An antiserum specific for cholinergic synaptic vesicles from electric organ

Rabbit antisera to highly purified synaptic vesicles from the electric organ of Narcine brasiliensis, an electric ray, reveal a unique population of synaptic vesicle antigens in addition to a population shared with other electric organ membranes. Synaptic vesicle antigens were detected by binding successively rabbit antivesicle serum and radioactive goat anti-rabbit serum. To remove antibodies directed against antigens common to synaptic vesicles and other electric organ fractions, the antivesicle serum was extensively preadsorbed against an electric organ membrane fraction that was essentially free of synaptic vesicles. The adsorbed serum retained 40% of its ability to bind to synaptic vesicles, suggesting that about half of the antigenic determinants are unique. Vesicle antigens were quantified with a radioimmunoassay (RIA) that utilized precipitation of antibody-antigen complexes with Staphylococcus aureus cells. By this assay, the vesicles, detected by their acetylcholine (ACh) content and the antigens detected by the RIA, have the same buoyant density after isopycnic centrifugation of crude membrane fractions on sucrose and glycerol density gradients. The ratio of ACh to antigenicity was constant across the vesicle peaks and was close to that observed for vesicles purified to homogeneity. Even though the vesicles make up only approximately 0.5% of the material in the original homogenate, the ratio of acetylcholine to vesicle antigenicity could still be measured and also was indistinguishable from that of pure vesicles. We conclude that synaptic vesicles contain unique antigenic determinants not present to any measurable extent in other fractions of the electric organ. Consequently, it is possible to raise a synaptic vesicle- specific antiserum that allows vesicles to be detected and quantified. These findings are consistent with earlier immunohistochemical observations of specific antibody binding to motor nerve terminals.     

Fine-structural study of the pineal body of the monkey (Macaca fuscata) with special reference to synaptic formations

Summary Various types of synaptic formations on pinealocytes and pineal neurons were found in the pineal body of Macaca fuscata. Axo-somatic synapses of the Gray type-II category were detected on the pinealocyte cell body. Gap junctions and ribbon synapses were observed between adjacent pinealocytes. About 70 nerve-cell bodies were detected in one half of the whole pineal body bisected midsagittally. They were localized exclusively deep in the central part. When examined electron-microscopically, they were found to receive ribbon-synapse-like contacts from pinealocytic processes. They also received synaptic contacts of the Gray type-I category on their dendrites, and those of the Gray type-II category on their cell bodies from nerve terminals of unknown origin. All these synapse-forming axon terminals contained small clear vesicles. Thus, the pineal neurons of the monkey, at least in part, are suggested to be derived from the pineal ganglion cells in the lower vertebrates and not from the postganglionic parasympathetic neurons. The functional significance of these observations is discussed in relation to the innervation of the pineal body of the monkey.     

Identification of a Synaptic Vesicle Antigen (Mr 86,000) Conserved Between Torpedo and Rat

Antisera were raised in guinea pigs to synaptic vesicles purified from the electric organ of Torpedo marmorata. In cholinergic nerve terminals from Torpedo the major antigens identified had Mr 300,000-150,000, 86,000, and 18,000. The Mr 86,000 antigen was conserved between Torpedo and rat, where it is neuron-specific and concentrated in nerve terminals. When rat brain synaptosomes are subfractionated the antigen is associated with synaptic vesicles. The antigen is not found in the cytoskeleton and in the vesicle-free cytosol. Immunohistochemical localization of the antigen in rat shows it to be associated with synapses in diaphragm, cerebellum, hippocampus, and cerebral cortex. The staining pattern of the antigen indicates that the antigen is not cholinergic-specific. The function of the Mr 86,000 antigen remains to be identified.     

