- Email: [email protected]
Morphological changes in the hippocampal CA3 region induced by non-invasive glucocorticoid administration: a paradox
Brain Research 809 Ž1998. 314–318
Short communication
Morphological changes in the hippocampal CA3 region induced by non-invasive glucocorticoid administration: a paradox Ana Marıa ´ Magarinos ˜ b
b, )
, Miles Orchinik a , Bruce S. McEwen
b
a Department of Biology, Arizona State UniÕersity, Tempe, AZ, USA The Rockefeller UniÕersity, 1230 York AÕenue, Box 165, New York, NY 10021, USA
Accepted 18 August 1998
Abstract Repeated stress induces atrophy, or remodeling, of apical dendrites in hippocampal CA3 pyramidal neurons. In rats, the stress effect is blocked by adrenal steroid synthesis inhibitors, and mimicked by daily injection of corticosterone. We report that non-invasive administration of corticosterone in the drinking water Ž400 mgrml. also produced atrophy of apical dendrites in CA3. Unexpectedly, the combination of daily stress and oral corticosterone negated the effects of either treatment alone, and no changes in the apical dendritic length or branching pattern of CA3 pyramidal neurons were observed compared to control unstressed rats. q 1998 Published by Elsevier Science B.V. All rights reserved. Keywords: Corticosterone; Dendritic atrophy; Golgi impregnation; Stress
In rats and tree shrews, repeated stress causes a remodeling of apical dendrites of CA3 pyramidal neurons in the hippocampus w13,23x. We have found that adrenal steroids and excitatory amino acids mediate this process, which thus constitutes a reversible remodeling of the apical dendritic tree w14x. This dendritic remodeling consists of an atrophy and reduction of dendritic branching and length of the apical, but not of the basal, dendrites, and is produced by repeated restraint stress in rats and repeated psychosocial stress in tree shrews w13,23x. The atrophy can be prevented by inhibiting adrenal steroid synthesis and by blocking NMDA receptors w14x. Also, daily treatment of stressed rats with the antiepileptic drug phenytoin or the atypical tricyclic antidepressant tianeptine, which enhances serotonin reuptake, is effective in blocking the stress-induced atrophy in rats w22,24x. We have recently demonstrated a stress-induced reorganization of synaptic vesicles within mossy fiber terminals that represents a presynaptic morphological counterpart of the postsynaptic atrophy of apical dendrites of CA3 pyramidal neurons w12x. The stress-induced postsynaptic atrophy, which is reversible over 7–10 days after the end of stress ŽConrad, Magarinos, ˜ LeDoux and McEwen, unpublished., is associated with
)
Corresponding author. Fax: [email protected]
q 1-212-327-8634;
E-mail:
impairments of hippocampus-dependent spatial learning tasks w4,11x. Mechanistically, this animal model of dendritic atrophy may be related to atrophy of the human hippocampus seen in Cushing’s syndrome and mild cognitive impairment in aging w5,9,21x. Daily injections of corticosterone produce dendritic atrophy and, similar to stress-induced remodeling, this effect is blocked by either phenytoin or tianeptine treatment w22,24,27x. However, the doses of glucocorticoid injected in those studies were supraphysiological and the injection regimen itself is probably stressful to the animal. We have previously reported that certain effects of corticosterone on hippocampal cyclic AMP formation are produced by injection but not by passive administration of corticosterone in the drinking water w8x. We therefore administered physiological levels of corticosterone in the drinking water and compared the effects of this treatment upon dendritic atrophy with that of 21 days of 6 hrday restraint stress and with the combination of corticosterone in the drinking water and daily stress. Male Sprague–Dawley rats ŽCD strain, Charles River, Kingston, NY. weighing 290–300 g at the beginning of the experiments were housed in groups of three per hanging metallic cage with ad libitum access to food and tap water. Animals were maintained in a temperature and light controlled environment Ž12r12 h lightrdark cycle, lights on from 7.00 to 19.00 h.. One week after delivery the rats,
0006-8993r98r$ - see front matter q 1998 Published by Elsevier Science B.V. All rights reserved. PII: S 0 0 0 6 - 8 9 9 3 Ž 9 8 . 0 0 8 8 2 - 8
A.M. Magarinos ˜ et al.r Brain Research 809 (1998) 314–318
Fig. 1. Effect of corticosterone ŽCort. in the drinking water Ž400 mgrml., daily restraint stress Ž R . and the combined treatments Ž RqCort. for 21 days on the number of dendritic branch points of CA3 apical dendrites. The vehicle ŽVeh. consisted of a 2.4% ethanol in the drinking water. ) p- 0.05 compared with controls. One-way ANOVA, Tukey HSD post-hoc analysis.
