Opsin-immunoreactive outer segments and acetylcholinesterase-positive neurons in the pineal complex of Phoxinus phoxinus (Teleostei,Cyprinidae)

Summary The pineal complex of the teleost, Phoxinus phoxinus L., was studied light-microscopically by the use of the indirect immunocytochemical antiopsin reaction and the histochemical acetylcholinersterase (AChE) method.Opsin-immunoreactive outer segments of photoreceptor cells were demonstrated in large numbers in all divisions of the pineal end-vesicle and in the pineal stalk. Moreover, they were found in the roof of the third ventricle, adjacent to the orifice of the pineal recess as well as scattered in the parapineal organ. These immunocytochemical observations provide direct evidence of the presence of an opsin associated with a photopigment in the photosensory cells of the pineal and parapineal organs of Phoxinus. By means of the AChE reaction (Karnovsky and Roots 1964) inner segments of pineal photoreceptors, intrinsic nerve cells, several intrapineal bundles of nerve fibers, and a prominent pineal tract were specifically marked. The pineal neurons can be divided into two types: one is located near the pineal lumen, the other near the basal lamina. The latter perikarya bear stained processes directed toward the photoreceptor layer. A rostral aggregation of two types of AChE-positive nerve cells occurs in the ventral wall of the pineal end-vesicle. The main portion of the AChE-positive pineal tract, which lies within the dorsal wall of the pineal stalk, can be followed to the posterior commissure where some of the nerve fibers course laterally. A few AChE-positive pineal nerve fibers run toward the lateral habenular nucleus via the habenular commissure. In the region of the subcommissural organ single AChE-positive neurons accompany the pineal tract. The nerve cells of the parapineal organ exhibit a moderate AChE activity.These findings extend the structural basis for the remarkable light-dependent activity of the pineal organ of Phoxinus phoxinus. To the memory of Professor Karl von Frisch, pioneer and master in the field of photoneuroendocrine systemsThis investigation was supported by grants from the Deutsche Forschungsgemeinschaft to A.O. (Ok 1/24; 1/25: Mechanismen biologischer Uhren) and to H.-W. K. (Ko 758/1; 758–2)On leave from the 2nd Department of Anatomy, SOTE, Budapest, Hungary     

GABA-immunoreactive neurons in the photosensory pineal organ of the rainbow trout: two distinct neuronal populations

Summary The distribution of putative GABA-ergic neurons in the photosensory pineal organ of the rainbow trout was investigated by use of a specific antiserum against -aminobutyric acid (GABA). GABA-immunoreactive (GABA-IR) neurons were located in the rostral portion of the pineal end-vesicle, presumably constituting a population of interneurons. GABA-IR neurons were also found in the pineal stalk. The axons of these neurons were traced along the pineal stalk toward the brain. The terminal areas of these axons could not be established. GABA-IR glial cells were observed in the pineal end-vesicle, but not in the pineal stalk.     

Cholinergic and GABAergic neuronal elements in the pineal organ of lampreys, and tract-tracing observations of differential connections of pinealofugal neurons

The putative cholinergic and GABAergic elements of the pineal organ of lampreys were investigated with immunocytochemistry to choline acetyltransferase (ChAT) and γ-aminobutyric acid (GABA), and by acetylcholinesterase (AChE) histochemistry. For comparison we also carried out immunocytochemistry to serotonin (5-HT) and a tract-tracing investigation of the two types of projecting cells, i.e., ganglion cells and long-axon photoreceptors. Most photoreceptors were ChAT-immunoreactive (ChAT-ir) and AChE-positive, while ganglion cells and the pineal tract were ChAT-negative and AChE-negative or only faintly positive. These results strongly suggest the presence of a cholinergic system of photoreceptors in the lamprey pineal organ. GABA-ir fibers that appear to originate from faintly to moderately stained ganglion cells were observed in the pineal stalk. Immunocytochemistry to 5-HT indicated the presence of two types of 5-HT-ir cells, bipolar cells and ganglion-like cells. The connections of the ganglion cells and long-axon photoreceptors were also studied by application of DiI to the pineal stalk in fixed brains or of biotinylated dextran amine (BDA) to one of the main targets of pinealofugal fibers (optic tectum or mesencephalic tegmentum) in isolated brains in vitro. Some long-axon photoreceptors and ganglion cells were labeled from the optic tectum. However, BDA application to the tegmentum exclusively labeled ganglion cells in the pineal organ. These results indicate that the two morphological types of afferent pineal neuron have different projections. No labeled cells were observed in the parapineal organ in BDA experiments, indicating that this organ and the pineal organ are involved in different neural circuits.     

