Neuropeptide Y blocks serotonergic phase shifts of the suprachiasmatic circadian clock in vitro

Brain Research 808 Ž1998. 31–41

Research report

Neuropeptide Y blocks serotonergic phase shifts of the suprachiasmatic circadian clock in vitro Rebecca A. Prosser

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Department of Biochemistry and Cellular and Molecular Biology, UniÕersity of Tennessee, M407 Walter’s Life Science Building, KnoxÕille, TN 37996, USA Accepted 4 August 1998

Abstract The mammalian circadian pacemaker in the suprachiasmatic nuclei ŽSCN. can be reset in vitro by various neurochemical stimuli. This study investigated the phase-shifting properties of neuropeptide Y ŽNPY. and serotonin Ž5-HT. agonists when applied alone, as well as their combined effects on clock resetting. These neurotransmitters have both been shown to advance the SCN clock in vitro when applied during the daytime. By monitoring the SCN neuronal activity rhythm in vitro, I first confirm that the 5HT1Ar5HT7 agonist Žq.DPAT maximally advances the SCN clock when applied at zeitgeber time 6 ŽZT6.. Conversely, NPY only phase advances the neuronal activity rhythm when applied at ZT 10. This effect occurs through stimulation of Y2 receptors. NPY, again acting through Y2 receptors, blocks Žq.DPAT-induced phase shifts at ZT 6, while neither Žq.DPAT nor 5-HT affect NPY-induced phase shifts at ZT 10. NPY appears to block Žq.DPAT-induced phase shifts by preventing increases in cyclic AMP. These data are the first to demonstrate in vitro interactions between daytime resetting stimuli in the rat, and provide critical insights into mechanisms controlling circadian clock phase. q 1998 Published by Elsevier Science B.V. All rights reserved. Keywords: Suprachiasmatic nucleus; Circadian rhythm; Serotonin; Neuropeptide Y; Cyclic AMP; Brain slice

1. Introduction Circadian rhythms are a characteristic shared by all organisms. The primary circadian clock in mammals is located in the suprachiasmatic nuclei ŽSCN. w33x. When isolated in a brain slice preparation, the SCN pacemaker generates 24 h rhythms of spontaneous neuronal activity that can be reset, or phase-shifted, by exogenous stimuli, including many of the neurotransmitters found in SCN afferents Žsee Ref. w12x for review.. Some, including glutamate and acetylcholine, act primarily at night, mimicking the phase-shifting effects of in vivo light pulses w5,11,26,55x. Other neurotransmitters, such as neuropeptide Y ŽNPY. w14,30,53x and serotonin Ž5-HT. w29,43,54x, phase-shift the SCN clock during the day, mimicking the ‘nonphotic’ phase-shifting effects of wheelrunning activity w37,50,51x.

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Fax: q1-423-974-6306; E-mail: [email protected]

Several SCN afferents, including glutamatergic input from the retina, 5-HT input from the raphe, and NPY input from the intergeniculate leaflet ŽIGL., synapse onto the same population of SCN neurons w16,20,34,35,58x. This anatomic arrangement suggests that these inputs could interact in modulating the SCN pacemaker. Recently, interactions between various photic and nonphotic stimuli have been explored. Studies investigating in vivo interactions between light Žor glutamate. and 5-HT have generally found that 5-HT and its agonists inhibit phase shifts w41,42,48,49x, Fos protein induction w49x, and neuronal activity increases w31,49,62,63x produced by light and glutamate. Other studies, investigating interactions between NPY and light, have found that these stimuli inhibit each other’s phase-shifts w4,8,60x. Still other studies have investigated interactions between light pulses and wheelrunning activity, the latter of which is thought to modulate the SCN clock through NPY w4,7,25,61x, 5-HT w10,40x, or a combination of both w28x. Interactions between these stimuli appear to be complex. Light pulses can both inhibit and enhance wheelrunning phase shifts w36x, and wheelrunning can both speed up w38,39x and inhibit w47x light-induced phase shifts.

0006-8993r98r$ - see front matter q 1998 Published by Elsevier Science B.V. All rights reserved. PII: S 0 0 0 6 - 8 9 9 3 Ž 9 8 . 0 0 8 0 8 - 7

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R.A. Prosserr Brain Research 808 (1998) 31–41

Recent in vitro studies have also investigated interactions between different stimuli. One study investigated interactions between NPY and glutamate, and found a reciprocally inhibitory relationship w5x: NPY inhibits glutamate-induced shifts at night and glutamate inhibits NPYinduced shifts during the day. Another study found that NPY inhibits daytime advances induced by pituitary adenylate cyclase-activating peptide ŽPACAP. w19x, a neuropeptide found in SCN retinal afferents w17x. These results are interesting, first, because they describe an interaction between two day-active neuromodulators, and second, because NPY blocks PACAP-induced shifts through a different receptor from the one associated with its own resetting w19x. In the present study, a series of experiments investigated interactions between 5-HTergic and NPYergic modulation of the SCN pacemaker. I determined the phaseshifting effects of 5-HT and NPY agonists applied individually, how they shift the circadian clock when applied simultaneously, and investigated the intracellular processes underlying their interactions. The results of these experiments indicate that 1. NPY and 5-HT agonists phase-shift the SCN clock at different times during the subjective day; 2. NPY blocks 5-HTergic phase advances, while 5-HTergic stimulation does not block NPY-induced shifts; 3. NPY-induced shifts and NPY inhibition of 5-HT-induced shifts occur through Y2 receptors; and 4. NPY antagonizes 5HTergic advances by preventing increases in cyclic AMP.