already adapted to daily handling, were weighed and randomly assigned to experimental groups: Ž1. Unstressed control animals Ž n s 6. remained in their cages. Ž2. Chronic restraint stress animals Ž n s 6. received restraint stress for 21 d. Restraint stress was applied for 6 hrday Ž10:00 h to 16:00 h. in wire mesh restrainers, secured at the head and tail ends with clips. During the restraint sessions, the rats were kept in their home cages. Ž3. Chronic corticosterone treated animals Ž n s 6. received 400 mg corticosteronerml of tap water over 21 days. Corticosterone ŽAldrich Chem., Milwaukee, WI. was dissolved in ethanol, and all other groups of rats received the same percentage of ethanol in their drinking water Ž2.4%.. Ž4. Chronic restraint stress plus corticosterone animals Ž n s 6. received the combined treatments. Blood samples were collected twice a day, at 09:00 h and just prior to lights off at 19:00 h on days 1, 7, 14 and 21 of the stress paradigm. Samples were obtained from the tail vein and less than 30 s elapsed from the moment the animals were handled until the blood was collected. Blood samples were centrifuged and the plasma immediately separated and frozen until use in corticosterone radioimmunoassay as previously described w14x. At the end of the treatment period, the rats were deeply anesthetized with Metofane ŽPitman-Moore, Mundelein, IL. and transcardially perfused with 150 ml 4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.4. Adrenals, thymus glands and spleens were removed, cleaned and weighed. Brains were postfixed in the perfusate overnight. Sections, 100 mm thick, were cut with a Vibratome into a bath of 3% potassium dichromate in distilled water. Sections were then processed according to a modified version of the ‘single’ section Golgi impregnation procedure as previously described w7,14x.
315
Slides containing brain sections were coded prior to quantitative analysis; the code was not broken until the analysis was complete. In order to be selected for analysis, Golgi impregnated neurons had to posses the following characteristics: Ž1. Location in the CA3c subregion of the dorsal hippocampus; Ž2. Dark and consistent impregnation throughout the extent of all of the dendrites; Ž3. relative isolation from neighboring impregnated cells which could interfere with analysis; and Ž4. a cell body in the middle third of the tissue section in order to avoid analysis of impregnated neurons which extended largely into other sections. For each brain, 6–8 pyramidal cells from CA3c were selected. Each selected neuron was traced at 400 = magnification using a light microscope with a camera lucida drawing tube attachment. From these drawings the number of dendritic branch Žbifurcation. points within a 100 mm thick section of each dendritic tree was determined for each selected neuron. In addition, the length of the dendrites present in a 100 mm thick section was determined for each dendritic tree using a Zeiss interactive digitizing analysis system. Means were determined for each variable for each brain and the resulting values were subjected to a One-way analysis of variance ŽANOVA. with Tukey HSD post-hoc comparisons. Organ weight data were analyzed by One-way ANOVA. Differences in adrenal weights between treatments were further evaluated using Tukey HSD post hoc comparisons. Corticosterone levels at 09:00 and 19:00 on days 7, 14 and 21 were separately analyzed by two-factor ANOVA and Tukey HSD post-hoc comparisons when appropriate. As shown in Fig. 1, chronic restraint stress or administration of corticosterone in the drinking water produced a decrease in the number of dendritic branch points in the
Fig. 2. Effect of corticosterone ŽCort. in the drinking water Ž400 mgrml., daily restraint stress Ž R . and the combined treatments Ž RqCort. for 21 days on the dendritic total length of CA3 apical dendrites. No significant differences were observed among experimental groups ŽOne-way ANOVA: F s 2.10, ps 0.135..
316
A.M. Magarinos ˜ et al.r Brain Research 809 (1998) 314–318
Fig. 3. Effect of corticosterone ŽCort. in the drinking water Ž400 mgrml., daily restraint stress Ž R . and the combined treatments Ž R q Cort. for 21 days on adrenal, thymic and splenic weights. )) p - 0.0001 compared with controls. One-way ANOVA, Tukey post-hoc analysis.