Histological,histochemical and electron microscopical studies on the nervous apparatus of the pineal organ in the tiger salamander,Ambystoma tigrinum

Summary 150–190 photoreceptor cells form a basic structural component of the pineal organ of Ambystoma tigrinum. Most of the outer and inner segments of these cells project into the lumen horizontally. Only 10 percent of the total number of photoreceptor cells are located within the pineal roof which is composed of a single cell layer. The photoreceptor cells are connected with nerve cells by synapses displaying characteristic ribbons. Different types of synaptic contacts, i.e. simple, tangential, dyad, triad and invaginated, are found. They are embedded in extended neuropil zones. A particular type of synapse indicates the presence of interneurons. The basal processes of some photoreceptor cells leave the pineal organ and make synaptic contacts with nervous elements located within the area of the subcommissural organ. Employing the method of Karnovsky and Roots (1964) for histochemical demonstration of acetylcholinesterase (AChE) approximately 70 neurons (intrapineal neurons) can be discerned in the pineal organ of Ambystoma tigrinum. In analogy to the distribution of photoreceptor cells only few nerve cells are observed in the roof portion of the pineal organ. Evidently, two different types of AChE-positive intrapineal neurons are present. About 40–50 AChE-positive neurons (extrapineal neurons) are scattered in the area of the subcommissural organ. In this area two types of nerve cells can be distinguished: 1) neurons which send pinealofugal (afferent) axons toward the posterior commissure and 2) neurons which emit pinealopetal (efferent) axons into or toward the pineal organ.The nervous pathways connecting the pineal organ with the diencephalomesencephalic border area are represented by a distinct pineal pedicle and several accessory pineal tracts.Granular nerve fibers run within the posterior commissure and establish synaptic contacts in the commissural region adjacent to the pineal organ. Some of these granular elements enter the pineal organ.The morphology of the nervous apparatus of the pineal organ of Ambystoma tigrinum is discussed in context with evidence from physiological experiments.In partial fulfillment of the requirements for the degree of Dr. med., Faculty of Medicine, Justus Liebig University, GiessenThe author is indebted to Professors A. Oksche and M. Ueck for their interest in this study. Thanks are due to Professor Ch. Baumann, Giessen, and Professor H. Langer, Bochum, for stimulating discussions. The technical assistance of Miss R. Liesner is gratefully acknowledgedDedicated to Professor Berta Scharrer on the occasion of her 70th birthday. Supported by grants from the Deutsche Forschungsgemeinschaft to A.O. and M.U.     

Molecular forms of acetylcholinesterases in Alzheimer's disease

In this study, we examined 26 cases of Alzheimer's disease (AD) and 14 age-matched controls. In Brodmann area 21 cerebral cortex of the AD cases, there was no change in soluble G1 and G4 acetylcholinesterase (AChE) (EC 3.1.1.7), a significant 40% decrease in membrane-associated G4 AChE, significant 342 and 406% increases in A12 and A8 AChE, and a significant 71% decrease in choline acetyltransferase (ChAT) (EC 2.3.1.6). Our working hypothesis to account for these changes postulates that soluble globular forms are unchanged because they are primarily associated with intrinsic cortical neurons that are relatively unaffected by AD, that ChAT and membrane-associated G4 AChE decrease because they are primarily associated with incoming axons of cholinergic neurons that are abnormal in AD, and that asymmetric forms of AChE increase because of an acrylamide-type impairment of fast axonal transport in diseased incoming cholinergic axons. In the nucleus basalis of Meynert (nbM) of the 26 AD cases, there was a significant 61% decrease in the number of cholinergic neurons, an insignificant 23% decrease in nbM ChAT, a significant 298% increase in nbM ChAT per cholinergic neuron, and a significant 7% increase in the area of cholinergic perikarya. To account for the increased ChAT in cholinergic neurons and the enlargement of cholinergic perikarya, we propose that slow axonal transport may be impaired in nbM cholinergic neurons in AD.     

