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Endocrinology Vol. 138, No. 8 3249-3258
Copyright © 1997 by The Endocrine Society

ARTICLES

Chronic Cold in Adrenalectomized, Corticosterone (B)-Treated Rats: Facilitated Corticotropin Responses to Acute Restraint Emerge as B Increases1

Susan F. Akana and Mary F. Dallman

Department of Physiology, University of California San Francisco, San Francisco, California 94143-0444

Address all correspondence and requests for reprints to: Susan F. Akana, Department of Physiology, Box 0444, University of California San Francisco, San Francisco, California 94143-0444. E-mail: akana{at}itsa.ucsf.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Small elevations in corticosterone (B) administered exogenously exert potent inhibitory effects on both basal and stress-induced ACTH secretion. However, under conditions of chronic stress with chronic elevations in B, the hypothalamo-pituitary-adrenal system appears to balance the negative feedback signal of B with central neural facilitation so that the system remains fully responsive to acute stressors. In these studies, we tested whether: 1) circulating B concentrations affect responses to acute restraint in rats exposed to 5 days at 5–7 C (cold), compared with room temperature (control); and 2) facilitated ACTH secretion can be explained by increased CRF or vasopressin messenger RNA (mRNA) levels in the hypothalamic parvocellular paraventricular nuclei (PVN). Rats were adrenalectomized and supplied with B in doses that fixed plasma B at constant levels between approximately 2 and 20 µg/dl; rats were placed in cold or remained as controls. Increasing concentrations of fixed B decreased basal ACTH similarly in both groups. By contrast, as B levels increased, ACTH responses to restraint also increased in cold vs. control rats. Semiquantitative analysis of CRF mRNA by in situ hybridization revealed decreases of similar magnitude in both groups with increasing fixed B. Vasopressin mRNA levels also decreased with increasing fixed B in both groups, but with slightly less sensitivity to inhibition by B in cold exposed rats. Taken together, the decreases in mRNA for these major ACTH neuropeptide secretogogues in the parvocellular PVN are unlikely to explain facilitated ACTH responses in chronically stressed rats. We conclude that a brain site is stimulated by B that is proximal to the PVN; feedforward, positive effects of B are thus implicated in mediation of prior stress-induced facilitation of acute hypothalamo-pituitary-adrenal responses to stress.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE PHENOMENON of facilitated responses in the hypothalamo-pituitary-adrenal (HPA) axis to acute stress in previously or chronically stressed rats has been well documented. Rats exposed to acute stress respond with normal or augmented ACTH when previously stressed, either within hours (1, 2) or persistently for days (3, 4, 5, 6, 7, 8, 9, 10, 11) despite the adequate glucocorticoid negative feedback signal induced by prior stress (12, 13, 14). Many, but not all, chronic stressors result in only minor elevations in AM trough corticosterone (B) and ACTH concentrations (4, 7, 8, 11, 15); however, such minor elevations when produced by exogenous B treatment reduce PM peak ACTH and B concentrations and also inhibit the magnitude of ACTH and B responses to acute restraint in the AM (14, 16, 17) .

There is a narrow range of average daily plasma B (4.5–6.0 µg/dl) that maintains normal physiological function as determined by comparison of variables in B-supplied, adrenalectomized rats to intact controls (18). Endpoints as varied as thymus (18), fat depot (19, 20), and body weights (18, 20), vertebral Ca++ content (13), plasma transcortin (21), and insulin (22) concentrations, brown adipose tissue variables (20), and blood pressure (23) are all normalized in adrenalectomized rats by this range of B. Chronically stressed rats frequently have mean plasma B levels that exceed this range (7, 10, 24, 25, 26), and the elevated B signal causes the expected effects on peripheral B target tissues (e.g. thymic atrophy). In contrast to glucocorticoid targets in the periphery, central neural sites involved in HPA responses to stress appear to be protected from the normally inhibitory effect of B through a matched facilitation signal of opposite sign, thus maintaining responsivity of the HPA axis to acute stress.

We hypothesized that if we treated adrenalectomized rats with constant B that caused steady-state B levels spanning the basal circadian range of intact rats and then exposed them to chronic stress, this might reveal new basic characteristics of facilitation. The studies were explicitly designed to reveal the effects of B on facilitated ACTH responses to acute stress in chronically stressed rats, and to determine whether the mechanism of facilitation could be attributed to changes in CRF and vasopressin (AVP) biosynthetic responses to B induced by chronic stress.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Surgery and corticosterone treatment
Ninety-nine young, male Sprague-Dawley rats (160–180 g) from Bantin and Kingman (Gilroy, CA), were delivered to our light (12-h light, 12-h dark; lights on 0000 h colony time)- and temperature- (22–23 C) controlled animal facility, pair-housed in hanging wire cages, and were supplied with food (Purina Rodent Chow, diet 5008) and tap water ad libitum. Two days later (day 0), between 0030–0200 h, the rats were anesthetized with ether and adrenalectomized by the dorsal approach. Adrenalectomy was followed immediately by sc implantation of one or two of a range of 100 mg pellets of corticosterone:cholesterol (12.5–100%, or 100% + 20%). The skin incisions were then closed. Additional comparison groups of sham-adrenalectomized (n = 30) and adrenalectomized rats (n = 6), both bearing placebo wax pellets, were prepared concurrently. All experiments were approved by the UCSF Committee on Animal Research.

