A double sequential immunofluorescence method demonstrating the co-localization of urotensins I and II in the caudal neurosecretory system of the teleost,Gillichthys mirabilis

Summary A double immunofluorescence method was devised to localize simultaneously urotensin-I (UI) and -II (UII) immunoreactivities in the caudal neurosecretory system of the goby, Gillichthys mirabilis. In a sequential fashion, sections of the posterior spinal cord and urophysis were treated with antiserum to corticotropin-releasing factor (CRF) that cross-reacts with UI, fluorescein-conjugated sheep anti-rabbit IgG, biotinylated anti-UII and rhodamine-conjugated avidin. UI and UII immunoreactivities appeared to coexist in some neurons and in most fibers and urophysial tissue; the remainder of the fibers and urophysis and the majority of neurons were immunoreactive for CRF/ UI only. No convincing evidence of immunoreactivity for UII only was found. A few nonreactive cells were seen, but these may not be neurosecretory neurons. The two immunoreactive cell types were not segregated topographically, and the intensity of perikaryal immunofluorescence for CRF/UI was variable. To explain these results a hypothesis that all caudal neurosecretory cells may synthesize both UI and UII and that immunoreactive differences may reflect different states of cellular activity, is suggested. This sequential double immunofluorescence method offers several advantages over other techniques and is especially useful for co-localization studies when primary antisera from different species are not available.     

Development of the caudal neurosecretory system of the nile tilapia Oreochromis niloticus: an immunohistochemical and electron microscopic study

The development of the caudal neurosecretory system (CNSS) of the Nile tilapia, Oreochromis niloticus, has been investigated by means of UI/oCRF (urotensin I/ovine corticotropin-releasing factor) immunohistochemistry and transmission electron microscopy. UI-like immunoreactive perikarya and fibers are first detected in the caudal spinal cord of larval fish about 4 days after hatching (stage 21). In the region of the future urophysis two bundles of strongly immunoreactive neurosecretory fibers are observed. At this stage, neurosecretory axons terminate on the meninx sheath of the spinal cord with immature neurosecretory terminals. The histogenesis of the urophysis begins at stage 24. The future neurohemal organ consists of a small ventral swelling of the spinal cord, which is associated with dilated vessels. At this stage, neurosecretory axons terminate on the basal lamina of the ingrowing blood vessels. Further development occurs by means of progressive branching of vessels and the concomitant increase in the number of neurosecretory terminals. In the caudal spinal cord, immunoreactive neurons also increase in number and progressively differentiate morphologically. Typical features of the mature CNSS are recognizable in 4-month-old juveniles. Data suggest that in tilapia both the synthesis and the release of urophysial hormones begin before morphogenesis of the neurohemal organ takes place.     

Ontogenetic Development of the Caudal Neurosecretory System in the Chum Salmon, Oncorhynchus keta, with Regard to Its Ultrastructural Changes and with Relation to Neuropeptide Y-immunoreactive Fibers

The ontogeny of the caudal neurosecretory cells (Dahlgren cells) in the caudal spinal cord of the chum salmon, Oncorhynchus keta, was examined by conventional electron microscopy and with immunohistochemistry for urotensins (U) and neuropeptide Y (NPY). The precursors of the Dahlgren cells first appeared as agranular ovoid cells in the caudal region of the neural tube of 40-day-old embryos about one week before hatching. The occurrence of cytoplasmic granules in the immature Dahlgren cells became evident by the 14th day after hatching. At this moment, the U-positive reaction was merely demonstrated in some of the granules. Close association of NPY-positive fibers with the caudal neurosecretory structures was recognizable in 1-month-old larvae. Thus, it is apparent that the salmon Dahlgren cells start their secretory activity (production of the secretory granules) in early larval stages and that, thereafter, NPYergic afferent innervation of the caudal neurosecretory system becomes evident.     

