The demonstration of close nerve-Purkinje fibre contacts in false tendons of sheep heart

Summary An observation of intimate nerve-Purkinje fibre associations in false tendons of sheep heart is reported. Nerve bundles were observed in deep clefts of Purkinje fibres, in channels running between coupled Purkinje cells and embedded within Purkinje cells, as well as in the outer connective tissue sheath. Most nerve terminals in these areas were filled with small clear vesicles and a few large dense-cored vesicles. Only a few axons with many small dense-cored vesicles were observed.Intimate associations (separation, 60 to 90 nm) between the Purkinje cell and nerve varicosity were observed in the deep clefts. Similar close appositions were also present where nerves were embedded in Purkinje cells. In these cases the Purkinje cell enclosing the nerve bundle formed intercellular junctions with its own sarcolemma.Elaborate sarcolemmal folds with multi-vesicular bodies were also frequently observed near nerve bundles and varicosities. The identity of the transmitter is unknown although the nerves forming intimate associations with Purkinje cells have a morphology typical of cholinergic nerves.     

Anatomy and physiology of neurons composing the commissural ring nerve of the cricket, Acheta domesticus

The commissural ring nerve (RN) of the cricket Acheta domesticus links together the two cercal motor nerves of the terminal abdominal ganglion. It contains the axons of almost 100 neurons including two bilateral clusters of eight to 13 ventrolateral neurons and approximately 75 neurons with midline somata within the terminal abdominal ganglion. The ventrolateral neurons have an ipsilateral dendritic arborization within the dorsal neuropil of the ganglion and their axons use the RN as a commissure in order to enter the contralateral nerves of the tenth ganglionic neuromere. In contrast, most midline neurons have bifurcating axons projecting bilaterally into the neuropil of the ganglion as well as into the RN where they often branch extensively before entering the contralateral tenth nerves. Most RN neurons have small, non-spiking somata with spike initiation zones distant from the soma. Many midline neurons also produce double-peaked spikes in their somata, indicative of multiple spike initiation zones. Spontaneous neuronal activity recorded extracellularly from the RN reveals several units, some with variable firing patterns, but none responding to sensory stimuli. The RN is primarily composed of small (50 nm diameter) axon profiles with a few large (0.5-1 microm diameter) profiles. Occasionally, profiles of nerve terminals containing primarily small clear vesicles and a few large dense vesicles are observed. These vesicles can sometimes be clustered about an active zone. We conclude that the primary function of the RN is to serve as a peripheral nerve commissure and that its role as a neurohemal organ is negligible. J. Exp. Zool. 286:350-366, 2000.Copyright 2000 Wiley-Liss, Inc.     

Quantitative analysis of the structure of the varicose dilatations in the nerve fibers of the rabbit coronary artery (according to the results of three-dimensional reconstruction)]

Axoplasmic vesicles and microtubes in varicosities of axonal plexus in the external sheath of the rabbit coronary artery have been studied. Comparing serial sections and examining three-dimensional reconstruction of small nerve plexus, it was demonstrated that various varicosities differed only in their correlation of the amount of small (30-80 nm) and large (80-180 nm) vesicles. Average diameters in profiles of small and large vesicles are 56.3 nm and 115.6 nm, respectively. There are varicosities containing about 250 or more than 1,000 vesicles. Evenly distributed vesicles throughout the volume of varicosities and lack of specialized structures on the axolemma are supposed to demonstrate the absence of special areas for the mediator removal in the axons studied. The microtubes in the varicosities are peripherally arranged, next to the axolemma and form 1-1.5 wide coils. A suggestion is made that the varicosities in neighbouring axons of the same nerve plexus, with specific structural organization, are special functional units and appear to be peculiar not only for nerve plexus of the coronary artery, but also for other parts of the peripheral vegetative nerve system.     

