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The immunohistochemical detection of bromodeoxyuridine (BrdU) was used to study the time of origin of the cells in the pineal gland of the rat. A study was made involving 17 groups of 4 rats each, administered with a single dose of bromodeoxyuridine (BrdU, 25 mg/kg) in 7 phases of the embryonic period (E15 to E21) and in 10 postnatal phases (between P0 and P30), followed by determination in each rat of the number of visible immune-labeled cells in the pineal gland 60 days after birth. The results show that approximately 60% of the pineal cells underwent the last division(s) prior to differentiation in the prenatal period between E18 and E21. The rest of the pineal cells originated after birth, particularly in the first 5 postnatal days.

The immunohistochemical detection of bromodeoxyuridine (BrdU) was used to study the cell proliferation in the developing rat pineal gland, from the appearance of pineal primordium in the embryonic day 15 (E15) until 30 days after birth. The results showed three different proliferative phases. From E15 to E21, the pineal gland shows a phase of rapid proliferation. The second phase corresponds to the first postnatal week, in which the number of labeled cells per surface unit decreases suddenly to values between 20% to 10% of those of embryonic period. From the second postnatal week onwards, the number of BrdU-positive cells progressively decreases.

The cellular proliferative activity in the adult rat pineal gland was studied using bromodeoxyuridine immunohistochemistry during the light and dark phases of an artificial 12L:12D photoperiod. The results showed statistically significant differences in the number of labeled cells between the light and dark phases, with the labeled cells being almost threefold more abundant in the light period. Minor changes were also found in the pineal gland volume between both periods. The decrease in the number of labeled cells in the dark phase of the cycle could be related to the well-documented antimitotic action of melatonin.

By means of teledischarge techniques from the database MEDLINE we selected those documents that contained in their title one or several of the following descriptors: pineal*, epiphys*, or melatonin*, in addition to the descriptor pineal-body in the MESH (Medical Subject Headings) section. A total of 7,617 original documents published between 1966 and 1994 were extracted that dealt with any aspect related with the pineal gland or its main secretary product, melatonin. The main bibliometric laws were applied: Price's Law on the increase in scientific literature, Bradford's Law on the dispersion of the scientific literature, and Lotka's Law on the author's productivity. Furthermore, we have analyzed the participation index (PaI) of the main countries within the global production, the productivity index of the authors (PI), and the number of authors/paper index. Our results demonstrate an exponential increase of the scientific literature on the pineal gland ("r" value = 0.983, in contrast with a "r" value = 0.966 after the linear adjustment). The number of publications on melatonin was less than those on other aspects of pineal research until 1991, when this situation was reversed. The journal with the largest number of original papers is Journal of Pineal Research (1st Bradford's zone) with 533 articles, followed by Journal of Neural Transmission (258) and Neuroendocrinology (221), which constituted the 2nd Bradford's zone. The total number of authors is 9,140, responsible for 23,524 authorships. 3.8% of the authors present a PI > or = 1 (large producers), and 64.9% a PI = 0 (occasional authors). Lotka's Law was widely fulfilled in this material since 10.3% of the authors are responsible of 50.2% of all the papers. The average number of authors per paper has changed from 2.29 in 1966 to 3.85 in 1994. The most productive country (during the interval between 1988-1994) was USA (PaI = 30.6), followed by Japan (7.15), United Kingdom (6.45), Germany (6.37), France (6.26), Italy (6.15) and Spain (5.34). Of the total number articles published, 86.9% are in English.

The ultrastructure of the pineal gland in the adult cat is described and compared with that of other mammals. Connective tissue spaces showed capillaries with nonfenestrated endothelia and numerous unmyelinated nerve fibers. In the proximal region of the gland, myelinated nerve fibers coming from the anterior commissure were also found. Cat pinealocytes showed a nucleus with prominent nucleoli, a well developed Golgi apparatus, centrioles, granular endoplasmic reticulum, ribosomes, abundant microtubuli and enlarged mitochondria. Pinealocytes showed several long processes with bulbous endings filled with clear vesicles and scarce "synaptic" ribbons. Pineal astrocytes and their processes were characterized by the presence of abundant filaments.

The immunocytochemical localization of basic fibroblast growth factor (b-FGF) during the postnatal development of the rat pineal gland was studied using a polyclonal antibody against the fraction 1-24 of bovine recombinant b-FGF. Basic FGF immunoreactivity was evident from day 20 after birth in the endothelial cells and perivascular spaces of the gland. Although b-FGF immunostaining showed its maximal expression at 30-45 days, it was maintained throughout the entire study period (up to 6 months), mainly in the distal zone of the gland. Pinealocytes did not show b-FGF immunoreactivity at any time. There were no differences in the localization patterns or the intensity of b-FGF immunostaining after the prenatal denervation with DSP-4, a neurotoxic amine. The physiological role of b-FGF in the adult pineal gland remains unknown; however, it does not seem to play a major role during the cytodifferentiation period of the parenchymal cells, or during the neovascularization in the early postnatal days. Furthermore, its immunocytochemical expression is not affected by the prenatal sympathetic denervation with DSP-4, in contrast with other neurotropic factors.

