Effect of different photoperiods on the ultrastructure of the specific secretory cells and α-subunit mRNA level in the chicken pars tuberalis

The effect of different photoperiods on the specific secretory cells of the pars tuberalis was examined in male chicks. Animals were placed in one of three different photoperiod regimens: (1) normal control (light:dark = 12 h:12 h), (2) continuous light (L:D = 24 h:0), and (3) extended darkness (L:D = 1 h:23 h). The levels of common alpha-subunit mRNA in the pars tuberalis were examined by Northern blot analysis and compared with those in the pars distalis. In chicks exposed to continuous light for 1 week, alpha-subunit mRNA level in the pars tuberalis was decreased, although the level in the pars distalis was increased. Exposure to continuous light for 30 days also induced a decrease in alpha-subunit mRNA level in the pars tuberalis. On the other hand, in chicks exposed to extended darkness for 1 week, the alpha-subunit mRNA level of the pars tuberalis was markedly increased. In situ hybridization with digoxigenin-labeled common alpha-subunit cRNA probe also showed that the hybridization signals for alpha-subunit mRNA in the pars tuberalis cells become weak under continuous light for 30 days but they are very intense under extended darkness. Thus, the synthesis of alpha-subunits in the chick pars tuberalis was inhibited by continuous light but stimulated by extended darkness. These results were confirmed by semiquantitative electron-microscopic analyses. After exposure to continuous light for 30 days, many pars tuberalis (PT)-specific cells were filled with enlarged secretory granules, showing the reduction of secretory activity. On the contrary, extended darkness for 30 days induced hypertrophy of the PT-specific cells; the areas of cytoplasm and nucleus were significantly increased. In addition, secretory granules became small in size and exocytotic features were more frequent. Mitochondria and lysosomes were also increased in number. Thus, the synthetic and secretory activities of the PT-specific cells were increased under extended darkness. The data indicate that the specific cells of the pars tuberalis are responsive to photoperiodic changes in the chick.     

Diurnal rhythms of common α-subunit mRNA expression in the pars tuberalis of hamsters and chickens

To clarify whether the common -subunit of glycoprotein hormones is involved in photic signal transduction, -subunit mRNA levels in the pars tuberalis (PT) of both hamsters and chickens were estimated at different time points of the day/night cycle by laser capture microdissection (LCM) and real-time quantitative polymerase chain reaction (PCR). Distinct diurnal rhythms were found for -subunit mRNA expression in both species. In the hamster PT, -subunit mRNA levels gradually increased during the dark phase; the diurnal peak was found at time (ZT) 21. The lowest value was obtained at ZT 5 during the day. In the chicken PT, -subunit mRNA levels were maintained at a low constant level at night between ZT 13 and 21. Thus, -subunit mRNA expression in the PT depends on the light–dark cycle and may be controlled by the pineal hormone melatonin. The effect of various photoperiods on the hamster PT was examined by real-time PCR, immunohistochemistry, and electron microscopy. In hamsters kept under short photoperiod (L/D=8 h:16 h) or complete darkness, a dramatic decrease of -subunit mRNA level was induced, and the PT-specific cells accumulated glycogen-like particles and enlarged secretory granules. Under long photoperiods (L/D=16 h:8 h), however, the -subunit mRNA level was elevated and the PT-specific cells exhibited highly active features, i.e., piles of lamellar cisternae of rough endoplasmic reticulum and well-developed Golgi complexes. The -subunit synthesized by the PT-specific cells may therefore participate in the circadian and seasonal regulation of endocrine activities.     

Pinealectomy increases and exogenous melatonin decreases leptin production in rat anterior pituitary cells: an immunohistochemical study

Melatonin, the main hormone of the pineal gland, informs the body about the environmental light and darkness regimen, which in turn contributes to the photoperiodic adaptation of several physiological functions. Leptin, the hormone secreted mainly by adipocytes and some other tissues including the pituitary, informs the brain about the mass of adipose tissue, which plays an important role in energy homeostasis. Melatonin has been shown to decrease circulating leptin levels. It is currently not known whether melatonin has an effect on leptin synthesis in the pituitary. The aim of this study was to immunohistochemically examine the effects of pinealectomy and administration of melatonin on leptin production in the rat anterior pituitary. The pituitary samples obtained from 18 male Wistar rats including sham-pinealectomized, pinealectomized and melatonin-injected pinealectomized groups were immunohistochemically evaluated. Immunostaining of leptin was moderate (3+) in sham-pinealectomized rats, heavy (5+) in pinealectomized rats and low (1+) in melatonin-treated pinealectomized rats, respectively. The present results indicate that pinealectomy induces leptin secretion in anterior pituitary cells, and this increase of leptin synthesis can be prevented by administration of melatonin. Thus, melatonin seems to have both physiological and pharmacological effects on leptin production in the anterior pituitary of male rats.     