Postsmolt change in numbers of acetylcholinesterase-positive cells in the pineal organ of the Pacific coho salmon

Summary We have examined the occurrence of acetylcholinesterase (AChE)-positive cells in the pineal organ of different developmental stages of the Pacific coho salmon. Large numbers of AChE cells were present in fresh-water living alevins, in all stages of presmolts (n=307–544), and in adult spawners (n=696–1774) whereas seawater-living postmolts displayed a total lack of labeled cells. The AChE-reactive cells were evently distributed within the pineal end-vesicle and stalk of the presmolts and adults. However, the AChE-positive cells that occurred in the pineal stalk were of a smaller type and more uniform in shape than the cells of the pineal endvesicle. The dense populations of AChE-stained cells in the alevins, were all situated in the caudal part of the pineal end-vesicle. We conclude that changes in pineal metabolism occur in postsmolt salmon that liver in saltwater. It is not clear whether the observed change in pineal AChE expression is an unspecific change caused by life in the sea, reflecting alterations that are related to aspects of osmoregulation, and/or is involved in the visual function of the pineal organ resulting from changes in the environmental lighting conditions, e.g., photoperiod, light-intensity, or spectral composition. This study adds to our previous findings of changes that occur in the central nervous system of the salmon during the time of the parr-smolt transformation and migration between limnic and marine environments and indicates a possible central role of the pineal organ in the control of these events.     

Substance P-like-immunoreactive neurons in the photosensory pineal organ of the rainbow trout,Salmo gairdneri Richardson (Teleostei)

Summary Restricted numbers of substance P-like-immuno-reactive (SPL-IR) neurons were demonstrated in the photosensory pineal organ of the rainbow trout. The small parapineal organ of this teleost species receives a distinct SPL-IR innervation via the habenular nuclei, but displays no intrinsic SPL-IR neurons. Intrapineal SPL-IR neurons were located in the rostral portion of the pineal end-vesicle. Neuronal somata were found in a lateral position with smooth axonal processes extending mediad. Immunoreactive somata and axonal processes were observed intraparenchymally as well as in the pineal lumen. The pattern of immunoreactivity was not changed in excised pineal organs that had been incubated in tissue culture medium in the dark for 18 h. The possibility that the intrapineal SPL-IR neurons are not part of the neural circuitry involved in the transduction of photic information, but may have other functions, is discussed.Fellow of the Alexander von Humboldt-Stiftung, Bonn, Federal Republic of GermanySupported by research funds from the Deutsche Forschungsge-meinschaft (Ko 758/2-4)     

Immunocytochemical localization of serotonin and photoreceptor-specific proteins (rod-opsin,S-antigen) in the pineal complex of the river lamprey,Lampetra japonica,with special reference to photoneuroendocrine cells

