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Endocrinology Vol. 139, No. 7 3165-3177
Copyright © 1998 by The Endocrine Society

ARTICLES

{alpha}, ß, and {gamma} Mineralocorticoid Receptor Messenger Ribonucleic Acid Splice Variants: Differential Expression and Rapid Regulation in the Developing Hippocampus1

Delia M. Vázquez, Juan F. López, María Inés Morano, Seung P. Kwak2, Stanley J. Watson and Huda Akil

Department of Pediatrics, Endocrine Division (D.M.V.), Mental Health Research Institute, Psychiatry Department (D.M.V., J.F.L., M.I.M., S.P.K., S.J.W., H.A.), and the University of Michigan, Ann Arbor, Michigan 48109

Address all correspondence and requests for reprints to: Dr. Delia M. Vázquez, 8240 Medical Science Research Building III, 1150 West Medical Center Drive, Ann Arbor, Michigan 48109-0646.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Two different types of corticoid receptor molecules bind circulating corticosterone in brain: mineralocorticoid receptors (MR) and glucocorticoid receptors. MR exhibit the highest affinity for the endogenous glucocorticoid in the rat, corticosterone. During development, low corticosterone levels influence neurogenesis, and these effects are probably MR mediated. Three MR complementary DNA clones, {alpha}, ß, and {gamma}, have been identified in the rodent. All of these MR complementary DNA clones have identical coding regions, but differ significantly at the 5'-untranslated end. Although the functional significance of these three messenger RNA (mRNA) species remains unknown, one hypothesis is that they reflect the ability of the brain to regulate the expression of MR, allowing multiple factors to differentially control transcription in a tissue- and time-specific manner. To investigate this possibility, we examined the presence of these distinct mRNA forms in the developing rat hippocampus (HC). In situ hybridization with specific {alpha}, ß, and {gamma} complementary RNA probes was performed in the HC of 3-, 5-, 7-, 12-, 14-, 28-, 35-, and 65-day-old animals. We found that there is differential expression of these forms in each of the HC subfields from infancy to adulthood. {gamma} expression appears to be associated with periods of cell birth and increased axonal sprouting. ß expression, on the other hand, may be best linked to periods of synaptogenesis, growth of commissural and associative terminal fields, and possibly active pruning. To explore the possibility that the differential gene expression may be related to corticosterone environment, adrenalectomy was performed. A rapid modulation of the MR mRNA variants (14 h) in an age- and site-specific fashion was seen. These findings suggest that the variation in expression and regulation during development of the multiple MR transcripts could reflect a complex pattern of developmental regulation that may involve a multitude of factors unique to each postnatal age and to the different neuronal populations within the hippocampal formation.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
TWO DIFFERENT types of corticoid receptor molecules bind circulating corticosterone in brain: mineralocorticoid receptors (MR) and glucocorticoid receptors (GR) (1). It is now well established that these two receptor subtypes differ in their affinity for corticosterone, their neuroanatomical distribution, their regulation, and their function (1, 2, 3). Although MR exhibits its highest affinity for aldosterone and for the glucocorticoid corticosterone, GR exhibits a lower affinity to corticosterone and a high affinity toward potent synthetic glucocorticoids such as dexamethasone (1, 4). Although in vitro MR binds both aldosterone and corticosterone with high affinity, in vivo the relative activation of this receptor by these two ligands is determined by the presence of the enzyme 11ß-hydroxysteroid dehydrogenase in specific target cells (5, 6). Thus, in the kidney, 11ß-hydroxysteroid dehydrogenase converts corticosterone to the less active compound cortisone, allowing aldosterone to bind to MR, which, in turn, leads to salt and water balance. In the brain, which expresses MR in many forebrain structures, 11ß- hydroxysteroid dehydrogenase activity is limited (7, 8), allowing MR to bind corticosterone avidly and to participate in the regulation of stress responsiveness.

Given the high affinity for corticosterone that characterizes MR and the lower affinity exhibited by GR, the two receptor systems complement each other to mediate a wide range of glucocorticoid effects. Thus, this dual system can optimally modulate the responses of the limbic-hypothalamic-pituitary-adrenal (LHPA) axis, both at rest across the circadian rhythm and under various stress conditions. In particular, the MR is operative at low corticosterone concentrations, and under these conditions it appears to stabilize neuronal activity (9), provide tonic inhibition to the axis at the nadir of the circadian rhythm (2), gate the threshold of the stress response, and contribute to the selection of appropriate behavioral responses (10).

Early in the postnatal life of the rodent, there is a delicate and critical balance in the activity of the LHPA stress system. This is particularly true of the first 2 weeks of life (day 4–14) when the LHPA axis is characterized by a "silent period" [stress-hyporesponsive period (SHRP)] (11, 12). During this time the developing infant rat maintains very low levels of circulating corticosterone, even under conditions that normally elicit corticosterone elevations in the adult. It is known that high glucocorticoid levels are predominantly catabolic, leading to inhibition of cell division, protein synthesis, and uptake of amino acids and glucose to cells (11, 12). Clearly, a catabolic state would be detrimental to growth and development, especially for the developing central nervous system. The combination of a hypofunctional adrenocortical stress response and a longer half-life of the circulating corticosterone present during the first few weeks of life provides a relatively stable corticoid environment for the developing brain (12, 13). Alteration of this environment leads to long lasting effects on neurogenesis and gliogenesis (14). Thus, rats treated with corticoids during the first week of life have permanently reduced brain weights, DNA content, formation of dendritic spines, axonal growth, and synaptogenesis (14, 15, 16, 17, 18). This is not surprising because low corticoid levels are linked to the induction of glycerol phosphate dehydrogenase, a glial-specific enzyme that is necessary for myelination (19). Glucocorticoids also play an important role in the expression of several transmitter phenotypes, directing a preferential development of chromaffin cells over sympathetic neurons (20) and accelerating the maturation and differentiation of a determined neural cell line (21).