Identification of a synaptic vesicle-specific membrane protein with a wide distribution in neuronal and neurosecretory tissue

Two different monoclonal antibodies, characterized initially as binding synaptic terminal regions of rat brain, bind a 65,000-dalton protein, which is exposed on the outer surface of brain synaptic vesicles. Immunocytochemical experiments at the electron microscope level demonstrate that these antibodies bind the vesicles in many different types of nerve terminals. The antibodies have been used successfully to purify synaptic vesicles from crude brain homogenates by immunoprecipitation onto the surface of polyacrylamide beads. The profiles of the structures precipitated by these beads are almost exclusively vesicular, confirming the vesicle-specificity of the antibodies. In SDS gels, the antibodies bind a single protein of 65,000 daltons. The two antibodies are not identical, but compete for binding sites on this protein. Immune competition experiments also demonstrate that the antigenic components on the 65,000-dalton protein are widely distributed in neuronal and neural secretory tissues. Detectable antigen is not found in uninnervated tissue--blood cells and extrajunctional muscle. Low levels are found in nonneural secretory tissues; it is not certain whether this reflects the presence of low amounts of the antigen on all the exocytotic vesicles in these tissues or whether the antigen is found only in neuronal fibers within these tissues. The molecular weight and at least two antigenic determinants of the 65,000-dalton protein are highly conserved throughout vertebrate phylogeny. The two antibodies recognize a 65,000-dalton protein present in shark, amphibia, birds, and mammals. The highly conserved nature of the determinants on this protein and their specific localization on secretory vesicles of many different types suggest that this protein may be essential for the normal function of neuronal secretory vesicles.     

Structure and innervation of the pineal gland of the rabbit,Oryctolagus cuniculus (L.)

In the rabbit pineal gland two types of postganglionic nerve endings were found which are characterized by the presence of small dense-core vesicles or small clear vesicles. Pharmacological and cytochemical experiments showed then to be noradrenergic and cholinergic, respectively. Both types were often present in the same nerve bundle, occasionally in close opposition. Intrapineal neurons were only rarely observed. They showed cholinergic synapses on their perikaryon and dendrites as well as noradrenergic axo-dendritic close contacts. Bilateral extirpation of the superior cervical ganglia revealed the postganglionic sympathetic origin of the pineal noradrenergic nerve fibres. Moreover, it appeared that these ganglia are hardly, if at all, involved in the pathway of pineal cholinergic innervation. The results obtained from lesions of both facial nerves, taken together with the results reported in the literature, led to the conclusion that the postganglionic cholinergic nerve fibers in the pineal are of parasympathetic origin. A model for the sympathetic and parasympathetic pineal innervation is proposed.     

Ultrastructural organization of the synapses in ganglia of the hen intestinal nerve under various conditions of its activity

The interneuronal connections in ganglia of the caudal part of the hen intestinal nerve of Remak are presented as axodendritic and axosomatic synapses and symmetric axo-axonal, dendro-dendritic and axodendritic contacts, often forming complicated complexes. Under conditions of preliminary decentralization or under certain disturbances of nervous connections with the intestine, a part of synapses remains, and a part of them degenerates, this demonstrates participation of peripheral afferent neurons in formation of the synaptic apparatus of the ganglia mentioned. The axonal terminals differentiate by composition of the synaptic vesicles: some contain mainly light agranular vesicles, others--a large amount of granular ones. The characteristic peculiarities of the hen intestinal nerve ganglia, in contrast to analogous mammalian ganglia, are abundant axosomatic synapses in some neurons, and presynaptic terminals, containing a large number of granular vesicles.     