face of a Hatton-style brain slice chamber w21x, where they were perfused continuously with warm Ž378C., oxygenated Ž95% O 2r5% CO 2 ., glucoserbicarbonate-supplemented Earle’s Balanced Salt Solution ŽEBSS; Sigma., pH 7.4–7.5. For experiments that continued for three days in vitro, gentamicin sulfate Ž0.05%. ŽSigma. was added to the perfusion medium. 2.2. Single unit recordings and data analysis

2. Materials and methods

Single unit recordings were obtained using methods described previously w43x. Briefly, the spontaneous activity of single SCN neurons was recorded using glass capillary microelectrodes filled with 3 M NaCl. Each neuron was recorded for 5 min, and the data stored for later determination of firing rate using a DataWaÕe system. These firing rates were then used to calculate 2 h running averages, lagged by 1 h, to obtain a measure of population neuronal activity. As in previous studies w43x, the time of peak neuronal activity was defined as the time of symmetrically highest activity, estimated to the nearest quarter hour. Thus, if the two highest 2-h means were equivalent, the time-of-peak would be calculated as being midway between them. Since previous studies have shown that medium exchange alone does not shift the time of peak neuronal activity, phase shifts were calculated as the difference in time-of-peak in drug-treated slices vs. the mean time-of-peak in untreated slices w43x. Student’s t-tests and ANOVAs were used, where appropriate, to test for significant differences between the means, with significance set at p - 0.05.

2.1. Brain slice preparation

2.3. Experimental treatments

Coronal brain slices Ž500 m . containing the SCN were prepared during the daytime from adult, male Sprague– Dawley rats housed in a 12:12 light–dark cycle as previously reported w43,44x. Slices were maintained at the inter-

All drugs were applied during the first day in vitro by stopping the perfusion and replacing the bathing medium in the slice chamber for 1 h with medium containing the test compound. At the end of the hour, the treated medium

Fig. 1. Control experiment, showing the firing rates of individual cells together with the population rhythm of neuronal activity. Firing rates of individual neurons Žsmall, open circles. are plotted vs. the zeitgeber time at which they were recorded. Large, filled circles denote the 2 h means" S.E.M. calculated from the individual firing rates. Neuronal activity peaks at ZT 6. Horizontal bars: lights-off in the donor colony.

R.A. Prosserr Brain Research 808 (1998) 31–41

was exchanged with normal medium, and perfusion was resumed. For blocking experiments, the perfusion medium was first replaced with medium containing the blocking compound. After 15 min this solution was replaced for 1 h with medium containing both compounds. This was fol-

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lowed by another 15 min treatment with medium containing the blocking agent, after which the normal medium was reintroduced to the slice chamber and perfusion was resumed. Chemicals used in the study wereŽq.-8hydroxy-dipropylaminotetralin HBr ŽŽq.DPAT. ŽResearch

Fig. 2. Žq.DPAT advances the rhythm in neuronal activity when applied at ZT 6, but not when applied at ZT 3 or 10. A. Žq.DPAT treatment at ZT 6–7 advances the peak by 4 h. B. Žq.DPAT treatment at ZT 3–4 induces a slight advance from control. C. Žq.DPAT treatment at ZT 10–11 does not shift the time of peak activity. All phase shifts are calculated as the difference in time-of-peak relative to control ŽZT 6.0 " 0.4, n s 3.. Dashed line: mean time-of-peak in untreated slices; Vertical bar: time of treatment. For other details see Fig. 1 legend.

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R.A. Prosserr Brain Research 808 (1998) 31–41

Biochemicals., neuropeptide YŽNPY., neuropeptide YŽ3– 36. ŽNPYŽ3–36.. and wLeu 31 ,Pro 34 xneuropeptide Y ŽwLeu31 ,Pro 34 xNPY. ŽPeninsula Laboratory., and 8-benzylaminoadenosine 3X :5X-cyclic monophosphate.Ž8BA-cAMP. ŽSigma..

3. Results 3.1. Control experiments Neuronal activity recorded from control slices on the second day in vitro peaked around the middle of the subjective day ŽFig. 1.. The mean Ž"S.E.M.. time of peak activity in control slices occurred at zeitgeber time ŽZT. 6.0 " 0.4 Ž n s 3. Žwhere ZT 0 refers to the time of lightson, and ZT 12 the time of lights-off in the donor animal colony.. These results are consistent with previous studies showing that the time-of-peak shows little variability across experiments, and is an accurate measure of the phase of the underlying circadian clock w12x. Therefore, in all subsequent experiments the effects of exogenous stimuli on the SCN clock were determined by calculating the extent to which the time of peak neuronal activity after experimental treatment differed from ZT 6.0. 3.2. Phase-shifting effects of 5-HT and NPY agonists when applied indiÕidually The effects of 5-HTergic stimulation were explored by applying the 5HT1A r5HT7 agonist Žq.DPAT at different times during the subjective day. Consistent with previous studies involving 5-HT and its agonists w29,43,46,54x, Žq.DPAT significantly advanced the time of peak activity primarily when applied at ZT 6 Ž p - 0.05., while application at ZT 3 and ZT 10 had no significant effect ŽFig. 2.. The effects of Žq.DPAT during subjective night were not investigated, since previous work has shown it does not induce phase shifts at night w43,54x. The results of these experiments are summarized in a phase response curve ŽFig. 3.. Next, the effects of NPYergic stimulation were investigated. NPY induced significant phase advances when applied at ZT 10 Ž p - 0.05., and had little or no effect at other times in the subjective day and subjective night ŽFigs. 3 and 4.. These results differ somewhat from those of previous studies, which have found NPY to advance the neuronal activity rhythm when applied at ZT 6-8 w2,14,22,30,53x. To investigate potential reasons for this difference, we first tested whether the change we observed after NPY application at ZT 10 reflected a permanent resetting of the SCN clock. To do this, the time of peak activity was determined two days after NPY application at