Cold
All rats were individually rehoused after surgery, given fresh food and 0.5% NaCl to drink, and allowed to recover for a minimum of 2 h before half of the rats were placed in the cold [5–7 C; (11)]. Rats in the cold were housed in plastic tubs with wood shavings for bedding. Fresh food was supplied to the rats each day.

Blood and tissue collections
Between 1100–1200 h on the evening of day 3, each rat was briefly placed in a restraint tube, at room temperature, for collection of 250 µl blood into heparinized microhematocrit tubes from a tail nick. Previous studies have established that tail nick blood collection at room temperature and tail cuts during restraint in the cold provoke similar ACTH and B responses (11). Blood was immediately expelled into chilled microcentrifuge tubes containing 10 µl 0.3 M EDTA for later separation of plasma and subsequent determination of B and ACTH. The rats were immediately returned to their home cages.

On the morning of day 5, between 0030–0200 h, each rat was removed from the home cage, placed in a restraint tube at room temperature, and 250 µl tail nick samples were collected at 0 and 15 min (as described above) followed by decapitation at 30 min. Five milliliters of trunk blood were collected after decapitation into chilled plastic centrifuge tubes with 100 µl 0.3 M EDTA. Plasma was separated by centrifugation and aliquotted for later determination of ACTH and corticosterone, and for separate report of insulin, glucagon and T3, leptin, and triglycerides.

Brains were rapidly removed and chilled on ice. A coronal slice that extended from the optic chiasm to mammillary bodies was prepared, positioned within a plastic mold (S22, Polysciences, Warington, PA) filled with embedding media (Tissue Tek, OCT compound, Miles Diagnostic/Sakura, Elkhart, IN), and frozen in a dry-ice/ethanol bath maintained at < -40 C. Frozen blocks were sealed in small plastic bags and stored at -70 C.

Anterior pituitary lobes were dissected and homogenized in 0.1 N HCl for later determination of ACTH and protein. Thymus glands and adrenals, when present, were collected and placed on saline-moistened filter paper within closed petri dishes for later weighing.

Three experiments, each of which included all treatment groups, were run to collect sufficient numbers of samples due to space constraints of the cold box. An additional fourth experiment with adrenalectomized rats bearing either low, moderate, or high corticosterone pellets (but no intact rats) was added to increase the number of brain and tissue samples. The data from the brains and tissue results from this fourth experiment were combined with the data from animals of the three stress collections. All experimental protocols received approval from the UCSF Committee on Animal Research.

Assays
Plasma corticosterone (27) and plasma and pituitary ACTH were measured by previously described assays (16). Pituitary protein was measured spectrophotometrically with the Bradford reagent (Biolab, Hercules, CA).

In situ hybridization
Tissue preparation.
Frozen brains were sliced on an American Optics Reichart (Buffalo, NY) cyrostat at 10 µm and thaw-mounted onto gelatin-dipped slides (Superfrost, Fisher, Pittsburgh, PA) previously washed in 100% ETOH. Brain sections were allowed to dry briefly, then were stored at -80 C with dessicant.

Brain sections retrieved from storage were immediately immersed in 4.0% paraformaldehyde at room temperature for 30 min. Two sequential 5-min rinses in PBS were followed by rinses in 70, 90, 100, and 100% ETOH:DEPC-prepared water). The slides were allowed to dry, the level of the paraventricular nucleus (PVN) noted (Paxinos and Watson, bregma -1.8 mm), three to five sections were designated for both CRF and AVP hybridization, and the slides were then restored with desiccant at -80 C.

AVP.
AVP messenger RNA (mRNA) was hybridized with a 35-bp oligomer of arginine vasopressin, generously provided by Drs. J. Barchas and J. Eberwine, by the method of Shivers et al. (29). The probe was labeled with 35S-dCTP (DuPont-New England Nuclear, Boston, MA) with terminal deoxynucleotidyl transferase (Collaborative Research, Bedford, MA) as previously reported (30) and diluted to 0.25 x 106 cpm/30 µl. Tissue selected for AVP hybridization with oligomers were removed from storage and were allowed to warm and dry at room temperature before prehybridization buffer (75 µl/section) was pipetted onto the section. Sections were placed in a humidity chamber with buffer-saturated paper towels and were allowed to incubate for 12–24 h at room temperature in the dark. Prehybridization solution was drained off each section, the surrounding glass dried with a kimwipe, and 30 µl hybridization mix was pipetted on, the section sealed with a baked glass cover slip, and the slide was then returned to the humid chamber for 72 h hybridization at room temperature in the dark. Sections hybridized for AVP were placed in baked glass holders and immersed in 2 x SSC with 0.05% sodium pyrophosphate (Sigma Chemical Co., St. Louis, MO) at room temperature with intermittent, mild agitation until all glass coverslips were detached. Two 5-min rinses in 2 x SSC were followed by placing slides in a fresh histology dish with 0.5 x SSC in a 35 C water bath for a minimum of 12 h. Slides were then allowed to cool to room temperature in fresh 0.5 x SSC and were then dehydrated through 70, 90, 100, and 100% ETOH.

