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Corticosterone Can Facilitate as Well as Inhibit Corticotropin-Releasing Hormone Gene Expression in the Rat Hypothalamic Paraventricular Nucleus¹

Program in Neural, Informational, and Behavioral Sciences, and Neuroscience Graduate Program, Department of Biological Sciences, University of Southern California, Los Angeles, California 90089-2520

Address all correspondence and requests for reprints to: Alan G. Watts, D. Phil, Department of Biological Sciences, Hedco Neuroscience Building, MC 2520, University of Southern California, Los Angeles, California 90089-2520. E-mail: watts{at}rcf.usc.edu

	Abstract

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We have used in situ hybridization to investigate how basal levels of circulating corticosterone modulate CRH gene transcription in the neuroendocrine parvicellular part of the hypothalamic paraventricular nucleus (PVHmpd) during sustained hypovolemia. In the absence of the stressor, the accumulation rate of the CRH primary transcript exhibited a dose dependency on low maintained levels of plasma corticosterone similar to that previously reported for the mature messenger RNA (mRNA); levels declined as plasma corticosterone increased. In response to hypovolemia, the absence of corticosterone compromised CRH gene transcription mechanisms to mount the activated response seen in intact animals. However, adrenalectomized rats with low doses of corticosterone (insufficient to normalize thymus weights) showed an augmented mRNA response compared with that in intact animals. When replaced corticosterone normalized thymus weights, the magnitude of the mRNA response was reduced to that seen in intact animals. In contrast to CRH gene regulation, PVHmpd proenkephalin mRNA levels were unaffected by corticosterone concentrations. These results suggest that corticosterone affects CRH gene transcription in the PVHmpd using two mechanisms: first, inhibition, which probably uses type II glucocorticoid receptor-dependent mechanisms and contributes to classic negative feedback; and second, facilitation, which is seen at low plasma concentrations and maintains gene transcription in the presence of sustained stress, possibly using type I mechanisms. This suggests that one reason why adrenal insufficiency severely compromises survival of sustained stress is that CRH gene transcription cannot be maintained without previous exposure to low levels of plasma corticosterone.

	Introduction

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THE HYPOTHALAMIC paraventricular nucleus (PVH) is pivotal for the endocrine response to hemodynamic stress in the rat. ACTH secretion from corticotropes is stimulated by the release of CRH from neuroendocrine neurons located in the dorsal aspect of the medial parvicellular part (PVHmpd) of the PVH (1, 2, 3). ACTH then enters the systemic circulation, stimulating corticosterone synthesis and release from the adrenal cortex, which, in turn, provides an inhibitory feedback signal to the system (for reviews, see Refs. 4, 5, 6). Despite the general appreciation for the structure of glucocorticoid feedback, the cellular mechanisms involved with corticosterone’s actions on CRH gene expression are unclear. For example, some abrupt transient stressors increase CRH messenger RNA (mRNA) levels (7, 8, 9) even though coincidentally increasing plasma corticosterone concentrations attain levels that would reduce its accumulation if persistently maintained in the unstimulated animal (6). This suggests that during abrupt stress the suppression of CRH mRNA in the PVHmp seen with chronically elevated corticosterone either does not occur because of the short duration of the corticosterone surge or is inhibited by other processes.

In intact animals the low levels of plasma corticosterone found in the early morning are sufficient to maintain levels of CRH mRNA in the PVHmp during the midpoint of the light phase (10), and these, presumably, position the transcriptional machinery in the CRH neuron to respond to ensuing stress at this time. Here, we posit that manipulating this antecedent corticosterone environment will help reveal the nature of its interaction with CRH gene regulatory mechanisms operating during a subsequent stress event.