A highly antigenic proteoglycan-like component of cholinergic synaptic vesicles

A monoclonal antibody, tor70, recognizes an antigenic determinant on the inside surface of synaptic vesicles, purified from the electric organ of Narcine brasiliensis. The antigenic determinant appears to be unique to vesicles since it co-purifies with vesicle content and is blocked by an antiserum specific for synaptic vesicle antigens. Immunoblotting of vesicle proteins after sodium dodecyl sulfate-polyacrylamide gel electrophoresis shows that the antigen has a low heterogeneous electrophoretic mobility and corresponds to a major protein component of pure synaptic vesicles. Synaptic vesicles contain a proteoglycan-like material since proteolytic digestion yields a ruthenium red-binding material that migrates during electrophoresis with a mammalian heparin standard. The only major vesicle component with which the proteoglycan-like material co-elutes during chromatography on Sepharose 6B is the material recognized by tor70. The antigen adsorbs specifically to beads coated with the lectin wheat germ agglutinin. Isolation of the tor70 antigen by velocity sedimentation in sodium dodecyl sulfate-sucrose gradients shows it to contain glucosamine (0.75 nmol/microgram of protein) and uronic acid but no galactosamine. Earlier work has shown that specific antiserum to pure synaptic vesicles could be used to identify nerve terminals, quantitate vesicle components, purify membranes, and monitor exocytosis. We now know that one of the components recognized by the antiserum is a molecule with properties of a proteoglycan, attached to the inside surface of vesicle membranes.     

VAT-1: an abundant membrane protein from Torpedo cholinergic synaptic vesicles

Expression screening was used to isolate cDNA clones encoding a synaptic vesicle membrane protein, VAT-1, which is specifically expressed in the electric lobe of marine rays. The predicted protein has a molecular weight of 41,572 daltons and contains several hydrophobic regions. An antibody raised against a fusion protein synthesized in E. coli recognizes an abundant 42 kd protein that copurifies largely with synaptic vesicles. Trypsin digestion of intact and lysed vesicles as well as membrane extractions suggests that VAT-1 is an integral membrane protein. The VAT-1 RNA is localized to the electromotor nucleus, and the fusion protein antibody stains the electric organ, demonstrating that the protein is transported to nerve terminals. These studies define a novel synaptic vesicle protein that is likely to play a central role in the functions mediated by specific classes of synaptic vesicles.     

Electron microscopic study on the innervation of the pancreas of the domestic fowl

Summary The innervation of the pancreas of the domestic fowl was studied electron microscopically. The extrapancreatic nerve is composed mostly of unmyelinated nerve fibers with a smaller component of myelinated nerve fibers. The latter are not found in the parenchyma. The pancreas contains ganglion cells in the interlobular connective tissue. The unmyelinated nerve fibers branch off along blood vessels. Their synaptic terminals contact with the exocrine and endocrine tissues. The synaptic terminals can be divided into four types based on a combination of three kinds of synaptic vesicles. Type I synaptic terminals contain only small clear vesicles about 600 Å in diameter. Type II terminals are characterized by small clear and large dense core vesicles 1,000 Å in diameter. Type III terminals contain small clear vesicles and small dense core vesicles 500 Å in diameter. Type IV terminals are characterized by small and large dense core vesicles. The exocrine tissue receives a richer nervous supply than the endocrine tissue. Type II and IV terminals are distributed in the acinus, and they contact A and D cells of the islets. B cells and pancreatic ducts are supplied mainly by Type II terminals, the blood vessels by Type IV terminals.This work was supported by a scientific research grant (No. 144017) and (No. 136031) from the Ministry of Education of Japan to Prof. M. Yasuda     

Innervation of the guinea pig spleen studied by electron microscopy

The innervation of the guinea pig spleen was investigated by electron microscopy. Unmyelinated nerve fibers in the capsulotrabecular and arterial systems were found to contain large and small granular and small agranular synaptic vesicles in their terminals and are thought to be sympathetic adrenergic in nature. They influence the contraction of the smooth muscle cells by diffusion innervation in these systems. These nerve terminals were also scattered in both the red and the white pulp. Pulp nerves wrapped by Schwann cells were further enclosed by myofibroblastic reticular cells. This condition revealed that the pulp nerves pass through the connective-tissue spaces of the reticular fibers, which contain elastic fibers, collagenous fibrils, and lamina densa-like materials of the usual basement laminae. One of the target cells for the pulp nerves is considered to be the myofibroblastic reticular cell in the reticular meshwork. Neurotransmitter substances released from the naked adrenergic nerve terminals travel through the reticular fibers and may play a role, by both close association innervation and diffusion innervation, in the contraction of reticular cells to expose the reticular fibers. At the exposed sides, connective-tissue elements of the reticular fibers are bathed with blood plasma, and the included naked nerve terminals, devoid of Schwann cells but with basement laminae of these cells, face free cells at some distance or are in close association with free cells, especially lymphocytes, macrophages, and plasma cells. The close ultrastructural relationship between the naked adrenergic nerve terminals and immunocytes strongly suggests that there is an intimate relationship between the immune system and the sympathetic nervous system through both close association innervation and diffusion innervation. Thus splenic adrenergic nerves of the guinea pig may play a triple role in 1) contraction of smooth muscle cells to regulate blood flow in the organ, 2) induction of the exposure of reticular fibers by contraction of the reticular cells in order to form a close relationship of the nerve terminals with the immunocytes, and 3) subsequent neuroimmunomodulation of the immunocytes.     

