Shoji Matsushima

Separation and assay methods for melatonin and its precursors

Melatonin is an indoleamine hormone that is synthesized from tryptophan via 5-hydroxytryptophan, serotonin and N-acetylserotonin in the vertebrate pineal gland. Many chromatographic and non-chromatographic techniques have been developed and improved for the determination and measurement of melatonin and its related indoleamines. At present, gas chromatography with mass spectrometry and reversed-phase high-performance liquid chromatography with fluorescence or electrochemical detection are widely used for indoleamine determinations in the pineal gland. This review will deal with methods for the separation and determination of the melatonin and its related indoleamines.

Circadian variations in pinealocytes of the Chinese hamster, Cricetulus griseus

SummaryCircadian morphological variations of pinealocytes in the superficial pineal of the Chinese hamster (Cricetulus griseus) were studied using quantitative electron-microscopic techniques. The volume of the nucleus and cytoplasm of pinealocytes exhibited similar circadian variations, with the maximum around the middle of the light period and the minimum during the first half of the dark period. Synaptic ribbons in pinealocytes were classified into three groups, type-1, −2 and −3 synaptic ribbons, which appeared as rods, round or irregular bodies and ring-shaped structures, respectively; a synaptic ribbon index was determined for the respective types. The synaptic ribbon index was expressed as the number of synaptic ribbons in the pinealocyte profile representing the cell size. The type-1 synaptic ribbon index, which was smallest during the second half of the light period, was increased during the dark period. The length of straight or slightly curved rods showed a 24-h change similar to that of the type-1 synaptic ribbon index; the length of the rods was maximal during the first half of the dark period and minimal at the end of the light period. There was no apparent circadian variation in the type-2 synaptic ribbon index. The type-3 synaptic ribbon index was higher during the light period than during the dark period; the index attained zero 3h after the onset of darkness and, thereafter, increased gradually.

Simultaneous determination of serotonin, N-acetylserotonin and melatonin in the pineal gland of the juvenile golden hamster by high-performance liquid chromatography with electrochemical detection

A simple and simultaneous determination of melatonin and its precursors, serotonin (5-HT) and N-acetylserotonin, was achieved by reversed-phase high-performance liquid chromatography with electrochemical detection. The addition of an ion-pairing agent, sodium 1-octanesulfonate, to the chromatographic mobile phase caused an increase of the retention time of 5-HT, and resulted in the successful simultaneous resolution of these three indoleamines. This method was used to quantitate these indoleamines in the pineal gland of juvenile golden hamsters.

Effects of acute cold exposure on the ultrastructure of the mouse pinealocyte

SummaryUltrastructural changes in pinealocytes were investigated qualitatively and quantitatively in mice exposed to low temperatures at 0° C or 5° C for 30 or 60 min. A quantitative estimation of the granulated vesicles of about 100 nm diameter in pinealocytes demonstrated that the number of these vesicles per pinealocyte in mice exposed to cold at 0° C or 5° C increased remarkably as compared with that of the controls. This finding indicates that cold stimuli may activate the formation of the granulated vesicles in pinealocytes. In mice exposed to cold at 0° C for 60 min, granulated vesicles were often found in groups close to the plasma membrane of the pinealocyte cell bodies or processes, indicating the release of the vesicle content into the extracellular spaces. Additionally, the pinealocytes of the mice exposed to cold at 0° C for 60 min were characterized by the frequent occurrence of lysosome-like bodies and filaments. The development of these structures in response to severe cold may represent a depressed functional state of pinealocytes.

Ultrastructure of pinealocytes of the cotton rat, Sigmodon hispidus.

SummaryFine structural features of pinealocytes of cotton rats (Sigmodon hispidus) were examined. Golgi complexes, mitochondria, endoplasmic reticulum and polysomes are usual organelles seen in the perikaryonal cytoplasm of pinealocytes. Many non-granulated vesicles (40 to 80 nm in diameter) and a few granulated vesicles (about 100 nm in diameter) are associated with the Golgi cisternae. Occasionally, the cisternae contain granular materials. The perikaryonal cytoplasm of pinealocytes is characterized by the presence of inclusion bodies. These bodies are usually round in shape, not bounded by a limiting membrane and composed of fine granular or filamentous materials of high electron-opacity, which are similar in appearance to the substance seen in the nucleolonema. Pinealocyte processes, filled with abundant non-granulated vesicles and some granulated vesicles, are mainly found within the parenchyma and occasionally in perivascular spaces.

