Introduction: The superficial pineal gland of the Sprague Dawley rat is a neuroendocrine structure secreting the hormone melatonin. By use of block face scanning electron microscopy, our aim here was to identify the 3-dimensional ultrastructure of the gland. Methods: A series of 2,731 block face images of the rat pineal tissue, 30 nm in thickness, was obtained in a Teneo volume scanning electron microscope and used for 3-dimensional reconstruction by use of the TrakEM2-plugin in the ImageJ software. Thin sections of the tissue were cut for transmission electron microscopy. Results: Our analyses revealed cellular bulbous processes, containing 50–100 nm clear vesicles, that emerged from a neck-like area at the cell body of the pinealocyte. These bulbous processes extend into small canaliculi located in the center of parenchymal folliculi of the gland as well as into the perivascular spaces. Junctional complexes, comprising both gap and tight junctions, connected the lateral cellular membranes of the pinealocytes, where the bulbous processes emerged from the cell bodies. The canaliculi were, via the extracellular space, connected to the perivascular spaces. Discussion: The junctional complexes reported here would prevent a substance, released from the vesicles in the bulbous processes, from targeting the cell body from which they emerge. In line with previous combined morphological and biochemical demonstrations of glutamate located in clear vesicles of bulbous processes in the rat pineal gland, our data ultrastructurally support the concept that bulbous processes could participate in a paracrine glutamatergic inhibition of the melatonin secretion in the pineal gland. Conclusion: Bulbous secretory projections separated from the cell body by a junctional complex represents a new feature of neuroendocrine cells.

In this ultrastructural investigation of the Sprague Dawley rat pineal gland, a series of 2,731 serial sections, obtained by block-face scanning electron microscopy, have been 3-dimensional reconstructed by using the Fiji software. The data show that the neuroendocrine pinealocyte is endowed with bulbous projections with clear vesicles, separated from the cell body by a junctional complex, preventing the secretory product to target the pinealocyte from which they originate. Together with biochemical evidence for the presence of glutamate in the clear vesicles, this morphology supports the concept of a paracrine inhibitory glutamatergic system, inhibiting melatonin production in the rat pineal gland.

The pineal gland is a neuroendocrine structure, developed from the neural tube [1], that secretes the lipophilic hormone melatonin [2, 3]. Melatonin is synthesized by the pinealocytes with a circadian rhythm peaking during the night. This circadian rhythm is generated by an endogenous pacemaker located in neurons of the suprachiasmatic nucleus in the hypothalamus [4‒6]. The pacemaker neurons contain an autoregulatory transcriptional 24-h machinery of clock genes [7] with clock gene products as the core elements [8]. Light input to the retina is transmitted to the suprachiasmatic nucleus to synchronize the endogenous pacemaker to the environmental day-night light cycle [9].

A neuronal chain from the suprachiasmatic nucleus projects via the hypothalamic paraventricular nucleus to sympathetic neurons in the intermedio-lateral nucleus of the thoracic spinal cord [10, 11]. Neurons in this nucleus project via the sympathetic trunk to the superior cervical ganglion [12]. Sympathetic nerve fibers with origin in perikarya of the superior cervical ganglion enter the brain following the internal carotid to stimulate the pinealocytes via adrenergic receptors on the pinealocyte cell membrane [6]. A parasympathetic innervation from neurons in the sphenopalatine ganglion [13, 14], inhibiting the melatonin secretion, is also present. Finally, anatomical tracing studies show an input to the pineal gland directly from the brain via the habenular area [15] and an input from neurons located in the trigeminal ganglion [16, 17]. However, these additional inputs to gland seem to be physiologically less important for the secretion of melatonin than the sympathetic innervation.

In addition to melatonin, the pinealocytes synthesize a peptidergic factor [18, 19] released from dense core granules [20]. Some of these dense core granules contain a low-molecular weight peptide with antigonadotrophic activity [21], but the molecular structure of this peptide is unknown. Interestingly, biochemical in vitro studies have indicated paracrine regulation of the pinealocytes via a release of glutamate, synthesized in the pinealocytes and released to the extracellular space in 50–100 nm clear vesicles [22].

The advances presented in this report are partially based on technological progress. The rat is the classical species for investigating pineal anatomy [11], physiology [23], biochemistry [6, 24, 25], and pharmacology [26, 27]. The ultrastructure of the rat pineal gland has been investigated and described in several studies [1, 28, 29]. However, the invention of serial block face scanning electron microscopy has made it possible to produce a detailed 3-dimensional ultrastructural analysis of the rat pinealocyte and its relation to the surrounding structures.

