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Dorothy Crowfoot Hodgkin Laboratories, Department of Medicine, University of Bristol, Bristol BS2 8HW, United Kingdom
Address all correspondence and requests for reprints to: Stafford Lightman, Ph.D., Dorothy Crowfoot Hodgkin Laboratories, Department of Medicine, University of Bristol, Marlborough Street, Bristol BS2 8HW, United Kingdom. E-mail: Stafford.Lightman{at}bristol.ac.uk
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Introduction |
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Recent studies have demonstrated that CRH heteronuclear RNA (hnRNA) and AVP hnRNA levels in the PVN increase rapidly in response to acute stress (23, 36, 37, 38). Because of the very short half-life of hnRNA, these changes reflect gene transcription much better than the more stable mRNA. We have recently published a detailed time course of the CRH and AVP hnRNA and mRNA responses to acute restraint stress (20) and have now gone on to detail how this response changes in animals previously exposed to 13 days of daily restraint.
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Materials and Methods |
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Animals were killed by decapitation. Trunk blood was collected into heparinized tubes and the brains removed and frozen on dry ice. Then 12-µm sections were cut through the medial parvocellular division of the PVN and thaw mounted on twice-gelatin-coated slides and stored at -80 C. Before hybridization, sections were air dried at room temperature and fixed with 4% formaldehyde for 5 min at room temperature, washed 3 times with PBS, and then incubated in 0.25% acetic anhydride in 0.1 M triethanolamine/0.9% NaCl (pH 8.0) for 10 min at room temperature. Sections were transferred through 70 (1 min), 80 (1 min), 95 (2 min), and 100% ethanol (1 min); 100% chloroform (5 min); and 100 (1 min) and 95% ethanol (1 min); and dried. All control and experimental sections were hybridized in the same hybridization reaction.
The AVP intronic probe, a synthetic 36-base oligonucleotide corresponding to the first 36 bases of rat AVP intron I (AVPin) (39), was made, as described (20). The AVP exonic probe (AVPex) was a 48-base oligonucleotide complementary to part of the exonic mRNA sequence coding for AVP. The specificity of AVPin (36, 40) and AVPex (40) probes has previously been determined. The probes were labeled with 35S-deoxyATP, as previously described (20). The hybridization procedures were carried out, as previously described (13, 40). In brief, sections were hybridized overnight at 37 C with 5 x 105 cpm labeled AVPin or 3 x 105 cpm AVPex probe per 2 sections, then washed in four 15-min rinses of 1 x SSC (1 x SSC = 0.15 M NaCl and 0.015 M sodium citrate, pH 7.0) on shaking waterbath (55 C), followed by two 30-min rinses in 1 x SSC at room temperature.
The rat CRH intron (CRHin) probe was generated from a 530-bp pvuII fragment of the CRH gene subcloned into PGem 3 and linearized by XbaI (which was kindly supplied by Dr. Bob Thompson). The rat CRH (CRHex 2) complementary DNA (41) used same source with CRHin) was a 770-bp BamH1 fragment subcloned in PGem3Z, linearized by HindIII. The 35S-labeled cRNA probes for CRHin and CRHex were produced as previously described (20). The specificity of CRHin and CRHex probes was confirmed by sense-strand cRNA probes, respectively (42). Sections were hybridized at 55 C with 1 x 106 cpm-labeled CRHin or CRHex probe per slide containing 2 sections; and then nonspecifically hybridized probe was removed by washing 2 times with 2 x SSC containing 50% formamide at 50 C, incubated with ribonuclease A for 30 min at 37 C, followed by 2 washings 3 times with 2 x SSC containing 50% formamide at 50 C, and 2 times with 2 x SSC at room temperature for 10 min. Finally, slides were dipped in water and air dried.
Plasma corticosterone assays
Total plasma corticosterone was determined in plasma (5 µl
diluted in 500 µl buffer) by RIA as described previously (16).
125I-corticosterone was used as the tracer with a specific
activity of 2–3 mCi/mg. Corticosterone antibody was a kind gift of Dr.
Gabor Makara, Budapest. The sensitivity of the assay was 25 ng/ml.
