Protein gene product 9.5 (PGP 9.5)

Summary The guinea pig uterus is supplied by different populations of nerves which can be demonstrated by specific immunocytochemical and histochemical techniques. So far, there has been no single marker displaying entire peripheral innervation patterns. Recently, protein gene product (PGP) 9.5, a cytoplasmic protein in neurons and neuroendocrine cells, was found to visualize both different populations and subtypes of nerves. This prompted the present study of using PGP 9.5 for visualization of the whole uterine innervation. This was performed by the indirect immunofluorescence method using antiserum to PGP 9.5 raised in rabbits.PGP-immunoreactivity was present in all neuronal parts of the extrinsic and intrinsic uterine innervation, including different subpopulations of nerves. This was verified by chemical sympathectomy and sensory denervation with 6-hydroxydopamine and capsaicin-treatment respectively, and double immunostaining.By term a disappearance of uterine PGP-nerve-immunoreactivity was observed which was almost complete in fetus-bearing uterine tissue and further strengthens previous assumptions of a general, pregnancy-induced uterine neuronal degeneration.The developmental time-course and morphology of PGP-immunoreactive nerve structures was similar to that for other neuronal markers and support the suggestion of PGP-immunoreactivity as a general marker for the entire uterine innervation, and suggests that the presence of PGP 9.5-immunoreactivity may coincide with functional maturation of uterine innervation.     

Protein gene product 9.5-immunoreactive nerve fibres and cells in human skin

Summary Sections of human skin were processed according to the indirect immunofluorescence technique with a rabbit antiserum against human protein gene product 9.5 (PGP 9.5). Immunoreactivity was detected in intraepidermal and dermal nerve fibres and cells. The intraepidermal nerves were varicose or smooth with different diameters, running as single processes or branched, straight or bent, projecting in various directions and terminating in the stratum basale, spinosum or granulosum. The density of the intraepidermal nerves varied between the different skin areas investigated. PGP 9.5-containing axons of the lower dermis were found in large bundles. They separated into smaller axon bundles within the upper dermis, entering this portion of the skin perpendicular to the surface. Then they branched into fibres mainly arranged parallel to the epidermal-dermal junctional zone. However, the fibres en route to the epidermis traversed the upper dermis more or less perpendicularly. Furthermore, immunoreactive dermal nerve fibres were found in the Meissner corpuscles, the arrector pili muscles, hair follicles, around the eccrine and apocrine sweat glands and around certain blood vessels. Such fibres were also observed around most subcutaneous blood vessels, sometimes heavily innervating these structures. Numerous weakly-to-strongly PGP 9.5-immunoreactive cells were found both in the epidermis and in the dermis.     

Cutaneous innervation in man visualized with protein gene product 9.5 (PGP 9.5) antibodies

Summary Using antibodies to the neuronal cytoplasmic protein, protein gene product 9.5 (PGP 9.5) the cutaneous innervation in man was investigated. The distribution of PGP 9.5 immunoreactive nerve fibers was compared with the distribution of nerve fibers immunoreactive to neuron specific enolase, neurofilament proteins, calcitonin gene related peptide, vasoactive intestinal polypeptide and neuropeptide Y. PGP 9.5 immunoreactive nerve fibers were found in the epidermis, dermis, in Meissner's corpuscles, innervating Merkel cells, around blood vessels, sweat glands and hair follicles. Merkel cells were also PGP 9.5 positive. The labelled nerve fibers included sensory and autonomic fibers, visualizing the whole innervation of the human skin. The number of positive fibers and the intensity of the fluorescence was greater with PGP 9.5 antibodies than with any of the other markers included. Thus, PGP 9.5 antibodies may serve as a tool for investigations of cutaneous innervation, reinnervation and nerve regeneration in different clinical conditions.     