Photoperiod-dependent changes in exocytotic activity in the hypophyseal pars tuberalis of the Djungarian hamster,Phodopus sungorus

The pars tuberalis of the hypophysis of the Djungarian hamster, Phodopus sungorus, was investigated with regard to secretory activity by applying the tannic acid-Ringer perfusion technique. Two groups were maintained under long photoperiods (16 h light: 8 h dark) or short photoperiods (8 h light: 16 h dark), respectively. Perfusion with tannic acid showed that specific pars tuberalis cells release some of their secretory granules as indicated by typical exocytotic figures. The percentage of cells displaying exocytotic activity was significantly higher in the pars tuberalis of hamsters kept under long photoperiods. The number of exocytotic figures per single cell was not increased. These results provide further evidence for a secretory activity of the pars tuberalis and support the hypothesis of its involvement as a mediator between photoperiodic stimuli and the endocrine system.     

Feeding rhythms in constant light and constant darkness: the role of the eyes and the effect of melatonin infusion

Exposure to constant light abolishes circadian behavioral rhythms of locomotion and feeding as well as circulating melatonin rhythms in pigeons (Columba livia). To determine if feeding rhythmicity could be maintained in pigeons exposed to constant light, periodic infusions (10h/day) of melatonin were administered to pinealectomized and bilaterally retinectomized/pinealectomized pigeons under conditions of both constant darkness and constant light. The infusions were sufficient to entrain rhythmicity in pinealectomized pigeons in constant darkness and to restore and maintain rhythmicity in bilaterally retinectomized/pinealectomized pigeons in constant darkness. On subsequent exposure to constant light, rhythmicity remained phase locked to the melatonin infusions in bilaterally retinectomized/pinealectomized pigeons but was abolished in sighted pinealectomized birds. These results suggest that while endogenous melatonin rhythms are both necessary and sufficient to maintain behavioral rhythms in DD, their effect can be overridden by constant light but only if perceived by the eyes. Thus, constant light may abolish behavioral rhythmicity in intact pigeons (and perhaps in other species) by a mechanism other than suppression of endogenous melatonin rhythmicity. Such a mechanism might involve direct stimulation of locomotor or feeding activity by retinally perceived (but not by extra-retinally perceived) light, or alternatively by suppression of a hypothalamic oscillator that receives its major light input from the retinae.Abbreviations PX pinealectomized - EX bilaterally enucleated - LD light:dark cycle - LL constant light - DD constant darkness - DD_b constant darkness before exposure to constant light - DD_a constant darkness after exposure to constant light     

Ultrastructural localization of thyrotropin (TSH)-like immunoreactivity in specific secretory cells of the hypophyseal pars tuberalis in the Djungarian hamster,Phodopus sungorus

Summary Specific secretory cells in the hypophyseal pars tuberalis of Djungarian hamsters maintained under different photoperiods were investigated immunocytochemically by means of the colloidal gold technique using antibodies against rat thyrotropin (TSH). Secretory cells of animals kept under long photoperiods (LD16:8) showed positive staining of secretory granules (diameters 90–130 nm), whereas other intracellular structures were free of immunoreactivity. In animals kept under short photoperiods (LD8:16) secretory cells displayed increased numbers of secretory granules, but these organelles were devoid of immunoreactivity. In contrast, immunoreactivity of thyrotropes in the pars distalis did not differ between the two groups of animals investigated. The present results confirm earlier light-microscopical studies that in the pars tuberalis specific secretory cells show TSH-like immunoreactivity; however, they differ in their reactivity pattern from classical thyrotropes in the pars distalis.     