Fig. 3. Phase response curves for Žq.DPAT and NPY. Shown are mean Ž"S.E.M.. shifts induced by Žq.DPAT treatment Žopen circles. and NPY treatment Žclosed diamonds. plotted according to the time of drug application. Žq.DPAT induces its largest effects at ZT 6, while NPY advances the rhythm only when applied at ZT 10.

ZT 10, and was found to occur at ZT 3.0 Ž n s 1.. This corresponds to an advance of 3 h, similar to the mean advance observed the first day following NPY treatment. These results support the conclusion that the underlying circadian pacemaker was permanently reset by NPY treatment at ZT 10. Next, we increased the concentration of NPY applied at ZT 6, to see if the inability of NPY to shift the clock was due to the concentration of NPY being too low. Application of 10 mM NPY at ZT 6 still did not advance the neuronal activity rhythm ŽFig. 5; Table 1.. Two NPY agonists were used to investigate the type of receptor associated with the NPY-induced phase shifts: wLeu31 ,Pro 34 xNPY, which preferentially binds the Y1 receptor, and NPYŽ3–36., which is more selective for the Y2 receptor w3x. Each of these agonists was tested at ZT 6 and ZT 10 to determine their ability to reset the SCN clock. As summarized in Fig. 5 and Table 1, NPYŽ3–36. significantly advanced the neuronal activity rhythm when applied at ZT 10 but not ZT 6 Ž p - 0.05., while wLeu31 ,Pro 34 xNPY did not affect the rhythm when applied at either time. These results suggest that NPY-induced phase shifts occur through stimulation of Y2 receptors. 3.3. Interactions between serotonergic and NPY agonists The time points ZT 6 and ZT 10 were chosen to investigate interactions between NPYergic and 5-HTergic stimulation. Co-administration of NPY with Žq.DPAT at ZT 6 inhibited the phase advance normally induced by Žq.DPAT at this time ŽFig. 6.. The difference in phase shifts induced by Žq.DPAT vs. Žq.DPATq NPY treat-

R.A. Prosserr Brain Research 808 (1998) 31–41

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Fig. 4. NPY advances the neuronal activity rhythm only when applied at ZT 10. A. NPY treatment at ZT 10 advances the rhythm by 4 h compared to controls. B. NPY treatment at ZT 6 does not change the time of peak activity. C. NPY treatment at ZT 18 does not change the time of peak activity from that of controls. See Fig. 1 and Fig. 2 legends for details.

ments was significant Ž p - 0.05.. This blocking effect of NPY was mimicked by NPYŽ3–36. Ž p - 0.05. but not by wLeu31 ,Pro 34 xNPY ŽFig. 6., suggesting that NPY inhibits 5-HTergic phase advances through stimulation of Y2 receptors ŽTable 1.. Next we investigated the effects of 5-HTergic stimulation on NPY-induced phase shifts. Co-administration of

Žq.DPAT with NPY at ZT 10 did not block NPY-induced phase shifts ŽFig. 6.. Similar results were obtained when the concentration of Žq.DPAT was increased 10-fold, in that NPY still advanced the neuronal activity rhythm 3.25 h Ž n s 1. ŽFig. 6.. While these results suggest that 5-HT does not modulate phase shifts induced by NPY, there are many types of 5-HT receptors in the SCN w27,32,43,52x, of

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R.A. Prosserr Brain Research 808 (1998) 31–41

Fig. 5. NPY and its agonists have no effect when applied at ZT 6, while NPY-induced phase shifts at ZT 10 are mimicked by NPYŽ3–36. but not wLeu31 ,Pro 34 xNPY. Histogram plots show mean Ž"S.E.M.. phase shifts induced by NPY and its agonists after application at either ZT 6 or ZT 10. None of the treatments induced significant changes when applied at ZT 6, while significant advances were seen after NPY and NPYŽ3–36. application at ZT 10 Ž p - 0.05 vs. time-of-peak in control slices.. Numbers below the bars indicate the number of experiments.

Table 1 Changes in SCN clock phase after different in vitro treatments Treatment a

Number of experiments

Time ŽZT. of treatment

Change in time-of-peak Žh.

