Comparative ultrastructure and cytochemistry of the avian pineal organ

The breeding of birds is expected to solve problems of nourishment for the growing human population. The function of the pineal organ synchronizing sexual activity and environmental light periods is important for successful reproduction. Comparative morphology of the avian pineal completes data furnished by experiments on some frequently used laboratory animals about the functional organization of the organ. According to comparative histological data, the pineal of vertebrates is originally a double organ (the "third" and the "fourth eye"). One of them often lies extracranially, perceiving direct solar radiation, and the other, located intracranially, is supposed to measure diffuse brightness of the environment. Birds have only a single pineal, presumably originating from the intracranial pineal of lower vertebrates. Developing from the epithalamus, the avian pineal organ histologically seems not to be a simple gland ("pineal gland") but a complex part of the brain composed of various pinealocytes and neurons that are embedded in an ependymal/glial network. In contrast to organs of "directional view" that develop large photoreceptor outer segments (retina, parietal pineal eye of reptiles) in order to decode two-dimensional images of the environment, the "densitometer"-like pineal organ seems to increase their photoreceptor membrane content by multiplying the number of photoreceptor perikarya and developing follicle-like foldings of its wall during evolution ("folded retina"). Photoreceptor membranes of avian pinealocytes can be stained by antibodies against various photoreceptor-specific compounds, among others, opsins, including pineal opsins. Photoreceptors immunoreacting with antibodies to chicken pinopsin were also found in the reptilian pineal organ. Similar to cones and rods representing the first neurons of the retina in the lateral eye, pinealocytes of birds possess an axonal effector process which terminates on the vascular surface of the organ as a neurohormonal ending, or forms ribbon-containing synapses on pineal neurons. Serotonin is detectable immunocytochemically on the granular vesicles accumulated in neurohormonal terminals. Pinealocytic perikarya and axon terminals also bind immunocytochemically recognizable excitatory amino acids. Peripheral autonomic fibers entering the pineal organ through its meningeal cover terminate near blood vessels. Being vasomotor fibers, they presumably regulate the blood supply of the pineal tissue according to the different levels of light-dependent pineal cell activity.     

Morphofunctional aspects of the mammalian pineal gland.

The primary aim of this review is to present the current state of knowledge of the ultrastructure of the mammalian pineal gland, with emphasis on its functional aspects. Basic ultrastructural features of the mammalian pinealocytes are presented with special attention paid to ultrastructural aspects of pineal secretion.     

Fine structure of the pinealopetal innervation of the mammalian pineal gland.

The mammalian pineal gland is innervated by peripheral sympathetic and parasympathetic nerve fibers as well as by nerve fibers originating in the central nervous system (central innervation). The perikarya of the sympathetic fibers are located in the superior cervical ganglia, while the fibers terminate in boutons containing small granular vesicles and a few large granular vesicles. Both noradrenaline and neuropeptide Y are contained in these neurons. The parasympathetic fibers originate from perikarya in the pterygopalatine ganglia. The neuropeptides, vasoactive intestinal peptide and peptide histidine isoleucine, are present in these fibers, the boutons of which contain small clear transmitter vesicles and larger granular vesicles. The fibers of the central innervation originate predominantly from perikarya located in hypothalamic and limbic forebrain structures as well as from perikarya in the optic system. These fibers terminate in boutons containing small clear and, in certain fibers, an abundant number of large granular vesicles. In rodents, the majority of the central fibers terminate in the deep pineal gland and the pineal stalk. From these areas impulses might be transmitted further caudally to the superficial pineal gland via neuronal structures or processes from pinealocytes. Several hypothalamic neuropeptides and monoamines might be contained in the central fibers. The intrapineal nerve fibers are located both in the perivascular spaces and intraparenchymally. The majority of the intraparenchymally located fibers terminate freely between the pinealocytes. However, some nerve terminals make synaptic contacts with the pinealocytes and in some species with intrapineal neurons. In fetal mammals, sympathetic, parasympathetic, and central fibers are also present. In addition, an unpaired nerve, connecting the caudal part of the pineal gland with the extreme rostral part of the mesencephalon, is present. This nerve is a homologue to the pineal nerve (nervus pinealis) observed in lower vertebrates.     