Structural changes in the pars intermedia of the cichlid teleost Sarotherodon mossambicus as a result of background adaptation and illumination

Summary The MSH producing cells in the pars intermedia of Sarotherodon mossambicus have been shown to be involved in background adaptation processes. Reflected light received by the eyes affects the activity of these cells. In the present study the hypothesis has been tested that also the pineal organ, as a second photoreceptor, is involved in regulation of the metabolic activity of the MSH cells. The pineal organ appears to contain photoreceptor cells and is considered to be capable of transferring information about light conditions to the animal. Removal of the pineal organ of fish kept on a black background has no effect on activity of MSH cells, whereas the activity of these cells in fish kept in darkness is increased. Thus it seems that the pineal organ exercises its influence on MSH cells only in darkness and that this influence results in a reduced activity of these cells. It is therefore concluded that the metabolic activity of MSH cells is inhibited not only by reflected light received by the eyes, but also by the action of the pineal organ as a result of the absence of illumination.No structural signs of secretory activity can be observed in the pineal, which might indicate synthesis or release of substances like melatonin. However, administration of melatonin reduces the activity of MSH cells. Neither pinealectomy nor treatment with melatonin has any influence on the second cell type of the pars intermedia, the PAS positive cells.     

Annual testicular cycle of the lizard Anolis carolinensis: effects of pinealectomy and melatonin

The effects of pinealectomy and exogenous melatonin treatment on the reproductive system of male anoles were examined at several different times of year. In September pinealectomy of anoles exposed to either a stimulatory LD 14:10 light cycle or a nonstimulatory LD 10:14 light cycle induced significant testicular growth and development over that observed in sham-operated anoles. At a nonphotosensitive time of year (December) pinealectomy also had a significant progonadal effect but no effect of pinealectomy was seen in February-March. Daily melatonin injections given either in the morning or afternoon (or both) failed to block gonadal growth either (1) in sham-operated or pinealectomized anoles exposed to LD 14:10 in the fall or (2) in pinealectomized lizards exposed to LD 10:14 in the fall. Continuous melatonin administration via subcutaneous silastic implants blocked the progonadal effects of pinealectomy in the winter (December). The results show that pinealectomy can have significant progonadal effects; these effects are seasonal but can encompass phases of the annual testicular cycle which are either photoperiod-dependent or temperature-dependent; and melatonin may be a reproductively active factor involved.     

Effect of pinealectomy on the circadian clock of the chick retina under different monochromatic lights

The avian circadian rhythm pacemaker is composed of the retina, pineal gland and suprachiasmatic nucleus. As an intact input-pacemaker-output system, each of these structures is linked within a neuroendocrine loop to influence downstream processes and peripheral oscillations. While our previous study found that monochromatic light affected the circadian rhythms of clock genes in the chick retina, the effect of the pineal gland on the response of the retinal circadian clock under monochromatic light still remains unclear. In this study, a total of 144 chicks, including sham-operated and pinealectomized groups, were exposed to white, red, green or blue light. After 2 weeks of light illumination, the circadian expression of six core clock genes (cClock, cBmal1, cCry1, cCry2, cPer2 and cPer3), melanopsin (cOpn4-1, cOpn4-2), Arylalkylamine N-acetyltransferase (cAanat) and melatonin was examined in the retina. The cBmal1, cCry1, cPer2, cPer3, cOpn4-1, cOpn4-2 and cAanat genes as well as melatonin had circadian rhythmic expression in both the sham-operated and pinealectomized groups under different monochromatic lights, while cClock and cCry2 had arrhythmic 24 h profiles in all of the light-treated groups. After pinealectomy, the rhythmicity of the clock genes, melanopsins, cAanat and melatonin in the chick retina did not change, especially the mesors, amplitudes and phases of cBmal1, cOpn4-1, cOpn4-2, cAanat and melatonin. Compared to the white light group, however, green light increased the mRNA expression of the positive-regulating clock genes cBmal1, cAanat, cOpn4-1 and cOpn4-2 as well as the melatonin content in pinealectomized chicks, whereas red light decreased their expression. These results suggest that the chick retina is a relatively independent circadian oscillator from the pineal gland, whose circadian rhythmicity (including photoreception, molecular clock and melatonin output) is not altered after pinealectomization. Moreover, green light increases ocular cAanat expression and melatonin synthesis by accelerating the expression of melanopsin and positive-regulating clock genes cBmal1 and cClock.     

Androgenic activity in 15-day-old male rats: role of the maternal pineal gland.

