- Email: [email protected]
Swim stress triggers the release of vasopressin within the suprachiasmatic nucleus of male rats
Brain Research 792 Ž1998. 343–347
Short communication
Swim stress triggers the release of vasopressin within the suprachiasmatic nucleus of male rats Mario Engelmann ) , Karl Ebner, Rainer Landgraf, Carsten T. Wotjak Max Planck Institute of Psychiatry, Munich, Germany Accepted 24 February 1998
Abstract The hypothalamic suprachiasmatic nucleus ŽSCN. is the predominant pacemaker of the mammalian brain that generates and controls circadian rhythms of various endocrine and behavioral processes. Different lines of evidence suggest that stress interferes with the maintenance of such rhythms. As a first approach to investigate whether the neuropeptide arginine vasopressin ŽAVP., which shows circadian rhythms of synthesis and release within the SCN, might contribute to this stress-induced alterations in circadian rhythms, we monitored acute effects of swim stress on the intra-SCN release of AVP in male rats by means of the microdialysis technique. A 10-min forced swimming session triggered a marked but relatively short-lasting increase in the intranuclear release of AVP Žto approx. 440%.. This effect was restricted to the area containing predominantly somata and dendrites of vasopressinergic neurons, since no changes in AVP release could be measured in one of their major projection areas, the nucleus of the dorsomedial hypothalamus. Our data provide evidence that the amount of AVP released within the SCN can vary widely not only in accordance with AVP’s intrinsically regulated circadian rhythm but also in response to a physiologically relevant stressor. In this way, the neuropeptide may contribute to the regulation of endocrine and behavioral rhythms particularly in challenging situations associated with resettings of the endogenous clock. q 1998 Elsevier Science B.V. Keywords: Circadian rhythm; Dorsomedial hypothalamus; Microdialysis
Common to central nervous systems of invertebrates and vertebrates is the intrinsic ability of distinct nerve cells to generate rhythms of activity. Although in vitro studies have shown that this ability is primarily independent of neuronal inputs w6,24x, in vivo the resetting of the respective endogenous clock is under the control of various neuronal afferents. Within the mammalian brain, for instance, the neurons of the suprachiasmatic nucleus ŽSCN., the predominant pacemakers for the generation of circadian rhythms of a variety of endocrine and behavioral processes, receive input from the retina w8,11x. Via the retina, light stimuli are known to control the activity of SCN neurons w28x. However, entrainment of the biological clock is possible not only with photic but also with nonphotic cues such as novelty-induced wheel running w22x and social interactions w21x. Additional evidence for an
) Corresponding author. Max Planck Institute of Psychiatry, Kraepelinstr. 2, D-80804 Munich, Germany. Fax: q49-89-30622-569; E-mail: [email protected]
0006-8993r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved. PII S 0 0 0 6 - 8 9 9 3 Ž 9 8 . 0 0 2 4 3 - 1
external influence on the endogenous clock comes from the field of stress research. The experience of stressful events may cause complex responses of the organism, including arhythmicities and desynchronizations in circadian rhythms in animals w17,18,29x and humans w4x. So far, little is known about the mechanisms underlying entrainments of the biological clock and stress-induced alterations in circadian rhythms. In particular, there is only sparse information on the extent to which neurons of the SCN are themselves actively involved. In this context, a single study investigated the influence of glucocorticoids on the diurnal expression of selected neuropeptides within the SCN. It could be shown that these steroid hormones influence the expression of arginine vasopressin ŽAVP. during a narrow window of time in the diurnal cycle coinciding with the time when the entrainment of the circadian pacemaker with non-photic cues is possible w16,21–23,27x. Under basal conditions, both the synthesis and the release of AVP within the SCN follow a circadian rhythm, with the intranuclear release reaching its peak level approx. 6 h after lights on Žzeitgeber time, ZT6 w12x.,
344
M. Engelmann et al.r Brain Research 792 (1998) 343–347
i.e., during the time period when glucocorticoids can affect the synthesis of this neuropeptide. Thus, taking into consideration the fact that AVP influences the firing activity of suprachiasmatic neurons w19x, stress-induced alterations in circadian rhythms might be mediated at least in part by the effects of glucocorticoids on the vasopressinergic neurons. In addition to these relatively long-lasting effects, stress could also have acute effects leading to an adjustment of endocrine and behavioral rhythms to the actual demands of the challenging situation. In this context, we were particularly interested in determining whether stress acutely affects the intra-SCN release of AVP. To address this issue, in the present study we monitored the effects of forced swimming on the release of AVP within the SCN in rats between ZT3 and ZT6. Moreover, because AVP released from axon terminals of suprachiasmatic neurons in the nucleus of the dorsomedial hypothalamus ŽDMH, w31x. seems to be critically involved in the regulation of the animals’ endocrine stress responses w1x, we also simultaneously monitored the effects of swim stress on AVP release within the DMH. Adult male Wistar rats Ž320–340 g b.wt. were purchased from a commercial supplier