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Single unit response of neurons within the hamster suprachiasmatic nucleus to neuropeptide Y
Brain Research Bullerin,
Vol. 27, pp. 825-828. c PergamonRess plc, 1991.Printedin the U.S.A.
0361-9230/91$3.00 + .@I
Single Unit Response of Neurons Within the Hamster Suprachiasmatic Nucleus to Neuropeptide Y S. Y. LIOU’ AND H. E. ALBERS2 Laboratory of Neuroendocrinology and Behavior, Departments of Biology and Psychology Georgia State University, Atlanta, GA 30303 Received 7 March 1991 LIOU, S. Y. AND H. E. ALBERS. Single unit response of neurons within the hamster suprachiasmatic nucleus to neuropeptide Y. BRAIN RBS BULL 27(6) 825-828, 19!X.-The effect of neuropeptide Y (NPY) on the spontaneous discharge of neurons within the hamster (Mesocricetus auratus) suprachiasmatic nucleus (SCN) (N = 83) was determined using the hypothalamic slice preparation. The discharge of neurons within the ventral SCN recorded during the day was either excited (6/42), immediately inhibited (17/42), or transiently excited and then inhibited (10/42) by NPY lo-‘. During the night, NF’Y produced excitatory effects in 2J23 neurons, inhibition in 7/23 and excitation followed by inhibition in 4/23. A higher percentage of neurons was found to be unresponsive to NF’Y during the night than during the day. This difference approached but did not reach statistical significance. In the 18 neurons recorded within the dorsal SCN, NPY had little effect on spontaneous discharge during the day or night. These data indicate that bath-application of NF’Ypredominately inhibits the spontaneous discharge of SCN neurons recorded in the hypothalamic slice preparation. Hypothalamic slice preparation
Extracellular recording
Peptides
Nevertheless, studies in which the GHT has been destroyed suggest that the GHT is not essential for the synchronization of circadian rhythms with the light-dark cycle. Electrical and chemical lesions of the intergeniculate leaflet produce some alterations in the pattern of circadian rhythms of rodents housed in light-dark cycles, but do not eliminate the ability of light-dark cycles to synchronize circadian rhythms (13, 18, 25). The effects of NPY on the spontaneous discharge of SCN neurons have been determined using the in vitro hypothalamic slice preparation. The first report examined the effects of pressure ejection of NPY on the spontaneous discharge of neurons in the hamster SCN (19). In the 65% of SCN neurons found to be responsive, NPY produced exclusively excitatory responses. Two subsequent studies have investigated NPY administered by bath application in the rat SCN (6,31). These studies found NPY’s effects to be predominately inhibitory. In some neurons, NPY had immediate inhibitory effects, while in other neurons, NPY produced a transitory excitation followed by a sustained inhibition. The inhibitory effect of NPY was not eliminated by low-calcium medium, suggesting that NPY produced inhibition by a direct effect on SCN neurons (31). If NPY has opposite effects on the spontaneous discharge of SCN neurons in the rat and hamster, it is possible that the function of the GHT may differ fundamentally between the two species. Alternatively, the species differences in the response of SCN neurons to NPY may simply be the result of differences in
CONSIDERABLE progress
has been made in identifying the neuroanatomical structures involved in controlling mammalian circadian rhythms (20,28). In rodents, neurons within the supra-
chiasmatic nucleus of the hypothalamus (SCN) appear to generate circadian rhythmicity and mediate the synchronization of circadian rhythms with the light-dark cycle. At least two major photic projections communicate light information to the SCN. The retinohypothalamic tract, which is a direct projection from retinal ganglion cells (15, 23, 26), appears to provide sufficient information about environmental lighting to maintain the synchronization of circadian rhythms with the light-dark cycle (17,21). A second photic pathway to the SCN, the geniculohypothalamic tract (GHT), projects to the SCN from the intergeniculate leaflet of the thalamus (27,34). While the GHT may participate in the synchronization of circadian rhythms with the light-dark cycle, its role in this function is not well understood. Immunocytochemical studies have localized neuropeptide Y (NPY) immunoreactivity in neurons of the GHT that end in a complex terminal field within the ventrolateral SCN (9, 12, 22). NPY and NPY-like peptides locally administered within the SCN produce a pattern of phase shifts in hamster circadian wheehunning rhythms (2,3) that are similar to the phase shifts produced by pulses of darkness (8,lO) and transitions from light to dark (1). Electrical and chemical stimulation of the GHT also produces phase shifts in hamster circadian rhythms like those seen following NPY microinjection (18,29).
