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Chronic stress exposure influences local cerebral blood flow in the rat hippocampus
Neuroscience Vol. 93, No.2, pp. 551–555, 1999 551 Copyright q 1999 IBRO. Published by Elsevier Science Ltd Printed in Great Britain. All rights reserved 0306-4522/99 $20.00+0.00
Chronic stress and local CBF in the hippocampus
Pergamon PII: S0306-4522(99)00176-1
CHRONIC STRESS EXPOSURE INFLUENCES LOCAL CEREBRAL BLOOD FLOW IN THE RAT HIPPOCAMPUS Y. ENDO,*† J.-I. NISHIMURA,* S. KOBAYASHI,‡§ and F. KIMURA* *Departments of Physiology, ‡Anatomy and §Oral and Maxillo-Facial Surgery, Yokohama City University School of Medicine, 3-9 Fuku-ura, Kanazawa-ku, Yokohama 236-0004, Japan
Abstract—To examine the influence of chronic stress on the brain, we measured local cerebral blood flow in the hippocampus of rats which had been exposed to chronic stress by the hydrogen clearance method in the freely moving status. Rats were exposed, once a day for 12 weeks, to stress of a 15-min immersion in cold water at 48C (the stress group) or slightly handled for about 1 min (the control group). Local cerebral blood flow values in the hippocampus, which were measured after a 12-week recovery period, were lower in rats in the stress group than those of rats in the control group only in the dark cycle, but not in the light cycle. Accordingly, local cerebral blood flow in the hippocampus of rats in the stress group did not have a daily fluctuation, i.e. lower in the light cycle and higher in the dark cycle, as was shown in rats in the control group. There were no significant changes in motor activity in rats in the stress group as compared to those in the control group. Severe structural damages were observed in the CA2 and CA3 cell fields of the hippocampus of rats in the stress group. We found that an increase in local cerebral blood flow in the hippocampus in the dark cycle was blunted following chronic stress exposure, suggesting that chronic stress exposure caused hippocampal neurons to be less responsive to environmental stimuli derived from motor activity during the dark cycle. q 1999 IBRO. Published by Elsevier Science Ltd. Key words: hippocampus, chronic stress, local cerebral blood flow, daily fluctuation, rats.
We have studied the daily fluctuation in the hippocampal function of rats, typical nocturnal animals. We measured local cerebral blood flow (CBF) and acetylcholine (ACh) release in the hippocampus of intact rats over a day, by means of the hydrogen clearance method and in vivo microdialysis method, respectively, and demonstrated that both parameters were not stable but fluctuated over a day, being lower in the light cycle and higher in the dark cycle, significantly correlating with the rat’s behavior. 5,20 In recent studies, it has been suggested that stress, or glucocorticoid (GC) secreted during stress, could accelerate agerelated changes in the brain. Histologically, chronic stress exposure or chronic GC treatment causes degenerative changes in the hippocampus, similar to the aged hippocampus, 13,23,27 probably due to the neurotoxicity of stress itself or stress-related GC. Exposure to an excess of GCs causes aging-like changes in the hippocampal neurochemical and electrophysiological parameters. 10,25 We have also demonstrated physiologically that long-term GC treatments for three months result in an impairment of maze learning and a decrease in local CBF in the hippocampus, 7,8 as observed in aged ones. 15,28 In accordance with these results, it has been reported that an exposure to elevated plasma GC levels causes hippocampus-dependent cognitive impairments, probably due to GC-induced hippocampal atrophy in human aging. 14 Conversely, reducing the exposure to GCs by means of adrenalectomy or behavioral manipulations protects the hippocampus from age-related neuron loss and dysfunctions. 13,17 Also, we found that adrenalectomy increased local CBF in
the hippocampus, in a direction that appeared to inhibit agerelated changes. 6 We hypothesized that prolonged elevation of endogenous GC through chronic stress exposure or stress itself would significantly affect local CBF in the hippocampus, probably due to histological damages in the hippocampus. We undertook the present study in order to investigate whether this hypothesis was correct. This issue was particularly interesting, considering that chronic stress exposure, not exogenous GC, could cause hippocampal dysfunctions. EXPERIMENTAL PROCEDURES
Animals Adult male Wistar–Imamichi rats obtained from the Animal Reproduction Research Co. (Urawa, Saitama, Japan) were used. Throughout the experiments, the animals were housed under standard conditions, with lights on 05.00–19.00 and controlled room temperature at 248C, and received food and water ad libitum. Stress sessions At 12 weeks-of-age, rats were divided into two groups, i.e. a chronically stressed group (stress group) and a control group (control group). For chronic stress, rats were immersed up to the neck in cold water at 48C for 15 min. This stress exposure was performed between 13.00 and 15.00 every day, over 12 weeks. Rats in the control group were handled slightly for about 1 min. Immediately after each of the stress exposures or handling procedures, rats were returned to their home cages. Measurement of local cerebral blood flow in the hippocampus At nine to 10 months-of-age, i.e. 12 weeks recovery after the stress sessions, local CBF in the hippocampus was measured. We measured local CBF in the hippocampus by means of the hydrogen clearance method in unanesthetized, freely moving rats, as previously reported. 5,6,8,21 One week prior to the measurement, a platinum electrode with a 1-mm bare tip was implanted stereotaxically into the dorsal hippocampus of the right hemisphere under ether anesthesia, according to the coordinates of Albe-Fessard et al. 1 We constructed a
