Trans-pineal microdialysis in the Djungarian hamster (Phodopus sungorus): a tool to study seasonal changes of circadian clock activities

The Djungarian hamster is a highly seasonal small mammal. The rhythmic secretion of melatonin by the pineal gland is under control of the circadian clock, conveying the photoperiodic message to the organism. Trans-pineal microdialysis permits the in vivo study of this well-defined and precise clock output by measuring melatonin release directly in the pineal gland. The aim of this study was to adapt this method to the Djungarian hamster in order to monitor clock properties during photoperiodic changes. Male adult Djungarian hamsters were kept in a long photoperiod (LD 16:8) and melatonin release was measured hourly during the dark period for several weeks. Melatonin showed a regular secretion between ZT 17 and ZT 23.5 whereas the amplitude became stable only after the third day of perfusion. To test how quickly changes in melatonin profile can be measured, 15-min light pulses were given at different time points throughout the scotophase. Light-pulses immediately interrupted melatonin secretion at any time point during the scotophase and the temporal resolution for measurement could be reduced to 30 min. In accordance with studies in the rat, long-term effects of light on the clock could only be observed when a light pulse was administered in the second half of the night. For the first time we established a method to measure precisely a direct and reliable clock-output in a highly seasonal species which allows us now to study the circadian and seasonal properties of the clock in detail.     

Urinary 6-sulphatoxymelatonin excretion reflects pineal melatonin secretion in the Djungarian hamster (Phodopus sungorus)

Abstract: To monitor pineal function in the Djungarian hamster (Phodopus sun-gorus), we measured the urinary excretion of the melatonin metabolite 6-sulpha-toxymelatonin (aMT6s) at 3-hr intervals by radioimmunoassay. Hamsters maintained in either long photoperiod (LP, LD 16: 8) or short photoperiod (SP, LD 8: 16) showed marked daily rhythms in aMT6s excretion, with elevated levels during the dark phase. In both photoperiods, we found large interindividual differ-ences, mainly in the amplitude of the signal. However, the amplitude as well as the duration of nocturnal aMT6s excretion was higher in SP than in LP. Light ex-posure at night (180 mW/m², 30 min) caused a decrease in aMT6s excretion, indicating that the pineal gland is the major source of urinary aMT6s. Moreover, there was a significant correlation between nocturnal pineal/plasma melatonin contents and 24-hr aMT6s excretion. We conclude that, measurements of aMT6s provide a valid and quantitative index of pineal melatonin synthesis in this hamster species. As an advantage in determining pineal melatonin contents, this approach will al-low noninvasive long-term studies of individual animals under varying environ-mental conditions.     

New sensitive serum melatonin radioimmunoassay employing the Kennaway G280 antibody: Syrian hamster morning adrenergic response

Abstract: A new procedure with the G280 antibody of Kennaway provides an assay for circulating melatonin (aMT) with a sample volume (200 μl), an analytic (0.33 pg/ml) and functional (0.62–0.80 pg/ml) detectability, a 50% displacement dose (6.4 pg/ml), a Kd (0.657 pM), and measured circulating daytime levels lower than reported for previous procedures, and 100% assay recovery. The normal daytime range in adult human and Syrian hamster serum was 0.4–4 pg/ml. The pattern of fall of the nocturnal surge of Syrian hamster serum aMT near the time of lights-on was unaltered by extended darkness. Isoproterenol (ISO) injection 1 hr after lights-on, when aMT had reached daytime levels, raised serum and pineal aMT dramatically 2 hr postinjection. The same dose of ISO injected 4 hr into light produced only a small detectable increase. Novel extension of nocturnal darkness did not affect the responses to ISO. Thus, when they are allowed to occur at the usual time on a 10-hr dark schedule, both the fall from the nocturnal aMT surge and the subsequent loss of pineal beta-adrenergic responsiveness in this species occur endogenously (probably entrained) rather than from gating by acute effects of morning light. Changes in daytime serum aMT consistent with concomitant changes in the pineal can be measured with a sufficiently sensitive radioimmunoassay.     