Nitric oxide synthase-immunoreactive axons innervating the guinea-pig lingual artery: an ultrastructural immunohistochemical study using elastic brightfield imaging

The ultrastructure of nitric oxide synthase-immunoreactive (NOS-IR) axons innervating the guineapig lingual artery was investigated by means of pre-embedding immunohistochemistry using an indirect peroxidase technique and diaminobenzidine. Sections ranging in thickness from 60 to 500 nm were ultrastructurally evaluated in elastic brightfield imaging mode. Thick sections (optimum at 300 nm) were advantageous for enhancement of the labelling intensity, whilst some subcellular details were better revealed by thin sections. NOS-IR axon terminals often contained aggregations of large, dense-cored vesicles, consistent with a previous light microscopical report on colocalization of NOS and vasoactive intestinal peptide-immunoreactivity in these fibres. NOS-IR axons formed direct neuro-muscular junctions (width less than 50 nm) at the outer surface of the tunica media, thus providing a structural basis for nitrergic vasodilation. In addition, NOS-IR axons made direct contacts with non-varicose and varicose segments of non-reactive axons, suggesting interneuronal communication between these elements.     

Nerve ultrastructure of the arteries of the brain stem]

Ultrastructure of the nerve apparatus in the arteries of the brain base has been studied in cats. The structure of peri- and adventitial nerves has been investigated electron microscopically. Three types of efferent axons and four types of synaptic vesicles (small agranular and granular, large granular, large electron opaque vesicles) have been revealed. Vesicle-containing axons in the brain arteries approach the external smooth muscle cells of about 80 nm. Terminal axonal dilatations possessing direct and mediated connections with muscular cells of the middle tunica have been revealed.     

Depletion of large dense-cored vesicles from parasympathetic nerve terminals in rat parotid glands after prolonged stimulation of the auriculotemporal nerve

Large dense-cored vesicles (60-100 nm in diameter) have been assessed electron-microscopically in terminal parasympathetic axons at acinar neuro-effector sites in rat parotid glands. Their numbers in control unstimulated glands have been compared with those in the contralateral glands of the same animals after prolonged nerve stimulation. Bilateral postganglionic sympathectomy had been undertaken 4-6 weeks previously to remove adrenergic axons from the glands. Stimulation of the postganglionic parasympathetic nerve to the gland--the auriculotemporal nerve--for 80 min at 40 Hz caused a significant depletion of large dense-cored vesicles from the terminal axons. This depletion corresponded in time and magnitude to the depletion of vasoactive intestinal peptide and substance P from the glands that had been found previously to occur under identical conditions. This adds support to the belief that the neuropeptides are stored in such vesicles and that these vesicles release their contents at neuro-effector sites as a result of propagated impulse formation in the axons.     

Nerve-purkinje fiber relationship in the moderator band of bovine and caprine heart

Summary The moderator band in the heart of the ox and goat contains bundles of Purkinje fibers and nerve fibers separated by connective tissue. The axons are mostly unmyelinated and embedded in the cytoplasm of Schwann cells.Small bundles of axons run close to the Purkinje fibers. The axons dilate into varicosities 0.5 to 1.6 in diameter (mean 0.95 ), containing three types of vesicles: 1) agranular vesicles with a diameter of 400–500 Å, 2) large dense-cored vesicles with a diameter of 800–1200 Å, 3) small dense-cored vesicles with a diameter of 500 Å. Most varicosities contain agranular vesicles together with a few large dense-cored vesicles.The gap between the varicosities and the nearest Purkinje fiber is unusually wide and normally varies between 0.3 and 0.8 . No intimate nerve-Purkinje fiber contacts, with a cleft of 200 Å, were observed.     

Ultrastructural localization of neuropeptides and GABA in rat dorsal horn: a comparison of different immunogold labeling techniques

