Kanjun Hirunagi

Light-induced hormone conversion of T4 to T3 regulates photoperiodic response of gonads in birds

Reproduction of many temperate zone birds is under photoperiodic control. The Japanese quail is an excellent model for studying the mechanism of photoperiodic time measurement because of its distinct and marked response to changing photoperiods. Studies on this animal have suggested that the mediobasal hypothalamus (MBH) is an important centre controlling photoperiodic time measurement. Here we report that expression in the MBH of the gene encoding type 2 iodothyronine deiodinase (Dio2), which catalyses the intracellular deiodination of thyroxine (T4) prohormone to the active 3,5,3′-triiodothyronine (T3), is induced by light in Japanese quail. Intracerebroventricular administration of T3 mimics the photoperiodic response, whereas the Dio2 inhibitor iopanoic acid prevents gonadal growth. These findings demonstrate that light-induced Dio2 expression in the MBH may be involved in the photoperiodic response of gonads in Japanese quail.

A mammalian neural tissue opsin (Opsin 5) is a deep brain photoreceptor in birds

It has been known for many decades that nonmammalian vertebrates detect light by deep brain photoreceptors that lie outside the retina and pineal organ to regulate seasonal cycle of reproduction. However, the identity of these photoreceptors has so far remained unclear. Here we report that Opsin 5 is a deep brain photoreceptive molecule in the quail brain. Expression analysis of members of the opsin superfamily identified as Opsin 5 (OPN5; also known as Gpr136, Neuropsin, PGR12, and TMEM13) mRNA in the paraventricular organ (PVO), an area long believed to be capable of phototransduction. Immunohistochemistry identified Opsin 5 in neurons that contact the cerebrospinal fluid in the PVO, as well as fibers extending to the external zone of the median eminence adjacent to the pars tuberalis of the pituitary gland, which translates photoperiodic information into neuroendocrine responses. Heterologous expression of Opsin 5 in Xenopus oocytes resulted in light-dependent activation of membrane currents, the action spectrum of which showed peak sensitivity (λmax) at ∼420 nm. We also found that short-wavelength light, i.e., between UV-B and blue light, induced photoperiodic responses in eye-patched, pinealectomized quail. Thus, Opsin 5 appears to be one of the deep brain photoreceptive molecules that regulates seasonal reproduction in birds.

T3 implantation mimics photoperiodically reduced encasement of nerve terminals by glial processes in the median eminence of Japanese quail

Photoperiodically generated triiodothyronin (T3) in the mediobasal hypothalamus (MBH) has critical roles in the photoperiodic response of the gonads in Japanese quail. In a previous study, we demonstrated seasonal morphological changes in the neuro-glial interaction between gonadotrophin-releasing hormone (GnRH) nerve terminals and glial endfeet in the median eminence (ME). However, a direct relationship between photoperiodically generated T3 and seasonal neuro-glial plasticity in the ME remained unclear. In the present study, we examined the effect of T3 implantation into the MBH on the neuro-glial interaction in the ME. T3 implantation caused testicular growth and reduced encasement of nerve terminals in the external zone of the ME. In contrast, no morphological changes were observed in birds given an excessive dose of T3, which did not cause testicular growth. These results support the hypothesis that thyroid hormone regulates photoperiodic GnRH secretion via neuro-glial plasticity in the ME.

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.

Effect of fasting and immobilization stress on estrogen receptor immunoreactivity in the brain in ovariectomized female rats

The present study examined the effect of 48-h fasting and 1-h immobilization on estrogen receptor immunoreactivity in selected hypothalamic areas and the nucleus of the solitary tract (NTS) in ovariectomized rats. Fasting induced an increase in ER-immunoreactive cells in the paraventricular nucleus (PVN), periventricular nucleus (PeVN) and NTS compared with the unfasted control group. Similarly, immobilization caused an increase in ER-positive cells in the same areas, PVN, PeVN and NTS, versus the non-immobilized group. There was no significant increase in the number of ER-immunoreactive cells in the preoptic area (POA), arcuate nucleus (ARC) or ventromedial hypothalamic nucleus (VMH) following fasting and immobilization. Our previous work in ovariectomized rats with estrogen microimplants in the brain revealed that the PVN and A2 region of the NTS are the feedback sites of estrogen in activating the neural pathway to suppress pulsatile LH secretion during 48-h fasting. The result in the food-deprived rats suggests that estrogen modulation of the suppression of LH secretion during fasting is partly due to the increase in estrogen receptors in the PVN and A2 region. The physiological significance of the increase in neural ER following immobilization remains to be elucidated.