Daily and photoperiodic 2- 125I-melatonin binding changes in the pars tuberalis of the Syrian hamster (Mesocricetus auratus): Effect of constant light exposure and pinealectomy

Abstract: Using quantitative autoradiography, 2¹²⁵I-melatonin binding was investigated throughout the light/dark cycle in the pars tuberalis (PT) of the pituitary of adult Syrian hamsters kept for 8 weeks either in long or short photoperiod (LP or SP, respectively). Melatonin receptor density in the PT displayed photoperiod dependent daily variations (maximal values in LP). Indeed, in LP, melatonin receptor density underwent strong daily variations with maximal values during the first half of the light period and minimal values at the end of the night. These variations depended on changes in the maximal binding (B_max) without differences in the dissociation constant (K_d). In contrast, PT melatonin receptor density was constant and at a very low level throughout the 1ight:dark cycle in SP exposed animals. Daily PT melatonin receptor density variations of LP exposed animals were abolished by pinealectomy or continuous light exposure. These results show clearly that both at the daily and at the seasonal level the regulation of PT melatonin receptors is strongly dependent on circulating melatonin concentrations in the Syrian hamster, but that other regulatory factors, yet unclarified, might also play a role.     

Serotonin in golden hamster testes: testicular levels, immunolocalization and role during sexual development and photoperiodic regression-recrudescence transition

Serotonin (5-HT) is found in the gonads and accessory reproductive organs of several species. The golden (Syrian) hamster is a seasonal breeder. Exposure of male adult hamsters to short days for 14 weeks results in a severe gonadal regression, while after a photoinhibition period of 22 weeks a spontaneous testicular recrudescence occurs. The aim of this study was to investigate the presence of 5-HT and its major metabolite 5-hydroxyindoleacetic acid (5-HIAA) in the gonads of golden hamsters, its immunolocation and its physiological role in the testis. The influence of age and photoperiod was also analyzed. Hamsters of 23, 36, 46, 60 and 90 days of age were kept in long photoperiod (LP: 14:10 h light/dark), and adult animals were exposed either to LP or to short photoperiod (SP: 6:18 h light/dark) for 14 and 22 weeks. Testicular parenchyma and capsule levels of 5-HT and 5-HIAA increased significantly at ages of 36 and 60-90 days, but decreased markedly during the exposure of adult hamsters to SP for 14 and 22 weeks. Mast cells were found exclusively in the testicular capsule. The testicular number of mast cells increased concomitantly with age, but decreased in adult hamsters exposed to SP. Mast and Leydig cells presented 5-HT-positive immunoreactivity. During sexual maturation as well as during the transfer of adult hamsters from LP to SP, the 5-HIAA/5-HT ratio showed the highest values in active adult animals, indicating that the increase in testicular 5-HT levels in adulthood is accompanied by an augment in 5-HT turnover. In vitro basal and hCG-stimulated testosterone production was significantly inhibited in presence of physiological concentrations of 5-HT. In conclusion, the present studies demonstrate the existence of 5-HT in mast cells and Leydig cells of hamster testes, as well as describe an inhibitory action of this neurotransmitter on gonadal testosterone production. Furthermore, the age-dependent and photoperiodic-related changes detected in testicular 5-HT levels suggest that this neurotransmitter might act as an important local modulator of the action of gonadotropins on steroidogenesis during sexual development and during the photoperiodic regression-recrudescence transition in the golden hamster.     

Pineal melatonin concentrations during day and night in the adult hedgehog: Effect of a light pulse at night and superior cervical ganglionectomy