Neuronal distribution and subcellular localization of HCNP-like immunoreactivity in rat small intestine

A novel peptide, hippocampal cholinergic neurostimulating peptide (HCNP), originally purified from young rat hippocampus, affects the development of specific cholinergic neurons of the central nervous system in vitro. In this study, HCNP-like-immunoreactive nerve processes and nerve cell bodies were identified by electron microscopic immunocytochemistry in the rat small intestine. Labeled nerve processes were numerous in the circular muscle layer and around the submucosal blood vessels. In the submucosal and myenteric plexuses, some HCNP-like-immunopositive nerve cell bodies and nerve fibers were present. The reaction product was deposited on the membranes of various subcellular organelles, including the rough endoplasmic reticulum, Golgi saccules, ovoid electron-lucent synaptic vesicles in axon terminals associated with submucosal and myenteric plexuses, and the outer membranes of a few mitochondria. The synaptic vesicles of HCNP-like-positive terminals were 60–85 nm in diameter. The present data provide direct immunocytochemical evidence that HCNP-like-positive nerve cell bodies and nerve fibers are present in the submucosal and myenteric plexuses of the rat small intestine. An immunohistochemical light microscopic study using mirror-image sections revealed that in both the submucosal and myenteric ganglia, almost all choline acetyltransferase (ChAT)-immunoreactive neurons were also immunoreactive for HCNP. These observations suggest (i) that HCNP proper and/or HCNP precursor protein is a membrane-associated protein with a widespread subcellular distribution, (ii) that HCNP precursor protein may be biosynthesized within neurons localized in the rat enteric nervous system, and (iii) that HCNP proper and/or HCNP precursor protein are probably stored in axon terminals.     

Substance P-containing axon terminals in the mucosa of the human urinary bladder: pre-embedding immunohistochemistry using cryostat sections for electron microscopy

The ultrastructure of substance P (SP)-containing axon terminals in the mucosa of the human urinary bladder was studied. Numerous SP-immunoreactive varicose nerve fibers were seen in the lamina propria, and most of them ran freely in the connective tissue. Many SP-immunoreactive nerve fibers were observed beneath the epithelium, and perivascular SP-immunoreactive nerves were also found in the submucosal layer. We observed a total of 305 SP-immunoreactive (IR) axon terminals, of which most (89.6%) were free nerve endings at the ultrastructural level; the rest of the SR-IR axon terminale were seen in the vicinity of the epithelium and blood vessels in the lamina propria. Varicose regions of SP-IR axon terminals contained large granular and small agranular synaptic vesicles, and most of them partially lacked a Schwann cell sheath. In some SP-IR varicosities, synaptic vesicles were concentrated in the region without any Schwann cell sheath. Long storage (for more than 1 month) of fixed-tissue pieces in sucrose before freezing has improved the ultrastructure of cryostat sections in pre-embedding immunohistochemistry. Trypsin digestion for the purpose of exposing antigenic sites was also employed before applying the first antiserum.     