Several immunogold techniques were used to determine the ultrastructural localization of calcitonin gene-related peptide (CGRP), tachykinin, somatostatin, and gamma-amino-butyric acid (GABA) immunoreactivity in the dorsal horn of rat spinal cord. The immunocytochemical reactions were carried out directly on ultrathin sections from non-osmicated frozen tissue, non-osmicated low temperature-embedded (Lowicryl K4M) tissue, and osmicated epoxy-embedded material. Preservation of ultrastructural morphology and immuno-labeling efficiency were compared. Morphology of subcellular organelles was relatively good in ultra-thin frozen sections, which showed the highest immunoreactivity. However, only very small samples of tissue could be examined. Although there was relatively good immunolabeling in the Lowicryl K4M-embedded tissue, the ultrastructure of the neuropil, and particularly that of synapses, was poorly maintained. In contrast, the osmicated epoxy-embedded material offered optimal morphological preservation together with accurate subcellular localization of all antigens under study. The latter approach thus enabled clear visualization of CGRP, tachykinin, and somatostatin immunoreactivity restricted to large dense-cored vesicles (90-150 nm diameter) in many axonal and synaptic profiles in the superficial layers of the dorsal horn. CGRP- and tachykinin-positive profiles were also present in the tract of Lissauer. GABA immunoreactivity was present mainly in axons and terminals, and less frequently in somatic and dendritic profiles. In terminals, which often formed symmetrical synapses on immunonegative dendritic profiles, it was associated with small (30-60 nm diameter) clear vesicles and mitochondria. Double immunolabeling was possible on all preparations, but the osmicated, epoxy-embedded material clearly showed co-localization of peptides, especially of CGRP and tachykinins, within the same dense-cored vesicles in axonal fibers and/or terminals. On the other hand, peptide and GABA immunoreactivity were consistently seen in different nerve profiles. In a few cases, GABAnergic terminals were seen to synapse on tachykinin-positive fibers.     

Catecholaminergic innervation of the rat adrenal cortex

Summary The zona glomerulosa of the rat adrenal gland is innervated by catecholaminergic nerves. Using histofluorescence techniques, we observed catecholaminergic plexuses surrounding adrenal capsular and subcapsular blood vessels. Individual varicose nerve fibers that branched off these plexuses were distributed among adrenal glomerulosa cells. This innervation was permanently eliminated after neonatal sympathectomy with guanethidine or 6-hydroxydopamine, but was not affected by ligation of the splanchnic nerve or extirpation of the suprarenal ganglion. At the ultrastructural level, axonal varicosities were commonly observed in close proximity to glomerulosa cells and blood vessels. Nerve fibers and varicosities were found to contain small (30–60 nm) clear vesicles as well as large (60–110 nm) and small (30–60 nm) dense-cored vesicles. In tissue fixed for the dichromate reaction with or without pretreatment with the false transmitter 5-hydroxydopamine, many nerve terminals contained numerous small dense-cored vesicles which are thought to contain catecholamines. These results establish the anatomical substrate for the catecholaminergic innervation of the rat adrenal cortex.     

Catecholamine-rich cells and varicosities in bovine splenic nerve, vesicle contents and evidence for exocytosis.

The bovine splenic nerve trunk contains mast cells, ganglion cells, small intensely fluorescent (SIF) cells, and varicosities which exhibit a brilliant fluorescence characteristic for noradrenaline (NA) and dopamine (DA) after formaldehyde exposure. All these catecholamine-rich structures could contribute particles to isolated nerve vesicle fractions. Mast cells are recognized ultrastructurally by their large (300-800 nm) dense granules. SIF cells may be represented by cells and processes containing dense cored vesicles (120-140 nm) which are larger than the typical vesicles in axons and terminals. Terminal-like areas with typical large dense cored vesicles (LDV, 75 nm) and small dense cored vesicles (SDV, 45-55 nm) probably correspond to the fluorescent varicosities. The LDV constitute about 40% of all vesicles in terminal-like areas and terminals. Their staining properties indicate the presence of protein, phospholipids, and ATP. Tyramine depletes NA without loss of matrix density. The LDV can fuse with the terminal membrane, and released material outside omega profiles is interpreted to depict exocytosis. Large and small vesicles are easily distinguished from the very large mast cell granules and the moderately dense Schwann cell vesicles. Neither appear to contaminate the LDV fractions but the latter may contain a small population of SIF cell vesicles. Golgi vesicles from the Schwann cells mainly occur in the lighter zones of the gradient.     