Female Sprague-Dawley rats, exposed to a long (18L:6D) or a short (6L:18D) photoperiod from 21 days of age, were mated when they reached 55 days of age. On Day 2 of gestation, dams were pinealectomized or sham-operated. Pre- and postnatal photoperiods were identical, and offspring were killed at 15 days of age. Maternal pinealectomy had no effect when rats were kept on 18L:6D. Rats born to sham-operated mothers and kept on 6L:18D had higher testicular testosterone and androstenedione content than offspring raised on the long photoperiod. This stimulatory effect of the short photoperiod was blocked by maternal pinealectomy and was not dependent on the offspring's own pineal since it was observed in both sham-operated and neonatally (on Day 5 after birth) pinealectomized rats. When sham-operated mothers housed on 18L:6D were treated daily during pregnancy and lactation by s.c. melatonin injection, there was an increase in the testicular testosterone content of offspring. It was concluded that when rats are maintained on a 6L:18D cycle the maternal pineal gland enhances the testicular testosterone and androstenedione content in 15-day-old offspring. This effect is probably mediated by maternally derived melatonin. At 15 days of age, the pineal of the offspring had no influence on testicular function.     

Effect of maternal pinealectomy and reverse photoperiod on the circadian melatonin rhythm in the sheep and fetus during the last trimester of pregnancy

The present study tested the hypothesis that the nocturnal melatonin rhythm in the fetal sheep results from transfer across the placenta of melatonin from maternal circulation. Pregnant ewes were exposed to an artificial reverse photoperiod at about 100 days gestation (n = 6; lights on 10 h, 2200-0800 h PST). This treatment tested for entrainment in the ewe and its fetus of the 24-h pattern of melatonin production from the pineal gland. Other ewes were pinealectomized at 55 days post-breeding (n = 6), and similarly treated. Catheters were implanted and blood samples were collected between 117 and 142 days gestation at two 48-h periods, about every 0.5-4 h, to assess the pattern of melatonin in maternal and fetal circulations. In pineal-intact ewes and their fetuses, melatonin rhythms conformed to the reverse photoperiod, i.e. plasma melatonin concentrations were relatively low during the light period and significantly increased for the duration of darkness. In contrast, maternal pinealectomy abolished the melatonin rhythms in both the ewe and fetus; melatonin concentrations remained at or below the limits of detection. Pineal-intact sheep gave birth about 139 +/- 2 days (mean +/- SE, n = 4) at 1915 +/- 0.7 h and pinealectomized ewes (n = 5 of 6) lambed at 149 +/- 2 days at 0424 +/- 0.5 h. Finally, in lambs (n = 3) born to pinealectomized ewes, typical melatonin rhythms were present within the first week of life. The findings indicate that the maternal pineal gland is responsible for the 24-h pattern of melatonin in the ewe and its fetus during the last trimester of pregnancy.     

Expression of the common α-subunit mRNA of glycoprotein hormones during the chick pituitary organogenesis, with special reference to the pars tuberalis

The expression of a common alpha-subunit mRNA of glycoprotein hormones was examined in the pituitary of chick embryos at various stages of development by in situ hybridization with a digoxigenin-labeled quail alpha-subunit cRNA probe. As a comparison with the expression of alpha-subunit mRNA, the onset of luteinizing hormone (LH) immunoreactivity was examined by immunohistochemical staining with a chicken LH antiserum. Both alpha-subunit mRNA and LH immunoreactivity began to appear in the basal-posterior region of the Rathke's pouch at embryonic day (E) 3.5. At E4.5 when the cephalic and caudal lobes of the pars distalis could be distinguished in the Rathke's pouch, intense signal for alpha-subunit mRNA was restricted to the cephalic lobe, consisting of a high columnar epithelium. At E6, gonadotrophs that were ovoid in shape, expressed intense signal for alpha-subunit mRNA, and revealed intense immunoreactivity for LH, were first detected in the cephalic lobe. At this stage, alpha-subunit mRNA expression became weak in the undifferentiated columnar cells of the cephalic lobe. At E8, the pars tuberalis primordium located close to the median eminence was formed at the lateral-apical end of the cephalic lobe. The primordium expressed intense signal for alpha-subunit mRNA. Gonadotrophs showing immunoreactivity for LH were densely distributed throughout the cephalic and caudal lobes in 8-day-old embryos. The pars tuberalis primordium expressing alpha-subunit mRNA progressively extended along the median eminence with embryonal age and reached the rostoral end by E14. Thus, both primordia of the pars distalis and pars tuberalis expressed intense signal for the common alpha-subunit mRNA. This subunit may play a role in the cytodifferentiation of the adenohypophysis.     

