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Circadian rhythm of [3H]imipramine binding in the rat suprachiasmatic nuclei
331
European Journal of Pharmacology, 87 (1983) 331-333 Elsevier Biomedical Press
Short communication
C I R C A D I A N R H Y T H M OF [ 3 H I I M I P R A M I N E B I N D I N G IN T H E RAT S U P R A C H I A S M A T I C NUCLEI A N N A WlRZ-JUSTICE, K U R T KRAUCHI, TADAOMI MORIMASA, ROSI WILLENER and HANS FEER
Psychiatrische Universiti~tsklinik, 4025 Basel Switzerland Received 7 December 1982, accepted 9 December 1982
A. WIRZ-JUSTICE, K. KRAUCHI, T. MORIMASA, R. WILLENER and H. FEER, Circadian rhythm of [-~H]imipramine binding in the rat suprachiasmatic nuclei, European J. Pharmacol. 87 (1983) 331-333. High affinity imipramine binding undergoes circadian variations of ca. 35% amplitude in many rat brain nuclei. The suprachiasmatic nuclei of the anterior hypothalamus (considered to be the circadian pacemaker driving many overt rhythms) has highest imipramine binding at the end of the dark and lowest at the end of the light phase. A similar circadian rhythm has previously been observed for serotonin uptake in the suprachiasmatic nuclei. In conjunction with other findings, these data indicate that serotonergic turnover in the suprachiasmatic nuclei decreases at lights on and increases at lights off. Circadian rhythm
Rat suprachiasmatic nuclei
Imipramine binding
1. Introduction
The suprachiasmatic nuclei (SCN) of the anterior hypothalamus are implicated in the generation of many circadian rhythms (Rusak and Zucker, 1979). It is not known which neurotransmitters/ neuromodulators in the SCN are essential for circadian timekeeping: we have reviewed elsewhere the evidence (a) for the existence in high concentrations of m a n y putative neurotransmitters, in particular serotonin (5HT), acetylcholine, and vasopressin; and (b) for their role in the generation or maintenance of circadian rhythmicity (Wirz-Justice et al., 1982). For example, depletion of 5 H T or absence of vasopressin do not affect circadian frequency. However, indirect evidence for an important role for 5 H T comes from neuropharmacology: antidepressant drugs (that increase 5 H T availability, albeit by different mechanisms) can slow circadian frequency (Wirz-Justice et al., 1982). Of particular * To whom all correspondence should be addressed: Psychiatrische Universit~itsklinik, Wilhelm Klein Strasse 27, CH4025 Basel, Switzerland. 0014-2999/83/0000-0000/$03.00 © 1983 Elsevier Biomedical Press
interest is that the tricyclic antidepressant imipramine can induce arrhythmicity when applied directly to the SCN but not in adjacent brain regions (Groos and Mason, 1982; Wirz-Justice et al., 1982). Recent studies have described specific high affinity binding of imipramine to brain homogenates (considered to be related to the 5HT presynaptic reuptake site), with highest density in the hypothalamus (Langer et al., 1982). We now report that imipramine binding in the SCN undergoes circadian variations, and that such rhythms are also present in other brain nuclei.