CRF.
For CRF, the protocol of Harbuz and Lightman (31) was adopted with use of the their characterized 48-bp oligomer that was synthesized in-house (Bio Molecular Resources, UCSF). The two modifications from their protocol was the use of 33P dATP instead of 35S dATP and of phenol-choloroform/ETOH precipitation as described for AVP (30) rather than the use of column separation. The hybridization mix was prepared as described by Harbuz and specific activity approximated 1.1 x 106 cpm/45 µl. For CRF mRNA hybridizations, brain sections were acetylated, chloroform-treated, hybridized overnight at 37 C and posthybridized exactly as described by Harbuz (24). The different concentrations of AVP and CRF probes used was based on prior saturation analyses (data not shown).

Signal detection
Sections hybridized for AVP were first opposed to x-ray film (Kodak X-OMAT, Eastman-Kodak, Rochester, NY) for 24 h before they were then dipped in nuclear track emulsion (Kodak NTB2). Sections hybridized for CRF were immediately prepared for emulsion dipping (NTB3) while a subset series of method control sections (ADX, RNAase pretreatment, dilution controls) were opposed to film (Hyperfilm MP, Amersham, Arlington Heights, IL). For both AVP and CRF, the appropriate emulsion was diluted 1:1 with distilled water, and the sections were dipped and dried at room temperature for 2 h and stored in light-tight slide boxes with desiccant. Slides were developed (3 days for AVP; 35 days for CRF) based on previously characterized timecourses (data not shown) with 4 min in Kodak D-19 developer, 10 seconds in distilled water, 4 min in Kodak fix at room temperature followed by a 30 min rinse of running water in the dark. Sections were counterstained with either neutral red or cresyl violet.

ISH quantitation
Brain sections were viewed with a 40x objective on a Leica (Foster City, CA) microscope using a Optronics 3 CCD video camera feeding into both a Scion LG-3 frame grabber card (Scion Corporation, Frederick, MD) and a MacIntosh Power PC (Apple Computer, Cupertino, CA). The computer imaging was run with the NIH Image 1.57 program developed by Rasband (Bethesda, MD).

Parameters of imaging PVN brain sections at 400x were a 1–10 particle pixel limit, density slice of 75–155 in series with a threshold set at 75, and fixed settings for the video capture. Contrast was set to distinguish small, highly dense silver grains distinct from paler counterstained cells. After an initial survey at 400x of 48 microscope fields from a PVN section of an adrenalectomized rat hybridized with CRF mRNA, six microcope fields corresponding to the neuroendocrine parvocellular cells (32) were chosen for individual cell imaging in subsequent treatment groups.

A pragmatic rule for eliminating magnocellular cells from the sampled population of neurons hybridized for AVP was adopted from Herman (33). Parvocellular cells were defined as discrete, small fusiform or ovoid cells confined within a 13-µm diameter circular template that had to lie entirely within the microscope field. Magnocellular cells were considered to be large, round cells > 13-µm in diameter, based on an average parvocellular cell diameter of 10 microns (34) and an 35S decay path through emulsion calculated at 0.5 microns. The identified magnocellular cells were intensely covered with silver grains and were subsequently centered within a 50-micron diameter circular template and masked to avoid grain spillover onto neighboring cells. These methods perforce underestimated the parvocellular population. Equal numbers of 109 ± 8 and 92 ± 8 AVP neurons were counted in brains from control (n = 12) and cold (n = 10) rats with no detectable effect of cold (F = 2.622; P = 0.118). By contrast, cold resulted in fewer CRF neurons in the fields examined: control; 111 ± 7 (n = 10); cold; 77 ± 6, (n = 8; F = 14.491, P = 0.002).

The total area covered by silver grains within a parvocellular template was converted from square pixels to square microns after calibration with a micrometer. Background was measured on three to five nonhybridizing tissue fields for each section, averaged, and subtracted from the cell counts. Three and 35 days were chosen for development of AVP and CRF emulsion, respectively, for optimal hybridization signal to background noise based on initial time course characterizations (data not shown). From an initial probe dilution series for both AVP and CRF, we chose a dilution that was double the titer that first produced plateau hybridization signal (data not shown).