Žq.DPAT Ž10 mM. NPY Ž1 mM. NPY Ž10 mM. NPYŽ3–36. Ž1 mM. wLeu31 ,Pro 34 xNPY Ž1 mM. Žq.DPAT Ž10 mM. q NPY Ž1 mM. Žq.DPAT Ž10 mM. q NPYŽ3–36. Ž1 mM. Žq.DPAT Ž10 mM. q wLeu31 ,Pro 34 xNPY Ž1 mM. 8BA-cAMP Ž1 mM. 8BA-cAMP Ž1 mM. q NPY Ž1 mM. 8BA-cAMP Ž1 mM. q NPY Ž10 mM. 8BA-cAMPŽ10 nM. q NPY Ž1 mM. Žq.DPAT Ž10 mM. 5-HT Ž10 mM. NPY Ž1 mM. NPYŽ3–36. Ž1 mM. wLeu31 ,Pro 34 xNPY Ž1 mM. NPY Ž1 mM. q Žq.DPAT Ž10 mM. NPY Ž1 mM. q Žq.DPAT Ž100 mM. NPY Ž1 mM. q 5-HT Ž10 mM.

3 2 1 1 1 3 2 2 3 3 2 2 2 1 4 3 2 3 1 2

6 6 6 6 6 6 6 6 6 6 6 6 10 10 10 10 10 10 10 10

3.67 " 0.5 b 0.5 " 0.4 y0.5 0.5 0.0 0.08 " 0.3 c 0.25 " 0.4 c 2.75 " 0.4 b 3.67 " 0.2 b 3.25 " 0.2 b 4.25 " 1.1b 3.63 " 0.2 b 0.25 " 1.1 0.0 3.38 " 0.3 b 3.17 " 0.4 b y0.25 " 0.4 2.5 " 0.7 b 2.75 3.25 " 0.4 b

a

All chemicals were bath applied for 1 h, and neuronal activity was recorded the day after treatment. All changes are relative to control Žno treatment. experiments, which peak at ZT 6.0 " 0.4 Ž n s 3.. b p - 0.05 vs. control. c p - 0.05 vs. Žq.DPAT.

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Fig. 6. Interactions between 5-HTergic and NPYergic stimulation in phase-shifting the SCN clock. Histograms on the left show mean phase shifts induced at ZT 6 by Žq.DPAT alone, and in the presence of various NPY agonists. The large advances induced by Žq.DPAT are blocked by co-application of NPY and NPYŽ3–36. Ž p - 0.05 vs. time-of-peak in control slices., but not by wLeu31 ,Pro 34 xNPY. Histograms on the right show mean phase shifts induced at ZT 10 by NPY treatment alone, and in the presence of 5-HT agonists. The large advances induced by NPY are not blocked by either Žq.DPAT or 5-HT, even when applied at concentrations well above those needed to reset the circadian clock. See Fig. 5 legend for details.

which only the 5HT1A and 5HT7 are stimulated by Žq.DPAT. Therefore, 5-HT was co-administered with NPY in order to stimulate all 5-HT receptors. NPY continued to phase-advance the neuronal activity rhythm in the presence of 10 mM 5-HT ŽFig. 6., a concentration at least 10-fold higher than that needed to phase-shift the clock when 5-HT is applied alone w43x. The results of these experiments are summarized in Table 1. 3.4. Signal transduction mechanism underlying NPY inhibition of 5-HTergic shifts The cellular mechanism underlying NPY inhibition of Žq.DPAT-induced phase shifts was investigated next. Previous work has shown that 5-HTergic phase advances require increases in cyclic AMP, activation of protein kinase A, and opening of Kq channels w45x. To determine the point in this pathway where NPY acts, we first investigated whether NPY acts by preventing increases in endogenous cyclic AMP. For this, we tested whether NPY blocks phase advances induced by a cyclic AMP analog, 8BA-cAMP, that is membrane-soluble, resists enzymatic

degradation, and mimics daytime phase advances induced by 5-HTergic stimulation w44,45x. 8BA-cAMP activates protein kinase A directly, thus bypassing the need for increases in endogenous cyclic AMP. Consistent with previous reports, application of 8BA-cAMPŽ1 mM. by itself at ZT 6 induced significant phase advances of the SCN circadian neuronal activity rhythm Ž p - 0.05. ŽFig. 7.. Co-application of NPY with 8BA-cAMP did not block these phase advances, even when the concentration of NPY was increased to 10 mM ŽFig. 7.. Co-administration of NPY Ž1 mM. also did not block advances induced by 10 nM 8BA-cAMP, a concentration of the analog much closer to its ED50 w44x Žsee Table 1.. Taken together, these results strongly support the conclusion that NPY blocks Žq.DPAT-induced phase shifts by inhibiting increases in cyclic AMP.

