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A 5-HT1B receptor agonist inhibits light-induced suppression of pineal melatonin production
Brain Research 858 Ž2000. 424–428 www.elsevier.comrlocaterbres
Short communication
A 5-HT1B receptor agonist inhibits light-induced suppression of pineal melatonin production Michael A. Rea
a,b,)
, Gary E. Pickard
c
a
BRAIN Research Institute, Air Force Research Laboratory, Brooks AFB, TX, USA Department of Biology and Biochemistry, UniÕersity of Houston, Houston, TX, USA Department of Anatomy and Neurobiology, Colorado State UniÕersity, Ft Collins, CO, USA b
c
Accepted 30 November 1999
Abstract Serotonin Ž5-HT. modulates the phase adjusting effects of light on the mammalian circadian clock through the activation of presynaptic 5-HT1B receptors located on retinal terminals in the suprachiasmatic nucleus ŽSCN.. The current study was conducted to determine whether activation of 5-HT1B receptors also alters photic regulation of nocturnal pineal melatonin production. Systemic administration of the 5-HT1B receptor agonist TFMPP attenuated the inhibitory effect of light on pineal melatonin synthesis in a dose-related manner with an apparent ED50 value of 0.9 mgrkg. The effect of TFMPP on light-induced melatonin suppression was blocked by the 5-HT1 receptor antagonist, methiothepin, but not by the 5-HT1A antagonist, WAY 100,635, consistent with the involvement of 5-HT1B receptors. The results are consistent with the interpretation that activation of presynaptic 5-HT1B receptors on retinal terminals in the SCN attenuates the effect of light on pineal melatonin production, as well as on circadian phase. q 2000 Elsevier Science B.V. All rights reserved. Keywords: Suprachiasmatic nucleus; Pineal; Melatonin; Circadian rhythm; Retinohypothalamic tract; 5-HT1B receptor
Vertebrates display robust circadian rhythms in pineal melatonin production Žfor review, see Cassone and Natesan w3x.. In many species, the pineal is an autonomous circadian oscillator capable of sustained rhythmic production of melatonin in the absence of external drivers w13,24,36,49x. In mammals, the pineal melatonin rhythm is dependent upon the output of a circadian clock located in the suprachiasmatic nucleus ŽSCN. of the hypothalamus w16,17,27x. SCN neurons drive nocturnal melatonin production through the multi-synaptic activation of sympathetic neurons in the superior cervical ganglion w16,20,26,37,44x. The elevated level of circulating melatonin that occurs during the night is thought to convey length-of-night information to several brain loci, including the SCN, and is important in determining the onset of seasonal changes in physiology and behavior w8,12,42,45x.
) Corresponding author. Circadian Neurobiology Laboratory, Department of Biology and Biochemistry, University of Houston, Houston, TX 77004, USA. Fax: q1-713-743-2636; e-mail: [email protected]
As with other SCN-driven circadian rhythms, pineal melatonin production remains rhythmic under constant environmental conditions w27x. However, the phase of circadian rhythms relative to the timing of dusk and dawn is dynamically regulated by ambient light w7,9,39x. The SCN circadian clock is sensitive to the phase adjusting effects of light only during the subjective nighttime w9x. In addition, light exposure during the subjective night suppresses pineal melatonin production w18x. The effects of light on both circadian phase and nocturnal pineal melatonin