young and aged rats.

This study examines (a) the effects of aging on plasma thyroid hormone concentration and (b) in vivo binding and cytoplasmic distribution of thyroid hormones as well as the conversion of thyroxine (T4) to triiodothyronine (T3) in liver and cerebral hemispheric tissue. The results show that (a) in male Long-Evans rats aging decreases plasma T4 concentration but does not affect plasma T3 concentration and (b) the in vivo nuclear T3 binding does not change significantly. However, nuclear T3 binding derived from T4 is decreased as a consequence of reduced T4 to T3 conversion in both tissues. The nuclear T4 binding is also depressed, perhaps due to the decrease in the T4 of the protein free cytoplasmic compartment. Aging was also found to change protein free and protein bound cytoplasmic distribution of T4. That is, an increase was observed in protein bound cytoplasmic T4 and a decrease in the protein free cytoplasmic T4 of both tissues. These results indicate an overall alteration in thyroid hormone production and peripheral tissue binding and processing of thyroid hormones with a consequent suboptimal thyroid state with aging.

[(3)H]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 [(3)H]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.

Characterization of nuclear triiodothyronine (T3) and tetraiodothyronine (T4) binding in developing brain tissue.

The main objective of this study was to characterize nuclear T3 and T4 binding in the developing rat brain. More specifically, we sought to determine (a) whether T3 and T4 bind to the same nuclear receptor, (b) whether there are multiple forms of nuclear T3 or T4 receptors, and (c) whether the above parameters are similar in nuclei of cerebral hemispheres and cerebellum of developing rat brain. From in vivo and in vitro binding experiments utilizing gel filtration techniques, we have shown that T3 binds to a main macromolecular fraction of molecular weight (M.W.) approx. 60 000 daltons; however, a minor binding component of M.W. greater than 100 000 daltons was also observed. Utilizing the same techniques it was shown that T4 does not bind with the main T3 binding macromolecule but only with the minor (M.W. greater than 100 000) binding component. Inasmuch as T4 competes with T3 for its binding, we have hypothesized that (a) the stability of the T4-receptor complex requires special stereochemical receptor-chromatin relationships that hold for in vivo or de novo conditions but not in the salt-extracted (0.4 M KCl) nuclear receptor preparation, or (b) T4 interacts with more than one receptor unit and forms unstable T4-receptor complexes corresponding to the high M.W. macromolecular fraction. The T3 and T4 binding characteristics described above were common to both brain regions at both developmental ages examined; however, these tissues were found to differ in quantitative aspects of T3 and T4 binding and with respect to the rate of the in vivo T4 to T3 conversion. We suggest that the nuclear T4 does not contribute to the end biological effects but, rather, it determines the number of free T3 binding sites. The end biological responses may thus be proportional to the binding of T3--derived from plasma and the local cellular conversion of T4 to T3--with its major nuclear binding protein and inversely proportional to the T4 nuclear concentration.

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 infected [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 monodeiodination 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 form 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.

Nuclear triiodothyronine receptors in the developing rat brain.

This study examines whether the high sensitivity of the developing brain to thyroid hormones and the purported decline in sensitivity in adulthood, are correlated with changes in the density and affinity characteristics of specific nuclear T3 receptors. The authors have found that the nuclei of cerebral hemispheres have a high density of T3 receptors at birth (212 +/-28 X 10(-17) mol/microgram DNA) which declines to adult levels by the end of the second postnatal week (115 +/- 7 X 10(-17) mol/microgram DNA), remaining at this level until 6 months of age. Even though no significant changes were detected in the equilibrium dissociation constant (Kd) during the early period of development, comparison neonatal with the adult brain reveals a decrease in Kd (neonatal, 3.9 X 10(-10) M; adult, 2.3 X 10(-10) M). In the developing animal, neonatal thyroidectomy increased the number of binding sites in the nucleus by 36--44%. It is concluded that the high number of nuclear T3 receptors in the first week of postnatal life is correlated with the high dependence of brain tissue on thyroid hormones and that the decline in brain sensitivity may be associated with the decline in nuclear T3 receptors. The high affinity and density of nuclear receptors in adult brain tissue relative to the developing brain and liver, respectively, point to a continued regulatory role of thyroid hormones in brain.

Effect of hypo- and hyperthyroidism on regional monoamine metabolism in the adult rat brain.

The effects of hypo- and hyperthyroidism were investigated on brain levels and accumulation rates (after pargyline) of 5-hydroxytryptamine (5-HT), norepinephrine (NE) and dopamine (DA) in discrete brain regions of the adult rat. Whereas NE remained unchanged in all brain areas except in the cerebellum, alterations in brain 5-HT and DA suggest that the behavioral abnormalities associated with thyroid dysfunction in adulthood may be related to neurotransmission disturbances. In hypothyroidism, 5-HT content decreased in cerebral hemispheres and mesodiencephalon and DA content decreased in these regions and also in cerebellum and pons-medulla. Concomitantly, accumulation rate of 5-HT was lower in pons-medulla whereas that of DA was increased in cerebral hemispheres and mesodiencephalon. In hyperthyroidism, 5-HT levels increased in cerebral hemispheres alone. Accumulation rate of 5-HT increased in pons-medulla and that of DA increased in mesodiencephalon. These data indicate that the influence of thyroid hormones on monoamines (MAs) in the adult brain varies with the neurotransmitter and the brain area considered.

Monoamine metabolism in the developing rat brain and effects of ionizing radiation.

Neonatal X-radiation induces profound changes in monoamine metabolism in the developing CNS. NE and 5-HT concentrations increase 7 days post-radiation in all CNS regions undergoing rapid axonal growth and proliferation, but not in the region of the cell bodies from which the respective neurotransmitters originate. The increase in NE and 5-HT levels is accompanied by a concomitant increase in the rate of synthesis. While these changes are evident as late as 22 days of age, the monoaminergic systems revert to normal by maturity. It is suggested that these alterations reflect an imbalance in the density of nerve endings to the region where these terminate. These regions are immature at birth and cell proliferation, a process which is affected by X-radiation, is still occurring at the time of exposure.