The fine structure of the submucous plexus of the guinea-pig ileum. I. The ganglia, neurons, Schwann cells and neuropil

Summary A fine structural study was made of the ganglia, neurons, Schwann cells and neuropil of the submucous plexus of the guinea-pig ileum. The arrangement of the plexus as seen by light microscopy is briefly described. Submucous ganglia are small, containing an average of eight neurons per ganglion (compared with 43 in myenteric ganglia) and are connected with each other by fine nerve strands.The cell bodies of neurons and Schwann cells and a neuropil consisting of neuronal and Schwann cell processes form the ganglia. No other cell types or blood vessels are found within the ganglia. Ganglia are surrounded by a continuous basal lamina but lack a well-defined connective tissue investment. The glial investment of neurons is incomplete: many neurons lie directly beneath the basal lamina with no intervening Schwann cell processes, and the plasma membranes of adjacent neurons are often directly apposed over large areas. Other areas of apposition occur between the cell bodies and processes of neurons and Schwann cells. Desmosome-like membrane specializations may be seen between neurons and other neurons or Schwann cells. Submucous neurons could not be categorized according to size, shape, organelle content or types of processes. Processes emerging from nerve-cell bodies were placed into four broad categories on the basis of shape and microtubule content.Many bundles of closely apposed small nerve profiles lacking intervening Schwann processes are found in the neuropil in addition to a large number of vesiculated varicosities, some of which are directly apposed to the plasma membranes of nerve-cell bodies. A small proportion of vesiculated profiles form synapses with nerve cell bodies, their processes and profiles in the neuropil. From their structure, submucous neurons appear to form a more homogeneous population than myenteric neurons. Because of their incomplete investment they are more likely to be freely exposed to substances diffusing in the extraganglionic tissue than are neurons of sympathetic ganglia.     

Exocytosis from large dense cored vesicles outside the active synaptic zones of terminals within the trigeminal subnucleus caudalis: a possible mechanism for neuropeptide release

It has been hypothesized that chemical interactions between neurons in the central nervous system can occur in the absence of well defined synaptic complexes, but morphological correlates have been difficult to find. The present study demonstrates exocytotic release from large (70-130 nm) dense cored vesicles at structurally nonspecialized areas along the plasmalemma of structurally different categories of terminals and occasionally from dendrites and axons within the neuropil of the trigeminal subnucleus caudalis. In rats, the marginal (lamina I) and substantia gelatinosa (lamina II) layers contain the central terminals of primary afferent fibers from the infraorbital nerve that supply the skin and whiskers (vibrissae). Different types of interneurons are also present and may modify the input being relayed to higher centers. While exocytotic profiles were present in control animals, they increased significantly (P less than 0.01) on the ipsilateral side 1-24 h after a unilateral skin lesion in the vibrissae area. A second increase (P less than 0.001) occurred 14-15 days after the lesion. Virtually all examples of large vesicle exocytosis were observed at structurally nonspecialized sites while those at the active synaptic zones involved small clear vesicles. Substance P-like immunofluorescence, present in controls and on the ipsilateral side during the first 6 days, subsequently declined until 4 weeks after surgery when some recovery was noted. The increase in large vesicle exocytosis and the decrease in substance P are interpreted to reflect functional adjustments of different neurons in response to the lesion. The exocytosis involving large dense cored vesicles may serve to deliver transmitters and/or neuropeptide modulators to appropriate receptors in a wider area than release into a specialized synaptic cleft would allow.     

Serotoninergic synapses on ependymal and hypendymal cells of the rat subcommissural organ