In this investigation, we have analyzed a consecutive series of 2,718 images (30 nm apart) from the Sprague Dawley rat superficial pineal gland, obtained by a FEI Teneo VolumeScope Scanning Electron Microscope (SEM). By image analysis using the software Fiji TrakEM, we show that pinealocytes − often polarized around an extracellular “canaliculus” − are endowed with cellular bulbous processes containing clear vesicles. Junctional complexes along the lateral membranes of these pinealocyte processes separate the released content from reaching the synthesizing pinealocyte. Via the intercellular or perivascular spaces, the secreted product can target other neighboring pinealocytes.

The secretory bulbous processes of the pinealocytes are collected in “secretory fields” located in the pineal parenchyma or in the perivascular spaces. This morphology supports the concept that some of the glutamate-containing bulbous processes might exert a paracrine inhibitory function on the secretion of melatonin in the pineal gland of the Sprague Dawley rat.

Animals, Fixation, and Embedding of Tissue

Five Sprague Dawley male rats weighing 200 g were kept in a 12:12 h light-dark cycle and sacrificed at Zeitgeber time 4–5. The rats were anaesthetized with tribromethanol (40 mg/100 g body weight) and vascularly perfused via the left ventricle of the heart with 100 mL phosphate buffered saline (PBS) with Heparin (1,500 units [6.72 mg]/100 mL; Sigma #H3393) for 1 min. The right atrium was opened for the venous efflux before starting the perfusion. This was followed by 2.5% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4) for 5 min. The superficial pineal gland was isolated and stored overnight at 4°C in the same fixative. The tissue was subsequently processed for serial block face SEM according to Deerinck et al. (https://ncmir.ucsd.edu/sbem-protocol).

The pineal tissue blocks were post-fixed in 2% OsO4 with 1.5% ferrocyanide in 0.15 M cacodylate buffer (pH 7.2) on ice for 2 h. Following 2 × 10 min rinse in double-distilled water (dd-water) at room temperature, the tissue blocks were impregnated with a 1% TCH-solution (0.1 g thiocarbohydrazide in 10 mL dd-water) for 40 min at room temperature, followed by impregnation in 2% OsO4 for 1 h. After 3 × 10 min rinse in dd-water, the tissue blocks were en-block stained in 0.02 M lead nitrate in 0.03 M sodium aspartate in dd-water (pH adjusted to 5.5) at 60°C for 60 min. Finally, the tissue was en-block stained with 1% uranyl acetate in dd-water at 4°C overnight. Following 3 × 10 min rinse in dd-water, the blocks were dehydrated in an increasing ethanol series (70, 90, 96, and 100% ethanol) and via a mixture of Epon Hard® and propylene oxide (EH/pro: 25, 50, and 75%) transferred to 100% Epon Hard® and polymerized for 48 h at 60°C.

TEM

Overview sections, 2 µm in thickness, were stained with toluidine blue. From the overview section, optimal fields were selected, and the blocks were trimmed accordingly for thin sectioning. Sections, 60 nm in thickness, were cut by use of an ultra-microtome (Leica UC7) and imaged in a CM 100 TEM (Philips) operated at 80 kV and equipped with a Veleta camera (Olympus) with a resolution of 2,048 × 2,048 pixels and the iTEM software package.

Serial Block Face SEM

This technique, based on the work of Denk and Horstman [30], allows a backscattered electron image of the block face to be collected following each diamond knife cut. Tissue blocks with the superficial pineal gland were trimmed to an approximately 300 μm × 300 μm block face with less than 2 mm depth using a razor blade and polished using a conventional ultra-microtome and a diamond knife. The blocks were mounted using Epo-Tek EE129-4 Adhesive (EMS #12670-EE, Ted Pella) to a 3 mm head diameter aluminum-mounting pin. The pinned sample was subsequently sputter-coated with 20 nm gold. The 30 nm serial images (16 k × 16 k, 8.4 nm x-y pixel resolution) were collected by a serial block face SEM (Teneo VolumeScope II, FEI Company), equipped with an in-chamber diamond knife, and using backscattered electron signals at an accelerating tension of 2.27 kV, at 26.4 e/nm2 electron dose, and 3 μs dwell time under high vacuum conditions. Two thousand seven hundred thirty-one images were recorded, giving an overall volume of 138 µm × 138 µm × 82 µm. This relates to 8.7 nm × 8.7 nm × 30 nm voxel size.