ŽCharles River, Sulzfeld, Germany.. Unless otherwise stated, the animals were housed in groups of 3–5 in standard rat cages under standard laboratory conditions Ži.e., 12 hr12 h lightrdark rhythm with lights on at 0700 h wZT0x; food and tap water ad lib.. For microdialysis experiments, the animals were implanted with microdialysis probes under halothane anesthesia as described in detail elsewhere w33x. Briefly, anesthetized rats were fixed in a stereotaxic frame and the microdialysis probe ŽU-shaped, home-made w25x. was implanted according to the coordinates of a stereotaxic atlas w26x into either the SCN Ž1.4 mm rostral to bregma, 1.1 mm lateral from midline, 9.9 mm beneath the surface of the skull, angle of 108 to the sagittal plane, nose bar at 5.0 mm. or the DMH Ž3.1 mm caudal to bregma, 1.7 mm, 9.4 mm, nose bar at y3.5 mm.. The implantation coordinates were chosen to minimize the risk of touching other brain nuclei known to contain large amounts of AVP. After surgery, the animals were housed singly and allowed to recover for 2 days. At 0800 h Ži.e., at ZT1. of the experimental day, the microdialysis probes were connected to milliliter syringes via pieces of PE-20 tubing and constantly perfused with 3.3 m lrmin of Ringer’s solution ŽFresenius, Bad Homburg, Germany.. After a 2-h period, which was allowed to establish an equilibrium between inside and outside of the probe, six 30-min microdialysates were collected directly into Eppendorf vials. During the third dialysis interval Ži.e., at ZT4., rats were forced to swim for 10 min in a Plexiglas cylinder that was 30 cm in diameter and 50 cm high and filled with tap water Ž208C " 18C. to a height of 35 cm. After swimming, the animals were removed from the tanks, carefully dried with a towel
and returned to their home cages. The microdialysates were transferred onto dry ice, lyophilized and stored at y208C until measurement of the AVP content w25x. All samples were measured in the same assay. At the end of the experiment, the animals were killed by an overdose of halothane. Their brains were removed and shock-frozen in dry ice-chilled n-methylbutane and processed for standard histology. After histological examination Žbut before radioimmunological analysis. the animals were assigned to one of two groups according to the localization of the tip of the microdialysis probe within Žhits; Fig. 1A and B. or outside Žoutsiders. the respective nucleus. Data are expressed as the percentage of the averaged two pre-stress values and are presented as means " SEM. Data were transformed by arc-tangent to fit into a Gaussian distribution and were submitted to a one-way ANOVA for repeated measures. Post hoc analysis with Fisher’s LSD test was done, if appropriate. Significance was set at P - 0.05. In only 6 out of 12 animals the microdialysis probe was judged to have hit the SCN ŽFig. 1A, Table 1.. As depicted in Fig. 1C, forced swimming caused a significant increase in AVP content in microdialysates collected from the SCN Žto 438% of the averaged baseline values, F5,25 s 3.29, P s 0.02; Table 1.. In contrast, it did not have a significant effect on the AVP content in microdialysates obtained from outsiders Ž F5,25 s 1.80, P s 0.15., independently of whether the microdialysis probe was localized dorsally, laterally or caudally to the nucleus Ždata not shown.. AVP was also clearly detectable in dialysates collected from the DMH under pre-stress conditions ŽTable 1.. However, forced swimming did not significantly influence the release of AVP within this nucleus Ž F5,30 s 1.40, P s 0.251; Fig. 1D.. Thus, our results demonstrate for the first time that an ethologically relevant stressor triggers a marked albeit brief increase in the release of AVP within the SCN without affecting the release of AVP within the DMH. Diurnal patterns of intra-SCN release of AVP have been observed in vitro w6x and in vivo w12x. As shown by the addition of depolarizing agents to the dialysis medium under in vivo conditions, the intra-SCN release of AVP can be potentiated even during the peak of the endogenous rhythm ŽZT4 and ZT5 w14x. and does not simply follow the circadian rhythm of AVP synthesis w2x by constitutive exocytosis. These findings are supported by the present study, which demonstrates a significant effect of swim stress on the intra-SCN release of AVP. As the relative increase in the stress-induced AVP release found here Žto approx. 440%; Fig. 1C. is similar to that after Kq stimulation Žto approx. 400% w14x., a 10-min swimming session has to be considered a powerful activator of SCN neurons under physiological conditions. This finding gains additional importance because the experiments were performed at ZT4, a time point at which Ži. relatively high pre-stress levels close to the diurnal peak have to be expected w12x
M. Engelmann et al.r Brain Research 792 (1998) 343–347
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Fig. 1. ŽA, B. Schematic drawings showing the reconstructed localizations of the dialysis membranes of those microdialysis probes judged to have hit either the suprachiasmatic nucleus ŽSCN; A. or the dorsomedial hypothalamus ŽDMH; B. ŽOX: optical chiasm, 3V: third ventricle.. ŽC, D. Effect of swim stress on AVP content of 30-min microdialysates obtained from either the SCN ŽC. or the DMH ŽD.. Data are expressed as the percentage of the two averaged baseline values Žsamples 1 and 2; 100%, dotted line.. Rats were exposed to a 10-min forced swimming session at the beginning of the third dialysis interval. ) P - 0.01 vs. samples 1, 2 and 6 and P - 0.05 vs. sample 5 ŽANOVA followed by Fisher’s LSD..