Sensory and endocrine characteristics of the avian pineal organ

The avian pineal organ represents a transitional type between a photosensory organ of lower vertebrates and the endocrine gland of mammals and shows remarkable changes in its innervation and structure during ontogeny. In the avian pineal organ the progressive reduction of the pinealofugal component and the spectacular increase in pinealopetal sympathetic innervation occur in parallel. In domestic fowl the number of intrapineal AChE-positive (afferent) neurons decreases rapidly during ontogenetic development, whereas the sympathetic innervation becomes more prominent. Furthermore, the end vesicle of the pineal organ is an anatomical entity fully separated from the brain in the adult domestic fowl, as observed in some mammalian pineals. The avian pineal organ contains several types of photoreceptors with different photopigments and the synthesis of melatonin, the pineal hormone, is controlled by light. Immunoreactivity for photopigments is reduced during the posthatching development of chicken, whereas neuron-specific enolase (NSE)-immunoreactive pinealocytes increase remarkably in number in the end-vesicle of the domestic fowl with age, followed by a gradual expansion toward the proximal portion. NSE is the most acidic isoenzyme of the glycolytic enzyme enolase and is useful as a cytoplasmic marker of neurons and neuroendocrine tissue. The above-mentioned findings reflect the sequence of changes leading from pineal sense organs to pineal gland. The demonstration of melatonin receptors in a variety of avian peripheral tissues suggest a possible direct action of melatonin on the physiological functions of different organ systems in response to internal and external stimuli.     

Distribution of GABA and glycine receptors on bipolar and ganglion cells in the mammalian retina

The amino acids GABA and glycine mediate synaptic transmission via specific neurotransmitter receptors. Molecular cloning studies have shown that there is a great diversity of GABA and glycine receptors. In the present article, the distribution of GABA and glycine receptors on identified bipolar and ganglion cell types in the mammalian retina is reviewed. Immunofluorescence obtained with antibodies against GABA and glycine receptors is punctate. Electron microscopy shows that the puncta represent a cluster of receptors at synaptic sites. Bipolar cell types were identified with immunohistochemical markers. Double immunofluorescence with subunit-specific antibodies was used to analyze the distribution of receptor clusters on bipolar axon terminals. The OFF cone bipolar cells seem to be dominated by glycinergic input, whereas the ON cone bipolar and rod bipolar cells are dominated by GABAergic input. Ganglion cells were intracellularly injected with Neurobiotin, visualized with Streptavidin coupled to FITC, and subsequently stained with subunit specific antibodies. The distribution and density of receptor clusters containing the alpha1 subunit of the GABA(A) receptor and the alpha1 subunit of the glycine receptor, respectively, were analyzed on midget and parasol cells in the marmoset (a New World monkey). Both GABA(A) and glycine receptors are distributed uniformly along the dendrites of ON and OFF types of parasol and midget ganglion cells, indicating that functional differences between these subtypes of ganglion cells are not determined by GABA or glycinergic input.     

Fine structure of a developing insect olfactory organ: morphogenesis of the silkmoth antenna.

The olfactory organ of the silkmoth Antheraea polyphemus is the feathered antenna which carries about 70,000 olfactory sensilla in the male. It develops within 3 weeks from a leaf-shaped epidermal sac by means of segmental primary and secondary indentations which proceed from the periphery towards the centerline. During the first day post-apolysis, the antennal epidermis differentiates into segmentally arranged, alternating sensillogenic and non-sensillogenic regions. Within the first 2 days post-apolysis, the anlagen of olfactory sensilla arise from electron-dense mother cells in the sensillogenic epidermis. The axons of the developing sensilla begin to form the primary innervation pattern during the second day. The sensilla develop approximately within the first 10 days to their final shape, while the indentations are completed during the same period of time. The indentations are most probably driven by long basal extensions of epidermal cells, the epidermal feet. Primary indentations follow the course of segmentally arranged tracheal bundles and form the segments of the antenna. The secondary indentations follow the course of the primary segmental nerves which are reconstructed by this process. During the remaining time of development, the cuticle of the antenna and the sensory hairs is secreted by the epidermal and the hair-forming cells.     

