Akihito Adachi

Identification of rhodopsin in the pigeon deep brain

We detected rhodopsin gene expression in the pigeon lateral septum, a photosensitive deep brain region that is responsible for the photoperiodic gonadal response. The nucleotide sequence of the deep brain rhodopsin cDNA clone exactly matched that of the retinal one, indicating that a single rhodopsin gene is transcribed in the two tissues. Immunohistochemical analysis localized rhodopsin in the cerebrospinal fluid‐contacting neurons, which have been assumed to be photoreceptive cells in the deep brain. Pigeon rhodopsin seems to play dual important roles in the visual and non‐visual systems, the latter of which contributes to the photoperiodic response.

Phase-relationship and mutual effects between circadian rhythms of ocular melatonin and dopamine in the pigeon.

In order to study the mechanisms of ocular circadian rhythms in the pigeon, we measured melatonin and dopamine simultaneously from the eye using in vivo microdialysis. In experiment 1, the phase relationship between circadian rhythms of ocular melatonin and dopamine under light-dark cycles (LD) and continuous dim light (LLdim) was examined. Under LD, melatonin was high during the dark and low during the light. On the other hand dopamine was high during the light and low during the dark. These rhythms with the anti-phase relationship were maintained after the birds were transferred from LD to LLdim. In experiment 2, effects of a single light pulse on melatonin and dopamine rhythms were examined. A light pulse at CT18 rapidly suppressed melatonin release to the daytime level, whereas it rapidly increased dopamine release to the daytime level. The light pulse also affected the phases of melatonin and dopamine rhythms, inducing phase advances of both rhythm without changing the anti-phase relationship before the light pulse. In experiment 3, effects of an intraocular injection of dopamine or melatonin on their circadian rhythms were examined. A dopamine injection during the subjective night suppressed melatonin release and induced a light-pulse type phase shift in both melatonin and dopamine rhythms. On the other hand, a melatonin injection during the subjective day suppressed dopamine release and induced a dark-pulse type phase shift. These results are compatible with either one or two oscillator models, but the interaction between melatonin and dopamine is, in either case considered as an important mechanism regulating ocular circadian rhythms of the pigeon.

Immunocytochemical identification of pinopsin in pineal glands of chicken and pigeon

Pinopsin is a blue-sensitive photoreceptive molecule possibly involved in photic entrainment of the circadian pacemaker in the chicken pineal gland. To characterize pinopsin as a circadian photoreceptor, antibodies were raised against the C-terminal portion of pinopsin. As expected from the divergence of the amino acid sequence of this region, the resultant antibody cross-reacted with neither chicken rhodopsin nor red-sensitive cone pigment (chicken red). In Western blot analysis, the antibody stained a single band of 42-kDa protein in a detergent-extract of chicken pineal membranes, suggesting that pinopsin (calculated molecular weight, 38187) might be glycosylated and/or palmitoylated. Immunocytochemical examination of pineal sections of the chicken and the pigeon with this antibody revealed strong positive images for most of the membrane structures in the lumen of the follicles. This antibody also stained string- and bulb-shaped structures of the chicken parafollicular cells, the morphology of which resembles those of retinal photoreceptor cells. In contrast to the predominant distribution of pinopsin, a monoclonal antibody specific for chicken red stained a smaller number of membrane structures in the lumen of chicken pineal follicles. These results strongly suggest that the chicken pineal gland contains at least two types of photoreceptive molecules, pinopsin (major) and chicken red (minor). We show that the former molecule is localized in parafollicular pinealocytes and in the outer segments of pinealocytes that make contact with the follicular lumen.