‘Present address: Upjohn Pharmaceuticals Limited, Tsukuba Research Laboratories, 23 Wadai, Tsukuba-shi, Ibaraki, 30-42, Japan. ‘Requests for reprints should be addressed to H. E. Albers.
825
826
LIOU AND ALBERS
the techniques used for NPY administration. The purpose of the present study was to discriminate between these possibilities by determining how NPY administered by bath application influences the spontaneous discharge of neurons in the hamster SCN. METHOD
Adult male hamsters obtained from Harlan Sprague-Dawley were housed in LD 14:lO for at least two weeks prior to experimental use. Hamsters were decapitated and coronal brain slices containing the SCN (400-450 pm in thickness) were cut on a vibratome. A dim red light (Kodak safelight filter No. 1A) was used to prepare brain slices from hamsters killed during the dark phase. Nine slices were obtained from hamsters sacrificed during the light phase and 14 slices from hamsters sacrificed during the dark phase. The slices were immediately placed in a modified Krebs solution (30) oxygenated with 95% 0, and 5% CO, at 35°C for at least one hour before they were transferred to the recording chamber. Extracellular single unit recordings were made with glass micropipettes (DC resistances of 25-40 M) filled with 0.5 M sodium acetate containing 2% Fast green. The micropipette was lowered into the slice with the aid of a dissecting microscope. At the end of the recording session, a current of 5 p,A was passed through the electrode for 3-5 min to mark the site of recording. After stable recordings were obtained, NPY was administered into the slice chamber for 2-5 min in a concentration of lop7 M, unless indicated otherwise. Peptide-containing medium reached the recording chamber 2-3 min after the switch from incubation medium. The responses to NPY were assessed in the integrated records obtained from a chart recorder by calculating the change in firing rate from the resting level (mean firing rate during the 5 min prior to peptide administration) to the peak level (mean firing rate during 1 min). The neuron was classified as excited or inhibited if its discharge increased or decreased by 20% following NPY application. Unless noted otherwise, analysis of variance was used to determine statistical significance (32). RESULTS The 83 spontaneously firing neurons recorded in this experiment exhibited either regular (N=54) or irregular (N= 29) discharge patterns according to established criteria (30). A statistically significant (p
TABLE 1 RESPONSE OF NEURONS RECORDED IN THE VENTRAL ASPECT OF THE SUPRACHIASMATIC NUCLEUS TO NEUROPEFTIDE Y DURING THE DAY AND NIGHT
Response
Day (42)
Night (23)
Excited Inhibited Excited/Inhibited None
14% (6) 40% (17) 24% (10) 21% (9)
9% (2) 30% (7) 17% (4) 43% (10)
Numbers of neurons are indicated in the parentheses.
