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Brain oxytocin augments stress-induced long-lasting plasma adrenocorticotropic hormone elevation in rats
Neuroscience Letters 321 (2002) 161–164 www.elsevier.com/locate/neulet
Brain oxytocin augments stress-induced long-lasting plasma adrenocorticotropic hormone elevation in rats Toshihiro Nakashima a,*, Tohru Noguchi a, Tomonori Furukawa a, Michiyo Yamasaki a, Shinya Makino b, Seiji Miyata a, Toshikazu Kiyohara a a
Department of Applied Biology, Kyoto Institute of Technology, Matsugasaki, Sakyo-ku, Kyoto 606-8585, Japan b 2nd Department of Internal Medicine, Kochi Medical School, Okoh-cho, Nankoku, Kochi, Japan Received 20 October 2001; received in revised form 30 November 2001; accepted 6 December 2001
Abstract Single stress induces long-lasting changes in the hypothalamo-pituitary–adrenal (HPA) axis of adult animals. Selective oxytocin (OXT) receptor antagonist was administrated into the cerebroventricle of male rats to test its influence on plasma adrenocorticotropic hormone (ACTH) responses induced by immobilization stress. The ACTH level is significantly higher than the control level (P , 0:05) up to 6 days after single stress. Although the OXT antagonist did not change the plasma ACTH level at the end of single stress (P ¼ 0:59), the antagonist significantly decreased the ACTH concentration at the end of repeated (3 days) stress and 2 days after single stress (P , 0:05) compared with controls. The results suggest that endogenous brain OXT enhances the long-lasting but not immediate HPA axis response to stress. q 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Stress; Immobilization; Oxytocin; Hypothalamo-pituitary–adrenal axis; Adrenocorticotropic hormone; Long-lasting effects
The hypothalamo-pituitary–adrenal (HPA) axis plays a pivotal role in the mammalian stress response, and the mechanisms regulating adrenocorticotropic hormone (ACTH) secretion in response to stress are multifactorial [1]. For decades, long-lasting changes in HPA axis were reported following a single stress. A single short session of footshock stress has long-lasting effects (14 days) on HPA responsiveness concomitant with behavioral alterations [15]. At the molecular level, single administration of interleukin-1 (IL-1) induces a long-lasting (at least 3 weeks) increase of vasopressin (AVP) stores in corticotropinreleasing hormone (CRH) terminals [14]. Single immobilization produces an increase in the AVP and CRH mRNA levels in the hypothalamic paraventricular nucleus (PVN) and these changes persisted for over 4 days after stress [2]. In parallel to these findings, there are growing evidences that central oxytocin (OXT) is involved in stress responses, especially in modification of HPA axis functions. OXT is released within the hypothalamus in response to shaker [11] and forced swimming [17] stress in rats. Study of oncogene expression has demonstrated that restraint stress activates * Corresponding author. Tel.: 181-75-724-7786; fax: 181-75724-7760. E-mail address: [email protected] (T. Nakashima).
oxytocinergic neurons in the PVN [7]. Moreover, OXT receptor mRNA is localized in the PVN [18], and OXT has been shown to influence the electrical activity of putative OXT neurons of the PVN or supraoptic nucleus [4,9]. More direct evidences have shown that centrally injected OXT antisense oligonucleotides block stress-induced tachycardia [8] and that OXT administrated intracerebroventricularly has been shown to reduce stress-induced corticosterone release and anxiety behavior in rats [16]. In the present study, we investigated whether a single immobilization stress can induce long-lasting change in the ACTH secretion and whether brain OXT modulates the ACTH responses induced by single or repeated stress. Experiments were carried out on adult male Wistar rats weighing 170–250 g. Animals were individually housed in plastic cages in a light (12 h on, 12 h off; lights on at 06:00 h) and temperature-controlled room (24 ^ 1 8C). Tap water and rodent chows were available ad libitum. Rats were implanted with sterile guide cannulae into the 3rd ventricle according to the atlas of Paxinos and Watson [12], under pentobarbital sodium (50 mg/kg i.p.) anesthesia. The rats were allowed to recover for more than 7 days before experiments. For the single stress experiment, rats were exposed to immobilization for 120 min (between 10:00 and 12:00 h), by taping the limbs of the rats to metal mounts attached to a
0304-3940/02/$ - see front matter q 2002 Elsevier Science Ireland Ltd. All rights reserved. PII: S03 04 - 394 0( 0 1) 02 54 8- 4
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T. Nakashima et al. / Neuroscience Letters 321 (2002) 161–164
Fig. 1. Plasma levels of ACTH in male rats 0, 2, 6 and 20 days after single stress. Data are expressed as means ^ SEM. #P , 0:05, compared with controls.