The expression of glial antigens vimentin (VIM) and glial fibrillary acidic protein (GFAP) is described in the pineal gland of cats and dogs from the first postnatal days to adulthood. VIM immunopositive cells were observed from the first postnatal days in both species. GFAP expression starts from the second postnatal week. In adults, a notable population of stellate cells immunopositive for GFAP and VIM was found dispersed throughout the gland. According to their immunocytochemical profile, these cells could be identified as astrocytes.

The developmental expression of the glial antigens, vimentin (VIM), glial fibrillary acidic protein (GFAP), and S-100 protein is described in the rat pineal gland from the first postnatal day to adulthood. Thick VIM immunopositive cell cords forming a network throughout the pineal gland were observed from the first postnatal days. These cords progressively disappeared during the first postnatal month as their cells dispersed into the pineal parenchyma. From 20 to 25 postnatal days, pineal glial cells appeared as isolated star-shaped VIM immunopositive cells. Immunostaining for GFAP and S-100 protein showed a similar developmental expression pattern. Both antigens appeared later than VIM (15-20 postnatal days) and were restricted to the pineal glial cells located in the proximal third of the gland, close to the pineal stalk.

In the present work, the presence and distribution of astrocytes in the rat pineal stalk is investigated applying an immunohistochemical technique for the demonstration of glial fibrillary acidic protein (GFAP) on Epon-embedded semithin sections (0.5 micron thick). GFAP-immunoreactive cells are evenly and regularly distributed along the entire pineal stalk. The GFAP-immunoreactive cells display a stellate shape showing variable numbers of cell processes that are mainly oriented parallel to the longitudinal stalk axis. Astrocytic processes show a clear tendency to encircle the remaining elements of the pineal stalk; i.e., pinealocytes, nerve fibres and blood vessels. Furthermore, glial processes form a cover layer separating the stalk from surrounding anatomical structures.

Sympathetic nerve fibers arising from the superior cervical ganglia are the main innervation of the rat pineal gland. Since most organs innervated by these ganglia contain nerve growth factor (NGF), the hypothetical existence of NGF in the pineal gland was investigated. The peroxidase anti-peroxidase technique was applied for the immunohistochemical demonstration of NGF using a polyclonal antiserum on Bouin-fixed, paraffin-embedded pineal glands from adult, young and 6-hydroxydopamine (6-OHDA)-treated rats. Few immunopositive cells were observed in the adult pineal gland. A more conspicuous population of immunoreactive cells was noted in young animals (20-45 days old), especially in those chemically denervated with 6-OHDA. NGF immunoreactive cells displayed a stellate shape resembling the interstitial or glial cells previously described in the rat pineal gland. Since NGF plays a trophic effect on sympathetic neurons during development and adulthood, we postulate that its presence in the pineal gland may exert a trophic role on its sympathetic innervation.

In the present work, coexpression of vimentin (VIM) and glial fibrillary acidic protein (GFAP) is demonstrated in the glial cells of the adult rat pineal gland. Serial consecutive Epon semithin sections (0.5 microns thick) were alternately immunostained for VIM and GFAP. GFAP positive cells and processes were found in the proximal region of the pineal gland, near the pineal stalk. Most of these cells were also immunostained for VIM in adjacent semithin sections. The significance of the coexpression VIM-GFAP and the restricted location of GFAP positive cells is discussed in relation with the maturation of pineal glial cells.

Light- and electron-microscopic features of pigmented cells in the cat pineal gland are described. These cells were observed throughout postnatal life from the second postnatal day to the oldest cats studied (up to 13 years old). No apparent relationship was observed among the amount of pigment and the animal age or sex. Pigmented cells showed a preferential localization at the ventral surface of the pineal gland near its distal end. The pineal pigment was histochemically identified as melanin. Pineal pigment granules showed ultrastructural features similar to melanocyte melanin granules.

Male and female parenchymal pineal cell types have been studied throughout postpubertal development to determine the existence of sex-related differences on a time basis. Six age groups (2, 3, 4, 8, 15 and 24 months) of eight rats (4 males and 4 females) were used in this study. Nuclei of both parenchymal pineal cell types were counted in 5 areas of 26.377 microns 2 per pineal gland on semithin sections. Nonparametric statistics of our results (Mann-Whitney U-test and Kruskal-Wallis H-test) demonstrated significant differences between male and female pinealocytes through the stages studied. In all age groups, the number of nuclei per unit area was larger in female rats. Pineoglial cells did not show significant sex-related differences.

Ultrastructural changes in the rat pineal gland were studied quantitatively 7 and 60 days after the sympathetic denervation by bilateral excission or decentralization of superior cervical ganglia. The surface occupied by pineal parenchymal cells decreased in rats of experimental groups with respect to the control group. Furthermore, profile areas of the cytoplasm, nucleus and nucleolus of the pinealocytes were also diminished. Cytoplasmic lipid droplets in the pinealocytes were markedly decreased in number and size in experimental rats. As demonstrated by the Kruskal-Wallis H test, statistically significant differences were found between rats of the control and operated groups. Rats treated by superior cervical ganglionectomy or decentralization showed morphological changes indicating a hypofunctional pineal gland, although differences were found between both groups.