4. Discussion These results show that 5-HTergic and NPYergic agonists reset the SCN pacemaker at different times during the

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R.A. Prosserr Brain Research 808 (1998) 31–41

Fig. 7. Interactions of NPY with 8BA-cAMP at ZT 6. Histograms show the mean phase shifts induced by 8BA-cAMP treatment alone Žsignificant change, p- 0.05., and in the presence of NPY at two different concentrations. NPY was unable to block advances induced by 8BA-cAMP. See Fig. 5 legend for details.

used varying methods and species, none of the differences clearly distinguish this study from the others. For example, one study w14x used hamsters, while the others used rats. Two studies w14,30x applied NPY via microdrops, while the other two, Ref. w53x and this study, used bath application. Perfusion medium in the different studies consisted of ACSF w14x, a Krebs solution w53x, and EBSS—Ref. w30x and this study. The lower concentration of NPY used here is the same as that Žin microdrop form. used by Medanic and Gillette Ž1993., and is between those used in the other studies. The differential effects of wLeu31 ,Pro 34 xNPY and NPYŽ3–36. indicate that NPY phase-shifts the SCN clock through stimulating Y2 receptors. These results are the first evidence in rats that NPY resets the circadian pacemaker through stimulating Y2 receptors, and are consistent with previous work in hamsters w14,23x. However, these results differ somewhat from rat SCN cell culture studies showing that stimulation of both Y1 and Y2 receptors can acutely inhibit evoked GABA release w9x, cause long-term depression of electrical activity, and severely inhibit cytosolic Ca2q oscillations w59x. This suggests that the cellular mechanisms associated with chronic resetting of the SCN pacemaker may be different from those capable of acutely modulating SCN cell activity.

4.2. Interactions between NPY and 5-HT agonists day. They also demonstrate a unidirectional interaction between NPYergic and 5-HTergic phase-shifting: NPY blocks 5-HTergic phase advances, while 5-HT stimulation does not block NPY-induced phase advances. The results further indicate that both the phase-shifting and blocking effects of NPY occur through stimulating Y2 receptors. Lastly, the results suggest that NPY inhibits 5-HTergic phase advances by preventing increases in cyclic AMP. 4.1. Differential sensitiÕity of the SCN circadian clock to serotonergic and NPY stimulation The time of sensitivity and magnitude of the phase shifts obtained here with Žq.DPAT are consistent with results from previous in vitro studies of 5-HTergic phase shifting w29,43,46,54x. This consistency indicates that the experimental conditions in this laboratory are similar to those of other laboratories conducting these experiments. In addition to adding support to previous studies, this consistency is relevant when evaluating data that do not match those of previous studies. The pattern of phase shifts induced by NPY is clearly different from that seen with Žq.DPAT. These results differ from previous in vivo w2,22x and in vitro w14,30,53x studies showing that NPY maximally advances the SCN clock when applied between ZT 6 and 8. The reasonŽs. for this discrepancy is not clear. While the in vitro studies

NPY inhibited 5-HTergic phase advances, while 5HTergic stimulation did not block NPY phase shifts. The latter result is unlikely to be due to the concentration of NPY being excessive, as this is the lowest concentration of NPY Žapplied as a microdrop. found to induce maximal shifts w30x. Therefore, these results suggest that the phase modulating effects of NPY dominate those of 5-HT. In other words, the SCN pacemaker is sensitive to resetting by 5-HT only in the absence of NPY, while NPY can reset the SCN pacemaker regardless of whether 5-HT is present. Whether similar interactions occur between other nonphotic stimuli is not known. In the only other study of nonphotic interactions, the effects of NPY on PACAP-induced shifts were investigated, but not the reverse w19x. However, this relationship is clearly different from those described for photicrnonphotic interactions. For example, NPY and glutamate form a mutually inhibitory relationship in vitro w5x which parallels the inhibitory relationship seen in vivo between NPY and light pulses w4,8,60x. Other in vivo studies have found that 5-HT and its agonists inhibit clock resetting, Fos protein induction, and increases in neuronal activity induced by glutamate and light w13,41,42,48,49,57x, but the reverse situation has not been investigated. There may be additional interactions between 5-HT and NPY that were not explored in these experiments.

R.A. Prosserr Brain Research 808 (1998) 31–41

Žq.DPAT is active primarily at 5HT1A r5HT7 receptors, and thus will not stimulate other 5-HT receptors known to be in the SCN w32,43,52x. 5-HT phase-delays the SCN clock at night through an unidentified 5-HT receptor type, and through intracellular processes that do not involve cyclic AMP w43x. Therefore, NPY may not inhibit nighttime 5-HTergic resetting. The ability of NPYŽ3–36. to mimic the inhibition by NPY, while wLeu31 ,Pro 34 xNPY does not, indicates that this inhibition occurs through stimulating Y2 receptors w3x. These results differ from those in the hamster, where NPY blocks PACAP-induced advances through a non-Y2 receptor. This might underscore differences between rats and hamsters, or it could indicate that different mechanisms underlie NPY inhibition of 5-HTergic vs. PACAP-induced shifts. The inability of NPY to inhibit advances by 8BA-cAMP, while it blocks advances by both Žq.DPAT and PACAP, suggests that NPY prevents neurotransmitter-induced increases in cyclic AMP. This is consistent with evidence from other systems showing that NPY can inhibit adenylate cyclase and decrease cyclic AMP levels w1,15,18,56x. Thus, NPY modulates at least two kinase systems in the SCN: it can activate PKC, and it can inhibit PKA. Together with the acute actions of NPY on SCN cells, these results demonstrate the complexity of signal transduction processing in the SCN. We do not know whether NPY acts pre- or postsynaptically to inhibit 5-HTergic phase shifts. NPY and 5-HT afferents synapse onto the same postsynaptic cells as well as synapsing onto each other’s terminals in the SCN w16x, which would allow for either site of action. With respect to its own resetting, some studies indicate that NPY-induced shifts are blocked by tetrodotoxin w24,53x, while others find NPY-induced shifts are insensitive to tetrodotoxin w6x. The acute effects of NPY on SCN cells have been shown to occur both pre- and postsynaptically w9,59x.