production are mediated through the activation of the retinohypothalamic tract w16x, a monosynaptic projection from retinal ganglion cells to the SCN w28x. An abundance of evidence suggests that the neurotransmitter serotonin, arising from neurons of the median raphe nucleus w25x, modulates retinohypothalamic tract ŽRHT. neurotransmission through both pre- and postsynaptic mechanisms within the SCN w10,29,33,38,41,46x. We recently reported that the 5-HT1B receptor agonist 1-w3-Žtrifluoromethyl. phenylx-piperazine ŽTFMPP. w22x attenuates the effects of retinal illumination on circadian phase in a dose-related manner, through the activation of
0006-8993r00r$ - see front matter q 2000 Elsevier Science B.V. All rights reserved. PII: S 0 0 0 6 - 8 9 9 3 Ž 9 9 . 0 2 4 6 2 - 2
M.A. Rea, G.E. Pickardr Brain Research 858 (2000) 424–428
presynaptic receptors on RHT terminals w33–35,38x, presumably resulting in a reduction in glutamate release w30x. In the current report, we tested the hypothesis that TFMPP administration also blocks light-induced suppression of nocturnal pineal melatonin synthesis. Male, Syrian hamsters Ž Mesocricetus auratus . obtained from Charles River Labs ŽWilmington, MA. were housed in groups of four in transparent cages under a light:dark ŽLD. cycle of 14 h of light Žapproximately 300 lux beginning at 0200. and 10 h of total darkness ŽLD 14:10.. Food and water were available ad libitum. Hamsters Ž160–180 g at the time of treatment. were maintained under LD14:10 for at least 4 weeks prior to experimentation to ensure that stable entrainment of circadian rhythmicity had been achieved. Groups of hamsters Ž n s 4–6. received i.p. injections of either TFMPP Ž0.5–10 mgrkg. or vehicle Ž200 ml of 0.9% saline. administered between 0830 and 0930 Ži.e., between 6.5 and 7.5 h after the onset of darkness. under infrared illumination Ž) 850 nm. and with the aid of night vision goggles ŽITT Night Vision, Roanoke, VA.. In some experiments, serotonin receptor antagonists were administered Ži.p.. 30 min prior to TFMPPrvehicle injection. Some animals were exposed to white light Ž20–40 lux. for 10 min beginning 30 min after TFMPPrvehicle injection. All hamsters were killed by decapitation 40 min after TFMPPrvehicle injection. Pineal glands were quickly removed, placed in microfuge tubes, frozen on dry ice, and stored at y808C. Pineal glands were sonicated to homogeneity in 200 ml of phosphate buffered saline ŽpH 7.0., diluted as required, and assayed for melatonin content by radioimmunoassay using a commercial kit ŽALPCO; Windham NH.. All samples from each experiment were analyzed in a single assay. Intra-assay coefficients of variation were between 5% and 15%.. TFMPP HCl, WAY100,635 maleate, and methiothepin were obtained from Research Biochemicals ŽNatick, MA..
Fig. 1. TFMPP completely blocks the inhibitory effect of nighttime light exposure on pineal melatonin content. Groups of hamsters received either vehicle injection without light exposure, vehicle injection with light exposure, 10 mgrkg TFMPP with light exposure, or 10 mgrkg TFMPP without light exposure. Filled bars indicate dark condition. Data represent the mean"S.E.M. of the four determinations. Asterisks ŽU . indicate statistically significant Ž p- 0.05. differences relative to the vehicleqdark group.
425
Fig. 2. Effect of TFMPP is dose-dependent. Data represent the mean" S.E.M. of between four and seven determinations. The apparent ED50 for TFMPP was 0.9 mgrkg.