Peptidergic peripheral nervous systems in the mammalian pineal gland.

The distribution and density of tyrosine hydroxylase (TH) and neuropeptide Y (NPY)‐immunoreactive, sympathetic fibers and calcitonin gene‐related peptide (CGRP)‐, substance P (SP)‐, and vasoactive intestinal polypeptide (VIP)‐immunoreactive, non‐sympathetic fibers in the pineal gland, the effects of superior cervical ganglionectomy (SCGX) on these fibers, and the location of their terminals in the pineal gland were compared between rodents and non‐rodents. A dense network of TH/NPY‐positive fibers is present all over the pineal gland. A less dense network of CGRP/SP‐ or VIP‐positive fibers occurs in the whole pineal gland of non‐rodents, but these fibers are usually confined to the superficial pineal gland in rodents. After SCGX, some TH/NPY‐fibers remain only in the deep pineal gland in rodents, whereas considerable numbers of these fibers persist throughout the gland in non‐rodents. Thus, the remaining fibers, probably originating from the brain, may be more numerous in non‐rodents. Since CGRP‐, SP‐ or VIP‐immunoreactive fibers in the pineal capsule can be traced to those in the gland, and since these fibers are ensheathed by Schwann cells, it is concluded that these fibers belong to the peripheral nervous system. However, the existence of SP‐positive central fibers cannot be denied in some species. In the superficial pineal gland of rodents, sympathetic terminals are mostly localized in perivascular spaces, whereas the parenchymal innervation by sympathetic fibers in the pineal gland is more dense in non‐rodents than in rodents. Synapses between sympathetic nerve terminals and pinealocytes occur occasionally in non‐rodents, but only rarely in the superficial pineal gland of rodents. The occurrence of the synapses may depend on the frequency of intraparenchymal sympathetic terminals. Microsc. Res. Tech. 46:265–280, 1999.

Comparisons of sizes of pinealocyte nuclei and pinealocytes in young and adult Chinese hamsters (Cricetulus griseus) under different photoperiod conditions

Semiquantitative electron microscopic observations on pinealocytes of the Chinese hamster (Cricetulus griseus) were made to compare the sizes of pinealocyte nuclei and pinealocytes as a whole, their nuclei and cytoplasm together, over a 24‐hr period, between young animals (60‐70 days old) and adult animals (120–130 days old) under LD 12: 12 and between adults under LD 12: 12 and LD 14: 10. Under LD 12: 12, similar 24‐hr rhythms exist in the nuclear area and the area of pinealocytes of young animals, whereas in adults these values exhibit no significant 24‐hr rhythm. In adults under LD 14: 10, there is no significant 24‐hr rhythm in the nuclear area, but the area of pinealocytes shows a statistically significant 24‐hr rhythm. Thus, in the Chinese hamster, the relationships between nuclear area and area of pinealocytes with time of day vary, depending on the age of animals as well as different photoperiodic conditions, although they differ only slightly.

Central GABAergic innervation of the mammalian pineal gland: a light and electron microscopic immunocytochemical investigation in rodent and nonrodent species.

Light and electron microscopic immunocytochemical observations were made to demonstrate central pinealopetal fibers immunoreactive for γ‐aminobutyric acid (GABA) and synapses between their terminals and pinealocytes in the pineal gland of four rodent (Wistar‐King rat; mouse; Syrian hamster, Mesocricetus auratus; Hartley strain guinea pig) and one nonrodent (tree shrew, Tupaia glis) species. GABA‐immunoreactive myelinated and unmyelinated fibers and endings were found in the parenchyma of the pineal gland of all the animals examined. In the rodent species, GABAergic fibers were mainly found in the intermediate and proximal portions of the pineal gland and were nearly or entirely absent in the distal portion of the gland. Abundant GABAergic fibers were evenly distributed throughout the gland of the tree shrew. In all the animals, the habenular and posterior commissures contained abundant GABA‐positive fibers, and some of them were followed to the pineal gland. GABA‐positive endings made synaptic contact with pinealocytes, occasionally in mice and guinea pigs, and frequently in tree shrews; no synapses were observed in Syrian hamsters and rats. In the pineal gland of all the animals, GABA‐immunoreactive cell bodies were not detected, and sympathetic fibers were not immunoreactive for GABA. These data indicate that GABAergic fibers are main pinealopetal projections from the brain. In view of the difference in the distribution of these fibers, central GABAergic innervation may play a more significant role in nonrodents than in rodents. The frequent occurrence of GABAergic synapses on pinealocytes in the tree shrew suggests that GABA released at these synapses directly controls activity of pinealocytes of this animal. J. Comp. Neurol. 430:72–84, 2001.

