Quantitation of fiber growth in transplanted central monoamine neurons

Summary Nerve fiber production by central noradrenaline (NA), dopamine (DA), and 5-hydroxytryptamine (5-HT) neurons was studied using immature brain tissue containing locus coeruleus, substantia nigra, or ventro-caudal medulla oblongata respectively, homologously grafted to the anterior chambers of rat eyes. A method was developed for quantitation of the fiber growth that occurs on the sympathetically denervated host irides as observed in whole mounts using Falck-Hillarp fluorescence histochemistry and by the uptake of ³H-metaraminol into the irides. Survival and growth in oculo of the three different areas were characterized by direct observations through the cornea in vivo for a number of pre- and postnatal stages of development of the donors, and the findings correlated to the degree of monoamine nerve fiber production on the host irides. The growth of fetal locus coeruleus transplants on irides was quantified using both fluorescence microscopical measurements of innervated areas and uptake of ³H-metaraminol. The uptake was well correlated to the histochemical measurements on individual irides, thus validating the fluorescence microscopical measurements of fiber production. The fiber growth of fetal locus coeruleus grafts on irides was followed for 20 weeks. The nerves increased in number and uptake capacity approximately linearly for 6 weeks whereafter the increase rapidly levelled off. On average, the final amount of nerve production by fetal locus grafts did not cover more than 1/3 of the host iris surface, and the average uptake of ³H-metaraminol by these nerves did not exceed 60% of that found in sympathetically intact control irides. The locus grafts produced a similar amount of fluorescent fibers in the host iris independent of the crown-rump length stage of the donor fetus and the final size of the transplants in oculo.The survival and growth of NA, DA and 5-HT neurons grafted from various postnatal donor rats was also followed by fluorescence microscopy. Locus coeruleus grafts produced markedly more fibers than the two other types of grafts when the donor was one week old or less, and DA grafts produced the least fibers of the three. Even from one month old donors some MA neurons survived grafting. Also, using prenatal donars, the locus coeruleus grafts produced many more fibers on the irides than did the DA grafts. It was concluded that the intraocular transplantation technique is very suitable for quantitative studies of nerve fiber production by immature monoamine neurons, and that it should be possible to study many other neuron systems in similar ways with this technique.Supported by the Swedish Medical Research Council (04X-03185), Magnus Bergvalls Stiftelse and Karolinska Institutets Fonder. The skilful technical assistance of Miss Ingrid Strömberg, Miss Maud Eriksson and Miss Gerd Boëtius is gratefully acknowledged. Thanks are due to Swedish Pfizer for the generous supply of Nialamid^®     

Brain tissue transplanted to the anterior chamber of the eye: 2. Fluorescence histochemistry of immature catecholamine and 5-hydroxytryptamine neurons innervating the rat vas deferens

Summary Small pieces of the wall of the rat vas deferens were homologously transplanted to the anterior chamber of the eye together with small pieces of embryonic brain stem containing either developing noradrenaline (NA) cells of the locus coeruleus or 5-hydroxytryptamine (5-HT) neurons of the developing raphe system. The eyes of the recipients were sympathetically denervated. The double transplants became rapidly vascularized from the host iris. After 31/2 months the irides, together with their two transplants were analyzed by Falck-Hillarp fluorescence microscopy. Both the NA and the 5-HT neurons had survived and matured in the eye. Fluorescent varicose nerve terminals of the NA and 5-HT type respectively were found in all three potential receptor areas, i.e. within the CNS transplants, in the host irides and in the vas deferens transplants. In the latter, the newly formed monoamine nerve terminals arborized mainly within a well developed smooth muscle layer. The density of such new fibres was higher than or similar to that of the normally present sympathetic plexus in areas of the transplant close to the CNS transplant and lower in areas at a distance from the CNS transplant. It is concluded that immature central NA and 5-HT fibres are able to grow simultaneously into different types of sympathetically denervated smooth muscle tissues to form networks of fibres in the receptor organs resembling the normal sympathetic innervation.Supported by the Swedish Medical Research Council (04X-3185), and by grants from Magnus Bergvalls Stiftelse, Tidningen Expressens Prenatalforskningsfond and Karolinska Institutets fonder. We thank miss Ingrid Strömberg for skilful technical assistance. Nialamide^® was generously donated by Swedish Pfizer AB.     