board as described previously [5,6]. Blood samples were collected 0, 2, 6 and 20 days after stress. The rats in the control groups were kept under non-stress conditions. A second experiment was conducted to evaluate the effect of OXT receptor antagonist treatment on plasma ACTH levels. The protocol was similar to that described above except that the animals were injected with OXT antagonist or saline into the cerebroventricle through the cannula 30 min before immobilizations. OXT receptor antagonist (d(CH2)5Tyr(de) 2OVT; 10 mg/5 ml; a generous gift from Dr Manning, Toledo, OH) was dissolved in physiological saline and injected in a volume of 5 ml. Repeatedly stressed rats were subjected to 2 h of immobilization for 3 consecutive days with pretreatment of OXT antagonist or saline 30 min before each immobilization. All blood samples were collected within 5 s after taking animals from their home cage and at 12:00 h to avoid the circadian plasma ACTH deviation. Immediately following decapitation, trunk blood
Fig. 2. Plasma levels of ACTH in male rats repeated (3 days) or 0, 2 days after stress. Rats pretreated with OXT antagonist (10 mg/5 ml intracerebroventricularly.; filled bars) or saline (open bars). Data are expressed as means ^ SEM. Significant (*P , 0:05) difference in rats pretreated with OXT antagonist or saline.
was collected in EDTA-coated tubes and centrifuged (5000 rpm, 5 min). The supernatant was collected and stored at 220 8C. ACTH was radioimmunoassayed by commercially available kits: ACTH IRMA (Mitsubishi Chemical, Tokyo, Japan). The sensitivity of the assay was 5 pg/ml. After the completion of experiments, the cannula tip was verified to locate in the 3rd ventricle by dye injection through the cannula. Data are expressed as means ^ SE. Statistical analysis was performed by a two-way analysis of variance. A 5% level of probability was accepted as statistically significant. The plasma ACTH responses elicited by an immobilization stress are shown in Fig. 1. At the end of immobilization (0 days), the plasma concentration of ACTH increased significantly (3.9-fold; Fð1;10Þ ¼ 66:5; P , 0:01) as compared with controls. Although the plasma ACTH concentration decreased at 2 days after the stress (Fð1;10Þ ¼ 40:6; P , 0:05), the ACTH level was still significantly higher than the control level (Fð1;10Þ ¼ 6:26; P , 0:05). It was also maintained at a higher level after 6 days of stress as compared with the control level (Fð1;10Þ ¼ 5:71; P , 0:05). The ACTH concentration had returned to the control level at 20 days after the stress exposure (Fð1;10Þ ¼ 1:34; P ¼ 0:27). Fig. 2 shows the effects of OXT antagonist injected into the cerebroventricle on the ACTH responses induced by immobilization stress. The plasma ACTH response to immobilization (0 days) was slightly enhanced by OXT antagonist pretreatment but there was no significant difference (Fð1;10Þ ¼ 0:30; P ¼ 0:59). On the other hand, repeated stress-induced elevation of plasma ACTH was significantly attenuated by the OXT antagonist (Fð1;10Þ ¼ 6:36; P , 0:05), indicating that OXT enhances ACTH secretion in response to acute stress when it is applied repeatedly. Furthermore, the elevated ACTH concentration induced by a stress 2 days before was decreased by pre-stress treatment of OXT antagonist (Fð1;19Þ ¼ 23:6; P , 0:05). The suppressed level of ACTH was not statistically different from the control level (Fð1;19Þ ¼ 4:75; P ¼ 0:054). Intracerebroventricular injection of the OXT antagonist did not cause a significant change in the resting level of plasma ACTH (Fð1;10Þ ¼ 0:13; P ¼ 0:73) compared with saline treatment. The obtained data show that a single immobilization stress enhanced ACTH secretion at least 6 days after single stress and that the elevated ACTH secretion was suppressed at the end of repeated (3 days) stress and 2 days after the single stress by pretreatment with central OXT receptor antagonist. The OXT antagonist did not change the plasma ACTH level at the end of single stress. The results suggest that endogenous brain OXT enhances the long-lasting but not immediate HPA axis response to stress. It has been shown that single exposure to acute stressful stimuli is followed by a rapid increase in plasma ACTH and decline to basal levels, depending on the nature and intensity of the stimulus [1]. These data, however, did not consider the