Third ventricular pineal gland grafts in the gerbil: an electron microscopical and immunohistochemical investigation.

The superficial pineal gland was grafted into the third ventricle of adult Mongolian gerbils. Donor pineal glands from both neonatal and 3-4 week old gerbils were able to survive for at least 6 months. The pinealocytes of the grafted superficial pineal glands maintained the morphology and the S-antigen immunoreactivity of the in situ pineal complex. Synaptic ribbons and spherules were present but rare. Unlike the in situ pinealocytes, glycogen accumulations were common in the graft pinealocytes. Site specific modulation of structure was indicated as the ventricular surface of the grafts became covered with cerebrospinal fluid (CSF)-contacting pinealocytes typical of those seen in the deep pineal. The CSF-contacting pinealocytes of the graft had numerous processes that extended along the ventricular surface of the graft. The blood vessels of the grafts had non-fenestrated endothelium and wide perivascular areas typical of those seen in the in situ pineal complex. Tyrosine hydroxylase-immunopositive nerve fibers were present in the grafted tissue indicating reinnervation of the graft. The source of the fibers was not determined. The nerve fibers were present both within the perivascular area and within the parenchyma where they were associated with pinealocytes. The results demonstrate that the cerebral ventricles are an ideal location for the survival of superficial pineal gland grafts. It is suggested that pineal grafts may be a means to further study pineal development and innervation.     

Ultrastructure of the mammalian pinealocyte under natural and experimental conditions: quantitative aspects.

This review briefly summarizes data accumulated on the quantitative aspects of the ultrastructure of the mammalian pinealocyte. Quantitative changes have been demonstrated under natural and experimental conditions in pinealocyte cell organelles in various species. Special attention is paid to two cytoplasmic components most frequently studied by means of quantitative electron microscopy, namely, dense-core vesicles and "synaptic" ribbons.     

Hydrogen peroxide in micropropagation of Lilium. A comparison with a traditional methodology.

The micropropagation of Lilium longiflorum requires adequate equipment which may not be afforded by small laboratories or producers. In this work we compared traditional methodology with a protocol that included easily available elements to sterilize materials and culture media, together with addition of hydrogen peroxide (H2O2) into the nutrient media as chemical sterilizer. A series of H2O2 concentrations (0.005, 0.010, 0.015 and 0.020% p/v) was used to control contamination during in vitro establishment and subsequent cultivation; the explant organogenic response was also examined and compared to the traditional micropropagation technique. The level of culture contamination was within acceptable limits in all treatments, though it was higher in the H2O2 treatments (40%) compared to the traditional methodology (20%). There were not significant differences in the number of bulblets per explant, and at the end of the multiplication phase, bulblets from 0.02% H2O2 treatment had greater biomass than from other treatments, indicating a beneficial effect. These bulblets also had a higher relative growth ratio with respect to the traditional method when cultivated for an additional period and showed the highest average bulblet fresh weight. It is expected that this higher bulblet mass would result in better performance during ex vitro cultivation.     

