Biological timekeeping is a fundamental feature of the avian physiology and behavior (Cassone et al., 2009; Kumar et al., 2010). It enables synchronization of numerous important biological processes, like migration and reproduction, with changes in the environment related to the Earth rotation and movement around the Sun. The participation of the pineal gland in the organization of the diurnal and seasonal rhythms is undisputed (Gwinner, 1989, 1996, 2003). The pineal is a directly photosensory organ in all investigated avian species. Moreover, it is one of two (beside the suprachiasmatic nucleus) major components of the circadian pace-making system in birds.
Migration is one of the most fascinating and complex phenomena of the avian behavior, which is closely related to the seasonal and diurnal rhythms. It involves marked changes in physiology to cope with the energetic demands (Kumar et al., 2010). When preparing for migration birds accumulate energy reserves that fuel their flights and help to regulate the migration phenomenon. The likelihood of any one individual succumbing in an extreme weather event may depend partly on its body condition at the time (Newton, 2006). It is known, that seabirds are marvelously adapted for enduring of migration difficulties. Migratory disposition is related to the nocturnal melatonin peak (Gwinner, 2003).
Anatomy, histology, and ultrastructure of the pineal organ in birds show large interspecies variability (Vollrath, 1981). Generally, the avian pineal organ is constituted of pinealocytes, supportive cells, and nerve cells. Classification of pinealocytes is not finally determined and their various types are identified dependent on the accepted criteria. Based on the ultrastructure and the presence of outer segment, pinealocytes are classified into three types: receptor cells, rudimentary–receptor cells and secretory cells (Vollrath, 1981; Collin et al., 1986; Lewczuk et al., 2000a, b). Taking into consideration the contact of pinealocytes with follicular lumina—the follicular and parafollicular cells were distinguished (Boya and Calvo, 1980). Pinealocytes show clear differences in morphology, although the results of cytochemical and cytological studies point to their large similarities (Goto et al., 1989; Ohshima and Matsuo, 1991b).
The current knowledge concerning histology and ultrastructure of the avian pineal organ derives mainly from the studies performed on the domestic birds: chicken (Boya and Calvo, 1979, 1980; Ohshima and Matsuo, 1984), quail (Ohshima and Matsuo, 1991a; Ohshima and Hiramatsu, 1993), goose (Prusik et al., 2006), turkey (Lewczuk et al., 2000a, Przybylska-Gornowicz et al., 2005) and muscovy duck (Lewczuk et al., 2000b). Their results show prominent interspecies differences in the morphological characteristics of rudimentary–receptor pinealocytes, which are the predominating cell type of the photoreceptory line in the pineals of the domestic birds. Ultrastructure of the pineal organs in free-living birds was infrequently investigated, with an exception for some tropical species (Haldar and Guchait, 2000; Haldar and Bishnupuri, 2001). The studies of tropical birds demonstrated common features in the morphology of the pineal organ of birds living in the same habitat (Haldar and Bishnupuri, 2001).
The common gull (Larus canus) is a medium-sized gull, which breeds on moorland and lakes in northern Europe and migrates further south in winter. In Poland, the species overwinters throughout the country, in particular on the coast. Interesting from our point of view—the researchers investigating the pineal gland—is the fact that the common gull is a water bird, wandering and living in the autumn and winter in harsh weather conditions of the Polish Baltic coast.
In this study morphology of the pineal gland in the common gull was investigated at the level of light and electron microscopy.
MATERIAL AND METHODS
Birds and Tissues
The studies were performed on the pineal organs of juvenile common gulls living in natural conditions of the Baltic Sea coast, which have been untreatably injured during strong storms in autumn and qualified for euthanasia. The birds were scarified by a lethal dose of sodium pentobarbital (Morbital, Biowet, Poland). The pineal organs with adjacent parts of the brains were collected immediately after stopping of the heartbeat and prepared to the investigations. All procedures were made with strict accordance with the Polish government regulations concerning animal welfare.