Summary The pineal complex of the river lamprey, Lampetra japonica, was examined by means of immunocytochemistry with antisera against serotonin, the precursor of melatonin, and two photoreceptor proteins, rod-opsin (the apoprotein of the photopigment rhodopsin) and S-antigen. Serotonin-immunoreactive cells were observed in both the pineal and the parapineal organ. The proximal portion of the pineal organ (atrium) comprised numerous serotonin-immunoreactive cells displaying spherical somata. In the distal end-vesicle of the pineal organ, the serotonin-immunoreactive elements resembled photoreceptors in their size and shape. These cells projecting into the pineal lumen and toward the basal lamina were especially conspicuous in the ventral portion of the end-vesicle. In addition, single serotonin-immunoreactive nerve cells were found in this location. Retinal photoreceptors were never seen to contain immunoreactive serotonin; amacrine cells were the only retinal elements exhibiting serotonin immunoreaction. Strong S-antigen immunoreactivity was found in numerous photoreceptors located in the pineal end-vesicle. In contrast, the S-antigen immunoreactivity was weak in the spherical cells of the atrium. Thus, the pattern of S-antigen immunoreactivity was roughly opposite to that of serotonin. Similar findings were obtained in the parapineal organ. The rod-opsin immunoreaction was restricted to the outer segments of photoreceptors in the pineal end-vesicle and parapineal organ. No rodopsin immunoreactive outer segments occurred in the proximal portion of the atrium. Double immunostaining was employed to investigate whether immunoreactive opsin and serotonin are colocalized in one and the same cell. This approach revealed that (i) most of the rodopsin-immunoreactive outer segments in the end-vesicle belonged to serotonin-immunonegative photoreceptors; (ii) nearly all serotonin-immunoreactive cells in the end-vesicle bore short rod-opsin-immunoreactive outer segments protruding into the pineal lumen; and (iii) the spherical serotonin-immunoreactive cells in the pineal stalk lacked rod-opsin immunoreaction and an outer segment. These results support the concept that multiple cell lines of the photoreceptor type exist in the pineal complex at an early evolutionary stage.     

The presence of acetylcholinesterase-positive nerve fibres in the deep pineal gland and the pineal stalk but not in the superficial part of adult albino rats

Application of the histochemical method for testing acetylcholinesterase (AChE, EC 3.1.1.7) showed the presence of AChE-positive nerve fibers in the deep pineal gland and the pineal stalk but not in the superficial part of adult albino rats. These findings may indirectly support the existence of the potentially cholinergic innervation of at least some of the rat pinealocytes present in these parts of the gland and augment the evidence of the heterogeneity of the rat pinealocytes. It is possible that cholinergic neurons in the medial habenular nuclei or in the parasympathetic sphenopalatine ganglion may be a source of these AChE-positive fibres. The examination was performed at the light microscope level.     

Acetylcholinesterase-positive intrapineal neuronal system in the palm squirrel Funambulus pennanti (Wroughton)

A well-developed acetylcholinesterase (AChE)-positive neuronal system could be demonstrated in the pineal organ of the palm squirrel. There are two longitudinal nerve tracts which run all along the margin of the pineal organ from its distal to proximal regions. These nerve tracts are confluent distally. Another short, but deep tract was seen in the middle part of the pineal organ which joins one of these tracts. A large number of AChE-positive neurons whose processes actually form the tracts are present all along the pineal organ. They are distinguished into multipolar and pseudounipolar/unipolar neurons. A few neurons seen outside the nerve tract form a network of nerve fibres among the pinealocytes and also link the main tracts. The nerve tracts appear wavy, irregular and tortuous. A large number of round ring-like bodies seen in close association with neuronal perikarya and nerves may represent the axo-somatic and axo-dendritic contacts.     

Development of acetylcholinesterase-positive neuronal pathways in the cochlea of the mouse

Brain Cell Biology - Development of the cholinergic enzymes, choline acetyltransferase (ChAT) and AChE, and of the AChE-positive innervation in the cochlea was studied biochemically and...     

Reformation of the severed septohippocampal cholinergic pathway in the adult rat by transplanted septal neurons