T. Nakashima et al. / Neuroscience Letters 321 (2002) 161–164
possible involvement of long-lasting effects following single stress and circadian variations of ACTH. In the present study, we collected all blood samples at 12:00 h to avoid the circadian plasma ACTH deviation. Some recent studies have investigated long-lasting changes in the HPA axis following single stress. For example, rats exposed to a single session of footshocks showed, 14 days later, an augmented ACTH response to a novel emotional stimulus [15]. Single administration of IL-1 induces a long-lasting increase of AVP stores in CRH terminals. A second IL-1 challenge at 11 days after IL-1 administration caused a marked increase in ACTH response [14]. Our data support their observations that single stress may lead to long-lasting change in the HPA axis, whereas they did not measure the basal ACTH level at 2 or 6 days after single stress. It is necessary to find out whether the basal ACTH level becomes elevated in response to a single session of footshock or IL-1 administration at 2 or 6 days before. Due to the ir-ACTH kit used in this study, it is possible that we detected not only ACTH but also the ACTH fragment corticotropin-like intermediate peptide (CLIP: ACTH 18–39). Although we do not exclude the possibility, the efficacy of CLIP is still confused because a fragment of CLIP (ACTH 20–24) is also effective on the rapid eye movement sleep episodes. The previous study by Neumann et al. [10] has demonstrated that OXT antagonist injected into the cerebral ventricle enhanced basal and stress-induced secretion of ACTH into blood. No statistical difference was shown between the OXT antagonist treated and saline treated animals in both basal and stress-induced ACTH secretion in this study. One possible reason for the discrepancy may be the difference in the nature of stress. They used elevated plus-maze stress compared with immobilization stress used in this study. The second possibility may depend on the time schedule of experimental protocols. They measured plasma ACTH at 10 min after OXT antagonist injection as the basal state. Although the plasma ACTH level of the OXT antagonist pretreated animal was higher than vehicle treated rats 5 min after the end of stress, no significant difference was shown by OXT antagonist treatment at 15 or 60 min after the end of stress. In contrast to their study, we measured the plasma ACTH concentration of controls 2.5 h after with or without OXT antagonist, and stressinduced ACTH secretions were measured just at the end of or 2 days after stress. Forced swimming and shaker stress were reported to enhance both intra-hypothalamic release of OXT and its secretion into the systemic circulation [11,17]. However, social defeat experience elicited the release within the SON but not peripheral release of OXT [3]. Although a 5day peripheral treatment of OXT without stress did not change the plasma level of ACTH, plasma levels of corticosterone were significantly lower in rats treated with OXT. This effect persisted for at least 10 days after the end of the treatment [13]. Further studies are needed to elucidate the