Melatonin Receptors in the Spinal Cord

The pineal hormone, melatonin, plays an important role in the regulation of diurnal and seasonal rhythms in animals. In addition to the well established actions on the brain, the possibility of a direct melatonin action on the spinal cord has to be considered. In our laboratory, we have obtained data suggesting that melatonin receptors are present in the spinal cords of birds and mammals. Using radioreceptor binding and quantitative autoradiography assays with 2-[125I]iodomelatonin as the specific melatonin agonist, melatonin binding sites have been demonstrated in the rabbit and chicken spinal cords. These sites are saturable, reversible, specific, guanosine nucleotide-sensitive, of picomolar affinity and femtomolar density. The linearity of Scatchard plots of saturation data and the unity of Hill coefficients indicate that a single class of melatonin binding sites is present in the spinal cord membranes studied. The picomolar affinity of these sites is in line with the circulating levels of melatonin in these animals suggesting that these sites are physiologically relevant. Autoradiography studies in the rabbit spinal cord show that melatonin binding sites are localized in the central gray substance (lamina X). In the chicken spinal cord, these binding sites are localized in dorsal gray horns (laminae I-V) and lamina X. As lamina X and laminae I-II have similar functions, melatonin may have comparable roles in the chicken and rabbit spinal cords. Moreover, in the chicken spinal cord, the density of 2-[125I]iodomelatonin binding in the lumbar segment was significantly higher than those of the cervical and thoracic segments. The densities of these binding sites changed with environmental manipulations. When chickens were adapted to a 12L/12D photoperiod and sacrificed at mid-light and mid-dark, there was a significant diurnal variation in the density (maximum number of binding sites; Bmax) of melatonin binding sites in the spinal cord. After constant light treatment or pinealectomy, the Bmax of melatonin receptors in the chicken spinal cord increased significantly in the subjective mid-dark period. Moreover, there was an age-related decrease in the 2-[125I]iodomelatonin binding to the chicken spinal cord. Our results suggest that melatonin receptors in the chicken spinal cord are regulated by environmental lighting and change with development. These receptors may play an important role in the chronobiology of spinal cord function. The biological responses of melatonin on spinal cords have also been demonstrated in vitro. Melatonin decreased the forskolin-stimulated cAMP production in the chicken spinal cord explant. Preincubation with pertussis toxin blocked the melatonin effect. Our results suggest that melatonin receptors in the chicken spinal cord are linked to the adenylate cyclase via a pertussis toxin-sensitive G protein and that melatonin binding sites in spinal cords are melatonin receptors with biological functions. These receptors may be involved in the regulation of spinal cord functions related to sensory transmission, visceral reflexes and autonomic activities.

The relationship between ocular melatonin and dopamine rhythms in the pigeon: effects of melatonin inhibition on dopamine release

Our previous study has shown that the phases of circadian rhythms of ocular melatonin and dopamine are always opposite and intraocular melatonin injection suppresses dopamine release. Therefore, it is possible that dopamine rhythms result from inhibitory action of melatonin. We have examined this possibility in the following experiments. In the first experiment effects of continuous light on melatonin and dopamine release were examined. The data indicated that continuous light exposure resulted in loss of circadian rhythmicity of melatonin and dopamine by suppressing melatonin and enhancing dopamine levels throughout the day. To further examine the effects of light in the second experiment, 2 h light pulse was applied during the night, then temporal changes of melatonin and dopamine release were studied. The light pulse rapidly suppressed melatonin release, whereas it rapidly increased dopamine release. These changes occurred within 30 min in both melatonin and dopamine. However, the recovery after the cessation of the light stimulus was slower in melatonin than dopamine. In the third experiment it was tested if dopamine release was increased by lowering melatonin release with an intraocular injection of the D2 agonist, quinpirol. Although quinpirol strongly inhibited melatonin release independently of the time of injection, dopamine did not always increase by the inhibition of melatonin. These results indicate that ocular dopamine rhythms are not simply produced by melatonin inhibitory action.

In vivo Microdialysis Studies of Pineal and Ocular Melatonin Rhythms in Birds

Pineal and retinal melatonin has an important role in the control of avian circadian rhythms. In order to study the mechanisms of circadian rhythms of melatonin synthesis in the pineal and in the eye, in vivo microdialysis was applied to these organs. In both pigeons and Japanese quails, pineal and ocular melatonin levels were high during the dark and low during the day under light-dark (LD) cycles. These rhythms persisted under constant dim light (LLdim) conditions indicating the circadian nature of pineal and ocular melatonin release. Light has two effects on melatonin synthesis. One is acute inhibition of melatonin synthesis and the other is entrainment of circadian melatonin rhythms. We have examined photoreceptors mediating these effects in the pigeon. The results have indicated that the eyes are not involved in light-induced suppression and photic entrainment of pineal melatonin release, and pineal photoreceptors themselves are likely to mediate these effects. Concerning ocular melatonin, retinal photoreceptors seem to mediate light-induced suppression and photic entrainment and no evidence supporting mediation of extraretinal photoreceptors was obtained. Because dopamine is implicated in retinal melatonin synthesis, we measured dopamine and melatonin release simultaneously from the eye of pigeon. In contrast to melatonin rhythms, dopamine increased during the day and decreased during the dark. This antiphase relationship between melatonin and dopamine persisted in LLdim, suggesting an interaction between these two rhythms. The results of an intraocular injection of dopamine or melatonin in the phase of melatonin and dopamine rhythms indicated that the interaction is required for maintaining the antiphase relationship between the two rhythms.