and Žii. an entrainment of the circadian pacemaker with non-photic cues is possible w21–23,27x. The data obtained in the outsiders ŽFig. 1C. clearly indicate that the release pattern observed is related to the release of the neuropeptide within the SCN, i.e., is not contaminated by AVP released in other hypothalamic nuclei located close to the SCN w34x. Within the SCN, AVP is likely to be released not only from axon terminals but also from cell bodies and dendrites of vasopressinergic neurons w3x, which have been shown to be located within the dorsomedial part of the SCN w30x. In the second part of the study, we investigated whether forced swimming affects the release of AVP from axon terminals of suprachiasmatic neurons located in the DMH. We chose this brain area because Ži. the majority of the
vasopressinergic neurons of the SCN project to this nucleus w31,32x, Žii. vasopressinergic innervation from other brain areas can be ignored w10x and Žiii. AVP originating Table 1 Averaged basal values of AVP content measured in microdialysates obtained from different brain regions Brain area
pgrsample
No. of animals
SCN Žhits. SCN Žoutsider, dorsal. SCN Žoutsider, lateral. SCN Žoutsider, caudal. DMH Žhits.
2.47"0.74 2.04 2.16 0.62 0.72"0.18
6 2 2 2 6
Abbreviations: SCN, suprachiasmatic nucleus; DMH dorsomedial hypothalamus.
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from the SCN and terminating in the DMH may participate in the regulation of corticosterone secretion w13x. As shown in Fig. 1D, we failed to detect stress-related changes in the release of AVP within this target area. Technical limitations are not likely to be responsible for this failure, as basal release in the DMH was measured in the very sensitive part of the standard curve of the radioimmunoassay, where even subtle alterations are clearly detectable. The AVP release from predominantly axon terminals in the DMH might be reflected by microdialysis with lower precision than, for instance, dendritic release patterns observed within hypothalamic nuclei w15x. However, this is not likely to be a major problem as the release of AVP from axon terminals in the septum was found to be significantly increased in response to forced swimming in a study using the same methodological approach w7x. Thus, one has to consider the possibility that the release of AVP within the SCN on the one hand and from axon terminals in the DMH on the other can be differentially regulated. Although at first glance this seems unlikely, we recently observed this phenomenon in magnocellular vasopressinergic neurons of the hypothalamic supraoptic and paraventricular nuclei during swim stress w34x. At the moment one can only speculate about the biological significance of the stress-induced release of AVP within the SCN. It has been demonstrated that administration of synthetic AVP excites neurons of the SCN w19x whereas administration of an AVP V1 receptor antagonist reduces their spontaneous activity w20x. Thus, intra-SCNreleased AVP may contribute to its own regulation and to the regulation of other neuroendocrine processes by influencing SCN neurons, which then affect neurons of the supraoptic w5x and paraventricular w9x nuclei, two central components of the hypothalamic-neurohypophysial and the hypothalamic-pituitary-adrenocortical systems w1x. In this way, AVP could be involved in the regulation of these neuroendocrine systems thereby contributing to longerlasting effects of stress on endocrine and behavioral rhythms observed, for instance, after excessive social defeat experience in rats w17,18,29x. In addition, AVP may be a candidate for mediating the entrainment of the circadian pacemaker at the level of the SCN. In summary, the results of the present study demonstrate that the release of AVP within the SCN, but not the DMH, varies widely not only following its intrinsically regulated circadian rhythm but also acutely in response to an ethologically relevant stressor. The marked and brief stress-induced release suggests that intra-SCN-released AVP contributes to the adaptation of endocrine and behavioral rhythms to the acute demands of challenging situations, including entrainments of the circadian pacemaker. To test this hypothesis, future studies have to monitor the effects of defined stressor exposure on the intra-SCN release of AVP and effects of local administration of selective AVP receptor antagonists.