apical dendrites of CA3 pyramidal neurons, relative to unstressed animals receiving vehicle. The restraint stress and corticosterone treatments resulted in a non-significant decrease in total apical dendritic length ŽFig. 2.. The combination of restraint stress and corticosterone administration negated the effects of each treatment alone ŽFig. 1.. Consistent with previous reports, basal dendritic length and branching were not affected by stress or corticosterone. As expected, corticosterone treatment produced a significant reduction of adrenal, spleen and thymus weight, corrected for body weight, but repeated stress produced a trend for increased adrenal weight and decreased thymus and spleen weights ŽFig. 3.. Body weight was also reduced by corticosterone treatment Ždata not shown.. Morning Ž09:00 h. and evening Ž19:00 h. levels of circulating corticosterone, sampled at days 7, 14 and 21 of the study, are given in Table 1. Administration of 400 mg corticosteronerml of drinking water produced circulating corticosterone levels in unstressed animals similar to those typically seen at the daily evening peak in untreated animals. Throughout the study, there were no significant differences in corticosterone levels between any groups obtained at 19:00 h. However, animals receiving corticosterone had significantly elevated steroid levels in the morning, relative to animals receiving vehicle Žat day 7: F s 23.00, p - 0.0001; at day 14: F s 39.89, p - 0.00001; at day 21: F s 16.65, p s 0.0003.. Further, since both groups receiving vehicle Žunstressed controls and chronic restraint stressed animals. showed typical low basal levels of corticosterone at 09:00 throughout the experimental period, the ethanol vehicle itself did not appear to activate the hypothalamic–pituitary–adrenal ŽHPA. axis. The results of this experiment confirm and extend our previous findings that repeated stress causes reorganization of the apical dendritic tree of CA3 pyramidal neurons and that chronic exposure to corticosterone mimics this effect w23,27x. The fact that the drinking water route of corticosterone administration works as well as injection of corticosterone to cause dendritic atrophy indicates that dendritic morphology is sensitive to increased levels of
corticosterone, even when delivered in a non-invasive manner and in spite of the different pharmacodynamics of corticosteroid clearance after subcutaneous administration of supraphysiological bolus in sesame oil, compared with the more continuous oral route. The drinking water route of administration of corticosterone potently reduced adrenal, spleen and thymus weight as well as body weight. The most surprising aspect of our present study is that the combination of repeated stress and corticosterone negated the effects of either treatment alone. The results seem paradoxical and we currently have no definitive explanation. One difference between the stressed group and group receiving stress and corticosterone is that the HPA axis was suppressed in the latter group, as indicated by the marked atrophy of adrenal glands ŽFig. 3.. It may be that CRF andror ACTH participate in the process of Table 1 Effect of corticosterone ŽCort. in the drinking water Ž400 mgrml., daily restraint stress Ž R . and the combined treatments Ž RqCort. for 21 days on AM and PM plasma levels of corticosterone Time of blood collection 09:00 h
19:00 h
C qVeh Day 7 Day 14 Day 21
2.28"0.39 2.58"0.43 1.30"1.01
18.67"2.37 14.17"3.52 26.14"3.66
C qCort Day 7 Day 14 Day 21
21.51"6.72 28.88"5.32 20.02"7.78
14.98"3.51 29.17"5.02 21.50"5.40
RqVeh Day 7 Day 14 Day 21
1.87"0.44 1.14"0.13 0.85"0.29
16.03"3.04 21.09"2.72 19.11"2.49
RqCort Day 7 Day 14 Day 21
33.97"8.31 39.87"8.80 16.94"3.33
13.69"3.92 26.49"7.03 30.00"4.47
A.M. Magarinos ˜ et al.r Brain Research 809 (1998) 314–318
dendritic atrophy, and these actions are canceled by oral corticosterone administration. Alternatively, suppression of the HPA axis by oral corticosterone may blunt stress-induced corticosterone secretion such that peak corticosterone levels during stress may be higher in animals receiving vehicle rather than corticosterone. Suppression of the HPA axis, by itself, cannot account for the difference in dendritic morphology between animals receiving the steroid or vehicle, because the HPA axis was suppressed in both the stressed and non-stressed groups. Another difference between animals receiving vehicle and those receiving corticosterone in the drinking water is that the daily rhythm of corticosteroid secretion is disrupted in the latter group. Multiple processes are involved in stress-induced atrophy of apical dendrites of CA3 pyramidal neurons ŽRefs. w15,16x for reviews.. Glucocorticoid-mediated dendritic atrophy involves the modulation of a number of hippocampal neurochemical systems w14x, including enhanced NMDA receptor subunit expression and receptor binding w10,26x, stress-induced glutamate release w2,6,19x, 5HT turnover w1x, the induction of presynaptic kainate receptors on mossy fiber terminals w25x, alterations of GABA A receptor subunit expression w20x, and the reduction of inhibitory 5HT1A receptor expression w3,17,18x. Taken together, these actions of adrenal steroids may contribute to a stress-induced enhancement of excitation and a reduction of inhibition in hippocampus. Simultaneous suppression of the HPA axis or the loss of rhythmicity in adrenal activity induced by exposure to oral corticosterone might impair the ability of the CA3 neurons to undergo dendritic remodeling. Future studies will look for the system or systems responsible from among those described above. In conclusion, corticosterone passively administered to rats in the drinking water mimics the effects of repeated restraint stress on dendritic branching in the apical dendrites of CA3 neurons in the rat hippocampus. Paradoxically, combining oral corticosterone with repeated stress negates the effects of both treatments. We suggest that the temporal pattern of exposure to corticosterone may alter neural responses to this steroid. Additionally, differences in the neuroendocrine environment between treatment groups—for example, differences in CRH, glutamate, vasopressin or monoamine systems—may alter the neural responses to corticosterone itself. One result appears to be that combining chronic restraint stress and chronic elevations of corticosterone suppresses some component of the neurochemical system that mediates the dendritic atrophy.