35S brain paste standards [prepared as previously described, (30)], 33P standards spotted on filters and commercial 14C microscales (catalog no. 146A, American Radiolabeled Chemicals, Inc., St. Louis, MO) were coexposed to Amersham Hyperfilm MP. Simultaneously, adjacent brain paste sections were solubilized (Solvable, DuPont-New England Nuclear), NaOH-neutralized, and counted in a Beckman (Fullerton, CA) LS233 counter along with the parallel series of 33P standards.

Background and sensitivity were optimal with Hyperfilm MP (Amershsam), and this was used for initial survey of method control and adrenalectomized rat brain sections for hybridization progress for CRF studies. All method control sections and adrenalectomized control sections fell within the linear range of all the radioactive standards. These comparisons were executed to allow continuity with past and future experiments using either film or emulsion. RNAase pretreatment (100 U/ml, 30 min at 37 C) of intervening brain sections from sections of adrenalectomized rat brain hybridized for CRF mRNA resulted in background levels over the PVN, similar to nonhybridizing tissue sections (data not shown).

As an a priori standard for AVP mRNA, a 13-micron diameter circle template over magnocellular cells in the SON had to register a mimimum silver gain area of 64 square microns for the hybridization to be scored a success and subsequent measurement of parvocellular hybrididzation undertaken.

CRF hybridizations were completed in two sets. AVP hybridizations were completed in six sets, each of which included at least one of each B treatment group in both control and cold conditions together with intact and adrenalectomized controls.

Statistics
Plasma corticosterone concentrations from adrenalectomized rats with corticosterone pellets were determined in four samples from each animal in the stress studies and two samples from rats in the basal collection; the values were averaged to produce a measure of mean corticosterone for subsequent use as the independent variable to assess thymus weight, ACTH and mRNA hybridizations.

We chose to evaluate most of the data in three groups containing different plasma corticosterone concentrations (low, medium and high B) using 2 way ANOVA followed by Newman-Keul’s post hoc analysis where appropriate ({alpha} = 0.05), for statistical analysis.

Stress-induced ACTH levels were analyzed by two-way ANOVA with correction for repeated measures, for both the effect of cold on basal AM and PM ACTH, and, separately, over time of restraint in the AM (0, 15, 30 min). Because of the wide range of ACTH levels determined by either B or stress, the data were log transformed for both statistical and display purposes. Best fit regressions were calculated using the curve-fitting programs of SlideWrite 3 (Advanced Graphics Software, Carlsbad, CA). Most data are presented as the mean ± SEM.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Lack of cold-induced B feedback on ACTH and B responses to acute stress in intact rats (Fig. 1Go, Table 1Go).



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  		Figure 1. There is no difference in ACTH and corticosterone (B) response of intact rats to restraint whether or not they have been exposed to the chronic stress of 5 days of cold (n = 12–18 rats/group).

 

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  		Table 1. Results in Sham-adrenalectomized rats maintained at room temperature (control) or at 5 C (cold) for 5 days

 
Intact rats exposed to continuous cold for 5 days had normal responses to acute restraint stress compared with control for both ACTH (cold P = 0.539; restraint P < 0.001; interaction P = 0.228; Fig. 1AGo) and B (cold P = 0.763; restraint P < 0.001; interaction P = 0.502; Fig. 1BGo). This confirms what we (4, 8, 9, 10, 11) and others (6, 35, 36, 37, 38, 39) have reported after cold and other chronic stimuli to the HPA axis. Normal responsivity in the HPA axis is maintained despite multiple direct [urinary B excretion, (8, 11)] and indirect (increased adrenal weight, decreased thymus and body weights) indices showing persistently elevated activity in the HPA axis elicited by the chronic stimuli. In this study, the intact rats in cold had significantly elevated adrenal weights, but there was not a significant decrease in thymus weight or alteration in pituitary ACTH content (Table 1Go).

Adrenalectomized rats do not survive in cold (40, 41) and we made no attempt to test this. Nonetheless, of 49 adrenalectomized rats prepared with B pellets, 7 rats that were targeted for the low range of B did not survive the 5-day cold exposure period; death occurred between 2 and 3 days. Death was probably not a consequence of the acute combination of adrenalectomy, steroid replacement, and cold exposure because recovery during the first day was excellent in all rats. The lowest concentration of B measured in rats that survived cold was 2.3 µg/dl. This concentration of B, necessary for survival in the cold, probably begins to enter the range at which glucocorticoid receptors are occupied (42).