Distribution of Thy-1-like immunoreactivity in normal,sympathetically denervated and grafted mouse irides

Summary The distribution of Thy-1-like immunoreactivity was studied in whole-mounts of adult mouse iris and intraocular iris grafts 4 days and 4 weeks postoperatively. After fixation in picric acid/paraformaldehyde, the irides were incubated with a polyclonal rabbit anti-mouse brain Thy-1 antibody. In the adult mouse iris, a dense network of thin bundles and individual fibres was seen on the dilator plate and in the sphincter. Fluorescence paucities, resembling Schwann cell nuclei, were frequently seen along the bundles. Numerous mast cells, stained specifically with the Thy-1 antibody, were scattered over the entire surface of the iris. The ciliary body contained several brightly fluorescent bundles, and some circularly running individual fibres. In 4-day iris grafts, the Thy-1-like immunoreactivity had disappeared, except in mast cells. After 4 weeks in oculo, a regular plexus of thin fibres had reappeared in the iris grafts. Sympathetic denervation of adult irides did not seem to affect the Thy-1 immunoreactivity in terms of either fluorescence intensity or fibre distribution. The present data suggest a distribution of the glycoprotein Thy-1 along nerve fibres in the iris.     

Distribution of Thy-1-like immunoreactivity in normal, sympathetically denervated and grafted mouse irides

The distribution of Thy-1-like immunoreactivity was studied in whole-mounts of adult mouse iris and intraocular iris grafts 4 days and 4 weeks postoperatively. After fixation in picric acid/paraformaldehyde, the irides were incubated with a polyclonal rabbit anti-mouse brain Thy-1 antibody. In the adult mouse iris, a dense network of thin bundles and individual fibres was seen on the dilator plate and in the sphincter. Fluorescence paucities, resembling Schwann cell nuclei, were frequently seen along the bundles. Numerous mast cells, stained specifically with the Thy-1 antibody, were scattered over the entire surface of the iris. The ciliary body contained several brightly fluorescent bundles, and some circularly running individual fibres. In 4-day iris grafts, the Thy-1-like immunoreactivity had disappeared, except in mast cells. After 4 weeks in oculo, a regular plexus of thin fibres had reappeared in the iris grafts. Sympathetic denervation of adult irides did not seem to affect the Thy-1 immunoreactivity in terms of either fluorescence intensity or fibre distribution. The present data suggest a distribution of the glycoprotein Thy-1 along nerve fibres in the iris.     

Evidence for coexistence of ATP and nitric oxide in non-adrenergic,non-cholinergic (NANC) inhibitory neurones in the rat ileum,colon and anococcygeus muscle

The possible coexistence of the two non-adrenergic, non-cholinergic (NANC) inhibitory neurotransmitters, adenosine 5-triphosphate and nitric oxide in the myenteric plexus was investigated using whole-mount preparations of rat ileum, proximal colon and anococcygeus muscle. The presence of adenosine 5-triphosphate in neurones was examined using the quinacrine fluorescence technique. After localizing and taking photographs of quinacrine-fluorescent neurones and nerve fibres, the same tissues were then fixed and processed for NADPH-diaphorase activity, a marker for nitric oxide-containing neurones. We have demonstrated for the first time that almost all quinacrine-fluorescent myenteric neurones in the proximal colon are also NADPH-diaphorase reactive, while only a subpopulation of quinacrine-fluorescent neurones in ileum and anococcygeus muscle were also NADPH-diaphorase reactive.     

Locus coeruleus fibre growth in oculo induced by trigeminotomy

Locus coeruleus from fetal donors was homologously grafted to the anterior eye chambers of adult rats whose eyes were sympathetically denervated. After intraocular maturation, outgrowth of noradrenaline-containing fibres from the locus coeruleus neurons on the host iris was studied by Falck--Hillarp fluorescence histochemistry.In control animals locus coeruleus grafts produce a halo of noradrenaline-containing nerve fibres around the graft, covering approximately one third of the surface of the host iris. Sensory denervation of host eyes carrying maturated locus coeruleus grafts was produced by intracranial lesions of the trigeminal nerve. Such lesions induced a rapid growth response in the grafted locus coeruleus neurons, leading within three weeks to complete innervation of the host iris. It was concluded that removal of non-sympathetic, non-parasympathetic nerve fibres on the host iris elicits a strong fibre-growth response in the grafted locus coeruleus.     