Acknowledgements The authors thank Gabriele Kohl for excellent technical assistance. This work was supported by the Deutsche Forschungsgemeinschaft. References w1x R.M. Buijs, J. Wortel, J.J. van Heerikhuize, A. Kalsbeek, Novel environment induced inhibition of corticosterone secretion: physiological evidence for a suprachiasmatic nucleus mediated neuronal hypothalamo-adrenal cortex pathway, Brain Res. 758 Ž1997. 229– 236. w2x J.P.H. Burbach, B. Liu, T.A.M. Voorhuis, H.H.M. van Tol, Diurnal variation in vasopressin and oxytocin messenger RNAs in hypothalamic nuclei of the rat, Brain Res. Mol. Brain Res. 4 Ž1988. 157–160. w3x M. Castel, J. Morris, M. Belenky, Non-synaptic and dendritic exocytosis from dense-core vesicles in the suprachiasmatic nucleus, Neuroreport 7 Ž1996. 543–547. w4x S. Checkley, The relationship between biological rhythms and the affective disorders, in: J. Arendt, D.S. Minors, J.M. Waterhouse ŽEds.., Biological Rhythms in Clinical Practice. Wright, London, 1989, pp. 160–183. w5x L.-N. Cui, K. Saeb-Parsy, R.E.J. Dyball, Neurones in the supraoptic nucleus of the rat are regulated by a projection from the suprachiasmatic nucleus, J. Physiol. 502 Ž1997. 149–159. w6x D.J. Earnest, C.D. Sladek, Circadian rhythms of vasopressin release from individual rat suprachiasmatic explants in vitro, Brain Res. 382 Ž1986. 129–133. w7x K. Ebner, C.T. Wotjak, R. Landgraf, M. Engelmann, Swim stress triggers the release of vasopressin within the septum in male rats, Exp. Brain Res. 117 Ž1997. S27, ŽAbstract.. w8x A.E. Hendrickson, N. Wagoner, W.M. Cowan, An autoradiographic and electron microscopic study of retino-hypothalamic connections, Z. Zellforsch. 135 Ž1972. 1–26. w9x M.L.H.J. Hermes, E.M. Coderre, R.M. Buijs, L.P. Renaud, GABA and glutamate mediate rapid neurotransmission from suprachiasmatic nucleus to hypothalamic paraventricular nucleus in rat, J. Physiol. 496 Ž1996. 749–757. w10x E.M.D. Hoorneman, R.M. Buijs, Vasopressin fiber pathways in the rat brain following suprachiasmatic nucleus lesioning, Brain Res. 243 Ž1982. 235–241. w11x Y. Ibata, Y. Takahashi, H. Okamura, F. Kawakami, H. Terubayashi, T. Kubo, N. Yanaihara, Vasoactive intestinal peptide ŽVIP.-like immunoreactive neurons located in the rat suprachiasmatic nucleus receive a direct retinal projection, Neurosci. Lett. 97 Ž1989. 1–5. w12x A. Kalsbeek, R.M. Buijs, M. Engelmann, C.T. Wotjak, R. Landgraf, In vivo measurement of a diurnal variation in vasopressin release in the rat suprachiasmatic nucleus, Brain Res. 682 Ž1995. 75–82. w13x A. Kalsbeek, J.J. van Heerikhuize, J. Wortel, R.M. Buijs, A diurnal rhythm of stimulatory input to the hypothalamo–pituitary–adrenal system as revealed by timed intrahypothalamic administration of the vasopressin V1 antagonist, J. Neurosci. 16 Ž1996. 5555–5565. w14x M. Kubota, R. Landgraf, C.T. Wotjak, Release of vasopressin within the rat suprachiasmatic nucleus: no effect of a V1rV2 antagonist, Neuroreport 7 Ž1996. 1933–1936. w15x R. Landgraf, Intracerebrally released vasopressin and oxytocin: measurement, mechanisms and behavioural consequences, J. Neuroendocrinol. 7 Ž1995. 243–253. w16x P.J. Larsen, N. Vrang, M. Moller, D.S. Jessop, S.L. Lightman, H.S. Chowdrey, J.D. Mikkelsen, The diurnal expression of genes encoding vasopressin and vasoactive intestinal peptide within the rat suprachiasmatic nucleus is influenced by circulating glucocorticoids, Mol. Brain Res. 27 Ž1994. 342–346.