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long-lasting central and peripheral OXT effects to stress responses. This work was supported in part by Grant-in-Aid for Scientific Research B12557006 and Special Coordination Funds for Promoting Science and Technology from the Ministry of Education, Culture, Sports, Science and Technology, the Japanese Government. [1] Aguilera, G., Regulation of pituitary ACTH secretion during chronic stress, Front. Neuroendocrinol., 15 (1994) 321–350. [2] Aubry, J.M., Bartanusz, V., Jeziva, D., Belin, D. and Kiss, J.Z., Single stress induces long-lasting elevations in vasopressin mRNA level in CRF hypophysiotrophic neurons, but repeated stress is required to modify AVP immunoreactivity, J. Neuroendocrinol., 11 (1999) 377–384. [3] Engelmann, M., Ebner, K., Landgraf, R., Holsboer, F. and Wotjak, C.T., Emotional stress triggers intrahypothalamic but not peripheral release of oxytocin in male rats, J. Neuroendocrinol., 11 (1999) 867–872. [4] Kuriyama, K., Nakashima, T., Kawarabayashi, T. and Kiyohara, T., Oxytocin inhibits nonphasically firing supraoptic and paraventricular neurons in the virgin female rat, Brain Res. Bull., 31 (1993) 681–687. [5] Kvetnansky, R. and Mikulaj, L., Adrenal and urinary catecholamines in rats during adaptation to repeated immobilization stress, Endocrinology, 87 (1970) 738–743. [6] Makino, S., Smith, M.A. and Gold, P.W., Increased expression of corticotropin-releasing hormone and vasopressin messenger ribonucleic acid (mRNA) in the hypothalamic paraventricular nucleus during repeated stress: association with reduction in glucocorticoid receptor mRNA levels, Endocrinology, 136 (1995) 3299–3309. [7] Miyata, S., Itoh, T., Lin, S.-H., Ishiyama, M., Nakashima, T. and Kiyohara, T., Temporal changes of c-fos expression in oxytocinergic magnocellular neuroendocrine cells of the rat hypothalamus with restraint stress, Brain Res. Bull., 37 (1995) 391–395. [8] Morris, M., Lucion, A.B., Li, P. and Callahan, M.F., Central oxytocin mediates stress-induced tachycardia, J. Neuroendocrinol., 7 (1995) 455–459. [9] Murai, T., Nakashima, T., Miyata, S. and Kiyohara, T., Different effect of oxytocin on membrane potential of supraoptic oxytocin neurons in virgin female and male rats in vitro, Neurosci. Res., 30 (1998) 35–41. [10] Neumann, I.D., Wigger, A., Torner, L., Holsboer, F. and Landgraf, R., Brain oxytocin inhibits basal and stressinduced activity of the hypothalamo-pituitary–adrenal axis in male and female rats: partial action within the paraventricular nucleus, J. Neuroendocrinol., 12 (2000) 235–243. [11] Nishioka, T., Anselmo-Franci, J.A., Li, P., Callahan, M.F. and Morris, M., Stress increases oxytocin release within the hypothalamic paraventricular nucleus, Brain Res., 781 (1998) 57–61. [12] Paxinos, G. and Watson, C., The Rat Brain in Stereotaxic Coordinates, Academic Press, San Diego, CA, 1986. [13] Peterson, M., Hulting, A. and Uvna¨s-Moberg, K., Oxytocin causes a sustained decrease in plasma levels of corticosterone in rats, Neurosci. Lett., 264 (1999) 41–44. [14] Schmidt, E.D., Janszen, A.W.J.W., Wouterlood, F.G. and Tilders, F.J.H., Interleukin-1-induced long-lasting changes in hypothalamic corticotropin-releasing hormone (CRH) neurons and hyperresponsiveness of the hypothalamus– pituitary–adrenal axis, J. Neurosci., 15 (1995) 7417–7426. [15] Van Dijken, H.H., de Goeij, D.C.E., Sutanto, W., Mos, J., de
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Kloet, E.R. and Tilders, F.J.H., Short inescapable stress produces long-lasting changes in the brain–pituitary–adrenal axis of adult male rats, Neuroendocrinology, 58 (1993) 57–64. [16] Windle, R.J., Shanks, N., Lightman, S.L. and Ingram, C.D., Central oxytocin administration reduces stress-induced corticosterone release and anxiety behavior in rats, Endocrinology, 138 (1997) 2829–2834. [17] Wotjak, C.T., Ganster, J., Kohl, G., Holsboer, F., Landgraf, R.