Analysis

Image analysis of the serial sections obtained in the serial block face scanning electron microscope was performed using the software package, ImageJ [31] with the plugin TrakEM (https://imagej.net/software/fiji/).

Cellular Morphology of the Rat Pineal Gland

The superficial pineal gland of the Sprague Dawley rat is an ellipsoid structure with a weight of about 1.5 mg, surrounded by a glial capsule [11]. From this capsule, the pial tissue continues into the gland itself as perivascular spaces, separating the pineal parenchyma (shown in Fig. 1). The perivascular spaces contain blood vessels, the majority of which are fenestrated capillaries, perivascular phagocytes (shown in Fig. 1), and many nerve fibers with terminal boutons as well as bulbous projections of the pinealocytes.

Fig. 1.

Photomicrograph of a 1-µm-thick Epon-embedded sections of a part of the superficial pineal gland of the Sprague Dawley rat. Perivascular spaces with capillaries (ca) separate the parenchyma. The pinealocytes (P) are seen as ellipsoid cells with lightly stained cytoplasm and nucleus. Many lipid droplets (Li) are present in the cytoplasm of the pinealocytes. An interstitial cell (in), triangular in shape, with a dark cytoplasm and nucleus, is located in the pineal parenchyma, separated from the perivascular spaces of an external limiting membrane. A perivascular phagocyte (ph) is located in the perivascular space itself. Toluidine blue staining. Scale bar = 20 µm.

Fig. 1.

Photomicrograph of a 1-µm-thick Epon-embedded sections of a part of the superficial pineal gland of the Sprague Dawley rat. Perivascular spaces with capillaries (ca) separate the parenchyma. The pinealocytes (P) are seen as ellipsoid cells with lightly stained cytoplasm and nucleus. Many lipid droplets (Li) are present in the cytoplasm of the pinealocytes. An interstitial cell (in), triangular in shape, with a dark cytoplasm and nucleus, is located in the pineal parenchyma, separated from the perivascular spaces of an external limiting membrane. A perivascular phagocyte (ph) is located in the perivascular space itself. Toluidine blue staining. Scale bar = 20 µm.

Close modal

The pineal parenchyma is made up of rosettes and cords of pinealocytes and interstitial cells (shown in Fig. 1-3). Nerve fibers also penetrate into the parenchyma. The ovoid-shaped pinealocytes, with a diameter of 7–10 µm, are characterized by a lightly stained cytoplasm and an ovoid nucleus (shown in Fig. 1-3). Darker-stained interstitial cells with a triangular nucleus are often located on the surface of the lobules and cords (shown in Fig. 1, 3) and are separated from the perivascular space by the perivascular external limiting membrane.

Fig. 2.

Survey block face image of the superficial pineal gland of the Sprague Dawley rat. The pineal tissue consists of lobules (lobule) and cords (cord) of pinealocytes and interstitial cells separated by perivascular spaces (ps) with fenestrated capillaries and perivascular cells. A few empty (embedding medium only) interstitial spaces suffer from charge artefacts (dark circular electron dense signal, arrows). Scale bar = 100 µm.

Fig. 2.

Survey block face image of the superficial pineal gland of the Sprague Dawley rat. The pineal tissue consists of lobules (lobule) and cords (cord) of pinealocytes and interstitial cells separated by perivascular spaces (ps) with fenestrated capillaries and perivascular cells. A few empty (embedding medium only) interstitial spaces suffer from charge artefacts (dark circular electron dense signal, arrows). Scale bar = 100 µm.

Close modal
Fig. 3.

Block face image of a parenchymal rosette in the superficial pineal gland of the Sprague Dawley rat. The pinealocytes (P) in the lobule are surrounded by interstitial cells (in). In the center of the rosette, a “canalicular-like” area of the extracellular space is seen (dark arrowhead). The white arrowhead mark a lipid droplet. ca, capillary; ps, perivascular space. Scale bar = 5 µm.

Fig. 3.

Block face image of a parenchymal rosette in the superficial pineal gland of the Sprague Dawley rat. The pinealocytes (P) in the lobule are surrounded by interstitial cells (in). In the center of the rosette, a “canalicular-like” area of the extracellular space is seen (dark arrowhead). The white arrowhead mark a lipid droplet. ca, capillary; ps, perivascular space. Scale bar = 5 µm.