Lack of interaction of B with cold on plasma B, thymus weight, and pituitary ACTH (Figs. 2Go and 3Go).
Corticosterone treatment of adrenalectomized rats produced similar levels of circulating B (Fig. 2Go, A and B) and thymic involution (Fig. 2Go, C and D) in both control and cold rats. Because there were no differences in circulating B between AM and PM (P = 0.627), or after restraint (P = 0.737), plasma B was effectively clamped in all adrenalectomized rats. The mean of two to four plasma corticosterone measurements/rat was therefore used to assign individual rats to low, medium, or high B subgroups (Fig. 2Go, B and D). Circulating B differed significantly among subgroups (B dose, P < 0.001, Fig. 2BGo), and there was neither a cold (P = 0.627) nor an interactive (P = 0.252) effect. Similarly, thymic involution was affected by the dose of B (P < 0.001) at concentrations well within the range of substantial glucocorticoid receptor occupancy (42). There was neither an effect of cold (P = 0.185) nor an interaction of B with cold (P = 0.335) on the bioefficacy of B on thymus weight (Fig. 2DGo). Pituitary ACTH content (Fig. 3Go) was decreased by B dose (P = 0.030) but not by cold (P = 0.617), and there was not a significant interaction of B with cold (P = 0.106). Because there were major effects of B, but no interactions between B and cold on these variables, we used the three B subgroups to assess effects of B on basal plasma ACTH and acute plasma ACTH responsivity to restraint in the control and cold-treated groups.



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  		Figure 2. A, Relationship between the % B pellet implanted 5 days previously and plasma corticosterone (B) in adrenalectomized rats. B, Assignment of the rats shown in A into low, medium, and high B subgroups. There is no effect of cold on the steady-state B levels. C, Relationship between steady-state plasma B on day 5 and thymus weight. D, When rats were assigned to low, medium, and high B subgroups, there was an inhibitory effect of B, but no effect of cold. Open stars show significant differences from the next lower subgroup of B. In panels B and D, open columns represent responses of control rats, hatched columns represent data from cold-exposed rats.

 


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  		Figure 3. Pituitary ACTH content in control and cold groups as a function of the level of corticosterone (B). There was a significant overall inhibitory effect of dose of corticosterone but no significant effects of cold or interaction between B and cold. Open columns represent responses of control rats, hatched columns represent data from cold-exposed rats.

 
ACTH responses to cold and subsequent restraint
Diurnal rhythm (Fig. 4Go).
Corticosterone inhibited basal ACTH secretion both in the AM and PM in control and cold-exposed rats (Fig. 4Go, A and B, see Table 2Go for statistics). In the a.m. (Fig. 4Go, top), plasma ACTH was elevated in cold rats at the low dose of B, but not at the higher doses. In the p.m., B inhibited basal ACTH with a main effect of cold but no interaction (Fig. 4Go, bottom). Although there were main effects of cold and B on the increase in plasma ACTH between a.m. and p.m., there was no cold x B interaction.



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  		Figure 4. Basal ACTH concentrations measured in samples collected in the a.m. (top) or p.m. (bottom) from the same rats. Open stars indicate differences between the same subgroup in the a.m. and p.m., closed stars indicate no a.m.-p.m. difference (see Table 2Go for statistics). Note the log scale for ACTH. Open columns represent responses of control rats; hatched columns represent data from cold-exposed rats.

 

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  		Table 2. Statistical results of the two-way ANOVAs, repeated in the dimension of time, of log basal ACTH in the a.m. and p.m. in control and cold rats at each corticosterone (B) treatment group (data shown in Figs. 4Go and 5Go).

 
Response to acute restraint in the AM (Fig. 5Go).
The interaction between 5 days of fixed levels of B and cold on the acute ACTH response to restraint is shown in Fig. 5Go. Initial, 0 min ACTH values repeat some of the AM data from Fig. 4Go. The profound sensitivity of ACTH to the inhibitory effects of increasing steady-state B levels is clearly discernible in controls, both under initial (0 min) conditions and in the magnitude of the response to restraint. In the low B control group, there was a marked increase in ACTH after restraint with a mean increase after 30 min of approximately 1000 pg/ml. In the medium B control group, the ACTH response to restraint was reduced, with a mean increase after 30 min of approximately 80 pg/ml. In the high B control group, the mean maximal ACTH at 30 min after restraint was 40 pg/ml, with a mean increase in ACTH over the 30 min of approximately 30 pg/ml.



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  		Figure 5. ACTH responses to acute restraint in control (dashed lines and open symbols) and cold-exposed (solid lines and symbols) rats. Facilitated ACTH responses to restraint emerge as steady-state corticosterone increases from low to high (left to right). Note the log scale for ACTH.

 
Although initial (0 time) ACTH levels decreased similarly in control and cold rats under the higher B conditions, the ACTH response to restraint with increasing B differed markedly between control and cold rats. First, although there was no difference in the ACTH response to restraint in the low B control and cold groups, the control response tended to be greater than the cold response (P = 0.071). As steady-state B levels increased, differences in the acute responses emerged. The ACTH response to restraint tended to be greater in the cold than control rats at medium B concentrations (P = 0.070), and at high B concentrations, cold rats had a greater ACTH response to restraint than controls (P = 0.049). Moreover, although the mean increase in ACTH in cold rats after 30 min was slightly less than controls in the low B group, the increase at 30 min was approximately 220 pg/ml in the medium B group and was also approximately 220 pg/ml in the high B group.

