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Section on Endocrine Physiology, Developmental Endocrinology Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892
Address all correspondence and requests for reprints to: Greti Aguilera, M.D., Section on Endocrine Physiology, DEB, NICHD, NIH Building 10, Room 10N 262, 10 Center Drive, MSC 1862, Bethesda, Maryland 20892-1862. E-mail: greti{at}helix.nih.gov
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Abstract |
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Introduction |
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The purpose of this study was to investigate the effects of glucocorticoids on CRH and VP transcription in parvocellular PVN neurons in rats subjected to adrenalectomy and corticosterone administration using sensitive intronic in situ hybridization techniques. Because in recent studies we show that CRH hnRNA levels increase between 12 and 72 h after ADX (20), the effect of glucocorticoids on CRH transcription was studied 48 h after ADX, a time at which the increase in CRH transcription reached a plateau. Because the stress of vehicle injection was sufficient to induce marked increases in CRH and VP hnRNA levels, the experiments also allowed determination of the effect of glucocorticoids on stress-induced transcription. In addition, the time course of the changes in VP hnRNA and mRNA during early ADX were compared with those previously observed for CRH.
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Materials and Methods |
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Rats were adrenalectomized bilaterally via dorsal approach or sham operated under ketamine/xylazine anesthesia. After recovery from anesthesia, rats were returned to their home cages, and ADX rats were given access to 0.9% NaCl in the drinking water. To determine the time course of changes in VP transcription following ADX, groups of 6 ADX or sham-operated rats were killed by decapitation at the times indicated in the figures, between 3 and 72 h after surgery. The effect of glucocorticoids was studied after injection of corticosterone (2.8 mg/100 g body weight in 100 µl of saline containing 50% ethanol, ip. Injection was performed 48 h after ADX, a time at which CRH hnRNA levels had reached maximal levels. Control rats received injections of the same volume of vehicle. Rats were killed by decapitation at the time point indicated in results and figures. Injections were performed between 0800 and 0900 h, and rats were killed between 0900 and 1200 h. Trunk blood was collected into ice-cold tubes containing EDTA for corticosterone determination by RIA using 50 µl of plasma and the rat corticosterone kit (DPC, Los Angeles, CA). The sensitivity of the assay was 7.5 ng/ml. Brains were removed, frozen on dry ice, and stored at -80 C until processed.
In situ hybridization
Serial 12-µm coronal brain sections were cut though the medial
parvocellular subdivision of the PVN (8 sections per rat) in a cryostat
at -20 C, thaw-mounted on poly-L-lysine
(Sigma, St. Louis, MO) coated slides, and stored at -80 C
until used for in situ hybridization.
The rat CRH intronic (CRHin) probe (kindly supplied by Dr. Robert Thompson, University of Michigan, Ann Arbor, MI), was a 530 bp pvu II fragment of the CRH gene subcloned into pGEM-3 (Promega Corp., Madison, WI) and linearized with XbaI. The rat CRH (CRHex 2) complementary DNA (cDNA) (Dr. Robert Thompson) was a 770 bp BamHI fragment subcloned in pGEM-3Z (Promega Corp.), linearized by HindIII. The VP exonic probe was an EcoRI-BamHI 200 bp fragment of the rat AVP cDNA (AVPex) kindly provided by Drs. Susan Wray and Harold Gainer (NINDS, NIH, Bethesda, MD), linerarized with BamHI. The AVP intron probe (kindly supplied by Dr. Thomas G. Sherman, Georgetown University, Washington, DC) was generated from a 735 bp PvuII fragment of AVP intron I subcloned into pGEM-3 and linearized by HindIII. High specific activity antisense complementary RNA (cRNA) probes for CRH and VP intronic and exonic probes were produced using 35S-ATP and 35S-UTP (21). In situ hybridization was performed as previously described (21). Briefly, before hybridization, stored sections were air-dried at room temperature, fixed with 4% formaldehyde for 5 min at room temperature, washed 3 times with PBS, and acetylated using 0.25% acetic anhydride in 0.1 M triethanolamine/0.9% NaCl (pH 8.0) for 10 min at room temperature. Slides used for mRNA stability evaluation followed the same prehybridization and hybridization procedures after fixation. Sections were dehydrated in serial ethanol solutions, defatted in chloroform, and dried before hybridization with the radiolabeled probes. Sections were hybridized overnight at 55 C with 2 x 106 cpm labeled CRHin or CRHex probe per slide containing four sections each. Nonhybridized probe was removed by washing with 50% formamide/250 mM NaCl at 60 C for 10–15 min, ribonuclease A treatment for 30 min at 37 C, followed by 3 washes with 0.1 x SSC.