Drug solutions were prepared fresh within 60 min of injection and stored in the dark. For statistical analysis, data were subjected to One-way ANOVA and differences between means were tested posthoc for statistical significance using the Newman–Keuls test. Ten minutes of light exposure during the middle of the subjective night reduced pineal melatonin content by more than 90% Žvehicleq dark s 1.6 " 0.3 ngrpineal; vehicle q light s 0.15 " 0.04 ngrpineal; p - 0.01; Fig. 1.. Administration of 10 mgrkg TFMPP 30 min prior to light exposure completely blocked the reduction in pineal melatonin content caused by light exposure Ž1.4 " 0.3 ngrpineal.. Administration of TFMPP without light exposure did not significantly alter pineal melatonin content ŽTFMPPq dark s 1.7 " 0.5 ngrpineal.. Blockade of the inhibitory effect of nocturnal light exposure on pineal melatonin content by TFMPP was dose dependent ŽFig. 2., with an apparent ED50 value of 0.9 mgrkg. The inhibitory effect of TFMPP Ž1.0 mgrkg. on photic suppression of pineal melatonin production was blocked by prior administration of the non-selective 5-HT1 antagonist, methiothepin Ž5 mgrkg., and this dose of the antagonist did not alter the effect of light on pineal melatonin content ŽFig. 3.. In a separate experiment, the selective 5-HT1A receptor antagonist WAY100,635 Ž5 mgrkg. failed to block the inhibition of light-induced suppression of pineal melatonin production by TFMPP ŽFig. 4.. In this study, we tested the hypothesis that activation of 5-HT1B receptors inhibits the effect of light on nocturnal melatonin production in a manner similar to that previously observed for light-induced phase shifts of circadian behavior. Systemic administration of the 5-HT1B agonist, TFMPP, dose-dependently attenuated the inhibitory effect of light on pineal melatonin production ŽFigs. 1 and 2.. Furthermore, the ED50 values for the attenuating effects of TFMPP on light-induced phase shifts w38x and on photic suppression of melatonin production are essentially identical. In addition, the effects of TFMPP on both light-induced phase shifts w38x and melatonin suppression are blocked by the 5-HT1 antagonist, methiothepin, but not by
426
M.A. Rea, G.E. Pickardr Brain Research 858 (2000) 424–428
the selective 5-HT1A antagonist, WAY 100,635, indicating that the receptors mediating these effects are pharmacologically similar. A least three serotonin receptor subtypes, 5-HT1A receptors, 5-HT1B receptors, and 5-HT7 receptors, have been implicated as mediators of the effects of serotonin on RHT neurotransmission w10,33,38,41,47,48x. TFMPP has moderate affinity for both 5-HT1A and 5-HT1B receptors w22x and very low affinity for the 5-HT7 receptor w21x. Thus, at the doses employed in the current study, the effect of TFMPP could have been mediated through either 5-HT1A receptors, andror 5-HT1B receptors. The observation that the effect of TFMPP was completely blocked by prior treatment with the non-selective 5-HT1 receptor antagonist, methiothepin, but not by the highly selective 5-HT1A antagonist, WAY100,635, strongly suggests the involvement of 5-HT1B receptors in this action of TFMPP. In rodents, the effects of light on both circadian phase and pineal melatonin production are dependent upon the integrity of the RHT w26x. Moore and Klein reported that disruption of the pre-chiasmatic optic projection prevents photic suppression of pineal melatonin production, while lesions of the post-chiasmatic optic tracts that spare the RHT do not block the effects of light on the pineal w16,27x. Similarly, Johnson et al. w19x demonstrated that selective transsection of the RHT completely blocked the effects of brief exposure to light at night on circadian phase, while lesions of the post-chiasmatic optic tracts did not alter this response to light. Recent reports of retrograde tracing studies using the pseudoradies virus as a probe to trace the multi-synaptic central pathway to the pineal gland suggest that retinorecipient cells within the SCN serve as the primary central relay of photic information to the pineal w20,44x. The results of these studies strongly argue that the RHT is uniquely responsible for conveying photic information, via the SCN, to the pineal gland.
Fig. 3. Methiothepin blocks the effect of TFMPP on photic suppression of pineal melatonin. Data represent the mean"S.E.M. of the number of determinations indicated in the bars. Antagonist or vehicle was administered 30 min prior to TFMPP. Filled bars indicate dark condition. Some animals were exposed to light for 15 min during a 1-h period beginning 7 h after dark onset Žstippled bars.. Asterisks ŽU . indicate statistically significant Ž p- 0.05. differences relative to both the vehicleqdark group and the TFMPPqlight group.