The hippocampus, which is particularly enriched with MR and GR, is directly affected by glucocorticoid exposure during the neonatal period. Glucocorticoid treatment inhibits neurogenesis of hippocampal granule cells and decreases the degree of normal cell death in the granule cell layer (22). Conversely, removal of corticoids by adrenalectomy (ADX) increases granule cell death and decreases the number of dendrites sprouting from granule cells within the dentate gyrus (DG) (23, 24). Thus, low circulating corticosterone levels seem to be an absolute requirement for the maturation of the central nervous system to proceed normally. Moreover, these studies suggest that different hippocampal regions may have different requirements at different times during development. Due to the characteristics of the corticosteroid receptors, MR is likely to be important for these trophic functions.

Although a single MR protein has been described to date, several MR messenger RNA (mRNA) species have been identified in brain (25, 26). The original three MR complementary DNA (cDNA) clones were isolated from human kidney, rat kidney, and hippocampal tissue. These were termed {alpha}, ß, and {gamma}, and they exhibit a specific expression pattern in adult brain and peripheral organs. Although these cDNAs share a high degree of homology throughout the translated domain and at the 3'-untranslated (3'UT) end, they vary significantly at their 5'UT region (25, 27, 28). Analysis of the rat MR gene has revealed that areas that correspond to each of the specific 5'UT regions are located on separate exons of a single gene (25). Each 5'UT exon has a splice donor consensus sequence that permits ligation onto the translated exon upstream of the ATG start site (25). The Kozak consensus sequence essential for translational efficiency is also present in each of these 5'UT mRNA variants (29). This suggests that the 5'UT exons are controlled by separate promoters and are alternatively spliced onto the protein-coding region. The result is three MR mRNAs that encode the same receptor protein (26, 29). These 5'UT MR mRNA species are differentially distributed and regulated in the adult hippocampus (25). The {alpha}-subtype is highly enriched in all hippocampal subregions, with higher expression in cytoarchitectural region 2 (CA2), dentate, and fasciculus cinereum. The ß- and {gamma}-subtypes are expressed at very low levels across all regions. In the adult, only one of these subtypes, {alpha}, appears to be regulated by endogenous corticosteroids (25).

The functional implications of the different MR 5'UT subtypes and the significance of their differential anatomical expression and regulation are unclear. However, based on what is known from other 5'UT variants that have the same open reading frame, the 5'UT feature may allow for fine control of gene expression through different cis-elements upstream from each promoter (30, 31). In addition, the 5'UT element could regulate the translational efficiency and mRNA stability (30, 31, 32). Judging from previous work on the significance of multiple 5'UT messenger RNAs of a single gene (26, 32), the transcription of MR 5'UT variants that are controlled by separate promoters in response to hormonal, tissue, or developmental specific factors may be of great advantage to the developing organism. Given the relatively stable corticosterone levels that "bathe" all organs during development and the neurotropic role of such low corticosterone levels in the immature brain, we postulated that MR 5'UT mRNA subtype expression and distribution within the developing hippocampus may differ from those in the adult animal. The presence of a differential distribution during hippocampal development could give us some insight regarding the significance of these MR 5'UT forms. Furthermore, as corticosterone levels are critical during development, alterations of this stable milieu might uncover differential regulation of the MR 5'UT forms that may not be evident in the adult. In the present study, we performed in situ hybridization in developing hippocampal tissue using specific complementary RNA (cRNA) probes for each distinct 5'UT MR mRNA variant. In addition, we determined their regulation by adrenal corticosteroids. Our results indicate the presence of an age-dependent differential distribution and a differential regulation of these 5'UT MR mRNA forms compared with those in the adult.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Sprague-Dawley female rats (Charles Rivers, Wilmington, MA) were mated in our facilities. Pregnant rats with free access to water and food were kept on a 14-h light, 10-h dark cycle. On the day after delivery, which was considered day 1, the litters were removed from the mother and randomly mixed, and 12 pups, 6 females and 6 males, were returned to a lactating mother. Pups were randomly selected from 42 litters for the ontogeny studies and from 30 litters for the regulation studies. The litters were undisturbed except for a brief period for ADX. Weaning from the mother was performed when the pups were 21 days old, at which time they were housed 6 animals/cage in male or female rooms accordingly. All animals were maintained in accordance with the NIH Guide for the Care and Use of Laboratory Animals.

Ontogeny study
For the ontogeny study, animals were killed in the morning (0800–1000 h) at 3, 5, 7, 12, 14, 28, and 35 days of age. Six to eight animals (1:1 sex ratio), each obtained from different litters, were killed from each age group over several days. Blood was obtained for corticosterone determination (see below), and brains were collected for in situ hybridization as described below. Adult male rats (65 days old; 300–400 g BW; n = 6–8/condition) were used as reference.

Regulation study
To ensure genetic diversity, 36 litters were used for the regulation study to determine both binding capacity and gene expression. As a litter consisted of 12 animals, ADX was performed on 6 animals from each litter (3 females and 3 males). The remaining 6 animals (3 females and 3 males) were sham operated. ADX was performed via a dorsal approach under metafane vapor anesthesia (Metafane, Pitman-Moore, Mundelein, IL). Sham animals had the same surgical procedure except that a small amount of suprarenal adipose tissue was removed. After recovery from the anesthesia, the animals were returned to their home cage and left undisturbed until 14 h later, when they were killed by decapitation. Animals were killed in the morning (0700–0800 h). Sham and adrenalectomized (ADX) animals came from the same litter. The animals were 10 and 28 days of age on the day they were killed. Adult ADX and sham-operated male rats, 65 days old (300–400 g BW), also from the colony were used as reference. Brain tissue was collected for in situ hybridization and for receptor binding capacity studies (see below).