The European hedgehog (Erinaceus europaeus L.) is a hibernating mammal and seasonal breeder in which numerous circadian and circannual rhythms are entrained and synchronized by photoperiod. The present study was undertaken in order to establish the involvement of the pineal gland in transducing the photoperiodic message in this species. Pineal melatonin concentrations were determined by radioimmunoassay in female hedgehogs kept under natural climatic conditions and killed during the light:dark (L:D) cycle in spring and autumn, after the interruption of darkness by a 45 min light pulse, and after bilateral superior cervical ganglionectomy (SCGx). Absolute melatonin concentrations were low (less than 100 pg/pineal) in the pineal gland. Under natural climatic conditions, in spring and in autumn, pineal melatonin concentrations exhibited a marked diurnal rhythmicity, with very low levels in the day (1200: less than 10 pg/pineal) and high levels during the night (2200: 71.9 +/- 18.6 pg/pineal; 0200: 42.5 +/- 15.6 pg/pineal). The 45 min light pulse during darkness depressed rapidly and significantly the melatonin content (dark + light [D + L]: less than 10 pg/pineal), but a subsequent return to darkness restored high melatonin content after approximately 2 h (D + L + D: 65.4 +/- 20.2 pg/pineal). After bilateral SCGx, melatonin concentrations were reduced and no increase during night could be observed, either in animals sacrificed 42 days after SCGx or in animals killed 2 years after SCGx. In the hedgehog, as in other mammals, pineal melatonin concentrations are related to the light:dark cycle. Such data indicate that during the year, in this species, the effects of light on seasonal endocrine rhythms may be mediated by the pineal gland.     

Decoding photoperiodic time through Per1 and ICER gene amplitude

The mammalian Per1 gene is expressed in the suprachiasmatic nucleus of the hypothalamus, where it is thought to play a critical role in the generation of circadian rhythms. Per1 mRNA also is expressed in other tissues. Its expression in the pars tuberalis (PT) of the pituitary is noteworthy because, like the suprachiasmatic nucleus, it is a known site of action of melatonin. The duration of the nocturnal melatonin signal encodes photoperiodic time, and many species use this to coordinate physiological adaptations with the yearly climatic cycle. This study reveals how the duration of photoperiodic time, conveyed through melatonin, is decoded as amplitude of Per1 and ICER (inducible cAMP early repressor) gene expression in the PT. Syrian hamsters display a robust and transient peak of Per1 and ICER gene expression 3 h after lights-on (Zeitgeber time 3) in the PT, under both long (16 h light/8 h dark) and short (8 h light/16 h dark) photoperiods. However, the amplitude of these peaks is greatly attenuated under a short photoperiod. The data show how amplitude of these genes may be important to the long-term measurement of photoperiodic time intervals.     

Clock genes in calendar cells as the basis of annual timekeeping in mammals--a unifying hypothesis

Melatonin-based photoperiod time-measurement and circannual rhythm generation are long-term time-keeping systems used to regulate seasonal cycles in physiology and behaviour in a wide range of mammals including man. We summarise recent evidence that temporal, melatonin-controlled expression of clock genes in specific calendar cells may provide a molecular mechanism for long-term timing. The agranular secretory cells of the pars tuberalis (PT) of the pituitary gland provide a model cell-type because they express a high density of melatonin (mt1) receptors and are implicated in photoperiod/circannual regulation of prolactin secretion and the associated seasonal biological responses. Studies of seasonal breeding hamsters and sheep indicate that circadian clock gene expression in the PT is modulated by photoperiod via the melatonin signal. In the Syrian and Siberian hamster PT, the high amplitude Per1 rhythm associated with dawn is suppressed under short photoperiods, an effect that is mimicked by melatonin treatment. More extensive studies in sheep show that many clock genes (e.g. Bmal1, Clock, Per1, Per2, Cry1 and Cry2) are expressed in the PT, and their expression oscillates through the 24-h light/darkness cycle in a temporal sequence distinct from that in the hypothalamic suprachiasmatic nucleus (central circadian pacemaker). Activation of Per1 occurs in the early light phase (dawn), while activation of Cry1 occurs in the dark phase (dusk), thus photoperiod-induced changes in the relative phase of Per and Cry gene expression acting through PER/CRY protein/protein interaction provide a potential mechanism for decoding the melatonin signal and generating a long-term photoperiodic response. The current challenge is to identify other calendar cells in the central nervous system regulating long-term cycles in reproduction, body weight and other seasonal characteristics and to establish whether clock genes provide a conserved molecular mechanism for long-term timekeeping.     