Brain tissue transplanted to the anterior chamber of the eye

Summary Knowing the ontogenesis of the central monoamine neurons of the rat it is possible to obtain, by free-hand dissection from embryos and newly born animals, pieces containing dopamine (DA), noradrenaline (NA), and 5-hydroxytryptamine (5-HT) neurons that are small enough to permit homologous transplantation to the anterior chamber of the eye of adult animals. With this technique it was established that all three types of immature monoamine neurons are able to survive in the anterior chamber. Fluorescence histochemical analysis of whole mount preparations of the sympathetically denervated host irides revealed that both the catecholamine- and the 5-HT-neurons are able to partly reinnervate the irides, forming networks of varicose nerve terminals similar to the normally present sympathetic adrenergic ground plexus.Monoamine nerve cell bodies are attached to the irides but the majority of fluorescent nerve cell bodies is located within the transplants. Serial sectioning of these transplants showed rather well organized brain tissue, containing groups of fluorescent and non-fluorescent cell bodies, many areas being innervated by monoamine nerve terminals. When brain tissue was transplanted before the normal appearance of fluorescent neuroblasts (embryos with a crown-rump length less than 8 mm) monoamine neurons developed and matured within the eye.The amount of newly formed nerves of central origin recovered on the irides increased with time between the 2nd and 4th postoperative week and persisted after 2 months. The yield of new fibers was better using transplants from embryos with a crown-rump length between 15 and 30 mm than using transplants from larger embryos and newly born animals.If embryonic brain tissue known to be devoid of monoamine nerve cell bodies but containing monoamine nerve terminals in the adult state (cortex cerebri and cerebelli, spinal cord) was transplanted to sympathetically non-denervated eyes, the sympathetic adrenergic fibers seemed to be able to innervate the transplants.This work was supported by grants from the Swedish Medical Research Council (14×–3185), Karolinska Institutets fonder, and Magnus Bergvalls Stiftelse. We thank Miss Monica Eliasson, Mrs. Ulla Flyger, Mrs. Barbro Norstedt and Miss Ingrid Strömberg for skilful technical assistance. The generous gifts of Nialamide, Pfizer, and Pargyline, Abbott are gratefully acknowledged.     

Ultrastructural localization of monoamines in the epidermis of Lumbricus terrestris (L.)

Summary Receptor cells in the epithelium and the basiepithelial nerve net of the prostomium of Lumbricus terrestris were investigated with electron microscope with special regard to the presence of monoamines. The receptor cells are found in groups of about 40 intermingled with supportive cells. After pretreatment with -methyl-noradrenaline and fixation with potassium permanganate a few receptor cells in each group and some nerve fibres in the basiepithelial nerve net contain small granular vesicles (about400 Å) characteristic for monoaminergic neurons. The distribution and relative number of these receptor cells and nerve fibres coincide well with previous reports on fluorescent receptor cells and varicose fibres. That the monoamine-storing small granular vesicles not are visualized until pretreatment with -methyl-noradrenaline is in accordance with recent microspectrofluorometric analysis, which shows that dopamine is the only primary monoamine present in the epithelium.In the epithelium there are occasional receptor cells and nerve fibres containing large vesicles (1000–1800 Å) which resemble the neurosecretory vesicles in the central nervous system. Photoreceptor cells having an intracellular cavity with microvilli and cilia have infrequently been observed at the base of the epithelium.No synapses on the mucous cells have been noticed. Nor have any synaptic specializations been observed in the basiepithelial nerve net. The morphological conditions necessary for the existence of possible axo-axonal synapses are briefly discussed.This work was supported by grants from the Helge Ax: son Johnson Foundation and the Magn. Bergvall Foundation.     

