Embryonic development of the bovine pineal gland (Bos taurus) during prenatal life (30 to 135 days of gestation)

The ontogenesis of the pineal gland of 30 bovine embryos (Bos taurus) has been analysed from 30 until 135 days of gestation by means of optical microscopy and immunohistochemical techniques. For this study, the specimens were grouped into three stages in accordance with the most relevant histological characteristics: Stage 1 (30 to 64 days of prenatal development); Stage 2 (70 to 90 days) and Stage 3 (106 to 135 days). In the cow, it is from 30 days of gestation that the first glandular outline becomes differentiated from the diencephalic ependyma of the third ventricle. This differentiation includes the phenomena of proliferation and multiplication of the ependymal cells that form the epithelium of the pineal outline in development. At 82 days of intrauterine life, in the interior of the pineal parenchyma, we witnessed some incipient pseudoglandular structures that at 135 days were well differentiated. The pineal parenchyma displays a cytology made up of two cellular types of structurally distinct characteristics: pinealoblasts and interstitial cells. Both cellular types begin differentiation at 70 days of embryonic development, the pinealoblasts being greater in number than the interstitial cells. The glandular stroma is formed from the capsular, trabecular and the perivascular connective tissue, filling the interparenchymal space. A dense network of capillaries, which drive across the trabecular connective tissue towards the central glandular zone where their density increases and their calibre is reduced, complete the glandular structure. GFAP positive cells were observed in the embryonic pineal parenchyma in stage 3. At 135 days of gestation, NPY positive fibers entered the pineal gland through the pineal capsule occupying a perivascular localization. Morphological studies of this nature are vital for future use as parameters, indicative of the functional activity of the bovine pineal gland during embryonic development.     

Innervation of the cat pineal gland by neuropeptide Y-immunoreactive nerve fibers: an experimental immunohistochemical study

An immunohistochemical study of the cat pineal gland was performed using a rabbit polyclonal antibody directed against neuropeptide Y (NPY) and an antibody directed against the C-terminal flanking peptide of neuropeptide Y (CPON). Numerous NPY- and CPON-immunoreactive (IR) nerve fibers were demonstrated throughout the gland and in the pineal capsule. The number of IR nerve fibers in the capsule was high and from this location fibers were observed to penetrate into the gland proper via the pineal connective tissue septa, often following the blood vessels. From the connective tissue septa IR fibers intruded into the parenchyma between the pinealocytes. Many IR nerve fibers were observed in the pineal stalk and in the habenular as well as the posterior commissural areas. The number of NPY/CPON-IR nerve fibers in pineal glands from animals bilaterally ganglionectomized two weeks before sacrifice was low. The source of most of the extrasympathetic NPY/CPONergic nerve fibers is probably the brain from where they enter the pineal via the pineal stalk. However, an origin of some of the fibers from parasympathetic ganglia cannot be excluded due to the presence of a few IR fibers in the pineal capsule of ganglionectomized animals. It is concluded that the cat pineal is richly innervated with NPYergic nerve fibers mostly of sympathetic origin. The posttranslational processing of the NPY promolecule results in the presence of both NPY and CPON in intrapineal nerve fibers.     

Pituitary adenylate cyclase-activating polypeptide-immunoreactive (PACAP-IR) nerve fibers in the pig pineal gland

The present study demonstrates the occurrence of PACAP-immunoreactive (PACAP-IR) nerve fibers in different compartments of the pig pineal gland, including glandular capsule (where they form a very dense network) and subependymal tissue close to the pineal recess (moderate to dense meshwork of varicose fibers). Furthermore, several varicose fibers penetrate from the capsule into the connective tissue septa and then into the parenchyma, where they form unequally distributed, fine network and, in some cases, basket-like structures around pinealocytes. Some of the PACAP-IR nerve fibers, observed both in the habenular and posterior epithalamic areas, extend to the pineal gland. PACAP-IR cells could be demonstrated neither in the pineal gland, nor in epithalamic areas.     