Effect of different photoperiods on concentrations of 5-methoxytryptophol and melatonin in the pineal gland of the Syrian hamster

Specific, sensitive and direct radioimmunoassays have been used to determine the daily patterns of 5-methoxytryptophol (ML) and melatonin in the pineal glands of Syrian hamsters kept in different photoperiods: 8 h light: 16 h darkness (8L:16D), 14L:10D and 16L:8D. A rhythm in pineal ML was evident in animals in all the photoperiods, with high daytime levels (641 +/- 35 (S.E.M.) fmol/gland; n = 162) which dropped to 119 +/- 16 fmol/gland (n = 44) 7.1-7.5 h after lights out. The duration of low night-time ML levels was proportional to the length of the dark phase (1.2 h in 16L:8D, 5.4 h in 14L:10D and 8.4 h in 8L:16D). A marked daily rhythm in melatonin was also present in hamsters in the different photoperiods, with daytime levels of 323 +/- 34 fmol/gland (n = 129) and night-time peak concentrations of 3676 +/- 336 fmol/gland (n = 22). The duration of high nocturnal melatonin levels was dependent upon the length of the dark phase (4.1 h in 16L:8D, 4.5 h in 14L:10D and 12.5 h in 8L:16D). Linear regression analysis revealed a statistically significant inverse relationship between pineal ML and melatonin levels in 8L:16D (P less than 0.001), 14L:10D normal (P less than 0.05) and 14L:10D shifted (P less than 0.001) photoperiods. After advancing the lighting schedule by 10 h (14L:10D, lights off at 04.00 h), pineal ML and melatonin rhythms became entrained to the new lighting regimen. The daily rhythms in pineal ML and melatonin in the Syrian hamster thus depend on the prevailing photoperiod, a reciprocal relationship existing between pineal ML and melatonin concentrations.     

Interactions of pinealectomy and short-photoperiod exposure on the neuroendocrine axis of the male Syrian hamster

The effect of pineal gland removal on neuroendocrine function of male Syrian hamsters housed under long (14 h light:10 h dark) or short (5 h light 19 h dark) photoperiod conditions was tested. In sham-operated, but not in pinealectomized, animals, exposure to the short photoperiod resulted in a significant reduction in testicular weight. Median eminence (ME), medial basal hypothalamus (MBH), and medial preoptic-suprachiasmatic (MPOA-SCN) norepinephrine (NE) turnover was significantly reduced in 5 L:19 D sham-operated animals as compared to 14 L:10 D sham-operated or 14 L:10 D pinealectomized controls. The effects of short photoperiod on ME and MPOA-SCN NE turnover were reversed by pinealectomy, but reductions in MBH NE turnover were not dependent on the presence of the pineal gland. Pineal-dependent decreases in MBH and increases in MPOA-SCN dopamine turnover were also observed after transfer of hamsters from long to short photoperiods. Both ME and MBH luteinizing hormone-releasing hormone (LHRH) levels were increased after short-photoperiod exposure, but pineal removal prevented these increases of LHRH levels only in the MBH of the 5 L:19 D hamsters. Levels of serotonin or its metabolite, 5-hydroxyindole-acetic acid, were not affected by pinealectomy and/or short-photoperiod exposure. We conclude that short-photoperiod-induced gonadal atrophy in the Syrian hamster is associated with pineal-dependent and pineal-independent changes in hypothalamic neurotransmitter turnover and hypothalamic LHRH content.     

The pineal gland and the photoperiodic control of luteinizing hormone secretion in intact and castrated Japanese quail

The effects of pinealectomy on a range of photoperiodic responses were investigated in male Japanese quail by measuring plasma LH concentrations in intact, sham-operated and pinealectomized birds in the following four experiments: (1) transfer of sexually quiescent birds from a short photoperiod of 8 h light: 16 h darkness (8L:16D) to a photostimulatory daylength of 16L:8D; (2) transfer of sexually mature birds from 16L:8D to 8L:16D; (3) castration in 16L:8D and exposure to 13L:11D; (4) castration in 8L:16D and exposure to 13L:11D. There was no evidence of effects of the pineal gland on the photoperiodically induced changes in LH secretion, the quantitative relationship between LH secretion and photoperiod in intact and castrated birds, or the induction of relative photorefractoriness by prolonged exposure to 16L:8D. This suggests that there is no pineal influence on the photoperiodic clock or its effectors in this bird.     