Histology and Histochemistry
Eight pineals were fixed in Bouin's solution, dehydrated and embedded in paraffin. Then, they were cut in sagittal and frontal planes into consecutive 7-μm-thick sections using a Microm HM 340E microtome (Carl Zeiss, Germany). The tissue sections were stained by turns with one of three methods: HE, Mallory's method and PAS (Gabe, 1976).The sections were studied in a Zeiss Axioimage microscope and photographed with a digital camera (AxioCam MRc5).
For the morphometrical analysis concerning distribution of the glycogen accumulations sections (three per each pineal) cut close to the median plane of the gland and stained with PAS method were digitalized using Mirax Desk scanner (Carl Zeiss, Germany). The measurements were performed with the use of AxioVision software (Carl Zeiss, Germany). The data were statistically analyzed using one-way ANOVA followed by Duncan test (Statistica 10.0, Statsoft, USA).
The pineal organs (N = 6) were cut into several parts, immersion fixed (2 hr, 4°C) in a mixture of 1% paraformaldehyde and 2.5% glutaraldehyde in 0.2 M phosphate buffer (pH 7.4), washed, postfixed in 2% osmium tetroxide (2 hr, at room temperature), and then embedded in Epon 812. Semithin sections were cut from each blocks of tissue, stained with toluidine blue and examined in light microscope in order to choose the places to cutting of ultrathin sections. The sections, stained with uranyl acetate and lead citrate, were examined in a Tecnai BioTwin transmission electron microscope operated at 80 KV (FEI, USA) equipped with two digital cameras: Veleta (Olympus, Japan) and Eage 4k (FEI, USA).
The gull pineal organ, located between the cerebrum and the cerebellum, consisted of a wide, triangular, superficially localized distal part closely attached to the dura mater and a narrow, elongated proximal part, attached via the choroid plexus to the intercommissural region of the diencephalon (Fig. 1). The accessory pineal tissue formed by several follicles was localized caudally to the choroid plexus. The pineal organ was covered by the connective tissue capsule, which was much thicker on the caudal than on the rostral surface. The septa originating from the capsule and penetrating inside the organ created delicate stroma. The structure of the pineal changed in a proximo-distal direction of the organ. The parenchyma of the distal part was mostly solid, but several elongated prominent follicles were also present. The proximal part and the accessory pineal tissue were predominantly formed by oval or round follicles.
The pineal parenchyma consisted of short columnar cells and oval cells. The follicular wall was formed by one layer of columnar cells contacting with the follicular lumen and one or more layers of oval cells located at the periphery. The areas of solid parenchyma occurring mainly in the distal part of the pineal organ were filled by oval cells, which created rosettes without a visible central lumen.
The common gull pineal organ contained numerous areas of glycogen accumulation, which were weekly stained with HE and showed intensive positive staining with PAS method (Fig. 2). They were usually visible as big round deposits with the diameter from 0.8 to 10 μm, but some smaller glycogen deposits were also observed. They were unevenly distributed in the pineal organ. In the distal part of the pineal only a few deposits of glycogen were observed (Fig. 2A). Numerous deposits of glycogen were found in the middle and proximal parts of the pineal body (Fig. 2B,C) and an average number of deposits was noted in the accessory pineal tissue (Fig. 2D). The deposits were mostly localized in the external layer, but some of them were also observed in the periluminal layer of the follicular wall (Fig. 2B). They were almost lacking in the areas of solid parenchyma (Fig. 2A).
The morphometrical studies demonstrated that both the number of glycogen accumulations per 1 μm2 and the percentage of the pineal section area covered by glycogen accumulations were significantly higher in the proximal than in the distal part of the gland (Fig. 3).
The cells limiting the follicular lumen and those situated in the central parts of the rosettes were classified as rudimentary-receptor pinealocytes and ependymal-like supporting cells. The oval cells, which formed the peripheral parts of the follicular wall or rosettes were represented by pinealocytes of secretory type and astrocyte-like supporting cells. The outermost layer of follicles or rosettes, closely attached to the basal lamina, was formed by the endings of the basal processes of all types of cells creating the pineal parenchyma.