Cutaneous innervation in man visualized with protein gene product 9.5 (PGP 9.5) antibodies

Using antibodies to the neuronal cytoplasmic protein, protein gene product 9.5 (PGP 9.5) the cutaneous innervation in man was investigated. The distribution of PGP 9.5 immunoreactive nerve fibers was compared with the distribution of nerve fibers immunoreactive to neuron specific enolase, neurofilament proteins, calcitonin gene related peptide, vasoactive intestinal polypeptide and neuropeptide Y. PGP 9.5 immunoreactive nerve fibers were found in the epidermis, dermis, in Meissner's corpuscles, innervating Merkel cells, around blood vessels, sweat glands and hair follicles. Merkel cells were also PGP 9.5 positive. The labelled nerve fibers included sensory and autonomic fibers, visualizing the whole innervation of the human skin. The number of positive fibers and the intensity of the fluorescence was greater with PGP 9.5 antibodies than with any of the other markers included. Thus, PGP 9.5 antibodies may serve as a tool for investigations of cutaneous innervation, reinnervation and nerve regeneration in different clinical conditions.     

Neuropeptide-containing nerve fibres in the human parotid gland: a semiquantitative analysis using an antibody against protein gene product 9.5

The occurrence and distribution of neuropeptide-containing fibres in the human parotid gland were examined by the peroxidase--antiperoxidase method with attention to the quality of fixation and the condition of patients. Many fibres immunoreactive for neuropeptide Y (NPY) and vasoactive intestinal polypeptide (VIP) and a moderate number of galanin- positive (GAL) fibres were distributed around the acini. A moderate number of NPY and VIP fibres were distributed around the intercalated ducts. The semiquantitative mean densities (_SD) of periacinar NPY, VIP and GAL fibres expressed as a percentage of the total protein gene product (PGP) 9.5 immunoreactive fibres were 75.62 _ 7.25%, 70.52 _ 9.33% and 41.76 _ 5.45%, respectively, whereas those of substance P (SP), calcitonin gene-related peptide (CGRP) and FMRF amide (FMRF) fibres were below 10%. The mean densities of NPY and VIP fibres around the intercalated ducts expressed as the percentage of PGP 9.5 fibres associated with these ducts were 52.37 _ 6.19% and 59.62 _ 7.02% respectively. Those of SP, CGRP, GAL, and FMRF fibres were below 10%. The densities of NPY, VIP, SP, CGRP, GAL and FMRF fibres around the striated and excretory ducts were also below 10%. In the vasculature, NPY fibres were the most prominent. Similarly, the mean density of perivascular NPY fibres was 93.76 _ 2.03%. No somatostatin or leucine or methionine enkephalin immunoreactivity was detected around the acini, duct system or blood vessels. These findings suggest that, in this gland, the periacinar NPY, VIP and GAL fibres may participate in regulating the synthesis of saliva and its secretion and that perivascular peptidergic fibres, especially NPY fibres, may be involved in controlling local blood flow This revised version was published online in November 2006 with corrections to the Cover Date.     

Neuropeptide Y-like immunoreactivity in perivascular nerve fibres of the guinea-pig

The distribution of perivascular nerve fibres displaying neuropeptide Y-like immunoreactivity was studied in the guinea-pig. Generally, neuropeptide Y fibres were numerous around arteries and moderate in number around veins. In the heart, immunoreactive fibres were numerous in the auricles and the atria (epi- and endocardium) whereas the ventricles had a more scarce supply. The coronary vessels were richly supplied with fibres. Around large elastic and muscular arteries the fibres formed well developed plexuses. Small arteries in the respiratory tract, the gastrointestinal tract and the genito-urinary tract received a particularly rich supply. In the liver, spleen and kidney only few perivascular fibres were seen. Since immunoreactive fibres around blood vessels disappeared upon surgical or chemical sympathectomy, and sequential immunostaining with antisera against dopamine-beta-hydroxylase (a marker for adrenergic neurons) and against neuropeptide Y revealed their co-existence, it is concluded that neuropeptide Y fibres around blood vessels are sympathetic and adrenergic.     