†To whom correspondence should be addressed at: Department of Physiology, University of Occupational and Environmental Health School of Medicine, 1-1 Iseigaoka, Yahatanishi-ku, Kitakyushu 807-8555, Japan. Abbreviations: ACh, acetylcholine; CBF, cerebral blood flow; GC, glucocorticoid. 551
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chamber for the measurement of CBF, in which an upper compartment held the rat and a lower compartment contained a duct that served to introduce the gas mixture. A 15% H2/85% air mixture was admitted into the chamber at 0.6 l/min for 3 min, and then the respired H2 was washed out. The H2 wash-out curve was recorded on an X–Y recorder, and the local CBF during the 2 min following the first 30 s of the washout curve was calculated with an analyser (MHG-D1, Unique Medical Co., Tokyo), based on the blood–tissue exchange theory of Kety and Schmidt. 11 Each rat was placed in the chamber for two to three days prior to the measurement, in order to adapt to the environment. Local CBF measurements were performed at 1-h intervals over 24 h on each rat. During the 24-h measurement period, the experimental room was maintained under the same conditions as noted above. Rats were allowed free access to food and water for the entire period in the chamber. At the end of CBF measurement, rats were perfused with 10% formalin under ether anesthesia and the brains were removed for the histologic identification of the site of each measuring electrode tip. We used the data of rats for statistical analysis only in cases where the electrode tip was found to be located mainly within the CA3 cell field of the hippocampus, since most studies elucidated that chronic stress or GC treatment could especially affect the CA3 cell field of the hippocampus histologically. 8,23,27
Fig. 1. Schematic illustration showing the location of 1-mm bare tip of electrodes in the rat brain. Each electrode is represented by a bar. Open bar, control group; closed bar, stress group. CA, Cornu ammonis; GD, gyrus dentatus.
Measurement of motor activity At 12 months-of-age, spontaneous motor activity was measured in rats different from those used in CBF measurement, with an automatic animal activity monitor (ACTMONITOR II, Dia Medical System Co., Tokyo, Japan). For measuring the activity, a plastic cage containing the experimental rat was placed on the instrument. The counts were recorded every 10 min and we analysed the data for every 1 h, as we did for local CBF data. Histology At 12 months-of-age, four rats (two rats in the stress group and two rats in the control group) in another series were anesthetized with an intraperitoneal injection of sodium pentobarbital (40 mg/kg body weight) and were transcardially perfused with 200 ml 0.9% saline added heparin (5 U/ml), followed by perfusion with 2.0 l of fixative (10% neutral buffered formalin), as previously reported. 8 The brains were removed carefully and postfixed in the same fixative. Each section was cut at 15 mm and mounted alternately on two series of glass slides. One series was stained with Hematoxylin–Eosin and another series was counterstained with Cresyl Violet. Fig. 2. Twenty-four hour profiles of local CBF in the hippocampus for rats in the control and the stress groups. Each point and its vertical line indicate the mean and S.E.M., respectively.
Statistics The significance of daily fluctuation was analysed by a repeated measures analysis of variance (ANOVA). Further, differences between light and dark cycles in each group were analysed by a paired t-test and differences between groups were analysed by Student’s t-test. The significance level was set at P,0.05. RESULTS
Effects of chronic stress on local cerebral blood flow in the hippocampus We observed that all the measuring electrode tips were located in the hippocampal formation, mainly within the CA3 cell field (Fig. 1). Figure 2 shows mean CBF values in each group, with respect to the time of day. ANOVA showed a significant fluctuation in mean CBF values of the hippocampus over a day in the control group (P,0.001), showing a daily fluctuation, higher in the dark cycle and lower in the light cycle, as shown in Fig. 3 (P,0.05 by paired t-test). However, there were no significant fluctuation and no significant difference between light and dark cycles in the stress group (Figs 2, 3). Furthermore, the dark/light ratio, i.e. the mean increase rate during the dark cycle (19.00–05.00) compared to the light
Fig. 3. Effects of chronic stress exposure on local CBF in the hippocampus during the light cycle, dark cycle, and 24 h. Each bar and its vertical line indicate the mean and S.E.M., respectively. *P,0.05, significantly different from rats in the control group.