Acknowledgements This work was supported by NIH Grant MH 41256 to BM, Servier ŽFrance. and The Health Foundation ŽNew York..
317
References w1x E. Azmitia, B.S. McEwen, Corticosterone regulation of tryptophan hydroxylase in rat midbrain, Science 166 Ž1969. 1274–1276. w2x D.L. Benson, P.A. Cohen, Activity-independent segregation of excitatory and inhibitory synaptic terminals in cultured hipocampal neurons, J. Neurosci. 16 Ž1996. 6424–6432. w3x D.T. Chalmers, S.P. Kwak, A. Mansour, H. Akil, S.J. Watson, Corticoids regulate brain hippocampal 5-HT1A receptor mRNA expression, J. Neurosci. 13 Ž3. Ž1993. 914–923. w4x C.D. Conrad, L.A.M. Galea, Y. Kuroda, B.S. McEwen, Chronic stress impairs rat spatial memory on the Y-Maze and this effect is blocked by tianeptine pre-treatment, Behav. Neurosci. 110 Ž1996. 1321–1334. w5x A. Convit, M.J. de Leon, C. Tarshish, S. De Santi, W. Tsui, H. Rusinek, A. George, Specific hippocampal volume reductions in individuals at risk for Alzheimer’s disease, Neurobiol. Aging 18 Ž1997. 131–138. w6x L.A. De Bruin, M.C. Schasfoort, A.B. Stefens, J. Korf, Effects of stress and excercise on rat hippocampus and striatum extracellular lactate, Am. J. Physiol. 259 Ž1994. R773–R779. w7x P.L. Gabbott, J. Somogyi, The ‘single’ section Golgi-impregnation procedure: methodological description, J. Neurosci. Methods 11 Ž1984. 221–230. w8x M. Gannon, B.S. McEwen, Calmodulin involvement in stress-and corticosterone-induced down-regulation of cyclic AMP-generating systems in brain, J. Neurochem. 55 Ž1990. 276–284. w9x J. Golomb, M.J. De Leon, A.E. George, A. Kluger, A. Convit, H. Rusinek, S. De Santi, A. Litt, S.H. Foo, S.H. Ferris, Hippocampal atrophy correlates with severe cognitive impairment in elderly patients with suspected normal pressure hydrocephalus, J. Neurol. Neurosurg. Psychiatry 57 Ž1994. 590–593. w10x M.T. Lowy, L. Gault, B.K. Yamamoto, Adrenalectomy attenuates stress-induced elevations in extracellular glutamate concentrations in the hippocampus, J. Neurochem. 61 Ž1993. 1957–1960. w11x V. Luine, M. Villegas, C. Martinez, B.S. McEwen, Repeated stress causes reversible impairments of spatial memory performance, Brain Res. 639 Ž1994. 167–170. w12x A.M. Magarinos, J.M. Garcia Verdugo, B.S. McEwen, Chronic ˜ stress alters synaptic terminal structure in the hippocampus, Proc. Natl. Acad. Sci. U.S.A. 94 Ž25. Ž1997. 14002–14008. w13x A.M. Magarinos, B.S. McEwen, G. Flugge, E. Fuchs, Chronic ˜ ¨ psychosocial stress causes apical dendritic atrophy of hippocampal CA3 pyramidal neurons in subordinate tree shrews, J. Neurosci. 16 Ž1996. 3534–3540. w14x A.M. Magarinos, ˜ B.S. McEwen, Stress-induced atrophy of apical dendrites of hippocampal CA3c neurons: involvement of glucocorticoid secretion and excitatory amino acid receptors, Neuroscience 69 Ž1995. 