Immunohistochemical investigation of urotensins in the caudal spinal cord of four species of elasmobranchs and the lamprey,Lampetra japonica

Summary In the four species of elasmobranchs examined (Triakis scyllia, Heterodontus japonicus, Scyliorhinus torazame, Dasyatis akajei), all identifiable caudal neurosecretory cells and their corresponding neurohemal areas showed urotensin II (UII)-immunoreactivity with varied intensity. To localize urotensin I (UI) in the caudal neurosecretory system of the dogfish, Triakis scyllia, h-CRF (1–20) antiserum that cross-reacts with UI was used in place of UI antiserum. CRF/UI-immunoreactivity was demonstrated in the neurosecretory cells and neurohemal areas. A considerable number of neurons showed both UII- and CRF/UI-immunoreactivities, suggesting that UII and UI are produced in the same neurosecretory cells. However, some neurons exhibited UII-immunoreactivity, but no CRF/UI-immunoreactivity. Cells immunoreactive only to CRF antiserum were not detected. At least two populations of neurons exist in the dogfish caudal neurosecretory system: (i) cells immunoreactive for both CRF/UI and UII, and (ii) cells immunoreactive for UII. The dorsal cells of the lamprey, Lampetra japonica, did not react with either UII or CRF antiserum.     

Immunohistochemical localization of urotensin I and other neuropeptides in the caudal neurosecretory system of three species of teleosts and two species of elasmobranchs

Summary In three species of teleosts — carp Cyprinus carpio; grass carp Ctenopharyngodon idella; and crucian carp Carassius auratus — the caudal neurosecretory system displays small, medium-sized and large neurons. Urotensin I (UI)-immunoreactive and UI-nonreactive neurons were found in all three groups; in general, the number of the latter neurons exceeded that of the former. Noteworthy are: (i) UI-immunoreactive fibers in the caudal spinal cord and (ii) dense accumulations of UI-immunoreactive product around the capillaries of the urophysis. In two species of elasmobranchs — cat shark Heterodontus japonicas and swell shark Cephaloscyllium umbratile — neurosecretory neurons decreased in size in rostro-caudal direction. Most of the neurosecretory perikarya, their axons and the corresponding neurohemal areas were UI-immunoreactive, but a small number of secretory neurons was devoid of immunoreaction. Oxytocin, arginine vasopressin, substance P, somatostatin, neurotensin, vasoactive intestinal polypeptide and gastrin-releasing peptide were not detected in the caudal neurosecretory system of the carp.     

Caudal neurosecretory system of the zebrafish: ultrastructural organization and immunocytochemical detection of urotensins

The caudal neurosecretory system is described here for the first time in the zebrafish, one of the most important models used to study biological processes. Light- and electron-microscopical approaches have been employed to describe the structural organization of Dahlgren cells and the urophysis, together with the immunohistochemical localization of urotensin I and II (UI and UII) peptides. Two latero-ventral bands of neuronal perikarya in the caudal spinal cord project axons to the urophysis. The largest secretory neurons (~20 μm) are located rostrally. UII-immunoreactive perikarya are much more numerous than those immunoreactive for UI. A few neurons are immunopositive for both peptides. Axons contain 75-nm to 180-nm dense-core vesicles comprising two populations distributed in two axonal types (A and B). Large dense vesicles predominate in type A axons and smaller ones in type B. Immunogold double-labelling has revealed that some fibres contain both UI and UII, sometimes even within the same neurosecretory granule. UII is apparently the major peptide present and predominates in type A axons, with UI predominating in type B. A surprising finding, not previously reported in other fish, is the presence of dense-core vesicles, similar to those in neurons, in astrocytes including their end-feet around capillaries. Secretory type vesicles are also evident in ependymocytes and cerebrospinal-fluid-contacting neurons in the terminal spinal cord. Thus, in addition to the urophysis, this region may possess further secretory systems whose products and associated targets remain to be established. These results provide the basis for further experimental, genetic and developmental studies of the urophysial system in the zebrafish.     

Immunohistochemical demonstration of urotensins I and II in the caudal neurosecretory system of the Japanese charr,Salvelinus leucomaenis,retained in sea water