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Fig. 4. Twenty-four hour profiles of motor activity for rats in the control and the stress groups. Each point and its vertical line indicate the mean and S.E.M., respectively.
cycle (06.00–18.00) in the stress group was significantly lower than that in the control group (control group, 1.21^0.05; stress group, 1.02^0.06, P,0.05 by Student’s t-test). The overall mean CBF value for the 24 h period in each group was in the range of 50.0–81.6 ml/min/100 g tissue in the control group and 50.9–72.1 ml/min/100 g tissue in the stress group, not showing a significant difference (Fig. 3). Effects of chronic stress on motor activity rhythm Figure 4 shows the motor activity rhythm in each group with respect to the time of day. ANOVA showed significant fluctuations in motor activity over a day (P,0.001) in both groups. There were significant differences between light and dark cycles (P,0.01); that is, higher in the dark cycle and lower in the light cycle in both groups. However, no significant differences between groups were seen in either cycle. Histological damages in the hippocampus There were severe damages in the hippocampus in both rats in the stress group. The most remarkable changes were found in pyramidal neurons in the CA2 and CA3 cell fields (Figs 5, 6), but not in these neurons in the CA1 and CA4. Also, no remarkable changes were found in granule cells of the dentate gyrus. Pyramidal neurons in the CA2 and CA3 were shrunken and sparse, and the zonal arrangement showed an irregularity. Neuropathological changes were characterized by soma shrinkage and condensation or nuclear pyknosis. These pyramidal neurons had irregularly-shaped perikarya associated with dispersed Nissl bodies. DISCUSSION
The present study demonstrated that a long-term stress
Fig. 5. Photomicrographs of 15 mm Hematoxylin–Eosin-stained sections of the dorsal hippocampus from rats in the control (upper, A) and the stress groups (lower, B).
exposure affected the hippocampal function, as assessed by measuring local CBF. Evidence of the influence of a longterm stress exposure is as follows: (i) no significant fluctuation of local CBF over a day in the stress group, and (ii) a significant decrease in mean CBF values in the stress group only during the dark cycle. There have been relatively few studies dealing with hippocampal dysfunctions after chronic stress exposures. Further, no study has reported the long-lasting or irreversible effects of chronic stress exposure on hippocampal functions, as far as we know. The present study clearly demonstrated for the first time a remarkable and long-lasting effect of chronic stress exposures on the hippocampal function. We have reported that local CBF in the hippocampus of intact rats was not stable but fluctuated considerably over a day, being lower in the light cycle and higher in the dark cycle. 5 The mechanism for this daily fluctuation was thought to be related to the rhythm of hippocampal neuronal activity, which is higher in the dark cycle, 9 since local CBF correlates with changes in energy metabolism, which further correlates with neuronal activity. 22 In this study, the loss of daily fluctuation of hippocampal CBF was mainly due to a decrease in local CBF values in the dark cycle. Accordingly, chronic stress exposure is likely to affect the increase in the neuronal activity in the hippocampus during the dark cycle. In the present study, it was found that pyramidal neurons in the CA2 and CA3 were shrunken and sparse in rats which had been exposed to chronic stress. This is the first demonstration that chronic stress exposure induces permanent damages to the hippocampal neurons, and, further, strongly suggests that
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Fig. 6. Photomicrographs of 15-mm Hematoxylin–Eosin-stained sections of the dorsal hippocampus from rats in the control (upper, A and C) and the stress groups (lower, B and D), showing CA2 (left side, A and B) and CA3 cell fields (right side, C and D). Scale bar100 mm.
functional CBF changes in the hippocampus due to chronic stress are related to the structural damage. It is reasonable to consider that the structural damage results in a decrease in the local metabolic demand. De la Torre et al. similarly reported that a significant decrease in local CBF in the hippocampus was observed only in rats which showed histological damages in the hippocampus following cerebrovascular insufficiency. 3 However, it was puzzling that the decrease in CBF was only seen in the dark cycle, but not in the light cycle. It is probable that the effect of these structural damages is distinct only during the active stage. Namely, during the dark cycle, hippocampal neurons of intact rats actively respond to the environmental stimuli and thus have an increased hippocampal CBF, 5 whereas hippocampal neurons of rats that have been exposed to chronic stress are less responsive to environmental stimuli and thus have a decreased CBF. Such a concept is in agreement with a less significant association of ACh release only during the dark cycle in aged rats (Mizuno and Kimura, unpublished observations). In contrast to CBF changes, locomotor activity of rats in the stress group fluctuated significantly over a day, being lower in the light cycle and higher in the dark cycle. This was not different from that of rats in the control group, i.e. there was no significant difference in the mean level of motor activity during the light or dark cycle between the control and stress groups. These results were also observed in GC-treated rats. 4 It has been reported that local CBF in the hippocampus was significantly correlated with rat’s behavior. 5 Further, nocturnal increase in locomotor activity was correlated with an increase in the ACh release in the hippocampus, 20 which is known to stimulate neuronal activity of the hippocampus. 12