Close modal

Some Pinealocytes Are Polarized around a “Canalicular-Like” Extracellular Space

The pinealocyte consists of a cell body from which 3–4 cellular processes emerge (shown in Fig. 4). By 3D-reconstruction of serial sections obtained by serial blockface SEM, many pinealocytes were found to be endowed with a long bulbous process, which terminated in the center of a parenchymal rosette (shown in Fig. 4). Transmission electron microscopy (TEM) of the parenchymal center of the rosette showed that the pinealocytes were polarized around a “canalicular-like” extracellular space in the center of the rosette (shown in Fig. 5a), and bulbous projections of the pinealocytes, each containing 50–100 clear vesicles, were found to enter the “canaliculus” (shown in Fig. 5a, b). Before terminating in a bulbous projection, the cell body of the pinealocytes was constricted in a neck-like structure, which continued in the projection (shown in online suppl. video S1; for all online suppl. material, see https://doi.org/10.1159/000535567). Junctional complexes (described below) were present in the constricted neck area of the pinealocytes. The bulbous projections contained 50–100 nm clear- and 100–300 nm (in diameter, respectively) granular vesicles (shown in Fig. 5a, b). Some of the 50–100 nm clear vesicles in the bulbous projections were attached to the cell membrane and opened towards the “canaliculus” (shown in Fig. 5b).

Fig. 4.

Orthogonal view of a 3-dimensional reconstructed pinealocyte (P) in the superficial pineal gland of the Sprague Dawley rat. One of the processes of the pinealocyte terminates in the center of a parenchymal rosette (arrow) in a “canalicular-like” area of the extracellular space (see Fig. 5a for a TEM image). The pinealocyte was reconstructed from 1,817 images (30 nm apart = 24.93 µm in total depth). The reconstruction was done by use of the software Fiji TrakEM. ps, perivascular spaces. Scale bar = 5 µm.

Fig. 4.

Orthogonal view of a 3-dimensional reconstructed pinealocyte (P) in the superficial pineal gland of the Sprague Dawley rat. One of the processes of the pinealocyte terminates in the center of a parenchymal rosette (arrow) in a “canalicular-like” area of the extracellular space (see Fig. 5a for a TEM image). The pinealocyte was reconstructed from 1,817 images (30 nm apart = 24.93 µm in total depth). The reconstruction was done by use of the software Fiji TrakEM. ps, perivascular spaces. Scale bar = 5 µm.

Close modal
Fig. 5.

TEM micrographs of “canalicular-like” extracellular spaces and the surrounding pinealocytes. a Electron micrograph of polarized pinealocytes (P) extending bulbous processes (b) with clear vesicles into the “canalicular-like” extracellular area. Li, lipid droplet. Scale bar = 2 µm. b A bulbous process with 50–100 nm clear vesicles (arrows) located in the “canalicular-like” extracellular space. A clear vesicle has fused with the cell membrane (fu). e, Golgi vesicle; Li, lipid droplet; Mi, mitochondrium. Scale bar = 0.5 µm.

Fig. 5.

TEM micrographs of “canalicular-like” extracellular spaces and the surrounding pinealocytes. a Electron micrograph of polarized pinealocytes (P) extending bulbous processes (b) with clear vesicles into the “canalicular-like” extracellular area. Li, lipid droplet. Scale bar = 2 µm. b A bulbous process with 50–100 nm clear vesicles (arrows) located in the “canalicular-like” extracellular space. A clear vesicle has fused with the cell membrane (fu). e, Golgi vesicle; Li, lipid droplet; Mi, mitochondrium. Scale bar = 0.5 µm.

Close modal

Pineal “Canaliculi” Are Connected with the Perivascular Spaces

By use of Fiji’s TrakEM2 and the plugin TreeLine segmentation, the connections of the intraparenchymal “canaliculi” were analyzed (shown in Fig. 6). This analysis showed that a “canaliculus” was a part of the extracellular space with neither a surrounding canalicular wall nor tight junctions confining the space. The “canaliculi” could be followed via the intercellular parenchymal space to the perivascular space of the gland (shown in Fig. 6).

Fig. 6.