2. Materials and methods
Male Fiallinsdorf Wistar rats (n = 336, 350 g, 6 - 8 months of age) that had been housed under 12 : 12 lighting conditions (lights on at 6 h), were kept in constant darkness during the day of sacrifice to prevent any direct influence of light. They were rapidly killed by decapitation at 3 h intervals throughout the 24 h, beginning at 11 h, and brains were immediately frozen in liquid nitrogen. Dissection of nuclei from frozen thin
332
sections (1 mm) followed the atlas of KOnig and Klippel. In the hypothalamic regions, pools from 9 animals, in other regions, pools from 3 animals wereused for assays. The tissue punches were homogenised in cold Tris buffer (500 /~1, 50 mM, pH 7.5, containing NaC1 (120 mM) and KCI (50 mM) with a small plastic pestle molded to fit the sample tubes, centrifuged (final volume 1 ml), and the washed pellet resuspended in buffer at a concentration of ca. 1 mg/200 ~1. Imipramine binding was measured by a minimethod modified from Langer et al. (1982) developed to eliminate tissue waste and reduce the amount of filter binding and unspecific binding. Briefly, triplicates of membrane homogenate (200 ~1) were incubated with [3H]imipramine (4.5 nM, 100/~1), with or without cold imipramine (100/LM, 100 /~1) for 1 h at 0°C. Filtration was carried out using Whatman G F / B filters (2.5 cm) that had been punched to a diameter of 1 cm in a filtration port also reduced to a diameter of 0.9 cm. The filter was first washed with 3 ml of Tris buffer containing cold imipramine (100 /~M), and the samples washed 4 × with 3 ml of the same imipramine-containing ice-cold buffer, and the radioactivity measured by liquid scintillation spectrometry. Specific binding of 4.5 nM [3H]imipramine under these conditions represented 80% of the total binding, and there was no specific binding to the filters.
3. Results
Significant circadian rhythms in [3H]imipramine binding were found in many rat brain regions. Imipramine binding in the SCN region was highest at the end of the dark and lowest at the end of the light phase (fig. 1). Further significant rhythms were found in the caudate putamen, medial preoptic area, and frontal cortex, all with similar amplitudes of ca. 35%. There were no significant rhythms in septum or hippocampus. Similar to the SCN, binding of imipramine in the caudate putamen and medial preoptic area was maximal in the dark phase, although peak binding occurred earlier. In the frontal cortex, imipramine
800
/r /r
"Jr
700
600
500
8
11
14
17
20
23
2
5
Fig. I. Specific binding of [3H]imipramine (at 4.5 nM) in membranes from the suprachiasmatic nucleic throughout 24 h. The hatched area indicates the dark phase of the light:dark cycle to which the animals had been adapted: the experiment itself was carried out in the dark to measure an endogenous rhythm. Each point represents the mean of 3 - 4 pools of 9 animals (+S.E.M.). The inter-individual variation coefficient per time point (mean of n = 8) was 10%, the amplitude of peak: trough % was 35%. Significance of the daily changes was established by one-way analysis of variance (FT.iZ = 3.40, P < 0.025); of differences between time points by linear contrasts: ** P < 0.01, * P < 0.05 peak compared with trough at 14 h or 17 h. Ordinate: [3H]imipramine bound ( f m o l / m g protein); abscissa: time of day (h).
binding showed a bimodal rhythm, with peaks in the early dark and early light phase. 24 h mean imipramine binding was highest in the SCN (657 + 17 f m o l / m g protein) and anterior ventral hypothalamus (684 + 24, data incomplete for rhythm characterisation), with lower binding in the septum (361 _+ 6), caudate putamen (285 _+ 6), frontal cortex (243 _+ 7), medial preoptic area (217 _+ 4) and hippocampus (105 _+ 4), n = 29-31 for each region.