The structure of the human gene encoding protein gene product 9.5 (PGP9.5), a neuron-specific ubiquitin C-terminal hydrolase.

Database search using a bovine thymus ubiquitin C-terminal hydrolase sequence indicated 54% sequence identity with the abundant human neuron-specific protein gene product 9.5 (PGP9.5), which was then shown to possess the same activity [Wilkinson, Lee, Deshpande, Duerksen-Hughes, Boss & Pohl (1989) Science 246, 670-673]. A yeast counterpart of the enzyme is also known. The human PGP9.5 gene, described here, spans 10 kb, contains nine exons and displays 5' features some common to many genes and some common with neurofilament neuron-specific enolase and Thy-1-antigen gene 5' regions.     

Neuropeptide Y (NPY)-like immunoreactivity in peripheral and central nerve fibres of the golden hamster (Mesocricetus auratus) with special respect to pineal gland innervation

Information on the ambient lighting conditions is conveyed from the retina to the pineal organ by a neuronal pathway involving the suprachiasmatic nucleus (SCN) which acts as a circadian pacemaker. In the hamster, circadian rhythms have been shown to be influenced by injection of neuropeptide Y (NPY) into the SCN. Since NPY-immunoreactive nerve fibres are present in the rat and guinea-pig pineal glands it appeared of interest to investigate the hamster pineal as part of the circadian rhythm generating/regulating system. For comparison kidney, small intestine and cerebral cortex were studied. Like in the other rodent species so far investigated only a few of the abundant sympathetic nerve fibres in the hamster pineal gland are NPY-immunoreactive, in contrast to the relatively rich innervation of the other organs. This speaks in favour of a possible central origin of pineal NPY-immunoreactive fibres. These may either exert vasoregulatory effects on pineal vasculature or be involved in the modulation of alpha-adrenergic receptor mediated regulation of pineal metabolism.     

Neuropeptide Y (NPY)-like immunoreactivity in peripheral and central nerve fibres of the golden hamster (Mesocricetus auratus) with special respect to pineal gland innervation

Summary Inforimation on the ambient lighting conditions is conveyed from the retina to the pineal organ by a neuronal pathway involving the suprachiasmatic nucleus (SCN) which acts as a circadian pacemaker. In the hamster, circadian rhythms have been shown to be influenced by injection of neuropeptide Y (NPY) into the SCN. Since NPY-immunoreactive nerve fibres are present in the rat and guinea-pig pineal glands it appeared of interest to investigate the hamster pineal as part of the circadian rhythm generating/regulating system. For comparison kidney, small intestine and cerebral cortex were studied. Like in the other rodent species so far investigated only a few of the abundant sympathetic nerve fibres in the hamster pineal gland are NPY-immunoreactive, in contrast to the relatively rich innervation of the other organs. This speaks in favour of a possible central origin of pineal NPY-immunoreactive fibres. These may either exert vasoregulatory effects on pineal vasculature or be involved in the modulation of alpha-adrenergic receptor mediated regulation of pineal metabolism.Supported by the Deutsche Forschungsgemeinschaft, grant Schr 283/1-1     

Distribution of growth associated protein (GAP-43) immunoreactivity in nerve fibers supplying the pig pineal gland

An immunohistochemical study of the pig pineal gland was carried out using monoclonal mouse antiserum against growth-associated protein GAP-43. The pineal glands were obtained from the 3, 5, 8 weeks old piglets. The immunopositive nerve fibers were observed in the pineal gland as well as in the habenular and the posterior comissural areas. They formed a dense network in the habenular area and the proximal part of the pineal gland. In the comissural area and in the apical part of the gland. single positive fibers were observed. The obtained results may suggest a difference in the plasticity of innervation between the particular regions of the pineal gland.     