Plasma corticosterone determination
Trunk blood was collected in tubes containing EDTA and spun at 2000 rpm for 7 min. to obtain plasma. Corticosterone was assayed to confirm the removal of the adrenal using a RIA as previously described (33, 34). The antibody cross-reacts 2.2% with cortisol and less than 1% with other endogenous steroids. The detection limit is 1 pg/ml, and the intra- and interassay coefficients of variation are 2% and 3%, respectively.

In situ hybridization
Brain tissue processing. Brains were rapidly removed, frozen in liquid isopentane (-42 C), and stored at -80 C. Subsequently, they were sectioned in coronal plane at 10 µm on a Bright-Hacker cryostat (Hacker Instruments, Fairfield, NJ) that was maintained at -20 C, and the sections were thaw-mounted onto polylysine-coated microscope slides. Brain sections were stored at -80 C until processed for in situ hybridization.

Riboprobe preparation. The antisense probes for the three MR 5'UT variants were prepared as previously described (25). They were synthesized from the 5'UT unique to each of the MR cDNAs. The lengths of the cRNAs were 210, 300, and 145 nucleotides for {alpha}, ß, and {gamma} probes, respectively (see Fig. 1Go). The {alpha} and ß probes were labeled using [35S]UTP (1000 Ci/mmol). The {gamma} cRNA was labeled using two radionucleotides, [35S]UTP and [35S]CTP, in the in vitro reaction to increase the specific activity and facilitate detection of the hybridization signal. Approximately 1 million dpm probe were used on each slide for hybridization. In addition, a cRNA probe that detects all MR mRNA forms (total 3'UT MR mRNA) was synthesized. It was a 347-nucleotide fragment of the MR clone, directed against the 3'UT of the MR mRNA (27). We have previously shown that the probes do not contain any regions of high homology with other RNAs, thus avoiding potential cross-hybridization. Controls consisting of hybridization using ribonuclease (RNase)-treated sections, sense probes, and high stringency conditions were used to confirm the specificity of these probes (25).



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  		Figure 1. MR gene 5'-flanking region and MR mRNA transcripts. An illustration of a 13.5-kb fragment isolated from an EMBL-3 Sprague-Dawley genomic DNA library is shown in the upper part of the figure. Splicing events are likely, as all products conform to the Kozak consensus sequence essential for translation, including guanine at -3 position (25 ). The ATG start codon is located at nucleotide 3 of exon III. The three MR mRNA variants have different 5'UT lengths derived from three different exons, i.e. {alpha} from exon II, ß from exon I, and {gamma} from exon {gamma}. However, the peptide-coding domains are identical in all three species. The 5'UT areas used to generate the specific MR cRNA probes are depicted. The {alpha} MR probe consists of 210 nucleotides (nts), ß is 300 nucleotides, and {gamma} is 145 nucleotides. Quantification of 5'UT variants in adult animals using protection assays revealed a greater proportion of {alpha} variant compared with the other variants (25 ). The proportions also suggest that other MR mRNA variants exist.

 
Hybridization conditions. Tissue sections were removed from the -80 C freezer and placed directly into glass tubes filled with 4% formaldehyde for 1 h. The sections were then incubated with 1 µg/ml of a solution of proteinase K to permeabilize the tissue and inactivate endogenous RNase. After this treatment, sections were incubated in succession in water (1 min), 0.1 M triethanolamine (pH 8.0; 1 min), and 0.25% acetic anhydride in 0.1 M triethanolamine (10 min). The tissue was then washed in 1 x SSC (0.3 mM NaCl and 0.03 nM Na citrate, pH 7.2; 5 min) and dehydrated through graded concentrations of ethanol. Sections were hybridized with 1.5 x 106 dpm [35S]UTP-labeled MR cRNA probes in 10 µl of a hybridization buffer containing 75% formamide, 10% dextran sulfate, 3 x SSC, 50 mM sodium phosphate buffer (pH 7.4), 1 x Denhart’s solution, 0.1 mg/ml yeast transfer RNA, and 0.1 mg/ml sheared salmon sperm DNA. Tissue sections were covered with coverslips. The slides were incubated overnight at 50 C. On the following day the coverslips were removed by soaking with 2 x SSC, and the tissue sections were washed for 10 min in fresh 2 x SSC solution. Single stranded probe not hybridized with endogenous mRNAs was removed by incubating the sections for 30 min in 200 µg/ml solution of RNase A at 37 C. The tissue was then washed in increasingly stringent SSC solutions (2, 1, and 0.5 x SSC; 10 min each), followed by a 1-h wash in 0.5 x SSC at 50 C. After this final wash, the slides were dehydrated, air-dried, and prepared for detection by x-ray autoradiography on Kodak XAR-5 film. A set of 14C-labeled radioactive standards was placed on the film (American Radioactive Chemicals, St. Louis, MO).

Microdensitometric analysis. Autoradiograms generated were analyzed using an automated image analysis system (MAC II/IMAGE, Dage camera, Michigan City, IN). The person analyzing the images was not aware of the treatment conditions under analysis. Hippocampal areas, corresponding to subfields 1) CA1, 2) CA2, 3) CA3–4, and 4) DG (see Fig. 2Go) were digitized from a given hippocampal section. The mean gray level of the region of interest was measured at x100 magnification. This measurement corrects for the size of structure analyzed (mean density per area). Background labeling was measured from the corresponding internal area of each section. Four sections per animal were analyzed. The mean of these was used as the individual value for a particular area in a given animal. Six to eight animals were analyzed per condition.