The isolation, from electromotor neurone perikarya, of messenger RNAs coding for synaptic proteins

Poly(A)-containing mRNA was isolated from the electric lobe, cerebellum and forebrain of Torpedo marmorata and from cholinergic electromotor perikarya isolated from the electric lobe. All the mRNA preparations were translated by a cell-free protein-synthesizing system from rabbit reticulocytes; no brain-specific factors were required. The highest stimulation rate was found with the perikaryal mRNA suggesting that this purely neuronal mRNA is a preferred template in the protein-synthesis system; the molecular basis of this phenomenon remains to be elucidated. The translation products of the perikaryal mRNA were analysed by two-dimensional gel electrophoresis and compared with the proteins of synaptosomes derived from the electromotor nerve terminals. The majority of the synaptosomal proteins comigrated with synthesized products. More than 100 synthesized proteins were detected as individual spots in the gel pattern, among them actin, subunits of neurofilamentous proteins and a protein considered to be a specific component of electromotor synaptic vesicles. Identities were confirmed in some cases by immunochemical methods. The results suggest that protein synthesis in the perikaryon of the electromotor neurone is largely directed to the production of proteins needed to maintain synaptic integrity. A comparison of the translation products of mRNA derived from the highly cholinergic electric lobe and a brain region, the cerebellum, which is non-cholinergic, revealed, as expected, some common translation products and others which appeared to be specific for the brain regions concerned. This approach may lead to the identification of protein specific for neurones of different transmitter types.     

Thyrotropin releasing hormone, somatostatin, and enkephalin: distribution studies using immunohistochemical techniques

Using the indirect immunofluorescence technique of Coons and collaborators, the localization of thyrotropin releasing hormone, somatostatin, and enkephalin was studied in the rat central nervous system. These three peptides were found in neurons throughout the brain and spinal cord; the latter two peptides were also found in the peripheral nervous system and in certain endocrine cells. Utilizing the peroxidase--antiperoxidase technique of Sternberger and collaborators the neuropeptides were also traced at the ultrastructural level. They seemed to be mainly confined to so-called large dense core vesicles (diameter around 1,000 A), localized within cell bodies and nerve terminals.     

Immunocytochemical localization of a chondroitin sulfate proteoglycan in nervous tissue. I. Adult brain, retina, and peripheral nerve

Monospecific antibodies were prepared to a previously characterized chondroitin sulfate proteoglycan of brain and used in conjunction with the peroxidase-antiperoxidase technique to localize the proteoglycan by immunoelectron microscopy. The proteoglycan was found to be exclusively intracellular in adult cerebellum, cerebrum, brain stem, and spinal cord. Some neurons and astrocytes (including Golgi epithelial cells and Bergmann fibers) showed strong cytoplasmic staining. Although in the central nervous system there was heavy axoplasmic staining of many myelinated and unmyelinated fibers, not all axons stained. Staining was also seen in retinal neurons and glia (ganglion cells, horizontal cells, and Muller cells), but several central nervous tissue elements were consistently unstained, including Purkinje cells, oligodendrocytes, myelin, optic nerve axons, nerve endings, and synaptic vesicles. In sympathetic ganglion and peripheral nerve there was no staining of neuronal cell bodies, axons, myelin, or Schwann cells, but in sciatic nerve the Schwann cell basal lamina was stained, as was the extracellular matrix surrounding collagen fibrils. Staining was also observed in connective tissue surrounding the trachea and in the lacunae of tracheal hyaline cartilage. These findings are consistent with immunochemical studies demonstrating that antibodies to the chondroitin sulfate proteoglycan of brain also cross-react to various degrees with certain connective tissue proteoglycans.     

Axonal Transport of Zinc Transporter 3 and Zinc Containing Organelles in the Rodent Adrenergic System