Summary The nervous input to the subcommissural organ (SCO) of the rat has been investigated with Falck—Hillarp fluorescence histochemistry and electron microscopical techniques. Previous fluorescence histochemical observations of a dense plexus of serotoninergic nerve fibres in relation to the basal SCO were confirmed. Electron microscopically, unmyelinated fine varicose axons ranging in size from 0.1–0.6 m were observed to penetrate into the SCO hypendyma. Boutons and presynaptic varicosities filled with a diversity of round and elongated clear vesicles, and occasional large dense cored vesicles establish asymmetric (Gray's type I) synaptic contacts with the basal processes and somata of the SCO ependymal and hypendymal cells. A typical varicosity in synaptic contact with an SCO cell contains a population of approximately 85% clear, elongated vesicles 45 × 60 nm in diameter, 15% clear, round vesicles 50 nm in diameter, and 1–2% large dense cored vesicles with a vesicle diameter of about 85 nm and a dense core diameter of 50–55 nm. The mean length of the postsynaptic membrane specialization was found to be 0.5 m.Experiments with specific neurotoxic drugs revealed that the nerve terminals in synaptic contact with the SCO cells are identical to the fibres of the serotoninergic plexus identified fluorescence histochemically. Thus, an intraventricular injection of either 5,6-dihydroxytryptamine or 5,7-dihydroxytryptamine induced typical degenerative changes in most of the boutons in synaptic contact with the SCO cells, and also a disappearance of the yellow fluorescent nerve plexus. It is concluded that the SCO of the rat receives a dense plexus of serotonin-containing nerve fibres which form typical synaptic contacts with the specialized ependymal cells of the SCO and that these fibres may constitute the only direct nervous input to the organ.The degeneration of the serotoninergic synapses elicited a long-lasting, pronounced increase in the secretory activity of the SCO. Despite long survival times after the treatment with neurotoxic drugs, we found no evidence of regenerative restitution of the serotoninergic innervation nor normalization of the secretory activity of the SCO. The observed inverse relationship between secretory activity and serotoninergic innervation is in line with previous observations which indicate that the 5-hydroxytryptamine input to the SCO ependymal and hypendymal cells exerts a powerful inhibition on their protein synthetic machinery.Presented in part at the symposium Organization and Function of Central Catecholamine Neurons, December 10, 1975, Lund, Sweden.     

Extracellular matrix of the superior olivary nuclei in the dog

Summary The extracellular matrix around nerve cell bodies in canine lateral and medial superior olivary nuclei was examined by conventional electron microscopy, Golgi impregnation and histochemical techniques. Each neuron is surrounded by a region of myelin-free neuropil embedded amongst the myelinated fibres of the trapezoid body. In the myelin-free neuropil there are astrocytes, axons, synaptic boutons and extracellular matrix. The extracellular matrix fills the spaces between slender axons near the terminals, synaptic boutons and glial processes, but not the synaptic cleft. Golgi impregnation selectively stains the perineuronal nets which cover some or all of the nerve cell bodies and dendrites. The Golgi-EM method revealed that the impregnated profiles of the nets are restricted to the extracellular matrix. Synaptic boutons are situated in the holes of the perineuronal nets. Peanut (PNA) and soybean (SBA) agglutinins bound the extracellular matrix but not the synaptic boutons, glial processes, nerve cell bodies or basal lamina of blood capillaries. Light microscopic immunohistochemistry of the glial fibrillary acidic protein (GFAP) and S-100 protein did not stain a layer corresponding to the extracellular matrix and synapses but showed an intensely positive reaction immediately outside this layer. These data suggest the existence of a unique microenvironment associated with glycoconjugates around nerve cell bodies in canine superior olivary nuclei.     

FMRFamide-like immunoreactivity in the brain of the honeybee (Apis mellifera). A light-and electron microscopical study

Peptide-FMRFamide-like immunoreactivity in the brain and suboesophageal ganglion of the honeybee Apis mellifera L. is demonstrated with the peroxidase-antiperoxidase technique. Immunoreactivity is found in about 120 perikarya of the brain and in about 30 of the suboesophageal ganglion. These cells are distributed in 13 paired clusters representing neurons of different types including neurosecretory neurons projecting to neurohemal organs. Immunoreactivity of different intensity is found in the non-glomerular neuropil around the mushroom bodies, in the lateral protocerebrum, the central body, the optic tubercles, the lobula and medulla of optic lobe, the ocellar neuropil, in multiglomerular elements of the antennal lobes and in the dorsal deuterocerebrum. In the mushroom bodies, immunoreactivity is located in layers of the lobes and stalks, corresponding to intrinsic fibre bundles of some Kenyon cell types. The somata of these intrinsic cells did not show FMRFamide-like immunoreactivity. Electron microscopy of immunostained somata and nerve fibres was performed employing a pre-embedding peroxidase-antiperoxidase technique. Fibres of optic lobes and the non-glomerular neuropil contain immunoreactive dense core vesicles (diameter 50-165 nm) accumulated in boutons besides small synaptic vesicles and synaptic membrane specializations. Immunoreactive layers of the mushroom body neuropil were analysed at the ultrastructural level. Axon profiles with dense-core vesicles of a small type (diameter 35-75 nm) show only faint immunoreactive products. Immunoreactivity of intrinsic mushroom body neurons does not appear to be specifically correlated with synaptic organelles. Our results indicate that FMRFamide or related peptides peptides may be neuroactive compounds in different classes of nerve cells in the bee brain.     