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  		Figure 2. A representative photomicrograph of in situ hybridization depicting the ontogenic progression of the MR in rat hippocampus at selected ages. {alpha}-specific cRNA, ß-specific cRNA, {gamma}-specific cRNA, and a 3'UT cRNA common to all forms were used, as described in Materials and Methods. The distributions of {alpha} and ß MR mRNA during development differed most in the CA2 and DG regions. As the animal matured, the differences among ß and {gamma} MR mRNA were most prominent in the CA1, CA2, and CA3–4 pyramidal cell regions. The exposure times on film were: 4 days for {alpha}, ß, and {gamma}, and 2 days for 3'UT total MR mRNA.

 
Receptor binding capacity: tissue preparation and binding conditions
In a separate experiment, hippocampi were dissected from animals 14 h after ADX or sham surgery. Hippocampi were dissected on ice and frozen immediately on dry ice. Later, estimates of the capacity of the mineralocorticoid and glucocorticoid receptor (Bmax) were determined by single near-saturation dose binding assay (34, 35, 36). This method is particularly useful when there is a limited amount of tissue, as is the case with infant rats. The procedure uses a saturating concentration of ligand, that is 10 nM [3H]dexamethasone, in combination with a specific concentration of GR agonist to approximate the x-intercept in the Scatchard plot. The parameters in this procedure that ensure that dexamethasone accurately labels MR and GR were replicated and further calibrated in our laboratory. The results of these calibrating experiments showed the Bmax estimates to be comparable to values obtained from Scatchard analysis using [3H]aldosterone as the labeling ligand (35) (our unpublished data). Thus, as reported by others, dexamethasone binds to both GR and MR present in the hippocampal cytosolic homogenate (in vitro), and the determination of Bmax values for both receptors is possible as explained below. Briefly, frozen hippocampi were homogenized in a hand-held glass homogenizer in 10 mM ice-cold Tris buffer, pH 7.4. The buffer also contained 1 mM EDTA, 10% glycerol, 40 mM molybdate, and 1 mM dithiothreitol. The homogenate was centrifuged in an ultracentrifuge at 40,000 rpm for 30 min, then the cytosol (supernatant) was transferred to another tube that remained on ice. A 50-µl aliquot of the cytosol was incubated with a saturating concentration of [3H]dexamethasone (10 nM; New England Nuclear, Boston, MA), with and without 500 nM of the GR agonist RU 26988 (Roussel-UCLAF, Romainville, France) and an excess of dexamethasone (2.5 mM, Sigma, St. Louis, MO). Thus, individual total binding (cytosol plus [3H]dexamethasone), MR binding (cytosol plus [3H]dexamethasone plus 500 nM RU 26988), and nonspecific binding (cytosol plus [3H]dexamethasone and an excess of 2.5 mM dexamethasone) were determined. The samples were incubated overnight (a minimum of 16 h) at 0 C. The bound [3H]ligand was separated from free ligand using charcoal suspension [2% Norit-A (J. T. Baker, Phillipsburg, NJ) and 0.2% dextran (mol wt, 7000; Sigma)]. A small sample of the original cytosol was assayed for protein content using a Bio-Rad protein assay (Bio-Rad Corp., Wilmington, DE), and the values are expressed as fentomoles bound per mg protein.

Statistical analysis
ANOVA procedures were used for the analysis of age and treatment interaction, with the level of significance set at P < 0.05. One-way ANOVA tests were followed by post-hoc comparisons using Fisher’s protected least significant difference test (PLSD). Results were analyzed independently for sex. When differences were determined to be nonsignificant, the data were collapsed across the respective variable.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Ontogeny study
Plasma corticosterone levels. Table 1Go depicts the total circulating corticosterone levels in the developing animal. The results obtained were analyzed independently for sex, but because the values did not reach significant differences, the data were collapsed. As shown in Table 1Go, plasma corticosterone levels decreased after postnatal day 3 (PN 3) of life and remained low until the end of the SHRP (PN 3, 2.1 ± 0.4; PN 5, 0.7 ± 0.3; PN 14, 1.3 ± 0.2). On day 35, which corresponds to the start of an active reproductive axis, corticosterone levels rose substantially (PN 35, 2.8 ± 0.1; P <= 0.05 compared with PN 5, 7, 12, and 14). However, these latter levels were not significantly different from the levels obtained in mature male rats.


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  		Table 1. Plasma corticosterone levels in the group of animals from which the ontogeny of MR 5'UT mRNA variants in situ hybridization is derived

 
Mineralocorticoid 5'UT mRNA variants in the developing hippocampus. The three 5'UT variant analyses relied on specific cRNA probes that were aimed at the unique {alpha}, ß, and {gamma} 5'UTs and avoided the protein-coding region of MR mRNA, thereby ensuring specificity for each receptor variant. A cRNA probe complementary to the 3'-end of the MR mRNA was used to ascertain changes in total MR mRNA content (see Fig. 1Go). Figure 2Go illustrates the anatomical expression of the various MR forms as well as the total pool of MR across various ages. Figures 3Go and 4Go depict the absolute levels of these 5'UT variants in the individual hippocampal regions. A two-way ANOVA considering age and hippocampal region for each cRNA probe was statistically significant for both of these factors (age, P < 0.0001; region, P < 0.0005). Age-region interactions were also present (P < 0.0001). Thus, we found a differential distribution within the pyramidal and granular cell regions that appears to be specific for the different developmental periods.



