In the present study the "interstitial" cells of the superficial pineal gland and the nonparenchymal cells of the pineal stalk in Sprague-Dawley rats were examined ultrastructurally with the aim of defining the cells more closely. The "interstitial" cells of the superficial pineal gland do not represent a homogeneous cell population. The most abundant cell type is the mononuclear phagocyte, most easily recognized by its dark appearance and its content of primary and conspicuous secondary lysosomes. Astrocytes can be distinguished by the typical appearance of their nuclei (i.e., a thin continuous rim of heterochromatin adjacent to the nuclear membrane), identical to that of astrocytes in the CNS. Depending on the absence or presence of glial filaments and their amount, a spectrum of astrocytic cells is present. Mature astrocytes with filaments throughout their cytoplasm are rare. Immature glial cells with few or no filaments predominate. In the vicinity of blood vessels pericytes are present. In view of the fact that the "interstitial" cells could generally be identified it is suggested to abandon the term interstitial for the cells in question. In the pineal stalk mature astrocytes predominate; they have some features in common with pinealocytes, i.e., the presence of intergrade endoplasmic reticulum and grumose bodies (lysosomes). Other unusual features are a relative abundance of coated pits and vesicles. Oligodendrocytes are restricted to the proximal part of the stalk, near the deep pineal, where myelinated axons are abundant. More distally a few Schwann cells were seen.
In view of the increasing interest in the central innervation of the mammalian pineal gland, this aspect was studied in depth in the rat. This species is especially suited since the nerve fibers in question form a distinct bundle running from the deep to the superficial pineal gland through the pineal stalk. The axons were counted and analysed ultrastructurally in the pineal stalks cut transversely at three levels (proximal, intermediate, and distal) relative to the neural axis and in longitudinal sections. The number of nerve fibers was highly variable, ranging from 551 to 1,132 proximally and from 110 to 448 distally, indicating that many fibers terminate in the stalk or leave the stalk after forming a loop. Large myelinated axons, which are abundant proximally, appear to lose their sheaths along their course through the stalk. Most of the axons were small and unmyelinated. A few of these had the appearance of sympathetic fibers and disappeared after sympathectomy. Others contained abundant neurosecretory granules, and, according to the literature, may originate in the hypothalamic paraventricular nuclei. The majority of the small axons which are apparently devoid of granules and dense-cored vesicles may come from the habenular nuclei and the stria medullaris. In addition to axons, the stalk contains astrocytes, a few oligodendrocytes and Schwann cells, as well as pinealocytes identical to those of the superficial pineal gland.
Cultured resident murine maaophages are incubated in the continuous presence of the fluorescent endocytic marker Lucifer Yellow and a phorbol ester that activates protein kinase C. Under these steady-state labeling conditions the fluorescent tracer was, for the most part, in a tubularlreticular compartment. Enzyme cytochemical localization of acid phosphatase in the same cells showed essentially a one-to-one correlation between the Lucifer Yellow-and acid phosphatase-
Thiamine pyrophosphatase (TPPase), nucleoside diphosphatase (NDPase), and glucose-6-phosphatase (G-6-Pase) were localized by the cerium technique in guinea pig pinealocytes and compared with the corresponding lead technique. NDPase and TPPase were also compared at different pH values using the cerium technique. Vibratome sections of perfusion-fixed tissue were incubated with cerium chloride or lead nitrate. Substrates used were thiamine pyrophosphate (for TPPase), sodium inosine diphosphate (NDPase), and disodium glucose-6-phosphate (G-6-Pase). The 1-2 trans saccules of the Golgi apparatus showed TPPase and NDPase activity but none for G-6-Pase. The endoplasmic reticulum (ER) cisternae and perinuclear space had NDPase and G-6-Pase activity but not TPPase. The abluminal plasmalemma of endothelial cells and the plasmalemma of Schwann cells demonstrated TPPase and NDPase activity but the luminal plasmalemma of the endothelial cells and the plasmalemma of pinealocyte processes showed only NDPase activity. TPPase was active at all pH values tested, but NDPase was most active at pH values of 6.5 and 7.0. Lead phosphate precipitate was frequently seen in nuclei, perinuclear space, ER cisternae, and "synaptic" vesicles when lead was used as the capturing agent. These sites were usually not labeled when cerium was used.