An essential role for ovarian inhibin in pineal gland-mediated anestrus in Syrian hamsters

ABSTRACT: When transferred from long photoperiod (LP) to short photoperiod (SP), female Syrian hamsters exhibit depressions of follicle stimulating hormone (FSH) and follicular development, cessation of ovulation, and marked ovarian interstitial tissue hyperplasia. Pinealectomy prevents these effects of SP. The object of this study was to determine the role of inhibin in the regulation of FSH during SP-induced anestrus. Adult LSH/SsLak hamsters maintained in LP (LD 14: 10 hr) were transferred to SP (LD 8: 16 hr) on the day of estrus, and groups of animals killed at either 16.00 hr on proestrus or 08.00 hr on estrus during each of five consecutive 4-day estrus cycles after transfer. Groups of females that became anestrus in SP were killed either at 08.00 or 16.00 hr, 12 days after the last observed estrus discharge. Compared to LP controls, serum FSH levels on estrus increased significantly ( P <0.01) during the first two cycles in SP before declining to concentrations that were significantly lower than control values ( P <0.01). Serum inhibin levels increased significantly by the third, fourth, and five days of estrus in SP. Regression analysis revealed a significant inverse correlation between serum inhibin and FSH levels on estrus during SP exposure (F=0.021) but not on proestrus. Relative levels of inhibin a- and_βA-subunit mRNAs were lower in ovaries from SP proestrus and anestrus females killed at 16.00 hr as compared to those from proestrus LP controls; they were elevated in ovaries from SP estrus and anestrus females killed at 08.00 hr compared to those from estrus LP controls. The absence of antral follicles on estrus in the last cycles of SP and anestrus suggests that the increase in circulating inhibin and inhibin mRNAs may be derived from hyperplastic interstitium. These observations suggest that inhibin may play an essential role in suppressing FSH secretion during pineal gland-mediated anestrus in Syrian hamsters.     

Vasoactive intestinal peptide stimulates N-acetyltransferase and hydroxyindole-O-methyltransferase activities and melatonin production in cultured rat but not in Syrian hamster pineal glands

The purpose of this study was to compare the responses of the Syrian hamster and rat pineal glands in organ culture to vasoactive intestinal peptide (VIP). The endpoints in these studies were the activities of pineal N-acetyltransferase (NAT) and hydroxyindole-O-methyltransferase (HIOMT), as well as pineal and medium melatonin levels. When rat pineal glands were incubated with either VIP (1 microM) or isoproterenol (1 microM), a beta-adrenergic agonist, a significant increase in NAT and HIOMT activities and melatonin levels were observed within 3 hr. Conversely, during the day, VIP (1 microM) was ineffective in stimulating these parameters in hamster pineal gland after incubation times of either 2, 4, 6, or 8 hr. In another experiment, hamster pineal glands were collected from animals killed in the late dark period (after 30 min light exposure). In these glands, isoproterenol promoted NAT activity and melatonin production; however, VIP was ineffective in stimulating either NAT or HIOMT activities; likewise, VIP had no stimulatory effect on pineal melatonin levels at night. Finally, when hamster pineal glands at night were incubated with either 0, 10 nM, 100 nM, 10 microM, or 100 microM VIP, no changes in any parameter of melatonin synthesis were measured. The results indicate that the hamster pineal gland, unlike that of the rat, may not respond to VIP with an increased melatonin production.     

The effects of near-ultraviolet light on serotonin N-acetyltransferase activity in the chick pineal gland

Effects of near-ultraviolet light (UV-A; 325-390 nm, peak at 365 nm) on the activity of the pineal serotonin N-acetyltransferase (NAT; a key regulatory enzyme in melatonin biosynthesis) were examined in chicks. Acute exposure of dark-adapted animals to UV-A radiation produced a marked decline in NAT activity of the pineal gland. The magnitude of this suppression was dependent upon duration of the light pulse and the age of the animals. The decrease in the nighttime NAT activity evoked by a 5 min pulse of UV-A light applied during the fourth hour of the dark phase of the 12 hr light:12 hr dark cycle (LD) gradually deepened during the first 40 min after the return of animals to constant darkness, then the enzyme activity began to rise, reaching control values by 2 hr. Exposure of chicks to a 5 min pulse of UV-A light during the ninth hour of the dark phase produced a marked decline in pineal NAT activity, which was reversible after 15 min of darkness. Pretreatment of animals with an inhibitor of catecholamine synthesis, alpha-methyl-p-tyrosine (300 mg/kg, i.p.), or with a blocker of alpha2-adrenergic receptors, yohimbine (2 mg/kg, i.p.), antagonized the suppressive effect of UV-A light on nighttime NAT activity of the chick pineal gland. It is concluded that UV-A irradiation, similar to visible light, potently suppresses melatonin biosynthesis in the chick pineal gland, with an alpha2-noradrenergic signal playing the role of an intermediate in this action.     