4. Discussion
In agreement with previous reports of the existence of circadian rhythms in adrenergic, cholinergic, dopaminergic and opiate receptor binding (summarised in Kafka et al., 1983), we
333
also found endogenous circadian rhythms (persisting under conditions without time cues) in imipramine binding to rat brain homogenates. The wave-form, phase, and amount of imipramine binding varied from region to region. Although we had insufficient tissue for kinetic studies when measuring binding in brain nuclei, no previous receptor rhythms have been associated with a change in affinity to the binding site (op cit.). Furthermore, the imipramine binding rhythms were of similar amplitude (ca. 35%) to that previously observed for other binding rhythms (op cit.). The SCN was chosen for its known role in circadian rhythm generation (Rusak and Zucker, 1979). The timing of peak imipramine binding is therefore as important as the finding of a significant rhythm. If imipramine binding is related to the presynaptic 5HT reuptake site, then the rhythm is parallel to that found by Meyer and Quay (1976), where 5HT uptake in the SCN region was high in the dark and low in the light phase. The 5HT metabolite 5HIAA (often used as an estimate of turnover rate), measured electrochemically in the SCN, is also high in the dark and low in the light phase (Faradji et al., 1983). Endogenous 5HT concentrations in the SCN are, in contrast, high in the light and low in the dark phase (Kordon et al., 1981). Since 5HT is an inhibitory neurotransmitter, these patterns can be understood in terms of functional activity: iontophoretically applied 5HT inhibits SCN firing, and iontophoresis of imipramine also depresses discharge as well as potentiating the 5HT effect (Groos and Mason, 1982). Thus these preliminary findings can be taken together as indicating that neuronal activity in the SCN should increase at lights on (when 5HT release is low) and decrease at lights off (when 5HT release is high). Two recent papers indeed show that single cell firing in SCN slices follows this pattern (Green and Gillette, 1982; Shibata et al., 1982). Although the SCN contains some of the highest concentrations of 5HT in the brain, the role of 5HT in circadian time-keeping is not yet clear. The few neuropharmacological agents that modify circadian frequency also increase 5HT availability (lithium, monoamine-oxidase inhibitors, imipramine), those that decrease 5HT availability (PCPA, 5,6-dihydroxytryptophan, or raphe lesions) do not
(Wirz-Justice et al., 1982). Thus it may be specifically the increased SCN 5HT turnover at dusk that is important in homeostasis of circadian rhythm frequency. Finally, the existence of circadian rhythms in imipramine binding in many brain regions is also of methodological significance. In any interpretation of changes in imipramine binding these considerable and rapid short-term fluctuations should be taken into account.
Acknowledgements We thank Dr. E. Bandle, Hoffmann LaRoche AG, Basel, for providing animals and facilities, and Brigitte Boeglin for joining the night shift.
References Faradji, H., M. Jouvet and R. Cespuglio, 1983, Electrochemical measurement of 5-hydroxyindole compounds in the suprachiasmatic nuclei: circadian fluctuations, Brain Res. (in press). Green, D.J. and R. Gillete, 1982, Circadian rhythm of firing rate recorded from single cells in the rat suprachiasmatic brain slice, Brain Res. 245, 198. Gross, G.A. and R. Mason, 1982, An electrophysiological study of the rat's suprachiasmatic nucleus: a locus for the action of antidepressants, J. Physiol. 330, 40P. Kafka, M.S., A. Wirz-Justice, D. Naber, R.Y. Moore and M.A. Benedito, 1983, Circadian rhythms in rat brain neurotransmitter receptors, Fed. Proc. (in press). Kordon, C., M. H6ry, A. Szafarczyk, G. Ixart and I. Assenmacher, 1981, Serotonin and the regulation of pituitary hormone secretion and of neuroendocrine rhythms, J. Physiol. Paris 77, 489. Langer, S.Z., E. Zarifian, M. Briley, R. Raisman and D. Sechter, 1982, High affinity 3H-imipramine binding: a new biological marker in depression, Pharmacopsychiat. 15, 4. Meyer, D.C. and W.B. Quay, 1976, Hypothalamic and suprachiasmatic uptake of serotonin in vitro: twenty-fourhour changes in male and prooestrus female rats, Endocrinol. 98, 1160. Rusak, B. and I. Zucker, 1979, Neural regulation of circadian rhythms, Physiol. Rev. 59, 449. Shibata, S., Y. Oomura, H. Kita and K. Hattori, 1982, Circadian rhythmic changes of neuronal activity in the suprachiasmatic nucleus of the rat hypothalamic slice, Brain Res. 247, 154. Wirz-Justice, A., G.A. Groos and T.A. Wehr, 1982, The neuropharmacology of circadian timekeeping in mammals, in: Vertebrate Circadian Systems, Structure and Physiology, eds. J. Aschoff, S. Daan and G.A. Groos, Springer Verlag Berlin, Heidelberg, p. 183.