Montage of serial block face images showing the connection, via the extracellular space, between a “canaliculus” surrounded by pinealocyte bulbous processes (located in image no. 1,422) and the perivascular space (located in image no. 1,959) in the pineal gland of the Sprague Dawley rat at by use of the plugin in TreeLine Fiji TrakEM2. A total of 537 serial block face images were used for the reconstruction (images no. 1,422–1,959. Distance between images = 30 nm. Total depth: 16.08 µm). a An orthogonal view of image no. 1,959 with the perivascular space (ps) to which the “canalicular-like” extracellular space (yellow ball) in image no. 1,422 is connected (connection shown by a ThreeLine in yellow). b Block face SEM micrograph of image no. 1,422. The “canalicular-like” extracellular space is marked with a yellow ball. Scale bar = 5 µm.

Fig. 6.

Montage of serial block face images showing the connection, via the extracellular space, between a “canaliculus” surrounded by pinealocyte bulbous processes (located in image no. 1,422) and the perivascular space (located in image no. 1,959) in the pineal gland of the Sprague Dawley rat at by use of the plugin in TreeLine Fiji TrakEM2. A total of 537 serial block face images were used for the reconstruction (images no. 1,422–1,959. Distance between images = 30 nm. Total depth: 16.08 µm). a An orthogonal view of image no. 1,959 with the perivascular space (ps) to which the “canalicular-like” extracellular space (yellow ball) in image no. 1,422 is connected (connection shown by a ThreeLine in yellow). b Block face SEM micrograph of image no. 1,422. The “canalicular-like” extracellular space is marked with a yellow ball. Scale bar = 5 µm.

Close modal

Some Pinealocytes Terminate with Bulbous Projections Directly in the Perivascular Space

Polarized pinealocytes with bulbous projections containing clear vesicles were also observed extending directly into the perivascular space (shown in Fig. 7a, b). Before terminating in bulbous projections, the pinealocytes with the projections into the perivascular spaces were also endowed with neck-like constrictions (shown in Fig. 7b). The lateral membranes of the pinealocytes in the constricted neck-like areas were also endowed with junctional complexes (shown in Fig. 7b; online suppl. Video S1). High-resolution TEM of this complex showed it to consist of both gap and tight junctions (shown in Fig. 8a, b). Often, many bulbous projections from different pinealocytes were located in the same perivascular area and constituted a “secretory field” (see paragraph below).

Fig. 7.

TEM micrographs showing bulbous processes from pinealocytes located in the perivascular space. a Pinealocytes with ovoid cell bodies (P) terminate with bulbous processes (arrow) directly in the perivascular space (ps). Scale bar = 5 µm. b High magnification of the bulbous processes showing the junctional complexes at the neck-like origin of the process (arrows). ps, perivascular space. Scale bar = 1 µm.

Fig. 7.

TEM micrographs showing bulbous processes from pinealocytes located in the perivascular space. a Pinealocytes with ovoid cell bodies (P) terminate with bulbous processes (arrow) directly in the perivascular space (ps). Scale bar = 5 µm. b High magnification of the bulbous processes showing the junctional complexes at the neck-like origin of the process (arrows). ps, perivascular space. Scale bar = 1 µm.

Close modal
Fig. 8.

TEM micrographs of junctional complexes between the lateral membanes of pinealocyte bulbous processes abutting the perivascular space. a Junctions between pinealocyte (P) and bulbous process (b) close to a perivascular space (ps). A gap junction-like structure is seen (gap). Scale bar = 0.5 µm. b Higher magnification of a junctional complexes between the lateral membranes of pinealocytes. Punctuate occlusions (tight junctions) are occluding the thin intercellular space (arrowheads). Scale bar = 250 nm.

Fig. 8.

TEM micrographs of junctional complexes between the lateral membanes of pinealocyte bulbous processes abutting the perivascular space. a Junctions between pinealocyte (P) and bulbous process (b) close to a perivascular space (ps). A gap junction-like structure is seen (gap). Scale bar = 0.5 µm. b Higher magnification of a junctional complexes between the lateral membranes of pinealocytes. Punctuate occlusions (tight junctions) are occluding the thin intercellular space (arrowheads). Scale bar = 250 nm.

Close modal

In addition, another pinealocyte-perivascular contact was observed. By following the shape of the pinealocytes in the serial block face images, it was observed that a pinealocyte with a bulbous projection emerging from one part of the cell body, in another part of the cell body could have direct contact with the external limiting membrane of the perivascular space, without membrane specializations and ultrastructural indication of exocytosis in this area (shown in Fig. 9a–d; online suppl. video S1).