Light and electron microscopic immunocytochemical study on the innervation of the pineal gland of the tree shrew (Tupaia glis), with special reference to peptidergic synaptic junctions with pinealocytes

Conventional and immunocytochemical, light- and electron-microscopic studies on the innervation of the pineal gland of the tree shrew (Tupaia glis) were made. Neuropeptide Y (NPY)-immunoreactive fibers, which were abundantly distributed in the gland, disappeared almost completely after superior cervical ganglionectomy, suggesting that these fibers are mostly postganglionic sympathetic fibers. By contrast, tyrosine hydroxylase (TH)-immunoreactive fibers, which were less numerous than NPY-fibers, remained in considerable numbers in ganglionectomized animals, indicating the innervation of TH-positive fibers from extrasympathetic sources. Bundles of substance P (SP)- or calcitonin gene-related peptide (CGRP)-immunoreactive fibers, entering the gland at its distal end, were left intact after ganglionectomy. SP-fibers were numerous, but CGRP-fibers were scarce in the gland. SP-immunoreactive fibers were myelinated and nonmyelinated, and were regarded as peripheral fibers because of the presence of a Schwann cell sheath. NPY- and SP-immunoreactive fibers and endings were mainly localized in the pineal parenchyma. NPY-immunoreactive endings synapsed frequently, and SP-positive ones did less frequently, with the cell bodies of pinealocytes. The results suggest that NPY and SP directly control the activity of pinealocytes. Sections stained for myelin showed that thick and less thick bundles of myelinated fibers entered the gland by way of the habenular and posterior commissures, respectively. Under the electron microscope, the bundles were found to contain also unmyelinated fibers. A considerable number of nerve endings synapsing with the cell bodies of pinealocytes remained in ganglionectomized animals; these endings were not immunoreactive for TH or SP. Such synaptic endings may be the terminals of commissural fibers.

Effects of melatonin on synaptic ribbons in pinealocytes of the Chinese hamster, Cricetulus griseus

SummaryThe effects of melatonin on synaptic ribbons (SR) in pinealocytes of the Chinese hamster (Cricetulus griseus) were examined. SR were classified into types 1, 2 and 3, which appear as rods, round or irregular bodies and ring-shaped structures, respectively; a synaptic ribbon index (SR index) was determined for the three types. Administration of two doses of 1.5 mg/kg melatonin at noon and 3 p.m. causes an increase in the type-1 and type-2 SR indices 3 h after the second injection in hamsters kept under alternating light and dark conditions (lights on from 7 a.m. to 7 p.m.). Likewise, in animals that are exposed to extended light for 6 h and receive two doses of melatonin at 7 p.m. and 10 p.m., an increase in the type-1 and type-2 SR indices occurs 3 h after the second injection. The increase in the type-2 SR index induced by melatonin administration to hamsters exposed to extended light is greater than the increase in the type-1 SR index under the same experimental conditions. Type-2 SR index, but not type-1 SR index, increases following bilateral superior cervical ganglionectomy. An increase in type-1 and type-2 SR indices occurs at 6 p.m. in ganglionectomized animals administered two doses of melatonin 6 h (noon) and 3 h (3 p.m.) before the time of sacrifice. No significant change is observed in type-3 SR index in animals subjected to any of the above treatments. The results indicate that exogenous melatonin may act directly on pinealocytes of the Chinese hamster to cause an increase in size and/or number of the type-1 and type-2 SR. Type 3-SR may have a role different from that of type-1 and type-2 SR; type-1 and type-2 SR may be functionally related.