Summary Transplants containing developing cholinergic neurons were obtained from the septum-diagonal band area of rat fetuses and were implanted into a lesion of the septohippocampal cholinergic pathway or into a cavity of the occipital cortex in adult recipient rats. The growth of new cholinergic fibres from the implant into the hippocampal formation was followed with choline acetyltransferase (ChAT) determinations and acetylcholine esterase (AChE) histochemistry. A fimbrial lesion alone, transecting the septohippocampal pathway, caused an almost complete cholinergic denervation of the hippocampal formation that persisted throughout the five month experimental period. A septal transplant implanted into the cavity of the fimbrial lesion restored a new AChE-positive innervation pattern in the hippocampus and the dentate gyrus that closely mimicked the original innervation removed by the lesion. In parallel, there was a progressive recovery in the ChAT levels, starting in the septal end, and progressing in a temporal direction. A new cholinergic fibre supply could be established in the hippocampal formation also along an abnormal route, i.e. from the transplants implanted into a cavity in the occipital cortex (involving also the dorsal part of the entorhinal cortex). Provided the hippocampus previously had been denervated of its normal cholinergic innervation, a partly normal AChE-positive terminal pattern was thus re-established also from this abnormal position. If, on the other hand, the cholinergic afferents were left intact, the ingrowing fibres were restricted mainly to the outer portion of the dentate molecular layer, i.e. the terminal zone of the lesioned entorhinal perforant path fibres. This suggests that the growth of the sprouting AChE-positive fibres into the normal cholinergic terminal fields was blocked by the presence of an intact cholinergic innervation. It is concluded that regrowing cholinergic axons can be guided over large distances within the hippocampal formation, and that their patterning within the terminal fields is very precisely regulated by mechanisms released by deafferentation.     

Ultrastructural localization of acetylcholinesterase in the synganglion of the tick,Dermacentor variabilis (Say)

Summary Light- and electron-microscopic enzyme cytochemistry was used to localize acetylcholinesterase (AChE) activity in the synganglion (brain) of the tick Dermacentor variabilis. High AChE activity was observed throughout the neuropil as well as adjacent to most neuronal perikarya. Intracellular activity was not observed by light microscopy. By electron microscopy, reaction product was localized at the plasma membrane of glia and neurons. Enzyme activity was not associated with the olfactory globuli neurons. In other types of neurons, small amounts of reaction product were observed in the Golgi apparatus and nuclear envelope. Large neurosecretory neurons contained activity that appeared to be associated with deep invaginations of the plasma membrane as well as intracellular membranes. AChE activity was also associated with processes of both neurons and glia. In most peripheral nerves AChE activity was associated with virtually all axons. Clearly then, AChE is associated with glia and non-cholinergic neurons as well as with presumed cholinergic neurons. The widespread localization and large amounts of AChE in the tick brain exceeds that reported for other invertebrates and vertebrates. As has been suggested for other animals, AChE in the tick brain may have functions in addition to its known role in cholinergic neurotransmission.     

Cholinergic neurons in the lamina ganglionaris of the blowfly Calliphora erythrocephala

Summary The distribution of putative cholinergic neurons in the lamina of the blowfly Calliphora erythrocephala was studied by immunocytochemical and histochemical methods. Three different antibodies directed against the AChsynthesizing enzyme, choline acetyltransferase (ChAT), revealed a cholinergic population of fibres running parallel to the laminar cartridges, which have branch-like structures at the distal lamina border. Cell bodies in the chiasma next to the lamina border were also labelled by the anti-ChAT antibodies. Monopolar cell bodies in the nuclear layer were faintly labelled. The distribution of the acetylcholine hydrolyzing enzyme, acetylcholine esterase (AChE), was revealed by histochemical staining and was similar to the ChAT immunocytochemistry. The arrangement of ChAT positive fibres in transverse and longitudinal sections and the distribution of AChE stained fibres indicate that the amacrine cells of the lamina are cholinergic cells.We dedicate this work to Prof. F. Zettler who passed away in fall 1988: K.-H. Datum, I. Rambold     

Acetylcholinesterase in central vocal control nuclei of the zebra finch (<Emphasis Type="Italic">Taeniopygia guttata</Emphasis>)