Demonstration of nerve fibers immunoreactive to peptide N-terminal histidine C-terminal isoleucine (PHI) and vasoactive intestinal peptide (VIP) in the pig pineal gland

The pineal functions are modulated by some neuropeptides including PHI and VIP. The presence of PHI-immunoreactive and VIP-immunoreactive nerve fibers in the pineal gland has been shown in several mammalian species. Both peptides influence the pineal serotonin N-acetyltransferase activity and melatonin synthesis. The aim of the present study was to examine the localization of PHI- and VIP-immunoreactive nerve fibers in the pig pineal gland. Four three-month old female pigs housed in natural light conditions, with free access to food and water, were used in the study. The pineals were fixed by perfusion with 4% paraformaldehyde in 0.1 M phosphate buffer. An immunohistochemical ABC streptavidin-biotin-complex method was used for the demonstration of PHI and VIP. PHI- and VIP-immunopositive nerve fibers were found in the pineal gland as well as in the habenular and posterior commissural areas. In the pineal gland, the density of PHI-immunoreactive nerve fibers was considerably higher than that of the fibers containing VIP. PHI- and VIP-immunopositive nerve fibers were more abundant in the cortical than in the medullary part of the gland. The nerve fibers formed bundles in the pineal capsule, from where they penetrated to the connective tissue septa and formed a dense meshwork surrounding blood vessels. In the parenchyma, PHI- and VIP-immunoreactive nerve terminals created baskets around clusters of pinealocytes. No PHI- or VIP-immunopositive cells were found in the pig pineal gland.     

Tyrosine hydroxylase- and neuropeptide Y-immunoreactive nerve fibers in the pineal complex of untreated rats and rats following removal of the superior cervical ganglia

Summary The distribution of tyrosine hydroxylase (TH)- and neuropeptide Y (NPY)-immunoreactive(IR) nerve fibers in the pineal complex was investigated in untreated rats and rats following bilateral removal of the superior cervical ganglia. In normal animals, a large number of TH- and NPY-IR nerve fibers were present in the pineal capsule, the perivascular spaces, and intraparenchymally between the pinealocytes throughout the superficial pineal and deep pineal gland. A small number of TH-IR and NPY-IR nerve fibers were found in the posterior and habenular commissures, a few fibers penetrating from the commissures into the deep pineal gland. To elucidate the origin of these fibers, the superior cervical ganglion was removed bilaterally in 10 animals, and the pineal complex was examined immunohistochemically. Two weeks after the ganglionectomy, the TH-IR and NPY-IR nerve fibers in the superficial pineal gland had almost completely disappeared. On the other hand, in the deep pineal and the pineal stalk, the TH-IR and NPY-IR fibers were still present after ganglionectomy. These data show that the deep pineal gland and the pineal stalk possess an extrasympathetic innervation by TH-IR and NPY-IR fibers. It is suggested that the extrasympathetic TH-IR and NPY-IR nerve fibers innervating the deep pineal and the pineal stalk originate from the brain.     

Tyrosine hydroxylase and neuropeptide-Y immunoreactivity in pineal glands developing in situ and in pineal grafts