Fig. 4. WAY100,635 does not block the effect of TFMPP on photic suppression of pineal melatonin. Data represent the mean"S.E.M. of the number of determinations indicated in the bars. Antagonist or vehicle was administered 30 min prior to TFMPP. Filled bars indicate dark condition. Some animals were exposed to light for 15 min during a 1-h period beginning 7 h after dark onset Žstippled bars.. Asterisks ŽU . indicate statistically significant Ž p- 0.05. differences relative to the vehicleqdark group.
Abundant evidence indicates that glutamate serves as the principal excitatory neurotransmitter released from the RHT w2,15,43x. In this regard, several laboratories have reported pharmacological evidence indicating that activation of NMDA receptors w5,6x in the SCN region w40x mediates the effects of retinal illumination on circadian phase and gene expression in the SCN w1,40x. Systemic administration of the non-competitive NMDA antagonist MK-801 blocks both light-induced phase shifts w6x and photic suppression of pineal melatonin w5,31x Žbut see also, Kennaway w14x.. These observations suggest that the chemistry of RHT neurotransmission is similar at synapses that convey photic information to the circadian clock and to the pineal. The current report provides additional evidence indicating that RHT neurochemistry is similar at synapses that mediate photic effects on the circadian clock and the pineal gland. Based upon these data, we propose that the inhibitory effect of TFMPP on photic suppression of pineal melatonin production and on light-induced alterations of circadian phase w34,38x are identical, and occur through the 5-HT1B receptor-mediated presynaptic inhibition of RHT input to the SCN w35x. The possibility that distinct populations of retinorecipient neurons in the SCN mediate the effects of light on circadian phase and suppression of pineal melatonin production remains to be determined. Indirect evidence suggests that, 5-HT1B receptors are also present on pre-synaptic terminals of the geniculohypothalamic tract w23x ŽGHT., a projection from the intergeniculate leaflet ŽIGL. that may convey photic information to the SCN w11,32x. Thus, it is possible that the effect of TFMPP on photic suppression of melatonin is mediated, at least in part, through modulation of GHT neurotransmission. However, bilateral lesions of the IGL failed to attenuate the suppression of pineal N-acetyltransferase activity
M.A. Rea, G.E. Pickardr Brain Research 858 (2000) 424–428
by 15 min light pulses in the rat w4x. Thus, under the conditions employed in the current study, it is unlikely that 5-HT1B receptors on GHT terminals played a role in the action of TFMPP. Activation of 5-HT1B autoreceptors, which inhibit the release of serotonin from terminals in the SCN region of the rat w30x may also contribute to the effects of TFMPP in the SCN. However, since these autoreceptors appear to comprise only a small fraction of the 5-HT1B receptor population in the SCN w23x their contribution to the effects of TFMPP in the present study is probably minimal. In summary, systemic administration of the 5-HT1B agonist TFMPP dose-dependently attenuates the inhibitory effect of light on pineal melatonin production. This effect is blocked by the 5-HT1 antagonist, methiothepin, but not by the selective 5-HT1A antagonist, WAY100,635, indicating that the effect of TFMPP is mediated by 5-HT1B receptors. The data suggest that the neurochemistry of RHT neurotransmission at synapses that mediate the effect of light on pineal melatonin production is similar to that at synapses that mediate light-induced adjustments of circadian phase. Furthermore, these observations raise the possibility that serotonin tonus in the SCN may modulate the effect of light exposure on melatonin production.
Acknowledgements The authors wish to thank Ms. Anna Marie Michel and Mr. Matthew Cato for excellent technical assistance. This work was supported by grants from the Air Force Office of Scientific Research AFOSR92-AL-004 ŽM.A.R.. and the National Institutes of Health NS 35615 ŽG.E.P... The animals involved in this study were procured, maintained and used in accordance with the Animal Welfare Act and the Guide for the Care and Use of Laboratory Animals prepared by the Institute for Laboratory Animal Resources, National Science Foundation.