5. Summary In conclusion, these experiments demonstrate a clear difference in the actions of NPY and 5-HT in the SCN, with NPY phase-shifting the clock later in the subjective day than 5-HT agonists. Further, they show that these two inputs interact within the SCN, such that NPY blocks 5-HTergic phase-advances, while 5-HTergic stimulation does not block NPY-induced phase shifts. Both the phaseshifting and blocking actions of NPY involve stimulating Y2 receptors, and the inhibition by NPY appears to occur through blocking 5-HTergic increases in cyclic AMP. Together, these data further our understanding of how the phase of the SCN circadian clock is modulated by its neuronal inputs.

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Acknowledgements This research was supported by NIH grant MH53317. I would like to thank Dr. Timothy Youngstrom and Kevin Biggs for technical assistance, and I would like to thank Dr. Norman Ruby for critical reading of previous versions of this manuscript.

References w1x L. Aakerlund, U. Gether, J. Fuhlendorff, T.W. Schwartz, O. Thastrup, Y1 receptors for neuropeptide Y are coupled to mobilization of intracellular calcium and inhibition of adenylate cyclase, FEBS Letters 260 Ž1990. 73–78. w2x H.E. Albers, C.F. Ferris, Neuropeptide Y: role in light dark cycle entrainment of hamster circadian rhythms, Neurosci. Lett. 50 Ž1984. 163–168. w3x A. Balasubramaniam, Neuropeptide Y family of hormones: receptor subtypes and antagonists, Peptides 18 Ž1997. 445–457. w4x S.M. Biello, Enhanced photic phase shifting after treatment with antiserum to neuropeptide Y, Brain Res. 673 Ž1995. 25–29. w5x S.M. Biello, D.A. Golombek, M.E. Harrington, Neuropeptide Y and glutamate block each other’s phase shifts in the suprachiasmatic nucleus in vitro, Neuroscience 77 Ž1997. 1049–1057. w6x S.M. Biello, D.A. Golombek, K.M. Schak, M.E. Harrington, Circadian phase shifts to neuropeptide Y in vitro: cellular communication and signal transduction, J. Neurosci. 17 Ž1997. 8468–8475. w7x S.M. Biello, D. Janik, N. Mrosovsky, Neuropeptide Y and behaviorally induced phase shifts, Neuroscience 62 Ž1994. 273–279. w8x S.M. Biello, N. Mrosovsky, Blocking the phase-shifting effect of neuropeptide Y with light, Proc. R. Soc. Lond. B 259 Ž1995. 179–187. w9x G. Chen, A.N. van den Pol, Multiple NPY receptors coexist in preand post-synaptic sites: inhibition of GABA release in isolated self-innervating SCN neurons, J. Neurosci. 16 Ž1996. 7711–7724. w10x R.A. Cutrera, A. Kalsbeek, P. Pevet, Specific destruction of the serotonergic afferents to the suprachiasmatic nuclei prevents triazolam-induced phase advances of hamster activity rhythms, Beh. Brain Res. 62 Ž1994. 21–28. w11x J.M. Ding, D. Chen, E.T. Weber, L.E. Faiman, M.A. Rea, M.U. Gillette, Resetting the biological clock: mediation of nocturnal circadian shifts by glutamate and NO, Science 266 Ž1994. 1713–1717. w12x M.U. Gillette, M. Medanic, A.J. McArthur, C. Liu, J.M. Ding, L.E. Faiman, E.T. Weber, T.K. Tcheng, E.A. Gallman, Intrinsic neuronal rhythms in the suprachiasmatic nuclei and their adjustment, CIBA Found. Symp. 183 Ž1995. 134–153. w13x J.D. Glass, M. Selim, G. Srkalovic, M.A. Rea, Tryptophan loading modulates light-induced responses in the mammalian circadian system, J. Biol. Rhythms 10 Ž1995. 80–90. w14x D.A. Golombek, S.M. Biello, R.A. Rendon, M.E. Harrington, Neuropeptide Y phase shifts the circadian clock in vitro via a Y2 receptor, NeuroReport 7 Ž1996. 1315–1319. w15x E. Grouzmann, F. Cressier, Ph. Walker, K. Hofbauer, B. Waeber, H.R. Brunner, Interactions between NPY and its receptor: assessment using anti-NPY antibodies, Reg. Pept. 54 Ž1994. 439–444. w16x J. Guy, O. Bosler, G. Dusticier, G. Pelletier, A. Calas, Morphological correlates of serotonin-neuropeptide Y interactions in the rat suprachiasmatic nucleus: combined radioautographic and immunocytochemical data, Cell Tissue Res. 250 Ž1987. 657–662. w17x J. Hannibal, J.M. Ding, D. Chen, J. Fahrenkrug, P.J. Larsen, M.U. Gillette, J.D. Mikkelsen, Pituitary adenylate cyclase-activating peptide ŽPACAP. in the retinohypothalamic tract: a potential daytime regulator of the biological clock, J. Neurosci. 17 Ž1997. 2637–2644.