Rudimentary-receptor pinealocytes differed with each other in size and shape. Most of them were longitudinal and showed structural polarity relative to the above-nuclear cytoplasm containing regularly distributed organelles—rough endoplasmic reticulum, Golgi apparatus and mitochondria (Fig. 4A). The pinealocytes possessed apical prolongations filled with the granular matrix and showed sporadically fragments of lamellae, mitochondria, clear, and granular vesicles (Fig. 4A–C). Some apical prolongations protruding into the lumen were partly separated out from the rest of the cell. In the apical part of rudimentary-receptor pinealocytes cilia without the central pair of microtubules were found.
Secretory pinealocytes formed rather uniform population of cells with a centrally localized nucleus and irregularly distributed organelles (Fig. 5). They composed external parts of follicles or rosettes. Secretory pinealocytes possessed several small processes penetrating between the adjacent cells and one process drawing towards the basement membrane, which surrounded the rosettes and the follicle.
The basal processes of both rudimentary-receptor and secretory pinealocytes formed bulbous endfeets close to the basal lamina (Fig. 6A). The processes' cytoplasm was occupied mostly by small electron-lucent vesicles, dense-core vesicles, microtubules, and synaptic ribbons (Fig. 6). The basal processes and their endfeets containing very high number of small electron-lucent vesicles of 30–40 nm in size showed the presence of paracrystalline formations (Fig. 6A–E). These formations were created from the small vesicles which combining with each other form a hexagonal system. The hexagonal structures were from 0.5 to 1.7 μm in diameter. The process of disintegration of these hexagonal structures and release of small vesicles was observed in some endfeets (Fig. 6B–D). In process endings, filled with small electron-lucent vesicles and usually situated close to the basal lamina, the synaptic ribbons were observed (Fig. 6B, F). Synaptic ribbons were formed by small electron-lucent vesicles surrounding an electron-dense plate, usually rod-like (up to 0.2 μm in length) and arranged perpendicularly to the plasma membrane.
A characteristic feature of both types of pinealocytes was the presence of many clusters of mitochondria arising as a result of close aggregation of three to eight individual mitochondria (Fig. 7). The clusters occurred in the cytoplasm of both the cell bodies (Fig. 7A,B) and the processes (Fig. 7C). They did not show any specific distribution patterns. The clusters of mitochondria were frequently observed nearby paracrystalline structures and glycogen accumulation areas, however at these localizations the aggregation of the mitochondria was not so close as in other parts of the cell (Fig. 7D,E).
The investigated pinealocytes possessed the areas of cytoplasm filled with numerous glycogen particles (Fig. 8). These areas were round or oval in shape (diameter from 0.2 to 10 μm) and they were usually not bordered by the membrane (the exception—infrequently occurring small membrane-bounded foci of glycogen accumulation). Most of them were localized in the perikarya (Fig. 8A,B) and process endings (Fig. 8C) of secretory cells, but some of them were also noted in the apical cytoplasm of rudimentary-receptor pinealocytes. Successive stages of glycogen accumulation were observed in the perikarya. At the early stage of this process small foci of densely packed glycogen particles were present in the central part of the cell body. At the following stage the foci of glycogen accumulation fused with each other and formed large round or oval aggregates containing glycogen particles with moderate electron density. In some cells glycogen aggregates increased in size and homogenously filled almost whole cytoplasm of pinealocyte. It caused destroying or/and shifting organelles at the periphery of the cell. The small accumulations of glycogen often surrounded by clusters of mitochondria were observed in pinealocyte processes. The areas of glycogen particles accumulation were visible at the level of light microscopy as PAS positive structures—see: Histological studies. 9
The pineal organ of the common gull included two types of supporting cells—ependymal-like cells and astrocyte-like cells (Fig. 9). The ultrastructural features of both cell types were similar to those described by us in the domestic goose (Prusik et al., 2006).