Ultrastructure of primary afferent fibres and terminals expressing alpha-galactose extended oligosaccharides in the spinal cord and brainstem of the rat

Summary The ultrastructural characteristics of primary afferent fibres, which express -galactose extended oligosaccharides recognized by LD2 and LA4 monoclonal antibodies, and the subcellular localization of these oligosaccharides were studied. LD2 and LA4 antibodies both label intensely the plasma membrane of primary afferent fibres, and with LD2 antibody all immunoreactive profiles also possessed strong intracellular staining. In contrast, intracellular staining with LA4 antibody was observed in only a subpopulation of stained profiles. LD2-immunoreactive fibres were detected in trigeminal and Lissauer tracts and in lamina I (LI) and lamina II (LII), and appeared as a mixture of unmyelinated and myelinated fibres. The highest density of LD2-immunoreactive synaptic boutons was found in lamina II outer (LII_o). Many of the terminals were simple dome-shaped terminals, making single asymmetric synapses over small and medium-sized dendritic shafts and dendritic spines. All LA4-immunoreactive fibres were unmyelinated. In addition, some small scalloped central-glomerular terminals contacting two or three dendrites were found. LA4-immunoreactive fibres were found more frequently than terminals and appeared most heavily immunostained in trigeminal and Lissauer tracts. In the neuropil of LI and LII, LA4 profiles were generally very weakly immunostained, although a small sample of immunostained synaptic boutons was detected. All LA4-immunoreactive terminals were found in lamina II inner (LII_i) and made simple asymmetric axodendritic synapses. In addition to axons and terminals, some dendrites exhibited LD2 immunoreactivity and this was most intense in the region of synaptic vesicles. In addition to neurons, some endothelial cells were immunostained with LD2 antibody and astrocytes were immunostained with LA4 antibody.     

Development of synaptic ultrastructure at neuromuscular contacts in an amphibian cell culture system

Summary Cultures of dissociated myotomal muscle and spinal cord derived from embryos ofXenopus laevis were grown in the presence of curare in order to abolish neuromuscular activity and were examined by electron microscopy. In one-day-old cultures a few of the neuromuscular contacts already displayed several synaptic specializations including 500 Å vesicles clustered against the axolemma, increased axolemmal densities, basal lamina in the cleft, an increased sarcolemmal density and subsarcolemmal filamentous material. Contacts with these specializations were observed more frequently in two and three-day-old cultures. Throughout the three-day culture period nerve fibres and neuromuscular contacts were devoid of Schwann cells. Isolated patches of basal lamina were relatively scarce and were usually accompanied by an increase in sarcolemmal density and subsarcolemmal filamentous material even in cultures in which spinal cord cells were not included. These observations indicate that the myotomal neuromuscular synapse differentiates in culture in much the same way as it does in vivo, that muscle contractions are not required for its differentiation, and that apparent postsynaptic specializations can develop in the absence of innervation.     

Immunohistochemical localization of a novel acidic calmodulin-binding protein, NAP-22, in the rat brain.

NAP-22 is a neuronal tissue-enriched acidic calmodulin-binding protein with a molecular mass of 22,000 and is recovered in the membrane fraction during biochemical fractionation. We observed the distribution pattern of this protein in the rat brain using an immunohistochemical method by light and electron microscopy. NAP-22 immunoreactivity was detected through the whole brain, and the most dense staining was observed in the forebrain including cerebral cortex, hippocampal formation, olfactory bulb, basal ganglia and thalamus. Immunoreactivity was distributed densely at the neuropil, whereas nerve cells and nerve fibres had little or no reaction. In the brain stem, immunonegative large nerve cell bodies were surrounded by immunopositive varicosities. In the cerebellar cortex, mossy fibre terminals and parallel fibres showed immunoreactivity, whereas Purkinje cells did not. Intracellular distribution was observed in the cerebral and cerebellar cortices. NAP-22 immunoreactivity was noted in the axon terminals, dendritic spines and thin nerve fibres. In these structures, reaction products were associated mainly with synaptic vesicles, pre- and postsynaptic membranes and microtubules. This study demonstrates that the immunoreactivity of NAP-22 is distributed widely in the brain, especially in the synapse, and suggests that this protein is involved in synaptic transmission both in the pre- and postsynaptic region.     

