Differential regulation of corticotropin-releasing hormone mRNA in rat brain.

Differential regulation of corticotropin-releasing hormone mRNA in rat brain D. M. FRIM,

B. G. ROBINSON,

K. B. PASIEKA,

AND

J. A. MAJZOUB

Division of Endocrinology, Department of Medicine, The Children’s Hospital; Endocrine-Hypertension Division, Department of Medicine, Brigham and Women’s Hospital; and Program in Neuroscience, Department of Neurobiology, Harvard Medical School, Boston, Massachusetts 02115

FRIM, D. M., B. G. ROBINSON, K. B. PASIEKA,AND J. A. MAJZOUB. Differential regulation of corticotropin-releasing hormone mRNA in rat brain. Am. J. Physiol. 258 (Endocrinol. Metab. 21): E686-E692, 1990.-Corticotropin-releasing hormone (CRH), a major hypothalamic component of the hypothalamic-pituitary-adrenal axis, has been localized to both the paraventricular nucleus (PVN) and cerebral cortex. Adrenalectomy causes an increase in PVN CRH content, whereas its effect on cortical CRH content is not clear. In the present study, adrenalectomy resulted in a threefold rise in the CRH mRNA content of anatomic micropunches of the PVN of individual rats (P < O.OOl),which was abolished by dexamethasone replacement. In parietal cortex, adrenalectomy did not affect CRH mRNA content, whereas hypophysectomy resulted in a twofold rise in CRH mRNA content (P < 0.02), which was not significantly reduced by dexamethasone replacement. These results demonstrate that the CRH gene is negatively regulated by glucocorticoid in the PVN but not in cerebral cortex and that the increase in cortical CRH mRNA content after hypophysectomy may be evidence for negative regulation of cortical CRH gene expression by a second pituitary-dependent factor other than glucocorticoid. glucocorticoid; hypophysectomy

CORTICOTROPIN-RELEASING hormone (CRH) is a 4lamino acid peptide released from the hypothalamus in response to a variety of stimuli including stress (29, 31). CRH regulates adrenocorticotropic hormone (ACTH) secretion (19, 20) from the pituitary and thus indirectly controls circulating glucocorticoid levels. CRH has been localized to the parvocellular division of the hypothalamic paraventricular nucleus (PVN) (3,14,16,27) where immunocytochemical CRH content rises after adrenalectomy (23, 27). After colchicine pretreatment, other areas of the rat brain, including the cortex, are also immunopositive for CRH (8, 14, 23, 27). Adrenalectomy has been reported to either have no effect (27) or to slightly increase (23) cortical CRH content. CRH mRNA has been localized to the rat hypothalamus (2, 12) and cerebral cortex (28) by Northern blot analysis and more precisely to the hypothalamic PVN by in situ histohybridization (10, 33). Adrenalectomy produces a 1.5 to twofold rise in hypothalamic CRH mRNA (12, 33) and no change in CRH mRNA levels in extrahypothalamic whole brain (12). We investigated CRH gene regulation in rat PVN and E686

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cerebral cortex by performing semiquantitative Northern blot analysis of CRH mRNA in response to adrenalectomy, hypophysectomy, and glucocorticoid treatment. Using paired samples of PVN and parietal cortical RNA isolated from the same animal, we found that CRH mRNA levels are differentially regulated in these two brain sites. METHODS