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  		Figure 3. Densitometric analysis of the MR mRNA 5'UT variants and total MR mRNA (3'UT) in the hippocampal pyramidal cell region of developing animals. Each panel presents the quantified signal taken from the hippocampal subfields CA1, CA2, and CA3–4. A depicts total MR mRNA (3'UT cRNA probe common to all MR forms), B shows {alpha} MR mRNA, C shows ß MR mRNA, and D shows {gamma} MR mRNA. Measurements are not comparable between subtypes as the cRNA probes differed in specific activities. A two-way ANOVA, with age and hippocampal region as the two variables for each cRNA probe, was statistically significant for both of these factors (age, P < 0.0001; region, P < 0.0001). An age-region interaction was also present (P < 0.0001). *, P <= 0.05 compared with adult. n = 6–8 animals/age.

 


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  		Figure 4. Semiquantification of the MR mRNA 5' UT variants and total MR mRNA (3'UT) in the hippocampal granular cell region or DG of developing animals. A, Total MR mRNA (3'UT cRNA probe common to all MR forms); B, {alpha} MR mRNA; C, ß MR mRNA; D, {gamma} MR mRNA. Again, measurements are not comparable between subtypes, as the cRNA probes differed in specific activities. *, P <= 0.05 compared with adult. n = 6–8 animals/age.

 
To better analyze the abundance of the three 5'UT MR forms and total MR, we compared the absolute mRNA levels (Figs. 3Go and 4Go), and we transformed these data to percentages (Table 2Go). For this purpose, the adult levels were normalized to 100%, and all other ages were expressed as a percentage, using the adult measurement of each respective cRNA as a reference.


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  		Table 2. Changes in MR mRNA levels during development

 
Total MR mRNA: Compared with that in the adult, total MR mRNA is less abundant in the CA1 area in PN 3 animals (P <= 0.05; Fig. 3AGo); it is roughly 50% the amount expressed in the adult (see Table 2Go). In contrast, abundant levels are seen in CA2 and CA3–4 during most of the first 2 weeks of life (PN 5, 7, and 14 vs. PN 35 and adult, P <= 0.05). These levels of total MR mRNA expression are 1.2–1.4 times higher than those in the adult (Table 2Go). In the DG, significantly lower levels are also seen on PN 3 (PN 3 vs. older ages, P <= 0.05; Fig. 4AGo and Table 2Go), whereas higher levels were evident on PN 14 and 28 (P <= 0.05, PN 14 and 28 vs. adult).

{alpha} MR mRNA: Animals that are clearly in the SHRP have less abundant {alpha} MR mRNA in the CA3–4 region than adults (PN 3, 5, and 7 vs. adult, P <= 0.05; n = 6–8 animals/age; see Fig. 3BGo). Significantly lower levels were also seen in the DG on PN 3 and 7 (PN 3 and 7 vs. older ages, except PN 35, P <= 0.05). As shown in Table 2Go, {alpha} MR mRNA expression is fairly constant in all hippocampal regions throughout development, that is close to 100% of the adult level (with some exceptions, e.g. CA3–4 and DG in 3-, 7-, and 35-day-old animals).

ß MR mRNA: ß MR mRNA appears to be a predominant MR variant from the first to the second week of life throughout the hippocampus, with levels diminishing as the animal is weaned from maternal care (PN 3, 5, 7, 12, and 14 vs. PN 28, PN 35, and adult, P <= 0.05; see Figs. 3CGo and 4CGo). Overall, ß MR mRNA expression is 2- to 10-fold greater than that in the adult in CA2, CA3–4, and DG during the first 2 weeks of life (Table 2Go; P <= 0.05).

{gamma} MR mRNA: The {gamma} MR mRNA pattern of abundance is similar to that of ß MR mRNA expression, but levels remain elevated for a longer period (i.e. until PN 35; P <= 0.05) in practically all areas analyzed (except CA2 and CA3–4 for animals older than PN 14; see Figs. 3DGo and 4DGo). When expressed as a percentage of the adult value, {gamma} MR mRNA predominates in the CA1 region in young and juvenile animals (2- to 9-fold greater than adult value; P <= 0.05) until adulthood when levels become less abundant (Table 2Go). In the DG, {gamma} predominates during the first week of life and postweaning to levels 2–2.6 higher than those in the adult (P <= 0.05). Interestingly, in the CA2 and CA3–4 regions, {gamma} MR mRNA is also a predominant variant during this period (2- to 5-fold greater than adult value; P <= 0.05).

Regulation study
Plasma corticosterone levels: 14 h after ADX. Next, we examined the effect of glucocorticoid removal on the expression of MR mRNA variants in the hippocampus. We also ascertained the MR-translated protein levels using the ligand binding capacity assay. As the binding assay requires ADX for 12–24 h before acquisition of the hippocampal tissue, we used animals 14 h after ADX for both the specific 5'UT in situ and Bmax receptor capacity measures (see below). Fourteen hours after bilateral ADX, the animals had undetectable total corticosterone levels. Sham-operated 10- and 28-day-old animals had low basal levels that were significantly different from those in the adult sham-operated animals (see Table 3Go; P <= 0.05).