and Engelmann, M., Dissociated central and peripheral release of vasopressin, but not oxytocin, in response to repeated swim stress: new insights into the secretory capacities of peptidergic neurons, Neuroscience, 85 (1998) 1209–1222. [18] Yoshimura, R., Kiyama, H., Kimura, T., Araki, T., Maeno, H., Tanizuka, O. and Tohyama, M., Localization of oxytocin receptor messenger ribonucleic acid in the rat brain, Endocrinology, 133 (1993) 1239–1246.
Brain oxytocin augments stress-induced long-lasting plasma adrenocorticotropic hormone elevation in rats Toshihiro Nakashima a,*, Tohru Noguchi a, Tomonori Furukawa a, Michiyo Yamasaki a, Shinya Makino b, Seiji Miyata a, Toshikazu Kiyohara a a
Department of Applied Biology, Kyoto Institute of Technology, Matsugasaki, Sakyo-ku, Kyoto 606-8585, Japan b 2nd Department of Internal Medicine, Kochi Medical School, Okoh-cho, Nankoku, Kochi, Japan Received 20 October 2001; received in revised form 30 November 2001; accepted 6 December 2001
Abstract Single stress induces long-lasting changes in the hypothalamo-pituitary–adrenal (HPA) axis of adult animals. Selective oxytocin (OXT) receptor antagonist was administrated into the cerebroventricle of male rats to test its influence on plasma adrenocorticotropic hormone (ACTH) responses induced by immobilization stress. The ACTH level is significantly higher than the control level (P , 0:05) up to 6 days after single stress. Although the OXT antagonist did not change the plasma ACTH level at the end of single stress (P ¼ 0:59), the antagonist significantly decreased the ACTH concentration at the end of repeated (3 days) stress and 2 days after single stress (P , 0:05) compared with controls. The results suggest that endogenous brain OXT enhances the long-lasting but not immediate HPA axis response to stress. q 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Stress; Immobilization; Oxytocin; Hypothalamo-pituitary–adrenal axis; Adrenocorticotropic hormone; Long-lasting effects
The hypothalamo-pituitary–adrenal (HPA) axis plays a pivotal role in the mammalian stress response, and the mechanisms regulating adrenocorticotropic hormone (ACTH) secretion in response to stress are multifactorial [1]. For decades, long-lasting changes in HPA axis were reported following a single stress. A single short session of footshock stress has long-lasting effects (14 days) on HPA responsiveness concomitant with behavioral alterations [15]. At the molecular level, single administration of interleukin-1 (IL-1) induces a long-lasting (at least 3 weeks) increase of vasopressin (AVP) stores in corticotropinreleasing hormone (CRH) terminals [14]. Single immobilization produces an increase in the AVP and CRH mRNA levels in the hypothalamic paraventricular nucleus (PVN) and these changes persisted for over 4 days after stress [2]. In parallel to these findings, there are growing evidences that central oxytocin (OXT) is involved in stress responses, especially in modification of HPA axis functions. OXT is released within the hypothalamus in response to shaker [11] and forced swimming [17] stress in rats. Study of oncogene expression has demonstrated that restraint stress activates * Corresponding author. Tel.: 181-75-724-7786; fax: 181-75724-7760. E-mail address: [email protected] (T. Nakashima).
