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Single or repeated mild stress increases synthesis and release of hypothalamic corticotropin-releasing factor
Brain Research, 461 (1988)230-237
230
Elsevier BRE 13948
Single or repeated mild stress increases synthesis and release of hypothalamic corticotropin-releasing factor Daniel A. Haas 2 and Susan R. George 1'2 Departments of 1Medicineand 2pharmacology, Universityof Toronto, Toronto, Ont. (Canada) (Accepted 19 April 1988)
Key words: Corticotropin-releasing factor; Immunoreactivity; Adrenocorticotropic hormone; Hypothalamus; Median eminence; Anisomycin; Stress
The effect of a specific mild stress on the levels of corticotropin-releasing factor immunoreactivity (CRF-ir) in the hypothalamus of adult male rats was determined using a radioimmunoassay specific for rat CRF. A single 5 min restraint significantlyincreased CRF-ir in the median eminence 24 h later compared to appropriate controls (P < 0.025), with no change detected earlier. Plasma ACTH, an indirect index of CRF release, was significantly elevated within 15 min (P < 0.025). Repetition of a mild stress daily for 9 days (P < 0.01), or a single episode of handling (P < 0.05), both resulted in significantlyincreased CRF-ir in the whole hypothalamus 24 h later. Blockade of axonal transport by intracisternal colchicine decreased CRF-ir in the median eminence 24 h later (P < 0.005). Inhibition of protein synthesis by anisomycin during a single 5 minute restraint resulted in significantly decreased CRF-ir in the median eminence 24 h later compared to vehicle-injected stressed rats (P < 0.005) or to anisomycin-injected unstressed controls (P < 0.025). These data show that mild stress increased net hypothalamic CRF content as a result of the balance between augmented synthesis and augmented release.
INTRODUCTION One of the prime mediators of the stress response 41 is corticotropin-releasing factor (CRF) 23'29 through its control of the hypothalamic-pituitary-adrenal (HPA) axis 36'37"46. C R F administration has also been shown to elicit other responses characteristic of stress, such as increased cardiac output and the levels of plasma catecholamines25, decreased gastrointestinal activity25, increased pituitary cyclic A M P 23, and increased general arousal 6. We were interested in investigating how stress affected C R F in the hypothalamus, where it is primarily localized in brain 27. Although the H P A axis in the rat is known to be sensitive to mild stresses such as handling39, their effect o n C R F immunoreactivity (CRF-ir) has not been reported. Studies examining the effect of relatively severe stresses 1s'43 have provided conflicting results re-
garding content change in CRF-ir or C R F bioactivity. One potential concern about studying CRF-ir following severe stressors is that they may involve parameters such as pain that could activate other neurotransmitter systems 26 and therefore a clear relation between stress and C R F may not be assumed. Selye 4° first showed that restraining the rat leads to a stress syndrome, and restraint has been widely used as a model of stress in biomedical research 31. It has been shown that as little as 2 min of restraint can elevate plasma adrenocorticotropic h o r m o n e ( A C T H ) concentrations12, a marker commonly used to study acute stress, and reflect endogenous C R F secretion. We therefore elected to study the effect of a specific but mild stress on hypothalamic CRF-ir by using the model of a 5 min restraint and in the present study report its effects on CRF-ir using a radioimmunoassay specific for rat CRF. We have examined the el-
Correspondence: S.R. George, Department of Pharmacology, University of Toronto, Medical Sciences Building, Room 4358, 1 King's College Circle, Toronto, Ont., M5S 1A8 Canada. 0006-8993/88/$03.50© 1988 Elsevier Science Publishers B.V. (Biomedical Division)
231 fects of both single and repeated stresses, and assessed the relative contribution of synthesis and release in the overall CRF changes induced by stress. MATERIALS AND METHODS
Animals Adult male Sprague-Dawley rats (Charles River, Canada), 220-300 g, were housed singly in environmental rooms under constant conditions of temperature, humidity, and maintained under a 12 h lightdark schedule with lights on at 08.00 and off at 20.00 h, with free access to food and water. It has been shown that rats housed singly show less individual variation in plasma corticosterone levels than those housed in groups 3. Sacrifice was carried out in an adjacent room by decapitation at approximately the same time on experimental days and alternated among the groups to diminish any diurnal effects. We have determined that between 09.00 and 14.00 h, during which all the sacrifice took place, there was no significant diurnal variation of hypothalamic CRF-ir (unpublished). Brains were quickly dissected on a chilled glass plate as previously described 15 and the tissues immediately frozen and stored at -70 °C until extraction. Trunk blood was collected in polypropylene tubes containing E D T A , spun at 2000 g for 15 min, and the plasma removed and stored at -20 °C until assay.
Stress treatments Stress treatments were alternated among groups to diminish any potential diurnal effect on CRF or A C T H 22,45.
Effect of single restraint. Rats were randomly assigned into 4 groups and handled daily for 12 days prior to the experiment. They were restrained for 5 min in a semi-cylindrical plexiglas restraint cage, 70 x 160 x 42 mm, immediately returned to their home cage and sacrificed either 15 min (n = 8), 2 h (n = 7) or 24 h (n = 7) afterwards. The control group (n = 7) was handled identically but not restrained. Median eminence was dissected separately from the rest of hypothalamus. Effect of repeated restraint. Rats were randomly assigned into 3 groups and handled every other day for one week prior to the experiment. The stressed group (n = 13) was restrained for 5 min in the re-
straint cage at approximately the same time daily for 9 consecutive days. As rats can habituate to the same stress 21, the environment in which subsequent restraints were performed was changed daily. Beginning on the second day, a second novel stress was added, in the following order: Day 2, s.c. injection of 250/A of saline; Day 3, exposure to vibration for 30 s using a tuning fork placed on the cranium; Day 4, light from a 100 W bulb placed 6 inches overhead; Day 5 the restraint cage was turned over during the 3rd minute of restraint. Beginning on the 6th day, two of these additional stresses were added to the restraint, with the stresses altered randomly, daily. A subgroup (n = 4) was stressed for the 9 days as described above then allowed 5 days of recovery by being handled daily with no restraint. The control group (n = 11) consisted of rats handled daily for the 9 days, with a subgroup (n = 3) handled for the 14 day duration of the study. All rats were sacrificed 25 h after the last stress or handling, and the whole hypothalamus dissected. Handling stress. One group of rats (n = 4) was not handled at all for the period described above until the 9th day, when they were handled and weighed. Twenty-four h later they were sacrificed and whole hypothalamus dissected.
Effect of protein synthesis inhibition on single restraint. Rats were randomly assigned into 4 groups and handled daily for one week prior to the experiment. Rats were either given one 5 min restraint stress 30 min following a subcutaneous injection of vehicle (n = 8) or anisomycin (50 mg/kg, n = 6), or were not handled following an injection of vehicle (n = 7) or anisomycin (n = 7). Rats were sacrificed 24 h later and median eminence was dissected separately from remaining hypothalamus. Anisomycin (Sigma, St. Louis, U.S.A.) was dissolved freshly in saline with 2% 1 N HC1.