Semiquantification of the CRH and VP transcripts was performed for films autoradiograms and emulsion dipped slides, respectively, as previously described (17, 22). While the sensitivity of both evaluation methods is similar under appropriate exposure conditions, it is necessary to use emulsion dipped slides to distinguish parvocellular neurons hybridized for VP from intermingled magnocellular neurons that are larger and express high levels of VP (17, 22). For analysis of CRH hnRNA and CRH mRNA in the PVN, sections were exposed to Kodak BIOMAX film (Kodak, Rochester, NY) together with 14C-labeled standards (American Radiochemical, St. Louis, MO), for 10 h (CRHex) or 15 days for (CRHin). For cellular localization of CRH mRNA or CRH hnRNA, slides were subsequently dipped in nuclear emulsion diluted 1:1 in distilled water (NTB2, Kodak), exposed for appropriate times (CRHin, 40 days; CRHex, 4 days) and counterstained with cresyl violet acetate (Sigma). The optical density of film autoradiographic images of parvocellular CRH mRNA and CRH hnRNA was measured in a computerized image analysis system (Imaging Research, Inc., St. Catherine, Ontario, Canada), using the public domain NIH Image Program (developed at the National Institutes of Health, and available on the Internet at: http://rsb.info.nih.gov/nih-image). Optical densities from two matched sections per rat were averaged after subtracting the background and used to calculate group means. The results are presented as mean and SEM of the percent of change from basal levels in sham-operated rats.
Analysis of grain density levels of VP hnRNA and mRNA and number of cells containing VP hnRNA and VP mRNA in the medial parvocellular of the PVN was carried out in the cresyl violet counterstained sections using a x40 objective with bright field condenser as described (17, 22). Medial parvocellular VP neurons in the PVN were differentiated histologically from magnocellular neurons on the basis of their overall size, their relatively low level of VP expression, and their small, dense-staining nuclei. The relative grain density levels of VP hnRNA and VP mRNA was quantified in the medial parvocellular subdivision of PVN using computerized densitometry as described above after subtracting the background measured in the immediate proximity of the cells. The grain density measurements for parvocellular VP hnRNA and VP mRNA were made on individual cells identified as parvocellular after excluding scattered magnocellular cells in the medial parvocellular subdivision of the PVN. For each animal, at least two sections were measured bilaterally, and the average value for each rat was used to calculate group means. The results of grain density measurement are presented as mean and SE of the percent change from the basal level in naive rats.
Statistical analyses were performed by one-way ANOVA followed by Fisher’s least significant difference procedure test to assess statistical significance between control and experimental groups at each time point. P < 0.05 was considered to be statistically significant.
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Results |
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, injection of corticosterone increased plasma levels rapidly to 3,479
ng/ml at 15 min, followed by a gradual decrease to levels still above
those in sham operated rats by 2 h (861 ng/ml, P
< 0.01 vs. sham). Vehicle injection had no effect on plasma
corticosterone in ADX rats but increased them to 307, 282, 282, and 129
ng/ml at 15 (P < 0.01), 30 (P <
0.05), 60 (P < 0.05) and 120 min, respectively, in
sham-operated rats.
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). In ADX rats, VP hnRNA grain density
was significantly higher than that in sham-operated controls 3 h
after ADX, and further increased by 6 h (Table 1, Figs. 2A and 3B and D). Unexpectedly, VP hnRNA started
to decrease at 12 h, and after reaching the lowest level at
18 h, increased again progressively from 24 to 72 h (Table 1;
Figs. 2A and 3, E and F). This biphasic pattern was different from the
changes in CRH hnRNA, which started to increase by 12 h from near
undetectable levels and reaching maximum levels by 48 h (Table 1).
As observed for VP hnRNA grain density, the changes in the number of
parvocellular neurons containing VP hnRNA were also biphasic. Values
were significantly higher than in sham-operated rats at 3 h and
6 h after ADX (403% and 320% of sham values, P
< 0.01, respectively), decreased at 18 h (200% of sham,
P < 0.05; 53% lower than ADX at 6 h,
P < 0.01 or 39% lower than ADX at 12 h,
P < 0.01), and increased again by 24 h (Fig. 2B).
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). The number of cells containing VP
mRNA increased slightly but significantly 3 h after ADX (133%,
P < 0.05 vs. sham at 3h), and remained at
values about 150% higher than sham-operated rats between 6 and 72
h (P < 0.01 vs. sham at all time points,
Fig. 4B).
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, Fig. 6B). CRH mRNA levels had a tendency to
increase in both vehicle and corticosterone injected rats but the
changes were not significant (Fig. 5B).
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, A and B). In 48-h ADX rats, levels of
VP hnRNA were 7.9-fold higher than in sham-operated rats
(P < 0.01, Fig. 8A), and
levels increased progressively after vehicle injection (19-, 22-, 24-,
and 28-fold at 15, 30, 60, and 120 min, respectively, Fig. 7A, Fig. 8B). In corticosterone injected rats, VP hnRNA increased significantly
at 15 min but declined markedly to levels below basal by 30 min, and
reaching levels not significantly different from sham-operated controls
by 120 min (Fig. 7A, Fig. 8B). In contrast to the minor effects of
corticosterone on CRH hnRNA, levels of VP hnRNA were significantly
lower in corticosterone injected rats compared with vehicle injected
rats at all time points.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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While glucocorticoid withdrawal sensitized the responses of both CRH and VP to stress, exogenous glucocorticoids had differential effects on these genes, markedly inhibiting VP but not CRH transcription. Corticosterone injection up to 2 h had no inhibitory effect on either stress-stimulated CRH transcription or adrenalectomy-induced basal levels of CRH hnRNA in spite of the rapid and massive increase in plasma corticosterone levels. The similar kinetics of CRH hnRNA in vehicle and corticosterone-injected rats suggests that glucocorticoids are not responsible for the usually self-limiting CRH hnRNA also observed in response to ether exposure, ip hypertonic saline injection, or restraint (16, 17, 22). While the present experiments show that stress induced-increases in CRH transcription are relatively refractory to glucocorticoid inhibition, it is not possible to rule out that prolonged exposure to glucocorticoids (for more than 2 h) may inhibit basal CRH transcription. Such a delayed effect of glucocorticoids would not explain the rapid decline of CRH transcription during stress, but it could contribute to the reduction in CRH mRNA known to occur following local or systemic administration of glucocorticoids (9, 12). Alternatively, or in addition, a decrease in CRH mRNA stability by glucocorticoids is likely to play a role on the inhibitory effect of glucocorticoids on CRH mRNA levels (20).