Resident peritoneal murine macrophages typically have acid phosphatase (AcPase) activity in lysosomes which are spherical in shape and have a perinuclear distribution.Upon stimulation with certain phorbol esters there is a rapid and dramatic modification of the AcPase-containing compartment into tubular structures which are often interconnected. The formation and maintenance of these tubular structures are dependent upon the presence of an intact microtubule system. Treatment of cells with the microtubule-depolymerizing agent nocodazole blocks the formation of the tubular structures and causes these tubular compartments to collapse toward the nucleus when nocodazole is administered after they are formed. We have employed whole mount electron microscopy and enzyme cytochemistry to document the association of the phorbol ester-induced AcPase-containing compartment and cytoplasmic microtubules. This methodological approach has the advantage of increased resolution when compared to optical microscopy and of an improved vantage point for analysis when compared to conventional thin section electron microscopy.
The ultrastructural localization of acetylcholinesterase (AChE) activity in guinea pig pineal gland was studied using the copper-glycine procedure. A small number of pinealocytes and bundles of unmyelinated nerve fibers were labeled by the AChE reaction. The AChE-positive pinealocytes were located near blood vessels and distributed in small groups. The AChE reaction product was localized in the perinuclear cistern, in the cisternae of the endoplasmic reticulum (ER), and in the saccules of the Golgi apparatus. These findings suggest that the AChE-positive pinealocytes synthesize AChE. The AChE reaction product was also seen in the intercellular space between pinealocyte processes. Besides pinealocytes, AChE activity was localized on the axolemma of myelinated and unmyelinated nerve fibers and in the basement membrane surrounding unmyelinated nerve fibers. Pseudocholinesterase activity was confined to Schwann cells, which showed the reaction product in their perinuclear cistern, in the cisternae of the ER, and on the plasmalemma.
Pineal "synaptic" ribbons are a heterogeneous population of organelles. "Synaptic" ribbons (SR) sensu stricto, "synaptic" spherules (SS), and intermediate forms (IMF) are present. Their function and origin are unknown, and a knowledge of their prenatal development is lacking. Thus the pineal glands of prenatal, neonatal, and adult guinea pigs were prepared for electron microscopy. "Synaptic" ribbons were studied morphologically and quantitatively. The three categories of "synaptic" ribbons reported in adult pineal glands were also present in prenatal pineal glands. Their structural features, distribution, grouping, and composition patterns are similar to those in adults. "Synaptic" ribbons were first detected in pinealocytes of the distal region of a 42-day postcoitus (PC) pineal gland and were comparable with those in adults. They increased in number with age and reached a peak at 63 days PC, followed by a steep decline at 66 and 67 days PC. By day 69 PC, the numbers increased again and showed a dramatic increase after birth. Several true ribbon synapses were seen at day 63 PC between pinealocyte cell processes or between pinealocyte cell process and pinealocyte cell body. Since true ribbon synapses have not been found in adult guinea pig pinealocytes, their synaptic nature could have been lost during development. No precursors for the "synaptic" ribbons were found. The endoplasmic reticulum cisternae may be the origin for the ribbon vesicles because of their close association with the "synaptic" ribbons.
Thiamine pyrophosphatase (TPPase) and nucleoside diphosphatase (NDPase) both have a similar localization in the endoplasmic reticulum (ER), the perinuclear space (PNS) and the Golgi apparatus (GA) in hepatocytes and other cell types. Whether they are two separate enzymes or a single one has not been determined. Recently cerium has been reported as an excellent substitute for lead. The study described here compared the activities of TPPase and NDPase in pinealocytes by the cerium-based method.Guinea pig pineal glands were fixed by perfusion with a mixture of 1% glutaraldehyde and 2% paraformaldehyde followed by immersion in the same fixative for a total of 30 min. Sections cut at 100μm on a vibratome were incubated in substrate-free incubation medium for 30 min, then for 3 hrs at room temperature in the complete medium, which contained thiamine pyrophosphate (1 mg/ml) for TPPase and inosine diphosphate (1 mg/ml) for NDPase, cerium chloride (0.7 mg/ml), manganese chloride (1 mg/ml), and 5% sucrose in buffer.
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