Effect of axonal flow blockade One hundred /zg colchicine (Sigma, St. Louis, U.S.A.) or vehicle (saline) was injected intracisternally in a volume of 25/tl under halothane anesthesia (5% for induction and 1.5% for maintenance in 95% 0 2 - 5 % CO2). Rats were then sacrificed after 4 h (n = 5 for vehicle, n = 3 for colchicine), 17 h (n = 4 for vehicle, n = 5 for colchicine) or 24 h (n = 4 for vehicle, n = 6 for colchicine).
232 RESULTS
Tissue extraction Hypothalamus (30-40 mg) and median eminence were extracted as described previously 17. Protein content of median eminence was d e t e r m i n e d by the method of B r a d f o r d 5.
CRF assay The lyophilized samples were assayed in triplicate using a double antibody r a d i o i m m u n o a s s a y specific for rat C R F , d e v e l o p e d for our use and described previously 17. The lower limit of assay detection was 4 pg p e r tube. The intra- and interassay coefficients of variation were 4.8% and 9.3% respectively. C R F - i r from each experiment was d e t e r m i n e d within the same assay.
A C TH assay Plasma A C T H concentrations were d e t e r m i n e d as described previously 17, using the r a d i o i m m u n o a s s a y m e t h o d of Nicholson et al. 3°.
Data analysis Statistical analyses utilized analysis of variance ( A N O V A ) and the Student's t-test, with P values of less than 0.05 judged to be significant. All data are p r e s e n t e d as the m e a n _+ S . E . M . of individual values of rats from each group. C R F - i r is expressed as either pg p e r mg wet weight for hypothalamus, or for median eminence as total pg or pg p e r ¢tg of protein.
The effect of a single 5 min restraint on C R F - i r in the median eminence and remaining h y p o t h a l a m u s is shown in Fig. 1. A s c o m p a r e d to unstressed controls a significant increase in median eminence C R F - i r w a s d o c u m e n t e d 24 h post-stress (P < 0.025), with no change detected after 15 min or 2 h. No change was noted in the remaining h y p o t h a l a m u s at any of the time points assessed. These differences were maintained whether C R F was expressed as C R F - i r per unit protein as shown in Fig. 1, o r as total C R F - i r per tissue. As shown in Fig. 2, plasma A C T H levels were found to be significantly increased 15 min post-stress (P < 0.025). These levels decreased significantly c o m p a r e d to baseline by 2 h post-stress ( P < 0.025), and r e m a i n e d near baseline levels 24 h later. The 5 min restraint was r e p e a t e d daily in a novel environment for 9 days and found to increase C R F - i r in total hypothalamus as c o m p a r e d to unstressed controis handled daily for the same time p e r i o d ( P < 0.01), as shown in Fig. 3. A subgroup of stressed rats was allowed a 5 day recovery period, and this resuited in no further change in CRF-ir. The stressed group had C R F - i r of 112.6 ___7.2 pg/mg as c o m p a r e d to 111.8 _+ 13.6 pg/mg for the group allowed recovery. The C R F - i r in the control rats also did not differ whether they were handled for 9 days (81.8 + 7.6
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Fig. 1. Effect of a single 5 min restraint on CRF-ir in median eminence (expressed as pg//~g protein) and remaining hypothalamus (expressed as pg/mg wet wt.). Significant difference from unstressed controls (0 h post-stress) is denoted by * for P < 0.025. n = 7 or 8 per group.
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Fig. 2. Effect of a single 5 min restraint on plasma ACTH. Significant difference from unstressed controls (0 hours poststress) is denoted by * for P < 0.025. n = 7 or 8 per group.
233 Whole Hypothalamus 120
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Fig. 3. Effect on hypothalamic CRE-ir of single episode of handling and weighing (n = 4) and repeated 5 min restraints daily for 9 days (n = 13) as compared to unstressed rats which were handled daily for 9 days (n = 11). Significant difference from control is denoted by * for P < 0.05 and ** for P < 0.01.
pg/mg) or 14 days (87.9 + 18.5 pg/mg). There were no statistically significant differences in ACTH levels in these rats (data not shown). The repeatedly stressed group was found to have a significantly lower gain in body weight (a mean increase 11.2 + 0.8%) compared to their handled controls (a mean increase of 14.1 + 0.6%, P < 0.025). Fig. 3 also shows that the effect of a single episode of handling and weighing was enough to cause a significant increase in hypothalamic CRF-ir 24 h later, compared to controls which were handled daily for 9 days (P < 0.05). The increase in plasma A C T H shown in Fig. 2 was not accompanied with a detectable decrease in CRFir (Fig. 1). This may have been due to rapid repletion of CRF transported from the site of synthesis, the paraventricular nucleus s, to the site of release, the median eminence. We were therefore interested in assessing the time interval required to detect a change in CRF-ir following inhibition of peptide transport. This was achieved by administering the axonal transport inhibitor colchicine intracisternally and then determining CRF-ir. As shown in Fig. 4, a progressive decrease in median eminence CRF-ir was apparent and became statistically significant 24 h after colchicine (P < 0.005). No change in the remaining hypothalamus was observed. The increase documented in CRF-ir content in the
Fig. 4. Effect of axonal blockade by colchicine on total CRF-ir in median eminence. Significant difference from vehicletreated controls is denoted by * for P < 0.005. n = 3 - 6 per group.
median eminence 24 h post-stress could be due to either an increase in synthesis or an inhibition of release. In order to investigate the mechanism involved, we repeated this restraint stress under conditions of protein synthesis inhibition induced by the administration of the protein synthesis inhibitor anisomycin, and then sacrificed the rats 24 h later. As shown in Fig. 5, anisomycin treatment resulted in a 13% decrease in CRF-ir in the median eminence in unstressed rats (not significant) and a 45% decrease in stressed rats (P < 0.005) compared to their respec20 ¸ Media
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Fig. 5. Effect of anisomycin-induced protein synthesis inhibition (Aniso) during a single 5 min restraint on CRF-ir in median eminence (expressed as pg/~tg protein) and remaining hypothalamus (expressed as pg/mg wet wt.). Anisomycin was administered at a dose of 50 mg/kg s.c., 30 min prior to restraint, and CRF-ir determined 24 h later. Significant difference from vehicle-treated controls (Saline) is denoted by * for P < 0.01, and significant difference from the vehicle-treated stressed group is denoted by ** for P < 0.005. n = 6 - 8 per group.