We have recently shown that a sustained viscerosensory stressor (colloid-induced hypovolemia) (11) is accompanied by a temporally ordered sequence of events at CRH neuroendocrine neurons, corticotropes, and adrenal cortical cells: first, stimulus onset; second, release of ACTH secretogogue, ACTH, and corticosterone; and finally, activation of CRH gene expression in the PVHmp (12). CRH gene transcription, and ACTH and corticosterone secretion all peak 4 h after stimulus onset (i.e. 5 h after injection). Here we have used this same viscerosensory stressor to investigate how the corticosterone environment preceding the stressor affects the subsequent response of CRH gene expression.

To this end we have used in situ hybridization to investigate how CRH gene expression responds to sustained hypovolemia in intact, adrenalectomized, or adrenalectomized animals with corticosterone replacement. Three doses of corticosterone were given to different groups of adrenalectomized animals 6 days before the stressor: first, a low dose that was not adequate to normalize thymus weights or CRH mRNA levels in the PVHmp; second, a dose that resulted in the same thymus weights and PVH CRH mRNA as those in intact animals; and finally, a higher dose that reduced thymus weights and PVH CRH mRNA to levels below those seen in intact animals at the time of maximum activation.

Because the size of the cytoplasmic pool of neuropeptide mRNAs is thought to depend primarily on the rate of transcription and mRNA degradation (13), it is difficult to determine how corticosterone interacts with CRH neurons by only measuring mRNA levels. Therefore, as the CRH gene only codes for a single intron (14), we have measured levels of the CRH heteronuclear (hn) RNA, the primary transcript from the gene, to address more directly the mechanisms associated with the activation of gene transcription (15, 16).

Some of these data were previously presented in abstract form (17, 18).

	Materials and Methods

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Adult male Sprague-Dawley rats (225–250 g BW at the beginning of the experiment) were maintained on a 12-h light, 12-h dark photoperiod (lights on at 0600 h) with unlimited water and rat chow and were allowed at least 5 days acclimation to animal quarters.

Five groups of rats were anesthetized with halothane and either bilaterally adrenalectomized (ADX; four groups) or sham ADX (one group) using flank incisions. While still under anesthesia, ADX animals were implanted sc with 0 mg (ADX/0), 25 mg (ADX/25), 50 mg (ADX/50), or 100 mg (ADX/100) slow release corticosterone pellets (Innovative Research of America, Sarasota Beach, FL) and allowed to recover for 6 days. The corticosterone pellets provide stable plasma concentrations of corticosterone for up to 21 days.

After surgery, animals were provided with unrestricted access to water, 0.9% saline, and rat chow. On the morning of day 7, water, saline, and food were removed, and rats were given sc injections of 5 ml 40% polyethylene glycol (PEG; mol wt, 8000; Sigma Chemical Co., St. Louis, MO) dissolved in saline or 5 ml 0.9% saline at room temperature under brief halothane anesthesia between 0700–0800 h, and left undisturbed for 5 h as described by Tanimura et al. (12).

Perfusion and tissue handling
Five hours after injection, rats were deeply anesthetized by ip injection of tribromoethanol, and a single 1- to 1.5-ml blood sample was taken from the external jugular vein into a heparinized syringe for hematocrit measurement and determination of plasma corticosterone concentrations. Animals were then perfused through the ascending aorta with a brief saline rinse, at which time the thymus was removed, dissected free of adjoining tissue and fluid, and weighed. The saline rinse was followed by 500 ml ice-cold 4% paraformaldehyde solution in 0.1 M borate buffer, pH 9.5. After perfusion, the brain from each animal was removed and postfixed overnight in the fixative containing 12% sucrose (wt/vol). Brains were frozen in powdered dry ice and immediately stored at -70 C until sectioning at a later date. Eight series of one in eight, 15-µm thick, frontal sections were cut through the rostral hypothalamus and saved in ice-cold 0.25% paraformaldehyde (pH 7.4), and sections were handled and stored as previously described (19). Serial sections were saved for thionin staining.