The initial excitatory effect of NPY was not found to be statistically significant @>0.05) in neurons recorded during the day (i.e., 3.7kO.6 to 5.OkO.6 impulses/s; N=9) or during the night (4.1kO.8 to 5.8r0.7; N=4). In contrast, the inhibition observed following the initial excitation was statistically significant (p
The present data demonstrate that NPY administered by bath application predominately inhibits the spontaneous discharge of hamster SCN neurons in a manner very similar to that previously repotted in rats. In agreement with these previous studies in rats (6,31), NPY was found to produce sustained inhibition of spontaneous discharge, and to influence more neurons recorded in the ventral SCN, than neurons recorded in the dorsal SCN. The major difference between the present data and those previously reported in the hamster (19) was that NPY produced primarily inhibitory effects in the these experiments, whereas NPY produced exclusively excitatory responses in the previous study. This difference in response to NPY would seem to be the result of the use of bath application versus pressure ejection. The reasons for these differences are unclear. One possibility is that the inhibitory effects of bath application are the result of indirect effects of NPY on SCN neurons. However, studies in the rat have found the inhibitory effects of NPY administered by bath application to persist in low-calcium medium (31), suggesting that NPY acts directly to inhibit SCN single-unit discharge. Other than this methodological difference, NPY had similar effects on SCN neurons in the hamster hypothalamic slice. In both studies, NPY had long-lasting effects, and altered the discharge of mom neurons recorded during the day than during the night. Although the GHT does not appear to be essential for the synchronization of circadian rhythms with the light-dark cycle, the GHT appears to influence how the circadian system responds to at least some forms of lighting stimuli. Destruction of the intergeniculate leaflet reduces phase advances produced by expo-
827
NPY AND SUPRACHIASMATIC DISCHARGE
2 lnln FIG. 1. Integrated tiring rate record illustrating the inhibitory effects of NPY on the spontaneous of a neuron recorded in the ventral part of the suprachiasmatic nucleus.
sure to pulses of light and darkness, shortens the free-running circadian period of hamsters housed in constant light, reduces the occurrence of “splitting” during exposure to constant light, and can alter the rate at which circadian rhythms become resynchronized following shifts in the timing of the light-dark cycle (13, 14, 18, 25). The mechanisms by which NPY influences the circadian response to light and phase shifts the circadian clock are not well understood. One possibility is that NPY influences circadian timing as a result of its effects on a subpopulation of SCN neurons that colocalize vasoactive intestinal peptide (VIP), peptide histidine isoleucine (PHI) and gastrin-releasing peptide ‘(CRP) (4,24). NPY immunoreactive terminals within the SCN appear to synapse directly on SCN neurons in the ventrolateral SCN that are immunopositive for VIP (16,35). Environmental lighting conditions influence the concentrations of VIP and PHI immu-
noreactivity within the SCN (5). mRNA encoding both VIP and PHI peaks during the dark phase (7, 11, ing GRP occurs in a 24-h rhythm
discharge
The cellular levels of the occur in a 24-h rhythm that 33), and the mRNA encodthat peaks during the light