In striking contrast to the lack of effect of corticosterone on CRH expression, there was a rapid and marked inhibition the VP hnRNA responses to the stress of vehicle injection. Corticosterone not only reduced the effect of stress but also decreased the elevated basal VP hnRNA levels observed after 48-h ADX to near levels observed in sham operated animals 2 h after injection. This is consistent with previous observations in 6-day adrenalectomized rats (18) and supports the hypothesis that VP mRNA levels in the parvocellular PVN are largely controlled at the level of transcription.
The increase in VP hnRNA, 3 to 6 h after ADX, preceded the increases in CRH hnRNA that occur 12 h after removal of the adrenals (20). This is consistent with the view that VP transcription is more sensitive to glucocorticoid feedback than CRH transcription. However, the biphasic pattern of change in VP transcription, with a marked decrease 18 h before a progressive rise between 24 and 72 h, suggests the involvement of more than one mechanism. Thus, the early rise in VP hnRNA could be the result of an exacerbated response to the surgical stress in the absence of glucocorticoids rather than to glucocorticoid withdrawal per se. The kinetics of the changes in VP hnRNA during the first 18 h are consistent with an effect of stress and are similar to the long-lasting VP hnRNA responses observed in other stress paradigms such as ip hypertonic saline injection (17). Although the effect of stress could mask the onset of the VP response to glucocorticoid withdrawal, the low VP hnRNA levels at 18 h suggests that, similar to CRH, release of VP transcription from glucocorticoid inhibition is not immediate following removal of glucocorticoids.
It has been postulated that differential sensitivity of CRH and VP to glucocorticoid feedback can explain the differential regulation in expression of both peptides during stress. This hypothesis is based on the demonstration that the declining phase of CRH hnRNA responses to ether exposure are preceded by the peak corticosterone response, whereas the VP hnRNA responses coincide with the decrease in plasma corticosterone (16). The present demonstration in 48-h adrenalectomized rats that CRH hnRNA responses to the stress of vehicle injection had declined to basal levels by 1 h in the absence of the stress-induced glucocorticoid surge indicates that this self limiting pattern of response is independent of glucocorticoid feedback. The fact that at the time of the plasma corticosterone peak VP hnRNA reaches levels similar to vehicle injected rats, is against the possibility that the stress-induced glucocorticoid surge causes the delay in VP transcription response described during ether exposure and ip hypertonic saline injection (16, 17). In stress models such as restraint and ip hypertonic saline injection, the onset of the VP transcription responses also occurs at the time of maximal increases in plasma corticosterone (17, 22), and VP hnRNA can be sustained for up to 4 h, despite equally sustained elevations in plasma corticosterone (17). While it is clear from these experiments and previous reports (18) that VP transcription is highly sensitive to glucocorticoid inhibition, stress can overcome this inhibition to different degrees, probably depending on the glucocorticoid levels and intensity of the stress.
The mechanism responsible for the modulatory effect of stress on glucocorticoid inhibition may involve interaction of glucocorticoids with neurotransmitters and neuropeptides activated by stress (24, 25). At the molecular level, second messenger-induced expression of intermediate early genes or transcription factors could modify glucocorticoid receptor activity by heterodimerization with the receptor or by binding to regulatory elements and by changing the conformation of the promoter (26, 27, 28, 29). In the parvicellular cell, increases in VP hnRNA induced by ether exposure are closely preceded by the induction of c-fos mRNA and fos protein (16), which has been shown to interact with the glucocorticoid receptor modifying its activity at the DNA responsive element (30).
In conclusion, the marked increases in CRH and VP hnRNA in response to the minor stress of vehicle injection in ADX rats, indicate that low prevailing levels of circulating glucocorticoids in normal intact rats reduce the responsiveness of parvicellular neurons. This effect of resting levels of glucocorticoids would prevent the onset of CRH and VP transcription and inappropriate activation of the HPA axis in response to minor stresses. While it is clear that glucocorticoids have distinct effects on CRH and VP transcription, the data show that increases in glucocorticoid levels alone cannot explain the differential regulation of CRH and VP transcription during stress.
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Footnotes |
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Received May 20, 1999.
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