Measurement of circadian rhythms of ocular melatonin in the pigeon by in vivo microdialysis.

Ocular melatonin rhythms were measured in pigeons (Columba livia) by in vivo microdialysis. The birds were placed in light–dark cycles with 12 h of light and 12 h of darkness (LD12:12) or continuous dim light (LLdim) after LD12:12. Under LD12:12, melatonin levels were low during the light and high during the dark. The rhythms persisted under LLdim with lower levels during the subjective day and higher levels during the subjective night. Two hours of light exposure in the middle of the night acutely suppressed melatonin to the daytime level. These results indicate that microdialysis is useful for studies investigating the mechanism regulating ocular melatonin rhythms.

Electron-microscopic investigations of vasoactive intestinal peptide (VIP)-like immunoreactive terminal formations in the lateral septum of the pigeon.

Vasoactive intestinal peptide (VIP)-like immunoreactive terminal fields were examined in the lateral septum of the pigeon by means of immunocytochemistry. According to light-microscopic observations, these projections originated from VIP-like immunoreactive cerebrospinal fluid (CSF)-contacting neurons, which are located in the ependymal layer of the lateral septum and form a part of the lateral septal organ. The processes of these cells gave rise to dense terminal-like structures in the lateral septum. Pre-embedding immuno-electron microscopy revealed that VIP-like immunoreactive axon terminals had synaptoid contacts with perikarya of small VIP-immunonegative neurons of the lateral septum, which were characterized by an invaginated nucleus, numerous mitochondria, a well-developed Golgi apparatus, endoplasmic reticulum and a small number of dense-core vesicles (about 100 nm in diameter). VIP-like immunoreactive axons were also seen in contact with immunonegative dendrites in the lateral septum. In both axosomatic and axodendritic connections, VIP-like immunoreactive presynaptic terminals contained large dense-core vesicles, clusters of small vesicles and mitochondria. These findings suggest that VIP-immunoreactive neurons of the lateral septal organ project to small, presumably peptidergic nerve cells of the lateral septum and that the VIP-like neuropeptide serves as a neuromodulator (-transmitter) in this area.

Melatonin Excretion Rhythms in the Cultured Pineal Organ of the Lamprey, Lampetra japonica

Pineal organ of the lamprey, Lampetra japonica, is essential to keep the circadian locomotor activity rhythm as previously reported. In this paper, we tried to show that an endogenous oscillator is located and is working in the pineal organ. When the pineal organs were excised and cultured in a plastic tube with M199 medium at 20 degrees C, melatonin secretion rhythms were clearly observed under both light-dark and continuous dark conditions. The circadian secretion of melatonin continued for more than five cycles under the continuous dark condition. This indicates that the pineal organ has an endogenous oscillator and that the melatonin secretion rhythm is controlled by this oscillator. These findings suggest the possibility that the locomotor activity rhythm of the lamprey is under the control of the oscillator in the pineal organ.

Retinally perceived light is not essential for photic regulation of pineal melatonin rhythms in the pigeon: studies with microdialysis

Using in vivo microdialysis, effects of retinally perceived light on pineal melatonin release and its rhythmicity was examined in the pigeon. In the first experiment, light-induced suppression of pineal melatonin release was studied. Although light given to the whole body during the dark strongly suppressed pineal melatonin release to a daytime level, light exclusively delivered to the eyes did not remarkably inhibit melatonin release. In the second experiment, in order to determine whether retinally perceived light has phase-shifting effects on pineal melatonin rhythms, pigeons were given a single light pulse of 2 h at circadian time (CT) 18 and the phases of the second cycle after the light pulse were compared with those of control pigeons without the light pulse. In this experiment, phase advances of pineal melatonin rhythms were observed when the light was given to the whole body but not when only the eyes were illuminated. In a third experiment, after entrainment to light-dark 12:12 (LD 12:12) cycles, birds whose heads were covered with black tapes were transferred into constant light (LL) conditions and only the eyes were exposed to new LD cycles for 7 days (the phase was advanced by 6 h from the previous cycles) using a patching protocol. This procedure, however, could not entrain pineal melatonin rhythms to the retinal LD cycles. These results indicate that the eyes are not essential for photic regulation of pineal melatonin release and its rhythmicity in the pigeon.