Synaptogenesis in the basilar papilla of the chick

Summary Synaptogenesis was studied in the basilar papilla of chicken embryos from days 7–21 of incubation. On the 9th day of incubation differentiating hair cells first appeared and a few growing nerve tips made contact with them, although no membrane specializations were apparent at this stage. Synaptic bodies associated with presynaptic membrane specializations were first observed on the 10th day. They lay opposite either supporting cells or afferent nerve processes; in the latter site slight membrane thickenings were occasionally found. During subsequent stages synaptic bodies and the surrounding vesicles increased in number. Synaptic bodies associated with presynaptic membrane specializations, but devoid of contact with afferent nerve endings, were often observed on the 14th day, whereas almost all the synaptic bodies associated with presynaptic specializations were in contact with afferent nerve processes by the 21st day. The efferent synapses were first recognized on the 14th day. These results suggest that in the hair cells of chicken basilar papilla the synaptic bodies and presynaptic membrane specializations appear first and after the synaptic sites are determined by the position of the synaptic bodies, the growing nerve tips seek out and establish synaptic contact at the pre-existing synaptic sites.     

Ultrastructural demonstration of oxytocin and vasopressin release sites in the neural lobe and median eminence of the rat by tannic acid and immunogold methods

We have investigated the localization of oxytocin (OXT) and vasopressin (AVP) release sites in the neural lobe and median eminence (ME) of the rat. Ultrastructural and post-embedding immunocytochemical studies with protein A-gold as a marker were used for the identification of OXT and AVP neurosecretory granules (NSG). Release of the contents of these OXT and AVP NSG by exocytosis has been clearly demonstrated with the tannic acid-Ringer incubation method in axon terminals of the neural lobe and in varicose fibres with OXT and AVP NSG in the internal zone of the ME. As these fibers lack synaptic specializations, and are not located in the direct vicinity of capillaries of the primary portal plexus, these observations suggest that OXT and AVP are released in a paracrine way in the ME of the rat.     

The fine structure of the ependymal surface of the recessus infundibularis in the rat

Summary The surface of the recessus infundibularis of the third ventricle has been studied with the scanning and transmission technique in normal and experimental material.Surface specializations such as microvilli, craters and areas of discontinuous lining are described. Supraependymal cells and fibres have been found; some of these cells form wide-meshed networks. The supraependymal fibres may be regular or varicose; the former seem to perforate the ependyma.With the transmission electron microscope the supraependymal cells are divided into three categories: nerve cells, lymphocytes and dense cells. Two fibre populations are distinguished: thin profiles (nerve fibres) and thick profiles (nerve terminals). Axosomatic and axoaxonic synapses are described.Synapses between supraependymal fibres and ependyma cells have also been found.     

Ultrastructure and synaptic relationships of calbindin-reactive, Dogiel type II neurons, in myenteric ganglia of guinea-pig small intestine

Summary Immunoreactivity for calbindin D 28K was localized ultrastructurally in nerve cell bodies and nerve fibres in myenteric ganglia of the guinea-pig small intestine. Reactive cell bodies had a characteristic ultrastructure: the cytoplasm contained many elongate, electron-dense mitochondria, numerous secondary lysosomes that were peripherally located, peripheral stacks of rough endoplasmic reticulum and dispersed Golgi apparatus. The cells were generally larger than other myenteric neurons and had mainly smooth outlines. The cytoplasmic features of these neurons were shared by a small group of immunonegative cells, but the majority of negative cells had clearly different ultrastructural appearances. Of 310 cells from 16 ganglia that were systematically examined, 38% were immunoreactive for calbindin, 10% were unreactive but similar in ultrastructure to the calbindin-reactive neurons and 51% were unreactive and dissimilar in the appearance of their cytoplasmic organelles. Immunoreactive varicosities with synaptic specializations were found on most unreactive neurons, but were markedly less frequent on the calbindin-immunoreactive cell bodies. Non-reactive presynaptic fibres were also more common on non-reactive neurons than on the calbindin-positive cell bodies. Numerous reactive varicosities, some showing synaptic specializations, were found adjacent to other fibres in the neuropil. Light microscopic studies show calbindin immunoreactive neurons to have Dogiel type-II morphology. Thus the present work links distinguishing ultrastructural features to a specific nerve cell type recognized by light microscopy in the enteric ganglia for the first time.     