Protein gene product 9.5 and ubiquitin immunoreactivities in rat epididymis epithelium

A quantitative immunohistochemical study was performed of the distribution of protein gene product 9.5 (PGP, a soluble protein localized in neurons and neuroendocrine cells as well as in some non-nervous cells) and ubiquitin along the rat epididymis. In the ductuli efferentes, PGP immunoreaction was observed in the whole cytoplasm of some columnar cells; a smaller number of columnar cells showed ubiquitin immunoreactivity with limited apical and basal cytoplasmic localization. In the proximal caput epididymidis, the whole cytoplasm of all columnar cells showed PGP immunoreactivity, ubiquitin immunostaining was negative in this region. In the middle and distal caput epididymidis and the distal cauda, the apical cytoplasm of some columnar cells and the whole cytoplasm of some basal cells showed immunoreactivity to PGP. In these regions, immunoreactivity to ubiquitin was positive in the supranuclear cytoplasm of some columnar cells but not in the basal cells. No immunoreactivity to PGP or ubiquitin was detected in the corpus epididymis and the proximal cauda. Double immunostaining revealed that all the epididymal ubiquitin immunoreactive cells were also PGP immunoreactive, whereas most PGP immunoreactive cells did not immunoreact to ubiquitin. In ubiquitin-PGP immunoreactive cells, the site of the PGP immunoreaction differed from that of the ubiquitin immunoreaction. PGP-ubiquitin immunoreactive cells also seemed to be immunoreactive to anti-AE1/AE3 keratin antibodies. The spermatozoal heads were immunoreactive to PGP antibodies in the epididymal regions from proximal caput to distal cauda but not in the ductuli efferentes. The findings suggest that non-ubiquitinated PGP immunoreactive proteins are secreted in the epididymis, mainly in the proximal caput, and attach to spermatozoa.     

Innervation of the rat pineal gland by pituitary adenylate cyclase-activating polypeptide (PACAP)-immunoreactive nerve fibres

Pituitary adenylate cyclase-activating polypeptide (PACAP)-immunoreactive nerve fibres were demonstrated in the rat pineal gland. These fibres entered the pineal gland through the conarian nerve at the distal tip of the gland. A high density of the fibres was observed in the capsule of the gland, from where the immunoreactive elements penetrated into the pineal perivascular spaces and parenchyma. The majority of PACAP-immunoreactive nerve fibres also contained calcitonin gene-related peptide (CGRP). Some PACAP-immunoreactive nerve fibres contained neuropeptide Y (NPY), but only occasionally was PACAP colocalized with vasoactive intestinal peptide (VIP). After removal of both superior cervical ganglia, a high number of PACAP-containing nerve fibres were still present in the gland. In the nervous system PACAP is present in two isoforms, PACAP-38 and PACAP-27. The concentration of PACAP-38 in the superficial pineal gland was determined by radioimmunoassay to be 20.4 pmol/g tissue at midday and 18.9 pmol/g tissue at midnight. The concentration of PACAP-27 was only about 3% of the concentration of PACAP-38. In summary, this study is the first demonstration of a PACAP-containing innervation of the rat pineal gland. The PACAP concentration in the pineal gland does not exhibit a day-night difference. The colocalization of PACAP with calcitonin gene-related peptide in the pincalopetal nerve fibres indicates that the majority of PACAP-immunoreactive nerve fibres might originate from the trigeminal ganglion.     

‘Neuron-specific’ protein gene product 9.5 (PGP 9.5) is also expressed in glioma cell lines and its expression depends on cellular growth state

Protein gene product 9.5 (PGP 9.5), which in the normal nervous system is restricted to certain neurons, has been detected in two glioma cell lines, rat C6 and human GL15, by immunoblotting and immunocytochemistry. Its expression in these cells depends on the cellular growth state, being maximal between the first and second post-plating day. Only a faint PGP 9.5 immunoreactivity can be observed in glioma cells after the eleventh post-plating day, i.e. about one week after confluency has been reached. The present results suggest that PGP 9.5 in cultured glial cells is maximally expressed during the growth phase and that the protein could play a role during brain development in glial cells, in reactive gliosis, or in tumorigenesis of the glial lineage.     