Progress in electron microscopic diagnostics: semi-quantitative determination of precipitable calcium in different cell types of the organ of Corti in the guinea-pig

Potassium antimonate was used to precipitate calcium in the cochlea of the guinea-pig. The distribution of the calcium antimonate precipitates was analysed by electron microscopy. The precipitate density was determined in different cell types in the organ of Corti by counting the number of calcium binding sites in a 10-μm² area. The size of the precipitates varied considerably, and thus the relative amount of the precipitable calcium was estimated only semi-quantitatively. As the prominent carbon signal is superimposed over the nearby small Ca²⁺ -edge signals, the combined signal of the antimony M_4,5-edge and the oxygen K-edge of the calcium antimonate salt formed was chosen for the semi-quantitative estimation. Images of the inelastically scattered electrons of the precipitates at ΔE = 570 eV were recorded by electron spectroscopic imaging. The area covered by the calcium precipitates within a given cell type was determined in different ultrathin sections of the same organ of Corti by an image processing system.     

Optimization and comparison of two different 3D culture methods to prepare cell aggregates as a bioink for organ printing

The ultimate goal of tissue engineering is to design and fabricate functional human tissues that are similar to natural cells and are capable of regeneration. Preparation of cell aggregates is one of the important steps in 3D tissue engineering technology, particularly in organ printing. Two simple methods, hanging drop (HD) and conical tube (CT) were utilized to prepare cell aggregates. The size and viability of the aggregates obtained at different initial cell densities and pre-culture duration were compared. The proliferative ability of the cell aggregates and their ability to spread in culture plates were also investigated. In both methods, the optimum average size of the aggregates was less than 500 μm. CT aggregates were smaller than HD aggregates. 5,000 cells per drop HD aggregates showed a marked ability to attach and spread on the culture surface. The proliferative ability reduced when the initial cell density was increased. Comparing these methods, we found that the HD method having better size controlling ability as well as enhanced ability to maintain higher rates of viability, spreading, and proliferation. In conclusion, smaller HD aggregates might be a suitable choice as building blocks for making bioink particles in bioprinting technique.     

Effects of loud noise exposure on mouse myocardium: a comparison with the rat

Loud noise is an environmental stressor of everyday life, which affects different organs and apparati, in particular the cardiovascular system. We have already reported that noise exposure produces significant alterations in the rat myocardium, consisting of mitochondrial damage, which is evident as lysis of the cristae and dilution of the matrix. Since there are high similarities between mouse and human species, the aim of our study was to investigate the effects of acute noise exposure on the mouse heart. We found that noise exposure affects mouse myocardium at similar subcellular sites to those already described in the rat; nonetheless, quantitative analysis of the percentage of altered mitochondria in both species disclosed a clear difference between mouse and rat myocardium, which strongly suggests a different sensitivity to noise stimulus. We hypothesize that the species differences on the extent of myocardial alterations here observed might be due to the zonal pattern of cardiac noradrenergic receptors, which should be the final effectors for noise-induced myocardial changes.     

Absorption microanalysis with a scanning soft X-ray microscope: mapping the distribution of calcium in bone

This article describes the scanning transmission X-ray microscope operated at the National Synchroton Light Source. The application of the instrument to elemental analysis is detailed. In particular, qualitative results on the calcium distribution in human skull tissue are presented.     

Onset of synaptogenesis in the plexiform layers of the chick retina: a transmission electron microscopic study

The presently acknowledged onset of synaptogenesis in the chick retina from embryonic day 12 (E12) onward stands in contrast with the appearance of spontaneous electrical activity, of presynaptic proteins, or of neurotransmitters during early formation of the inner (E6-E8) and outer (E9) plexiform layers. Therefore, we investigated the chick retina from E6 to E12 at which age first synapses appear by transmission electron microscopy (TEM). The study provides evidence that synaptogenesis in the chick retina begins shortly after the plexiform layers have started to emerge. The first synapses are electrical synapses, which appear on E7, one day after the future inner plexiform layer emerged, and towards the end of E8 in the nascent outer plexiform layer. Conventional chemical synapses appear in both plexiform layers on E8, in the inner plexiform layer (stage 34) only a few hours earlier than in the outer plexiform layer (stage 35). The first synapses are formed close to the apex of the optic fissure and their frequency increases rapidly with age. The onset, the topography, and the developmental course of synaptogenesis correlate with the chronotopic course of maturation of retinal neurons and the age when spontaneous electrical activity occurs in the retina.     