Experimental animals and experimental design. Male Sprague-Dawley rats were prepared at the Charles River Laboratories. Siblings were divided into five surgical groups on day 21 of life: normal nonoperated (n = lo), adrenalectomized (n = 30), hypophysectomized (n = 21), sham-adrenalectomized (n = 12), or sham-hypophysectomized (n = 13). We received these animals 7 days after surgery and maintained the normal and sham-operated animals on tap water and standard rat chow and the operated animals on 0.9% saline in 5% sucrose solution and rat chow. All animals received daily intraperitoneal injections for the 7 days between arrival and death. Animals in the normal group received a daily injection of 0.5 ml normal saline. All other groups were subdivided into two groups, one receiving a daily injection of 0.5 ml normal saline and the other receiving an injection of 400 pg of dexamethasone in 0.5 ml saline. All animals were maintained on a 12:12 h light-dark cycle, with lights on at 0600 h and lights off at 1800 h; all injections were delivered between 1700 and 1800 h daily. Tissue preparation. On the 8th day after arrival, animals were killed by decapitation between 0900 and 1000 h. Trunk blood was collected in tubes containing EDTA (Monoject, St. Louis, MO), spun, and plasma was stored at -80°C for ACTH and corticosterone radioimmunoassay (RIA). Brains were removed, and a 3-mm slab of tissue was obtained by coronal sectioning between the anterior margin of the optic chiasm and the posterior aspect of the mammillary bodies. Adequate reproducibility of sectioning and angle of cutting were achieved with a custom-made rat brain mold. Slabs of tissue were fast frozen to -80°C in hexane in dry ice and stored at -8OOC. Brain slabs were mounted on cryostat chucks, and 20pm sections were cut in a cryostat (Hacker Instruments, Fairfield, NJ) at -20°C, from the anterior extent of the slab (level of the anterior chiasm) to a point (-200 PM caudad) where both the chiasm and anterior commissure

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were visible in the plane of section (Fig. 1). PVN tissue was then removed in three punches using a blunt-ended 18-g needle and stored at -80°C. Extent of removal of PVN tissue was assessed by methyl green staining of sections cut before and after punching (Fig. 1). Because quantitation depended on the collection of complete PVN, samples not collected in their entirety were ex-

FIG. 1. Photomicrographs of coronal section through region of third ventricle before (A) and after (B) micropunching of PVN. Region of PVN (P) is delimited by dashed line in A. 0, optic chiasm; 3V, third ventricle. Magnification ~25.

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eluded from further analysis. Cortex was obtained from brain slabs after PVN tissue was obtained as described above by removal of tissue above a horizontal cut -1 mm dorsal to the corpus callosum. These semilunar slabs of tissue, weighing -100 mg each, contained parietal and cingulate cortex from the anterior to posterior level of the posterior hypothalamus. Cortical slabs were separately stored at -80°C for further analysis. Skeletal muscle was obtained by removal of rectus abdominus muscle from Sprague-Dawley rats killed by CO2 intoxication. Tissue from several animals was fast frozen at -80°C in hexane and dry ice and powdered while frozen to produce one homogeneous pool of skeletal muscle. This pool of tissue was separated into l-g aliquots and stored at -80°C. Isolation of RNA and Northern blot analysis. For PVN RNA extraction, 1 g of skeletal muscle was added to 10 ml of guanidine thiocyanate solution (5) containing 4 ng of a synthetic CRH cRNA standard (see below). This mixture was homogenized in a polytron (Brinkmann) for two 15-s bursts, and then a l-ml aliquot was mixed with each PVN punch and sonicated (Sonifier) for two 10-s bursts. Cortical RNA was prepared by sonication of lOOmg slabs of cortical tissue in 1 ml of guanidine thiocyanate. After underlayering with 1 ml of 5.7 M cesium chloride, all samples were spun for 18 h at 65,000 rpm in a TL1OO.l ultracentrifuge rotor (Beckman). RNA pellets were resuspended in 0.3 M sodium acetate, ethanol precipitated, and quantitated by ultraviolet spectroscopy. Skeletal muscle RNA devoid of PVN punches was also prepared as a control. As described (9), 10 pg of total RNA from each PVN sample, cortical sample, or skeletal muscle control sample were subjected to electrophoresis through 1.4% agarose containing 2.2 M formaldehyde and were transferred to Genescreen (Du Pont). Hybridizations were performed using 1 X lo7 32P-labeled antisense cRNA probes (see below) at 65°C for 24 to 36 h. Blots were washed twice at 65°C in 0.1% sodium dodecyl sulfate (SDS), 15 mM NaCl, and 1.5 mM sodium citrate for 20 min, air dried, and exposed at -80°C to Kodak XAR film with an intensifying screen. Blots were serially hybridized to two rat CRH probes (rCRH-1, then rCRH-2) followed by a mouse @-actin probe. Semiquantitative assessment of the relative amounts of CRH mRNA (1,700 nucleotides), brain P-actin mRNA (2,100 nucleotides), and CRH cRNA standard (400 nucleotides) content was performed using an Ultroscan densitometer (LKB 2400 gelscan XL). The amount of CRH cRNA sense-strand standard detected was used to correct for the recovery of PVN CRH mRNA, which was expressed in arbitrary units. Reliable quantitation depended on 1) accurate, complete punching of hypothalamic nuclei; 2) co-isolation of RNA from hypothalamic nuclear tissue and a constant amount of carrier tissue that does not express the mRNA of interest; 3) the addition of an exogenous RNA recovery marker, because endogenous RNA recovery markers such as @-actin mRNA are contained in both PVN as well as in the variable amount of non-PVN tissue present in each