40

R.A. Prosserr Brain Research 808 (1998) 31–41

w18x A. Harfstrand, B. Fredholm, K. Fuxe, Inhibitory effects of neuropeptide Y on cyclic AMP accumulation in slices of the nucleus tractus solitarius region of the rat, Neurosci. Lett. 76 Ž1987. 185–190. w19x M.E. Harrington, S. Hoque, NPY opposes PACAP phase shifts via receptors different from those involved in NPY phase shifts, NeuroReport 8 Ž1997. 2677–2680. w20x M.E. Harrington, D.M. Nance, B. Rusak, Double-labeling of neuropeptide Y-immunoreactive neurons which project from the geniculate to the suprachiasmatic nuclei, Brain Res. 410 Ž1987. 275–282. w21x G.I. Hatton, A.D. Doran, A.K. Salm, C.D. Tweedle, Brain slice preparation: hypothalamus, Brain Res. Bull. 5 Ž1980. 405–414. w22x K.L. Huhman, H.E. Albers, Neuropeptide Y microinjected into the suprachiasmatic region phase shifts circadian rhythms in constant darkness, Peptides 15 Ž1994. 1475–1478. w23x K.L. Huhman, C.F. Gillespie, C.L. Marvel, H.E. Albers, Neuropeptide Y phase shifts circadian rhythms in vivo via a Y2 receptor, NeuroReport 7 Ž1996. 1249–1252. w24x K.L. Huhman, C.L. Marvel, C.F. Gillespie, E.M. Mintz, H.E. Albers, Tetrodotoxin blocks NPY-induced but not muscimol-induced phase advances of wheel-running activity in Syrian hamsters, Brain Res. 772 Ž1997. 176–180. w25x D. Janik, N. Mrosovsky, Intergeniculate leaflet lesions and behaviorally-induced shifts of circadian rhythms, Brain Res. 651 Ž1994. 174–182. w26x C. Liu, M.U. Gillette, Cholinergic regulation of the suprachiasmatic nucleus circadian rhythms via a muscarinic mechanism at night, J. Neurosci. 16 Ž1996. 744–751. w27x T.W. Lovenberg, B.M. Baron, L. de Lecea, J.D. Miller, R.A. Prosser, M.A. Rea, P.E. Foye, M. Racke, A.L. Slone, B.W. Siegel, P.E. Danielson, J.G. Sutcliffe, M.G. Erlander, A novel adenylate cyclase-activating serotonin receptor Ž5-HT7 . implicated in the regulation of mammalian circadian rhythms, Neuron 11 Ž1993. 449–458. w28x E.G. Marchant, N.V. Watson, R.E. Mistlberger, Both neuropeptide Y and serotonin are necessary for entrainment of circadian rhythms in mice by daily treadmill running schedules, J. Neurosci. 17 Ž1997. 7974–7987. w29x M. Medanic, M.U. Gillette, Serotonin regulates the phase of the rat suprachiasmatic circadian pacemaker in vitro only during the subjective day, J. Physiol. 450 Ž1992. 629–642. w30x M. Medanic, M.U. Gillette, Suprachiasmatic circadian pacemaker of rat shows two windows of sensitivity to neuropeptide Y in vitro, Brain Res. 620 Ž1993. 281–286. w31x J.D. Miller, C.A. Fuller, The response of suprachiasmatic neurons of the rat hypothalamus to photic and serotonergic stimulation, Brain Res. 515 Ž1990. 155–162. w32x S.M. Molineaux, T.M. Jessell, R. Axel, D. Julius, 5-HT1c receptor is a prominent serotonin receptor subtype in the central nervous system, Proc. Natl. Acad. Sci. 86 Ž1989. 6793–6797. w33x R.Y. Moore, Organization of the mammalian circadian system, CIBA Found. Symp. 183 Ž1995. 88–106. w34x R.Y. Moore, N.J. Lenn, A retinohypothalamic projection in the rat, J. Comp. Neurol. 146 Ž1972. 1–14. w35x L.P. Morin, J. Blanchard, R.Y. Moore, Intergeniculate leaflet and suprachiasmatic nucleus organization and connections in the golden hamster, Vis. Neurosci. 8 Ž1992. 219–230. w36x N. Mrosovsky, Double-pulse experiments with nonphotic and photic phase-shifting stimuli, J. Biol. Rhythms 6 Ž1991. 167–179. w37x N. Mrosovsky, Locomotor activity and non-photic influences on circadian clocks, Biol. Rev. 71 Ž1996. 343–372. w38x N. Mrosovsky, P.A. Salmon, A behavioral method for accelerating re-entrainment of rhythms to new light–dark cycles, Nature 330 Ž1987. 372–373. w39x N. Mrosovsky, P.A. Salmon, Triazolam and phase-shifting acceleration re-evaluated, Chronobiol. Internatl. 7 Ž1990. 35–41. w40x P.D. Penev, F.W. Turek, P.C. Zee, A serotonin neurotoxin attenuates