234 tive controls. Anisomycin-treated stressed rats had significantly reduced CRF-ir in the median eminence compared to vehicle-treated stressed rats (P < 0.005) which would reflect the effect of protein synthesis inhibition. These values were also lower when compared to anisomycin-treated unstressed rats (P < 0.025), which would reflect the effect of stress. Analysis utilizing one and two-way A N O V A demonstrated a significant difference among the groups (F3,24 = 6.58, P < 0.005) with the main effect of anisomycin treatment being significant (F1,22 = 10.92, P < 0.005). These differences are maintained whether C R F is expressed as CRF-ir//~g protein as shown in Fig. 5, or as total CRF-ir per tissue. Anisomycin treatment showed no effect on the remaining hypothalamus whereas the vehicle-treated stressed group did show a significant increase in CRF-ir compared to vehicle-treated unstressed rats (P < 0.01). Plasma A C T H levels showed no statistically significant differences between any of the groups (data not shown). DISCUSSION These data show that mild stresses consisting of a single episode of handling, or a 5 min restraint, whether applied once or repeatedly, altered hypothalamic CRF synthesis and release. The advantages of this investigation compared to previous reports are that a discrete, reproducible mild stress was used thereby diminishing non-stress effects and CRF-ir was directly measured to assess effect on synthesis, transport and release. Previous reports of the effects of stress on the H P A axis have shown alteration of rat plasma corticosterone and A C T H levels by stresses of varying intensity ranging from severe, such as ether-laparotomy TMor tibial fracture 19 to mild, such as handling and weighing 3. C R F bioactivity has variably been shown to increase TM, demonstrate a biphasic response 19 or remain unchanged 47. Recently, more direct assessments measuring immunoreactivity have examined the effects of severe stressors such as ether-laparotomy 43, or 3 h of restraint during cold exposure 1° on hypothalamic CRF-ir. In contrast, shorter periods of restraint have variably been reported to have no effect on CRF-ir in the median eminence 44 or to decrease and then increase hypothalamic CRF-ir 28. The
previous studies 1°'43 reporting stress-induced effects on CRF-ir have utilized stresses more severe than those used in our studies. The drawback to severe and multiple forms of stress is the potential that the pain of laparotomy, the pharmacological effects of ether, or the alteration of body temperature may activate other neurotransmitter or neuropeptide systems that secondarily influence C R F levels. Therefore a direct relationship between a unitary form of stress and C R F dynamics cannot be assumed. Also, it will not be possible to eventually elucidate the precise neural mechanisms involved in mediating C R F release in various types of stress. The older studies served to establish that the c o m m o n mediator of severe stress was C R F release stimulating A C T H secretion. Since we do not believe that all stresses release C R F through the same mechanisms, we wished to employ a relatively pure unitary stress. One other drawback to the previous studies on CRF-ir is that two of these l°'28 utilized antisera to ovine CRF in their radioimmunoassay for rat CRF. These two peptides differ by 7 amino acids 38'46 and it has been shown that the CRF-ir measured in rat brain can vary greatly depending on which antiserum is used 42. Our study has the advantage of using an antiserum raised against rat C R F and should therefore be a more accurate reflection of C R F levels in rat hypothalamus. It is possible that discrepancies among the studies could be due to different antisera as well as different stress models used. The single 5 min restraint caused a significant increase in plasma A C T H within 15 min. This confirms previous studies on the effects of acute restraint on rat A C T H 12'16 and is evidence of this restraint model being an adequate and effective stressful event. A C T H levels act as an indirect index of C R F release and our data imply that C R F was released from the median eminence within this time period. This was not detected as a change in CRF-ir content either in whole hypothalamus or median eminence, indicating either that the release was so small as to be undetectable as a change in content, or that the turnover was so rapid that the released C R F was repleted as fast as it was depleted. The latter explanation is less likely given CRF-ir could not be detected to increase until 24 h later, implying a slow rate of turnover. An indication of the time required to detect a change in turnover of C R F was obtained by deter-
235 mining the time course of CRF-ir change following blockade of axonal transport induced by the administration of intracisternal colchicine. The results showed that a significant change could not be documented until 24 h later. This is consistent with previous studies that utilized immunocytochemical visualization to show a colchicine-induced decrease in CRF-ir at 48 h ~. it was noted that A C T H levels declined significantly below baseline 2 h post-stress, a possible consequence of increased glucocorticoid feedback induced by the initial ACTH peak. By 24 h post-stress ACTH levels were not different from baseline in any of the stressed groups assessed in this study, suggesting a normalization of plasma ACTH after this interval. Our studies demonstrated that any of the mild stresses employed increased CRF-ir in the hypothalamus after 24 h. The exact time of significant CRF content change following the single restraint could have occurred any time between 2 and 24 h post-stress. This change could be due to either increased CRF synthesis with subsequent transport to the median eminence, or inhibition of release, such as that mediated by increased glucocorticoid secretion, although the latter possibility is less likely. In order to investigate the mechanism involved we repeated the acute restraint stress while blocking protein synthesis with the general protein synthesis inhibitor anisomycin. We then inferred changes of CRF synthesis on the basis of the measurement of CRF-ir, coupled with the measurements of plasma ACTH. Anisomycin was selected as it has been shown to effectively inhibit cerebral protein synthesis with very little systemic toxicity4A3, especially compared to other inhibitors such as cycloheximide. This factor is important in order to minimize non-specific stress effects on CRF. We chose the regimen of 50 mg/kg s.c., 30 min prior to restraint, which would inhibit protein synthesis in the hypothalamus by 80-90% as based on previous reports 32'35. Anisomycin administration resulted in a 13% decrease in median eminence CRF-ir in the unstressed rats and a 45% decrease in restrained rats (Fig. 5). The rate of protein synthesis has been shown to return to baseline levels within 6 hours following injection of anisomycin 35. Our studies determined CRF-ir 24 h after its administration, thereby allowing at least partial re-