Starting the zebrafish pineal circadian clock with a single photic transition

The issue of what starts the circadian clock ticking was addressed by studying the developmental appearance of the daily rhythm in the expression of two genes in the zebrafish pineal gland that are part of the circadian clock system. One encodes the photopigment exorhodopsin and the other the melatonin synthesizing enzyme arylalkylamine N-acetyltransferase (AANAT2). Significant daily rhythms in AANAT2 mRNA abundance were detectable for several days after fertilization in animals maintained in a normal or reversed lighting cycle providing 12 h of light and 12 h of dark. In contrast, these rhythms do not develop if animals are maintained in constant lighting or constant darkness from fertilization. In contrast to exorhodopsin, rhythmicity of AANAT2 can be initiated by a pulse of light against a background of constant darkness, by a pulse of darkness against a background of constant lighting, or by single light-to-dark or dark-to-light transitions. Accordingly, these studies indicate that circadian clock function in the zebrafish pineal gland can be initiated by minimal photic cues, and that single photic transitions can be used as an experimental tool to dissect the mechanism that starts the circadian clock in the pineal gland.     

Circadian rhythm of melatonin in the pineal gland of the Japanese quail (Coturnix coturnix japonica)

Melatonin was measured by radioimmunoassay in homogenates of pineal glands from quail (Coturnix coturnix japonica) kept under different photoperiods and in darkness. Under 8-, 12- and 16-h daylengths melatonin levels were increased during the dark period, the duration of the increase depending on the duration of the dark period. As the daylength was increased the peak occurred closer to lights-off, reflecting the more rapid melatonin rise under the longer photoperiods. The pineal melatonin rhythm continued in darkness with an amplitude relative to that seen under a light/dark cycle of slightly less than one-half after 2 days in darkness and one-third after 6 days in darkness. The corresponding average periods of the rhythm were 25.5 h and 25.7 h. These results show that there is a circadian rhythm of melatonin in the pineal gland of the quail which is entrained by light/dark cycles and which continues in darkness.     

Phototransduction-related circadian changes in indoleamine metabolism in the chick pineal gland in vivo

Abstract: The purpose of this study was to examine the day/night levels of pineal melatonin and its rate limiting enzyme N-acetyltransferase (NAT) in relationship to the ratio of 11-cis-to all-trans-retinal. Three-week-old chicks were placed in 12:12 light: dark (LD 12:12) cycle for one week, pineals were collected during the light phase at 1500 (i.e., after 10 hr light), during the dark phase at 1900 (i.e., 2 hr after dark), at 2100 (i.e., 4 hr after dark), and at 2300 (i.e., 6 hr after dark) and after light extension to 1900. The results show that light-sensitive 11-cis-retinal in the chick pineal has the same diurnal rhythm as NAT and melatonin; all constituents increased within 2 hr of darkness onset (at 1900) and reached their peak after 4 hr of dark. All values were lowest during the light phase at 1500. Low values for 11-cis-retinal, NAT, and melatonin were also seen in the group of chicks which experienced light extension to 1900. The data indicate that in vivo light plays a major role in triggering rhodopsin-bound 11-cis-retinal production within 2–4 hr after darkness onset; this change likely serves as the signal for the subsequent formation of the hormonal product of the pineal gland, melatonin.     