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  		Table 3. Plasma corticosterone levels and cytosolic binding capacity of MR receptors 14 h after adrenlectomy or sham surgery

 
Regulation of MR 5'UT mRNA variants in the developing hippocampus: 14 h after ADX
We quantified total MR mRNA and the specific 5'UT MR mRNA variants 14 h after ADX or sham surgery. A three-way ANOVA considering age, treatment and hippocampal region for each cRNA probe was statistically significant for both age and treatment (age, P < 0.006; treatment, P < 0.002). Age-treatment interactions were present for ß and total MR mRNA (P < 0.005). Hippocampal region effects were found for ß and {gamma} MR mRNA (P < 0.05), with treatment-hippocampal region interaction observed for ß MR mRNA only.

In agreement with a previous observation (34), total MR mRNA increased significantly in CA1, CA2, and DG in the ADX adult animals compared with that in sham-operated animals (Fig. 5Go and Table 4Go; P <= 0.05). In these same adult animals, {alpha} MR mRNA levels increased in CA1, CA2, CA3–4, and DG (P <= 0.05). ß MR mRNA also increased in CA2 and DG (P <= 0.05), yet {gamma} remained unchanged in all subregions.



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  		Figure 5. Densitometric analysis of the MR mRNA 5'UT {alpha}, ß, {gamma}, and 3'UT total MR mRNA digitized images of sham and ADX animals. The CA1, CA2, CA3–4, and DG subfields of the hippocampal formation are shown in A–D.. A three-way ANOVA revealed age and treatment effects (P < 0.006). Hippocampal region effects were present for ß and {gamma} (P < 0.05). *, P <= 0.05, sham vs. ADX. Values are the mean ± SE; for each age and condition, n = 6–8. The SEs not shown, as the error bar is too small to be visualized.

 

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  		Table 4. Changes in MR mRNA content after short term adrenalectomy in the three ages studied

 
The developing animal had a region-specific and MR variant-specific pattern of response that was different from that in the adult animal. In addition, up-regulation of a given 5'UT MR variant did not necessarily lead to an elevation of the total MR mRNA measurement of that region. {gamma} MR mRNA levels were particularly inefficient at increasing the total MR mRNA content. For example, total MR mRNA hippocampal levels were not altered in the hippocampal pyramidal cell regions of the 10-day-old ADX animal (see Table 4Go), although {gamma} MR mRNA was significantly elevated in the CA1 subfield (Fig. 5Go and Table 4Go; {gamma} 5'UT, day 10, 131 ± 7% of sham ± SE; P <= 0.05). In contrast, ß and {alpha} MR mRNA elevations, alone or in combination, led to increases in total message. This was observed in the 28-day-old ADX animal, in which total MR mRNA increased significantly in CA1 and CA2 compared with that in sham controls (Table 4Go, total MR, day 28; CA1, 149 ± 5; CA2, 166 ± 3; P <= 0.05). In these animals, both {alpha} and ß MR mRNA transcriptions were increased in CA2, whereas only ß MR message was increased in the CA1 region. In contrast, {gamma} MR mRNA remained unaltered in all hippocampal subfields. Thus, the total MR mRNA increase observed in the CA1 region of the ADX 28-day-old animal is primarily due to ß MR mRNA up-regulation, whereas CA2 up-regulation results from both {alpha} and ß responses to decreasing steroid levels.

MR binding capacity: 14 h after ADX
To ascertain the effect of 14 h of ADX on receptor expression, binding capacity of GR and MR were measured. Fourteen hours is within the time period that allows for the clearance of circulating endogenous corticosterone that would otherwise interfere with the binding assay (35). The binding assay used was one developed as a GR and MR microassay by Landfield and co-workers (37). This method offers two advantages. First, it allows for the determination of both GR and MR binding capacity with a single radioactive ligand. Secondly, minimal tissue is required because multiple ligand concentration Scatchard plots are not generated. We have also validated this method in our laboratory (data not shown), and the extrapolated Bmax estimates obtained correlated closely with those obtained from Scatchard plots (r2 = 0.86 for MR; r2 = 0.98 for GR). For the purpose of this study, we only report the MR binding capacity values. Table 3Go shows the amount of MR receptor per mg protein obtained by the microbinding assay method. As can be seen for the sham animal values, the developing hippocampus has a significantly higher MR binding capacity than that of the adult, which correlates with the lower basal corticosterone levels (PN 10, 84.5 ± 15.7; PN 28, 76.8 ± 19.4; adult, 19.2 ± 6.3; mean ± SE; P <= 0.05). Fourteen hours after ADX, all of the ADX animals studied had an increased MR binding capacity to significant or nearly significant statistical levels. Values in the PN 28 and adult animals reached significance (Fig. 6Go; adult, 186% above sham; PN 28, 131% above sham; PN 10, 57% above sham; P <= 0.05). It is important to point out that as the developing sham hippocampus has significantly higher MR binding capacity at basal levels compared with that of the adult, the percentage of up-regulation observed is greatest in the adult animal (see Table 3Go and Fig. 6Go). However, the actual binding capacity is significantly greater in the developing hippocampus.



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  		Figure 6. MR binding capacity in the PN 10, PN 28, and adult animal. Animals were killed 14 h after ADX. Binding capacity analyses were performed using a single point binding assay from a hippocampus for individual animals as described in Materials and Methods. Values were expressed as femtomoles per mg protein ± SE, then normalized using sham values as 100%. *, P <= 0.05, sham vs. ADX; {dagger}, P <= 0.05, age vs. adult. Values are the mean ± SE; for each age and condition, n = 6.

 
The receptor binding capacity data correlate with the gene expression pattern described above.