phase (36). Taken together, these data suggest that NPY could be at least one of the factors that serve to regulate VIP, PHI and GRP gene expression, concentration and possibly release within the SCN. It is possible that NPY influences circadian timing by altering the corelease of VIP, PHI and GRP, since microinjection of a cocktail containing VIP, PHI and GRP into the SCN phase shifts circadian rhythms (4). _ ACKNOWLEDGEMENT
This work was supported by NOOO14-89-J-1640 Naval Research.
from the Office of
REFERENCES 1. Albers, H. E. Response of the hamster circadian system to transitions between light and darkness. Am. J. Physiol. 250:R708-R711; 1986. 2. Albers, H. E.; Ferris, C. F. Neuropeptide Y: Role in light-dark cycle entrainment of hamster circadian rhythm. Neurosci. Lea. 50: 163-168; 1984. 3. Albers, H. E.; Ferris, C. F.; Leeman, S. E.; Goldman, B. D. Avian pancreatic polypeptide phase shifts hamster circadian rhythms when microinjected into the suprachiasmatic region. Science 223:833-835; 1984. 4. Albers, H. E.; Liou, S. Y.; Stopa, E. G.; Zoeiler, R. T. Interaction of co-localized neuropeptides: Functional significance in the circadian timing system. J. Neurosci. 11:84&851; 1991. 5. Albers, H. E.; Minamitani, N.; Stopa, E.; Ferris, C. F. Light selectively alters vasoactive intestinal peptide and peptide histidine isoleucine immunoreactivity with the rat suprachiasmatic nucleus. Brain Res. 437:189-192; 1987. 6. Albers, H. E.; Gttenweller, J. E.; Liou, S. Y.; Lumpkin, M. D.; Anderson, E. R. Neuropeptide Y in the hypothalamus: Effect on corticosterone and single-unit activity. Am. J. Physiol. 258:R376 R382; 1990. I. Albers, H. E.; Stopa, E. G.; Zoeller, R. T.; Kauer, J. S.; King, J. C.; Fink, J. S.; Mobtaker, H.; Wolfe, H. Day-night variation in prepro vasoactive intestinal peptide/peptide his&dine isoleucine mRNA within the rat suprachiasmatic nucleus. Mol. Brain Res. 7:85-89; 1990. 8. Boulos, 2.; Rusak, B. Circadian phase response curves for dark pulses in the hamster. J. Comp. Physiol. 146:41l-417; 1982. 9. Card, J. P.; Moore, R. Y. Ventral lateral geniculate nucleus effer-
ents to the rat suprachiasmatic nucleus exhibit avian pancreatic polypeptide-like immunoreactivity. J. Comp. Neural. 206:39&396; 1982. 10. ElJis, G. B.; McKlveen, R. E.; Tnrek, F. W. Dark pulses affect the
circadian rhythm of activity in hamsters kept in constant light. Am. J. Physiol. 242:R&R50; 1982. 11. GAZES, I.; Shard, Y.; Liu, B.; Burbach, J. P. H. Diurnal variation in vasoactive intestinal peptide messenger RNA in the suprachias-
matic nucleus of the rat. Neurosci. Res. Commun. 5:83-86; 1989. 12. Harrington, M. E.; Name, D. M.; Rusak, B. Neuropeptide Y immunoreactivity in the hamster geniculo-suprachiasmatic tract. Brain Res. Bull. 15465-472; 1985. 13. Harrington, M. E.; Rusak, B. Lesions of the thalamic intergeniculate leaflet alter hamster circadian rhythms. J. Biol. Rhythms 1:309325; 1986. 14. Harrington, M. E.; Rusak, B. Ablation of the geniculo-hypothalamic tract alters circadian activity rhythms of hamster housed under constant light. Physiol. Behav. 42:183-189; 1987. A. E.; Wagoner, N.; Cowan, W. M. An autoradio15. Hendrickson, graphic and electron microscopic study of retino-hypothalamic connections. Z. Zell. 135:1-26; 1972. 16. Hisano, S.; Chikamori-Aoyama, M.; Katoh, S.; Kagotani, Y.; Daikoku, S.; Chihara, K. Suprachiasmatic nucleus neurons immunoreactive for vasoactive intestinal peptide have synaptic contacts with axons immunoreactive for neuropeptide Y: An immunoelectromnicroscopic study in the rat. Neurosci. Lett. 88:145-150; 1988. 17. Johnson, R. F.; Moore, R. Y.; Morin, L. P. Loss of entrainment and anatomical plasticity after lesions of the hamster retinohypothalamic tract. Brain Res. 460:297-313; 1988. 18. Johnson, R. F.; Moore, R. Y.; Morin, L. P. Lateral geniculate lesions alter circadian activity rhythms in the hamster. Brain Res. 22: 411422; 1989. 