Therefore, the present finding in the stress group that the nocturnal rise in the motor activity does not correlate with an increase in CBF is very interesting. Similar phenomenon was observed in ACh release in the hippocampus of aged rats: motor activity of 24-month-old male rats was less significantly correlated with ACh release. 19 Restriction of the environmental space of rats also produced a less significant correlation of motor activity with ACh release in the hippocampus. 18 Together with these latter findings, it is probable that some conditions, such as aging, chronic stress, restriction etc., are characterized by a lower correlation of the hippocampal neuronal activity and motor activity. A uniform factor involved through these conditions may be dysfunction and/ or destruction of hippocampal neurons. As to the mechanism for the structural damages of hippocampal neurons, it has been demonstrated that a prolonged elevation of blood levels of GC caused by either repeated stress or GC treatment damages hippocampal neurons, 8,13,23,27 a principal neural target site for the steroid. 16 Therefore, in our study, an elevation of GC level might have caused hippocampal damages and reduced GC receptors, resulting in further hypersecretion of GC, in accordance with the glucocorticoid cascade hypothesis for the mechanism of hippocampal aging. 24 There is another possibility that chronic stress exposure affects hippocampal CBF by damaging the septohippocampal projections which regulate local CBF in the hippocampus. 2 Tizabi et al. reported that GC administration for two months caused degeneration of the septohippocampal cholinergic neurons. 26 Since the septohippocampal projection might provide the hippocampal CBF with a feature of daily fluctuation,
Chronic stress and local CBF in the hippocampus
it is probable that the local CBF in the hippocampus of rats in the stress group does not show a daily fluctuation mainly due to damages of the septohippocampal projections. CONCLUSIONS
This report demonstrates, that chronic stress exposure for 12 weeks resulted in structural damages of the hippocampal neurons and disturbances of daily fluctuation of local CBF in the hippocampus, with a significant decrease only during the dark cycle. It is also reported that chronic stress exposure does
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not disrupt the daily rhythm of motor activity. It is assumed that hippocampal neurons of rats following chronic stress exposures are less responsive to environmental stimuli derived from motor activity during the dark cycle, probably due to the structural damages, and a decrease in local CBF in the hippocampus occurs in the dark cycle. Acknowledgements—Supported in part by a Grant-in-Aid for Encouragement of Young Scientists from the Ministry of Education, Science, Sports and Culture, Japan (No 05770738) to YE, and by a grant from the Promotional Foundation for Life Science to FK.
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Albe-Fessard D., Stutinsky F. and Libouban S. (1966) Atlas Stereotaxique du Dience´phale du Rat Blanc. Centre National de la Recherche Scientifique, Paris. Cao W.-H., Inanami O., Sato A. and Sato Y. (1989) Stimulation of the septal complex increases local cerebral blood flow in the hippocampus in anesthetized rats. Neurosci. Lett. 107, 135–140. de la Torre J. C., Fortin T., Park G. A. S., Butler K. S., Kozlowski P., Pappas B. A., de Socarraz H., Saunders J. K. and Richard M. T. (1992) Chronic cerebrovascular insufficiency induces dementia-like deficits in aged rats. Brain Res. 582, 186–195. Endo Y. (1999) Do long-term glucocorticoid treatments induce behavioral rhythm disturbances in rats? Physiol. Behav. (in press). Endo Y., Jinnai K., Endo M., Fujita K. and Kimura F. (1990) Diurnal variation of cerebral blood flow in rat hippocampus. Stroke 21, 1464–1469. Endo Y., Nishimura J.-I. and Kimura F. (1994) Adrenalectomy increases local cerebral blood flow in the rat hippocampus. Pflu¨gers Arch. 426, 183–188. Endo Y., Nishimura J.-I. and Kimura F. (1996) Impairment of maze learning in rats following long-term glucocorticoid treatments. Neurosci. Lett. 203, 199–202. Endo Y., Nishimura J.-I., Kobayashi S. and Kimura F. (1997) Long-term glucocorticoid treatments decrease local cerebral blood flow in the rat hippocampus, in association with histological damage. Neuroscience 79, 745–752. Kawakami M. and Kimura F. (1978) The limbic forebrain structures and reproduction. In Perspectives in Endocrine Psychobiology (eds Brambilla F., Bridges P. K., Endroczi E. and Heuser G.), pp. 101–156. Akademiai Kiado, Budapest. Kerr D. S., Campbell L. W., Applegate M. D., Brodish A. and Landfield P. W. (1991) Chronic stress-induced acceleration of electrophysiologic and morphometric biomarkers of hippocampal aging. J. Neurosci. 