oxytocinergic neurons in the PVN [7]. Moreover, OXT receptor mRNA is localized in the PVN [18], and OXT has been shown to influence the electrical activity of putative OXT neurons of the PVN or supraoptic nucleus [4,9]. More direct evidences have shown that centrally injected OXT antisense oligonucleotides block stress-induced tachycardia [8] and that OXT administrated intracerebroventricularly has been shown to reduce stress-induced corticosterone release and anxiety behavior in rats [16]. In the present study, we investigated whether a single immobilization stress can induce long-lasting change in the ACTH secretion and whether brain OXT modulates the ACTH responses induced by single or repeated stress. Experiments were carried out on adult male Wistar rats weighing 170–250 g. Animals were individually housed in plastic cages in a light (12 h on, 12 h off; lights on at 06:00 h) and temperature-controlled room (24 ^ 1 8C). Tap water and rodent chows were available ad libitum. Rats were implanted with sterile guide cannulae into the 3rd ventricle according to the atlas of Paxinos and Watson [12], under pentobarbital sodium (50 mg/kg i.p.) anesthesia. The rats were allowed to recover for more than 7 days before experiments. For the single stress experiment, rats were exposed to immobilization for 120 min (between 10:00 and 12:00 h), by taping the limbs of the rats to metal mounts attached to a
0304-3940/02/$ - see front matter q 2002 Elsevier Science Ireland Ltd. All rights reserved. PII: S03 04 - 394 0( 0 1) 02 54 8- 4
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T. Nakashima et al. / Neuroscience Letters 321 (2002) 161–164
Fig. 1. Plasma levels of ACTH in male rats 0, 2, 6 and 20 days after single stress. Data are expressed as means ^ SEM. #P , 0:05, compared with controls.
board as described previously [5,6]. Blood samples were collected 0, 2, 6 and 20 days after stress. The rats in the control groups were kept under non-stress conditions. A second experiment was conducted to evaluate the effect of OXT receptor antagonist treatment on plasma ACTH levels. The protocol was similar to that described above except that the animals were injected with OXT antagonist or saline into the cerebroventricle through the cannula 30 min before immobilizations. OXT receptor antagonist (d(CH2)5Tyr(de) 2OVT; 10 mg/5 ml; a generous gift from Dr Manning, Toledo, OH) was dissolved in physiological saline and injected in a volume of 5 ml. Repeatedly stressed rats were subjected to 2 h of immobilization for 3 consecutive days with pretreatment of OXT antagonist or saline 30 min before each immobilization. All blood samples were collected within 5 s after taking animals from their home cage and at 12:00 h to avoid the circadian plasma ACTH deviation. Immediately following decapitation, trunk blood
Fig. 2. Plasma levels of ACTH in male rats repeated (3 days) or 0, 2 days after stress. Rats pretreated with OXT antagonist (10 mg/5 ml intracerebroventricularly.; filled bars) or saline (open bars). Data are expressed as means ^ SEM. Significant (*P , 0:05) difference in rats pretreated with OXT antagonist or saline.
was collected in EDTA-coated tubes and centrifuged (5000 rpm, 5 min). The supernatant was collected and stored at 220 8C. ACTH was radioimmunoassayed by commercially available kits: ACTH IRMA (Mitsubishi Chemical, Tokyo, Japan). The sensitivity of the assay was 5 pg/ml. After the completion of experiments, the cannula tip was verified to locate in the 3rd ventricle by dye injection through the cannula. Data are expressed as means ^ SE. Statistical analysis was performed by a two-way analysis of variance. A 5% level of probability was accepted as statistically significant. The plasma ACTH responses elicited by an immobilization stress are shown in Fig. 1. At the end of immobilization (0 days), the plasma concentration of ACTH increased significantly (3.9-fold; Fð1;10Þ ¼ 66:5; P , 0:01) as compared with controls. Although the plasma ACTH concentration decreased at 2 days after the stress (Fð1;10Þ ¼ 40:6; P , 0:05), the ACTH level was still significantly higher than the control level (Fð1;10Þ ¼ 6:26; P , 0:05). It was also maintained at a higher level after 6 days of stress as compared with