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Short communication
A 5-HT1B receptor agonist inhibits light-induced suppression of pineal melatonin production Michael A. Rea
a,b,)
, Gary E. Pickard
c
a
BRAIN Research Institute, Air Force Research Laboratory, Brooks AFB, TX, USA Department of Biology and Biochemistry, UniÕersity of Houston, Houston, TX, USA Department of Anatomy and Neurobiology, Colorado State UniÕersity, Ft Collins, CO, USA b
c
Accepted 30 November 1999
Abstract Serotonin Ž5-HT. modulates the phase adjusting effects of light on the mammalian circadian clock through the activation of presynaptic 5-HT1B receptors located on retinal terminals in the suprachiasmatic nucleus ŽSCN.. The current study was conducted to determine whether activation of 5-HT1B receptors also alters photic regulation of nocturnal pineal melatonin production. Systemic administration of the 5-HT1B receptor agonist TFMPP attenuated the inhibitory effect of light on pineal melatonin synthesis in a dose-related manner with an apparent ED50 value of 0.9 mgrkg. The effect of TFMPP on light-induced melatonin suppression was blocked by the 5-HT1 receptor antagonist, methiothepin, but not by the 5-HT1A antagonist, WAY 100,635, consistent with the involvement of 5-HT1B receptors. The results are consistent with the interpretation that activation of presynaptic 5-HT1B receptors on retinal terminals in the SCN attenuates the effect of light on pineal melatonin production, as well as on circadian phase. q 2000 Elsevier Science B.V. All rights reserved. Keywords: Suprachiasmatic nucleus; Pineal; Melatonin; Circadian rhythm; Retinohypothalamic tract; 5-HT1B receptor
Vertebrates display robust circadian rhythms in pineal melatonin production Žfor review, see Cassone and Natesan w3x.. In many species, the pineal is an autonomous circadian oscillator capable of sustained rhythmic production of melatonin in the absence of external drivers w13,24,36,49x. In mammals, the pineal melatonin rhythm is dependent upon the output of a circadian clock located in the suprachiasmatic nucleus ŽSCN. of the hypothalamus w16,17,27x. SCN neurons drive nocturnal melatonin production through the multi-synaptic activation of sympathetic neurons in the superior cervical ganglion w16,20,26,37,44x. The elevated level of circulating melatonin that occurs during the night is thought to convey length-of-night information to several brain loci, including the SCN, and is important in determining the onset of seasonal changes in physiology and behavior w8,12,42,45x.
) Corresponding author. Circadian Neurobiology Laboratory, Department of Biology and Biochemistry, University of Houston, Houston, TX 77004, USA. Fax: q1-713-743-2636; e-mail: [email protected]
As with other SCN-driven circadian rhythms, pineal melatonin production remains rhythmic under constant environmental conditions w27x. However, the phase of circadian rhythms relative to the timing of dusk and dawn is dynamically regulated by ambient light w7,9,39x. The SCN circadian clock is sensitive to the phase adjusting effects of light only during the subjective nighttime w9x. In addition, light exposure during the subjective night suppresses pineal melatonin production w18x. The effects of light on both circadian phase and nocturnal pineal melatonin production are mediated through the activation of the retinohypothalamic tract w16x, a monosynaptic projection