w41x

w42x

w43x

w44x

w45x

w46x

w47x w48x

w49x

w50x

w51x

w52x

w53x

w54x

w55x

w56x

w57x

w58x

w59x

the phase-shifting effects of triazolam on the circadian clock in hamsters, Brain Res. 669 Ž1995. 207–216. G.E. Pickard, M.A. Rea, TFMPP, a 5HT1B receptor agonist, inhibits light-induced phase shifts of the circadian activity rhythm and c-Fos expression in the mouse suprachiasmatic nucleus, Neurosci. Lett. 231 Ž1997. 95–98. G.E. Pickard, E.T. Weber, P.A. Scott, A.F. Riberdy, M.A. Rea, 5-HT1B receptor agonists inhibit light-induced phase shifts of behavioral circadian rhythms and expression of the immediate-early gene c-fos in the suprachiasmatic nucleus, J. Neurosci. 16 Ž1996. 8208– 8220. R.A. Prosser, R.R. Dean, D.M. Edgar, H.C. Heller, J.D. Miller, Serotonin and the mammalian circadian system: I. In vitro phase shifts by serotonergic agonists and antagonists, J. Biol. Rhythms 8 Ž1993. 1–16. R.A. Prosser, M.U. Gillette, The mammalian circadian clock in the suprachiasmatic nuclei is reset in vitro by cAMP, J. Neurosci. 9 Ž1989. 1073–1081. R.A. Prosser, H.C. Heller, J.D. Miller, Serotonergic phase advances of the mammalian circadian clock involve protein kinase A and Kq channel opening, Brain Res. 644 Ž1994. 67–73. R.A. Prosser, J.D. Miller, H.C. Heller, A serotonin agonist phaseshifts the circadian clock in the suprachiasmatic nuclei in vitro, Brain Res. 534 Ž1990. 336–339. M.R. Ralph, N. Mrosovsky, Behavioral inhibition of circadian responses to light, J. Biol. Rhythms 7 Ž1992. 353–359. M.A. Rea, J. Barrera, J.D. Glass, R.L. Gannon, Serotonergic potentiation of photic phase shifts of the circadian activity rhythm, NeuroReport 6 Ž1995. 1289–1292. M.A. Rea, J.D. Glass, C.S. Colwell, Serotonin modulates photic responses in the hamster suprachiasmatic nuclei, J. Neurosci. 14 Ž1994. 3635–3642. S.G. Reebs, R.J. Lavery, N. Mrosovsky, Running activity mediates the phase-advancing effects of dark pulses on hamster circadian rhythms, J. Comp. Physiol. 165 Ž1989. 811–818. S.G. Reebs, N. Mrosovsky, Effects of induced wheel running on the circadian activity rhythms of Syrian hamsters: entrainment and phase response curve, J. Biol. Rhythms 4 Ž1989. 39–48. A.L. Roca, D.R. Weaver, S.M. Reppert, Serotonin receptor gene expression in the rat suprachiasmatic nuclei, Brain Res. 608 Ž1993. 159–165. S. Shibata, R.Y. Moore, Neuropeptide Y and optic chiasm stimulation affect suprachiasmatic nucleus circadian function in vitro, Brain Res. 615 Ž1993. 95–100. S. Shibata, A. Tsuneyoshi, T. Hamada, K. Tominaga, S. Watanabe, Phase-resetting effect of 8-OH-DPAT, a serotonin 1A receptor agonist, on the circadian rhythm of firing rate in the rat suprachiasmatic nuclei in vitro, Brain Res. 356 Ž1992. 19203–19356. S. Shibata, A. Watanabe, T. Hamada, M. Ono, S. Watanabe, Nmethyl-D-aspartate induces phase shifts in circadian rhythm of neuronal activity of rat SCN in vitro, Am. J. Physiol. 267 Ž1994. R360–R364. V. Simonneaux, A. Ouichou, C. Craft, P. Pevet, Presynaptic and postsynaptic effects of neuropeptide Y in the rat pineal gland, J. Neurochem. 62 Ž1994. 2464–2471. G. Srkalovic, M. Selim, M.A. Rea, J.D. Glass, Serotonergic inhibition of extracellular glutamate in the suprachiasmatic nuclear region assessed using in vivo brain microdialysis, Brain Res. 656 Ž1994. 302–308. M. Tanaka, Y. Ichitani, H. Okamura, Y. Tanaka, Y. Ibata, The direct retinal projection to VIP neuronal elements in the rat SCN, Brain Res. Bull. 31 Ž1993. 637–640. A.N. van den Pol, K. Obrietan, G. Chen, A.B. Belousov, Neuropeptide g-mediated long-term depression of excitatory activity in suprachiasmatic nucleus neurons, J. Neurosci. 16 Ž1996. 5883–5895.

R.A. Prosserr Brain Research 808 (1998) 31–41 w60x E.T. Weber, M.A. Rea, Neuropeptide Y blocks light-induced phase advances but not delays of the circadian activity rhythm in hamsters, Neurosci. Lett. 231 Ž1997. 159–162. w61x C. Wickland, F.W. Turek, Lesions of the thalamic intergeniculate leaflet block activity-induced phase shifts in the circadian activity rhythm of golden hamster, Brain Res. 660 Ž1994. 293–300.

41

w62x S.-W. Ying, B. Rusak, Effects of serotonergic agonists on firing rates of photically responsive cells in the hamster suprachiasmatic nucleus, Brain Res. 651 Ž1994. 37–46. w63x S.-W. Ying, B. Rusak, 5-HT7 receptors mediate serotonergic effects on light-sensitive suprachiasmatic nucleus neurons, Brain Res. 755 Ž1997. 246–254.