covery of normal synthesis. The depletion documented in both groups of rats was apparent even after this interval, suggesting there may have been a greater reduction in CRF-ir within the first few hours following administration. The 13% decrease documented in unstressed rats would likely reflect the loss of CRF due to normal physiologic release. Anisomycin treatment would have prevented the normal CRF turnover by blocking synthesis, and possibly transport as well, resulting in the observed decrease. The greater depletion of CRF-ir documented in anisomycin-treated stressed rats compared to vehicle-treated stressed controls implies that restraint induced an increased release in CRF and that protein synthesis was involved even in the early maintenance of CRF levels post-stress. This was indirectly confirmed by our ACTH data which showed a significant increase in plasma ACTH within 15 min of restraint. As CRF is the primary regulator of ACTH 46, increased plasma levels imply an increase in CRF release from the median eminence to allow stimulation of release of ACTH from the anterior pituitary. Therefore it appears that restraint stress induced an increase in CRF release, and anisomycin treatment prevented the expected increase in CRF-ir as found with either the single restraint, repeated restraint, or handling. As total content increased 24 h following these stresses, it suggests that this increased synthesis overcompensated the initial reduction in CRF. Combining the restraint stress with the intraperitoneal vehicle injection was sufficient to induce an increase in CRF-ir in remaining hypothalamus compared to non-restrained controls (Fig. 5). This increase was not apparent in remaining hypothalamus when comparing the two anisomycin-treated groups (Fig. 5) or when restraint was executed without the intraperitoneal injection (Fig. 1). These data suggest that this combination of stresses was more severe than restraint alone and subsequently altered CRF dynamics. The result was an increase in content detected in the remaining hypothalamus as opposed to the median eminence, where an increase was observed with the single restraint alone. This increase was apparently due to increased synthesis of CRF as evidenced by the lack of effect when comparing the anisomycintreated groups. The increase in CRF-ir documented following the single episode of handling has not been reported be-
236 fore. The activation of C R F in as simple a stress as being handled and weighed could have been speculated as handling of rats has been shown to increase plasma corticosterone 3'39, and epinephrine 24, both of which are under the indirect regulation of C R F TM. Our data would imply that hypothalamic C R F could be involved in the mediation of these responses. O u r results also emphasize the importance of matching and handling controls identically when studying the effect of various treatments on C R F - i r since simple handling and weighing of the animal can alter C R F ir. The same degree of increase in C R F - i r was found with the r e p e a t e d restraint stress model. That these r e p e a t e d stresses were effective is confirmed by the inhibition of body weight gain in the stressed group, a finding consistent with stress 2A1,33. A n i m a l s adapt to a r e p e a t e d stress 9,2° but the habituation is specific to the one stressor used 21. Stress that is unpredictable has been shown to have a m o r e p r o n o u n c e d effect on corticosterone levels 34, and is therefore considered more effective. We avoided habituation to the 5 min restraint by changing the environment in which the stress was applied daily, thereby diminishing its predictability. It is of interest to note that this r e p e a t e d stress administered for only 5 min a day resulted in an increase in hypothalamic C R F - i r similar to that found with the single restraint. This 9 day exposure to restraint was enough to induce increased C R F levels which persisted after the rats were allowed a 5 day REFERENCES 1 Alonso, G., Szafarczyk, A., Balmefrezol, M. and Assenmacher, I., Immunocytochemical evidence for stimulatory control by the ventral noradrenergic bundle of parvocellular neurons of the paraventricular nucleus secreting corticotropin releasing hormone and vasopressin in rats, Brain Research, 397 (1986) 297-307. 2 Armario, A., Restrepo, C., Castellanos, J.M. and Balasch, J., Dissociation between adrenocorticotropin and corticosterone responses to restraint after previous chronic exposure to stress, Life Sci., 36 (1985) 2085-2092. 3 Barrett, A.M. and Stockham, M.A., The effect of housing conditions and simple experimental procedures upon the corticosterone level in the plasma of rats, J. Endocrinol., 26 (1963) 97-105. 4 Bennett, E.L., Orme, A. and Herbert, M., Cerebral protein synthesis inhibition and amnesia produced by scopolamine, cycloheximide, streptovitacin A, anisomycin and emetine in rat, Fed. Proc., 31 (1972) 838. 5 Bradford, M.M., A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding, Anal. Biochem,. 72 (1976)
period of recovery. The final 5 days of handling in these restraint-stressed rats may have now served as a new daily stress and acted to maintain increased C R F - i r levels. This study has d e m o n s t r a t e d that the specific mild stresses of handling or a 5 min restraint can increase hypothalamic C R F - i r content. This was d e p e n d e n t on the synthesis of new peptide which was transp o r t e d to the median eminence to replace C R F initially released due to stress. The newly synthesized C R F more than c o m p e n s a t e d for this reduction, leading to an increase in C R F - i r content. The stress models used in this study do not involve activation of pain pathways, pharmacological effects or alteration of body t e m p e r a t u r e , and can therefore be considered to be a relatively pure unitary form of stress. Thus, the effects on C R F - i r d o c u m e n t e d herein can be assumed to be due to stress alone. W e can therefore conclude that a single mild stress alone can stimulate the synthesis and release of hypothalamic C R F . ACKNOWLEDGEMENTS This work was s u p p o r t e d by funds from the Medical Research Council of Canada. S . R . G . is an M R C Scholar and D . A . H . an M R C Fellow. W e gratefully acknowledge the generous assistance of Dr. J. Tong and Dr. W. Sturtridge in setting up the C R F radioimmunoassay and Dr. L. R o l d a n , B. Borgundvaag and R. Ycas for their assistance with the experiments. 248-254. 6 Britton, D.R., Koob, G.F., Rivier, J. and Vale, W., Intraventricular corticotropin-releasing factor enhances behavioral effects of novelty, Life Sci., 31 (1982) 363-367. 7 Brown, M.R, and Fisher, L.A., Corticotropin-releasing factor: effects on the autonomic nervous system and visceral systems, Fed. Proc., 44 (1985) 243-248. 8 Bruhn, T.O., Plotsky, P.M. and Vale, W.W., Effect of paraventricular lesions on corticotropin-releasing factor (CRF)-like immunoreactivity in the stalk-median eminence: studies on the adrenocorticotropin response to ether stress and exogenous CRF, Endocrinology, 114 (1984) 57-62. 9 Burchfield, S.R., Woods, S.C and Elich, M.S., Pituitary adrenocortical response to chronic intermittent stress, Physiol. Behav., 24 (1980) 297-302. 10 Chappell, P.B., Smith, M.A., Kilts, C.D., Bissette, G., Ritchie, J., Anderson, C. and Nemeroff, C.B., Alterations in corticotropin-releasing factor-like immunoreactivity in discrete rat brain regions after acute and chronic stress, J. Neurosci., 6 (1986) 2908-2914. 11 Daniels-Severs, A., Goodwin, A., Keil, L.C. and Vernikos-Danellis, J., Effect of chronic crowding and cold on the
