Thomas C. Rainbow

Steroid hormones: humoral signals which alter brain cell properties and functions.

Publisher Summary The steroid hormones influence the brain to affect behavior. This chapter discusses the intracellular steroid receptors in neural tissue; their anatomical distribution and properties, their role in carrying hormone to the cell nucleus, and their effects upon gene expression. It also describes the direct effects of steroids upon neural tissue which do not appear to be mediated by intracellular steroid receptors. It provides a more integrated picture of how steroid hormones influence patterns of behavior during the development and in adult life, using as an example the effects of estradiol in the rat brain. The brain contains receptor sites for all five classes of steroid hormones. Brain regions that contain such cytosol steroid receptors also display translocation of labeled hormone to the cell nuclear compartment, and this phenomenon underlies autoradiographic localization of steroid hormone concentrating cells, because the presence of label over the cell nucleus provides a visually striking and quantifiable endpoint. Cell nuclear translocation of steroid hormones in neural tissues is not necessarily accompanied by the extensive cytosol receptor depletion even when nuclear sites are loaded to capacity. There are other, indirect demonstrations of genomic involvement in steroid action in the brain.

Quantitative densitometry of neurotransmitter receptors

An autoradiographic procedure is described that allows the quantitative measurement of neurotransmitter receptors by optical density readings. This procedure is a modification of the method of Young and Kuhar (1979a). Frozen brain sections are labeled in vitro with [3H]ligands under conditions that maximize specific binding to neurotransmitter receptors. The labeled sections are then placed against the 3H-sensitive LKB Ultrofilm to produce the autoradiograms. These autoradiograms resemble those produced by [14C]deoxyglucose autoradiography (Sokoloff, 1977) and are suitable for quantitative analysis with a densitometer. Muscarinic cholinergic receptors in rat and zebra finch brain and 5-HT receptors in rat brain were visualized by this method. When the proper combination of ligand concentration and exposure time are used, the method provides quantitative information about the amount and affinity of neurotransmitter receptors in brain sections. This was established by comparisons of densitometric readings with parallel measurements made by scintillation counting of sections.

Quantitative receptor autoradiography with tritium-labeled ligands: comparison of biochemical and densitometric measurements

Tritium-labeled receptor ligands are commonly used for quantitative receptor autoradiography with LKB Ultrofilm. In this study, we compared estimates of tritium concentrations by optical density measurements with direct measurements of tritium concentrations by scintillation counting. We found that predominantly grey matter regions of rat brain showed similar tritium concentrations when measured by either method, but that optical density reading of white matter regions indicated a lower tritium concentration than determined by direct measurement with scintillation counting. We also compared measurements of receptor density by quantitative autoradiography with radioligand-binding measurements on microdissected brain regions. Higher per milligram protein levels of muscarinic receptors were obtained by quantitative autoradiography, comparable to the specific activity of receptors found in purified membranes.

Regulation of Glucocorticoid Receptors in Brain by Corticosterone Treatment of Adrenalectomized Rats

Binding of (3H)-corticosterone in cytosol of hippocampus and hypothalamus has been measured in adrenalectomized (ADX) rats in the presence or absence of corticosterone replacement therapy (suspended shortly before receptor analysis). Corticosterone pellet implantation into female rats or oral corticosterone administration in salinized drinking water given to males for 3 weeks reduced (3H)-corticosterone binding by half in the hippocampus. This reduction was observed whether corticosterone or dexamethasone was employed as competitor to determine nonspecific binding, thus eliminating transcortin as the cause of the corticosterone effect on binding. Scatchard analysis of binding data revealed that the reduction was mostly due to decreased number of receptors. Animals pretreated with corticosterone had a reduction in thymus weight, indicating further the biological effectiveness of the treatment. Further, serum corticosterone in ADX rats pretreated with corticosterone (but with therapy suspended for 24 h) was very low and similar to that of untreated ADX rats. Uptake studies after injection of (3H)-corticosterone intravenously into ADX rats showed that the injected hormone was absent from blood and brain tissues 1 day later, ruling out (in addition to the measurement of serum corticosterone) that the reduction in binding was due to occupation of receptor sites by exogenous corticosterone remaining after withdrawal from therapy. It is suggested that down-regulation of glucocorticoid receptors in brain follows the chronic corticosterone administration. These data are discussed in relation with evidence for down-regulation of other classes of steroid receptors in several tissues, and the consequence that changes in receptor binding in brain may have on the feedback mechanism of corticoids at the central level.