11, 1316–1324. Kety S. S. and Schmidt C. F. (1945) The determination of cerebral blood flow in man by the use of nitrous oxide in low concentrations. Am. J. Physiol. 143, 53–66. Krnjevic K., Reiffenstein R. J. and Ropert N. (1981) Disinhibitory action of acetylcholine in the rat’s hippocampus: extracellular observations. Neuroscience 6, 2465–2474. Landfield P. W., Baskin R. K. and Pitler T. A. (1981) Brain aging correlates: retardation by hormonal–pharmacological treatments. Science 214, 581–584. Lupien S. J., de Leon M., de Santi S., Convit A., Tarshish C., Nair N. P. V., Thakur M., McEwen B. S., Hauger R. L. and Meaney M. J. (1998) Cortisol levels during human aging predict hippocampal atrophy and memory deficits. Nature Neurosci. 1, 69–73. Martin A. J., Friston K. J., Colebatch J. G. and Frackowiak R. S. J. (1991) Decreases in regional cerebral blood flow with normal aging. J. cerebr. Blood Flow Metab. 11, 684–689. McEwen B. S., de Kloet E. and Rostene W. (1986) Adrenal steroid receptors and actions in the nervous system. Physiol. Rev. 66, 1121–1188. Meaney M. J., Aitken D. H., van Berkel C., Bhatnagar S. and Sapolsky R. M. (1988) Effect of neonatal handling on age-related impairments associated with the hippocampus. Science 239, 766–768. Mitsushima D., Yamanoi C. and Kimura F. (1998) Restriction of environmental space attenuates locomotor activity and hippocampal acetylcholine release in male rats. Brain Res. 805, 207–212. Mizuno T., Arita J. and Kimura F. (1991) Spontaneous acetylcholine release in the hippocampus exhibits a diurnal variation in both young and old rats. Neurosci. Lett. 178, 271–274. Mizuno T., Endo Y., Arita J. and Kimura F. (1991) Acetylcholine release in the rat hippocampus as measured by the microdialysis method correlates with motor activity and exhibits a diurnal variation. Neuroscience 44, 607–612. Nishimura J.-I., Endo Y. and Kimura F. (1992) Increases in cerebral blood flow in rat hippocampus after medial septal injection of naloxone. Stroke 23, 1325–1330. Raichle M. E. (1987) Circulatory and metabolic correlates of brain function in normal humans. In Handbook of Physiology. The Nervous System. Higher Functions of the Brain (eds Mountcastle D. B., Plum F. and Geiger S. R.), Sect. 1, Vol. 5, pt. 2, Chap. 16, pp. 643–674. American Physiology Soc., Bethesda, MD. Sapolsky R. M., Krey L. C. and McEwen B. S. (1985) Prolonged glucocorticoid exposure reduces hippocampal neuron number: implication for aging. J. Neurosci. 5, 1222–1227. Sapolsky R. M., Krey L. C. and McEwen B. S. (1986) The neuroendocrinology of stress and aging: the glucocorticoid cascade hypothesis. Endocr. Rev. 7, 284–301. Talmi M., Carlier E. and Soumireu-Mourat B. (1993) Similar effects of aging and corticosterone treatment on mouse hippocampal function. Neurobiol. Aging 14, 239–244. Tizabi Y., Gilad V. H. and Gilad G. M. (1989) Effects of chronic stressors or corticosterone treatment on the septohippocampal cholinergic system of the rat. Neurosci. Lett. 105, 177–182. Uno H., Tarara R., Else J., Suleman M. and Sapolsky R. M. (1989) Hippocampal damage associated with prolonged and fatal stress in primates. J. Neurosci. 9, 1705–1711. Wallace J. E., Krauter E. E. and Campbell B. A. (1980) Animal models of decline memory in the aged: short-term and spatial memory in the aged rat. J. Gerontol. 35, 355–363. (Accepted 16 March 1999)
Chronic stress and local CBF in the hippocampus
Pergamon PII: S0306-4522(99)00176-1
CHRONIC STRESS EXPOSURE INFLUENCES LOCAL CEREBRAL BLOOD FLOW IN THE RAT HIPPOCAMPUS Y. ENDO,*† J.-I. NISHIMURA,* S. KOBAYASHI,‡§ and F. KIMURA* *Departments of Physiology, ‡Anatomy and §Oral and Maxillo-Facial Surgery, Yokohama City University School of Medicine, 3-9 Fuku-ura, Kanazawa-ku, Yokohama 236-0004, Japan
Abstract—To examine the influence of chronic stress on the brain, we measured local cerebral blood flow in the hippocampus of rats which had been exposed to chronic stress by the hydrogen clearance method in the freely moving status. Rats were exposed, once a day for 12 weeks, to stress of a 15-min immersion in cold water at 48C (the stress group) or slightly handled for about 1 min (the control group). Local cerebral blood flow values in the hippocampus, which were measured after a 12-week recovery period, were lower in rats in the stress group than those of rats in the control group only in the dark cycle, but not in the light cycle. Accordingly, local cerebral blood flow in the hippocampus of rats in the stress group did not have a daily fluctuation, i.e. lower in the light cycle and higher in the dark cycle, as was shown in rats in the control group. There were no significant changes in motor activity in rats in the stress group as compared to those in the control group. Severe structural damages were observed in the CA2 and CA3 cell fields of the hippocampus of rats in the stress group. We found that an increase in local cerebral blood flow in the hippocampus in the dark cycle was blunted following chronic stress exposure, suggesting that chronic stress exposure caused hippocampal neurons to be less responsive to environmental stimuli derived from motor activity during the dark cycle. q 1999 IBRO. Published by Elsevier Science Ltd. Key words: hippocampus, chronic stress, local cerebral blood flow, daily fluctuation, rats.