from retinal ganglion cells to the SCN w28x. An abundance of evidence suggests that the neurotransmitter serotonin, arising from neurons of the median raphe nucleus w25x, modulates retinohypothalamic tract ŽRHT. neurotransmission through both pre- and postsynaptic mechanisms within the SCN w10,29,33,38,41,46x. We recently reported that the 5-HT1B receptor agonist 1-w3-Žtrifluoromethyl. phenylx-piperazine ŽTFMPP. w22x attenuates the effects of retinal illumination on circadian phase in a dose-related manner, through the activation of
0006-8993r00r$ - see front matter q 2000 Elsevier Science B.V. All rights reserved. PII: S 0 0 0 6 - 8 9 9 3 Ž 9 9 . 0 2 4 6 2 - 2
M.A. Rea, G.E. Pickardr Brain Research 858 (2000) 424–428
presynaptic receptors on RHT terminals w33–35,38x, presumably resulting in a reduction in glutamate release w30x. In the current report, we tested the hypothesis that TFMPP administration also blocks light-induced suppression of nocturnal pineal melatonin synthesis. Male, Syrian hamsters Ž Mesocricetus auratus . obtained from Charles River Labs ŽWilmington, MA. were housed in groups of four in transparent cages under a light:dark ŽLD. cycle of 14 h of light Žapproximately 300 lux beginning at 0200. and 10 h of total darkness ŽLD 14:10.. Food and water were available ad libitum. Hamsters Ž160–180 g at the time of treatment. were maintained under LD14:10 for at least 4 weeks prior to experimentation to ensure that stable entrainment of circadian rhythmicity had been achieved. Groups of hamsters Ž n s 4–6. received i.p. injections of either TFMPP Ž0.5–10 mgrkg. or vehicle Ž200 ml of 0.9% saline. administered between 0830 and 0930 Ži.e., between 6.5 and 7.5 h after the onset of darkness. under infrared illumination Ž) 850 nm. and with the aid of night vision goggles ŽITT Night Vision, Roanoke, VA.. In some experiments, serotonin receptor antagonists were administered Ži.p.. 30 min prior to TFMPPrvehicle injection. Some animals were exposed to white light Ž20–40 lux. for 10 min beginning 30 min after TFMPPrvehicle injection. All hamsters were killed by decapitation 40 min after TFMPPrvehicle injection. Pineal glands were quickly removed, placed in microfuge tubes, frozen on dry ice, and stored at y808C. Pineal glands were sonicated to homogeneity in 200 ml of phosphate buffered saline ŽpH 7.0., diluted as required, and assayed for melatonin content by radioimmunoassay using a commercial kit ŽALPCO; Windham NH.. All samples from each experiment were analyzed in a single assay. Intra-assay coefficients of variation were between 5% and 15%.. TFMPP HCl, WAY100,635 maleate, and methiothepin were obtained from Research Biochemicals ŽNatick, MA..
Fig. 1. TFMPP completely blocks the inhibitory effect of nighttime light exposure on pineal melatonin content. Groups of hamsters received either vehicle injection without light exposure, vehicle injection with light exposure, 10 mgrkg TFMPP with light exposure, or 10 mgrkg TFMPP without light exposure. Filled bars indicate dark condition. Data represent the mean"S.E.M. of the four determinations. Asterisks ŽU . indicate statistically significant Ž p- 0.05. differences relative to the vehicleqdark group.
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Fig. 2. Effect of TFMPP is dose-dependent. Data represent the mean" S.E.M. of between four and seven determinations. The apparent ED50 for TFMPP was 0.9 mgrkg.