Fig. 9.

a Orthogonal view of a 3-dimensional reconstructed pinealocyte (P) in the superficial pineal gland of the Sprague Dawley rat projected on serial image no. 1,742. The pinealocyte is reconstructed from 796 serial block face images (distance between images: 30 nm; images no. 2,418–1,622). A yellow bulbous process (arrow) is emerging from the cell body (green color) of the pinealocyte (P). A part of the pinealocyte (marked with a white rectangle), containing the nucleus of the pinealocyte, is apposing the perivascular space (seen in b). Scale bar: 10 µm. b The inset shows the block face image no. 2,193 corresponding to the area in the white rectangle in (a). At this location, the pinealocyte (green) cell membrane is directly apposing the perivascular space (ps). Scale bar = 5 µm. c Drawing of the block face image no. 1,742 showing the bulbous process (yellow color) of the reconstructed pinealocyte (green color) (P). Arrows points towards junctional complexes (yellow lines) connecting the lateral membranes of the pinealocytes. The two spaces, delineated by the red lines, are extracellular spaces. Scale bar = 500 nm. d Drawing of the block face image no. 2,193 shown in (b). The red line illustrates the location of the external limiting membrane of the perivascular space (ps). P, pinealocyte. Scale bar = 5 µm.

Fig. 9.

a Orthogonal view of a 3-dimensional reconstructed pinealocyte (P) in the superficial pineal gland of the Sprague Dawley rat projected on serial image no. 1,742. The pinealocyte is reconstructed from 796 serial block face images (distance between images: 30 nm; images no. 2,418–1,622). A yellow bulbous process (arrow) is emerging from the cell body (green color) of the pinealocyte (P). A part of the pinealocyte (marked with a white rectangle), containing the nucleus of the pinealocyte, is apposing the perivascular space (seen in b). Scale bar: 10 µm. b The inset shows the block face image no. 2,193 corresponding to the area in the white rectangle in (a). At this location, the pinealocyte (green) cell membrane is directly apposing the perivascular space (ps). Scale bar = 5 µm. c Drawing of the block face image no. 1,742 showing the bulbous process (yellow color) of the reconstructed pinealocyte (green color) (P). Arrows points towards junctional complexes (yellow lines) connecting the lateral membranes of the pinealocytes. The two spaces, delineated by the red lines, are extracellular spaces. Scale bar = 500 nm. d Drawing of the block face image no. 2,193 shown in (b). The red line illustrates the location of the external limiting membrane of the perivascular space (ps). P, pinealocyte. Scale bar = 5 µm.

Close modal

Anatomical Localization of “Secretory Fields” in Relation to the Pinealocyte

As shown in shown in Figure 10a, a pinealocyte is surrounded by several “secretory fields,” defined as areas containing bulbous projections from the pinealocytes. By analysis of serial block face images with Fiji’s TrakEM, it could be shown that the reconstructed pinealocyte contributed with a secretory bulbous process to several, but not all, surrounding “secretory fields” (shown in Fig. 10a–c.

Fig. 10.

Montage of a 3-dimensional reconstructed pinealocyte and five “secretory fields” surrounding the pinealocyte. One thousand five hundred fifty one serial block face images were used for the 3-D reconstruction of the pinealocyte (49.53 µm in depth, sections no. 867–2,518). a Block face image no. 2,340 of the pinealocyte (P) extending a bulbous process into a surrounding “secretory field” (the area covered by the blue circle) and shown in (c). Scale bar = 5 µm. b Orthogonal view of the pinealocyte seen in (a). The pinealocyte is in this montage 3-dimensional reconstructed (P, blue) and orthogonal projected on image no. 2,513. The pinealocyte is surrounded by five “secretory fields” (seen as light green ellipsoid structures, and marked by arrowheads) plus a secretory field located in the red rectangle (also seen in a, c). Scale bar = 10 µm. c Parallel block face image (no. 2,336) to the section in (a) showing two pinealocyte bulbous processes (arrows) with 60–100 nm clear vesicles projecting into an intraparenchymal “canalicular-like” extracellular space (green circle). The pinealocyte (P) corresponds to the pinealocyte in (a). Scale bar = 2.5 µm.

Fig. 10.