Serotonin-immunoreactive neurones in the brain of Locusta migratoria innervating the corpus cardiacum

Summary The serotoninergic innervation of the corpus cardiacum (CC) of Locusta migratoria was investigated using two antisera against serotonin. A dense network of immunoreactive nerve fibres was present in the storage lobe of the CC. Immunopositive fibres only sporadically crossed the border between the storage lobe and the glandular lobe of the CC. Immunopositive fibres entered the storage lobe of the CC via the nervus corporis cardiaci I (NCCI); NCCII was immunonegative. Unilateral retrograde fillings of the NCCI with the fluorescent tracer Lucifer yellow, followed by antiserotonin immunocytochemistry, revealed about 20 double-labelled neurones in the anterior part of the pars intercerebralis. The double-labelled neurones were scattered between fluorescent non-immunoreactive neurones. Additionally, 5–7 neurones labelled only with Lucifer yellow were found at the ventrolateral side of the tritocerebrum. No immunopositive neurones were observed in the hypocerebral ganglion. Immunopositive fibres from neurones in the frontal ganglion ran via the recurrent nerve and the neuropile of the hypocerebral ganglion into the paired oesophageal nerve. At most, a few immunopositive nerve fibres occurred in the cardiostomatogastric nerves II, which connect the storage lobe of the CC with the paired oesophageal nerve at the caudal end of the hypocerebral ganglion.     

Ultrastructural localization of monoamines in the central nervous system of Lumbricus terrestris (L.) with remarks on neurosecretory vesicles

Summary The cerebral ganglion and the ventral nerve cord of Lumbricus terrestris have been studied with the electron microscope. The results are as follows: In the neuropile small granular vesicles (300 to 500 Å) occur in some varicose nerve fibres after fixation with potassium permanganate. This indicates the presence of noradrenaline. Sometimes only a few of the vesicles produce a positive reaction. After incubation with -methyl-noradrenaline the numbers of nerve terminals with small granular vesicles greatly increase, indicating the presence of dopamine and/or 5-hydroxytryptamine. In this case the reaction is now complete. The number of small granular vesicles is largest in the terminal swellings.These findings are consistent with histofluorescence, chemical, and microspectrofluorometric analyses, which have demonstrated noradrenaline, dopamine, and 5-hydroxytryptamine in neurones in the central nervous system.Large granular vesicles (600 to 900 Å) are to be found in some perikarya, not identical with neurosecretory cell bodies. In this case the granular vesicles in the axon are smaller and fewer. This indicates a simultaneous proximo-distal transport and gradual decrease in size of the granular vesicles. The intraneuronal distribution of the vesicles is in agreement with the distribution of the fluorophores in the fluorescent neurones.Neurosecretory neurones are found most likely not to contain monoamines.This work was supported by grants from the Helge Axison Johnson Foundation, the Magn. Bergvall Foundation, and the Roy. Physiographic Society at Lund.I am greatly indebted to Mrs. Lena Eriksson, Miss Rita Jönsson, Miss Inger Norling, Mrs. Lena Svenre, and Mr. Henryk Keff for their excellent technical assistance.     

Fate of intraocular chromaffin cell suspensions: role of initial nerve growth factor support

Summary Adrenal medullary tissue from adult rats was dissociated into cell suspensions and injected into the anterior chamber of the eye, where the cells were made to attach to the previously sympathectomized irides with the use of fibronectin. Short- and long-term survival of the chromaffin cells was examined in whole mounts of irides using Falck-Hillarp fluorescence histochemistry or indirect immunohistochemistry with antibodies against adrenaline and dopamine--hydroxylase (DBH). After 6 days in oculo all cells were immunoreactive for adrenaline; almost none displayed processes even if -nerve growth factor (NGF) was given at grafting. One month after weekly intraocular injections of NGF, many cells were surrounded by nerve fiber net-works and all cells were DBH-immunoreactive. Eight months postgrafting and 7 months after the last injection of NGF almost the entire iris was reinnervated and resembled a normal, sympathetically innervated iris. Both at 1 and 8 months, chromaffin cells, ganglion cells and transitional cell forms (chromaffin cells transforming towards ganglion-like cells) were found in irides from the NGF-treated eyes. The number of ganglion cells was remarkably increased with time by NGF, while the number of chromaffin cells decreased compared to controls. A single treatment with NGF at grafting had no marked effects as examined up to 3 months; at this time there was a certain outgrowth of nerve terminals, which, however, was not as pronounced as 1 month after repeated NGF injections. In conclusion, it is shown that some cells in a chromaffin cell suspension attach to the iris, transform to ganglion cells after an induction with exogenous NGF, and reinnervate the sympathically denervated iris. Such cells remain ganglion-like in character and continue to form processes even after cessation of exogenous NGF treatment.     