89–98. w15x B.S. McEwen, D. Albeck, H. Cameron, H.M. Chao, E. Gould, N. Hastings, Y. Kuroda, V. Luine, A.M. Magarinos, ˜ C.R. McKittrick, M. Orchinik, C. Pavlides, P. Vaher, Y. Watanabe, N. Weiland, Stress and the brain: a paradoxical role for adrenal steroids, in: G.D. Litwack ŽEd.., Vitamins and Hormones, Academic Press, 1995, pp. 371–402. w16x B.S. McEwen, Possible mechanisms for atrophy of the human hippocampus, Mol. Psychiatry 2 Ž1997. 255–262. w17x O.C. Meijer, R.V. Van Oosten, E.R. de Kloet, Elevated basal trough levels of corticosterone suppress hippocampal 5-hydroxytryptamine1A receptor expression in adrenally intact rats: implication for the pathogenesis of depression, Neuroscience 80 Ž1997. 419–426. w18x S.D. Mendelson, B.S. McEwen, Autoradiographic analyses of the effects of adrenalectomy and corticosterone on 5-HT1A and 5-HT1B receptors in the dorsal hippocampus and cortex of the rat, Neuroendocrinology 55 Ž1992. 444–450. w19x B. Moghaddam, M.L. Bolinao, B. Stein-Behrens, R. Sapolsky,
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A.M. Magarinos ˜ et al.r Brain Research 809 (1998) 314–318 Glucocorticoids mediate the stress-induced extracellular accumulation of glutamate, Brain Res. 655 Ž1994. 251–254. M. Orchinik, N.G. Weiland, B.S. McEwen, Chronic exposure to stress levels of corticosterone alters GABA A receptor subunit mRNA levels in rat hippocampus, Brain Res. Mol. Brain Res. 34 Ž1995. 29–37. M.N. Starkman, S.S. Gebarski, S. Berent, D.E. Schteingart, Hippocampal formation volume, memory dysfunction, and cortisol levels in patients with Cushing’s syndrome, Biol. Psychiatry 32 Ž1992. 756–765. Y. Watanabe, E. Gould, H.A. Cameron, D.C. Daniels, B.S. McEwen, Phenytoin prevents stress- and corticosterone-induced atrophy of CA3 pyramidal neurons, Hippocampus 2 Ž1992. 431–436. Y. Watanabe, E. Gould, B.S. McEwen, Stress induces atrophy of apical dendrites of hippocampus CA3 pyramidal neurons, Brain Res. 588 Ž1992. 341–344.
w24x Y. Watanabe, E. Gould, D.C. Daniels, H. Cameron, B.S. McEwen, Tianeptine attenuates stress-induced morphological changes in the hippocampus, Eur. J. Pharmacol. 222 Ž1992. 157–162. w25x Y. Watanabe, N.G. Weiland, B.S. McEwen, Effects of adrenal steroid manipulations and repeated restraint stress on dynorphin mRNA levels and excitatory amino acid receptor binding in hippocampus, Brain Res. 680 Ž1995. 217–225. w26x N. Weiland, M. Orchinik, P. Tanapat, Chronic corticosterone treatment induces parallel changes in N-methyl-D-aspartate receptor subunit messenger RNA levels and antagonist binding sites in the hippocampus, Neuroscience 78 Ž1997. 653–662. w27x C.S. Woolley, E. Gould, B.S. McEwen, Exposure to excess glucocorticoids alters dendritic morphology of adult hippocampal pyramidal neurons, Brain Res. 531 Ž1990. 225–231.