European Journal of Pharmacology, 87 (1983) 331-333 Elsevier Biomedical Press
Short communication
C I R C A D I A N R H Y T H M OF [ 3 H I I M I P R A M I N E B I N D I N G IN T H E RAT S U P R A C H I A S M A T I C NUCLEI A N N A WlRZ-JUSTICE, K U R T KRAUCHI, TADAOMI MORIMASA, ROSI WILLENER and HANS FEER
Psychiatrische Universiti~tsklinik, 4025 Basel Switzerland Received 7 December 1982, accepted 9 December 1982
A. WIRZ-JUSTICE, K. KRAUCHI, T. MORIMASA, R. WILLENER and H. FEER, Circadian rhythm of [-~H]imipramine binding in the rat suprachiasmatic nuclei, European J. Pharmacol. 87 (1983) 331-333. High affinity imipramine binding undergoes circadian variations of ca. 35% amplitude in many rat brain nuclei. The suprachiasmatic nuclei of the anterior hypothalamus (considered to be the circadian pacemaker driving many overt rhythms) has highest imipramine binding at the end of the dark and lowest at the end of the light phase. A similar circadian rhythm has previously been observed for serotonin uptake in the suprachiasmatic nuclei. In conjunction with other findings, these data indicate that serotonergic turnover in the suprachiasmatic nuclei decreases at lights on and increases at lights off. Circadian rhythm
Rat suprachiasmatic nuclei
Imipramine binding
1. Introduction
The suprachiasmatic nuclei (SCN) of the anterior hypothalamus are implicated in the generation of many circadian rhythms (Rusak and Zucker, 1979). It is not known which neurotransmitters/ neuromodulators in the SCN are essential for circadian timekeeping: we have reviewed elsewhere the evidence (a) for the existence in high concentrations of m a n y putative neurotransmitters, in particular serotonin (5HT), acetylcholine, and vasopressin; and (b) for their role in the generation or maintenance of circadian rhythmicity (Wirz-Justice et al., 1982). For example, depletion of 5 H T or absence of vasopressin do not affect circadian frequency. However, indirect evidence for an important role for 5 H T comes from neuropharmacology: antidepressant drugs (that increase 5 H T availability, albeit by different mechanisms) can slow circadian frequency (Wirz-Justice et al., 1982). Of particular * To whom all correspondence should be addressed: Psychiatrische Universit~itsklinik, Wilhelm Klein Strasse 27, CH4025 Basel, Switzerland. 0014-2999/83/0000-0000/$03.00 © 1983 Elsevier Biomedical Press
interest is that the tricyclic antidepressant imipramine can induce arrhythmicity when applied directly to the SCN but not in adjacent brain regions (Groos and Mason, 1982; Wirz-Justice et al., 1982). Recent studies have described specific high affinity binding of imipramine to brain homogenates (considered to be related to the 5HT presynaptic reuptake site), with highest density in the hypothalamus (Langer et al., 1982). We now report that imipramine binding in the SCN undergoes circadian variations, and that such rhythms are also present in other brain nuclei.