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cance were two tailed. Graphic data are displayed as the punched sample; 4) Northern blot analysis with a probe present in vast excess compared with the mRNA of mean value for each determination + the SE of the mean. interest; and 5) densitometric quantitation of autoradiographic bands in the linear range of the dose-response RESULTS curve for signal vs. band intensity. With this technique Isolation of total RNA from PVN and cerebral cortex. it is possible to quantitate the level of CRH mRNA We validated our technique for the isolation of total present in a fraction of an individual animal’s paravenRNA from punches of paired PVN in the following way. tricular nuclei, allowing for the analysis of sufficient Brain tissue sections were cut before and after PVN numbers of animals in each experiment to reach statispunching as shown in Fig. 1. The entire PVN, both tically sound conclusions. This same technique has been magnocellular and parvocellular divisions, was removed. used previously to study the expression of the vasopressin Serial sections in all animals used in this study assessed gene in discrete hypothalamic nuclei (22). The amount the accuracy of the punching technique and resulted in of brain P-actin mRNA detected was used to correct for the exclusion of two from further study. We had the recovery of cortical CRH mRNA. In prior studies, /3- determined previouslyanimals by in situ histohybridization (10) actin mRNA levels have been found to be unaffected by that within the PVN punches there were no CRH glucocorticoid status (18). Because of the different methmRNA-containing cells outside of the PVN. Thus any ods used to prepare PVN and cortical RNA, CRH mRNA CRH mRNA signal detected in these tissue samples was in these two sites could not be directly compared in a derived from PVN. quantitative manner. Total RNA from paired PVN punches of individual Construction of probes. rCRH1, the 700 nucleotideanimals mixed with 100 mg of skeletal muscle was prelong Rsa I fragment of the coding region of a rat hy- pared. Ten micrograms (-0.25) of each preparation was pothalamic CRH cDNA isolated in our laboratory, en- assayed for CRH mRNA content by Northern blot analycompassing bases 451-1,151 of the rat CRH gene (28), sis. In Fig. 2A, lane I, such a sample from an adrenalecwas subcloned into the polylinker Rsa I site of Bluescribe tomized animal is displayed. CRH mRNA appeared as a (Stratagene, San Diego, CA). rCRH2, consisting of the single band -1,700 nucleotides long, as demonstrated 280 nucleotides upstream from the Kpn I site in the 5’ previously (9, 10). Ten micrograms of skeletal muscle untranslated region of the rat CRH gene, was inserted RNA by itself contained no detectable CRH mRNA (Fig. into the Kpn I/Eco RI site of Bluescribe as described 2A, lane 3). Thus the CRH mRNA signal present in the previously (10). A full-length mouse /3-actin cDNA, a PVN/skeletal muscle RNA preparations was derived generous gift of B. Spiegelman (Harvard University), from the PVN punch alone. An equal amount of total was cloned into the Pst I site of Bluescribe. To make cortical RNA contained a CRH mRNA band similar in “‘P-labeled cRNA transcripts, all three plasmids were size to that found in PVN (Fig. 2A, lane 2). linearized with Eco RI and transcribed with T:z RNA These blots were also hybridized to an actin probe. polymerase (Stratagene) using [01-32P]UTP (Amersham, RNA from the PVN/skeletal muscle preparations conArlington Heights, IL) as described (“Protocols for use tained two actin mRNA species (Fig. 2B, lane 1). As with SP6 system,” Code RPN.1506, Amersham) to spe- expected, the larger of these species comigrated with the cific activities of -1.5 X lo7 cpm/pmol. Then lo7 cpm (-0.67 pmol) were added to each blot for hybridization. A E3 Sense-strand CRH cRNA standard was generated from 123. 123 the Xmn I/Pst I fragment of the human CRH gene (l), encompassing bases 87-462 of the human CRH gene (25). This fragment, cloned into the corresponding polylinker region of Bluescribe, was linearized with Hind III and transcribed with T7 RNA polymerase (without addition of a radiolabeled nucleotide) to produce a sensestrand human CRH cRNA 400 bases in length. Radioimmunoassay. Serum samples were assayed for ACTH and corticosterone by RIA using commercial kits (Nichols Institute, San Juan Capistrano, CA and Cambridge Medical Diagnostics, Billerica, MA, respectively). Sensitivity was 7 rig/ml plasma for corticosterone and 70 pg/ml plasma for ACTH. Statistical analysis. Two-way analysis of variance with post hoc comparison of means using the Student-NewFIG. 2. Northern blot analysis of CRH mRNA in rat PVN and man-Keuls test was performed with a SAS computer parietal cortex. Ten micrograms of total RNA from a PVN punch t 1) program (SAS Institute, Cary, NC) to determine the or parietal cortex of an adrenalectomized rat (2), or skeletal muscle significance of differences found between control and alone (3) were analyzed usmg either a CRH probe, rCRH1 (A), or an (R). Top and bottom arrows denote positions of migration experimental groups. Five outlying data points (of 344 ofactin28s probe and 1% ribosomal RNA, respectively. Apparent band just below total observations) were rejected at a 1% significance 2% rRNA in R, lane 1 is most likely a compression artifact caused by 28s rRNA. level using the method of Grubbs (11). Tests of signifiDownloaded from www.physiology.org/journal/ajpendo at Midwestern Univ Lib (132.174.254.157) on February 13, 2019.