Analysis and quantification
For quantification of CRH hnRNA and CRH mRNA in the PVN, the
sections and 14C-labeled standards of known radioactivity
(American Radiochemical, St. Louis, MO) were placed in x-ray cassettes,
then exposed to Hyperfilm MP autoradiography film (Amersham
International Plc, Amersham, UK) for 3 days (CRHex) and 15 days
(CRHin). For cellular localization of AVP and CRH hybrids, the slides
were subsequently dipped in nuclear emulsion (Ilford Scientific Product
K-5, Ilford, Essex, UK) and exposed for appropriate times (AVPin, 50
days; AVPex, 9 days; CRHin, 35 days; CRHex, 12 days). The slides were
counterstained with cresyl violet acetate (Sigma, Dorset, UK). The
optical density of autoradiographic images was measured by using a
computerized image analysis system, as described (16). In brief, the
autoradiographic image of probe bound to parvocellular CRH mRNA and CRH
hnRNA, together with 14C-labeled standards, were measured
using a computerized image analysis system (Image 1.22, developed by W.
Rasband, NIH) on an Apple Mac IICi computer. The optical densities were
obtained in two consecutive sections per rat, the average value for
each rat was used to calculate group means, and the results are
presented as means percentage change from control with SEM.
Analysis of changes in the prevalence of AVP transcripts in the medial
parvocellular and posterior magnocellular subdivisions of the PVN was
carried out in the cresyl violet counterstained sections using a x40
objective for AVP mRNA and a x100 oil immersion objective for AVP
hnRNA, with brightfield condenser, as described (20, 23). Briefly, the
medial parvocellular AVP neurons in the PVN were differentiated
histologically from magnocellular neurons on the basis of their overall
size, their relatively low level of AVP expression, and their small,
dense-staining nuclei. Magnocellular cells in the medial parvocellular
subdivision were characterized by large numbers of superimposed silver
grains, relatively faint nuclear and Nissl cytoplasmic staining, and
their large size. The neurons in the medial parvocellular subdivision
of the PVN were considered to be positive for AVP hnRNA if overlying
grain densities were at least three times that of background, measured
in the vicinity of the labeled cells. The relative levels of AVP hnRNA
and mRNA were quantified in the medial parvocellular and posterior
magnocellular subdivisions of PVN using computerized densitometry, as
described above on coronal sections, subtracting background from the
proximity of the measured cells. Scattered magnocellular cells in the
medial parvocellular subdivision of the PVN were excluded from the
analysis on the basis of their histological appearance. For each
animal, at least 4 PVNs on two sections were assessed; the average
value of each section for each rat was used to calculate group means.
The results are presented as means percentage change from control with
SEM.
Statistical analysis was performed by one-way ANOVA, followed by the Fisher least significant difference procedure (PLSD) test, to assess statistical significance between the 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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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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), although the levels
themselves were less than those seen in the control animals (control
animals: time 0 min, 62.6 ± 12.3; 30 min, 569.62 ± 41.9; 60
min, 522.9 ± 48.9; 120 min, 41.9 ± 5.03 ng/ml) (repeatedly
immobilized animals: time 0 min, 73.7 ± 11.0; 30 min, 273.5
± 51.03; 60 min, 162.42 ± 19.68; 120 min, 47.43 ± 11.47
ng/ml).
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). No silver grains were observed in
the parvocellular neurons hybridized with CRH sense riboprobe (Fig. 2b). There was no significant difference between basal CRH hnRNA or
mRNA levels, after 13 days of repeated stress, and the levels found in
the control animals. After restraint, there was no significant increase
in either CRH hnRNA or mRNA levels in these repeatedly stressed animals
(Fig. 2, C and D; Fig. 4; also see Fig. 5).
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, A and
B). There was no significant difference
between basal AVP hnRNA or mRNA levels in the parvocellular subdivision
of the PVN, after 13 days of repeated stress, and the levels found in
control animals. After restraint of these repeatedly stressed animals,
there was a very prompt increase in AVP hnRNA levels in the
parvocellular PVN, which was significant at 15 min, peaked at 60 and 90
min, and rapidly returned to basal levels at 2 h (within 30 min of
return to their home cage from the restrainer) (Fig. 3, C and D; also
see Fig. 6). AVP mRNA responded more slowly, reaching a significant
increase at 90 min and remaining raised at 4 h (Figs. 4
and 6).