Periterminal synaptic organization of primary afferents in laminae I and IIo of the rat spinal cord, as shown after anterograde HRP labelling

Summary The fine structure and periterminal synaptology of the primary afferent terminations in laminae I and IIo are examined in the rat, following anterograde labelling with horseradish peroxidase applied to the right C₅-dorsal root. Labelled varicosities observed along the terminal arbors in parasagittal thick sections were relocated in ultrathin sections by electron microscopy. The labelled terminal profiles generated by the three primary afferent plexuses which can be identified by light microscopy in laminae I-IIo had similar fine structural features, except that axo-axonal contacts, although rare, were more frequent in the medial network plexus. Primary boutons were packed with agranular spherical vesicles and some large granular vesicles, and were mostly presynaptic to profiles of dendritic trunks of marginal cells. Unlabelled axonal profiles, either light with some flattened vesicles, or dense with round vesicles, were also presynaptic at symmetrical or asymmetrical contacts, respectively, to those dendritic profiles. It is suggested that such knobs of intrinsic origin are responsible for postsynaptic modulation of the primary noxious input. Although the 20 m wide lamina IIo belongs cytoarchitectonically to lamina II and can be distinguished from lamina I by a decreased amount of myelinated fibres and large dendritic profiles, the periterminal synaptology was here found to be the same as in lamina I.     

Visceral and somatic afferent origin of calcitonin gene-related peptide immunoreactivity in the lower thoracic spinal cord of the rat

The origin of calcitonin gene-related peptide in the thoracic spinal cord of the rat was investigated by radioimmunoassay and immunohistochemistry. In transverse sections from normal animals there was a dense staining of calcitonin gene-related peptide-immunoreactivity in laminae I, II and V of the dorsal horn. In parasagittal sections this was found to consist of rostrocaudally orientated fibres in laminae I and II and longitudinal bundles of fibres interspersed with a plexus of immunoreactivity in lamina V. After sectioning the thoracic spinal nerves there was a significant reduction in immunoreactivity in the dorsal horn of the spinal cord which was seen as a marked reduction of staining in lamina II and in the bundles of fibres in lamina V. Section of the splanchnic nerve slightly reduced staining in lamina I and virtually abolished the plexuses of immunoreactivity in lamina V. However, measurement of calcitonin gene-related peptide in samples from coeliac-ganglionized rats revealed an increase in immunoreactivity in regions of the spinal cord containing lamina V. These results provide evidence of a visceral and somatic afferent origin of calcitonin gene-related peptide in the thoracic spinal cord of the rat.     

Effect of puromycin treatment on the regeneration of hemisected and transected rat spinal cord

Summary The effect of puromycin on spinal cord regeneration was studied following implantation into the site of spinal cord hemi- or transection of Gel-foam saturated with puromycin (1mM) in a saline carrier, implantation of Gel-foam sponge saturated with saline (carrier control), or lesion alone (lesion control). The spinal cords of 107 rats were studied with light and electron microscopy 7, 14, 30, 60 and 90 days postoperative (DPO). Spinal cord hemisected animals developed a dense cicatrix at the site of lesion replete with connective tissue, blood vessels, and myelinated and unmyelinated nerve fibres which could be traced to peripheral sources. Rostrally at the C.N.S.-cicatrix interface, there were reactive neuroglial cells, occasional nerve fibres and finger-like projections of spinal cord (due to cavitation lesions) which contained neuroglia, axons and dendrites. Implantation of saline in Gel-foam resulted in the same morphology as in hemisected animals except for increased lesion size due to mechanical factors and decreased cicatrix density during the first 30 DPO. Puromycin treatment resulted in a cicatrix with initial decreased cell density but which contained a new class of nerve fibres at 30 DPO. These nerve fibres were oriented in a rostro—caudal direction, were unmyelinated, 0.1–0.2 m in diameter and had expanded smooth endoplasmic reticulum. Some of these nerve fibres were degenerating at 30 DPO and all were absent by 60 DPO. The puromycin-treated spinal cord within 200 m rostral to the basal lamina contained nerve terminal conglomerates, which resembled boutons, in fascicles from 30–90 DPO (duration of experiment). Hemisection of the spinal cord by crushing 1-11/2 segments rostral to the site of puromycin implantation at 30 DPO resulted in degeneration of these nerve fibres in the cicatrix as well as the degeneration of nerve terminal conglomerates just rostral to the basal lamina. The regenerative capacity of the spinal cord is discussed in relationship to these findings.     