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2.1-kb @actin mRNA present in brain (Fig. 2B, lane 2; Ref. 18), and the smaller of these species comigrated with the 1.5kb skeletal muscle-specific cY-actin mRNA (Fig. 2B, lane 3; Ref. 24). Hormonal manipulations. Plasma corticosterone levels were similar in the untreated control, the sham-adrenalectomized, and the sham-hypophysectomized groups (299 + 17 rig/ml). Corticosterone was undetectable (2,000 pg/ml. These two adrenalectomized animals, which had ACTH values of 380 and 689 pg/ml, were most likely only partially adrenalectomized and were therefore excluded from further study. All other animals subjected to surgical or hormonal manipulations demonstrated the appropriate changes in plasma corticosterone and ACTH and were analyzed as described below. CRH mRNA regulation in the PVN and cerebral cortex.

CRH mRNA content of PVN punches was assessed in several hormonal states. The amount of synthetic sensestrand CRH cRNA standard added to each RNA preparation before extraction was measured in each sample (Fig. 3, bottom) and used to correct for the recovery of

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PVN CRH mRNA (see Fig. 3, top and Fig. 5, left). Animals in the three untreated control groups, normal, sham adrenalectomy, and sham hypophysectomy, had no significant differences in plasma corticosterone, plasma ACTH, or CRH mRNA content in PVN and cortex (data not shown) and were therefore combined into a single normal untreated group (n = 18). The mean value of PVN CRH mRNA in this group was arbitrarily adjusted to 1.0 units, and PVN CRH mRNA contents of all other groups were expressed relative to this group. The dexamethasone-treated sham-adrenalectomy and dexamethasone-treated sham-hypophysectomy groups, which were indistinguishable in all of the parameters examined, were also analyzed together as a normal treated group (n = 11, 0.9 + 0.4). Compared with the normal group, PVN CRH mRNA levels increased approximately threefold (P < 0.001) in adrenalectomized animals (n = 12, 2.8 & 0.3). This increase was abolished in adrenalectomized animals treated with dexamethasone (n = 13, 1.2 + 0.3). Hypophysectomy, either without (n = 10, 1.6 + 0.4) .or with (n = 11, 1.0 + 0.4) dexamethasone treatment was not associated with a statistically significant change in PVN CRH mRNA compared with untreated normal rats. Changes in cortical CRH mRNA levels in response to hormonal manipulations were quite different from those observed in the PVN of these same animals (Fig. 4; and Fig. 5, right). Compared with normal animals (n = 18, mean value arbitrarily adjusted to 1.0 units with all other cortical CRH mRNA values expressed relative to this group), adrenalectomized animals, either without (n = 12, 1.0 + 0.3) or with (n = 13, 1.0 + 0.3) dexamethasone

+

FIG. 3. Northern blot analysis of PVN CRH mRNA levels in different hormonal states. Ten micrograms of RNA from PVN punches of rats that were normal untreated (N-); adrenalectomized untreated (A-); hypophysectomized untreated (H-); adrenalectomized, dexamethasone-treated (A+); or hypophysectomized, dexamethasone (Dx)treated (H+) were analyzed using either rCRH1, complementary to rCRH mRNA coding region (top), or rCRH2, complementary to-the CRH sense-strand cRNA standard (bottom). Ton and bottom arrows. positions of migration of 2% and 1% ribosomal RNA, respectively. * Position of migration of the CRH standard (0.4 kb)

FIG. 4. Northern blot analysis of parietal cortical CRH mRNA levels in different hormonal states. Ten micrograms of cortical RNA from rats that were adrenalectomized untreated (A-); hypophysectomized untreated (H-); adrenalectomized dexamethasone-treated (A+); hypophysectomized dexamethasone-treated (H+); or normal, untreated (N-) were analyzed using either rCRH1 (top) or an actin probe (bottom), showing position of migration of 2.1-kb-long o-actin mRNA.


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FIG. 5. Graphic representation of CRH mRNA levels in different hormonal states in PVN (left) and parietal cortex (right). Normal (N), adrenalectomized (A), and hypophysectomized (H) rats were either untreated (no Dex, open bars) or treated with dexamethasone (Dex, solid bars). Left: * P < 0.001 compared with untreated normal. ** P < 0.05 compared with untreated adrenalectomy group. Right: * P < 0.015 compared with untreated normal.