the control level (Fð1;10Þ ¼ 5:71; P , 0:05). The ACTH concentration had returned to the control level at 20 days after the stress exposure (Fð1;10Þ ¼ 1:34; P ¼ 0:27). Fig. 2 shows the effects of OXT antagonist injected into the cerebroventricle on the ACTH responses induced by immobilization stress. The plasma ACTH response to immobilization (0 days) was slightly enhanced by OXT antagonist pretreatment but there was no significant difference (Fð1;10Þ ¼ 0:30; P ¼ 0:59). On the other hand, repeated stress-induced elevation of plasma ACTH was significantly attenuated by the OXT antagonist (Fð1;10Þ ¼ 6:36; P , 0:05), indicating that OXT enhances ACTH secretion in response to acute stress when it is applied repeatedly. Furthermore, the elevated ACTH concentration induced by a stress 2 days before was decreased by pre-stress treatment of OXT antagonist (Fð1;19Þ ¼ 23:6; P , 0:05). The suppressed level of ACTH was not statistically different from the control level (Fð1;19Þ ¼ 4:75; P ¼ 0:054). Intracerebroventricular injection of the OXT antagonist did not cause a significant change in the resting level of plasma ACTH (Fð1;10Þ ¼ 0:13; P ¼ 0:73) compared with saline treatment. The obtained data show that a single immobilization stress enhanced ACTH secretion at least 6 days after single stress and that the elevated ACTH secretion was suppressed at the end of repeated (3 days) stress and 2 days after the single stress by pretreatment with central OXT receptor antagonist. The OXT antagonist did not change the plasma ACTH level at the end of single stress. The results suggest that endogenous brain OXT enhances the long-lasting but not immediate HPA axis response to stress. It has been shown that single exposure to acute stressful stimuli is followed by a rapid increase in plasma ACTH and decline to basal levels, depending on the nature and intensity of the stimulus [1]. These data, however, did not consider the
T. Nakashima et al. / Neuroscience Letters 321 (2002) 161–164
possible involvement of long-lasting effects following single stress and circadian variations of ACTH. In the present study, we collected all blood samples at 12:00 h to avoid the circadian plasma ACTH deviation. Some recent studies have investigated long-lasting changes in the HPA axis following single stress. For example, rats exposed to a single session of footshocks showed, 14 days later, an augmented ACTH response to a novel emotional stimulus [15]. Single administration of IL-1 induces a long-lasting increase of AVP stores in CRH terminals. A second IL-1 challenge at 11 days after IL-1 administration caused a marked increase in ACTH response [14]. Our data support their observations that single stress may lead to long-lasting change in the HPA axis, whereas they did not measure the basal ACTH level at 2 or 6 days after single stress. It is necessary to find out whether the basal ACTH level becomes elevated in response to a single session of footshock or IL-1 administration at 2 or 6 days before. Due to the ir-ACTH kit used in this study, it is possible that we detected not only ACTH but also the ACTH fragment corticotropin-like intermediate peptide (CLIP: ACTH 18–39). Although we do not exclude the possibility, the efficacy of CLIP is still confused because a fragment of CLIP (ACTH 20–24) is also effective on the rapid eye movement sleep episodes. The previous study by Neumann et al. [10] has demonstrated that OXT antagonist injected into the cerebral ventricle enhanced basal and stress-induced secretion of ACTH into blood. No statistical difference was shown between the OXT antagonist treated and saline treated animals in both basal and stress-induced ACTH secretion in this study. One possible reason for the discrepancy may be the difference in the nature of stress. They used elevated plus-maze stress compared with immobilization stress used in this study. The second possibility may depend on the time schedule of experimental protocols. They measured plasma ACTH at 10 min after OXT antagonist injection as the basal state. Although the plasma ACTH level of the OXT antagonist pretreated animal was higher than vehicle treated rats 5 min after the end of stress, no significant difference was shown by OXT antagonist treatment