Ultrastructural identification of VIP-containing nerve fibres in the myenteric plexus of the rat ileum

Summary Vasoactive intestinal polypeptide (VIP)-containing nerve fibres of myenteric plexus of the rat ileum were investigated using the pre-embedding peroxidase-antiperoxidase technique for electron microscopy. The VIP-positive fibres were characterized by a predominance of spherical agranular vesicles with a diameter of 40–55 nm. The immunoprecipitate was largely localized on the vesicle membrane. Large granular vesicles (100–144 mm) containing labelled dense cores were also present within labelled nerve profiles. Quantitative data indicate that the vesicles appeared in the following proportions: 93% small agranular vesicles and 7% large granular vesicles.     

血管周肾上腺素能神经密度与管壁组成成分的关系

本文应用组织化学技术和电镜方法对家兔、豚鼠和大鼠血管周的肾上腺素能神经密度与管壁组成成分的关系进行了观察。实验结果证明,血管周神经分布于外膜层,中膜内未见有神经分布,肌性动脉(以肠系膜动脉为代表)较弹性动脉(以颈总动脉为代表)的血管周神经密度和含膨体数都较高。神经肌肉间隔近(0.05—3微米),最近者神经与肌肉间除基板外无其它组织成分。弹性动脉神经肌肉间隔较远(1—12微米),神经肌肉间隔以外弹力膜、成纤维细胞和板层状的结缔组织。股动脉和肾动脉周的神经分布特点介于上二者之间。静脉较相应动脉神经分布稀疏。但也存在部位的特殊性和种属差异性。如脐动脉虽属肌性动脉,但动脉周并无神经分布,豚鼠肾动脉周的神经密度远较兔及大鼠稀疏。作者认为血管周的神经密度与血管壁中平滑肌的含量有关。本文并对肌性动脉周神经分布致密的原因,不同类型动脉的神经分布特点与生理功能的关系进行了讨论。     

The primary afferent projection of the greater petrosal nerve to the solitary complex in the rat, revealed by transganglionic transport of horseradish peroxidase

The primary afferent projection of the greater petrosal nerve (GPN) to the solitary complex was studied following application of horseradish peroxidase (HRP) to the GPN just distal to the geniculate ganglion. Labeled fibers were traced to the most rostral part of the solitary tract. Numerous collaterals entered the solitary complex from its dorsal and lateral aspects, and formed a dense plexus. They terminated in the dorsal half of the medial solitary nucleus at the level of the rostral half of the solitary complex, and in the ventrolateral and commissural nuclei at the level of the caudal half. The densest termination was observed in the medial solitary nucleus. Labeled terminals were found to contain round, clear synaptic vesicles and to make asymmetrical synaptic contacts with dendritic profiles.     

Basal lamina and the organization of neuromuscular synapses

Fast chemical synapses are comprised of presynaptic and postsynaptic specializations precisely aligned across a protein-filled synaptic cleft. At the vertebrate neuromuscular junction (NMJ), the synaptic cleft contains a structured form of extracellular matrix known as a basal lamina (BL). Synaptic BL is molecularly differentiated from the BL that covers the extrasynaptic region of the myofiber. This review summarizes current understanding of the morphology, composition, and function of the synaptic BL at the vertebrate NMJ. Considerable evidence supports the conclusion that the synaptic BL organizes and maintains pre- and postsynaptic specializations during development and regeneration, and promotes robust neurotransmission in the adult.