M. Engelmann et al.r Brain Research 792 (1998) 343–347 w17x P. Meerlo, S.F. de Boer, J.M. Koolhaas, S. Daan, R.H. van den Hoofdakker, Changes in daily rhythms of body temperature and activity after a single social defeat in rats, Physiol. Behav. 59 Ž1996. 735–739. w18x P. Meerlo, G.J.F. Overkamp, M.A. Benning, J.M. Koolhaas, R.H. van den Hoofdakker, Long-term changes in open field behaviour following a single social defeat in rats can be reversed by sleep deprivation, Physiol. Behav. 60 Ž1996. 115–119. w19x R. Mihai, M. Coculescu, J.B. Wakerley, C.D. Ingram, The effects of wArg 8 xvasopressin and wArg 8 xvasotocin on the firing rate of suprachiasmatic neurons in vitro, Neuroscience 62 Ž1994. 783–792. w20x R. Mihai, T.S. Juss, C.D. Ingram, Supression of suprachiasmatic nucleus neurone activity with a vasopressin receptor antagonist: possible role for endogenous vasopressin in circadian activity cycles in vitro, Neurosci. Lett. 179 Ž1994. 95–99. w21x N. Mrosovsky, Phase response curves for social entrainment, J. Comp. Physiol. 162 Ž1988. 35–46. w22x N. Mrosovsky, P. Salmon, A behavioral method for accelerating re-entrainment of rhythms to new light–dark cycles, Nature 330 Ž1987. 372–373. w23x N. Mrosovsky, S.G. Reebs, G.I. Honrado, P.A. Salmon, Behavioural entrainment of circadian rhythms, Experientia 45 Ž1989. 696–702. w24x N. Murakami, M. Takamure, K. Takahashi, K. Utunomiya, H. Kuroda, T. Etoh, Long-term cultured neurons from rat suprachiasmatic nucleus retain the capacity for circadian oscillation of vasopressin release, Brain Res. 545 Ž1991. 347–350. w25x I. Neumann, M. Ludwig, M. Engelmann, Q.J. Pittman, R. Landgraf, Simultaneous microdialysis in blood and brain: oxytocin and vasopressin release in response to central and peripheral osmotic stimulation and suckling in the rat, Neuroendocrinology 58 Ž1993. 637–645. w26x G. Paxinos, C. Watson, The Rat Brain in Stereotaxic Coordinates, Academic Press, Sydney, 1986. w27x S.G. Reebs, R.J. Lavery, N. Mrosovsky, Running activity mediates
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the phase-advancing effects of dark pulses on hamster circadian rhythms, J. Comp. Physiol. 165 Ž1989. 811–818. K. Shinohara, K. Tominaga, Y. Isobe, S.-I.T. Inouye, Photic regulation of peptides located in the ventrolateral subdivision of the suprachiasmatic nucleus of the rat: daily variations of vasoactive intestinal polypeptide, gastrin-releasing peptide, and neuropeptide Y, J. Neurosci. 13 Ž1993. 793–800. W. Tornatzky, K.A. Micek, Long-term impairment of autonomic circadian rhythms after brief intermittent social stress, Physiol. Behav. 53 Ž1993. 983–993. A.N. van den Pol, Gamma-aminobutyrate, gastrin releasing peptide, serotonin, somatostatin, and vasopressin: ultrastructural immunocytochemical localization in presynaptic axons in the suprachiasmatic nucleus, Neuroscience 17 Ž1986. 643–659. A.G. Watts, L.W. Swanson, Efferent projections of the suprachiasmatic nucleus: II. Studies using retrograde transport of fluorescent dyes and simultaneous peptide immunohistochemistry in the rat, J. Comp. Neurol. 258 Ž1987. 230–252. A.G. Watts, L.W. Swanson, G. Sanchez-Watts, Efferent projections of the suprachiasmatic nucleus: I. Studies using anterograde transport of Phaseolus Õulgaris leucoagglutinin in the rat, J. Comp. Neurol. 258 Ž1987. 204–229. C.T. Wotjak, M. Kubota, G. Liebsch, A. Montkowski, F. Holsboer, I. Neumann, R. Landgraf, Release of vasopressin within the rat paraventricular nucleus in response to emotional stress: a novel mechanism of regulating adrenocorticotropic hormone secretion?, J. Neurosci. 16 Ž1996. 7725–7732. C.T. Wotjak, J. Ganster, G. Kohl, F. Holsboer, R. Landgraf, M. Engelmann, Dissociated central and peripheral release of vasopressin, but not oxytocin, in response to repeated swim stress: new insights into the regulatory capacities of peptidergic neurons, Neuroscience 84 Ž1998. .