Short communication
Morphological changes in the hippocampal CA3 region induced by non-invasive glucocorticoid administration: a paradox Ana Marıa ´ Magarinos ˜ b
b, )
, Miles Orchinik a , Bruce S. McEwen
b
a Department of Biology, Arizona State UniÕersity, Tempe, AZ, USA The Rockefeller UniÕersity, 1230 York AÕenue, Box 165, New York, NY 10021, USA
Accepted 18 August 1998
Abstract Repeated stress induces atrophy, or remodeling, of apical dendrites in hippocampal CA3 pyramidal neurons. In rats, the stress effect is blocked by adrenal steroid synthesis inhibitors, and mimicked by daily injection of corticosterone. We report that non-invasive administration of corticosterone in the drinking water Ž400 mgrml. also produced atrophy of apical dendrites in CA3. Unexpectedly, the combination of daily stress and oral corticosterone negated the effects of either treatment alone, and no changes in the apical dendritic length or branching pattern of CA3 pyramidal neurons were observed compared to control unstressed rats. q 1998 Published by Elsevier Science B.V. All rights reserved. Keywords: Corticosterone; Dendritic atrophy; Golgi impregnation; Stress
In rats and tree shrews, repeated stress causes a remodeling of apical dendrites of CA3 pyramidal neurons in the hippocampus w13,23x. We have found that adrenal steroids and excitatory amino acids mediate this process, which thus constitutes a reversible remodeling of the apical dendritic tree w14x. This dendritic remodeling consists of an atrophy and reduction of dendritic branching and length of the apical, but not of the basal, dendrites, and is produced by repeated restraint stress in rats and repeated psychosocial stress in tree shrews w13,23x. The atrophy can be prevented by inhibiting adrenal steroid synthesis and by blocking NMDA receptors w14x. Also, daily treatment of stressed rats with the antiepileptic drug phenytoin or the atypical tricyclic antidepressant tianeptine, which enhances serotonin reuptake, is effective in blocking the stress-induced atrophy in rats w22,24x. We have recently demonstrated a stress-induced reorganization of synaptic vesicles within mossy fiber terminals that represents a presynaptic morphological counterpart of the postsynaptic atrophy of apical dendrites of CA3 pyramidal neurons w12x. The stress-induced postsynaptic atrophy, which is reversible over 7–10 days after the end of stress ŽConrad, Magarinos, ˜ LeDoux and McEwen, unpublished., is associated with
)
Corresponding author. Fax: [email protected]
q 1-212-327-8634;
E-mail:
impairments of hippocampus-dependent spatial learning tasks w4,11x. Mechanistically, this animal model of dendritic atrophy may be related to atrophy of the human hippocampus seen in Cushing’s syndrome and mild cognitive impairment in aging w5,9,21x. Daily injections of corticosterone produce dendritic atrophy and, similar to stress-induced remodeling, this effect is blocked by either phenytoin or tianeptine treatment w22,24,27x. However, the doses of glucocorticoid injected in those studies were supraphysiological and the injection regimen itself is probably stressful to the animal. We have previously reported that certain effects of corticosterone on hippocampal cyclic AMP formation are produced by injection but not by passive administration of corticosterone in the drinking water w8x. We therefore administered physiological levels of corticosterone in the drinking water and compared the effects of this treatment upon dendritic atrophy with that of 21 days of 6 hrday restraint stress and with the combination of corticosterone in the drinking water and daily stress. Male Sprague–Dawley rats ŽCD strain, Charles River, Kingston, NY. weighing 290–300 g at the beginning of the experiments were housed in groups of three per hanging metallic cage with ad libitum access to food and tap water. Animals were maintained in a temperature and light controlled environment Ž12r12 h lightrdark cycle, lights on from 7.00 to 19.00 h.. One week after delivery the rats,
0006-8993r98r$ - see front matter q 1998 Published by Elsevier Science B.V. All rights reserved. PII: S 0 0 0 6 - 8 9 9 3 Ž 9 8 . 0 0 8 8 2 - 8
A.M. Magarinos ˜ et al.r Brain Research 809 (1998) 314–318
Fig. 1. Effect of corticosterone ŽCort. in the drinking water Ž400 mgrml., daily restraint stress Ž R . and the combined treatments Ž RqCort. for 21 days on the number of dendritic branch points of CA3 apical dendrites. The vehicle ŽVeh. consisted of a 2.4% ethanol in the drinking water. ) p- 0.05 compared with controls. One-way ANOVA, Tukey HSD post-hoc analysis.
already adapted to daily handling, were weighed and randomly assigned to experimental groups: Ž1. Unstressed control animals Ž n s 6. remained in their cages. Ž2. Chronic restraint stress animals Ž n s 6. received restraint stress for 21 d. Restraint stress was applied for 6 hrday Ž10:00 h to 16:00 h. in wire mesh restrainers, secured at the head and tail ends with clips. During the restraint sessions, the rats were kept in their home cages. Ž3. Chronic corticosterone treated animals Ž n s 6. received 400 mg corticosteronerml of tap water over 21 days. Corticosterone ŽAldrich Chem., Milwaukee, WI. was dissolved in ethanol, and all other groups of rats received the same percentage of ethanol in their drinking water Ž2.4%.. Ž4. Chronic restraint stress plus corticosterone animals Ž n s 6. received the combined treatments. Blood samples were collected twice a day, at 09:00 h and just prior to lights off at 19:00 h on days 1, 7, 14 and 21 of the stress paradigm. Samples were obtained from the tail vein and less than 30 s elapsed from the moment the animals were handled until the blood was collected. Blood samples were centrifuged and the plasma immediately separated and frozen until use in corticosterone radioimmunoassay as previously described w14x. At the end of the treatment period, the rats were deeply anesthetized with Metofane ŽPitman-Moore, Mundelein, IL. and transcardially perfused with 150 ml 4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.4. Adrenals, thymus glands and spleens were removed, cleaned and weighed. Brains were postfixed in the perfusate overnight. Sections, 100 mm thick, were cut with a Vibratome into a bath of 3% potassium dichromate in distilled water. Sections were then processed according to a modified version of the ‘single’ section Golgi impregnation procedure as previously described w7,14x.
