Electron microscopic and histochemical observations on different types of nerve endings in the extraocular muscles of the rat

Summary Nerve endings in the extraocular muscles of the rat were submitted to histochemical tests for formalin-induced fluorescence and carboxylic esterases. Acetylthiocholine, butyrylthiocholine and -naphthyl acetate were used as substrates and iso-OMPA, 284C51, eserine and E-600 as inhibitors. The ultrastructure of the endings was studied with the electron microscope.Both single and multiple nerve terminals were observed in all six extraocular muscles. The single terminals of myelinated axons were comparable in their light and electron microscopic structure with the typical motor end plates of other striated muscles, and like these they exhibit acetylcholinesterase (AChE), non-specific cholinesterase (ns. ChE) and non-specific esterase (ns. E) activity. These endings were apposed to twitch-type muscle fibres.The multiple terminals were classified with the light microscope into two types. The larger type was 1/3 of the size of the motor end plate; 2–5 endings innervated the same muscle fibre; subneural infoldings were weakly developed and possessed only slight AChE and ns. ChE and probably no ns. E activity. No subneural lamellae were visible under the light microscope in the smaller type, which also possessed AChE and ns. ChE and was composed of 10–20 small dots dispersed along a single muscle fibre. The Schwann cells along nerve fibres leading to these two types of multiple endings exhibited ns. ChE but not AChE and ns. E activity.The ultrastructure of the two types of multiple endings was principally similar. The main difference, compared with the motor end plate, was that these endings were derived from unmyelinated axons which either make synaptic contacts along their course with the muscle fibre at variable distances (smaller-type) or these terminals were grouped closely together (larger-type).A few dense-core vesicles were observed in these unmyelinated nerves and in their terminals which were considerably smaller than those in the motor end plate. They were not always separated from each other by sarcoplasm and teloglia (larger-type) and contained also empty vesicles. The secondary synaptic clefts were often sparse and irregular or even absent, but the typical myoneural postsynaptic electron density was always observed. These multiple endings, in contrast to the motor end plate, were apposed only to muscle fibres with slow contraction.No catecholamine containing nerve endings were observed in the extraocular muscles. These observations indicate that the rat extraocular muscles have a double cholinergic innervation.The author wishes to express his gratitude to Professor Antti Telkkä, M. D., Head of the Electron Microscope Laboratory, University of Helsinki, for permission to avail himself of the electron microscope facilities.     

Electron microscopic observations on the reactional changes occurring in insect nerve fibers after transection

Summary An electron microscopic study of the interganglionic connectives of an orthopteran (Laplatacris dispar) demonstrated that a reaction starts in the sectioned fibers very soon after transection (30 minutes). This reaction is closely similar to that observed in sectioned nerves of vertebrates and consists in the appearance of microvesicles and the proliferation of mitochondria. Sectioned connectives were studied from 30 minutes to 88 hours after section. The reaction mentioned increases progressively during the indicated lapse of time.This research was supported by the United States Air Force under Contract No AF 49 (638) 585 and Grant Af 61-64, monitored by the Air Force Office of Scientific Research, Air Force Research Division of the Air Research and Development Command.     

Substance P-like immunoreactive nerve fibers in the pars distalis of the anterior pituitary in the dog

Summary The pars distalis of the anterior pituitary is known to be regulated by hypothalamic hormones. Recently, we have discovered the presence of substance P-like immunoreactive nerve fibers in the pars distalis of the monkeys. Substance P-like immunoreactivity in the pars distalis of the dog was investigated in this study. A substantial amount of substance P-like immunoreactive nerve fibers with a large amount of varicosities were found. They were widely distributed in the gland, more abundant along its periphery. Most of them were closely related to the glandular tissue, some were located on vascular walls. Substance P-like immunoreactive nerve fibers were also found in the meningeal sheath of the anterior pituitary. They could be followed into the parenchyma of the gland.     

Electron microscopic studies on the pars intermedia in normal and in mice with hereditary nephrogenic diabetes insipidus

Summary The ultrastructure of the pars intermedia (PI) of the normal VII +/+ and hereditary nephrogenic diabetes inspidus DI Os/+ mice has been studied with particular reference to the morphology of the glandular cells and their innervation. Four types of cells were observed in both the genotypes of mice, 1) the light glandular cell, 2) the dark cell, 3) a type of cell similar to ependymal cells and 4) a small percentage of typical ACTH cells, observed mostly on the PI border of the cleft and rarely in the centre of PI. The predominant light glandular cells contain mainly two types of membrane bound granules: 1) electron dense core granules, which measure 1500–2500 Å and 2) electron lucent vesicles, which measure 3000–4000 Å in diameter. Granules of intermediate size with various density are also present in both types of mice. The electron dense core granules are predominant in DI Os/+ mice, whereas, electron lucent vesicles are predominant in the normal VII +/+ mice. Similar uniform size membrane bound electron dense granules have been observed in ACTH cells of PI and pars distalis. From earlier experimental evidences and the present observations, it is concluded that the dense core granules in PI may be synthesizing ACTH or ACTH-like substance. It is also discussed that these dense core granules may further mature and give rise to MSH in the form of electron lucent vesicles. If it is so, PI light glandular cells may have dual functions, of producing MSH and ACTH. One of the functions of ependymal-like cells, may be the transport of PI secretion.Three types of nerve endings are observed throughout the PI, making synaptic contact with the predominant cell type. The innervation is more in DI Os/+ mice than in normal mice. The classification of these nerves is according to Bargmann and co-workers 1) peptidergic neurosecretory fibers, contain mainly membrane bound dense core granules, measuring 1200 to 1800 Å, and are the classic neurosecretory granules; 2) adrenergic fibers, measuring 700–900 Å; 3) cholinergic fibers, measuring 300–400 Å. Adrenergic and cholinergic fibers are more towards the hypophysial cleft. The increased innervation, the synaptic contact, the extremely hypertrophied PI and the greater activity of its light glandular cells in the DI Os/+ mice show the PI is under the influence of the nervous system.This study was supported by MRC of Canada Grant No. MA-3759.     