The distribution of acetylcholinesterase (AChE) in the central vocal control nuclei of the zebra finch was studied using enzyme histochemistry. AChE fibres and cells are intensely labelled in the forebrain nucleus area X, strongly labelled in high vocal centre (HVC) perikarya, and moderately to lightly labelled in the somata and neuropil of vocal control nuclei robust nucleus of arcopallium (RA), medial magnocellular nucleus of the anterior nidopallium (MMAN) and lateral magnocellular nucleus of the anterior nidopallium (LMAN). The identified sites of cholinergic and/or cholinoceptive neurons are similar to the cholinergic presence in vocal control regions of other songbirds such as the song sparrow, starling and another genus of the zebra finch (Poephila guttata), and to a certain extent in parallel vocal control regions in vocalizing birds such as the budgerigar. AChE presence in the vocal control system suggests innervation by either afferent projecting cholinergic systems and/or local circuit cholinergic neurons. Co-occurrence with choline acetyltransferase (ChAT) indicates efferent cholinergic projections. The cholinergic presence in parts of the zebra finch vocal control system, such as the area X, that is also intricately wired with parts of the basal ganglia, the descending fibre tracts and brain stem nuclei could underlie this circuitry’s involvement in sensory processing and motor control of song.     

Pattern of synaptic connections in the pineal organ of the ayu,Plecoglossus altivelis (Teleostei)

Summary Synaptic connections were studied by means of electron microscopy in the sensory pineal organ of the ayu, Plecoglossus altivelis, a highly photosensitive teleost species. Three types of specific contacts were observed in the pineal end-vesicle: 1) symmetrically organized gap junctions between the basal processes of adjacent photoreceptor cells; 2) sensory synapses endowed with synaptic ribbons, formed by basal processes of photoreceptor cells and dendrites of pineal neurons; 3) conventional synapses between pineal neurons, containing both clear and dense-core vesicles at the presynaptic site. Based on these findings, the following interpretations are given: (i) The gap junctions may be involved in an enhancement of electric communication and signal encoding between pineal photoreceptor cells. (ii) The sensory synapses transmit photic signals from the photoreceptor cells to pineal nerve cells. (iii) The conventional synapses are assumed to be involved in a lateral interaction and/or summation of information in the sensory pineal organ. A concept of synaptic relationships among the sensory and neuronal elements in the pineal organ of the ayu is presented.Fellow of the Alexander von Humboldt Foundation, Federal Republic of Germany     

The pineal organ of Raja clavata: Opsin immunoreactivity and ultrastructure

Summary The pineal organ of Raja clavata was studied by light and electron microscopy, including the immunocytochemical antiopsin reaction. The pineal organ of the ray consists of three portions: (i) a large proximal pineal, (ii) a long tube-like connecting stalk, and (iii) a short distal terminal enlargement. This latter end-vesicle lies in the deep connective tissue layers of the braincase. All portions of the pineal are composed of pinealocytes, intrinsic neurons, ependymal/glial cells, and bundles of nerve fibers embedded in thin neuropil formations. The inner segments of the pinealocytes protrude into the lumen in all parts of the organ and usually contain basal bodies and numerous mitochondria. Often, two outer segments were found to arise from the basal bodies of a single inner segment. By means of light-microscopic immunocytochemistry the outer segments showed a strong antiopsin reaction.The axons of the pinealocytes form ribbon-containing synapses on dendritelike profiles, which appear to belong to the intrinsic pineal neurons. There are other axo-dendritic synapses established by presynaptic terminals lacking ribbons and containing granular and synaptic vesicles. Pineal neurons may contain granular vesicles approximately 60–100 nm in diameter; their processes contribute to the bundles of unmyelinated axons.The fine structural organization of the pineal organ and the opsin immunoreactivity of the outer segments of the pinealocytes indicate a photoreceptive capacity of the organ. The double outer segments represent a peculiar multiplication of the photoreceptor structures.This investigation was supported by grants from the Deutsche Forschungsgemeinschaft to A. Oksche (Ok 1/24; 1/25: Mechanismen biologischer Uhren)On leave from the 2nd Department of Anatomy, Semmelweis OTE, Budapest, Hungary     