315
Slides containing brain sections were coded prior to quantitative analysis; the code was not broken until the analysis was complete. In order to be selected for analysis, Golgi impregnated neurons had to posses the following characteristics: Ž1. Location in the CA3c subregion of the dorsal hippocampus; Ž2. Dark and consistent impregnation throughout the extent of all of the dendrites; Ž3. relative isolation from neighboring impregnated cells which could interfere with analysis; and Ž4. a cell body in the middle third of the tissue section in order to avoid analysis of impregnated neurons which extended largely into other sections. For each brain, 6–8 pyramidal cells from CA3c were selected. Each selected neuron was traced at 400 = magnification using a light microscope with a camera lucida drawing tube attachment. From these drawings the number of dendritic branch Žbifurcation. points within a 100 mm thick section of each dendritic tree was determined for each selected neuron. In addition, the length of the dendrites present in a 100 mm thick section was determined for each dendritic tree using a Zeiss interactive digitizing analysis system. Means were determined for each variable for each brain and the resulting values were subjected to a One-way analysis of variance ŽANOVA. with Tukey HSD post-hoc comparisons. Organ weight data were analyzed by One-way ANOVA. Differences in adrenal weights between treatments were further evaluated using Tukey HSD post hoc comparisons. Corticosterone levels at 09:00 and 19:00 on days 7, 14 and 21 were separately analyzed by two-factor ANOVA and Tukey HSD post-hoc comparisons when appropriate. As shown in Fig. 1, chronic restraint stress or administration of corticosterone in the drinking water produced a decrease in the number of dendritic branch points in the
Fig. 2. Effect of corticosterone ŽCort. in the drinking water Ž400 mgrml., daily restraint stress Ž R . and the combined treatments Ž RqCort. for 21 days on the dendritic total length of CA3 apical dendrites. No significant differences were observed among experimental groups ŽOne-way ANOVA: F s 2.10, ps 0.135..
316
A.M. Magarinos ˜ et al.r Brain Research 809 (1998) 314–318
Fig. 3. Effect of corticosterone ŽCort. in the drinking water Ž400 mgrml., daily restraint stress Ž R . and the combined treatments Ž R q Cort. for 21 days on adrenal, thymic and splenic weights. )) p - 0.0001 compared with controls. One-way ANOVA, Tukey post-hoc analysis.
apical dendrites of CA3 pyramidal neurons, relative to unstressed animals receiving vehicle. The restraint stress and corticosterone treatments resulted in a non-significant decrease in total apical dendritic length ŽFig. 2.. The combination of restraint stress and corticosterone administration negated the effects of each treatment alone ŽFig. 1.. Consistent with previous reports, basal dendritic length and branching were not affected by stress or corticosterone. As expected, corticosterone treatment produced a significant reduction of adrenal, spleen and thymus weight, corrected for body weight, but repeated stress produced a trend for increased adrenal weight and decreased thymus and spleen weights ŽFig. 3.. Body weight was also reduced by corticosterone treatment Ždata not shown.. Morning Ž09:00 h. and evening Ž19:00 h. levels of circulating corticosterone, sampled at days 7, 14 and 21 of the study, are given in Table 1. Administration of 400 mg corticosteronerml of drinking water produced circulating corticosterone levels in unstressed animals similar to those typically seen at the daily evening peak in untreated animals. Throughout the study, there were no significant differences in corticosterone levels between any groups obtained at 19:00 h. However, animals receiving corticosterone had significantly elevated steroid levels in the morning, relative to animals receiving vehicle Žat day 7: F s 23.00, p - 0.0001; at day 14: F s 39.89, p - 0.00001; at day 21: F s 16.65, p s 0.0003.. Further, since both groups receiving vehicle Žunstressed controls and chronic restraint stressed animals. showed typical low basal levels of corticosterone at 09:00 throughout the experimental period, the ethanol vehicle itself did not appear to activate the hypothalamic–pituitary–adrenal ŽHPA. axis. The results of this experiment confirm and extend our previous findings that repeated stress causes reorganization of the apical dendritic tree of CA3 pyramidal neurons and that chronic exposure to corticosterone mimics this effect w23,27x. The fact that the drinking water route of corticosterone administration works as well as injection of corticosterone to cause dendritic atrophy indicates that dendritic morphology is sensitive to increased levels of