2. Materials and methods
Male Fiallinsdorf Wistar rats (n = 336, 350 g, 6 - 8 months of age) that had been housed under 12 : 12 lighting conditions (lights on at 6 h), were kept in constant darkness during the day of sacrifice to prevent any direct influence of light. They were rapidly killed by decapitation at 3 h intervals throughout the 24 h, beginning at 11 h, and brains were immediately frozen in liquid nitrogen. Dissection of nuclei from frozen thin
332
sections (1 mm) followed the atlas of KOnig and Klippel. In the hypothalamic regions, pools from 9 animals, in other regions, pools from 3 animals wereused for assays. The tissue punches were homogenised in cold Tris buffer (500 /~1, 50 mM, pH 7.5, containing NaC1 (120 mM) and KCI (50 mM) with a small plastic pestle molded to fit the sample tubes, centrifuged (final volume 1 ml), and the washed pellet resuspended in buffer at a concentration of ca. 1 mg/200 ~1. Imipramine binding was measured by a minimethod modified from Langer et al. (1982) developed to eliminate tissue waste and reduce the amount of filter binding and unspecific binding. Briefly, triplicates of membrane homogenate (200 ~1) were incubated with [3H]imipramine (4.5 nM, 100/~1), with or without cold imipramine (100/LM, 100 /~1) for 1 h at 0°C. Filtration was carried out using Whatman G F / B filters (2.5 cm) that had been punched to a diameter of 1 cm in a filtration port also reduced to a diameter of 0.9 cm. The filter was first washed with 3 ml of Tris buffer containing cold imipramine (100 /~M), and the samples washed 4 × with 3 ml of the same imipramine-containing ice-cold buffer, and the radioactivity measured by liquid scintillation spectrometry. Specific binding of 4.5 nM [3H]imipramine under these conditions represented 80% of the total binding, and there was no specific binding to the filters.
3. Results
Significant circadian rhythms in [3H]imipramine binding were found in many rat brain regions. Imipramine binding in the SCN region was highest at the end of the dark and lowest at the end of the light phase (fig. 1). Further significant rhythms were found in the caudate putamen, medial preoptic area, and frontal cortex, all with similar amplitudes of ca. 35%. There were no significant rhythms in septum or hippocampus. Similar to the SCN, binding of imipramine in the caudate putamen and medial preoptic area was maximal in the dark phase, although peak binding occurred earlier. In the frontal cortex, imipramine
800
/r /r
"Jr
700
600
500
8
11
14
17
20
23
2
5
Fig. I. Specific binding of [3H]imipramine (at 4.5 nM) in membranes from the suprachiasmatic nucleic throughout 24 h. The hatched area indicates the dark phase of the light:dark cycle to which the animals had been adapted: the experiment itself was carried out in the dark to measure an endogenous rhythm. Each point represents the mean of 3 - 4 pools of 9 animals (+S.E.M.). The inter-individual variation coefficient per time point (mean of n = 8) was 10%, the amplitude of peak: trough % was 35%. Significance of the daily changes was established by one-way analysis of variance (FT.iZ = 3.40, P < 0.025); of differences between time points by linear contrasts: ** P < 0.01, * P < 0.05 peak compared with trough at 14 h or 17 h. Ordinate: [3H]imipramine bound ( f m o l / m g protein); abscissa: time of day (h).
binding showed a bimodal rhythm, with peaks in the early dark and early light phase. 24 h mean imipramine binding was highest in the SCN (657 + 17 f m o l / m g protein) and anterior ventral hypothalamus (684 + 24, data incomplete for rhythm characterisation), with lower binding in the septum (361 _+ 6), caudate putamen (285 _+ 6), frontal cortex (243 _+ 7), medial preoptic area (217 _+ 4) and hippocampus (105 _+ 4), n = 29-31 for each region.