Thyroidectomy induces coated pit formation on cerebellar mossy fiber terminals

Summary The influence that thyroid hormones have on the development of the cerebellar cortex is well known. Their absence in adults leads to important functional changes probably related to abnormalities in nerve terminal activity, although no morphological alterations have hitherto been described. Using ultrastructural morphometric methods, we have studied the effects of thyroidectomy on the cerebellar cortex mossy fiber terminals of adult rats with different survival periods. No significant changes in the volume and surface area of these terminals were found. The numbers of synaptic vesicles in groups 7, 15 and 30 days after thyroidectomy were significantly greater than in controls. A significant increase in coated pit formation was found when thyroidectomized groups were compared with controls. This increase is due to the lack of thyroxin since it can be counteracted by thyroxin administration. Whether this increased coated pit formation is a membrane retrieval mechanism or is related to the uptake of extracellular molecules remains to be determined.     

Fluorescence-microscopical demonstration of a population of gastro-intestinal nerve fibres with a selective affinity for Quinacrine

Summary A population of nerve fibres in the gastro-intestinal tract of mice showing a high affinity for quinacrine was revealed by fluorescence microscopy. Similar results were obtained in rats and guinea pigs. Whole-mounts of sheets of the smooth muscle layer following incubation in 10^-6-10^-7 M quinacrine for 15–60 min revealed fine fluorescent varicose nerve fibers in the myenteric plexus of Auerbach both around nerve cell bodies and in the interconnecting strands. Many fibers were also present between the strands of the plexus, especially running parallel to the circular muscle layer. Such fibers were not seen in similarly quinacrine-incubated irides. A proportion of the cell bodies in Auerbach's plexus also showed quinacrine accumulation. These cells were apparently smaller neurons, sometimes with fluorescent processes. Intraperitoneal injections of quinacrine failed to demonstrate nerve fibers, but some cell bodies in Auerbach's plexus were positive. Subsequent paraformaldehyde treatment for monoamine visualization showed persistent adrenergic nerve terminals in the intestine and iris. These nerves seemed to be fewer and had a more yellow fluorescence than normally. The identity of the quinacrine-positive fibers is discussed with respect to recent suggestions that purinergic, substance P, enkephalin, and somatosin-containing nerves, in addition to adrenergic and cholinergic nerves, are present in the gut wall.Supported by the Swedish Medical Research Council (04X-03185). Magnus Bergvalls Stiftelse and Karolinska Institutets Fonder. For generous gifts of Mepacrine we thank Winthrop, Skärholmen, Stockholm, Sweden. The skilful technical assistance of Miss Gerd Boetius and Miss Maud Eriksson is gratefully acknowledged     

Adrenergic innervation of the mouse heart revealed at an early stage using α-methylnoradrenaline

Summary The development of noradrenergic innervation was studied in the mouse heart using fluorescence histochemistry. Following incubation of hearts with -methylnoradrenaline fluorescent nerve fibres were seen as early as 13 days in utero. It is suggested that the neuronal uptake mechanism for noradrenaline is functional at an early stage.     

Sexual dimorphism in the visual system of flies: The divided brain of male Bibionidae (Diptera)

Summary The mapping of the compound eyes onto the visual neuropils and the cell types in the lamina and the lobula complex of Bibionidae (Diptera) were studied by means of extracellular cobalt injections and Golgi impregnations. Dorsal and ventral eyes in males map into separate dorsal-and ventral neuropils up to the level of the lobula complex. The dorsal-eye lamina is unilayered, while the ventral-eye lamina in males and the lamina in females are multilayered: layers A and C are invaded by en-passant terminals of long visual fibres, layer B by the terminals of short visual fibres. Long visual fibres have a short and a long terminal in the ventral medulla with terminal specialisations in three distinct layers. Only one type of receptor ending exists in the dorsal medulla, the terminal branches of which are restricted to one layer only. Arrays of contralateral neurones are found in the medial part of the dorsal lobula, which receives input from the zone of binocular vision of the ipsilateral dorsal eye, and in the posterior dorsal lobula and lobula plate. The dorsal lobula plate contains large tangential neurones, the dendritic arborisations of which are revealed by cobalt injection into the thoracic ganglia. The divided brain of male bibionids offers the opportunity to investigate separately the nervous systems involved in sex-specific visually guided flight behaviour and in general visually guided flight control.     