Summary Postnatal development of the innervation of the pineal gland in situ as well as the reinnervation of pineal grafts by tyrosine hydroxylase (TH)- and neuropeptide Y (NPY)-immunoreactive nerve fibers were examined using the avidin-biotin-peroxidase immunohistochemical technique. TH-immunoreactive nerve fibers appeared in the pineal gland on the second postnatal day (P2) in both hamsters and gerbils. NPY-immunoreactive nerve fibers first appeared in the pineal gland of gerbils on P2 and in the hamsters on P3. By the seventh postnatal day (P7), the pineal glands of both hamsters and gerbils were richly innervated by TH- and NPY-fibers that appeared as smooth fibers or fibers with sporadic varicosities. By the age of 4 weeks, the innervation of the pineal glands of hamsters and gerbils by TH-and NPY-fibers was fully developed. Abundant TH- and NPY-fibers formed a dense meshwork in the parenchyma of the superficial and deep pineals. The great majority of the fibers bore a large number of varicosities. More NPY-fibers were found in the pineal glands of gerbils than hamsters. NPY fibers were distributed evenly throughout the pineal glands of the gerbil, but they were more often located in the central region of the superficial pineal of the hamster. For the pineal grafts, superficial pineals from neonatal and 4-week-old hamsters were transplanted to different sites in the third cerebral ventricle (infundibular recess, posterior third ventricle) or beneath the renal capsule. The pineal grafts from 4-week-old donors appeared to undergo severe degeneration and eventually disappeared. The pineal grafts from neonatal hamsters, however, successfully survived and became well integrated into their new locations. Abundant TH-and NPY-fibers in the host brain were found surrounding the pineal grafts placed in the third cerebral ventricle, but were only rarely seen entering the parenchyma of the grafts. A few TH-fibers were demonstrated in the renal grafts 4 weeks after transplantation. These studies describe the postnatal development of the innervation of the pineal glands in situ by TH-and NPY-nerve fibers, and demonstrate a lack of reinnervation of cerebroventricular pineal grafts by TH and NPY fibers from adjacent host brain.Portions of the results of this paper were previously reported in abstract form at the 1990 Meeting of The American Association of Anatomists (Anat Rec 226:57A)     

Innervation of the mink pineal gland with neuropeptide Y (NPY)-containing nerve fibers

Summary An immunohistochemical investigation of the mink pineal gland was performed by use of antibodies raised in rabbits against neuropeptide Y (NPY) and Cys-NPY (32–36)-amide recognizing neuropeptide Y with an amidation at position 36 (NPYamide). NPY-immunoreactive nerve fibers were located predominantly in the rostral part of the pineal gland and in the pineal stalk. Immunoreactive nerve fibers were found throughout the pineal gland, but the number of fibers in the caudal part of the gland was low. The fibers were present both in the perivascular spaces and between the pinealocytes. Many NPY-immunoreactive fibers were also located in the posterior and habenular commissures; some of these fibers were connected with the fibers in the rostral part of the mink pineal gland, indicating that at least some of the NPY-immunoreactive nerve fibers are of central origin. The nerve fibers immunoreactive to amidated NPY were distributed in a similar manner. However, the number of fibers immunoreactive to NPYamide was lower than the number of fibers immunoreactive to NPY itself. After removal of the superior cervical ganglia bilaterally 22 days or 12 months before sacrifice, NPY-immunoreactive nerve fibers remained in the gland. This immunohistochemical study of the mink pineal gland therefore shows that the NPY/NPYamide-immunoreactive nerve fibers innervating the pineal gland in this spegcies are a component of the central innervation or originnate from extracerebral parasympathetic ganglia.     

Specific distribution pattern of nerve fibers containing catecholamine-synthesizing enzymes, neuropeptide Y (NPY) and C-terminal flanking peptide of NPY (CPON) in the pineal gland of the chinchilla (Chinchilla laniger)--an immunohistochemical study

The sympathetic nerve fibers originating from the superior cervical ganglia and supplying the pineal gland play the most important role in the control of the pineal activity in mammals. NPY and CPON are also present in the majority of the pinealopetal sympathetic neurons. In this study, immunohistochemical techniques were used to demonstrate the existence and coexistence of tyrosine hydroxylase (TH), dopamine beta-hydroxylase (DbetaH) as well as NPY and CPON in the nerve fibers supplying the chinchilla pineal gland. Ten two-year-old female chinchillas housed in natural light conditions were used in the study. The pineals were fixed by perfusion. ABC immunohistochemical technique and immunofluorescence labelling method were employed. TH-immunoreactive (TH-IR) varicose nerve fibers were observed in the pineal gland as well as in the posterior commissural area. Within the chinchilla pineal gland, TH-IR nerve fibers were located in the capsule and connective tissue septa. Numerous varicose TH-IR branches penetrated into the parenchyma and formed a network showing the highest density in the proximal region of the gland. In the central and distal parts of the pineal parenchyma, a subtle network, composed of thin varicose nerve branches, was observed. Double immunostaining revealed that the majority of TH-IR nerve fibers was positive for DbetaH or NPY. TH- and DbetaH-positive neuron-like cells were observed in the proximal region of the gland. The pattern of pineal innervation immunoreactive to CPON was similar to the innervation containing NPY, TH and DbetaH. The chinchilla intrapineal innervation containing TH, DbetaH, NPY and CPON is characterized by the higher density in the proximal part of the gland than in the middle and distal ones. The specific feature of the chinchilla pineal is also the presence of single TH/DbetaH-immunoreactive neuron-like cells in the proximal part of the gland.     