Zinc is the second most abundant trace metal (after iron) in mammalian tissues, and it is an essential element for growth, development, DNA synthesis, immunity, and other important cellular processes. A considerable amount of zinc in the brain exists as a pool of free or loosely bound zinc ions in synaptic vesicles with zinc transporter 3 (ZnT3) in their membranes. Here we demonstrate that also in the peripheral sympathetic nervous system zinc handling neurons exist. In autonomic ganglia of rats and mice a subset of neuronal cell bodies contain zinc, visualized by the autometallographic (AMG) and TSQ histochemical methods. The Zn-transporter 3 is, as shown by immunofluorescence, also present in tyrosine hydroxylase (TH)-positive neurons, but rarely in cell bodies with neuropeptide Y (NPY)-immunoreactivity (IR). In axons of crush-operated sciatic nerves a rapid bidirectional accumulation of AMG granules occurred. Also ZnT3-IR was found to accumulate rapidly in anterograde as well as retrograde direction, colocalized with TH-IR. So far nerve terminals with ZnT3-IR have not been observed. The functional significance of zinc ions in the sympathetic system is not known. Special issue article in honor of Dr. Anna Maria Giuffrida-Stella.     

ATP-Dependent calcium uptake by cholinergic synaptic vesicles isolated fromtorpedo electric organ

Summary Cholinergic synaptic vesicles were purified fromTorpedo electric organ to near morphological homogeneity. They were isolated in a K⁺ environment. A method is described for the preparation of concentrated synaptic vesicles that allows uptake studies by conventional techniques. An ATP-Mg-dependent calcium uptake associated with synaptic vesicles is characterized. The uptake system transports calcium against a high concentration gradient. The maximum accumulation rate is obtained for the calcium, Mg⁺⁺ and ATP concentrations likely to be found in the nerve terminal cytoplasm. It is suggested that synaptic vesicles are implicated in the removal of the calcium entering the nerve terminal during synaptic activity.     

Neural Organization of the Median Ocellus of the Dragonfly : II. Synaptic structure

Two types of presumed synaptic contacts have been recognized by electron microscopy in the synaptic plexus of the median ocellus of the dragonfly. The first type is characterized by an electron-opaque, button-like organelle in the presynaptic cytoplasm, surrounded by a cluster of synaptic vesicles. Two postsynaptic elements are associated with these junctions, which we have termed button synapses. The second synaptic type is characterized by a dense cluster of synaptic vesicles adjacent to the presumed presynaptic membrane. One postsynaptic element is observed at these junctions. The overwhelming majority of synapses seen in the plexus are button synapses. They are found most commonly in the receptor cell axons where they synaptically contact ocellar nerve dendrites and adjacent receptor cell axons. Button synapses are also seen in the ocellar nerve dendrites where they appear to make synapses back onto receptor axon terminals as well as onto adjacent ocellar nerve dendrites. Reciprocal and serial synaptic arrangements between receptor cell axon terminals, and between receptor cell axon terminals and ocellar nerve dendrites are occasionally seen. It is suggested that the lateral and feedback synapses in the median ocellus of the dragonfly play a role in enhancing transients in the postsynaptic responses.     

Ultrastructural cytochemistry of biogenic amines in nervous tissue: methodologic improvements.

A new fixation method has been developed for localizing biogenic amines in nervous tissue. The method is a modification of the chromaffin reaction in which all fixation steps are buffered with mixtures of sodium chromate and potassium dichromate. In this way the fixation and cytochemical reaction are carried out almost simultaneously. Using the rat vas deferens as a model tissue, it was found that the preservation of electron dense (chromaffin) cores in the vesicles of adrenergic nerve terminals depended on several factors: a short primary fixation using low concentrations of aldehydes, the presence of the chromate/dichromate buffer during all fixation steps and, finally, a long incubation period in a slightly acidic (pH 6.0) solution of this buffer before postfixation in osmium tetroxide. Using this method it was possible to identify not only small and large dense-cored vesicles as storage sites for amines but also a tubular reticulum (neuronal endoplasmic reticulum), the latter especially in nerve terminals of mesenteric arteries and iris. Biogenic amines were also visualized in sympathetic ganglion cells and in the central nervous system e.g., supraependymal nerve terminals, tissues that up to now proved the most difficult in terms of amine localization. In all the tissues examined the cytochemical reaction was highly selective and present in well preserved tissue, which is a significant advance over previously available techniques. It therefore offers new opportunities for further studies on the role of biogenic amines as neurotransmitters.