replacement had no change in cortical CRH mRNA increase in PVN CRH mRNA, although there was a content. In contrast, hypophysectomy (n = 9, 2.0 t 0.3) trend in this direction. The lack of increase was somewas associated with a twofold rise (P < 0.015)in cortical what surprising in light of the findings within the PVN CRH mRNA content, which was not significantly lower after adrenalectomy. However, this difference could be after dexamethasone treatment of hypophysectomized caused by several factors. There was probably a shorter animals (n = 11, 1.4 t 0.3). duration of glucocorticoid insufficiency in our hypophysectomized compared with adrenalectomized animals, DISCUSSION although by death both groups had undetectable corticosterone levels. Furthermore, hypophysectomy might We have demonstrated that the CRH gene is regulated have resulted in the damage, by retrograde degeneration, quite differently in two regions of the brain, the hy- of CRH mRNA-containing neurons within the PVN if pothalamic PVN and the parietal cortex. In the PVN, the median eminence was also disrupted. rats subjected to adrenalectomy 2 wk before death susIn contrast to the findings in PVN, adrenalectomy had tained a threefold rise in CRH mRNA content. This rise no on CRH mRNA content within parietal cortex. in PVN CRH mRNA after adrenalectomy was similar to Thiseffect observation is consistent with a prior immunocythat reported in prior studies (2, 12, 33). Jingami et al. tochemical study that did not find an increase in CRH (l2), who described only a M-fold increase in CRH peptide within rat cerebral cortex after adrenalectomy mRNA in the whole hypothalamus after adrenalectomy, (27), although another study by this same group (23) suggested that the rather modest increase they observed found that adrenalectomy caused a slight increase in might have been caused by the presence of CRH mRNAimmunoreactive cortical CRH neurons. The difference containing cells not responsive to glucocorticoid within in CRH mRNA expression in PVN and cortex taken the hypothalamus. This suggestion is supported by the from the same adrenalectomized animals suggests that finding of CRH mRNA within the supraoptic nucleus of the CRH gene in these two sites responded differently the rat hypothalamus (2, 10, 32). Of interest, other CRH A rise in PVN CRH mRNA content was not seen in to glucocorticoid withdrawal. mRNA-containing brain sites outside of the PVN have sham-adrenalectomized animals or in adrenalectomized also been shown to be unresponsive to glucocorticoid animals treated for 1 wk before death with dexamethawithdrawal (2). However, in our study, as well as these sone, suggesting that the rise seen in adrenalectomized CRH mRNA regulation animals was caused by glucocorticoid withdrawal. These other studies of differential within and outside of the PVN, relatively few time points data confirm those of Beyer et al. (2) and are consistent after surgical or hormonal manipulations were examined. with the finding that CRH peptide increases severalfold within the parvocellular region of the PVN after adre- It is possible that some of the observed differences could nalectomy (15, 23, 27), as well as with the recent dem- be caused by differences in the kinetics of CRH mRNA onstration of negative regulation by