Rhythmic Melatonin Response of the Syrian Hamster Pineal Gland to Norepinephrine In Vitro and In Vivo

Norepinephrine (NE, 10(-6) M) stimulated melatonin accumulation in the incubation medium of rat (but not Syrian hamster) pineals taken at the end of the light phase. However, NE elevated melatonin accumulation in the medium of pineals taken after 20 min of light exposure of animals of either species at 6 h into the 10-h dark phase. A dose response to 10(-7)-10(-5) M NE was observed in both the medium and pineals upon incubation of pineals taken from rats at 4 h into the light phase and from hamsters after 20 min light exposure at 6 h into the dark phase. Approximately 95% of the melatonin present was in the medium. The incubation time was 4 h in all cases. Subcutaneous injection of 1 microgram/g NE (either at the end of the light phase or after 30 min of light at 6 h into the dark phase) did not stimulate in vivo Syrian hamster pineal melatonin content determined 1 or 2 h after injection, whether the hamsters were placed in light or darkness after the injection. However, after 30 min of light beginning at 6 h into dark, injection of 5 micrograms/g desipramine (DMI, a blocker of catecholamine uptake into nerve endings) allowed a dramatic hamster pineal melatonin response to additional injection of 1 microgram/g NE, observed at 1 and 2 h in light after injection. A small effect of DMI alone was seen. DMI also potentiated the effect of NE (each 10(-6) M) on melatonin accumulation in the medium of incubated hamster pineals taken after a short light exposure at night. No significant stimulatory effect of NE and/or DMI was seen in vivo or in vitro near the middle of the light phase. Measurement of melatonin in the incubation medium is a useful method for studying pineal function. The Syrian hamster pineal has rhythm of sensitivity to NE (sensitivity evident at night) and even at night is protected by neuronal uptake from circulating NE-induced stimulation of melatonin production. NE appears to be the neurotransmitter for stimulation of pineal melatonin production in the Syrian hamster. The sensitivity rhythm and uptake protection might provide specificity of control of the nightly melatonin signal by reducing the chance of a melatonin response during the day or a response to circulating catecholamines from general sympathetic stimuli.     

A test of the coincidence and duration models of melatonin action in Siberian hamsters: the effects of 1-hr melatonin infusions on testicular development in intact and pinealectomized prepubertal Phodopus sungorus

The pineal hormone melatonin is known to play an important role in mediating photoperiodic messages to the reproductive system in seasonal breeding animals. Our goal was to test, in a single experimental paradigm, two hypotheses that have been forwarded to describe how the circadian rhythm of pineal melatonin transmits photoperiodic information to the reproductive system: 1) induction, i.e., a short-day effect, occurs when secreted melatonin and a circadian rhythm of sensitivity to melatonin coincide in time; 2) induction occurs following exposure to elevated circulating melatonin levels for a prescribed duration. In order to determine the relative validity of these hypotheses, we investigated the testicular maturation response to 1-hr daily infusions of 10, 25, and 50 ng of melatonin in pinealectomized intact and prepubertal Siberian hamsters (Phodopus sungorus). Animals received, beginning on day 15 of life, programmed subcutaneous infusions of melatonin or vehicle at one of five time points (19:00-20:00, 20:00-21:00, 21:00-22:00, 24:00-01:00, and 03:00-04:00 hr) for 15 days. In animals gestated and raised in a long photoperiod (LD16:8 = 16L, where L is the duration of light in hours, and D that of dark), melatonin infusion right after lights off (20:00-21:00 hr) significantly retarded gonadal maturation; this dose was ineffective at other times tested. Doses of 10 and 25 ng melatonin were ineffective at all time points. Identical results were obtained in prepubertal hamsters gestated in a short photoperiod (LD10:14 = 10L) and raised in 16L; these results were independent of the presence or absence of the pineal gland. In animals gestated and raised in 10L, melatonin infusions failed to suppress testicular development beyond that induced by the photoperiod; testicular development was maximally suppressed in all groups. The results of these investigations are best explained under the experimental conditions employed here: 1) the photoperiodic gonadal response in juvenile Siberian hamsters is regulated by the coincidence in time of exogenously administered melatonin with an intrinsic rhythm of sensitivity to melatonin, which, under the constraints imposed by our experimental design, occurred at 20:00-21:00 hr; and 2) the duration of the melatonin signal alone, equal in all groups, cannot explain the results.