Localization of NADPH-diaphorase activity in the pineal gland of the domestic pig

The study showed the presence of NADPH-diaphorase containing structures in the pineal gland of the domestic pig. NADPH-diaphorase activity was found in the nerve fibers and in the endothelial cells of the vasculature. The nerve fibers were localized in the capsule, around the blood vessels as well as in the parenchyma. The positive staining was not observed in the pig pinealocytes.     

Characteristic pattern of monoaminergic nerve fibers in the pineal organ of the monkey,Macaca fuscata

Summary Monoaminergic nerve fibers were studied in the pineal organ of the monkey, Macaca fuscata, by use of fluorescence and immunohistochemical procedures. Abundant formations of noradrenergic nerve fibers were observed in the pineal organ. They entered the parenchyma in the form of several coarse bundles via the capsule in the distal portion of the organ and spread throughout the organ after branching into smaller units. The density of the autonomic innervation decreased gradually toward the proximal portion of the organ. In the distal portion, numerous nerve fibers formed perivascular plexuses around the blood vessels and some fibers ran as bundles unrelated to the blood vessels in the stroma. Fine varicose fibers and bundles derived from these plexuses penetrated among the pinealocytes. However, only a few intraparenchymal fluorescent fibers were detected in the proximal third of the gland. With the use of serotonin antiserum serotonin-immunoreactive nerve fibers were clearly restricted to the ventroproximal part of the pineal organ. Although the somata of the pinealocytes showed intense immunoreactivity, their processes were not stained. In one exceptional case, clusters of pinealocytes displaying very intense immunoreactivity were found in an area extending from the distal margin of the ventral portion of the pineal stalk to the proximal portion of the pineal organ proper; these cells were bipolar or multipolar and endowed with well-stained processes.     

Central innervation of the pineal organ of the Mongolian gerbil

Nerve fibers connecting the brain with the pineal gland of the Mongolian gerbil (central pinealopetal fibers) were investigated by means of light and electron microscopy. Several myelinated fibers penetrate from the brain into the deep pineal gland, extend further into the pineal stalk and continue to the superficial portion of the pineal gland. In the centripetal direction these fibers were traced to the stria medullaris and to the habenular nuclei, where they turned laterad and then occupied a position immediately ventral to the optic tract. As shown in electron micrographs, lesions of the habenular area led to degeneration of myelinated fibers and nerve boutons in the deep pineal gland, the pineal stalk and the superficial pineal gland. Only boutons containing clear transmitter vesicles (devoid of a dense core) were observed to degenerate after the habenular lesions. On the other hand, removal of the superior cervical ganglia resulted in degeneration of boutons containing small (40 to 60 nm in diameter) dense-core vesicles. Several of the nerve fibers that penetrate into the deep pineal directly from the brain (central fibers) exhibited a positive reaction for acetylcholinesterase (AChE). AChE-positive perikarya were located in the projections of the stria medullaris, the lateral portions of the deep pineal, the area of the posterior commissure, and the periventricular gray of the mesencephalon. Such perikarya were found neither in the pineal stalk nor in the superficial pineal gland. These results present anatomical evidence that the pineal organ of the Mongolian gerbil receives multiple nervous inputs mediated by peripheral autonomic (i.e., sympathetic) nerve fibers, on the one hand, and by central fibers, on the other.     