Development of interscapular brown adipose tissue in the hamster. II - Differentiation of transplants in the anterior chamber of the eye: role of the sympathetic innervation

The relationship between brown adipose tissue (BAT) and its sympathetic innervation during development was investigated by transplantation of undifferentiated (white fat-like) hamster BAT into the anterior eye chamber of adult hamsters. Such transplants are known to be revascularized and reinnervated by the vessels and the nerves of the host iris. The morphology of the BAT transplants was analysed during the post-operative weeks by light and electron microscopy, and the ingrowth of sympathetic nerve fibres from the iris was followed by radioautography. BAT appeared to differentiate in oculo, i.e. presented increasing amounts of adipocytes with multilocular fat deposits and abundant, well-developed mitochondria, but only after a delay of approx. 10 days, and remained much fatter than in situ. The establishment of the sympathetic innervation was not synchronous with the revascularization process. It occurred simultaneously with the morphological differentiation of the BAT transplants, and the nerve fibre density remained low. In the absence of sympathetic innervation, i.e. when the host irides were sympathectomized prior to transplantation, BAT still differentiated, but the process was further delayed and the proportion of differentiated brown adipocytes after 20 days in oculo was clearly lower than in control transplants. It is concluded that the sympathetic innervation in BAT is involved in the regulation of differentiating activity in the tissue, but is not obligatory for differentiation to occur.     

Second messenger and protein phosphorylation mechanisms underlying opiate addiction: Studies in the rat locus coeruleus

We have studied the role of second messenger and protein phosphorylation pathways in mediating changes in neuronal function associated with opiate addiction in the rat locus coeruleus. We have found that chronic opiates increase levels of the G-protein subunits Gi and Go, adenylate cyclase, cyclic AMP-dependent protein kinase, and a number of phosphoproteins (including tyrosine hydroxylase) in this brain region. Electrophysiological data have provided direct support for the view that this up-regulation of the cyclic AMP system contributes to opiate tolerance, dependence, and withdrawal exhibited by these neurons. As the adaptations in G-proteins and the cyclic AMP system appear to occur at least in part at the level of gene expression, current efforts are aimed at identifying the mechanisms, at the molecular level, by which opiates regulate the expression of these intracellular messenger proteins in the locus coeruleus. These studies will lead to an improved understanding of the biochemical basis of opiate addiction.Special issue dedicated to Dr. Paul Greengard     

Production and characterization of a monoclonal antibody against human calcitonin gene-related peptide (CGRP) and its immunohistochemical application to salivary glands