Short communication
Swim stress triggers the release of vasopressin within the suprachiasmatic nucleus of male rats Mario Engelmann ) , Karl Ebner, Rainer Landgraf, Carsten T. Wotjak Max Planck Institute of Psychiatry, Munich, Germany Accepted 24 February 1998
Abstract The hypothalamic suprachiasmatic nucleus ŽSCN. is the predominant pacemaker of the mammalian brain that generates and controls circadian rhythms of various endocrine and behavioral processes. Different lines of evidence suggest that stress interferes with the maintenance of such rhythms. As a first approach to investigate whether the neuropeptide arginine vasopressin ŽAVP., which shows circadian rhythms of synthesis and release within the SCN, might contribute to this stress-induced alterations in circadian rhythms, we monitored acute effects of swim stress on the intra-SCN release of AVP in male rats by means of the microdialysis technique. A 10-min forced swimming session triggered a marked but relatively short-lasting increase in the intranuclear release of AVP Žto approx. 440%.. This effect was restricted to the area containing predominantly somata and dendrites of vasopressinergic neurons, since no changes in AVP release could be measured in one of their major projection areas, the nucleus of the dorsomedial hypothalamus. Our data provide evidence that the amount of AVP released within the SCN can vary widely not only in accordance with AVP’s intrinsically regulated circadian rhythm but also in response to a physiologically relevant stressor. In this way, the neuropeptide may contribute to the regulation of endocrine and behavioral rhythms particularly in challenging situations associated with resettings of the endogenous clock. q 1998 Elsevier Science B.V. Keywords: Circadian rhythm; Dorsomedial hypothalamus; Microdialysis
Common to central nervous systems of invertebrates and vertebrates is the intrinsic ability of distinct nerve cells to generate rhythms of activity. Although in vitro studies have shown that this ability is primarily independent of neuronal inputs w6,24x, in vivo the resetting of the respective endogenous clock is under the control of various neuronal afferents. Within the mammalian brain, for instance, the neurons of the suprachiasmatic nucleus ŽSCN., the predominant pacemakers for the generation of circadian rhythms of a variety of endocrine and behavioral processes, receive input from the retina w8,11x. Via the retina, light stimuli are known to control the activity of SCN neurons w28x. However, entrainment of the biological clock is possible not only with photic but also with nonphotic cues such as novelty-induced wheel running w22x and social interactions w21x. Additional evidence for an
) Corresponding author. Max Planck Institute of Psychiatry, Kraepelinstr. 2, D-80804 Munich, Germany. Fax: q49-89-30622-569; E-mail: [email protected]
0006-8993r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved. PII S 0 0 0 6 - 8 9 9 3 Ž 9 8 . 0 0 2 4 3 - 1
external influence on the endogenous clock comes from the field of stress research. The experience of stressful events may cause complex responses of the organism, including arhythmicities and desynchronizations in circadian rhythms in animals w17,18,29x and humans w4x. So far, little is known about the mechanisms underlying entrainments of the biological clock and stress-induced alterations in circadian rhythms. In particular, there is only sparse information on the extent to which neurons of the SCN are themselves actively involved. In this context, a single study investigated the influence of glucocorticoids on the diurnal expression of selected neuropeptides within the SCN. It could be shown that these steroid hormones influence the expression of arginine vasopressin ŽAVP. during a narrow window of time in the diurnal cycle coinciding with the time when the entrainment of the circadian pacemaker with non-photic cues is possible w16,21–23,27x. Under basal conditions, both the synthesis and the release of AVP within the SCN follow a circadian rhythm, with the intranuclear release reaching its peak level approx. 6 h after lights on Žzeitgeber time, ZT6 w12x.,
344
M. Engelmann et al.r Brain Research 792 (1998) 343–347
i.e., during the time period when glucocorticoids can affect the synthesis of this neuropeptide. Thus, taking into consideration the fact that AVP influences the firing activity of suprachiasmatic neurons w19x, stress-induced alterations in circadian rhythms might be mediated at least in part by the effects of glucocorticoids on the vasopressinergic neurons. In addition to these relatively long-lasting effects, stress could also have acute effects leading to an adjustment of endocrine and behavioral rhythms to the actual demands of the challenging situation. In this context, we were particularly interested in determining whether stress acutely affects the intra-SCN release of AVP. To address this issue, in the present study we monitored the effects of forced swimming on the release of AVP within the SCN in rats between ZT3 and ZT6. Moreover, because AVP released from axon terminals of suprachiasmatic neurons in the nucleus of the dorsomedial hypothalamus ŽDMH, w31x. seems to be critically involved in the regulation of the animals’ endocrine stress responses w1x, we also simultaneously monitored the effects of swim stress on AVP release within the DMH. Adult male Wistar rats Ž320–340 g b.wt. were purchased from a commercial supplier ŽCharles River, Sulzfeld, Germany.. Unless otherwise