19. Mason, R.; Harrington, M. E.; Rusak, B. Electrophysiological responses of hamster suprachiasmatic neurones to neuropeptide Y in the hypothalamic slice preparation. Neurosci. Lett. 80:173-179; 1987. 20. Meijer, J. H.; Rietveld, W. J. Neurophysiology of the suprachiasmatic circadian pacemaker in rodents. Physiol. Rev. 69671-707; 1989. 21. Moore, R. Y. Visual pathways and the central neural control of diurnal rhythms. In: Circadian oscillations and organization in nervous systems, Cambridge, MA: MIT Press; 1975:537-542. 22. Moore, R. Y.; Gustafson, E. L.; Card, J. P. Identical immunoreactivity of afferents to the rat suprachiasmatic nucleus with antisera against avian pancreatic polypeptide, molluscan cardioexcitatory
LIOU ANDALBERS
828
peptide and neuropeptide Y. Cell Tissue Res. 236~4146; 1984. 23. Moore, R. Y.; Lenn, N. J. A ~tinoh~~al~~c projection in the rat. J. Comp. Neuroi. 1461-14; 1972. 24. Okamura, H.; Murakami, S.; Uda, K.; Sugano, T.; Takahashi, Y.; Yanaihara, C.; Yanaihara, N.; Ibata. Y. Coexistence of vasoactive intestinal peptide (VIP)-, peptide histidine isoleucine amide (PHI)-, and gastrin releasing peptide (GRP)-like immunoreactivity in neurons of the rat supmchiasmatic nucleus. Biomed. Res. 7:295-299: 1986. 25. Pickard, G. E.; Ralph, M. R.; Menaker, M. The intergeniculate leaflet partially mediates effects of light on circadian rhythms. J. Biol. Rhythms 2:35-56; 1987. 26. Pickard, G. E. The afferent connections of the suprachiasmatic nucleus of the golden hamster with emphasis on the retinohypothalamic projection. J. Comp. Neurol. 21165-83; 1982. 27. Ribak, C. E.; Peters, A. An autoradiographic study of the projections from the lateral geniculate body of the rat. Brain Res. 92:261294; 1975. 28. Rosewasser, A. M.; Adler, N. T. Structure and function in circadian timing systems: Evidence for multiple coupled oscillators. Neurosci. Biobehav. Rev. 10:431-448; 1986. 29. Rusak, B.; Meijer, J. H.; Harrington. M. E. Hamster circadian rhythms are phase-shifted by electrical stimulation of the geniculohy~~~a~c tract. Brain Res. 493283-291: 1989. 30. Shibata, S.; Lieu, S. Y.; Ueki, S. Effect of amino acids and mono-
3 I.
32.
33.
34.
35.
amines on the neuronal activity of suprachiasmatic nucleus in hypothalamic slice pre~ations. Jpn. J. Pharmacol. 33:1225-1231; 1983. Shibata, S.; Moore, R. Y. Neuropeptide Y and vasopressin effects on rat suprachiasmatic nucleus neurons in vitro. J. Biol. Rhythms 3~265-276: 1988. Steinmetz, J. E.; Romano, A. G.; Patterson, M. M. Statistical programs for the Apple II microcomputer. Behav. Res. Methods Instrum. 13:702-702; 1981. Stopa, E. G.; Minami~ni, N.; Jonassen, J. A.; King, J. C.; Wolfe, H.; Mobtaker, H.; Albers, H. E. Localization of vasoactive intestinal peptide and peptide histidine isoleucine i~unoreactivity and mRNA within the rat suprachiasmatic nucleus. Mol. Brain Res. 4:319-325; 1988. Swanson, L. W.; Cowan, W. M.; Jones, E. Cl. An autoradiographic study of the efferent connections of the ventral lateral geniculate nucleus in the albino rat and cat. J. Comp. Neural. 1.56~143-163; 1974. Wyatt, L. M.; Norgren, R. B.; Lehman, M. N. Retinal and neu-
ropeptide Y innervation of the hamster suprachiasmatic nucleus: light and electron microscopic observations. Sot. Neurosci. Abstr. 1450; 1988. 36. Zoeller, R. T.; Lickhder, N. R.; Anderson, E. R.; Albers, H. E. Cellular levels of messenger RNAs encoding VIP/PHI and gastrin releasing peptide (GRP, Bombesinf exhibit different 24 hr rhythms in the rat SCN. .I. Neu~nd~~noI.~ in press.
Vol. 27, pp. 825-828. c PergamonRess plc, 1991.Printedin the U.S.A.