This paper is concerned with part of the role and function of the caudal neurosecretory system of the charr,Salvelinus leucomaenis, studied by immunohistochemistry. In order to elucidate the different histologic changes, we examined the immunoreactivities of urotenisn I (UI) and urotensin II (UII) in 3 experimental groups: the feral (river) fish, the fresh-water aquarium-, and sea water aquarium-retained fish. Coexistence of UI and UII was demonstrated in most of the smaller and larger neurons distributed in and near the urophyseal system of all 3 groups. However, some of the larger neurons were immunoreactive only to a single hormone, UI or UII. Merely a few neurons indicated no reactivity for either UI or UII. No such clearcut differences were encountered immunohistochemically in the 3 groups. Neuronal and urophysial immuno-reactivity to UI of feral and fresh-water-retained fish was slightly stronger than that of sea water-retained fish. Moreover, in sea water-retained fish, the intensity of immunoreactivity for UI was variable, and the number of neurons positive for UII only was somewhat larger than that in feral and fresh-water-retained fish. A series of UII-positive cerebrospinal fluid (CSF)-contacting neurons were seen in the ependymal and subependymal layers ventral to the central canal of the spinal cord in every group. These CSF-contacting neurons might constitute another neurosecretory system aside from the ordinary caudal neurosecretory system equipped with urophysis. In contrast to the hypothalamohypophysial neurosecretory system, the caudal neurosecretory system did not show any significant changes among the 3 groups. This suggests that urotensins I and II have no essential role in osmoregulation of the charr.     

Immunohistochemical investigation of neuropeptides in the central nervous system of the amphioxus,Branchiostoma belcheri

The immunohistochemical localization of nine different neuropeptides was studied in the central nervous system of the amphioxus, Branchiostoma belcheri. In the brain, perikarya immunoreactive for urotensin I and FMRFamide were localized in the vicinity of the central canal. One of the processes of each of these perikarya was found to cross the dorso ventral slit-like lumen of the central canal. Oxytocin-immunoreactive short fibers, but not perikarya, were detected in the ventral part of the brain. Perikarya immunoreactive for arginine vasopressin/vasotocin, oxytocin and FMRFamide were widely distributed in the spinal cord. Arginine vasopressin/vasotocin-immunoreactive fibers often made contacts with Rohde cell axons. Angiotensin II-immunoreactive perikarya were observed in the posterior half of the spinal cord, and urotensin I-immunoreactive perikarya were found in the caudal region of the spinal cord. Cholecystokinin/gastrin-immunoreactive fibers, but not perikarya, were detected in the spinal cord; some extended as far as the ependymal layer of the cerebral ventricle. No colocalization of the peptides examined was observed. No immunoreactivity for atrial and brain natriuretic peptides nor for urotensin II was detected. The present study indicates that there are at least six separate neuronal systems that contain different peptides, respectively, in the central nervous system of the amphioxus. Their functions remain to be determined.Part of this investigation has previously been presented in abstract form (Uemura et al. 1989)     

Monoamine oxidase in the hypothalamo-hypophysial region of the teleosts,Anguilla japonica and Oryzias latipes

Summary Distribution of monoamine oxidase (MAO) was histochemically examined in the hypothalamo-hypophysial region of the eel (Anguilla japonica) and the medaka (Oryzias latipes) with a modified Glenner's tryptamine-tetrazolium method. The hypothalamic neurosecretory cells showed very weak MAO activity in their perikarya. MAO-positive fibers were present in close contact with the neurosecretory cells, suggesting that monoaminergic fibers participate in the control of neurosecretory cell activity. The nucleus lateralis tuberis (NLT) contained cells exhibiting strong MAO activity. These cells must be monoaminergic neurons.In the anterior region of the neurohypophysis of both eel and medaka, two bundles of MAO-positive fibers originating from the NLT proceed down along each side of the third ventricle into the pars distalis. This suggests that monoaminergic neurons of the NLT are involved in the release of hormones from the pars distalis. In addition to these tracts, numerous MAO-positive fibers proceed backward from the post-optic area and end around the blood capillaries located between the neurohypophysis and the pars intermedia in both species.I wish to express my gratitude to Prof. H. Kobayashi for his valuable advice during the course of this study. I am indebted to Prof. S. Uchida, Ocean Research Institute, University of Tokyo, for supplying the eels.     

Terminal processes of serotonin neurons in the caudal spinal cord of the molly,Poecilia latipinna,project to the leptomeninges and urophysis