237 pituitary-adrenal system: responsiveness to an acute stimulus during chronic stress, Pharmacology, 9 (1973) 348-356. 12 De Souza, E.B. and Van Loon, G.R., Stress-induced inhibition of the plasma corticosterone response to a subsequent stress in rats: a nonadrenocorticotropin-mediated mechanism, Endocrinology, 110 (1982) 23-33. 13 Flood, J.F., Bennett, E.L., Rosenzweig, M.R. and Orme, A.E., The influence of duration of protein synthesis inhibition on memory, Physiol. Behav., 10 (1973) 555-562. 14 Fujieda, K. and Hiroshige, T., Changes in rat hypothalamic content of corticotropin-releasing factor (CRF) activity, plasma ACTH and corticosterone under stress and the effect of cycloheximide, Acta EndocrinoL, 89 (1978) 10-19. 15 George, S.R. and Van Loon, G.R., Characterization of high affinity dopamine uptake into the dopamine neurons of the hypothalamus, Brain Research, 234 (1982) 339-355. 16 Gibbs, D.M., Dissociation of oxytocin, vasopressin and corticotropin secretion during different types of stress, Life Sci., 35 (1984) 487-491. 17 Haas, D.A. and George, S.R., Neuropeptide Y administration acutely increases hypothalamic corticotropin-releasing factor immunoreactivity: lack of effect in other rat brain regions, Life Sci., 41 (1987) 2725-2731. 18 Hiroshige, T., Fujieda, K., Kaneko, M. and Honma, K., Assays and dynamics of corticotropin-releasing factor activity in rat hypothalamus, Ann. NYAcad. Sci., 297 (1977) 436-454. 19 Hiroshige, T., Sato, T. and Abe, K., Dynamic changes in the hypothalamic content of corticotropin-releasing factor following noxious stimuli: delayed response in early neonates in comparison with biphasic response in adult rats, Endocrinology, 89 (1971) 1287-1294. 20 Kant, G.J., Bunnell, B.N., Mougey, E.H., Pennington, L.L. and Meyerhoff, J.L., Effects of repeated stress on pituitary cyclic AMP, and plasma prolactin, corticosterone and growth hormone in male rats, Pharmacol. Biochem. Behav., 18 (1983) 967-971. 21 Kant, G.J., Eggleston, T., Landman-Roberts, L., Kenion, C.C., Diver, G.C. and Meyerhoff, J.L., Habituation to repeated stress is stressor specific, Pharmacol. Biochem. Behay., 22 (1985) 631-634. 22 Kant, G.J., Mougey, E.H. and Meyerhoff, J.L., Diurnal variation in neuroendocrine response to stress in rats: plasma ACTH, fl-endorphin, fl-LPH, corticosterone, prolactin and pituitary cyclic AMP responses, Neuroendocrinology, 43 (1986) 383-390. 23 Kant, G.J., Oleshansky, M.A., Walczak, D.D., Mougey, E.H. and Meyerhoff, J.L, Comparison of the effects of CRF and stress on levels of pituitary cyclic AMP and plasma ACTH in vivo, Peptides, 7 (1986) 1153-1158. 24 Kvetnansky, R., Sun, C.L., Lake, C.R., Thoa, N., Torda, T. and Kopin, I.J., Effect of handling and forced immobilization on rat plasma levels of epinephrine, norepinephrine, and dopamine-fl-hydroxylase, Endocrinology, 103 (1978) 1868-1874. 25 Lenz, H.J., Raedler, A., Greten, H. and Brown, M.R., CRF initiates biological actions within the brain that are observed in response to stress, Am. J. Physiol., 252 (1987) R34-R39. 26 Mason, J.W., A re-evaluation of the concept of 'non-specificity' in stress theory, J. Psychiatry Res., 8 (1971) 323-333. 27 Merchenthaler, I., Vigh, S., Petrusz, P. and Schally, A.V., Immunocytochemical localization of corticotropin-releasing factor (CRF) in the rat brain, Am. J. Anat., 165 (1982) 385-396. 28 Moldow, R.L., Kastin, A.J., Graf, M. and Fischman, A.J., Stress mediated changes in hypothalamic corticotropin re-
leasing factor-like immunoreactivity, Life Sci., 40 (1987) 413-418. 29 Nakane, T., Audhya, T., Kanie, N. and Hollander, C.S., Evidence for a role of endogenous corticotropin-releasing factor in cold, ether, immobilization, and traumatic stress, Proc. Natl. Acad. Sci. U.S.A., 82 (1985) 1247-1251. 30 Nicholson, W.E., Davis, D.R., Sherell, B.J. and Orth, D.N., Rapid radioimmunoassay for corticotropin in unextracted human plasma, Clin. Chem., 30 (1984) 259-265. 31 Par6, W.P. and Glavin, G.B., Restraint stress in biomedical research: a review, Neurosci. Biobehav. Rev., 10 (1986) 339-370. 32 Parsons, B., Rainbow, T.C., Pfaff, D.W. and McEwen, B.S., Hypothalamic protein synthesis essential for the activation of the lordosis reflex in the female rat, Endocrinology, 110 (1982) 620-624. 33 Pfeiffer, C.J., The physiologic effect of restricted activity in the rat: stress effects of chronic restraint, Exp. Med. Surg., 25 (1967) 201-217. 34 Quirce, C.M., Odio, M. and Solano, J.M., The effects of predictable and unpredictable schedules of physical restraint upon rats, Life Sci., 28 (1981) 1897-1902. 35 Rainbow, T.C., Davis, P.G. and McEwen, B.S., Anisomycin inhibits the activation of sexual behavior by estradiol and progesterone, Brain Research, 194 (1980) 548-555. 36 Rivier, C. and Vale, W., Modulation of stress-induced ACTH release by corticotropin-releasing factor, catecholamines and vasopressin, Nature (Lond.), 305 (1983) 325-327. 37 Rivier, J., Rivier, C. and Vale, W., Synthetic competitive antagonists of corticotropin-releasing factor; effect on ACTH secretion in the rat, Science, 224 (1984) 889-891. 38 Rivier, J., Spiess, J. and Vale, W., Characterization of rat hypothalamic corticotropin-releasing factor, Proc. Natl. Acad. Sci. U.S.A., 80 (1983) 4851-4855. 39 Seggie, J.A. and Brown, G.M., Stress response patterns of plasma corticosterone, prolactin, and growth hormone in the rat, following handling or exposure to novel environment, Can. J. PhysioL Pharmacol., 53 (1975) 629-637. 40 Selye, H., Thymus and adrenals in the response of the organism to injuries and intoxications, Br. J. Exp. Pathol., 17 (1936) 234-248. 41 Selye, H., A syndrome produced by diverse nocuous agents, Nature (Lond.), 138 (1936) 32. 42 Skofitsch, G. and Jacobowitz, D., Distribution of corticotropin-releasing factor-like immunoreactivity in the rat brain by immunohistochemistry and radioimmunoassay, Peptides, 6 (1985) 319-336. 43 Suemaru, S., Hashimoto, K. and Ota, Z., Brain corticotropin-releasing factor (CRF) and catecholamine responses in acutely stressed rats, Endocrinol. Jpn., 32 (1985) 709-718. 44 Tilders, F.J.H. and Berkenbosch, F., CRF and catecholamines; their place in the central and peripheral regulation of the stress response, Acta Endocrinol., 276 (Suppl.) (1986) 63-75. 45 Torrellas, A., Guaza, C., Borrell, J. and Borrell, S., Adrenal hormones and brain catecholamines responses to morning and afternoon immobilization stress in rats, Physiol. Behav., 26 (1981) 129-133. 46 Vale, W., Spiess, J., Rivier, C. and Rivier, J., Characterization of a 41-residue ovine hypothalamic peptide that stimulates secretion of corticotropin and fl-endorphin, Science, 213 (1981) 1394-1397. 47 Yasuda, N. and Greer, M.A., Rat hypothalamic corticotropin-releasing factor (CRF) content remains constant despite marked acute or chronic changes in ACTH secretion, Neuroendocrinology, 22 (1976) 48-56.
230
Elsevier BRE 13948
Single or repeated mild stress increases synthesis and release of hypothalamic corticotropin-releasing factor Daniel A. Haas 2 and Susan R. George 1'2 Departments of 1Medicineand 2pharmacology, Universityof Toronto, Toronto, Ont. (Canada) (Accepted 19 April 1988)
Key words: Corticotropin-releasing factor; Immunoreactivity; Adrenocorticotropic hormone; Hypothalamus; Median eminence; Anisomycin; Stress
The effect of a specific mild stress on the levels of corticotropin-releasing factor immunoreactivity (CRF-ir) in the hypothalamus of adult male rats was determined using a radioimmunoassay specific for rat CRF. A single 5 min restraint significantlyincreased CRF-ir in the median eminence 24 h later compared to appropriate controls (P < 0.025), with no change detected earlier. Plasma ACTH, an indirect index of CRF release, was significantly elevated within 15 min (P < 0.025). Repetition of a mild stress daily for 9 days (P < 0.01), or a single episode of handling (P < 0.05), both resulted in significantlyincreased CRF-ir in the whole hypothalamus 24 h later. Blockade of axonal transport by intracisternal colchicine decreased CRF-ir in the median eminence 24 h later (P < 0.005). Inhibition of protein synthesis by anisomycin during a single 5 minute restraint resulted in significantly decreased CRF-ir in the median eminence 24 h later compared to vehicle-injected stressed rats (P < 0.005) or to anisomycin-injected unstressed controls (P < 0.025). These data show that mild stress increased net hypothalamic CRF content as a result of the balance between augmented synthesis and augmented release.