glucocorticoid of the regulation in these sites. To further investigate the response of cortical CRH human CRH gene transfected into a heterologous mouse anterior pituitary cell line (1). Whether the increase in mRNA to glucocorticoid withdrawal, hypophysectomized resulted in a CRH mRNA content in PVN after adrenalectomy is animals were studied. Hypophysectomy caused by increased transcription of the CRH gene or significant, twofold increase in CRH mRNA levels within cerebral cortex. Because adrenalectomy did not yield increased stability of CRH mRNA cannot be predicted. similar results, this increase was caused, at least in part, Glucocorticoid has been found to affect both gene transcription rate (4, 7, 13) as well as mRNA stability (17, by a factor other than adrenal insufficiency. Glucocorticoid-treated hypophysectomized animals had cortical 30) Hvpophvsectomv was not associated with a significant CRH mRNA levels midwav between normal and hvDownloaded from www.physiology.org/journal/ajpendo at Midwestern Univ Lib (132.174.254.157) on February 13, 2019.

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pophysectomized values. Although these data do not indicate whether glucocorticoid has a negative regulatory effect on cortical CRH mRNA expression, the fact that adrenalectomy, either with or without dexamethasone replacement, did not affect CRH mRNA expression in cortex suggests that glucocorticoid may not contribute to the negative regulation of the CRH gene in this site. The effects of hypophysectomy on CRH mRNA levels in cortex may be evidence for negative regulation of cortical CRH gene expression by a pituitary-dependent factor other than glucocorticoid. ACTH, which we found to rise after adrenalectomy, has been previously shown to negatively regulate the secretion of CRH from hypothalamic explants in vitro (26). Alternatively, other pituitarydependent factors that might more easily cross the bloodbrain barrier, such as thyroid hormone or gonadal steroids, might affect cortical CRH gene expression. Animals in this study were treated with a pharmacologic dose of dexamethasone. Although this high dose is catabolic, the animals receiving dexamethasone did not display any overt signs of systemic illness. This dose was chosen to determine whether markedly supraphysiologic levels of glucocorticoid are capable of completely suppressing the expression of CRH mRNA in vivo. We had previously shown in vitro that high levels of dexamethasone (1 ,uM) resulted in only a 50-70% fall in CRH mRNA in heterologous cells transfected with the human CRH gene (l), and Beyer et al. (2) found that high-dose dexamethasone administration to rats did not completely suppress CRH mRNA expression. Those data, together with the present data, suggest that CRH gene expression cannot be completely inhibited by dexamethasone even at high doses. Thus the CRH gene appears to be negatively regulated by glucocorticoid in brain PVN and by an additional factor in cortex. The differential tissue-specific regulation of CRH in these sites, together with the widespread distribution of CRH and its receptor outside of the hypothalamus (2, 6, 8, 27), suggest that CRH outside of the PVN may play an important role other than in the modulation of the hypothalamic-pituitary-adrenal axis.