0361-9230/91$3.00 + .@I
Single Unit Response of Neurons Within the Hamster Suprachiasmatic Nucleus to Neuropeptide Y S. Y. LIOU’ AND H. E. ALBERS2 Laboratory of Neuroendocrinology and Behavior, Departments of Biology and Psychology Georgia State University, Atlanta, GA 30303 Received 7 March 1991 LIOU, S. Y. AND H. E. ALBERS. Single unit response of neurons within the hamster suprachiasmatic nucleus to neuropeptide Y. BRAIN RBS BULL 27(6) 825-828, 19!X.-The effect of neuropeptide Y (NPY) on the spontaneous discharge of neurons within the hamster (Mesocricetus auratus) suprachiasmatic nucleus (SCN) (N = 83) was determined using the hypothalamic slice preparation. The discharge of neurons within the ventral SCN recorded during the day was either excited (6/42), immediately inhibited (17/42), or transiently excited and then inhibited (10/42) by NPY lo-‘. During the night, NF’Y produced excitatory effects in 2J23 neurons, inhibition in 7/23 and excitation followed by inhibition in 4/23. A higher percentage of neurons was found to be unresponsive to NF’Y during the night than during the day. This difference approached but did not reach statistical significance. In the 18 neurons recorded within the dorsal SCN, NPY had little effect on spontaneous discharge during the day or night. These data indicate that bath-application of NF’Ypredominately inhibits the spontaneous discharge of SCN neurons recorded in the hypothalamic slice preparation. Hypothalamic slice preparation
Extracellular recording
Peptides
Nevertheless, studies in which the GHT has been destroyed suggest that the GHT is not essential for the synchronization of circadian rhythms with the light-dark cycle. Electrical and chemical lesions of the intergeniculate leaflet produce some alterations in the pattern of circadian rhythms of rodents housed in light-dark cycles, but do not eliminate the ability of light-dark cycles to synchronize circadian rhythms (13, 18, 25). The effects of NPY on the spontaneous discharge of SCN neurons have been determined using the in vitro hypothalamic slice preparation. The first report examined the effects of pressure ejection of NPY on the spontaneous discharge of neurons in the hamster SCN (19). In the 65% of SCN neurons found to be responsive, NPY produced exclusively excitatory responses. Two subsequent studies have investigated NPY administered by bath application in the rat SCN (6,31). These studies found NPY’s effects to be predominately inhibitory. In some neurons, NPY had immediate inhibitory effects, while in other neurons, NPY produced a transitory excitation followed by a sustained inhibition. The inhibitory effect of NPY was not eliminated by low-calcium medium, suggesting that NPY produced inhibition by a direct effect on SCN neurons (31). If NPY has opposite effects on the spontaneous discharge of SCN neurons in the rat and hamster, it is possible that the function of the GHT may differ fundamentally between the two species. Alternatively, the species differences in the response of SCN neurons to NPY may simply be the result of differences in
CONSIDERABLE progress
has been made in identifying the neuroanatomical structures involved in controlling mammalian circadian rhythms (20,28). In rodents, neurons within the supra-
chiasmatic nucleus of the hypothalamus (SCN) appear to generate circadian rhythmicity and mediate the synchronization of circadian rhythms with the light-dark cycle. At least two major photic projections communicate light information to the SCN. The retinohypothalamic tract, which is a direct projection from retinal ganglion cells (15, 23, 26), appears to provide sufficient information about environmental lighting to maintain the synchronization of circadian rhythms with the light-dark cycle (17,21). A second photic pathway to the SCN, the geniculohypothalamic tract (GHT), projects to the SCN from the intergeniculate leaflet of the thalamus (27,34). While the GHT may participate in the synchronization of circadian rhythms with the light-dark cycle, its role in this function is not well understood. Immunocytochemical studies have localized neuropeptide Y (NPY) immunoreactivity in neurons of the GHT that end in a complex terminal field within the ventrolateral SCN (9, 12, 22). NPY and NPY-like peptides locally administered within the SCN produce a pattern of phase shifts in hamster circadian wheehunning rhythms (2,3) that are similar to the phase shifts produced by pulses of darkness (8,lO) and transitions from light to dark (1). Electrical and chemical stimulation of the GHT also produces phase shifts in hamster circadian rhythms like those seen following NPY microinjection (18,29).
‘Present address: Upjohn Pharmaceuticals Limited, Tsukuba Research Laboratories, 23 Wadai, Tsukuba-shi, Ibaraki, 30-42, Japan. ‘Requests for reprints should be addressed to H. E. Albers.