at 15 or 60 min after the end of stress. In contrast to their study, we measured the plasma ACTH concentration of controls 2.5 h after with or without OXT antagonist, and stressinduced ACTH secretions were measured just at the end of or 2 days after stress. Forced swimming and shaker stress were reported to enhance both intra-hypothalamic release of OXT and its secretion into the systemic circulation [11,17]. However, social defeat experience elicited the release within the SON but not peripheral release of OXT [3]. Although a 5day peripheral treatment of OXT without stress did not change the plasma level of ACTH, plasma levels of corticosterone were significantly lower in rats treated with OXT. This effect persisted for at least 10 days after the end of the treatment [13]. Further studies are needed to elucidate the
163
long-lasting central and peripheral OXT effects to stress responses. This work was supported in part by Grant-in-Aid for Scientific Research B12557006 and Special Coordination Funds for Promoting Science and Technology from the Ministry of Education, Culture, Sports, Science and Technology, the Japanese Government. [1] Aguilera, G., Regulation of pituitary ACTH secretion during chronic stress, Front. Neuroendocrinol., 15 (1994) 321–350. [2] Aubry, J.M., Bartanusz, V., Jeziva, D., Belin, D. and Kiss, J.Z., Single stress induces long-lasting elevations in vasopressin mRNA level in CRF hypophysiotrophic neurons, but repeated stress is required to modify AVP immunoreactivity, J. Neuroendocrinol., 11 (1999) 377–384. [3] Engelmann, M., Ebner, K., Landgraf, R., Holsboer, F. and Wotjak, C.T., Emotional stress triggers intrahypothalamic but not peripheral release of oxytocin in male rats, J. Neuroendocrinol., 11 (1999) 867–872. [4] Kuriyama, K., Nakashima, T., Kawarabayashi, T. and Kiyohara, T., Oxytocin inhibits nonphasically firing supraoptic and paraventricular neurons in the virgin female rat, Brain Res. Bull., 31 (1993) 681–687. [5] Kvetnansky, R. and Mikulaj, L., Adrenal and urinary catecholamines in rats during adaptation to repeated immobilization stress, Endocrinology, 87 (1970) 738–743. [6] Makino, S., Smith, M.A. and Gold, P.W., Increased expression of corticotropin-releasing hormone and vasopressin messenger ribonucleic acid (mRNA) in the hypothalamic paraventricular nucleus during repeated stress: association with reduction in glucocorticoid receptor mRNA levels, Endocrinology, 136 (1995) 3299–3309. [7] Miyata, S., Itoh, T., Lin, S.-H., Ishiyama, M., Nakashima, T. and Kiyohara, T., Temporal changes of c-fos expression in oxytocinergic magnocellular neuroendocrine cells of the rat hypothalamus with restraint stress, Brain Res. Bull., 37 (1995) 391–395. [8] Morris, M., Lucion, A.B., Li, P. and Callahan, M.F., Central oxytocin mediates stress-induced tachycardia, J. Neuroendocrinol., 7 (1995) 455–459. [9] Murai, T., Nakashima, T., Miyata, S. and Kiyohara, T., Different effect of oxytocin on membrane potential of supraoptic oxytocin neurons in virgin female and male rats in vitro, Neurosci. Res., 30 (1998) 35–41. [10] Neumann, I.D., Wigger, A., Torner, L., Holsboer, F. and Landgraf, R., Brain oxytocin inhibits basal and stressinduced activity of the hypothalamo-pituitary–adrenal axis in male and female rats: partial action within the paraventricular nucleus, J. Neuroendocrinol., 12 (2000) 235–243. [11] Nishioka, T., Anselmo-Franci, J.A., Li, P., Callahan, M.F. and Morris, M., Stress increases oxytocin release within the hypothalamic paraventricular nucleus, Brain Res., 781 (1998) 57–61. [12] Paxinos, G. and Watson, C., The Rat Brain in Stereotaxic Coordinates, Academic Press, San Diego, CA, 1986. [13] Peterson, M., Hulting, A. and Uvna¨s-Moberg, K., Oxytocin causes a sustained decrease in plasma levels of corticosterone in rats, Neurosci. Lett., 264 (1999) 41–44. [14] Schmidt, E.D., Janszen, A.W.J.W., Wouterlood, F.G. and Tilders, F.J.H., Interleukin-1-induced long-lasting changes in hypothalamic corticotropin-releasing hormone (CRH) neurons and hyperresponsiveness of the hypothalamus– pituitary–adrenal axis, J. Neurosci., 15 (1995) 7417–7426. [15] Van Dijken, H.H., de Goeij, D.C.E., Sutanto, W., Mos, J., de
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