In situ hybridization
Sections were hybridized with [³⁵S]UTP-labeled complementary RNA (cRNA) probes transcribed from either a 700-bp complementary DNA (cDNA) sequence coding for part of the mRNA encoding prepro-CRH, a 935-bp cDNA sequence coding for the entire coding sequence of preproenkephalin, or a 536-bp PvuII fragment complementary to the sequence within the single CRH intron. All probes were synthesized using the Promega Gemini kit (Promega, Madison, WI) and the appropriate RNA polymerase. In situ hybridization with the ³⁵S-labeled cRNA probes was performed as described previously (10) with posthybridization modifications for CRH hnRNA as follows. After the ribonuclease incubation and room temperature washes from 0.1–4 x SSC (standard saline citrate), slides were incubated at 70 C for 30 min with slight agitation every 10 min. Controls for all three in situ hybridization probes consisted of incubating sections with cRNAs synthesized from cDNA sense strands or the incubating sections pretreated with ribonuclease and then hybridizing with antisense-generated probes. In all cases no hybridization signal was seen. Sections were exposed to Cronex Microvision x-ray film (DuPont, Wilmington, DE) for appropriate exposure periods (4–21 days), then dipped in nuclear track emulsion (Kodak NTB-2, Eastman Kodak, Rochester, NY; diluted 1:1 with distilled water), exposed for 5–25 days, developed, and counterstained with thionin.

RIA
Plasma corticosterone concentrations were measured in duplicate unextracted samples by double antibody RIAs as described previously (10) using an [¹²⁵I]corticosterone double antibody RIA supplied in kit form (ICN Biochemicals, Costa Mesa, CA). The lower sensitivity limit was 15 ng/ml, and the intraassay coefficient of variation was less than 8%. All samples were measured in single assays.

Semiquantitation of [³⁵S]UTP cRNA hybridization
Mean gray levels (MGL) of the hybridization signal in the Nissl-defined PVHmpd were measured from images on Cronex microvision x-ray film as described by Watts and Sanchez Watts (19). The response linearity of the image analysis system used for measuring MGL of film images from in situ hybridization was confirmed using a series of ¹⁴C-labeled microscales (Amersham International, Aylesbury, UK) (20). The response of the film and camera system over the signal range used in this experiment was linear (r² = 0.9946; F = 464; P < 0.0001)

Statistical analysis
The significance of differences between dependent variables across treatment groups was determined using multifactorial ANOVA, followed by Tukey’s or Dunnett’s two-tailed post-hoc test, with intact values taken as the control. P < 0.05 was regarded as statistically significant for all tests. The significance of differences in dependent variables between saline-injected and PEG-treated animals within each steroid treatment group was determined using Student’s t test, assuming unequal variances. All statistical analyses were performed using Excel (Mac version 4.0, Microsoft, Redmond, WA) and Systat (Mac version 5.2, Systat, Evanston, IL).

	Results

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Hematocrits were not significantly different between saline-injected animals of any group (Table 1); however, hematocrits were significantly elevated in all PEG-treated groups compared with their respective control values (P < 0.001 in all cases). Table 1 also shows that plasma concentrations of corticosterone were markedly elevated 5 h after PEG treatment in intact animals (P < 0.0001), whereas there was no significant increase after PEG injection in ADX animals given exogenous corticosterone. There were no significant differences in thymus weights between animals injected with vehicle or PEG (data not shown). Values from vehicle- and PEG-injected animals in each steroid treatment group were pooled for both plasma corticosterone concentrations and thymus weights for subsequent comparisons across groups. Except for the ADX/25 and ADX/50 groups, mean pooled plasma corticosterone concentrations in each adrenalectomized group were all significantly different from each other (Fig. 1A; P < 0.01 or greater). Figure 1b shows that plasma corticosterone levels in the ADX/50 animals reduced mean thymus weights of ADX/0 animals to those seen in intact animals. Thymus weights in ADX/0 animals, or ADX/25, or ADX/100 animals were significantly different from those in any other group (Fig. 1B; P < 0.025 or greater).