Pineal cells enhance choline acetyltransferase activity in sympathetic neurons

Cells derived from the neonatal rat pineal gland were cocultured with cells derived from neonatal rat superior cervical ganglia (SCG) in an attempt to determine whether a sympathetic target organ with only adrenergic properties could enhance the development of adrenergic transmitter properties in sympathetic neurons in tissue culture. Choline acetyltransferase was measured as an index of cholinergic differentiation, and tyrosine hydroxylase was measured as an index of adrenergic differentiation. As indices of total cell number and cellular volume, DNA and protein, respectively, were also measured. We found that the pineal-SCG cocultures contained ten times greater choline acetyltransferase activity than sister neuronal cultures cultured without pineal cells, thus indicating that the pineal cells enhanced cholinergic properties in the sympathetic neurons. This cholinergic enhancement was dependent upon the presence of nerve growth factor and could not be obtained with pineal-conditioned medium. Tyrosine hydroxylase activity, measured on cultures sister to those mentioned above, was low in all cultures and decreased somewhat in SCGs cultured alone. TH activity in the pineal-SCG cocultures, however, increased slightly. Some tyrosine hydroxylating activity developed in pineals cultured alone, however, and may have been responsible for the small increase in tyrosine hydroxylase activity noted in the pineal-SCG cocultures. The implications of these results for a determination of the role that target organ plays in the development of the transmitter properties of sympathetic neurons are discussed.     

Cholinergic neurons of the pelvic autonomic ganglia and uterus of the female rat: distribution of axons and presence of muscarinic receptors

Acetylcholine (ACh) stimulates contraction of the uterus and dilates the uterine arterial supply. Uterine cholinergic nerves arise from the paracervical ganglia and were, in the past, characterized based on acetylcholinesterase (AChE) histochemistry. However, the histochemical reaction for acetylcholinesterase provides only indirect evidence of acetylcholine location and is a nonspecific marker for cholinergic nerves. The present study: (1) reevaluated cholinergic neurons of the paracervical ganglia, (2) examined the cholinergic innervation of the uterus by using retrograde axonal tracing and antibodies against molecules specific to cholinergic neurons, choline acetyltransferase and the vesicular acetylcholine transporter, and (3) examined muscarinic receptors in the paracervical ganglia using autoradiography and a radiolabeled agonist. Most ganglionic neurons were choline acetyltransferase- and vesicular acetylcholine transporter-immunoreactive and were apposed by choline acetyltransferase/vesicular acetylcholine transporter-immunoreactive terminals. Retrograde tracing showed that some cholinergic neurons projected axons to the uterus. These nerves formed moderately dense plexuses in the myometrium, cervical smooth muscle and microarterial system of the uterine horns and cervix. Finally, the paracervical ganglia contain muscarinic receptors. These results clearly reveal the cholinergic innervation of the uterus and cervix, a source of these nerves, and demonstrate the muscarinic receptor content of the paracervical ganglia. Cholinergic nerves could play significant roles in the control of uterine myometrium and vasculature.     

Colocalization of GLUT3 and choline acetyltransferase immunoreactivity in the rat retina

Toward elucidating the functional aspects ofGLUT3, a primary neuronal glucose transporter isoform in the vertebrate central nervous system, this study examined its expression in cholinergic amacrine cells made identifiable by the presence of acetylcholine-synthesizing enzyme, choline acetyltransferase (ChAT), in the rat retina. Double-immunofluorescence staining of adult rat retinal tissue with anti-GLUT3 and anti-ChAT antibodies revealed characteristic stratified GLUT3 immunoreactivity (GLUT3-IR) in the inner plexiform layer (IPL) that was identical to the arborization pattern of ChAT-positive neuronal processes there. In addition, approximately 30-50% of intensely GLUT3-immunoreactive cell bodies in the inner nuclear layer and ganglion cell layer showed ChAT-IR, while the majority of ChAT-positive cell bodies were also intensely GLUT3 immunoreactive. Analysis at the cellular level using retinal cells in culture revealed similar findings. These results collectively indicate that cholinergic amacrine cells constitute the major component of GLUT3-expressing cells in the rat retina. It is expected that the link demonstrated here between GLUT3 expression and cholinergic amacrine cell population will provide clues for further analyzing GLUT3 function in the retina.