Ultrastructure of melatonin-responsive cells in the ovine pars tuberalis

Summary Functional receptors for melatonin have been localized and characterized on the pars tuberalis (PT) of a number of mammalian species, but the cell-type responsive to melatonin is unknown. The ultrastructure of the ovine pars tuberalis has been examined and these findings correlated with the functional response of the gland to melatonin. This study revealed that two secretory cell types predominate in the ovine PT, which differ in the abundance of dense-core granules. The most abundant of the cells are either agranular or very sparsely granulated and represent 90% of the total population, with the remaining 10% being composed of cells with abundant dense-core vesicles. Few follicular cells were observed. This ratio of secretory cell-types persisted in primary culture, with the two types non-separable by Percoll gradient centrifugation. Using forskolin, as a non-specific stimulant of adenylate cyclase, melatonin was shown to inhibit the formation of cyclic AMP by 80–90% in cells both before and after Percoll centrifugation. The results demonstrate that the agranular secretory cells of the ovine pars tuberalis are the melatonin responsive cell-type of this gland.     

Melatonin and the synthesis of vasopressin in pinealectomized male rats

The pineal hormone, melatonin, is known to modify, under different experimental conditions, neurohypophysial hormone secretion in the rat. The aim of this study was to investigate the effect of melatonin on the vasopressin biosynthesis rate in the hypothalamus of either pinealectomized or sham-operated rats, using the colchicine method. To estimate whether colchicine affects the function of the neurohypophysis in these animals, the neurohypophysial and plasma vasopressin levels were also measured. The vasopressin synthesis rate was increased after pineal removal, when compared with sham-operated animals, and melatonin strongly inhibited the rise in the hormone synthesis due to pinealectomy. After pineal removal plasma vasopressin concentration was significantly elevated, and melatonin attenuated this effect. On the contrary, the neurohypophysial vasopressin content was significantly decreased after pinealectomy, but it was not further modified by melatonin.Thus, melatonin suppresses the synthesis and secretion of vasopressin in pinealectomized rats. The present results confirm our previous reports as to the inhibitory impact of the pineal on both vasopressin synthesis and release and suggest that melatonin may mediate the effect of the pineal gland on vasopressinergic neuron activity.     

Melatonin stimulates thyroid-stimulating hormone accumulation in the thyrotropes of the rat pars tuberalis

We have reported that the unique thyroid-stimulating hormone-immunoreactive cells (TSH cells) in the intact adult and fetal rat pars tuberalis (PT) show an intense spot-like TSH immunoreaction in the perinuclear region. The present study was designed to investigate the relationship between melatonin and these unique TSH cells. We classified TSH cells in the PT (PT-TSH cells), on the basis of immunoreactivity, into spot-like stained cells (SC) and whole cytoplasm stained cells (WC), and estimated the proportion of each TSH cell type to total cells in the experimental rats by morphometry. Chronic administration of melatonin to control rats leads to an increase of WC in number but a decrease of SC. On the other hand, the intensity of TSH immunoreactivity and the number of rat PT-TSH cells significantly decreased after pinealectomy and recovered by melatonin administration. Radioimmunoassay showed that melatonin treatment increased the TSH content in the PT. Moreover, electron microscopy showed that the number of TSH secretory granules in the PT-TSH cells increased in the melatonin-replaced rats. These results demonstrated that melatonin stimulates the accumulation of TSH in the rat PT-TSH cells via secretory granule formation and suggest that melatonin regulates TSH release from PT-TSH cells.     

Localization and development of chromogranin A and luteinizing hormone immunoreactivities in the secretory-specific cells of the hypophyseal pars tuberalis of the chicken