stated, the animals were housed in groups of 3–5 in standard rat cages under standard laboratory conditions Ži.e., 12 hr12 h lightrdark rhythm with lights on at 0700 h wZT0x; food and tap water ad lib.. For microdialysis experiments, the animals were implanted with microdialysis probes under halothane anesthesia as described in detail elsewhere w33x. Briefly, anesthetized rats were fixed in a stereotaxic frame and the microdialysis probe ŽU-shaped, home-made w25x. was implanted according to the coordinates of a stereotaxic atlas w26x into either the SCN Ž1.4 mm rostral to bregma, 1.1 mm lateral from midline, 9.9 mm beneath the surface of the skull, angle of 108 to the sagittal plane, nose bar at 5.0 mm. or the DMH Ž3.1 mm caudal to bregma, 1.7 mm, 9.4 mm, nose bar at y3.5 mm.. The implantation coordinates were chosen to minimize the risk of touching other brain nuclei known to contain large amounts of AVP. After surgery, the animals were housed singly and allowed to recover for 2 days. At 0800 h Ži.e., at ZT1. of the experimental day, the microdialysis probes were connected to milliliter syringes via pieces of PE-20 tubing and constantly perfused with 3.3 m lrmin of Ringer’s solution ŽFresenius, Bad Homburg, Germany.. After a 2-h period, which was allowed to establish an equilibrium between inside and outside of the probe, six 30-min microdialysates were collected directly into Eppendorf vials. During the third dialysis interval Ži.e., at ZT4., rats were forced to swim for 10 min in a Plexiglas cylinder that was 30 cm in diameter and 50 cm high and filled with tap water Ž208C " 18C. to a height of 35 cm. After swimming, the animals were removed from the tanks, carefully dried with a towel
and returned to their home cages. The microdialysates were transferred onto dry ice, lyophilized and stored at y208C until measurement of the AVP content w25x. All samples were measured in the same assay. At the end of the experiment, the animals were killed by an overdose of halothane. Their brains were removed and shock-frozen in dry ice-chilled n-methylbutane and processed for standard histology. After histological examination Žbut before radioimmunological analysis. the animals were assigned to one of two groups according to the localization of the tip of the microdialysis probe within Žhits; Fig. 1A and B. or outside Žoutsiders. the respective nucleus. Data are expressed as the percentage of the averaged two pre-stress values and are presented as means " SEM. Data were transformed by arc-tangent to fit into a Gaussian distribution and were submitted to a one-way ANOVA for repeated measures. Post hoc analysis with Fisher’s LSD test was done, if appropriate. Significance was set at P - 0.05. In only 6 out of 12 animals the microdialysis probe was judged to have hit the SCN ŽFig. 1A, Table 1.. As depicted in Fig. 1C, forced swimming caused a significant increase in AVP content in microdialysates collected from the SCN Žto 438% of the averaged baseline values, F5,25 s 3.29, P s 0.02; Table 1.. In contrast, it did not have a significant effect on the AVP content in microdialysates obtained from outsiders Ž F5,25 s 1.80, P s 0.15., independently of whether the microdialysis probe was localized dorsally, laterally or caudally to the nucleus Ždata not shown.. AVP was also clearly detectable in dialysates collected from the DMH under pre-stress conditions ŽTable 1.. However, forced swimming did not significantly influence the release of AVP within this nucleus Ž F5,30 s 1.40, P s 0.251; Fig. 1D.. Thus, our results demonstrate for the first time that an ethologically relevant stressor triggers a marked albeit brief increase in the release of AVP within the SCN without affecting the release of AVP within the DMH. Diurnal patterns of intra-SCN release of AVP have been observed in vitro w6x and in vivo w12x. As shown by the addition of depolarizing agents to the dialysis medium under in vivo conditions, the intra-SCN release of AVP can be potentiated even during the peak of the endogenous rhythm ŽZT4 and ZT5 w14x. and does not simply follow the circadian rhythm of AVP synthesis w2x by constitutive exocytosis. These findings are supported by the present study, which demonstrates a significant effect of swim stress on the intra-SCN release of AVP. As the relative increase in the stress-induced AVP release found here Žto approx. 440%; Fig. 1C. is similar to that after Kq stimulation Žto approx. 400% w14x., a 10-min swimming session has to be considered a powerful activator of SCN neurons under physiological conditions. This finding gains additional importance because the experiments were performed at ZT4, a time point at which Ži. relatively high pre-stress levels close to the diurnal peak have to be expected w12x
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Fig. 1. ŽA, B. Schematic drawings showing the reconstructed localizations of the dialysis membranes of those microdialysis probes judged to have hit either the suprachiasmatic nucleus ŽSCN; A. or the dorsomedial hypothalamus ŽDMH; B. ŽOX: optical chiasm, 3V: third ventricle.. ŽC, D. Effect of swim stress on AVP content of 30-min microdialysates obtained from either the SCN ŽC. or the DMH ŽD.. Data are expressed as the percentage of the two averaged baseline values Žsamples 1 and 2; 100%, dotted line.. Rats were exposed to a 10-min forced swimming session at the beginning of the third dialysis interval. ) P - 0.01 vs. samples 1, 2 and 6 and P - 0.05 vs. sample 5 ŽANOVA followed by Fisher’s LSD..