Montage of a 3-dimensional reconstructed pinealocyte and five “secretory fields” surrounding the pinealocyte. One thousand five hundred fifty one serial block face images were used for the 3-D reconstruction of the pinealocyte (49.53 µm in depth, sections no. 867–2,518). a Block face image no. 2,340 of the pinealocyte (P) extending a bulbous process into a surrounding “secretory field” (the area covered by the blue circle) and shown in (c). Scale bar = 5 µm. b Orthogonal view of the pinealocyte seen in (a). The pinealocyte is in this montage 3-dimensional reconstructed (P, blue) and orthogonal projected on image no. 2,513. The pinealocyte is surrounded by five “secretory fields” (seen as light green ellipsoid structures, and marked by arrowheads) plus a secretory field located in the red rectangle (also seen in a, c). Scale bar = 10 µm. c Parallel block face image (no. 2,336) to the section in (a) showing two pinealocyte bulbous processes (arrows) with 60–100 nm clear vesicles projecting into an intraparenchymal “canalicular-like” extracellular space (green circle). The pinealocyte (P) corresponds to the pinealocyte in (a). Scale bar = 2.5 µm.

Close modal

This ultrastructural 3D study of the pineal gland of the Sprague Dawley rat describes that the pinealocytes are endowed with clear vesicle-containing bulbous processes connected to the cell body by a neck-like process with a junctional complex. Such a cellular appendage on a neuroendocrine cell has not been reported before.

By analyzing the block face images by using the software Fiji TrakEM [32], we also showed that the histological arrangement of the pinealocytes is polarized with bulbous cellular projections of the pinealocytes extending both towards the extracellular space in the canaliculi and the perivascular spaces. The junctional complexes located in the neck-like area contained both gap and tight junctions. The tight junctions make a barrier between the extracellular space surrounding the bulbous processes and the extracellular space surrounding the cell body of the pinealocyte from which the processes emerge. Accordingly, this morphology prevents a hydrophilic secretory product to target the product-secreting pinealocyte. On the other hand, the secretory product can reach surrounding pinealocytes via the release to a “canalicular-like” extracellular space in the lobules or in the perivascular spaces, followed by diffusion to neighboring pinealocytes.

It is also of interest to notice that other parts of a pinealocyte can be in direct contact with the external limiting membrane of the perivascular space, but in this contact area, the cell membrane has no ultrastructural indications of exocytosis. However, the pineal hormone, melatonin, can be released from this area because melatonin is a lipophilic molecule, and a release from the pinealocytes to the perivascular space occurs by diffusion without involving exocytosis [33].

Several biochemical and molecular biological studies have strongly indicated that microvesicle-mediated exocytosis of glutamate is a paracrine-like chemical transduction mechanism inhibiting melatonin secretion in rat pineal gland [34‒36]. Thus, the 50–80 nm clear microvesicles in mammalian pineal gland, including the vesicles in the bulbous projection, have been shown to contain glutamate [37] which is taken up in the microvesicles by an l-glutamate transporter in the microvesicles [38, 39]. The microvesicles also contain proteins important for release of content from the vesicle. Accordingly, proteins belonging to the SNARE complex, e.g., synaptotagmin, synaptobrevin [22], and Snap-25 [40], all involved in fusion of the vesicle with the cell membrane, have been demonstrated.

Release of glutamate from microvesicles in the rat pinealocyte is induced by a parasympathetic cholinergic stimulation of nicotinic receptors on the pinealocyte cell membrane [36, 41]. Binding to this receptor opens calcium channels, followed by release of glutamate into the intercellular space [42, 43]. The released glutamate can diffuse to neighboring pinealocytes and bind to a metabotropic glutamate receptor 3 (mGluR3) [34], resulting in a decreased synthesis of melatonin [35]. The mechanism behind this decrease is due to activation of a Gi-protein, reducing the intracellular level of cAMP and resulting in an inhibition of the activity of the enzyme aralkylamine N-acetyltransferase (AANAT) [34, 44], the rate-limiting enzyme in the melatonin synthesis [6].

In addition to the mglutR3, the rat pinealocytes also possess an AMPA-type ionotropic glutamate receptor (GlutR1) [45]. This receptor opens a calcium channel followed by release of glutamate from the pinealocytes to the extracellular space. The junctional complex at the origin of the secretory bulbous processes would prevent glutamate from targeting the AMPA receptor on the cell body of the pinealocytes from which the bulbous process emerges [46]. Thereby, hyperstimulation of the pinealocyte will be prevented.

Although the junctional complexes prevent the released glutamate to directly target the pinealocyte, releasing the transmitter, glutamate can diffuse in the extracellular space and finally reach the receptors on the glutamate synthesizing pinealocyte via a longer diffusion in the extracellular space [46]. However, such a diffusion process is physiologically a slow regulatory process, and during the diffusion, the glutamate will be diluted, possibly below the saturation level of the receptor.