 The pars tuberalis mainly consists of the secretory cells specific to this portion of the pituitary. We examined the localization and development of luteinizing hormone (LH) and chromogranin A in the chicken pars tuberalis by immunohistochemistry. The vast majority of the chicken pars tuberalis was occupied by cells immunoreactive for both LH and chromogranin A. Furthermore, immunoblot analysis of chicken pars tuberalis extracts with LH antiserum demonstrated that two bands, the large α-subunit and small β-subunit of the LH molecule, were expressed in this tissue as well as in the pars distalis. A band for chromogranin A was also detected in pars tuberalis extracts with chromogranin A antiserum. In contrast to the cells of mammalian species that contain only a few small secretory granules, the specific cells of the chicken pars tuberalis were characterized by the presence of many secretory granules ranging from 90 to 400 nm in diameter. Postembedding immunogold labeling showed that gold particles representing immunoreactivity for LH were densely located on all secretory granules of the secretory-specific cells. Many secretory granules, especially the large ones, of the cells were also loaded with immunogold particles for chromogranin A. Double immunogold labeling confirmed that LH and chromogranin A were colocalized on the same secretory granules. During embryonic development, the primordium of the pars tuberalis was first detected at 8 days of incubation as a small group of cells containing LH- and chromogranin-immunoreactive cells. In the pars distalis, the onset of LH and chromogranin expression occurred earlier, at 6 days of incubation. At 10 days of incubation, the pars tuberalis primordium became large cell masses consisting of LH- and chromogranin-immunoreactive cells, which were located close to the median eminence. Subsequently, the primordium extended along the median eminence progressively with age. At 14 days of incubation, it reached to the rostral end and surrounded the median eminence as slender cell cords. These results indicate that specific cells of the chicken pars tuberalis synthesize a glycoprotein hormone related to the LH molecule, which is stored in the secretory granules together with chromogranin A. The pars tuberalis may be involved in the regulation of gonadal function in a different way from that of the pars distalis. Accepted: 26 August 1997     

Influence of light and darkness on the ultrastructure of the pineal organ in the blind cave fish,Astyanax mexicanus

Ultrastructural changes of the pineal organ were investigated in the blind cave fish, Astyanax mexicanus, kept under continous artificial light (5000 lux), in continuous darkness, and under natural light conditions. The pineal end-vesicle of the fish kept under natural photoperiod consisted of photoreceptor cells and supporting cells mixed with a few ganglion cells. The photoreceptor cells possessed well-developed outer segments with regularly arranged lamellar membranes. The supporting cells contained a number of lipid droplets and large globular cisternae filled with fine granules. In the fish kept under continuous light or in darkness, the pineal end-vesicle displayed a dilated lumen, and the outer segments of the receptors showed signs of degeneration. Furthermore, alterations of cell organelles were observed in the photoreceptor and supporting cells.     

Diurnal Variations in [125I]Melatonin Binding by Rat Thymus Membranes: Effects of Continuous Light Exposure and Pinealectomy

Binding of melatonin by rat thymus membranes exhibited diurnal changes. Binding increased during the daytime and reached maximal values before entering the dark period. Then, binding decreased rapidly during the dark phase. In rats kept in light at night, binding of [¹²⁵I]melatonin by membranes was significantly higher than in animals that entered the normal dark period. Neonatal pinealectomy, which suppresses the circadian rhythm of melatonin, led to an increase in melatonin binding of 106%. Moreover, in animals maintained under continuous light exposure, which corresponds to functional pinealectomy, binding of melatonin by thymus membranes also increased in a time-dependent manner. The results support the hypothesis of a regulatory role of melatonin in the thymus in which melatonin downregulates its own binding sites.     

The effects of pinealectomy and blinding on the circadian locomotor activity rhythm in the Japanese newt,Cynops pyrrhogaster

We examined the effects of pinealectomy and blinding (bilateral ocular enucleation) on the circadian locomotor activity rhythm in the Japanese newt, Cynops pyrrhogaster. The pinealectomized newts were entrained to a light-dark cycle of 12 h light and 12 h darkness. After transfer to constant darkness they showed residual rhythmicity for at least several days which was gradually disrupted in prolonged constant darkness. Blinded newts were also entrained to a 12 h light/12 h dark cycle. In subsequent constant darkness they showed free-running rhythms of locomotor activity. However, the freerunning periods noticeably increased compared with those observed in the previous period of constant darkness before blinding. In blinded newts entrained to the light/dark cycle the activity rhythms were gradually disrupted after pinealectomy even in the presence of the light/dark cycle. These results suggest that both the pineal and the eyes are involved in the newt's circadian system, and also suggest that the pineal of the newt acts as an extraretinal photoreceptor which mediates the entrainment of the locomotor activity rhythm.Abbreviations circadian period - DD constant darkness - LD cycle, light-dark cycle - LD 12:12 light-dark cycle of 12 h light and 12 h darkness