Total MR gene expression and binding capacity are significantly up-regulated by 14 h ADX in PN 28 and adult animals. The increased MR levels appear to correspond to contributions of {alpha} and ß MR mRNA translation.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The present study focused on the ontogeny of the three variants of MR mRNA, which encode the same protein but diverge in their 5'UT sequence and have been isolated from rat and human kidney and hippocampus (25, 38, 39). In the adult animal, these MR mRNA variants are expressed in a tissue-dependent manner, and only one appears to be regulated by endogenous steroids. As nothing was known about the ontogeny of these receptor variants, we first determined the expression of MR mRNA species in the developing rat hippocampus and compared it to what is known about that in the adult hippocampus. We found a differential distribution within the pyramidal and granular cell regions that is unique for each variant and is distinctive for each developmental period. Our second goal was to ascertain which MR mRNA subtype was regulated by the steroid environment during development. We found that in the developing animal, two mRNA subtypes, {alpha} and ß, appear to be regulated by endogenous steroids. Furthermore, the increases in mRNA levels observed with ADX result in increased MR binding capacity, suggesting that increases in receptor protein correspond to individual or combined contributions of {alpha} and ß mRNA translation.

The distribution of MR mRNA has been previously reported in the developing animal (34, 40). However, these studies did not focus on the multiple RNA species, as the probes were generated from the 3'-end of the MR cDNA, thereby detecting the entire MR population. Compared with that in the adult animal, the developing hippocampus has a relative abundance of MR message in the DG and CA2 regions. The adult pattern of MR mRNA expression is seen after PN day 35 (CA2 > DG > CA1) (34). In the present study, we reexamined MR mRNA distribution in the hippocampus using probes specific to the 5'UT and determined their differential distribution within the hippocampal structure. In agreement with a previous study, we found that in the adult animal, the expression of the {alpha} variant is consistent with the distribution pattern observed after hybridization with the 3'UT probe. This is consonant with the low level of expression of the other MR mRNA forms, suggesting that total MR expression in the adult is primarily comprised of the {alpha} variant (25). However, during the first 2 weeks of life, the pyramidal formation and DG of the developing animal appear to be enriched with ß and {gamma} MR mRNA gene expression, whereas {alpha} mRNA remains relatively constant across ages. Furthermore, the relative abundance patterns of the ß and {gamma} mRNA variants were specific to various subfields, with {gamma} predominating in the CA1 region, ß predominating in the CA2 and CA3–4, and {gamma} and ß being expressed in relatively equal proportions in the DG.

The functional significance of the differential expression of the MR 5'UT isoforms within the hippocampus is unclear. However, the differential expression of the mRNA isoforms within the developing hippocampus along with the pattern of organization at the genomic level first suggested to us the possibility that they may be under the control of different promoter sequences. Heterogeneity in the 5'UT region may also regulate translational efficiencies or mRNA stability, thus playing an active role in posttranscriptional control at the translational level (29). These mechanisms controlling gene activation are particularly significant during early life when precise control of gene expression, both tissue and stage specific, become critical for development to proceed normally (30).

Promoter diversity could allow for very precise and selective regulation of MR gene expression at the transcriptional level. There is direct and indirect evidence that supports the possibility that the rat {alpha} and ß MR mRNA 5'UT variants are controlled by separate promoters (26, 32, 38, 41). Although a third promoter region has not been identified for the {gamma} 5'UT variant, the structure of the MR gene is such that this 5'UT exon is likely to be controlled by a separate promoter. Different cis elements upstream of each promoter can conceivably provide a mechanism for hormonal and tissue-specific factors to regulate each subtype independently in the context of tissue specificity or of temporal events unique to developmental programming. In fact, sequencing of areas flanking the {alpha} and ß exons has revealed promoter elements characteristic of constitutively expressed genes. These characteristics include a rich GC region, an Sp1 site, and the absence of TATA or CAAT boxes interdigitated with regulatory elements (26, 38). In addition to these elements, the {alpha} promoter contains a weak glucocorticoid response element site (half-glucocorticoid response element) and several consensus sequences for the following cis-acting enhancers: GCF (negative regulator of epidermal growth factor), AP-2 and PEA-3 (sites for growth factor, cAMP, phorbol ester, and oncogene regulatory elements), LF-A1 (site that directs the expression of hepatic genes), and a heat shock-responsive element (26). The promoter directing ß MR mRNA expression contains a consensus sequence for an AP-2 site, a site that mediates gene activation in response to cAMP and phorbol esters (42). This suggests that the signal transduction pathway modulates activity of the exon encoding the ß 5'-flanking region. Thus, the structure of these regions contains potential areas where regulatory factors interact with constitutive factors. These regulatory elements can allow or impede transcription in a tissue-specific or developmentally inducible pattern of expression (30). Direct evidence of a developmentally inducible pattern of expression of MR mRNA variants has been reported by Castrèn and co-workers (43). Using the {alpha} promoter, flanking the first untranslated exon of the rat MR gene, Castrèn and co-workers have shown transcriptional induction by progesterone and corticosterone in primary hippocampal neurons (43, 44). Hormonal changes of this nature may have a marked impact on the anatomical organization and function of the developing hippocampus.