View larger version (20K): [in this window] [in a new window]   Figure 1. Mean (+SEM) plasma corticosterone concentrations (nanograms per ml) in each steroid replacement group (A) and thymus weights (milligrams) in intact and steroid treatment groups (B). See text for levels of significance.

View larger version (19K): [in this window] [in a new window]   Figure 2. A, Mean (+SEM) MGL expressed in arbitrary units of CRH mRNA hybridization in the PVHmpd of saline-injected groups and each corticosterone treatment group. B, Mean (+SEM) change in the MGL of the CRH mRNA response to sc injection of 40% PEG in intact and steroid replacement groups. See text for levels of significance.

CRH hnRNA and pENK mRNA levels in the PVHmpd
To investigate the mechanisms responsible for the regulation of CRH mRNA in ADX animals with and without low replacement doses of corticosterone, CRH hnRNA and pENK mRNA levels were determined in the PVHmpd of intact, ADX/0, and ADX/25 animals (Fig. 3). In all cases, the group trends in the CRH mRNA response to saline and PEG (Figs. 3A and 4) were paralleled by those of CRH hnRNA (Figs. 3B and 4). Thus, in saline-injected animals there was a significant increase in CRH hnRNA in the PVHmpd of ADX/0 animals compared with those in intact (P < 0.005) and ADX/25 (both P < 0.02) groups. Although not significantly different from those in intact animals, values for ADX/25 animals were intermediate between those for intact and ADX/0 animals. In response to PEG injections, both intact and ADX/25 animals showed significant increases in CRH hnRNA signal compared with saline-injected animals (intact, P < 0.005; ADX/25, P < 0.02). However, in the ADX/0, CRH hnRNA was significantly lower after PEG injection than the corresponding saline-injected control value (P < 0.02).

View larger version (19K): [in this window] [in a new window]   Figure 3. Mean (+SEM) of CRH mRNA (A), CRH hnRNA (B), and pENK (C) mRNA hybridization signal of the dorsal aspect of the medial parvicellular part of the hypothalamic paraventricular nucleus seen 5 h after sc injections of either vehicle (0.9% saline: open bars) or 40% PEG (black bars), expressed as a percentage of the mean intact control (saline-injected) value. The number of animals per group is shown in Table 1. See text for levels of significance.

View larger version (109K): [in this window] [in a new window]   Figure 4. CRH mRNA, CRH hnRNA, or pENK mRNA response to sustained hypovolemia. Images from Cronex microvision x-ray film of three serial sections hybridized for CRH mRNA, CRH hnRNA, or pENK mRNA in the PVHmpd of representative intact and ADX animals 5 h after sc injection of 0.9% saline or 40% PEG.

	Discussion

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Colloid-induced hypovolemia in intact animals leads to sustained activation of CRH gene transcription and consequent elevations in CRH mRNA in PVHmpd neurons (12). We now show that the magnitude of CRH gene response seen 5 h after PEG injection in intact animals is dependent on the corticosterone environment preceding the stressor. This dependency has three aspects, each related to prevailing circulating corticosterone concentrations. First, in the absence of corticosterone, the CRH gene response is severely compromised to the extent that mRNA levels are reduced 5 h after injection; second, low doses of corticosterone apparently facilitate the CRH gene response beyond that seen in intact animals; and third, the response is normalized by plasma corticosterone concentrations that also normalize thymus weights and basal levels of CRH mRNA (10).