corticosterone, even when delivered in a non-invasive manner and in spite of the different pharmacodynamics of corticosteroid clearance after subcutaneous administration of supraphysiological bolus in sesame oil, compared with the more continuous oral route. The drinking water route of administration of corticosterone potently reduced adrenal, spleen and thymus weight as well as body weight. The most surprising aspect of our present study is that the combination of repeated stress and corticosterone negated the effects of either treatment alone. The results seem paradoxical and we currently have no definitive explanation. One difference between the stressed group and group receiving stress and corticosterone is that the HPA axis was suppressed in the latter group, as indicated by the marked atrophy of adrenal glands ŽFig. 3.. It may be that CRF andror ACTH participate in the process of Table 1 Effect of corticosterone ŽCort. in the drinking water Ž400 mgrml., daily restraint stress Ž R . and the combined treatments Ž RqCort. for 21 days on AM and PM plasma levels of corticosterone Time of blood collection 09:00 h
19:00 h
C qVeh Day 7 Day 14 Day 21
2.28"0.39 2.58"0.43 1.30"1.01
18.67"2.37 14.17"3.52 26.14"3.66
C qCort Day 7 Day 14 Day 21
21.51"6.72 28.88"5.32 20.02"7.78
14.98"3.51 29.17"5.02 21.50"5.40
RqVeh Day 7 Day 14 Day 21
1.87"0.44 1.14"0.13 0.85"0.29
16.03"3.04 21.09"2.72 19.11"2.49
RqCort Day 7 Day 14 Day 21
33.97"8.31 39.87"8.80 16.94"3.33
13.69"3.92 26.49"7.03 30.00"4.47
A.M. Magarinos ˜ et al.r Brain Research 809 (1998) 314–318
dendritic atrophy, and these actions are canceled by oral corticosterone administration. Alternatively, suppression of the HPA axis by oral corticosterone may blunt stress-induced corticosterone secretion such that peak corticosterone levels during stress may be higher in animals receiving vehicle rather than corticosterone. Suppression of the HPA axis, by itself, cannot account for the difference in dendritic morphology between animals receiving the steroid or vehicle, because the HPA axis was suppressed in both the stressed and non-stressed groups. Another difference between animals receiving vehicle and those receiving corticosterone in the drinking water is that the daily rhythm of corticosteroid secretion is disrupted in the latter group. Multiple processes are involved in stress-induced atrophy of apical dendrites of CA3 pyramidal neurons ŽRefs. w15,16x for reviews.. Glucocorticoid-mediated dendritic atrophy involves the modulation of a number of hippocampal neurochemical systems w14x, including enhanced NMDA receptor subunit expression and receptor binding w10,26x, stress-induced glutamate release w2,6,19x, 5HT turnover w1x, the induction of presynaptic kainate receptors on mossy fiber terminals w25x, alterations of GABA A receptor subunit expression w20x, and the reduction of inhibitory 5HT1A receptor expression w3,17,18x. Taken together, these actions of adrenal steroids may contribute to a stress-induced enhancement of excitation and a reduction of inhibition in hippocampus. Simultaneous suppression of the HPA axis or the loss of rhythmicity in adrenal activity induced by exposure to oral corticosterone might impair the ability of the CA3 neurons to undergo dendritic remodeling. Future studies will look for the system or systems responsible from among those described above. In conclusion, corticosterone passively administered to rats in the drinking water mimics the effects of repeated restraint stress on dendritic branching in the apical dendrites of CA3 neurons in the rat hippocampus. Paradoxically, combining oral corticosterone with repeated stress negates the effects of both treatments. We suggest that the temporal pattern of exposure to corticosterone may alter neural responses to this steroid. Additionally, differences in the neuroendocrine environment between treatment groups—for example, differences in CRH, glutamate, vasopressin or monoamine systems—may alter the neural responses to corticosterone itself. One result appears to be that combining chronic restraint stress and chronic elevations of corticosterone suppresses some component of the neurochemical system that mediates the dendritic atrophy.
Acknowledgements This work was supported by NIH Grant MH 41256 to BM, Servier ŽFrance. and The Health Foundation ŽNew York..
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