Summary The caudal neurosecretory complex of poeciliids has previously been shown to be innervated by extranuclear and intrinsic serotonergic projections. In the present study, immunohistochemical techniques were used to characterize fibers originating from serotonin neurons intrinsic to the caudal spinal cord. Bipolar and multipolar neurons were oriented ventromedially, and contained numerous large granular vesicles. Three types of serotonergic fibers were distinguished based on their distribution and morphology. Intrinsic Type-A fibers branched into varicose segments near the ventrolateral surface of the spinal cord and contacted the basal lamina beneath the leptomeninges. Type-B fibers coursed longitudinally to enter the urophysis, where they diverged and terminated around fenestrated capillaries. Labelled vesicles in Type-A and Type-B terminals were the same size as those in labelled cells and in unlabelled neurosecretory terminals in the urophysis. Type-C small varicose fibers branched within the neuropil of the caudal neurosecretory complex. Serotonin may be secreted into the submeningeal cerebrospinal fluid, the urophysis, and the caudal vein by Type-A and Type-B fibers, whereas, Type-C fibers may be processes of serotonergic interneurons in the neuroendocrine nucleus. The possibility that urotensins I and II or arginine vasotocin were colocalized in the processes of the intrinsic serotonin neurons was investigated immunohistochemically. The negative results of these experiments suggest that serotonin-containing neurons may represent a neurochemically distinct subpopulation in the caudal neurosecretory complex.     

Immunocytochemical localization of a substance in the eyestalk of the prawn,Palaemon serratus,reactive with an anti-FMRF-amide rabbit serum

Summary By use of a specific antiserum against the molluscan cardio-excitatory tetrapeptide FMRF-amide in combination with the PAP-method it was possible to obtain positive immunocytochemical reactions in several neurosecretory regions of the eyestalk of the prawn Palaemon serratus. FMRF-amide-like material was found in perikarya and nerve fibers of the medulla terminalis and in neurons in the lamina ganglionaris. The immunoreactivity observed in the glandular tissue located at the basal insertion of the eyestalk muscles must be ascribed to a non-specific reaction. The identification of immunopositive nerve fibers, ending on a nerve bundle in the medulla terminalis, and the fact that immunoreactive material was absent in the neurohemal sinus gland seem to indicate a neurotransmitter/neuromodulator function.     

Immunocytochemical localization of vasotocin-like immunoreactivity in the brain of the cartilaginous fish,Scyliorhinus caniculus

Summary The distribution of vasotocin-like peptides in the central nervous system of the cartilaginous fish Scyliorhinus canicula was determined by indirect immunofluorescence and peroxidase anti-peroxidase techniques, using a specific antiserum raised in rabbits against synthetic vasotocin. Immunoreactive perikarya were mainly detected in the anterior hypothalamus, within the midcaudal part of the preoptic nucleus. The most rostral positive cell bodies were located in the dorso-lateral parts of the preoptic area, whereas at a more caudal level, they took a ventro-medial position within the deepest layers of the nucleus. Throughout the preoptic region these cells varied in shape according to their location. Occasionally, scattered vasotocin-like immunopositive cells were also identified in the nucleus periventricularis hypothalami. Vasotocin immunoreactivity was detected in numerous varicose nerve fibers of the preopticohypophysial tract. These fibers were seen to course through the medio-basal hypothalamus and caudally, after having passed the hypophysial stem, they reached the neurointermediate lobe of the pituitary. Numerous immunoreactive fibers were also observed within the rostro-medial region of the median eminence. At this level the fibers were in close proximity to the capillary loops. In the preoptic region, some stained cells exibited short processes that appeared to contact non-reactive perikarya. By comparing the distribution of vasotocin- and corticotropin-releasing factor immunoreactivity on adjacent then serial sections, it was revealed that these peptides, in S. canicula, do not coexist in the same perikarya. The present results, are compared with those obtained in other vertebrate groups, and their possible functional implications are discussed.     

Immunocytochemical localization of CCAP,a novel crustacean cardioactive peptide,in the nervous system of the shore crab,Carcinus maenas L.