Our CRH hnRNA data show that these differences in the accumulation rate of CRH mRNA most likely involve alterations in gene transcription. In the absence of corticosterone, the decreased CRH mRNA accumulation seen 5 h after PEG injection is paralleled by decreased hnRNA levels. These data indicate that a substantial modification of the mechanisms associated with stress-induced CRH gene activation occurs in the absence of corticosterone. This modification cannot be accounted for by a radical alteration in afferent signals because there were still significant increases in hematocrit and pENK mRNAs in the same PVH neuronal cell group that expressed CRH mRNA 5 h after PEG injection in ADX groups. Thus, PVHmpd neurons in ADX animals still apparently receive afferent signals conveying stimulus information, and the transcriptional machinery of these neurons can still respond to the stressor. When ADX animals are given a dose of exogenous corticosterone that is insufficient to normalize thymus weights or CRH mRNA levels in the PVHmpd, the ability of the CRH neuron to accumulate mRNA in response to the stressor not only returns but actually appears to be facilitated, as rates of mRNA accumulation actually exceed those in intact animals. At least part of this increase in mRNA accumulation is most likely due to facilitated transcription, because there is now a significant elevation in CRH hnRNA levels at 5 h in ADX/25 animals. In this manner, corticosterone-dependent facilitated CRH gene expression is perhaps analogous to the corticosterone-dependent facilitated ACTH secretory responses to novel stressors recently reported by Akana and Dallman (21) and Murakami et al. (22).

Although the nature of this facilitatory process is currently unclear, two possibilities can account for our data. First, in the absence of corticosterone, gene transcription is never activated by hypovolemia; second, gene activation occurs initially, but cannot be maintained. Data from other groups suggest that corticosterone modifies the CRH neuron using the second mechanism; corticosterone allows CRH gene transcription to be maintained in the event that a particular stressor becomes prolonged. However, it should be noted that this interpretation derives from stressors that undoubtedly use afferent mechanisms different from those activated in the present study by sustained hypovolemia (12). After abrupt transient stressors, many responses of the hypothalamo-pituitary-adrenal axis are augmented by adrenalectomy. For example at the pituitary, adrenalectomy or adrenalectomy with low level corticosterone replacement produces hypersecretion of ACTH in response to a stressor (23, 24, 25). In PVHmp CRH neurons of adrenalectomized rats, Lightman and Young (26) reported increased CRH mRNA levels 4 h after ip injections of hypertonic saline. Furthermore, Imaki et al. (27) reported an exaggerated response in CRH hnRNA 30 min after restraint stress in adrenalectomized rats. Our data now show that if gene transcription is activated by PEG in ADX/0 animals, it cannot be maintained in the absence of corticosterone, ultimately leaving the animal with reduced ability to cope with the stress event. Our results suggest that in the presence of a maintained stressor of high intensity, low levels of plasma corticosterone before the stress event (as would occur in intact animals) can facilitate the mechanisms of CRH gene transcription and prolong the ability of the CRH neuron to respond and maintain secretion. The reason CRH hnRNA and mRNA levels actually decline in the absence of corticosterone is currently unclear, but may well be related to a subsequent alteration in the turnover rates of these components. Faced with increased CRH secretion, the increased rates of CRH translation coupled with a reduced transcription rate may well result in the reduced accumulation rates of hnRNA and mRNA we observed. This does not occur in intact animals, in which CRH hnRNA levels are elevated above control values from 3 h until at least 6 h after PEG injection (12).

Two observations suggest that the amount of corticosterone required to facilitate CRH gene transcription during hypovolemia is rather low. Thus, both thymus weights and levels of CRH mRNA in the PVHmpd in saline-injected ADX/25 animals were significantly greater than those in intact animals. We have recently shown that ADX animals with an exogenous corticosterone treatment producing plasma concentrations of 20–50 ng/ml have thymus weights that are 76% of those in adrenalectomized rats (10). In the present study, thymus weights of the ADX/25 animals were 82% of those measured in the saline-treated ADX/0 group. This indicates that these animals had plasma corticosterone levels within or below the lower part of this range required for normalization of these variables (5, 6).

The fact that only low plasma concentrations of corticosterone are required to facilitate CRH gene expression suggests that it is possibly a predominantly type I glucocorticoid (mineralocorticoid) receptor-mediated event. In support of this assertion, it is significant to note that ADX rats given aldosterone (a type I agonist) alone, at a dose adequate to normalize sodium appetite, had significantly increased CRH mRNA levels compared with adrenalectomized animals with no steroid replacement (10), showing that in some circumstances type I occupation can be facilitatory to CRH gene expression.