In birds, the pineal organ generally consists of the primary pineal organ and, in some species, the accessory pineal tissue (Quay and Renzoni, 1967; Przybylska-Gornowicz et al., 2005). The avian pineal organ presents high variability as regards size, shape and localization (Vollrath, 1981; Fejér et al., 2001; Haldar and Bishnupuri, 2001; Przybylska-Gornowicz et al., 2005; Prusik et al., 2006). According Vollrath (1981) five anatomical types of the pineal organs could be distinguished taking into consideration their shape and localization. On the other hand, based on the histological structure three principal morphological types were acknowledged: saccular, tubulofollicular, and solid. More recently, a solid-follicular transitional type (an intermediate between tubulofollicular and solid lobular type) of the pineal organ has been distinguished in birds (Ohshima and Hiramatsu, 1993; Haldar and Bishnupari, 2001).
In this study the localization and shape of the pineal in the common gull agree well with those attributed to the third anatomical type. This type includes organs having the prominent distal part superficially localized, tapering parenchymal pineal stalk, which is proximally reduced and definitely attached to the intercommissural region. The details of the histological structure of the pineal organ in the common gull enable its classification as the solid follicular type, because the parenchyma of the examined pineals comprised two parts with different arrangement of cells—the solid tissue (localized mainly in the distal part of the organ) and the follicular tissue (localized mainly in the middle and proximal parts of the organ).
The parenchymal cells of the avian pineal organ belong to pinealocytes, supporting cells and nerve cells. Pinealocytes have been classified into three main types—receptor, rudimentary-receptor, and secretory pinealocytes (Vollrath, 1981; Collin et al., 1986). The presence of rudimentary-receptor and secretory pinealocytes was determined in this study.
The rudimentary-receptor pinealocyte of the gull pineal gland is characterized by a reduced outer segment in the form of an apical prolongation, stratified distribution of organelles and nucleus situated in the basal position. The above features are typical of cells creating the subluminal layers of the follicles in the avian pineal gland (Collin et al., 1986; Tabecka et al., 1999; Lewczuk et al., 2000a). It is worth to note that rudimentary-receptor pinealocytes of the common gull possess relatively small apical prolongations filled with the granular matrix and sporadically occurring cytoplasmic structures. The presence of similar apical prolongations has been reported in some other avian species (Boya and Calvo, 1980; Korf and Vigh-Teichman, 1984; Vigh and Vigh-Teichman, 1999). The chemical nature of the granular cytoplasm filling the apical prolongation remains unknown (Fejér et al., 2001). The ultrastructural findings of secretory pinealocytes are in accordance with those described in the chicken (Boya and Calvo, 1980), the goose (Prusik et al., 2006), and the turkey (Tabecka et al., 1999).
The variable structural composition of the basal processes of pinealocytes and their bulbous endings has been observed in birds. In the chicken, the basal processes contained numerous clear and some granular vesicles as well as occasionally—dense bodies (Calvo and Boya, 1979; Ohshima and Matsuo, 1984). The presence of a bunch of empty vesicles, dense bodies, and granular vesicles in Athene brama, as well as a high number of dense bodies and granular vesicles in Euroloncha punchulata was reported (Haldar and Guchhait, 2000; Haldar and Bishnupuri, 2001). Our observations point to the presence of numerous dense core vesicles and a few small electron-lucent vesicles in the basal processes of both rudimentary-receptor and secretory pinealocytes in the pineal organ of the domestic goose (Prusik et al., 2006). On the other hand, we observed opposite picture of the basal processes composition in the turkey pineal gland (Tabecka et al., 1999). An equal number of electron-lucent vesicles and dense core vesicles were found in the basal processes of the pineal gland of the Muscovy duck (Lewczuk et al., 2000b).