When PM basal ACTH concentrations were correlated with the 30-min restraint ACTH concentrations in the same rats, there was a highly significant positive correlation between the values in the 2 samples (r2 = 0.81, F = 85.29, P < 0.001). This result suggests that regulation of PM basal and AM stress-induced ACTH levels may involve similar, or the same CNS processes.

CRF and AVP mRNA in the parvocellular PVN (Figs. 6Go and 7Go).
Because of a freezer failure and some methodological problems, we could only analyze a subset of the brains from the rats in this study. The individual rat brain CRF and AVP mRNA data shown in Fig. 6Go may not be from the same brain, and no attempt was made to match adjacent sections.



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  		Figure 6. Semiquantitation of in situ hybridization for CRF (left) and AVP (right) mRNA in the hypothalamic dorsomedial paraventricular nucleus. Symbols represent the mean (± SEM) square microns/cell occupied by silver grains (see Materials and Methods). CRF mRNA (left panel), Dashed and solid lines on the CRF plot represent the best fitted curves for CRF mRNA in control (open symbols) and cold (closed symbols), respectively. AVP mRNA (right panel), Dotted lines represent the 95% confidence limits for the fitted curve for the cells in control rats.

 


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  		Figure 7. Brightfield micrographs of cells (400 x magnification) with silver grains showing AVP mRNA in the most different control (left) and cold (right) rats with nearly equivalent plasma corticosterone concentrations (the individual rats can be seen in Fig. 7Go as the control at 8.6 µg B/dl, and the cold at 9.6 µg/dl). The large neurons with heavy silver grains were assumed to be magnocellular, AVP-expressing cells and were excluded from analysis (see Materials and Methods). The four less densely labeled, circled neurons in each photograph also lie in the dorsal medial PVN and show more silver grains/cell in the cold rat (right) than the control rat (left). Scale is provided by the diameter of the circle (13 microns) around each of the four neurons in each figure.

 
CRF mRNA.
CRF mRNA levels, as measured by the density of hybridization over a standard cell template (see Materials and Methods), were markedly inhibited as steady-state B levels increased in both control and cold groups. There was a sharp decrease in CRF mRNA expression that occurred between 0 and approximately 2 µg/dl B in the control rats (Fig. 6Go, left). Because rats in cold did not survive with such low B levels, there are no comparison brains in this range of B. However, above this range, there were no differences in CRF mRNA expression in parvocellular PVN cells of control and cold-exposed rats, as shown by the best-fit power equations for the two sets of results. The data for control rat CRF mRNA confirm several reports of the relationship between CRF mRNA and steady-state B levels measured by in situ hybridization (33, 43) and solution protection assays (43, 44), although the apparent effective inhibitory concentration of B (Fig. 6Go, left) is lower than the majority of reports. The control data were best fit with the equation: 1/y = 0.121 - 0.114x + 0.127x2 - 0.031x3 + 0.003x4 - 0.001x5 (r2 = 0.835, F = 2.535, P < 0.01). The equation describing the cold data set was: y = -76 + 83.21x - 22.59x2 + 2.79x3 - 0.17x4 + 0.006x5 (r2 = 0.9768, F = 7.038, P < 0.01). The curves are shown in Fig. 6Go, left; the fits of the control and cold data sets could not be statistically distinguished. Therefore, chronic cold compared with control had no effect on expression of CRF mRNA/cell. However, the number of CRF mRNA-expressing cells was significantly reduced in rats in the cold (see Materials and Methods).

AVP mRNA.
AVP mRNA hybridization was also decreased with increasing steady-state B levels in parvocellular PVN cells (Fig. 6Go, right). The pattern of inhibition of AVP mRNA may have differed slightly between control and cold-exposed rats and is certainly more complex than that seen in CRF mRNA hybridization. CRF and AVP cannot be directly compared in these studies because there were differences in isotope, emulsion, and some uncertainty about the relative hybridization efficiencies between the two sets of results. The control AVP mRNA data were best described by the equation: y = 10.37 + 8.32x - 5.59x2 + 1.34x3 - 0.155x4 + 0.008x5 (r2 = 0.9168, F = 9.19, P < 0.001). Three of the ten rats sampled in cold had AVP mRNA levels with SEs that fell either above or to the right of the 95% confidence interval for control rats (indicated by the dotted lines in Fig. 6Go, right), suggesting that the chronically stressed rats with circulating B levels approximately similar to controls may have been somewhat less sensitive to glucocorticoid inhibition of AVP mRNA levels. The effect of B on AVP mRNA levels was certainly more impressive than that of cold. Unlike the results for CRF mRNA, the number of parvocellular PVN cells expressing AVP mRNA did not differ between control and cold rats (see Materials and Methods).