Immunohistochemical localization of substance P in the pineal gland of the domestic pig

An immunohistochemical study of the pig pineal gland was carried out using polyclonal rabbit antiserum raised against substance P (SP). The pineal glands were taken from the newborn, 21-day- and 7-month-old female pigs. Immunoreactive nerve fibers were observed in the pineal gland as well as in the posterior commissure and habenular areas. The bundles of SP-immunoreactive fibers were also seen in the subependymal layer of the pineal tissue. The single SP-immunoreactive nerve fibers and few small bundles of nerve fibers were located with equal density throughout the pineal gland, in the connective tissue septa and in the parenchyma. SP-immunoreactive cell bodies were observed in the medial habenular nucleus. The obtained results point to this nucleus as one of the central sources of SP innervation in the pig pineal gland. The study did not show any differences in the distribution and the density of SP-immunoreactive nerve fibers between newborn, 21-day- and 7 month-old pigs.     

Development of the circadian melatonin rhythm and the effect of PACAP on melatonin release in the embryonic chicken pineal gland. An in vitro study

Pituitary adenylate cyclase activating polypeptide (PACAP) has been shown to participate in modulation of circadian rhythm and to stimulate melatonin (MT) secretion in both the rat and chicken pineal glands. In contrast to mammals, the main regulator of circadian rhythm in birds is the pineal gland, which begins its rhythmic MT production already during embryonic life. In the present study, we investigated the development of MT secretion in explanted embryonic chicken pineals and their responsiveness to PACAP in a perifusion system. Our results show that: (1) the circadian clock and/or the intracellular signal transduction system connecting the clock to MT synthesizing apparatus develop between the embryonic days 16-18 (E16-18), even in vitro. (2) Exposure of the embryonic chicken pineal gland to PACAP induces transitory increase in MT secretion but does not induce visible phase shift in the circadian rhythm. (3) Cyclic AMP (cAMP) efflux also responds to PACAP at or before day E13 in embryonic chicken pineal gland in vitro.     

Cell proliferation in the developing rat pineal gland. A bromodeoxyuridine immunohistochemical study

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 anatomy and innervation of the mammalian pineal gland

The parenchymal cells of the mammalian pineal gland are the hormone-producing pinealocytes and the interstitial cells. In addition, perivascular phagocytes are present. The phagocytes share antigenic properties with microglial and antigen-presenting cells. In certain species, the pineal gland also contains neurons and/or neuron-like peptidergic cells. The peptidergic cells might influence the pinealocyte by a paracrine secretion of the peptide. Nerve fibers innervating the mammalian pineal gland originate from perikarya located in the sympathetic superior cervical ganglion and the parasympathetic sphenopalatine and otic ganglia. The sympathetic nerve fibers contain norepinephrine and neuropeptide Y as neurotransmitters. The parasympathetic nerve fibers contain vasoactive intestinal peptide and peptide histidine isoleucine. Recently, neurons in the trigeminal ganglion, containing substance P, calcitonin gene-related peptide, and pituitary adenylate cyclase-activating peptide, have been shown to project to the mammalian pineal gland. Finally, nerve fibers originating from perikarya located in the brain containing, for example, GABA, orexin, serotonin, histamine, oxytocin, and vasopressin innervate the pineal gland directly via the pineal stalk. Biochemical studies have demonstrated numerous receptors on the pinealocyte cell membrane, which are able to bind the neurotransmitters located in the pinealopetal nerve fibers. These findings indicate that the mammalian pinealocyte can be influenced by a plethora of neurotransmitters.     

Innervation of the rat pineal gland by PACAP-immunoreactive nerve fibers originating in the trigeminal ganglion: a degeneration study

In order to establish that the pineal gland is innervated by pituitary adenylate cyclase-activating polypeptide (PACAP)-immunoreactive nerve fibers originating in the trigeminal ganglion, ophthalmic and maxillary nerves were transected by using a subtemporal fossa approach. The number of PACAP-immunoreactive nerve fibers in the pineal gland of rats with a total transection of the nerve was compared with that of rats without surgery. In the operated rat, PACAP-immunoreactive nerve fibers in the superficial pineal decreased remarkably, indicating that the trigeminal ganglion was the origin of these nerve fibers. This research provides evidence supporting the hypothesis that PACAP-immunoreactive nerves regulate the synthesis and/or secretion of melatonin in the pineal gland.     