and Žii. an entrainment of the circadian pacemaker with non-photic cues is possible w21–23,27x. The data obtained in the outsiders ŽFig. 1C. clearly indicate that the release pattern observed is related to the release of the neuropeptide within the SCN, i.e., is not contaminated by AVP released in other hypothalamic nuclei located close to the SCN w34x. Within the SCN, AVP is likely to be released not only from axon terminals but also from cell bodies and dendrites of vasopressinergic neurons w3x, which have been shown to be located within the dorsomedial part of the SCN w30x. In the second part of the study, we investigated whether forced swimming affects the release of AVP from axon terminals of suprachiasmatic neurons located in the DMH. We chose this brain area because Ži. the majority of the
vasopressinergic neurons of the SCN project to this nucleus w31,32x, Žii. vasopressinergic innervation from other brain areas can be ignored w10x and Žiii. AVP originating Table 1 Averaged basal values of AVP content measured in microdialysates obtained from different brain regions Brain area
pgrsample
No. of animals
SCN Žhits. SCN Žoutsider, dorsal. SCN Žoutsider, lateral. SCN Žoutsider, caudal. DMH Žhits.
2.47"0.74 2.04 2.16 0.62 0.72"0.18
6 2 2 2 6
Abbreviations: SCN, suprachiasmatic nucleus; DMH dorsomedial hypothalamus.
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from the SCN and terminating in the DMH may participate in the regulation of corticosterone secretion w13x. As shown in Fig. 1D, we failed to detect stress-related changes in the release of AVP within this target area. Technical limitations are not likely to be responsible for this failure, as basal release in the DMH was measured in the very sensitive part of the standard curve of the radioimmunoassay, where even subtle alterations are clearly detectable. The AVP release from predominantly axon terminals in the DMH might be reflected by microdialysis with lower precision than, for instance, dendritic release patterns observed within hypothalamic nuclei w15x. However, this is not likely to be a major problem as the release of AVP from axon terminals in the septum was found to be significantly increased in response to forced swimming in a study using the same methodological approach w7x. Thus, one has to consider the possibility that the release of AVP within the SCN on the one hand and from axon terminals in the DMH on the other can be differentially regulated. Although at first glance this seems unlikely, we recently observed this phenomenon in magnocellular vasopressinergic neurons of the hypothalamic supraoptic and paraventricular nuclei during swim stress w34x. At the moment one can only speculate about the biological significance of the stress-induced release of AVP within the SCN. It has been demonstrated that administration of synthetic AVP excites neurons of the SCN w19x whereas administration of an AVP V1 receptor antagonist reduces their spontaneous activity w20x. Thus, intra-SCNreleased AVP may contribute to its own regulation and to the regulation of other neuroendocrine processes by influencing SCN neurons, which then affect neurons of the supraoptic w5x and paraventricular w9x nuclei, two central components of the hypothalamic-neurohypophysial and the hypothalamic-pituitary-adrenocortical systems w1x. In this way, AVP could be involved in the regulation of these neuroendocrine systems thereby contributing to longerlasting effects of stress on endocrine and behavioral rhythms observed, for instance, after excessive social defeat experience in rats w17,18,29x. In addition, AVP may be a candidate for mediating the entrainment of the circadian pacemaker at the level of the SCN. In summary, the results of the present study demonstrate that the release of AVP within the SCN, but not the DMH, varies widely not only following its intrinsically regulated circadian rhythm but also acutely in response to an ethologically relevant stressor. The marked and brief stress-induced release suggests that intra-SCN-released AVP contributes to the adaptation of endocrine and behavioral rhythms to the acute demands of challenging situations, including entrainments of the circadian pacemaker. To test this hypothesis, future studies have to monitor the effects of defined stressor exposure on the intra-SCN release of AVP and effects of local administration of selective AVP receptor antagonists.
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