Fixation of mammalian tissues in different fixatives and its influence on the staining with methyl green-pyronin.

This investigation is a study on the role of fixation of different mammalian tissues in different fixatives as well as in 10% neutral formalin containing different metal cations and its effect on the staining with the dye-mixture, methyl green-pyronin. The results indicate variation in the colour of the nuclei in tissues fixed in the different fixatives. The possible role of the fixatives on nuclear colouration has been discussed.     

Experimental comparison of the performance of a journal bearing with a single and a twin axial groove configuration

It is often assumed that a journal bearing will operate more effectively with a twin groove arrangement than a single groove one, but little evidence has been presented in support of this.An experimental assessment of a journal bearing with either one or two axial grooves located perpendicularly to the load line was performed.It was found that under heavy loaded operation the twin groove configuration might actually deteriorate the bearing performance when compared with the single groove arrangement, namely due to uneven lubricant feed through each groove. It is concluded that the knowledge of the feed flow rates through each groove can be used to improve bearing performance under specific regimes by implementing groove deactivation or flow balancing strategies.     

Characterization of several invertebrate muscle cell types: a comparison with vertebrate muscles

Ultrastructural classification of invertebrate muscles is complex and not always clear. The aim of the present paper was to establish some criteria that might be useful for classification of invertebrate muscles and for a better understanding of the differences between them. The procedures used were: (1) immunochemical evaluation of those proteins that differentiated striated from smooth muscle (troponin, caldesmon, and calponin), and (2) calculations of several myofilament parameters to establish differences among muscles. The muscles studied were: striated muscles from the rat, Drosophila, the crab Callinectes, and the snail Helix (heart); obliquely striated muscles from the earthworm Eisenia foetida and Helix (mouth); and smooth muscles from the rat, and Helix (retractor, body wall, and intestinal wall). Immunochemical studies revealed that troponin was only present in the striated muscles and the obliquely striated muscle from Eisenia, whereas caldesmon and calponin were only present in the smooth muscles and the obliquely striated muscle from Helix. The highest thick filament/thin filament volume ratio was found in the striated muscles, followed by the obliquely striated muscles, and the smooth muscles. This suggests the order in which the contraction strength decreases. The myofilament length is inversely related to the contraction speed, which was higher in the striated muscles than in the obliquely striated muscles. In vertebrates, the smooth muscle seems to be less rapid than the striated muscle because their myofilaments are longer. This assertion cannot be generalized for invertebrate smooth muscle, because myofilament lengths vary widely in both striated and smooth muscles. In smooth muscles, the presence of apparently unordered electron-dense bodies instead of ordered Z lines and the absence of true sarcomeres permit a certain overlapping of thin filaments increasing the range of shortening.     

High-resolution autoradiography as a tool for the localization of nucleic acid synthesis and distribution in the mammalian cell nucleus.

Several examples of the application of high resolution autoradiography to the study of nucleic acid distribution in ultrathin sections of fixed and plastic embedded or frozen material are presented. Newly-synthesized DNA, labelled by very short pulses of 3H-thymidine is found to be localized throughout the nucleus. In the blastomeres of early mouse embryos developing after fertilization by 3H-thymidine-labelled spermatozoa, the labelled paternal DNA is distributed non-homogeneously in the nucleus. Finally some results obtained by using a cytochemical enzymatic technique for visualization of DNA directly in ultrathin sections of fixed and embedded cells are demonstrated. Concerning the distribution of RNA, perichromatin localization of rapidly transcribed RNA is described in different cell systems. This label can be attributed to perichromatin fibrils. In cells labelled for longer periods of time, sometimes followed by prolonged postincubations, a residual label is always found over the nucleus. Clusters of interchromatin granules are usually only weakly labelled or unlabelled. These structures probably contain a limited amount of rather slowly labelled RNA. The present results are discussed in the context of some biochemical evidence and of the data described by other investigators.