MRNA

3.

4.

5.

6.

7.

8.

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11. 12.

13.

14, ,

15.

16. We thank Dr. R. Gleason for help with statistical analyses; 0. Cortez, J. Gleason, and T. Shepard for excellent technical assistance; B. Spiegelman for the gift of a mouse ,&actin cDNA; and M. Jacobs for excellent secretarial assistance. This work was supported in part by National Institute of Child Health and Human Development Grant ROl HD-24704 (to J. A. Majzoub) and by grants from the Harvard Medical Scientist Training Program (National Institute of General Medical Services 2T 32GM07753-07 to D. Frim), the Medical Foundation, the University of Sydney, and the Royal Australasian College of Physicians (to B. Robinson). Address for reprint requests: J. A. Majzoub, Division of Endocrinology, Dept. of Medicine, The Children’s Hospital, Boston, MA 02115.

17. 18.

19.

20. Received

29 September

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accepted

in final

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5 December

1989. 21.

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Differential regulation of corticotropin-releasing factor mRNA in rat brain regions by glucocorticoids and stress.

neurogranin mRNA.

Differential regulation of rat AT1a and AT1b receptor mRNA.

Differential regulation of thyrotropin-releasing hormone receptor mRNA levels by thyroid hormone in vivo and in vitro (GH3 cells).

Expression of c-fos mRNA in rat brain after intracerebroventricular administration of corticotropin-releasing hormone.

Effect of growth hormone on rat hepatic cytochrome P-450f mRNA: a new mode of regulation.

Differential regulation of glucose transporter isoforms by thyroid hormone in rat heart.

Diurnal regulation of per repeat mRNA in the suprachiasmatic nucleus in rat brain.

Intrahippocampal colchicine alters hypothalamic corticotropin-releasing hormone and hippocampal steroid receptor mRNA in rat brain.

Expression and regulation of growth hormone (GH) receptor messenger ribonucleic acid (mRNA) in rat adipose tissue, adipocytes, and adipocyte precursor cells: GH regulation of GH receptor mRNA.

Thyroid hormone regulation of NGF, NT-3 and BDNF RNA in the adult rat brain.

Dopamine regulation of gonadotropin-releasing hormone (GnRH) gene expression in the female rat brain.

Dopaminergic regulation of cholecystokinin mRNA content in rat striatum.

Second messengers involved in the regulation of corticotropin-releasing hormone mRNA and peptide in cultured rat fetal hypothalamic primary cultures.

In situ localization of tau mRNA in developing rat brain.

The effects of pituitary stalk transection, hypophysectomy and thyroid hormone status on insulin-like growth factor 2-, growth hormone releasing hormone-, and somatostatin mRNA prevalence in rat brain.

Differential distribution of the mRNA encoding two isoforms of the catalytic subunit of calcineurin in the rat brain.

Presence of luteinizing hormone-releasing hormone (LHRH) mRNA in rat spleen lymphocytes.

chorionic gonadotropin receptor mRNA in rat ovarian cells during follicular development and luteinization.

Induction of interleukin-1 beta mRNA in adult rat brain.

Distribution of D5 dopamine receptor mRNA in rat brain.

Differential regulation of brain beta-adrenoceptor subpopulations in aging rodents.

BAG3 mRNA is present in synaptosomal polysomes of rat brain.

Ontogeny of dopamine D2 receptor mRNA in rat brain.