Summary Polyclonal antibodies were raised in rabbits against synthetic crustacean cardioactive peptide (CCAP) conjugated to bovine thyroglobulin, and were used to map CCAP-immunoreactive structures in the central nervous system of Carcinus maenas. As expected, the neurohemal pericardial organs (PO) displayed abundant immunoreactivity in nerve fibers and terminals. In addition, immunoreactive neurons were demonstrated in other parts of the nervous system. At least some of them do not appear to terminate in neurohemal structures and may have a non-endocrine, as yet unknown function. Immunoreactive perikarya with a diameter of 25–30 m occur in the brain. They project into the optic and antennary neuropil, and into the eyestalk. One cell was found in the medulla terminalis of the eyestalk and in the connective ganglion, respectively. From the latter, axonal branches could be traced into the brain and the thoracic ganglia (TG). In the TG, small-diameter perikarya give rise to extensive networks of varicose fibers. Some of the perikarya occur in a characteristic paired arrangement with larger CCAP-immunoreactive somata (diameter 40–50 m). These pairs of one small and one large cell occur in all mouthpart and leg segments of the TG, except the abdominal ganglia (AG), where only large cells were found. The main projections of the large neurons comprise one or more fibers in each of the seven segmental nerves (SN), leading to neurosecretory terminals in the PO. The fibers in the SN are joined by branches of an ascending axonal tract from the large perikarya in the AG. The large-type perikarya are considered to be the principal source of CCAP in the PO. The optic ganglia in the eyestalk, except the medulla terminalis, the neurohemal sinus gland and the stomatogastric nervous system are devoid of CCAP-immunoreactivity.In axon terminals of the PO, CCAP is not colocalized with other PO-neuropeptides, i.e. proctolin-, FMRFamide-like, and Leu-enkephalin-like immunoreactive materials. Electron-microscopic immunocytochemistry revealed a distinct CCAP-containing granule type in specific axon profiles and terminals in the PO.The architecture of CCAP-immunoreactive neurons is discussed with respect to previous morphological studies on the origin and pathways of fibers terminating in the PO.Dedicated to Professor K.E. Wohlfarth-Bottermann, Bonn, on the occasion of his 65^th birthday     

The caudal neurosecretory system of the teleostean Clupea melanostoma

Summary The caudal neurosecretory system of Clupea melanostoma is described. The urophyseal area in this species is merely a spinal cord enlargement divided into two distinct zones: a ventral and ventrolateral vascular zone where neurosecretory material is concentrated, and a dorsal cell-rich area where the perikarya of the neurosecretory cells are found.The hypothesis is advanced that the first-named vascular area has developed into the more differentiated urophysis of the less primitive teleosts while the dorsal cell-rich area has become part of the filum terminale. Two main types of neurosecretory cells are described.This work was supported by grant L 96 Z from the Consejo Nacional de Investigaciones Cientificas y Técnicas.     

Nitric oxide and neuromodulation in the caudal neurosecretory system of teleosts

Although evidence exists that nitric oxide (NO) mediates neuroendocrine secretion in mammals, the involvement of NO in the neuroendocrine regulation of non-mammalian vertebrates has yet to be investigated in detail. The present review conveys several recent data, suggesting that NO plays a modulatory role in the caudal neurosecretory system (CNSS) of teleosts. The presence and distribution of neuronal NO synthase (nNOS) was demonstrated in the CNSS of the Nile tilapia Oreochromis niloticus by means of NADPHd histochemistry, NOS immunohistochemistry, NOS immunogold electron microscopy, the citrulline assay for NOS activity and Western blot analysis. NO production by the caudal spinal cord homogenates was also evaluated by the oxyhemoglobin assay. On the whole, these findings indicate that caudal neurosecretory cells express NOS enzymes and presumably produce NO as a cotransmitter. Moreover, the comparison of the nNOS distribution with that of urotensins I and II (UI and UII) suggests that neurosecretory Dahlgren cells belong to two different functional subpopulations: a population of UI/UII secreting nitrergic neurons and a population of non-nitrergic neurons, which principally secrete UII. These results implicate NO as a putative modulator of the release of urotensins from the neurosecretory axon terminals. Therefore, like in mammals, NO appears to influence neuroendocrine secretion in teleosts.     

Immunocytochemical localization of somatostatin in the brain of the lizard,Ctenosauria pectinata

Summary The brain of the lizard, Ctenosauria pectinata, was studied light microscopically using an immunocytochemical staining method that is specific for neurohypophysial hormones and somatostatin. It was shown that the telencephalon and particularly the diencephalon contain somatostatin-producing perikarya, while somatostatinergic fibers occur in the entire brain. Similar to the situation in other vertebrates, somatostatin neurons in Ctenosauria pectinata form a population distinct from the neurohypophysial hormone-producing neurons. The small-sized somatostatin neurons were found in the cortex and the hypopallium of the telencephalon, and in two distinct clusters in the diencephalon: (1) ventral from, and partially overlapping with, the classical neurosecretory para ventricular nucleus; and (2) in the region of the infundibular (tuberal) nucleus. Somatostatin fibers were found among the classical neurosecretory fibers of the supraoptico-paraventricular system (tract, median eminence, neural lobe), near to and within the epiphysis, in the septum, in the vicinity of the tectum opticum and the cerebellum, and in the tegmentum.This investigation was supported by a grant from the Belgian Nationaal Fonds voor Geneeskundig Wetenschappelijk Onderzoek     

Immunofluorescence of somatostatin-producing sites in the hypothalamus of the tadpole,Alytes obstetricans Laur.