INTRODUCTION One of the prime mediators of the stress response 41 is corticotropin-releasing factor (CRF) 23'29 through its control of the hypothalamic-pituitary-adrenal (HPA) axis 36'37"46. C R F administration has also been shown to elicit other responses characteristic of stress, such as increased cardiac output and the levels of plasma catecholamines25, decreased gastrointestinal activity25, increased pituitary cyclic A M P 23, and increased general arousal 6. We were interested in investigating how stress affected C R F in the hypothalamus, where it is primarily localized in brain 27. Although the H P A axis in the rat is known to be sensitive to mild stresses such as handling39, their effect o n C R F immunoreactivity (CRF-ir) has not been reported. Studies examining the effect of relatively severe stresses 1s'43 have provided conflicting results re-
garding content change in CRF-ir or C R F bioactivity. One potential concern about studying CRF-ir following severe stressors is that they may involve parameters such as pain that could activate other neurotransmitter systems 26 and therefore a clear relation between stress and C R F may not be assumed. Selye 4° first showed that restraining the rat leads to a stress syndrome, and restraint has been widely used as a model of stress in biomedical research 31. It has been shown that as little as 2 min of restraint can elevate plasma adrenocorticotropic h o r m o n e ( A C T H ) concentrations12, a marker commonly used to study acute stress, and reflect endogenous C R F secretion. We therefore elected to study the effect of a specific but mild stress on hypothalamic CRF-ir by using the model of a 5 min restraint and in the present study report its effects on CRF-ir using a radioimmunoassay specific for rat CRF. We have examined the el-
Correspondence: S.R. George, Department of Pharmacology, University of Toronto, Medical Sciences Building, Room 4358, 1 King's College Circle, Toronto, Ont., M5S 1A8 Canada. 0006-8993/88/$03.50© 1988 Elsevier Science Publishers B.V. (Biomedical Division)
231 fects of both single and repeated stresses, and assessed the relative contribution of synthesis and release in the overall CRF changes induced by stress. MATERIALS AND METHODS
Animals Adult male Sprague-Dawley rats (Charles River, Canada), 220-300 g, were housed singly in environmental rooms under constant conditions of temperature, humidity, and maintained under a 12 h lightdark schedule with lights on at 08.00 and off at 20.00 h, with free access to food and water. It has been shown that rats housed singly show less individual variation in plasma corticosterone levels than those housed in groups 3. Sacrifice was carried out in an adjacent room by decapitation at approximately the same time on experimental days and alternated among the groups to diminish any diurnal effects. We have determined that between 09.00 and 14.00 h, during which all the sacrifice took place, there was no significant diurnal variation of hypothalamic CRF-ir (unpublished). Brains were quickly dissected on a chilled glass plate as previously described 15 and the tissues immediately frozen and stored at -70 °C until extraction. Trunk blood was collected in polypropylene tubes containing E D T A , spun at 2000 g for 15 min, and the plasma removed and stored at -20 °C until assay.
Stress treatments Stress treatments were alternated among groups to diminish any potential diurnal effect on CRF or A C T H 22,45.
Effect of single restraint. Rats were randomly assigned into 4 groups and handled daily for 12 days prior to the experiment. They were restrained for 5 min in a semi-cylindrical plexiglas restraint cage, 70 x 160 x 42 mm, immediately returned to their home cage and sacrificed either 15 min (n = 8), 2 h (n = 7) or 24 h (n = 7) afterwards. The control group (n = 7) was handled identically but not restrained. Median eminence was dissected separately from the rest of hypothalamus. Effect of repeated restraint. Rats were randomly assigned into 3 groups and handled every other day for one week prior to the experiment. The stressed group (n = 13) was restrained for 5 min in the re-
straint cage at approximately the same time daily for 9 consecutive days. As rats can habituate to the same stress 21, the environment in which subsequent restraints were performed was changed daily. Beginning on the second day, a second novel stress was added, in the following order: Day 2, s.c. injection of 250/A of saline; Day 3, exposure to vibration for 30 s using a tuning fork placed on the cranium; Day 4, light from a 100 W bulb placed 6 inches overhead; Day 5 the restraint cage was turned over during the 3rd minute of restraint. Beginning on the 6th day, two of these additional stresses were added to the restraint, with the stresses altered randomly, daily. A subgroup (n = 4) was stressed for the 9 days as described above then allowed 5 days of recovery by being handled daily with no restraint. The control group (n = 11) consisted of rats handled daily for the 9 days, with a subgroup (n = 3) handled for the 14 day duration of the study. All rats were sacrificed 25 h after the last stress or handling, and the whole hypothalamus dissected. Handling stress. One group of rats (n = 4) was not handled at all for the period described above until the 9th day, when they were handled and weighed. Twenty-four h later they were sacrificed and whole hypothalamus dissected.
Effect of protein synthesis inhibition on single restraint. Rats were randomly assigned into 4 groups and handled daily for one week prior to the experiment. Rats were either given one 5 min restraint stress 30 min following a subcutaneous injection of vehicle (n = 8) or anisomycin (50 mg/kg, n = 6), or were not handled following an injection of vehicle (n = 7) or anisomycin (n = 7). Rats were sacrificed 24 h later and median eminence was dissected separately from remaining hypothalamus. Anisomycin (Sigma, St. Louis, U.S.A.) was dissolved freshly in saline with 2% 1 N HC1.
Effect of axonal flow blockade One hundred /zg colchicine (Sigma, St. Louis, U.S.A.) or vehicle (saline) was injected intracisternally in a volume of 25/tl under halothane anesthesia (5% for induction and 1.5% for maintenance in 95% 0 2 - 5 % CO2). Rats were then sacrificed after 4 h (n = 5 for vehicle, n = 3 for colchicine), 17 h (n = 4 for vehicle, n = 5 for colchicine) or 24 h (n = 4 for vehicle, n = 6 for colchicine).
232 RESULTS
Tissue extraction Hypothalamus (30-40 mg) and median eminence were extracted as described previously 17. Protein content of median eminence was d e t e r m i n e d by the method of B r a d f o r d 5.
CRF assay The lyophilized samples were assayed in triplicate using a double antibody r a d i o i m m u n o a s s a y specific for rat C R F , d e v e l o p e d for our use and described previously 17. The lower limit of assay detection was 4 pg p e r tube. The intra- and interassay coefficients of variation were 4.8% and 9.3% respectively. C R F - i r from each experiment was d e t e r m i n e d within the same assay.
A C TH assay Plasma A C T H concentrations were d e t e r m i n e d as described previously 17, using the r a d i o i m m u n o a s s a y m e t h o d of Nicholson et al. 3°.
Data analysis Statistical analyses utilized analysis of variance ( A N O V A ) and the Student's t-test, with P values of less than 0.05 judged to be significant. All data are p r e s e n t e d as the m e a n _+ S . E . M . of individual values of rats from each group. C R F - i r is expressed as either pg p e r mg wet weight for hypothalamus, or for median eminence as total pg or pg p e r ¢tg of protein.