Aldosterone Effects on Salt Appetite in Adrenalectomized Rats

In order to establish the in vivo specificity of the mineralocorticoid recognition system of the rat brain, we investigated the potencies of aldosterone (ALDO) and corticosterone (CORT) in suppressing salt intake in adrenalectomized (ADX) rats. Increasing doses of ALDO (25, 50 and 100 ng/h), administered by Alzet minipump, suppress salt intake in a two-bottle preference test. CORT in doses up to 50 micrograms/h failed to mimic this effect of ALDO or to block it. Using the 50 micrograms/h dose of CORT, we demonstrated that the forebrain uptake of 3H-ALDO in vivo is suppressed by 60-75% when measured by isolation of cell nuclei or by quantitative autoradiography. The suppression is especially marked in the hippocampal formation, amygdala and septum, sites which also accumulate high levels of 3H-CORT. The uptake of 3H-ALDO by ADX rat forebrain can be suppressed approximately 95% by infusion of a specific antimineralocorticoid, RU 28318, at a dose of 50 micrograms/h. This dose also blocks ALDO action on salt intake. Lower doses of RU 28318 fail to block ALDO action or brain 3H-ALDO uptake. We conclude that: (1) ALDO is at least 500-fold more potent in vivo than CORT as a mineralocorticoid. (2) High uptake of 3H-ALDO in vivo by hippocampal formation, amygdala and septum in ADX rats is due in large part to binding to sites preferentially suppressed by CORT. The 3H-ALDO uptake (ca. 30%) after suppression by stress levels of CORT shows a regional distribution in which uptake is slightly higher in circumventricular structures than in hippocampal formation, septum or amygdala.(ABSTRACT TRUNCATED AT 250 WORDS)

Quantitative autoradiography of serotonin receptors in the rat brain

The distribution of serotonin receptors in the rat brain was studied using a new, quantitative autoradiographic technique utilizing tritium-sensitive LKB sheet film, and densitometric analysis of the film images. OD values above 13 were found in the dentate gyrus, subiculum, substantia nigra and ependymal tissue throughout the brain. ODs between 7 and 11 were found in the globus pallidus, anterior hippocampus and CA1 field, dorsal raphe nucleus and interpeduncular nucleus. The lowest densities were observed in the thalamus, cerebellum, and over white matter, between 0.1 and 0.4 OD units. Other brain nuclei have intermediate values. Serotonin receptor distribution is found to correlate with 5-HT levels, corticosteroid and estrogen receptors in some but not all brain regions.

Quantitative autoradiography of binding sites for [3H]AMPA, a structural analogue of glutamic acid

Binding sites for the potent glutamate agonist [3H] alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) were localized in rat brain frozen sections by quantitative autoradiography. Highest levels of binding were seen in stratum radiatum and stratum oriens of the CA1 hippocampal subfield and in the dorsal subiculum. Substantially less but still high amounts of [3H]AMPA binding occurred in other hippocampal subfields and in rostral forebrain structures. The heterogeneous nature of [3H]AMPA binding is discussed in relation to [3H]glutamate binding visualized by similar methods. From these data it is suggested that [3H]AMPA may label a particular subclass of the glutamate receptor population which exhibits a high affinity for quisqualic acid.

Further studies of brain aldosterone binding sites employing new mineralocorticoid and glucocorticoid receptor markers in vitro

We have used synthetic markers of the glucocorticoid (GC) receptor (RU 28362) and of the mineralocorticoid (MC) receptors (RU 26752 and RU 28318) to characterize the specificity of the sites binding aldosterone (ALDO), dexamethasone (DEX) and corticosterone (CORT) in cytosol of hippocampus. The results obtained suggest that ALDO was bound mostly to a MC receptor, as the relative binding affinity (RBA) of the GC receptor marker (and that of the previously studied RU 26988) was negligible for this site, in contrast to the high RBA displayed by RU 26752. DEX was bound for a large part to a GC receptor, as RU 28362 competed for this site, although the MC receptor marker still showed some affinity. An intermediate effect of both marker types was obtained with CORT. RU 28318 was a weak competitor for either the GC or the MC binding site. Thus, RU 28362 and RU 26752 allowed the discrimination of two to three receptors in the hippocampus, similarly to those described in the kidney. Finally, we have demonstrated the usefulness of these synthetic markers in identifying MC binding sites in several brain regions and also in the hippocampus during ontogenetic development.

Corticosterone modulation of neurotransmitter receptors in rat hippocampus: A quantitative autoradiographic study

The effect of adrenalectomy (ADX) and corticosterone (CORT) replacement on neurotransmitter receptors was studied in dorsal hippocampus of rat using quantitative autoradiography. ADX for one week causes an increase in [3H]5-HT binding to 5-HT1 receptors which is significant in the CA1 cell field. CORT treatment of ADX rats for 3-5 days results in localized reductions of [3H]5-HT binding including a partial reversal of the increase observed after ADX in CA1. CORT treatment of ADX animals also decreases binding of [3H]QNB to muscarinic receptors in the dorsal hippocampus, with a significant effect in an area designated as subiculum. No influence of CORT was detected on [3H]prazosin binding to alpha 1 adrenergic receptors in dorsal hippocampus. Possible mechanisms for hormone effects on neurotransmitter receptor levels are discussed.

Quantitative autoradiography of [3H]corticosterone receptors in rat brain

We have quantified corticosterone receptors in rat brain by optical density measurements of tritium-film autoradiograms. Rats were injected i.v. with 500 microCi [3H]corticosterone to label brain receptors. Frozen sections of brain were cut with a cryostat and exposed for 2 months against tritium-sensitive sheet film (LKB Ultrofilm). Tritium standards were used to convert optical density readings into molar concentrations of receptor. High levels of corticosterone receptors were present throughout the pyramidal and granule cell layers of the hippocampus. Moderate levels of receptors were found in the neuropil of the hippocampus, the lateral septum, the cortical nucleus of the amygdala and the entorhinal cortex. All other brain regions had low levels of receptors. These results extend previous non-quantitative autoradiographic studies of corticosterone receptors and provide a general procedure for the quantitative autoradiography of steroid hormone receptors in brain tissue.