Post-natal development of the pineal organ in the hamsters Phodopus sungorus and Mesocricetus auratus

Summary In Phodopus sungorus, as in other mammals, the pineal organ forms an important link in the transduction of photoperiodic information to the endocrine system. The sympathetic innervation, via the superior cervical ganglion, controls the metabolism of serotonin and melatonin in the pineal, which in turn is involved in the control of the gonads. In the present study, the post-natal development of this system was investigated. Specimens 1, 5, 10, and 15 days post partum (p.p.) and adults were treated with monoamine-oxidase-inhibitor and perfused under ether anesthesia via the aorta with a buffer containing glyoxylic acid, formaldehyde and Mg⁺⁺. The brains were then dissected out and treated according to Falck-Hillarp for fluorescence microscopy and microspectrofluorometry. Day 1: The nervi conarii had reached the pineal capsule, but only in a few cases was the pineal organ invaded and then only by a few fibers. Day 5: A rich green-fluorescing net of fibers was present in the entire organ, stalk and lamina intercalaris. No 5-HT fluorescence was observable. Day 10: Similar to the stage at 5 days a rich green-fluorescing nerve fiber net was observed throughout the pineal and a yellow fluorescence in the pineal perikarya. Day 15: The general appearance resembles the adult. The nerve fibers are masked by the intense yellow fluorescence of the pineal perikarya. Fading of the latter, however, allows the catecholamine fluorescence to be seen. Golden hamsters at an age of 15 days p.p. show a similar appearance to Phodopus at an age of 15 days. Microspectrofluorometric determinations indicated the catecholamine to be noradrenaline, and confirmed a 5-HT/5-HTP origin of the yellow fluorescence appearing between day 5 and day 10. The amount of 5-HT/5-HTP was considerably less at day 10 than at day 15 or in adults. Sympathectomy by extirpation of the superior cervical ganglion abolished the catecholamine fluorescence completely in the pineal body, stalk and lamina intercalaris.Supported by grants from the Swedish Natural Science Research Council (to P. Meurling and Th. van Veen), and the Royal Physiographic Society of Lund     

Development of rhythmic melatonin secretion from the pineal gland of embryonic mummichog (Fundulus heteroclitus)

The pineal gland of vertebrates produces and secretes the hormone melatonin in response to changes in the light-dark cycle, with high production at night and low production during the day. Melatonin is thought to play an important role in synchronizing daily and/or seasonal physiological, behavioral, and developmental rhythms in vertebrates. In this study, the functional development of the pineal melatonin-generating system was examined in the mummichog, Fundulus heteroclitus, an euryhaline teleost. In this species, the pineal gland contains an endogenous oscillator, ultimately responsible for timing the melatonin rhythm. Oocytes from gravid females were collected and fertilized in vitro from sperm collected from mature males. Skull caps containing attached pineal glands were obtained from F. heteroclitus embryos at different embryonic stages and placed in static or perfusion culture under various photoperiodic regimes. Rhythmic melatonin secretion from pineal glands of embryonic F. heteroclitus embryos exposed to a 12L:12D cycle in static culture was observed at five days post-fertilization. The ontogeny of circadian-controlled melatonin production from F. heteroclitus pineal glands exposed to constant darkness for five days was also seen at day five post-fertilization. These data show that early development of the pineal melatonin-generating system in this teleost occurs prior to hatching. Pre-hatching development of the melatonin-generating system may confer some selective advantage in this species in its interactions with the environment.     

Distribution of NADPH-diaphorase activity in the pineal gland of the Turkey

NADPH-diaphorase activity was histochemically demonstrated in the nerve fibers, neuronal-like cell bodies and in the endothelial cells of the vasculature in the pineal gland of the turkey. The nerve fibers were localized in the choroid plexus, connecting the pineal gland with the diencephalon as well as inside the pineal gland, where they formed basket-like structures around the pineal follicles. A group of neuronal-like cell bodies was observed in the proximal part of the gland. The positive staining was not observed in the pinealocytes of rudimentary-photoreceptor type and in the supporting cells.