We have studied the daily fluctuation in the hippocampal function of rats, typical nocturnal animals. We measured local cerebral blood flow (CBF) and acetylcholine (ACh) release in the hippocampus of intact rats over a day, by means of the hydrogen clearance method and in vivo microdialysis method, respectively, and demonstrated that both parameters were not stable but fluctuated over a day, being lower in the light cycle and higher in the dark cycle, significantly correlating with the rat’s behavior. 5,20 In recent studies, it has been suggested that stress, or glucocorticoid (GC) secreted during stress, could accelerate agerelated changes in the brain. Histologically, chronic stress exposure or chronic GC treatment causes degenerative changes in the hippocampus, similar to the aged hippocampus, 13,23,27 probably due to the neurotoxicity of stress itself or stress-related GC. Exposure to an excess of GCs causes aging-like changes in the hippocampal neurochemical and electrophysiological parameters. 10,25 We have also demonstrated physiologically that long-term GC treatments for three months result in an impairment of maze learning and a decrease in local CBF in the hippocampus, 7,8 as observed in aged ones. 15,28 In accordance with these results, it has been reported that an exposure to elevated plasma GC levels causes hippocampus-dependent cognitive impairments, probably due to GC-induced hippocampal atrophy in human aging. 14 Conversely, reducing the exposure to GCs by means of adrenalectomy or behavioral manipulations protects the hippocampus from age-related neuron loss and dysfunctions. 13,17 Also, we found that adrenalectomy increased local CBF in
the hippocampus, in a direction that appeared to inhibit agerelated changes. 6 We hypothesized that prolonged elevation of endogenous GC through chronic stress exposure or stress itself would significantly affect local CBF in the hippocampus, probably due to histological damages in the hippocampus. We undertook the present study in order to investigate whether this hypothesis was correct. This issue was particularly interesting, considering that chronic stress exposure, not exogenous GC, could cause hippocampal dysfunctions. EXPERIMENTAL PROCEDURES
Animals Adult male Wistar–Imamichi rats obtained from the Animal Reproduction Research Co. (Urawa, Saitama, Japan) were used. Throughout the experiments, the animals were housed under standard conditions, with lights on 05.00–19.00 and controlled room temperature at 248C, and received food and water ad libitum. Stress sessions At 12 weeks-of-age, rats were divided into two groups, i.e. a chronically stressed group (stress group) and a control group (control group). For chronic stress, rats were immersed up to the neck in cold water at 48C for 15 min. This stress exposure was performed between 13.00 and 15.00 every day, over 12 weeks. Rats in the control group were handled slightly for about 1 min. Immediately after each of the stress exposures or handling procedures, rats were returned to their home cages. Measurement of local cerebral blood flow in the hippocampus At nine to 10 months-of-age, i.e. 12 weeks recovery after the stress sessions, local CBF in the hippocampus was measured. We measured local CBF in the hippocampus by means of the hydrogen clearance method in unanesthetized, freely moving rats, as previously reported. 5,6,8,21 One week prior to the measurement, a platinum electrode with a 1-mm bare tip was implanted stereotaxically into the dorsal hippocampus of the right hemisphere under ether anesthesia, according to the coordinates of Albe-Fessard et al. 1 We constructed a
†To whom correspondence should be addressed at: Department of Physiology, University of Occupational and Environmental Health School of Medicine, 1-1 Iseigaoka, Yahatanishi-ku, Kitakyushu 807-8555, Japan. Abbreviations: ACh, acetylcholine; CBF, cerebral blood flow; GC, glucocorticoid. 551
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chamber for the measurement of CBF, in which an upper compartment held the rat and a lower compartment contained a duct that served to introduce the gas mixture. A 15% H2/85% air mixture was admitted into the chamber at 0.6 l/min for 3 min, and then the respired H2 was washed out. The H2 wash-out curve was recorded on an X–Y recorder, and the local CBF during the 2 min following the first 30 s of the washout curve was calculated with an analyser (MHG-D1, Unique Medical Co., Tokyo), based on the blood–tissue exchange theory of Kety and Schmidt. 11 Each rat was placed in the chamber for two to three days prior to the measurement, in order to adapt to the environment. Local CBF measurements were performed at 1-h intervals over 24 h on each rat. During the 24-h measurement period, the experimental room was maintained under the same conditions as noted above. Rats were allowed free access to food and water for the entire period in the chamber. At the end of CBF measurement, rats were perfused with 10% formalin under ether anesthesia and the brains were removed for the histologic identification of the site of each measuring electrode tip. We used the data of rats for statistical analysis only in cases where the electrode tip was found to be located mainly within the CA3 cell field of the hippocampus, since most studies elucidated that chronic stress or GC treatment could especially affect the CA3 cell field of the hippocampus histologically. 8,23,27
Fig. 1. Schematic illustration showing the location of 1-mm bare tip of electrodes in the rat brain. Each electrode is represented by a bar. Open bar, control group; closed bar, stress group. CA, Cornu ammonis; GD, gyrus dentatus.