Summary Three sites of somatostatin-synthesizing perikarya, or a related antigen, were determined by immunofluorescence in the hypothalamus of the tadpole, Alytes obstetricans (Amphibia, Anura). Two sites of neurosecretory perikarya were localized in the preoptic nuclei of the anterior hypothalamus; the axons extended either to the anterior diencephalon or to the median eminence and the pituitary. The third site was found in the posterior hypothalamus. These neurosecretory cells showed a strong immunofluorescent reaction; their axons all terminated at the level of the median eminence. Somatostatin cells were only found in intact or hypophysectomized tadpoles given somatotropin (STH). The strong reaction observed in hypophysectomized tadpoles was possibly due to the loss of the terminal portion of the neurosecretory pathway (median eminence and pituitary) by which the agent is transported to the site of discharge.     

Morphological aspects of feeding and improvement in feeding ability in early stage larvae of the milkfish,Chanos chanos

The osteological development of elements comprising the oral cavity and fins was examined in early stage larvae of laboratory-reared milkfish,Chanos chanos, from hatching to 200 hours after hatching. Fundamental elements of the oral cavity had developed by the time of initial mouth opening, 54 hours after hatching. The oral cavity was long and cylindrical, with a short, robust Meckel's cartilage, and robust quadrate and symplectic-hyomandibular cartilages. The initial ossification of existing elements and addition of new elements occurred between 120–146 hours after initial mouth opening (HAMO), whereas the cartilaginous basihyal and caudal fin-supports appeared at 37.5 and 61.5 HAMO, respectively. Based on the morphology and developmental patterns of characters examined in this study, the feeding mode of early stage larval milkfish was considered to be “straining,” with an improvement in feeding ability occurring between 120–146 HAMO.     

Adrenergic neurons in the spinal cord of the pike (Esox lucius) and their relation to the caudal neurosecretory system

Summary The lower spinal cord including the caudal neurosecretory system of the pike (Esox lucius) was investigated by means of light and electron microscopy and also with the fluorescence histochemical method of Falck and Hillarp for the visualization of monoamines. A system of perikarya displaying a specific green fluorescence of remarkably high intensity is disclosed in the basal part of the ventrolateral and lateral ependymal lining of the central canal. The area corresponding to the upper half of the urophysis has most cells; their number decreases caudally and cranially. A considerable number of their beaded neurites reach the neurosecretory neurons by different routes but are only occasionally present in the actual neurohemal region. An intensely fluorescent dendritic process is sometimes observed terminating with a bulbous enlargement at the ependymal surface in the central canal. Besides small, electron lucid vesicles in the terminal parts of the axons, the neurons contain numerous large dense-core vesicles which can apparently take up and store 5-hydroxydopa (5-OH-dopa) and 5-hydroxydopamine (5-OH-DA). These neurons are thought to be adrenergic and to contain a primary catecholamine, possibly noradrenaline.The varicosities of the adrenergic terminals are repeatedly observed contiguous to some of the neurosecretory axons, the membrane distance at places of contacts generally ranging from 150–200 Å. Another type of nerve terminals that contain only small empty vesicles, also after pretreatment with 5-OH-dopa or 5-OH-DA, are frequent among the neurosecretory neurons. These axons establish synaptic contacts with membrane thickenings on most of the neurosecretory neurons. Thus it seems that the neurosecretory neurons are innervated by neurons morphologically similar to cholinergic neurons and that part of them receive an adrenergic innervation, which supports the view hat the caudal neurosecretory cells do not constitute a functionally homogeneous population.Supported by the Deutsche Forschungsgemeinschaft and the Joachim-Jungius Gesellschaft zur Förderung der Wissenschaften, Hamburg.Supported by the Swedish Natural Research Council (No. 99-35). This work was in part carried out within a research organization sponsored by the Swedish Medical Research Council (Projects No. B70-14X-56-06 and B70-14X-712-05).Supported by the Deutsche Forschungsgemeinschaft and USPHS Research Grant TW 00295-02.