Figure 7Go shows a bright field micrograph of silver grains resulting from AVP mRNA expression over parvocellular cells in a control (left) and a cold-exposed (right) rat that had mean AVP mRNA expression above the 95% confidence interval for the control B-AVP mRNA curve (Fig. 6Go, right). Inspection shows that there are more silver grains over the circled cells of the cold-exposed rat than over the control rat. Nonetheless, AVP mRNA expression in most of the cold group lay well within the 95% confidence limits of the curve describing B-dependent inhibition in control rats.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The results of these studies show that, as expected, the acute stressor of restraint provokes ACTH responses of similar magnitude in chronically cold stressed and control intact rats, despite increased endogenous B secretion in the cold stressed rats. They also show that exogenously supplied corticosterone exerts the expected, marked inhibitory effects on CRF and AVP mRNA in parvocellular neurons of the PVN and on both basal and restraint-induced increases in ACTH in adrenalectomized control rats. Beyond this, however, the data show that although the chronic stress of cold does not modify, to any notable extent, expression of the major ACTH secretogogues CRF and AVP, cold does cause facilitated basal p.m. and restraint-induced ACTH responses in the a.m. Moreover, and unexpectedly, the magnitude of facilitated ACTH responses in cold stressed rats increases, compared with control, as steady-state B increases.

The mechanism by which chronically stressed rats that have had previously or chronically elevated corticosterone feedback signals maintain normal responses of ACTH to acute stress is elusive. In this study and in others (11), we have shown that previously or chronically stressed rats exhibit normal responses to acute stress despite the elevated [and effective (2, 14, 45)] B feedback signal stimulated by the prior stressor. When B responses to either repeated acute (2) or chronic (11) stimuli are prevented, frank hypersecretion of ACTH to acute stimuli occurs in rats that have experienced prior stress. Similar findings (12) prompted the hypothesis that stress induces a memory in central neural components controlling the HPA axis such that the increased negative feedback signal provided by stress-induced endogenous B secretion is balanced by facilitation of responses to acute stress.

B feedback
We confirm the results of others (30, 33, 46, 47, 48, 49) that there is a sensitive negative feedback effect of increasing corticosterone on secretogogue mRNA expression in parvocellular neurons of the PVN in control, nonstressed rats. Because it usually requires more than 30 min to observe an increase in concentrations of cytosolic mRNAs (30, 33, 47, 48, 49, 50), we felt comfortable including data from rats after 30 min restraint in our sample.

Although chronic stressors have been shown by others to elevate CRF- or AVP-mRNA expression or peptide in intact rats (5, 33, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60), this is the first study of which we are aware to directly compare these in control and cold-exposed rats across a range of similar plasma B concentrations. We supplied constant B by pellet because the alternative phasic replacement paradigm, using B in the water (61), would have provided more B to rats in the cold because they drink more than their room temperature counterparts (unpublished data from 11 . The effect of chronically elevated but controlled B in cold exposed rats compared with controls on CRF and AVP mRNA is mixed. We found fewer CRF mRNA-expressing cells in cold exposed rats, and more AVP mRNA expression/cell in 30% of the cold exposed rats. Although our studies do not identify whether the measured CRF and AVP mRNAs were colocalized in the same parvocellular PVN cells as has been shown by others (55, 62), we suspect that colocalization occurred in most cases. As a consequence of these results, we can determine no pronounced effects of chronic cold on the biosynthesis of these secretogogues in the PVN.

CRF mRNA is not the only standard by which inhibitory effects of B at the PVN should be evaluated. However, in other studies (63) using a similar B-treatment paradigm, with similar steady-state B levels, CRF peptide content in the median eminence of nonstressed rats also decreased with increasing corticosterone [CRF (ng/mg protein): low B, 11.23 ± 0.93, medium B, 8.28 ± 0.88, and high B, 6.42 ± 0.47; F = 9.05, P = 0.001]. Therefore, in separate but equivalent studies the effects of B on CRF mRNA and protein content in control rats were similar, and it is likely that the inhibition of CRF mRNA by B also resulted in equivalent decreases in CRF peptide in both control and cold-exposed rats. This conclusion is reinforced by the finding that pituitary ACTH content was similarly inhibited by B in control and cold rats. Furthermore, these results with the chronic stimulus of cold are corroborated by previous findings in rats chronically stimulated with streptozotocin-induced diabetes mellitus, in which both K+-stimulated CRF secretion from hypothalami in vitro and ACTH responses to CRF, AVP and the combination in vivo was normal (7, 8). Similar to the results with cold, diabetic rats that were adrenalectomized and treated with a variety of doses of B had a similar CRF mRNA inhibition curve as that of the nonstressed, vehicle-treated rats (64) .

In the presence of some CRF, other potential secretogogues than AVP, such as oxytocin, catecholamines, and angiotensin II, can potentiate ACTH responses from the pituitary (65). However, evidence that these other agents do stimulate ACTH secretion during any but hypertonic stress is slight (66, 67). During acute restraint stress, treatment with antisera to AVP and to CRF and to both antisera together inhibit ACTH responses (68), suggesting that these secretogogues are responsible for ACTH responses to restraint.