Measurement of motor activity At 12 months-of-age, spontaneous motor activity was measured in rats different from those used in CBF measurement, with an automatic animal activity monitor (ACTMONITOR II, Dia Medical System Co., Tokyo, Japan). For measuring the activity, a plastic cage containing the experimental rat was placed on the instrument. The counts were recorded every 10 min and we analysed the data for every 1 h, as we did for local CBF data. Histology At 12 months-of-age, four rats (two rats in the stress group and two rats in the control group) in another series were anesthetized with an intraperitoneal injection of sodium pentobarbital (40 mg/kg body weight) and were transcardially perfused with 200 ml 0.9% saline added heparin (5 U/ml), followed by perfusion with 2.0 l of fixative (10% neutral buffered formalin), as previously reported. 8 The brains were removed carefully and postfixed in the same fixative. Each section was cut at 15 mm and mounted alternately on two series of glass slides. One series was stained with Hematoxylin–Eosin and another series was counterstained with Cresyl Violet. Fig. 2. Twenty-four hour profiles of local CBF in the hippocampus for rats in the control and the stress groups. Each point and its vertical line indicate the mean and S.E.M., respectively.
Statistics The significance of daily fluctuation was analysed by a repeated measures analysis of variance (ANOVA). Further, differences between light and dark cycles in each group were analysed by a paired t-test and differences between groups were analysed by Student’s t-test. The significance level was set at P,0.05. RESULTS
Effects of chronic stress on local cerebral blood flow in the hippocampus We observed that all the measuring electrode tips were located in the hippocampal formation, mainly within the CA3 cell field (Fig. 1). Figure 2 shows mean CBF values in each group, with respect to the time of day. ANOVA showed a significant fluctuation in mean CBF values of the hippocampus over a day in the control group (P,0.001), showing a daily fluctuation, higher in the dark cycle and lower in the light cycle, as shown in Fig. 3 (P,0.05 by paired t-test). However, there were no significant fluctuation and no significant difference between light and dark cycles in the stress group (Figs 2, 3). Furthermore, the dark/light ratio, i.e. the mean increase rate during the dark cycle (19.00–05.00) compared to the light
Fig. 3. Effects of chronic stress exposure on local CBF in the hippocampus during the light cycle, dark cycle, and 24 h. Each bar and its vertical line indicate the mean and S.E.M., respectively. *P,0.05, significantly different from rats in the control group.
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Fig. 4. Twenty-four hour profiles of motor activity for rats in the control and the stress groups. Each point and its vertical line indicate the mean and S.E.M., respectively.
cycle (06.00–18.00) in the stress group was significantly lower than that in the control group (control group, 1.21^0.05; stress group, 1.02^0.06, P,0.05 by Student’s t-test). The overall mean CBF value for the 24 h period in each group was in the range of 50.0–81.6 ml/min/100 g tissue in the control group and 50.9–72.1 ml/min/100 g tissue in the stress group, not showing a significant difference (Fig. 3). Effects of chronic stress on motor activity rhythm Figure 4 shows the motor activity rhythm in each group with respect to the time of day. ANOVA showed significant fluctuations in motor activity over a day (P,0.001) in both groups. There were significant differences between light and dark cycles (P,0.01); that is, higher in the dark cycle and lower in the light cycle in both groups. However, no significant differences between groups were seen in either cycle. Histological damages in the hippocampus There were severe damages in the hippocampus in both rats in the stress group. The most remarkable changes were found in pyramidal neurons in the CA2 and CA3 cell fields (Figs 5, 6), but not in these neurons in the CA1 and CA4. Also, no remarkable changes were found in granule cells of the dentate gyrus. Pyramidal neurons in the CA2 and CA3 were shrunken and sparse, and the zonal arrangement showed an irregularity. Neuropathological changes were characterized by soma shrinkage and condensation or nuclear pyknosis. These pyramidal neurons had irregularly-shaped perikarya associated with dispersed Nissl bodies. DISCUSSION
The present study demonstrated that a long-term stress
Fig. 5. Photomicrographs of 15 mm Hematoxylin–Eosin-stained sections of the dorsal hippocampus from rats in the control (upper, A) and the stress groups (lower, B).