Drug solutions were prepared fresh within 60 min of injection and stored in the dark. For statistical analysis, data were subjected to One-way ANOVA and differences between means were tested posthoc for statistical significance using the Newman–Keuls test. Ten minutes of light exposure during the middle of the subjective night reduced pineal melatonin content by more than 90% Žvehicleq dark s 1.6 " 0.3 ngrpineal; vehicle q light s 0.15 " 0.04 ngrpineal; p - 0.01; Fig. 1.. Administration of 10 mgrkg TFMPP 30 min prior to light exposure completely blocked the reduction in pineal melatonin content caused by light exposure Ž1.4 " 0.3 ngrpineal.. Administration of TFMPP without light exposure did not significantly alter pineal melatonin content ŽTFMPPq dark s 1.7 " 0.5 ngrpineal.. Blockade of the inhibitory effect of nocturnal light exposure on pineal melatonin content by TFMPP was dose dependent ŽFig. 2., with an apparent ED50 value of 0.9 mgrkg. The inhibitory effect of TFMPP Ž1.0 mgrkg. on photic suppression of pineal melatonin production was blocked by prior administration of the non-selective 5-HT1 antagonist, methiothepin Ž5 mgrkg., and this dose of the antagonist did not alter the effect of light on pineal melatonin content ŽFig. 3.. In a separate experiment, the selective 5-HT1A receptor antagonist WAY100,635 Ž5 mgrkg. failed to block the inhibition of light-induced suppression of pineal melatonin production by TFMPP ŽFig. 4.. In this study, we tested the hypothesis that activation of 5-HT1B receptors inhibits the effect of light on nocturnal melatonin production in a manner similar to that previously observed for light-induced phase shifts of circadian behavior. Systemic administration of the 5-HT1B agonist, TFMPP, dose-dependently attenuated the inhibitory effect of light on pineal melatonin production ŽFigs. 1 and 2.. Furthermore, the ED50 values for the attenuating effects of TFMPP on light-induced phase shifts w38x and on photic suppression of melatonin production are essentially identical. In addition, the effects of TFMPP on both light-induced phase shifts w38x and melatonin suppression are blocked by the 5-HT1 antagonist, methiothepin, but not by
426
M.A. Rea, G.E. Pickardr Brain Research 858 (2000) 424–428
the selective 5-HT1A antagonist, WAY 100,635, indicating that the receptors mediating these effects are pharmacologically similar. A least three serotonin receptor subtypes, 5-HT1A receptors, 5-HT1B receptors, and 5-HT7 receptors, have been implicated as mediators of the effects of serotonin on RHT neurotransmission w10,33,38,41,47,48x. TFMPP has moderate affinity for both 5-HT1A and 5-HT1B receptors w22x and very low affinity for the 5-HT7 receptor w21x. Thus, at the doses employed in the current study, the effect of TFMPP could have been mediated through either 5-HT1A receptors, andror 5-HT1B receptors. The observation that the effect of TFMPP was completely blocked by prior treatment with the non-selective 5-HT1 receptor antagonist, methiothepin, but not by the highly selective 5-HT1A antagonist, WAY100,635, strongly suggests the involvement of 5-HT1B receptors in this action of TFMPP. In rodents, the effects of light on both circadian phase and pineal melatonin production are dependent upon the integrity of the RHT w26x. Moore and Klein reported that disruption of the pre-chiasmatic optic projection prevents photic suppression of pineal melatonin production, while lesions of the post-chiasmatic optic tracts that spare the RHT do not block the effects of light on the pineal w16,27x. Similarly, Johnson et al. w19x demonstrated that selective transsection of the RHT completely blocked the effects of brief exposure to light at night on circadian phase, while lesions of the post-chiasmatic optic tracts did not alter this response to light. Recent reports of retrograde tracing studies using the pseudoradies virus as a probe to trace the multi-synaptic central pathway to the pineal gland suggest that retinorecipient cells within the SCN serve as the primary central relay of photic information to the pineal w20,44x. The results of these studies strongly argue that the RHT is uniquely responsible for conveying photic information, via the SCN, to the pineal gland.
Fig. 3. Methiothepin blocks the effect of TFMPP on photic suppression of pineal melatonin. Data represent the mean"S.E.M. of the number of determinations indicated in the bars. Antagonist or vehicle was administered 30 min prior to TFMPP. Filled bars indicate dark condition. Some animals were exposed to light for 15 min during a 1-h period beginning 7 h after dark onset Žstippled bars.. Asterisks ŽU . indicate statistically significant Ž p- 0.05. differences relative to both the vehicleqdark group and the TFMPPqlight group.