825
826
LIOU AND ALBERS
the techniques used for NPY administration. The purpose of the present study was to discriminate between these possibilities by determining how NPY administered by bath application influences the spontaneous discharge of neurons in the hamster SCN. METHOD
Adult male hamsters obtained from Harlan Sprague-Dawley were housed in LD 14:lO for at least two weeks prior to experimental use. Hamsters were decapitated and coronal brain slices containing the SCN (400-450 pm in thickness) were cut on a vibratome. A dim red light (Kodak safelight filter No. 1A) was used to prepare brain slices from hamsters killed during the dark phase. Nine slices were obtained from hamsters sacrificed during the light phase and 14 slices from hamsters sacrificed during the dark phase. The slices were immediately placed in a modified Krebs solution (30) oxygenated with 95% 0, and 5% CO, at 35°C for at least one hour before they were transferred to the recording chamber. Extracellular single unit recordings were made with glass micropipettes (DC resistances of 25-40 M) filled with 0.5 M sodium acetate containing 2% Fast green. The micropipette was lowered into the slice with the aid of a dissecting microscope. At the end of the recording session, a current of 5 p,A was passed through the electrode for 3-5 min to mark the site of recording. After stable recordings were obtained, NPY was administered into the slice chamber for 2-5 min in a concentration of lop7 M, unless indicated otherwise. Peptide-containing medium reached the recording chamber 2-3 min after the switch from incubation medium. The responses to NPY were assessed in the integrated records obtained from a chart recorder by calculating the change in firing rate from the resting level (mean firing rate during the 5 min prior to peptide administration) to the peak level (mean firing rate during 1 min). The neuron was classified as excited or inhibited if its discharge increased or decreased by 20% following NPY application. Unless noted otherwise, analysis of variance was used to determine statistical significance (32). RESULTS The 83 spontaneously firing neurons recorded in this experiment exhibited either regular (N=54) or irregular (N= 29) discharge patterns according to established criteria (30). A statistically significant (p
TABLE 1 RESPONSE OF NEURONS RECORDED IN THE VENTRAL ASPECT OF THE SUPRACHIASMATIC NUCLEUS TO NEUROPEFTIDE Y DURING THE DAY AND NIGHT
Response
Day (42)
Night (23)
Excited Inhibited Excited/Inhibited None
14% (6) 40% (17) 24% (10) 21% (9)
9% (2) 30% (7) 17% (4) 43% (10)
Numbers of neurons are indicated in the parentheses.
The initial excitatory effect of NPY was not found to be statistically significant @>0.05) in neurons recorded during the day (i.e., 3.7kO.6 to 5.OkO.6 impulses/s; N=9) or during the night (4.1kO.8 to 5.8r0.7; N=4). In contrast, the inhibition observed following the initial excitation was statistically significant (p
The present data demonstrate that NPY administered by bath application predominately inhibits the spontaneous discharge of hamster SCN neurons in a manner very similar to that previously repotted in rats. In agreement with these previous studies in rats (6,31), NPY was found to produce sustained inhibition of spontaneous discharge, and to influence more neurons recorded in the ventral SCN, than neurons recorded in the dorsal SCN. The major difference between the present data and those previously reported in the hamster (19) was that NPY produced primarily inhibitory effects in the these experiments, whereas NPY produced exclusively excitatory responses in the previous study. This difference in response to NPY would seem to be the result of the use of bath application versus pressure ejection. The reasons for these differences are unclear. One possibility is that the inhibitory effects of bath application are the result of indirect effects of NPY on SCN neurons. However, studies in the rat have found the inhibitory effects of NPY administered by bath application to persist in low-calcium medium (31), suggesting that NPY acts directly to inhibit SCN single-unit discharge. Other than this methodological difference, NPY had similar effects on SCN neurons in the hamster hypothalamic slice. In both studies, NPY had long-lasting effects, and altered the discharge of mom neurons recorded during the day than during the night. Although the GHT does not appear to be essential for the synchronization of circadian rhythms with the light-dark cycle, the GHT appears to influence how the circadian system responds to at least some forms of lighting stimuli. Destruction of the intergeniculate leaflet reduces phase advances produced by expo-
827
NPY AND SUPRACHIASMATIC DISCHARGE
2 lnln FIG. 1. Integrated tiring rate record illustrating the inhibitory effects of NPY on the spontaneous of a neuron recorded in the ventral part of the suprachiasmatic nucleus.