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.

Immunocytochemical demonstration of serotonin-immunoreactive cerebrospinal fluid-contacting neurons in the paraventricular organ of pigeons and domestic chickens.

The paraventricular organs (PVO) of the pigeon and domestic chicken contain at least three types of serotonin-immunoreactive (serotonin-ir) CSF-contacting neurons. Type 1 neurons were predominant. They had two bipolar extending processes. The somata were mostly found in the pars hypendymalis. Type 2 neurons were characterized by thin and long apical processes. Their perikarya were found in the pars distalis of the PVO or the more lateral area of this organ. Type 3 neurons were considerably smaller and had round somata. They were mostly bipolar with thin and short dendritic processes and thin basal processes. A small number of this type was conspicuous along the cranial peripheral region of the PVO. In addition to the PVO area, aggregations of small, bipolar serotonin-ir CSF-contacting neurons were shown in the most caudal wall of the third ventricle of both species, distributed medially or paramedially. Immunoelectron microscopy revealed many dense granules in apical ventricular processes and perikarya. Synaptic connections were frequently observed on basal processes.

Confocal laser scanning and electron-microscopic analyses of the relationship between VIP-like and GnRH-like-immunoreactive neurons in the lateral septal-preoptic area of the pigeon

Abstract The lateral septum and the preoptic area of birds comprise neurons immunoreactive (ir) for vasoactive intestinal polypeptide (VIP) and gonadotropin-releasing hormone (GnRH). By use of immunohistochemical single- and double-labeling techniques, we have investigated the distribution and the connections of these two types of peptidergic neurons in the lateral septal-preoptic area of the pigeon at both the light- and electron-microscopic levels. An accumulation of VIP-like-ir neurons, some of which are cerebrospinal fluid-contacting neurons, is found in the area adjacent to the ventromedial walls of the lateral ventricles in the lateral septum corresponding to the medial part of the lateral septal organ. VIP-like-ir terminals are scattered throughout the lateral septal-preoptic area, which also contains GnRH-like-ir cell bodies. The number of GnRH-like-ir cell bodies in the lateral septum is smaller than that of the VIP-like-ir neurons. GnRH-like-ir cells have a simple bipolar or multipolar shape and a beaded axon that emerges from the soma or one of the proximal dendrites. Confocal laser scanning microscopy has shown VIP-like-ir terminals in close apposition to GnRH-like-ir cell bodies in the lateral septal-preoptic area. Furthermore, the electron-microscopic double-immunolabeling has revealed synaptic contacts between VIP-like-ir axon terminals and GnRH-like-ir cell bodies or dendrites. These contacts, however, do not show synaptic specializations. The present results suggest that functional interactions take place between VIP and GnRH neurons in the lateral septal-preoptic area of the pigeon and that these interactions are involved in mediating photoperiodic responses.

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.

Immunoelectron-microscopic investigation of the subcellular localization of pinopsin in the pineal organ of the chicken

Abstract.Pinopsin is a photoreceptive molecule cloned from the chicken pineal organ. An antibody highly specific for pinopsin was applied in light- and electron-microscopic immunocytochemical studies of the pineal organ of 1 to 2-month-old chickens. Intense immunoreactivity was found in the follicular lumen at the light-microscopic level. In addition, small immunoreactive spherical or fibrous structures were diffusely distributed at the parafollicular aspect of the pineal organ. To identify immunoreactive elements precisely, we used pre-embedding immunoelectron microscopy. These studies revealed immunoreactive outer segments of pinealocytes arranged closely side by side in the follicular lumina. The thin initial portion of the outer segment arose from a basal body located in the inner segment. Immunoreactive pear-shaped outer segments occupied small lumina. Follicular lumina displayed immunonegative arrays of whorl-like lamellar membranes. Occasionally, these immunonegative structures were surrounded by immunoreactive concentric lamellar complexes. In the parafollicular pineal parenchyma, long slender cilium-like structures or enlarged cilia and concentric lamellar arrays showed intense immunoreactivity. All immunoreactive structures observed in this study were considered to represent outer segments of pinealocytes of the chicken pineal organ.