Fig. 4. WAY100,635 does not block the effect of TFMPP on photic suppression of pineal melatonin. Data represent the mean"S.E.M. of the number of determinations indicated in the bars. Antagonist or vehicle was administered 30 min prior to TFMPP. Filled bars indicate dark condition. Some animals were exposed to light for 15 min during a 1-h period beginning 7 h after dark onset Žstippled bars.. Asterisks ŽU . indicate statistically significant Ž p- 0.05. differences relative to the vehicleqdark group.
Abundant evidence indicates that glutamate serves as the principal excitatory neurotransmitter released from the RHT w2,15,43x. In this regard, several laboratories have reported pharmacological evidence indicating that activation of NMDA receptors w5,6x in the SCN region w40x mediates the effects of retinal illumination on circadian phase and gene expression in the SCN w1,40x. Systemic administration of the non-competitive NMDA antagonist MK-801 blocks both light-induced phase shifts w6x and photic suppression of pineal melatonin w5,31x Žbut see also, Kennaway w14x.. These observations suggest that the chemistry of RHT neurotransmission is similar at synapses that convey photic information to the circadian clock and to the pineal. The current report provides additional evidence indicating that RHT neurochemistry is similar at synapses that mediate photic effects on the circadian clock and the pineal gland. Based upon these data, we propose that the inhibitory effect of TFMPP on photic suppression of pineal melatonin production and on light-induced alterations of circadian phase w34,38x are identical, and occur through the 5-HT1B receptor-mediated presynaptic inhibition of RHT input to the SCN w35x. The possibility that distinct populations of retinorecipient neurons in the SCN mediate the effects of light on circadian phase and suppression of pineal melatonin production remains to be determined. Indirect evidence suggests that, 5-HT1B receptors are also present on pre-synaptic terminals of the geniculohypothalamic tract w23x ŽGHT., a projection from the intergeniculate leaflet ŽIGL. that may convey photic information to the SCN w11,32x. Thus, it is possible that the effect of TFMPP on photic suppression of melatonin is mediated, at least in part, through modulation of GHT neurotransmission. However, bilateral lesions of the IGL failed to attenuate the suppression of pineal N-acetyltransferase activity
M.A. Rea, G.E. Pickardr Brain Research 858 (2000) 424–428
by 15 min light pulses in the rat w4x. Thus, under the conditions employed in the current study, it is unlikely that 5-HT1B receptors on GHT terminals played a role in the action of TFMPP. Activation of 5-HT1B autoreceptors, which inhibit the release of serotonin from terminals in the SCN region of the rat w30x may also contribute to the effects of TFMPP in the SCN. However, since these autoreceptors appear to comprise only a small fraction of the 5-HT1B receptor population in the SCN w23x their contribution to the effects of TFMPP in the present study is probably minimal. In summary, systemic administration of the 5-HT1B agonist TFMPP dose-dependently attenuates the inhibitory effect of light on pineal melatonin production. This effect is blocked by the 5-HT1 antagonist, methiothepin, but not by the selective 5-HT1A antagonist, WAY100,635, indicating that the effect of TFMPP is mediated by 5-HT1B receptors. The data suggest that the neurochemistry of RHT neurotransmission at synapses that mediate the effect of light on pineal melatonin production is similar to that at synapses that mediate light-induced adjustments of circadian phase. Furthermore, these observations raise the possibility that serotonin tonus in the SCN may modulate the effect of light exposure on melatonin production.
Acknowledgements The authors wish to thank Ms. Anna Marie Michel and Mr. Matthew Cato for excellent technical assistance. This work was supported by grants from the Air Force Office of Scientific Research AFOSR92-AL-004 ŽM.A.R.. and the National Institutes of Health NS 35615 ŽG.E.P... The animals involved in this study were procured, maintained and used in accordance with the Animal Welfare Act and the Guide for the Care and Use of Laboratory Animals prepared by the Institute for Laboratory Animal Resources, National Science Foundation.
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