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).
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Discussion |
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We chose the simple stimulus of daily restraint and carried out these studies in the Wistar strain of rat, which has been extensively studied in stress research and which has very similar CRH mRNA, AVP mRNA, proenkephalin A mRNA, and corticosterone responses to the other frequently studied strain (the Sprague-Dawley) (16). We have previously shown that in naive Wistar rats, acute restraint stress results in a very rapid increase of CRH hnRNA level, to more than 1,000% (as reflected by probe binding) of control values, with CRH mRNA only increasing significantly at 4 h. AVP hnRNA approximately doubled at 60 min, and AVP mRNA showed a significant increase at 4 h (20). After 13 days of restraint, we found a very marked change in the pattern of the hypothalamic response to stress. The CRH hnRNA and mRNA responses were completely abolished, whereas the response of AVP gene transcription showed no diminution and, if anything, increased. AVP hnRNA was significantly elevated within 15 min and was increased about 3-fold at 60 and 90 min, before falling to baseline values at 2 h (just 30 min after the animal returned to its home cage). AVP mRNA was significantly increased by 90 min and remained raised up to 4 h, reaching peak levels of approximately 3 times basal value. The up-regulation of parvocellular AVP gene transcription in this experimental model is consistent with previous studies showing that parvocellular AVP mRNA levels rise significantly after repeated immobilization (19, 27) or repeated foot shock (44). The response does, however, differ from that seen after multiple varied stressors, which would not be expected to result in the same degree of adaptation (26). These data suggest that AVP synthesized in the parvocellular PVN plays an important role in activating the HPA axis under conditions of chronic stress and may be necessary for the resistance of the HPA axis to adaptation in response to such repeated stress (9).
The significant increase of AVP transcription, but not CRH transcription, in the parvocellular PVN represents a remarkable transcript-specific alteration in gene regulation and supports the hypothesis that parvocellular AVP and CRH gene expression in the PVN may have quite different transcriptional control mechanisms (26). It is interesting that vasopressin-immunoreactive parvocellular neurons in the PVN are identical to CRH neurons, and vasopressin-positive parvocellular neurons are always stained for CRH (45, 46). Under normal resting conditions, AVP is expressed in a subpopulation of CRH-positive neurons in the PVN and colocalizes with approximately 50% of CRH-positive terminals in the median eminence (6, 7). Repeated immobilization increases the proportion of CRH parvocellular neurons containing AVP to 90% and results in a 2-fold increase in the number of AVP positive parvocellular neurons, whereas the number of CRH parvocellular neurons remains at a normal level (27). In addition, repeated immobilization does not affect CRH stores but significantly increases the AVP stores and colocalization of AVP with CRH in the median eminence (34). The proportion of CRH-expressing cells in the parvocellular PVN that also express AVP increases substantially in response to repeated stress (35). Furthermore, repeated immobilization induces an increase in AVP-binding affinity and AVP V1b receptor mRNA in the pituitary (47). Our results, taken together with these published data, suggest that repeated restraint does activate CRH parvocellular neurons to express AVP but not CRH (an adaptive change whose mechanism is not understood).
Another interesting result is the fact that after repeated restraint, there is still a marked corticosterone response to stress, in the absence of any change in hypothalamic CRH gene transcription. Although we cannot be sure that the lack of change in CRH RNA reflects lack of peptide release from the median eminence, we have shown previously that the reduction of CRH mRNA in chronic arthritis does correlate with reduced levels of hypothalamic pituitary portal CRH-41 (30). It certainly seems likely that the HPA response seen in our repeated restraint paradigm results predominantly from the hypothalamic release of AVP rather than CRH.
In conclusion, after a repeated restraint, there is a transcript-specific alteration in gene regulation within the parvocellular cells of the PVN, in which CRH gene transcription is desensitized, but AVP gene transcription is maintained. These data provide further evidence of the importance of AVP in the activation of the HPA axis during chronic or repeated stress.
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Acknowledgments |
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Footnotes |
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2 Present address: Xin-Ming Ma, Developmental Endocrinology Branch,
National Institute of Child Health and Human Development, National
Institute of Health, Building 10, Room 10N262, Bethesda, Maryland
20892.
Received April 24, 1997.
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