4. Discussion
In agreement with previous reports of the existence of circadian rhythms in adrenergic, cholinergic, dopaminergic and opiate receptor binding (summarised in Kafka et al., 1983), we
333
also found endogenous circadian rhythms (persisting under conditions without time cues) in imipramine binding to rat brain homogenates. The wave-form, phase, and amount of imipramine binding varied from region to region. Although we had insufficient tissue for kinetic studies when measuring binding in brain nuclei, no previous receptor rhythms have been associated with a change in affinity to the binding site (op cit.). Furthermore, the imipramine binding rhythms were of similar amplitude (ca. 35%) to that previously observed for other binding rhythms (op cit.). The SCN was chosen for its known role in circadian rhythm generation (Rusak and Zucker, 1979). The timing of peak imipramine binding is therefore as important as the finding of a significant rhythm. If imipramine binding is related to the presynaptic 5HT reuptake site, then the rhythm is parallel to that found by Meyer and Quay (1976), where 5HT uptake in the SCN region was high in the dark and low in the light phase. The 5HT metabolite 5HIAA (often used as an estimate of turnover rate), measured electrochemically in the SCN, is also high in the dark and low in the light phase (Faradji et al., 1983). Endogenous 5HT concentrations in the SCN are, in contrast, high in the light and low in the dark phase (Kordon et al., 1981). Since 5HT is an inhibitory neurotransmitter, these patterns can be understood in terms of functional activity: iontophoretically applied 5HT inhibits SCN firing, and iontophoresis of imipramine also depresses discharge as well as potentiating the 5HT effect (Groos and Mason, 1982). Thus these preliminary findings can be taken together as indicating that neuronal activity in the SCN should increase at lights on (when 5HT release is low) and decrease at lights off (when 5HT release is high). Two recent papers indeed show that single cell firing in SCN slices follows this pattern (Green and Gillette, 1982; Shibata et al., 1982). Although the SCN contains some of the highest concentrations of 5HT in the brain, the role of 5HT in circadian time-keeping is not yet clear. The few neuropharmacological agents that modify circadian frequency also increase 5HT availability (lithium, monoamine-oxidase inhibitors, imipramine), those that decrease 5HT availability (PCPA, 5,6-dihydroxytryptophan, or raphe lesions) do not
(Wirz-Justice et al., 1982). Thus it may be specifically the increased SCN 5HT turnover at dusk that is important in homeostasis of circadian rhythm frequency. Finally, the existence of circadian rhythms in imipramine binding in many brain regions is also of methodological significance. In any interpretation of changes in imipramine binding these considerable and rapid short-term fluctuations should be taken into account.
Acknowledgements We thank Dr. E. Bandle, Hoffmann LaRoche AG, Basel, for providing animals and facilities, and Brigitte Boeglin for joining the night shift.
References Faradji, H., M. Jouvet and R. Cespuglio, 1983, Electrochemical measurement of 5-hydroxyindole compounds in the suprachiasmatic nuclei: circadian fluctuations, Brain Res. (in press). Green, D.J. and R. Gillete, 1982, Circadian rhythm of firing rate recorded from single cells in the rat suprachiasmatic brain slice, Brain Res. 245, 198. Gross, G.A. and R. Mason, 1982, An electrophysiological study of the rat's suprachiasmatic nucleus: a locus for the action of antidepressants, J. Physiol. 330, 40P. Kafka, M.S., A. Wirz-Justice, D. Naber, R.Y. Moore and M.A. Benedito, 1983, Circadian rhythms in rat brain neurotransmitter receptors, Fed. Proc. (in press). Kordon, C., M. H6ry, A. Szafarczyk, G. Ixart and I. Assenmacher, 1981, Serotonin and the regulation of pituitary hormone secretion and of neuroendocrine rhythms, J. Physiol. Paris 77, 489. Langer, S.Z., E. Zarifian, M. Briley, R. Raisman and D. Sechter, 1982, High affinity 3H-imipramine binding: a new biological marker in depression, Pharmacopsychiat. 15, 4. Meyer, D.C. and W.B. Quay, 1976, Hypothalamic and suprachiasmatic uptake of serotonin in vitro: twenty-fourhour changes in male and prooestrus female rats, Endocrinol. 98, 1160. Rusak, B. and I. Zucker, 1979, Neural regulation of circadian rhythms, Physiol. Rev. 59, 449. Shibata, S., Y. Oomura, H. Kita and K. Hattori, 1982, Circadian rhythmic changes of neuronal activity in the suprachiasmatic nucleus of the rat hypothalamic slice, Brain Res. 247, 154. Wirz-Justice, A., G.A. Groos and T.A. Wehr, 1982, The neuropharmacology of circadian timekeeping in mammals, in: Vertebrate Circadian Systems, Structure and Physiology, eds. J. Aschoff, S. Daan and G.A. Groos, Springer Verlag Berlin, Heidelberg, p. 183.