exposure affected the hippocampal function, as assessed by measuring local CBF. Evidence of the influence of a longterm stress exposure is as follows: (i) no significant fluctuation of local CBF over a day in the stress group, and (ii) a significant decrease in mean CBF values in the stress group only during the dark cycle. There have been relatively few studies dealing with hippocampal dysfunctions after chronic stress exposures. Further, no study has reported the long-lasting or irreversible effects of chronic stress exposure on hippocampal functions, as far as we know. The present study clearly demonstrated for the first time a remarkable and long-lasting effect of chronic stress exposures on the hippocampal function. We have reported that local CBF in the hippocampus of intact rats was not stable but fluctuated considerably over a day, being lower in the light cycle and higher in the dark cycle. 5 The mechanism for this daily fluctuation was thought to be related to the rhythm of hippocampal neuronal activity, which is higher in the dark cycle, 9 since local CBF correlates with changes in energy metabolism, which further correlates with neuronal activity. 22 In this study, the loss of daily fluctuation of hippocampal CBF was mainly due to a decrease in local CBF values in the dark cycle. Accordingly, chronic stress exposure is likely to affect the increase in the neuronal activity in the hippocampus during the dark cycle. In the present study, it was found that pyramidal neurons in the CA2 and CA3 were shrunken and sparse in rats which had been exposed to chronic stress. This is the first demonstration that chronic stress exposure induces permanent damages to the hippocampal neurons, and, further, strongly suggests that
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Fig. 6. Photomicrographs of 15-mm Hematoxylin–Eosin-stained sections of the dorsal hippocampus from rats in the control (upper, A and C) and the stress groups (lower, B and D), showing CA2 (left side, A and B) and CA3 cell fields (right side, C and D). Scale bar100 mm.
functional CBF changes in the hippocampus due to chronic stress are related to the structural damage. It is reasonable to consider that the structural damage results in a decrease in the local metabolic demand. De la Torre et al. similarly reported that a significant decrease in local CBF in the hippocampus was observed only in rats which showed histological damages in the hippocampus following cerebrovascular insufficiency. 3 However, it was puzzling that the decrease in CBF was only seen in the dark cycle, but not in the light cycle. It is probable that the effect of these structural damages is distinct only during the active stage. Namely, during the dark cycle, hippocampal neurons of intact rats actively respond to the environmental stimuli and thus have an increased hippocampal CBF, 5 whereas hippocampal neurons of rats that have been exposed to chronic stress are less responsive to environmental stimuli and thus have a decreased CBF. Such a concept is in agreement with a less significant association of ACh release only during the dark cycle in aged rats (Mizuno and Kimura, unpublished observations). In contrast to CBF changes, locomotor activity of rats in the stress group fluctuated significantly over a day, being lower in the light cycle and higher in the dark cycle. This was not different from that of rats in the control group, i.e. there was no significant difference in the mean level of motor activity during the light or dark cycle between the control and stress groups. These results were also observed in GC-treated rats. 4 It has been reported that local CBF in the hippocampus was significantly correlated with rat’s behavior. 5 Further, nocturnal increase in locomotor activity was correlated with an increase in the ACh release in the hippocampus, 20 which is known to stimulate neuronal activity of the hippocampus. 12
Therefore, the present finding in the stress group that the nocturnal rise in the motor activity does not correlate with an increase in CBF is very interesting. Similar phenomenon was observed in ACh release in the hippocampus of aged rats: motor activity of 24-month-old male rats was less significantly correlated with ACh release. 19 Restriction of the environmental space of rats also produced a less significant correlation of motor activity with ACh release in the hippocampus. 18 Together with these latter findings, it is probable that some conditions, such as aging, chronic stress, restriction etc., are characterized by a lower correlation of the hippocampal neuronal activity and motor activity. A uniform factor involved through these conditions may be dysfunction and/ or destruction of hippocampal neurons. As to the mechanism for the structural damages of hippocampal neurons, it has been demonstrated that a prolonged elevation of blood levels of GC caused by either repeated stress or GC treatment damages hippocampal neurons, 8,13,23,27 a principal neural target site for the steroid. 16 Therefore, in our study, an elevation of GC level might have caused hippocampal damages and reduced GC receptors, resulting in further hypersecretion of GC, in accordance with the glucocorticoid cascade hypothesis for the mechanism of hippocampal aging. 24 There is another possibility that chronic stress exposure affects hippocampal CBF by damaging the septohippocampal projections which regulate local CBF in the hippocampus. 2 Tizabi et al. reported that GC administration for two months caused degeneration of the septohippocampal cholinergic neurons. 26 Since the septohippocampal projection might provide the hippocampal CBF with a feature of daily fluctuation,
Chronic stress and local CBF in the hippocampus
it is probable that the local CBF in the hippocampus of rats in the stress group does not show a daily fluctuation mainly due to damages of the septohippocampal projections. CONCLUSIONS
This report demonstrates, that chronic stress exposure for 12 weeks resulted in structural damages of the hippocampal neurons and disturbances of daily fluctuation of local CBF in the hippocampus, with a significant decrease only during the dark cycle. It is also reported that chronic stress exposure does
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not disrupt the daily rhythm of motor activity. It is assumed that hippocampal neurons of rats following chronic stress exposures are less responsive to environmental stimuli derived from motor activity during the dark cycle, probably due to the structural damages, and a decrease in local CBF in the hippocampus occurs in the dark cycle. Acknowledgements—Supported in part by a Grant-in-Aid for Encouragement of Young Scientists from the Ministry of Education, Science, Sports and Culture, Japan (No 05770738) to YE, and by a grant from the Promotional Foundation for Life Science to FK.
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