The reduction in the magnitude of the stress-induced accumulation of CRH mRNA seen as plasma corticosterone concentrations increase from ADX/25 to ADX/50 and ADX/100 PEG-injected animals is consistent with the data of Kovács and Sawchenko (28). Here, dexamethasone or corticosterone 5-day pretreatment inhibits accumulation of CRH hnRNA after a brief transient stressor. However, it should be noted that differences in this stress model (ether anesthesia) caution against more detailed comparisons with our data at this time. Considering our present and previously published data (10), it is tempting to speculate that type I receptor occupation facilitates, whereas type II receptor occupation inhibits, CRH mRNA accumulation in the PVHmpd of both unstressed and stressed animals. This dual nature of corticosterone action on CRH gene expression is consistent with the coordinate action of type I and type II receptors in regulating common sets of genes first suggested by Evans and Arriza (29). However, it is important to emphasize that these data do not provide information about the neural circuits and mechanisms through which these events occur.

That plasma corticosterone values determined 5 h after injections in ADX corticosterone-replaced animals were higher than we have previously reported (10) is somewhat puzzling, but may be a consequence of the halothane anesthesia at the time of injection on the hepatic clearance of corticosterone; halothane anesthesia is known to affect some aspects of liver metabolism in this manner (30, 31, 32). Nonetheless, the values of the other measured indicators of corticosterone bioactivity in saline-injected controls, i.e. thymus weight and CRH mRNA levels in the PVH, show that exposure to low levels of plasma corticosterone before the stress event is all that is required to restore CRH gene activation to that seen in intact animals.

Finally, two other points supported by our data are worthy of mention. First, the significant differences in the levels of CRH hnRNA in saline-injected intact, ADX/0, and ADX/25 animals are consistent with the idea that CRH gene transcription is suppressed by corticosterone acting in the slow feedback time domain (4) and is thus an important component of normal negative feedback inhibition. Second, the fact that CRH mRNA accumulation in ADX/50 animals was indistinguishable from that in intact animals shows that the significant elevation of plasma corticosterone concentrations that occurs as a consequence of hypovolemia does not modify concomitant CRH gene activation, because there was no stress-induced increase in plasma corticosterone in the pellet-treated animals. These data show that in intact hypovolemic rats, the pronounced stress activation of corticosterone secretion in itself does not compromise CRH gene transcription and mRNA accumulation during the stress event (12), a conclusion that confirms previous findings from more abrupt stressors (26, 27). Taken together with these data, our results suggest that the continued presence of low levels of plasma corticosterone before the stress, but not the stress-mediated elevation in plasma corticosterone, is required to maintain CRH gene transcription during a prolonged viscerosensory stress event. If corticosterone is absent before the stress event, mechanisms antecedent to transcription are altered in such a manner that activated CRH gene expression is either never initiated or cannot be maintained.

In summary, by removing corticosterone and then presenting a sustained stressor, we have revealed a subtle property that has not been recognized previously at the CRH neuroendocrine neuron, that of a facilitatory agent in the regulation of CRH gene transcription and mRNA accumulation. The facilitatory nature of these low levels of plasma corticosterone appears to allow the formulation of a normal stress event and signifies that reduced adrenal function will have profound consequences on mechanisms of CRH gene expression and the subsequent ability of an animal to mount an adequate stress response.

	Acknowledgments

 
We are very grateful Graciela Sanchez-Watts for careful technical support, and to Drs. Robert Thompson, Joseph Majzoub, and Steven Sabol for the cDNAs used to generate the riboprobes.

	Footnotes

 
¹ This work was supported by Grants NS-29728 and KO-4–01833 (to A.G.W.) from the NINDS, NIH.

Received March 27, 1998.

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