Electron microscopic histochemistry of lysosomes in neurosecretory nerve endings and pituicytes of rat posterior pituitary

Summary Electron microscopic localization of acid phosphatase (AcPase) was carried out on posterior pituitary glands from rats. An estimated 5% of the neurosecretory nerve terminals contained structures which showed reaction product. Most of the lysosomes were small dense bodies, often with a membranous substructure. Other lysosomes were larger in size or were found within vacuoles. AcPase was also localized to lysosomes and the Golgi apparatus of pituicytes. Evidence is presented which would associate the large lipid droplets characteristic of pituicytes with AcPase-positive dense bodies. The present study indicates that hydrolytic activity by lysosomes occurs within the terminals of neurosecretory cells, and adds further support to the concept that this process represents a normal phenomenon of cells and their extensions in general.Supported by the Medical Research Council of Canada.Medical Research Associate of the Medical Research Council of Canada.     

Neuroglandular contacts between vasopressin-reactive fibers and cells of the pars tuberalis in the rat

Summary In electron micrographs fibers containing vasopressin-immunoreactive elementary granules 100–120 nm in diameter are observed within the basal lamina of the adenohypophyseal pars tuberalis adjacent to the rostral portion of the median eminence. The concept of a neuroglandular transmitter function of vasopressin is discussed.Supported by the Deutsche Forschungsgemeinschaft (Grant Nr. Kr. 569/2) and the Stiftung VolkswagenwerkThe excellent technical assistance of Mrs. Helga Prien is thankfully acknowledged     

Electron microscopic identification of spoon-like contacts in the oculomotor nerve of the trout]

In the nc. n. oculomotorii of the trout exist different types of axonal terminals of second order vestibular neurons. In semithin sections there are observed seldom very large end-knobs (calyciferous endings). Electron microscopic pictures of the same terminal show typical spoon-like endings. A random sample like analysis of the mitochondrial density refers to functional differences between the different types of endings. A intramitochondrial cristaline inclusion in the axoplasma of a neuron is shown.     

Electron microscopic investigation of adrenergic and non-adrenergic axons in the rabbit SA-node

Summary The rabbit SA-node was outlined electrophysiologically and its adrenergic and cholinergic innervation patterns were studied with the electron microscope. Differentiation between adrenergic and cholinergic terminals was achieved by fixation of the specimens in KMnO₄ which produces dense-cored synaptic vesicles in adrenergic terminals, whereas synaptic vesicles in cholinergic terminals are empty. It was found that adrenergic and cholinergic nerve terminals often come in close apposition to each other, the distance between adjoining membranes being in the order of 250 Å. At times, faint membrane thickenings could be seen in these places. The available pharmacological, physiological and morphological evidence leaves little room for doubt that cholinergic terminal fibers can influence the adrenergic ones. From mainly morphological evidence it is also postulated that adrenergic terminals influence cholinergic terminals.This work was supported by grants from Åhlén-Stiftelsen, the Faculty of Medicine, University of Lund, Lund, Sweden, the United States Public Health Service (project 06701-04) and the Swedish Medical Research Council (B70-14X-2321-03 and B70-14X-712-05).     

Electron microscopic studies on the pars intermedia of the ferret

Summary The structure of the pars intermedia of the ferret has been studied with the electron microscope, with particular reference to the morphology of the glandular cells and their innervation. Two types of cell were found. The predominant cell is ovoid in shape and contains membrane-bound vesicles of varying size (1,000–5,000 Å) and density, the most electron-dense of which are associated with the Golgi region. The nucleus is indented and the cytoplasm contains rough endoplasmic reticulum. The second cell type is often associated with the colloid material and is elongated or stellate-shaped with long processes which extend between the predominant cells. It is devoid of cytoplasmic vesicles and has a poorly defined Golgi apparatus. Certain other structural features of this cell such as microvilli, cilia or cytoplasmic microfilaments are reminiscent of ependymal cells.Numerous nerve endings are observed throughout the pars intermedia, making synaptic contact with the predominant cell type. The majority contain vesicles with an electron-dense core measuring 750 Å; less frequently terminals contain dense granules measuring 1,000 A or more. Both also contain small electron-lucent vesicles (200–400 Å); occasionally terminals containing only the latter type are found. The pattern of innervation in the ferret is thus comparable to that previously observed in the cat, rather than that seen in rodents or monkeys, and the implications of this finding are discussed.We are indebted to Prof. Sir Solly Zuckerman, O. M., K. C. B., F. R. S., for his help and guidance and to Mr. J. Wallington for his unfailing technical assistance.     

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