Differences between species and a limited number of the data concerning ultrastructure of the processes caused the lack of generally accepted classification of these effective poles. According Haldar and Bishnupuri (2001), the effector poles of avian pinealocytes are represented by processes of axonal type terminating on other pinealocytes and neurohormonal terminals forming endings close to the capillaries. Processes of axonal type contain small electron-lucent vesicles, whereas processes of neurohormonal type additionally comprise a significant number of dense-core vesicles. On the other hand, Fejér et al. (2001) considered that pinealocyte axons either form a neurohormonal ending on the basal lamina of the pineal vascular surface or terminates on pineal neurons.
Our present findings show the presence of two morphological types of the basal processes of gull pinealocytes: (1) processes with occurrence of numerous small electron-lucent vesicles and synaptic ribbons, and (2) processes containing both types of vesicles—small electron-lucent vesicles and dense core vesicles (usually with the predominance of electron-lucent vesicles). It is apparent that in the common gull, pinealocytes form both neural and neurohormonal endings of the basal (effectory) processes. The presence of synaptic ribbons or their variants is typical of presynaptic specialization (Collin and Meiniel, 1968; Ekström and Meissl, 2003).
Major findings of this study are as follows—occurrence of specific hexagonal, paracrystalline structures in the basal processes of pinealocytes and their endings, storage of glycogen in the form of large accumulations in both types of pinealocytes localized in particular in the middle and proximal parts of the pineal gland and a characteristic arrangement of mitochondria in the form of clusters.
The presence of the paracrystalline structures was the most striking feature of neural endings in pinealocytes of the common gull. These peculiar structures have not been previously described in the pineal organs of vertebrates. Mechanism of the creation of the paracrystalline structures suggested by us involves: (1) the accumulation of small electron-lucent vesicles in cell process or its ending in situation of their overproduction, (2) the association of vesicles into a hexagonal structure to enable effective storage. We believe that the hexagonal structures are disintegrated by the successive release of vesicles, followed by their exocytosis. The question is: which factors cause the storage of vesicles and what type of signal causes their release? It is reasonable to suspect that hexagonal structures, their formation and collapse result from circadian and circuannual programs in the migration cycle, the role played by the pineal gland in this rhythmic phenomenon and the pineal gland's adaptation to the environmental changes.
Glycogen comprises the main readily mobilized energy resource and is distributed in high-energy-consuming organs (Richards et al., 2002; Gruetter, 2003; Chang et al., 2007; Tseng et al., 2007). It has been well documented that glycogen serves as an emergency fuel supply for the brain (Choi et al., 2003, Choi and Gruetter, 2004; Chang et al., 2007; Tseng and al., 2007). In the brain, glycogen is found predominantly in astrocytes (Cataldo and Broadwell, 1986). In the retina, where glycogen is also an immediate accessible energy reserve, the presence of glycogen was reported in all retinal layers (Coffe et al., 2004). In ultrastructural studies of the mammalian pineal glands, glycogen granules were observed in pinealocytes as well as in glial cells (Kachi et al., 1975; Kachi and Ito, 1977; Vollrath, 1981). In birds, an abundance of glycogen particles was described in pinealocytes of the Japanese quail (Ohshima and Matsuo, 1991a). During the embryonic development, glycogen aggregates were found in the pinealocytes of the chicken, but their presence was transitory and restricted to certain days of incubation (Ohshima and Matsuo, 1988).
Changes in the glycogen content in the vertebrate pineal glands have been studied mainly in the mouse and the rat. The diurnal rhythm of glycogen content in the mouse was observed starting from 22 days of age and it persisted until the age of two years (Kachi et al., 1975). Marked seasonal changes in glycogen levels with bimodal level—lower in fall and spring and higher in winter and summer were noted in the white-footed mouse (Peromyscus leucopus). In winter, the glycogen level in pinealocytes was very high and did not show of time-of-day differences (Kachi and Quay, 1984). Glycogen metabolism and its diurnal rhythm in the pinealocytes are regulated by the sympathetic innervations. Norepinephrine causes a marked decrease in glycogen content in the pineal gland of the mouse (Kachi and Ito, 1977) and the rat (Eugenin et al., 1997).