It would have been of interest if a circadian change, e.g., a decreased number of bulbous processes or junctional complexes during the night, could be observed, indicating a lower glutamatergic inhibitory activity during darkness. However, this is not possible to determine with the block-face scanning electron microscopical technique and also difficult to evaluate with classical transmission electron microcopy due to the paucity of the above ultrastructural features. Quantitative biochemical determination of the amount of tight junction proteins in the total pineal during day and night could provide data regarding such possible changes [47].

A glutamatergic signaling system in the brain has been known for many years, controlling important functions such as long-term potentiation, depression, and synaptic plasticity [48]. However, glutamate has now also been shown in several endocrine tissues, such as the islets of Langerhans, the intestine, the stomach, and the testis, to be a paracrine regulator [36]. Our study opens up the possibility that the channeling system described in the current report represents a general feature of paracrine signaling glutamatergic signaling in endocrine tissues.

In addition to the glutamatergic paracrine system, morphological studies of intrapineal peptidergic cells also indicate the presence of a paracrine system in the pineal of some species involving peptidergic cells [11, 49]. Most of the pineal neuropeptides are located in nerve fibers and terminals innervating the gland, but originating from neurons with perikarya located outside the pineal [50]. However, some of the neuropeptides are confined to intrapineal neurons, e.g., somatostatin in the rat [51], substance P in the cotton rat [52], and leu-enkephalin in the human [53]. These intrapineal peptidergic neurons are to be regarded as ganglionic neurons in the parasympathetic nervous system. However, in the European hamster, light- and ultrastructural immunocytochemistry has shown that leu-enkephalin is located in some of the pinealocytes and that processes from these leu-enkephalin immunoreactive pinealocytes make synapse-like contacts with other pinealocytes [54]. The physiological function of the enkephalinergic paracrine signaling in the European hamster is still enigmatic but could in addition to the direct cellular contact also make use of the canalicular-like system reported here.

The bulbous processes in the pineal gland extending from the cell body of the secretory pinealocytes were observed in some of the first TEM studies of the rat pineal gland [55]. However, the polarization of these processes towards the “canalicular-like” extracellular space in a lobule or toward the perivascular space was impossible to visualize with classical TEM. Similar processes have also been described in the pineal of the guinea pig [56], rabbit [57], cow [58], and sheep [58]. Whether the processes emerged from a neck-like structure with junctional complexes was not described, but these reports collectively point towards a high degree of conservation of pinealocyte morphology among mammals that could involve the presence of a canalicular-like system.

In summary, this study shows a hitherto undescribed morphology of a neuroendocrine cell with polarized secretory bulbous processes, separated from the cell body of the pinealocyte by occluding junctions. Microvesicles in the bulbous projections with a clear vesicular content release the content into either a “canalicular-like” extracellular space in the center of a pineal lobule or to the perivascular space. This morphology supports biochemical data suggestions of the presence of a paracrine system in the rat pineal gland with release of glutamate from the microvesicles in the gland, and inhibition of the enzymatic machinery synthesizing the melatonin secreted by the gland.

We thank Zhila Nikrozi, electron microscopy laboratory manager, for excellent technical assistance with the preparation of the tissues for serial block face SEM, and Michael Johnson, technical director for electron microscopy, for assistance with TEM.

All animal experiments were performed in accordance with the guidelines of EU directive 2010/63/EU. The study was approved by the Danish Animal Experiments Inspectorate (authorization number 2017-15-0201-01190) and by the Faculty of Health and Medical Sciences, University of Copenhagen (authorization number P21-146).

On behalf of all authors, the corresponding author declares that there is no financial or non-financial conflict of interest.

This work was supported by the Aage og Johanne Louis-Hansen’s Foundation, Grant No. 22-2B-11198 to M. Møller and The Independent Research Fund Denmark, Grant No. 8020-00037B to M.F. Rath, and the Lundbeck Foundation, Grant No. R344-2020-261 to M.F. Rath.

Conceptualization: M. Møller, M.F. Rath, J. Midtgaard, and K. Qvortrup. Investigation: M. Møller, J. Midtgaard, and K. Qvortrup. Analysis: M. Møller and J. Midtgaard. Writing: M. Møller, M.F. Rath, K. Qvortrup, and J. Midtgaard.

The size and number of data files prevent availability in a public online repository. However, all data underlying the research presented in this manuscript are available from the corresponding author (M. Møller).

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