Cold-induced facilitation of subsequent HPA responses to restraint
From the above results, we believe that there is strong evidence that the chronic stressors of cold or diabetes do not act at or below the level of neuroendocrine neurons in the PVN to cause facilitated HPA responses to acute stress. At these regulatory levels, the corticosterone feedback signal appears to dominate. Nonetheless, the results of these studies provide clear evidence that facilitated ACTH responses to acute restraint occur in the AM in cold-exposed rats. Importantly, facilitation only emerges as steady-state corticosterone concentrations increase, until at high B levels, only the cold-exposed rats have appreciable ACTH responses to restraint. We have previously shown that facilitated ACTH responses do not occur in adrenalectomized rats without B in either the a.m. or p.m. (8, 69); this is also true with low B in the a.m. in this study.

After prior stress in intact rats, facilitated ACTH responses do occur to acute stress in the a.m. but not the p.m.; however, in the p.m., basal activity in the HPA axis is increased as a consequence of prior stress (2, 15). In this study, the elevation in PM basal ACTH levels in cold-exposed rats was strongly correlated to the amplitude of the ACTH responses to acute restraint in the AM, suggesting that a similar, or the same control pathway mediates these facilitated responses to chronic stress.

Both its diurnal complexity and the finding of little effect of chronic stress (2, 15) at or distal to the PVN motor neurons of the HPA axis suggest strongly that stress-induced facilitation is mediated by neural substrates proximal to the PVN. Recently, Bhatnagar (70) has shown that a few neural structures demonstrate increased numbers of fos-like immunoreactive (fos-lir) cells in intermittently cold-stressed compared with control rats after acute restraint stress. One site that expresses increased numbers of fos-lir cells is the posterior portion of the paraventricular nuclei of the thalamus, a nucleus that also receives major input from the suprachiasmatic nuclei (71) providing time-of-day information. The outputs from the posterior paraventricular thalamus are discrete and primarily innervate those parts of the amygdala (71, 72), which Bhatnagar also finds to contain more fos-lir cells after acute restraint in cold-stressed rats. Thus, the paraventricular nuclei of the thalamus may provide time-of-day information to the facilitation circuit induced by prior stress.

Moreover, facilitation executed by the amygdala may resolve the puzzling fact that stress-induced facilitation of HPA function increases with increasing corticosterone. The autonomic and endocrine output of the amygdala is from the central nucleus, which contains a large group of CRF-synthesizing cells (32). Several studies have shown that as circulating levels of B increase, CRF mRNA in the central nucleus of the amygdala also increases, in contrast to the decrease observed in CRF mRNA in the PVN (46, 48, 49). Watts and Sanchez-Watts (49) have elegantly shown that as PVN CRF mRNA decreases, amygdalar CRF mRNA increases as a function of steady-state B levels in rats. Functionally, central CRF injections mimic the effects of stress in rats, and lesions of the amygdala or injection of a CRF antagonist block HPA responses to many stimuli (reviewed in Refs. 74, 75). Therefore, it seems likely that facilitation induced by prior or chronic stress, which balances so precisely the inhibitory effects of B on the PVN in intact rats, may be mediated by direct (76) or indirect [through the bed nucleus of the stria terminalis, (77)] amygdalar CRF pathways to the PVN and other sites. Herman has proposed that cell groups in the bed nucleus of the stria terminalis are critical to acute HPA responses to processive stressors, like restraint (78). Because the amygdala project to the bed nucleus, this may comprise the pathway taken by chronic stress-induced facilitation to cause augmented ACTH responses to acute stress. These specific hypotheses about the site at which B acts to produce facilitated responses and the pathway by which facilitation reaches the PVN remain to be tested.

In summary, we have shown that facilitated ACTH responses to restraint that are induced by chronic exposure of rats to cold do not occur at or below the motor neuroendocrine neurons in the PVN. We have also shown that facilitation emerges as steady state corticosterone concentrations increase. We hypothesize that this effect is mediated by the positive effects of B on CRF synthesis (and secretion) from the central nuclei of the amygdala.


   Acknowledgments
 
The authors thank Simon Hanson, Drs. Alison M. Strack and Seema Bhatnagar, Cydney J. Horsley, and Erin D. Milligan for their essential cooperation.


   Footnotes
 
1 This work was supported, in part, by United States Public Health Service Grant DK-28172. Portions of these data have been presented in abstract form at the Annual Meeting of The Endocrine Society, June 14–17, 1995, Washington, D.C.; the Annual Meeting of The Society for Neuroscience, November 11–16, 1995, San Diego, California; and the 10th International Congress of Endocrinology, June 12–16, 1996, San Francisco, California. Back

Received February 24, 1997.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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