The use of histochemical method and transmission electron microscopy enabled us to demonstrate the presence of abundant glycogen accumulations in the pineal organ of the common gull. Two arrangement patterns of glycogen have been distinguished in pinealocytes: the single, dispersed glycogen particles, and large accumulations of glycogen particles. Both types of structures were found in the cytoplasm of the perikaryon and the processes. Glycogen accumulations were very often surrounded by clusters of mitochondria. The presence of such large amount of glycogen particles and occurrence of mitochondria in their vicinity we considered as a large energy reserve that can be used by pinealocytes in time of living of common gulls in variable environmental conditions of the cold sea. It should be emphasized that distribution of glycogen accumulations in the pineal gland, as it is pointed by results of histochemical studies is not equal. A higher number of accumulations were observed in the middle and proximal parts than in the distal part of the gland. The biological significance of the observed distribution pattern remains unexplained. Considering several descriptions of the glycogen in the pineal glands of mammals and birds, the content and distribution of glycogen in the pinealocytes of the common gull should be regarded as unusual phenomenon. An open question is whether the form of glycogen observed in the common gull is the species-specific or characteristic for the pineal glands of water birds during migration and dwelling in harsh weather conditions of cold seas.
The mitochondria exist in two interconverting forms: as small isolated particles and as extended filaments, networks or clusters connected with intermitochondrial junctions (Skulachev, 2001). The physiological states of mitochondria are clearly related to their distinctive morphology (Perkins and Ellisman, 2011). This includes considerable structural diversity, in particular related to the cristae (Frey et al., 2002; Perkins and Ellisman, 2007). Furthermore the positioning of mitochondria in the cytoplasm may also be essential for energy homeostasis (Chada and Hollenbeck, 2003). The mitochondria have been reported to be highly dynamic and mobile (Müller et al., 2005). A direct correlation between energy usage in local areas and mitochondrial movements was demonstrated (Mironov, 2007).
In regions with high density of mitochondria they often form clusters (Mironov, 2007; Perkins and Ellisman, 2011). The clusters of mitochondria were observed in Schwann cells (Perkins and Ellisman, 2011), in cell bodies of neurons (Dedov et al., 2000a, b) and in synapses (Wimmer et al., 2006). Local coupling between neighboring mitochondria causes their communication and synchronization in clusters (Kurz et al., 2010). Clustering of mitochondria could be the first step leading by their oxidative metabolism to the local generation of reactive oxygen species, which, in turn, could link mitochondrial functions with cellular functions (Skulachev, 2001). It has been suggested that in neurons clusters of mitochondria may be storage pools of mobile mitochondria able to be mobilized to provide energy for axonal transport during neuronal regeneration and neuronal outgrowth (Dedov et al., 2000a, b).
This study shows a high number of mitochondria in both types of pinealocytes. Two structural forms of mitochondria occur in pinealocytes: isolated particles and clusters with various degrees of interconnections. Mitochondria in the form of isolated particles are randomly distributed in the cytoplasm (secretory pinealocytes) or accumulated in the region of cytoplasm above the nucleus (rudimentary-receptor pinealocytes). However, many pinealocytes contain structurally diverse clusters of mitochondria. Clusters containing three to eight closely connected mitochondria are a regular feature of the perinuclear cytoplasm of secretory pinealocytes, however they are also present in rudimentary-receptor pinealocytes. It can be interpreted that arrangement of mitochondria in the pineal gland of the common gull is not random, but it results from a regulatory process that controls energy status of a cell and correlates positioning of mitochondria with areas of higher energy consumption.
Summing up, pinealocytes of the common gull are characterized by unusual features, which have not been previously described in avian pinealocytes: the presence of paracrystalline structures in the basal processes and their endings, the storage of glycogen in the form of large accumulations, and the arrangement of mitochondria in clusters. Further studies on other species of wild water birds dwelling in conditions of the cold sea are necessary to explain if the described features of pinealocytes are specific for genus Larus, family Laridae or a larger group of water birds living in similar environmental conditions.