Summary A monoclonal antibody (mAb), 129CD8 was raised against a C-terminal fragment (aa28–37) of -human calcitonin gene-related peptide (CGRP) coupled to bovine serum albumin. The specificity of the monoclonal antibody 129CD8 was corroborated by dot immunobinding experiments, enzyme-linked immunoassay and immunostaining of tissue sections. In vitro studies showed that the mAb 129CD8 readily recognized the fragment 28–37 of -human CGRP and to a slightly lesser degree whole -human CGRP and the fragments containing the C-terminal part of the molecule. The mAb 129CD8 also recognized the -human CGRP but not the -rat CGRP. The mAb 129CD8 did not react with substance P, katacalcin, calcitonin, amylin or fragments of -human CGRP lacking the C-terminal part of the molecule.Immunocytochemical staining was performed on human skin, guinea-pig thyroid and salivary glands and the trigeminal ganglion, and rat thyroid gland. Our findings demonstrate, in keeping with previous studies, that in human skin, nerve fibres containing CGRP immunoreactivity are found in both epidermis and dermis. In accordance with previous investigators, the Merkel cells were immunoreactive for CGRP. In the guinea-pig and rat thyroid gland CGRP immunoreactivity was localized in the C-cells. The distribution of CGRP immunoreactivity in the guinea-pig salivary glands is different from that previously reported for rat salivary glands. In the guinea-pig trigeminal ganglion, CGRP immunoreactivity was localized mainly in small-sized neurons and fibres traversing the ganglion. Double staining with substance P performed on guinea-pig trigeminal ganglion revealed four types of sensory neurons, those containing both peptides, those containing only substance P or CGRP and those lacking both peptides. Guinea-pig parotid gland, but not the submandibular or sublingual glands, contained periacinar fibres exhibiting both immunoreactivities. Substance P-positive, CGRP-positive fibres were also seen around parotid and submandibular, but not around sublingual, gland ducts. All glands received perivascular innervation showing immunoreactivities for both peptides. The present results support the idea that in the peripheral nervous system only a subpopulation of sensory neurons contains both substance P and CGRP. Consequently, colocalization of substance P and CGRP indicates a sensory nerve, while those containing either substance P or CGRP may be sensory or parasympathetic.     

Correlation of neuronal size and peptide immunoreactivity in the guinea-pig trigeminal ganglion

Summary Frequency and size of guinea-pig trigeminal neurones immunoreactive with antisera to -neo-endorphin(-neo-END), dynorphin A-(DYN), vasoactive intestinal polypeptide-(VIP), somatostatin-(SOM), and substance P-(SP) are reported. Co-localisation of the various peptides to the same ganglion cells was investigated immunocytochemically in consecutive 7-m thick paraffin sections. According to their size, all peptide-immunoreactive neurones belong to the class of small ganglion cells. Within this cell group, SP-immunoreactive neurones appear to be the largest, followed by SOM-, VIP-, -neo-END- and DYN-immunoreactive ganglion cells. The observed differences in size are statistically significant with the exception of that between -neo-END and DYN. This finding correlates well with the observed co-occurrence of the two immunoreactive peptides. All -neo-END-immunoreactive perikarya are also reactive to VIP antisera. These neurones are significantly smaller than those containing VIP-immunoreactivity exclusively. Ganglion cells displaying co-existence of -neoEND- and SP-immunoreactivity or VIP- and SP-immunoreactivity are found too infrequently to allow morphometric analysis. Some non-immunoreactive ganglion cells are shown to be approached by dense baskets of VIP-, -neo-END- or SP-immunoreactive varicose fibres, indicating the presence of intraganglionic modulation sites. The combination of immunohistochemistry and morphometry presented in this study allows the differentiation of diverse populations of primary afferent neurones exhibiting peptide immunoreactivity, most likely reflecting their involvement in different central and peripheral reflex arcs and sensory modalities.     

Distribution of sensory neurones of the pudendal nerve in the dorsal root ganglia and their projection to the spinal cord

Summary The morphology and distribution of the sensory neurones of the pudendal nerve within the spinal ganglia of rats were investigated by use of horseradish peroxidase (HRP). The labelling was visualized in diaminobenzidine (DAB) or tetramethyl-benzidine (TMB)-stained sections. Injection of HRP directly into the pudendal nerve labelled perikarya predominantly in the sixth lumbar DRG (L6). Following injection of HRP into the scrotal skin, however, additional cells were labelled in L5 and SI. Labelling was invariably unilateral. Approximately equal numbers of small (<30 m) and large neurones (>40 m) were labelled following subcutaneous injections although injections into the nerve marked twice as many small cells as large cells. This suggests that, in the rat, most of the small-diameter fibres within the pudendal nerve ascend through L6. Although a cluster of neurones was observed in one experiment, the remaining 25 experiments did not reveal any somatotopic arrangement since the labelled perikarya were distributed evenly throughout the ganglion. Similar numbers of retrogradely labelled neurones (somatopetal transport of the tracer) were observed in both DAB- and TMB-stained sections, although TMB allowed the demonstration of anterograde (somatofugal) HRP transport by terminal labelling in the superficial laminae of the lumbar spinal cord, extending into laminae II–IV.Partially supported by grants from the DFG to HWK (Ko 758/1)