The anatomical organization of the hippocampal formation resembles two interlocking Cs. The first C contains the pyramidal cell fields of the hippocampus proper (Ammon’s horn), and the second contains the granular cell layer of the DG. In adult and developing animals, there are marked differences in the characteristics of the cell populations that comprise the hippocampal formation. It is of interest to relate the patterns of increase in multiple MR forms with the established patterns of neurogenesis, axonal and dendrite formation of multiple cell populations within the hippocampus. Neurogenesis of the pyramidal cell layer occurs during prenatal life in an environment of relatively high corticosterone levels compared with those present postnatally. At birth, Ammon’s horn is clearly delineated with mature pyramidal cells (23). In contrast, considerable neurogenesis and granular cell migration are evident in the DG during the first 2 weeks of life (23). Unlike the pyramidal cell regions, DG granular cell neurogenesis and migration are particularly favored by the low corticosterone environment seen in the first week of life (23). However, the development of axons, dendrites, and mossy fiber synaptic contacts that characterize regional wiring of both pyramidal and granular cells is not completed until the fourth week of life (23, 45, 46). Rapid elaboration of pyramidal axons and dendrites occurs most rapidly during the second week of life (45, 47). Similarly, in the DG, the greatest increase in synaptogenic density occurs between PN 4 and 14 and then rises more slowly to adult values (48). The extent of these processes varies across hippocampal regions (49). For example, CA1 pyramidal cells send relatively few local axon collaterals to neighboring neurons (mostly interneurons) compared with CA3. In the CA3 region, axons extend a large network of local collaterals to neighboring neurons, including other pyramidal cells. In addition, active trimming of initial collateral sprouting (pruning) is part of the neuronal plasticity observed during development. It is evident from our study that these morphological changes may parallel the maximal rate of change in {gamma} and ß 5'UT MR mRNA expression in particular hippocampal regions. For example, {gamma} expression appears to be directly linked to periods of cell birth, as seen in DG during the first week of life. It also appears to be linked to periods of increased axonal sprouting, as {gamma} expression also predominates in the CA1 pyramidal region where collateral sprouting takes place during postnatal life (49). ß expression, on the other hand, may be best linked to synaptogenesis, growth of commissural and associative terminal fields, perhaps in combination with periods of active trimming. These processes predominate during the first 2 weeks of life in CA2 and CA3 pyramidal regions, where we observed increased ß MR mRNA expression. A similar relationship is observed in the DG beginning on the final days of the first week of life. Studies in which MR genomic transcripts are coexpressed with specific growth factors, markers of synapse formation, and apoptosis markers may clarify these initial impressions.

The second objective of this study was to determine whether the different MR mRNA variants were differentially regulated by adrenal steroids. We were surprised to find that removal of endogenous corticosteroids for a short period of time resulted in an increase in both {alpha} and ß 5'UT MR mRNA variants in adult and 28-day-old animals. Our laboratory had previously reported that in the adult animal, only the {alpha} 5'UT MR mRNA form was sensitive to corticoid environment (25). The discrepancy between these two studies is probably due to the ADX time: 7 days post-ADX vs. 14 h post-ADX in the present report. Thus, the ß 5'UT MR mRNA variant appears to respond rapidly to changes induced by adrenocortical steroid environment in the adult animal. However, it remains the case that in the adult animal, the {alpha} form was consistently elevated in those areas that also exhibited up-regulation when analyzed with the total MR mRNA probe. This is probably due to the fact that in the adult, the {alpha} form consistently represents the highest proportion of the total MR mRNA found in any given hippocampal region (25, 27), whereas this is not necessarily the case at other ages. In the developing animal, the ß and {gamma} 5'UT MR mRNA are not only relatively more abundant than in the adult, but they are also sensitive to the absence of corticosterone levels in a site-specific manner. The ß and {gamma} forms in the CA1 pyramidal cells appear to be a particularly sensitive target of corticosterone transcriptional modulation. In this region, the 10-day-old animal up-regulates {gamma} 5'UT MR mRNA, whereas the 28-day-old animal increases the expression of ß 5'UT MR mRNA. In contrast to the adult animal, the significant hippocampal subfield elevations of 5'UT MR mRNA forms in the developing animal were not always reflected in the total MR mRNA probe measurement. Thus, in the 28-day-old animal ß 5'UT MR mRNA is the only 5'UT MR mRNA up-regulated in the CA1 region, and the total MR mRNA analysis reflects this up-regulation. In contrast, short term ADX increases transcription of the {gamma} 5'UT MR mRNA in the CA1 hippocampal region of the 10-day-old animal. However, this increase is not reflected in the total MR mRNA measurement. This is likely because the change is modest in magnitude, and this single form may not represent a very large proportion of the total of all the mRNA species in the young animal. Thus, although the 5'UT MR mRNA variants appear to respond rapidly to a changing corticosteroid environment, their contribution to the total MR mRNA and mineralocorticoid receptor binding levels differs depending on the developmental period, their relative proportion within the total pool, and possible factors that we have not explored in this study, such as mRNA stability.

In conclusion, the results of the present study indicate that multiple 5'UT variants of the MR message exist in the developing hippocampus. The level of expression of these variants appears to be site specific within the hippocampus and appears to follow the pattern of salient developmental events within this structure. Increases in hippocampal transcriptional and translational levels observed after short term ADX suggest that increases in receptor binding correspond to individual or combined contributions of {alpha} and ß MR mRNA translation in both adults and developing animals. Different promoters are likely to play a role in the expression of these mRNA variants. Thus, the multiple MR forms might simply represent vestiges or indicators of a complex pattern of developmental regulation that may involve a multitude of factors unique to each time and brain region, revealing the critical and differential role of MR in the various tissues. The present findings add to a body of evidence that points to the great complexity and exquisite precision of neurogenesis and regulation of multiple populations of neurons within the hippocampal formation.


   Acknowledgments
 
The authors thank Aleda Nash and Lisa Montney for her technical assistance.


   Footnotes
 
1 This work was supported by NIDA Grants DA-02265, DA-00250 (to D.M.V. and H.A.), and PO1-MH-42251 (to S.J.W., H.A., and D.M.V.). Back

2 Present address: Molecular Genetics, Wyeth-Ayerst Research, 865 Ridge Road, Monmouth Junction, New Jersey 08852. Back

Received January 23, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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