The presence of vasoactive intestinal polypeptide (VIP)-like-immunoreactive nerve fibres and VIP-receptors in the pineal gland of the Mongolian gerbil (Meriones unguiculatus)

Summary The photocytes and other endodermal cells composing the wall of the meridional canals of the comb-jelly, Mnemiopsis leidyi, were investigated by transmission electron microscopy. Although many of these cells possess distinctive features such as a ciliary apparatus, lysosome-like bodies or vacuoles, they share with photocytes the presence of a network of rough endoplasmic reticulum (RER) whose cisternae enwrap large mitochondria and are aligned along the subsurface of the plasma membrane. A stereological analysis of organelle content in photocytes confirms the prominence of the RER in these cells and a shift of RER from mitochondria to plasma membrane subsurface in photocytes induced to luminesce by the mitochondrial inhibitor dinitrophenol. Photocytes and other endodermal cells of the meridional canals are interconnected by numerous gap junctions which, among photocytes, often form symmetrical triads with cortical cisternae and mitochondria. The gap junctions and RER/mitochondria assemblages are interpreted as possible substrates for, respectively, conduction of luminescence excitation along the canals and for excitation-luminescence coupling. Neuntes occasionally make synapses with photocytes and other endodermal cells lying adjacent to the mesoglea.     

The presence of opioidergic pinealocytes in the pineal gland of the European hamster (Cricetus cricetus): an immunocytochemical study

By use of antibodies raised against leu-enkephalin and met-enkephalin immunoreactive, opioidergic bi- and multipolar cells were demonstrated in the pineal gland of the European hamster. Ultrastructural analysis of these opioidergic cells revealed them to be pinealocytes. Processes emerged from the cell bodies and terminated in club-shaped swellings containing many small clear and some larger granular vesicles. Some of the terminals made synapse-like contacts with non-immunoreactive pinealocytes. The presence of the opioidergic pinealocytes strongly indicates that the pineal gland of the European hamster, in addition to its pinealopetal nervous regulation, is regulated by intrapineal peptidergic pinealocytes via a synaptic mechanism. A possible paracrine role of the opioidergic cells must also be considered.     

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.     

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.     

Melatonin-like immunoreactivity in the pineal gland of the cow: an immunohistochemical study

With a view to checking the presence of melatonin in the pineal gland of the cow, in the present work we used six adult animals, ranging in age from one to six years, which were sacrificed at dawn. Sections of 6 micro m thickness of Bouin-fixed and paraffin-embedded pineal glands were incubated in an anti-melatonin serum, which was provided by the Institute for Molecular and Cellular Recognition, Gunma University, Maebshi, Japan. After incubation and successive washings in PBS, some of the sections were treated with the avidin-biotin-peroxidase complex (ABC) technique using antisera from Sigma, and developed with the method of Graham and Karnovsky (which employs 3,3'-diaminobenzidine and H2O2 as developer). Other sections were incubated in a goat-anti-rabbit IgG (H+L) bound to fluorochrome Cy5 for immunofluorescence studies. An intense reaction for melatonin was observed in the cytoplasm but not in the nucleus of melatonin secreting pinealocytes located in peripheral and intermediate zones of the pineal gland. Immunoabsorption of the antimelatonin primary antibody with melatonin at a dilution of 10 mM per 0.1 ml of serum prevented the reaction, as happened when any of the antisera used in the procedure were used. Immunoabsorption of anti-melatonin serum with different amounts of bovine albumin (ranging between 1/5 to 1/50) failed to inhibit the immunoreactivity. When a bovine anti-albumin antibody was employed, working with the above methods, no immunoreaction was detected. Our data suggest that the pinealocytes of cows sacrificed at dawn contain immunoreactive melatonin.