sure to pulses of light and darkness, shortens the free-running circadian period of hamsters housed in constant light, reduces the occurrence of “splitting” during exposure to constant light, and can alter the rate at which circadian rhythms become resynchronized following shifts in the timing of the light-dark cycle (13, 14, 18, 25). The mechanisms by which NPY influences the circadian response to light and phase shifts the circadian clock are not well understood. One possibility is that NPY influences circadian timing as a result of its effects on a subpopulation of SCN neurons that colocalize vasoactive intestinal peptide (VIP), peptide histidine isoleucine (PHI) and gastrin-releasing peptide ‘(CRP) (4,24). NPY immunoreactive terminals within the SCN appear to synapse directly on SCN neurons in the ventrolateral SCN that are immunopositive for VIP (16,35). Environmental lighting conditions influence the concentrations of VIP and PHI immu-
noreactivity within the SCN (5). mRNA encoding both VIP and PHI peaks during the dark phase (7, 11, ing GRP occurs in a 24-h rhythm
discharge
The cellular levels of the occur in a 24-h rhythm that 33), and the mRNA encodthat peaks during the light
phase (36). Taken together, these data suggest that NPY could be at least one of the factors that serve to regulate VIP, PHI and GRP gene expression, concentration and possibly release within the SCN. It is possible that NPY influences circadian timing by altering the corelease of VIP, PHI and GRP, since microinjection of a cocktail containing VIP, PHI and GRP into the SCN phase shifts circadian rhythms (4). _ ACKNOWLEDGEMENT
This work was supported by NOOO14-89-J-1640 Naval Research.
from the Office of
REFERENCES 1. Albers, H. E. Response of the hamster circadian system to transitions between light and darkness. Am. J. Physiol. 250:R708-R711; 1986. 2. Albers, H. E.; Ferris, C. F. Neuropeptide Y: Role in light-dark cycle entrainment of hamster circadian rhythm. Neurosci. Lea. 50: 163-168; 1984. 3. Albers, H. E.; Ferris, C. F.; Leeman, S. E.; Goldman, B. D. Avian pancreatic polypeptide phase shifts hamster circadian rhythms when microinjected into the suprachiasmatic region. Science 223:833-835; 1984. 4. Albers, H. E.; Liou, S. Y.; Stopa, E. G.; Zoeiler, R. T. Interaction of co-localized neuropeptides: Functional significance in the circadian timing system. J. Neurosci. 11:84&851; 1991. 5. Albers, H. E.; Minamitani, N.; Stopa, E.; Ferris, C. F. Light selectively alters vasoactive intestinal peptide and peptide histidine isoleucine immunoreactivity with the rat suprachiasmatic nucleus. Brain Res. 437:189-192; 1987. 6. Albers, H. E.; Gttenweller, J. E.; Liou, S. Y.; Lumpkin, M. D.; Anderson, E. R. Neuropeptide Y in the hypothalamus: Effect on corticosterone and single-unit activity. Am. J. Physiol. 258:R376 R382; 1990. I. Albers, H. E.; Stopa, E. G.; Zoeller, R. T.; Kauer, J. S.; King, J. C.; Fink, J. S.; Mobtaker, H.; Wolfe, H. Day-night variation in prepro vasoactive intestinal peptide/peptide his&dine isoleucine mRNA within the rat suprachiasmatic nucleus. Mol. Brain Res. 7:85-89; 1990. 8. Boulos, 2.; Rusak, B. Circadian phase response curves for dark pulses in the hamster. J. Comp. Physiol. 146:41l-417; 1982. 9. Card, J. P.; Moore, R. Y. Ventral lateral geniculate nucleus effer-
ents to the rat suprachiasmatic nucleus exhibit avian pancreatic polypeptide-like immunoreactivity. J. Comp. Neural. 206:39&396; 1982. 10. ElJis, G. B.; McKlveen, R. E.; Tnrek, F. W. Dark pulses affect the
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