The effect of a single 5 min restraint on C R F - i r in the median eminence and remaining h y p o t h a l a m u s is shown in Fig. 1. A s c o m p a r e d to unstressed controls a significant increase in median eminence C R F - i r w a s d o c u m e n t e d 24 h post-stress (P < 0.025), with no change detected after 15 min or 2 h. No change was noted in the remaining h y p o t h a l a m u s at any of the time points assessed. These differences were maintained whether C R F was expressed as C R F - i r per unit protein as shown in Fig. 1, o r as total C R F - i r per tissue. As shown in Fig. 2, plasma A C T H levels were found to be significantly increased 15 min post-stress (P < 0.025). These levels decreased significantly c o m p a r e d to baseline by 2 h post-stress ( P < 0.025), and r e m a i n e d near baseline levels 24 h later. The 5 min restraint was r e p e a t e d daily in a novel environment for 9 days and found to increase C R F - i r in total hypothalamus as c o m p a r e d to unstressed controis handled daily for the same time p e r i o d ( P < 0.01), as shown in Fig. 3. A subgroup of stressed rats was allowed a 5 day recovery period, and this resuited in no further change in CRF-ir. The stressed group had C R F - i r of 112.6 ___7.2 pg/mg as c o m p a r e d to 111.8 _+ 13.6 pg/mg for the group allowed recovery. The C R F - i r in the control rats also did not differ whether they were handled for 9 days (81.8 + 7.6
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Fig. 1. Effect of a single 5 min restraint on CRF-ir in median eminence (expressed as pg//~g protein) and remaining hypothalamus (expressed as pg/mg wet wt.). Significant difference from unstressed controls (0 h post-stress) is denoted by * for P < 0.025. n = 7 or 8 per group.
Hours Post-Stress
Fig. 2. Effect of a single 5 min restraint on plasma ACTH. Significant difference from unstressed controls (0 hours poststress) is denoted by * for P < 0.025. n = 7 or 8 per group.
233 Whole Hypothalamus 120
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Fig. 3. Effect on hypothalamic CRE-ir of single episode of handling and weighing (n = 4) and repeated 5 min restraints daily for 9 days (n = 13) as compared to unstressed rats which were handled daily for 9 days (n = 11). Significant difference from control is denoted by * for P < 0.05 and ** for P < 0.01.
pg/mg) or 14 days (87.9 + 18.5 pg/mg). There were no statistically significant differences in ACTH levels in these rats (data not shown). The repeatedly stressed group was found to have a significantly lower gain in body weight (a mean increase 11.2 + 0.8%) compared to their handled controls (a mean increase of 14.1 + 0.6%, P < 0.025). Fig. 3 also shows that the effect of a single episode of handling and weighing was enough to cause a significant increase in hypothalamic CRF-ir 24 h later, compared to controls which were handled daily for 9 days (P < 0.05). The increase in plasma A C T H shown in Fig. 2 was not accompanied with a detectable decrease in CRFir (Fig. 1). This may have been due to rapid repletion of CRF transported from the site of synthesis, the paraventricular nucleus s, to the site of release, the median eminence. We were therefore interested in assessing the time interval required to detect a change in CRF-ir following inhibition of peptide transport. This was achieved by administering the axonal transport inhibitor colchicine intracisternally and then determining CRF-ir. As shown in Fig. 4, a progressive decrease in median eminence CRF-ir was apparent and became statistically significant 24 h after colchicine (P < 0.005). No change in the remaining hypothalamus was observed. The increase documented in CRF-ir content in the
Fig. 4. Effect of axonal blockade by colchicine on total CRF-ir in median eminence. Significant difference from vehicletreated controls is denoted by * for P < 0.005. n = 3 - 6 per group.
median eminence 24 h post-stress could be due to either an increase in synthesis or an inhibition of release. In order to investigate the mechanism involved, we repeated this restraint stress under conditions of protein synthesis inhibition induced by the administration of the protein synthesis inhibitor anisomycin, and then sacrificed the rats 24 h later. As shown in Fig. 5, anisomycin treatment resulted in a 13% decrease in CRF-ir in the median eminence in unstressed rats (not significant) and a 45% decrease in stressed rats (P < 0.005) compared to their respec20 ¸ Media
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Fig. 5. Effect of anisomycin-induced protein synthesis inhibition (Aniso) during a single 5 min restraint on CRF-ir in median eminence (expressed as pg/~tg protein) and remaining hypothalamus (expressed as pg/mg wet wt.). Anisomycin was administered at a dose of 50 mg/kg s.c., 30 min prior to restraint, and CRF-ir determined 24 h later. Significant difference from vehicle-treated controls (Saline) is denoted by * for P < 0.01, and significant difference from the vehicle-treated stressed group is denoted by ** for P < 0.005. n = 6 - 8 per group.
234 tive controls. Anisomycin-treated stressed rats had significantly reduced CRF-ir in the median eminence compared to vehicle-treated stressed rats (P < 0.005) which would reflect the effect of protein synthesis inhibition. These values were also lower when compared to anisomycin-treated unstressed rats (P < 0.025), which would reflect the effect of stress. Analysis utilizing one and two-way A N O V A demonstrated a significant difference among the groups (F3,24 = 6.58, P < 0.005) with the main effect of anisomycin treatment being significant (F1,22 = 10.92, P < 0.005). These differences are maintained whether C R F is expressed as CRF-ir//~g protein as shown in Fig. 5, or as total CRF-ir per tissue. Anisomycin treatment showed no effect on the remaining hypothalamus whereas the vehicle-treated stressed group did show a significant increase in CRF-ir compared to vehicle-treated unstressed rats (P < 0.01). Plasma A C T H levels showed no statistically significant differences between any of the groups (data not shown). DISCUSSION These data show that mild stresses consisting of a single episode of handling, or a 5 min restraint, whether applied once or repeatedly, altered hypothalamic CRF synthesis and release. The advantages of this investigation compared to previous reports are that a discrete, reproducible mild stress was used thereby diminishing non-stress effects and CRF-ir was directly measured to assess effect on synthesis, transport and release. Previous reports of the effects of stress on the H P A axis have shown alteration of rat plasma corticosterone and A C T H levels by stresses of varying intensity ranging from severe, such as ether-laparotomy TMor tibial fracture 19 to mild, such as handling and weighing 3. C R F bioactivity has variably been shown to increase TM, demonstrate a biphasic response 19 or remain unchanged 47. Recently, more direct assessments measuring immunoreactivity have examined the effects of severe stressors such as ether-laparotomy 43, or 3 h of restraint during cold exposure 1° on hypothalamic CRF-ir. In contrast, shorter periods of restraint have variably been reported to have no effect on CRF-ir in the median eminence 44 or to decrease and then increase hypothalamic CRF-ir 28. The
previous studies 1°'43 reporting stress-induced effects on CRF-ir have utilized stresses more severe than those used in our studies. The drawback to severe and multiple forms of stress is the potential that the pain of laparotomy, the pharmacological effects of ether, or the alteration of body temperature may activate other neurotransmitter or neuropeptide systems that secondarily influence C R F levels. Therefore a direct relationship between a unitary form of stress and C R F dynamics cannot be assumed. Also, it will not be possible to eventually elucidate the precise neural mechanisms involved in mediating C R F release in various types of stress. The older studies served to establish that the c o m m o n mediator of severe stress was C R F release stimulating A C T H secretion. Since we do not believe that all stresses release C R F through the same mechanisms, we wished to employ a relatively pure unitary stress. One other drawback to the previous studies on CRF-ir is that two of these l°'28 utilized antisera to ovine CRF in their radioimmunoassay for rat CRF. These two peptides differ by 7 amino acids 38'46 and it has been shown that the CRF-ir measured in rat brain can vary greatly depending on which antiserum is used 42. Our study has the advantage of using an antiserum raised against rat C R F and should therefore be a more accurate reflection of C R F levels in rat hypothalamus. It is possible that discrepancies among the studies could be due to different antisera as well as different stress models used. The single 5 min restraint caused a significant increase in plasma A C T H within 15 min. This confirms previous studies on the effects of acute restraint on rat A C T H 12'16 and is evidence of this restraint model being an adequate and effective stressful event. A C T H levels act as an indirect index of C R F release and our data imply that C R F was released from the median eminence within this time period. This was not detected as a change in CRF-ir content either in whole hypothalamus or median eminence, indicating either that the release was so small as to be undetectable as a change in content, or that the turnover was so rapid that the released C R F was repleted as fast as it was depleted. The latter explanation is less likely given CRF-ir could not be detected to increase until 24 h later, implying a slow rate of turnover. An indication of the time required to detect a change in turnover of C R F was obtained by deter-
235 mining the time course of CRF-ir change following blockade of axonal transport induced by the administration of intracisternal colchicine. The results showed that a significant change could not be documented until 24 h later. This is consistent with previous studies that utilized immunocytochemical visualization to show a colchicine-induced decrease in CRF-ir at 48 h ~. it was noted that A C T H levels declined significantly below baseline 2 h post-stress, a possible consequence of increased glucocorticoid feedback induced by the initial ACTH peak. By 24 h post-stress ACTH levels were not different from baseline in any of the stressed groups assessed in this study, suggesting a normalization of plasma ACTH after this interval. Our studies demonstrated that any of the mild stresses employed increased CRF-ir in the hypothalamus after 24 h. The exact time of significant CRF content change following the single restraint could have occurred any time between 2 and 24 h post-stress. This change could be due to either increased CRF synthesis with subsequent transport to the median eminence, or inhibition of release, such as that mediated by increased glucocorticoid secretion, although the latter possibility is less likely. In order to investigate the mechanism involved we repeated the acute restraint stress while blocking protein synthesis with the general protein synthesis inhibitor anisomycin. We then inferred changes of CRF synthesis on the basis of the measurement of CRF-ir, coupled with the measurements of plasma ACTH. Anisomycin was selected as it has been shown to effectively inhibit cerebral protein synthesis with very little systemic toxicity4A3, especially compared to other inhibitors such as cycloheximide. This factor is important in order to minimize non-specific stress effects on CRF. We chose the regimen of 50 mg/kg s.c., 30 min prior to restraint, which would inhibit protein synthesis in the hypothalamus by 80-90% as based on previous reports 32'35. Anisomycin administration resulted in a 13% decrease in median eminence CRF-ir in the unstressed rats and a 45% decrease in restrained rats (Fig. 5). The rate of protein synthesis has been shown to return to baseline levels within 6 hours following injection of anisomycin 35. Our studies determined CRF-ir 24 h after its administration, thereby allowing at least partial re-
covery of normal synthesis. The depletion documented in both groups of rats was apparent even after this interval, suggesting there may have been a greater reduction in CRF-ir within the first few hours following administration. The 13% decrease documented in unstressed rats would likely reflect the loss of CRF due to normal physiologic release. Anisomycin treatment would have prevented the normal CRF turnover by blocking synthesis, and possibly transport as well, resulting in the observed decrease. The greater depletion of CRF-ir documented in anisomycin-treated stressed rats compared to vehicle-treated stressed controls implies that restraint induced an increased release in CRF and that protein synthesis was involved even in the early maintenance of CRF levels post-stress. This was indirectly confirmed by our ACTH data which showed a significant increase in plasma ACTH within 15 min of restraint. As CRF is the primary regulator of ACTH 46, increased plasma levels imply an increase in CRF release from the median eminence to allow stimulation of release of ACTH from the anterior pituitary. Therefore it appears that restraint stress induced an increase in CRF release, and anisomycin treatment prevented the expected increase in CRF-ir as found with either the single restraint, repeated restraint, or handling. As total content increased 24 h following these stresses, it suggests that this increased synthesis overcompensated the initial reduction in CRF. Combining the restraint stress with the intraperitoneal vehicle injection was sufficient to induce an increase in CRF-ir in remaining hypothalamus compared to non-restrained controls (Fig. 5). This increase was not apparent in remaining hypothalamus when comparing the two anisomycin-treated groups (Fig. 5) or when restraint was executed without the intraperitoneal injection (Fig. 1). These data suggest that this combination of stresses was more severe than restraint alone and subsequently altered CRF dynamics. The result was an increase in content detected in the remaining hypothalamus as opposed to the median eminence, where an increase was observed with the single restraint alone. This increase was apparently due to increased synthesis of CRF as evidenced by the lack of effect when comparing the anisomycintreated groups. The increase in CRF-ir documented following the single episode of handling has not been reported be-
236 fore. The activation of C R F in as simple a stress as being handled and weighed could have been speculated as handling of rats has been shown to increase plasma corticosterone 3'39, and epinephrine 24, both of which are under the indirect regulation of C R F TM. Our data would imply that hypothalamic C R F could be involved in the mediation of these responses. O u r results also emphasize the importance of matching and handling controls identically when studying the effect of various treatments on C R F - i r since simple handling and weighing of the animal can alter C R F ir. The same degree of increase in C R F - i r was found with the r e p e a t e d restraint stress model. That these r e p e a t e d stresses were effective is confirmed by the inhibition of body weight gain in the stressed group, a finding consistent with stress 2A1,33. A n i m a l s adapt to a r e p e a t e d stress 9,2° but the habituation is specific to the one stressor used 21. Stress that is unpredictable has been shown to have a m o r e p r o n o u n c e d effect on corticosterone levels 34, and is therefore considered more effective. We avoided habituation to the 5 min restraint by changing the environment in which the stress was applied daily, thereby diminishing its predictability. It is of interest to note that this r e p e a t e d stress administered for only 5 min a day resulted in an increase in hypothalamic C R F - i r similar to that found with the single restraint. This 9 day exposure to restraint was enough to induce increased C R F levels which persisted after the rats were allowed a 5 day REFERENCES 1 Alonso, G., Szafarczyk, A., Balmefrezol, M. and Assenmacher, I., Immunocytochemical evidence for stimulatory control by the ventral noradrenergic bundle of parvocellular neurons of the paraventricular nucleus secreting corticotropin releasing hormone and vasopressin in rats, Brain Research, 397 (1986) 297-307. 2 Armario, A., Restrepo, C., Castellanos, J.M. and Balasch, J., Dissociation between adrenocorticotropin and corticosterone responses to restraint after previous chronic exposure to stress, Life Sci., 36 (1985) 2085-2092. 3 Barrett, A.M. and Stockham, M.A., The effect of housing conditions and simple experimental procedures upon the corticosterone level in the plasma of rats, J. Endocrinol., 26 (1963) 97-105. 4 Bennett, E.L., Orme, A. and Herbert, M., Cerebral protein synthesis inhibition and amnesia produced by scopolamine, cycloheximide, streptovitacin A, anisomycin and emetine in rat, Fed. Proc., 31 (1972) 838. 5 Bradford, M.M., A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding, Anal. Biochem,. 72 (1976)
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