Ultrastructure of the larval firefly light organ as related to control of light emission

The firefly larva has a pair of light organs consisting of a layer of interdigitating, light emitting cells, covered dorsally with a layer of opaque, white cells. Each light organ is ventilated by one large and several smaller tracheal branches and is innervated by a branch of the segmental nerve containing two axons. These axons branch profusely in the photocyte layer so that several nerve profiles are seen around any photocyte. Nerve terminals contain large dense-core vesicles and small light-core vesicles. Clusters of light-core vesicles surrounding irregularly shaped membrane densifications, presumably the synapses between nerve and photocyte, are common in nerve terminals. Light emitting cells in insects characteristically contain photocyte vesicles. In the larva there are both full and empty photocyte vesicles; the full vesicles contain a matrix with tubular membrane invaginations in contrast to the empty vesicles which contain amorphous membrane invaginations.     

Ultrastructural localization of acetylcholinesterase (AChE) activity in the chicken Harderian gland

Localization of acetylcholinesterase (AChE) was investigated in the chicken Harderian gland at the electron microscopic level. Nerve cells in the pterygopalatine ganglion showed AChE activity. They had a pale and large nucleus which was round or oval in shape. Reaction product of AChE was detected between the nuclear envelopes; in the cisterna of rough endoplasmic reticulum and the lumen of the Golgi lamellae, and on the plasma membrane of the nerve cell. In the interstitium of the gland, nerve fibers showing AChE activity were easily found. They were often seen in the perivascular space and between plasma cells. These nerve fibers had varicosities in contact with plasma cells and the endothelium or the smooth muscle fiber of the blood vessels. AChE-positive varicosities or terminals contained many small clear vesicles (about 50nm in diameter) and a few large dense-cored vesicles (about 100 nm in diameter). No contacts of nerve fibers with acinar cells or the ductal epithelium were observed in the present study. Our data indicate that cholinergic nerves play distinct roles in the regulation of the immune function of the chicken Harderian gland.     

Fine structure and composition of the submucous nerve plexus of the guinea-pig trachea

Summary The ultrastructure of the nerves forming the submucous plexus of cervical and thoracic parts of the trachea was studied in the guinea-pig. Specimens were obtained from 6 animals perfused with warm fixative and from 6 animals in which pieces of trachea were incubated in buffer containing 5-hydroxydopamine before being immersed in cold fixative. Of the two types of axonal terminal identified in the nerves, one contained mainly large dense-cored vesicles, and the second contained numerous small vesicles. In specimens incubated in 5-hydroxydopamine, the small vesicles of the latter terminals exhibited the electron-dense cores which are characteristic of adrenergic axonal terminals. Counts made on perfused specimens showed that, in both the thoracic and cervical parts of the trachea, the density of adrenergic terminals was higher than that of terminals containing mainly large dense-cored vesicles. Overall terminal density was, however, higher in the thoracic than in the cervical part of the trachea, and estimates of nerve size showed that this was associated with the presence in the thoracic plexus of a substantially greater proportion of nerves with less than 6 axons. The possible function of the nerves in the control of the calibre of the submucous blood vessels was discussed.     

Fine structure of dense-cored vesicles in glomus cells of rat carotid body after fixation with permanganate or glutaraldehyde.

Glomus (Type I) cells of the carotid body of adult rats were studied electron microscopically after fixation with potassium permanganate or with glutaraldehyde and osmium tetroxide. Two permanganate fixation methods (using Krebs-Ringer-glucose, pH 7.0, or acetate buffer, pH 5.0) were compared. Numerous dense-cored vesicles were observed only in about one tenth of the glomus cells when neutral permanganate was used for fixation, although all glomus cells showed such vesicles after fixation with glutaraldehyde and osmium tetroxide. Numerous vesicles with a dense core were observed in about one third of the cells after fixation with acid potassium permanganate. With this fixation, small dense-cored vesicles similar to those in adrenergic nerve terminals were occasionally seen in the cytoplasm of glomus cells. It is tentatively concluded that the amine-storing vesicles of the carotid body are different from those in the small intensely fluorescent (SIF) cells and those in adrenergic nerve terminals.     

Fine structure of dense-cored vesicles in glomus cells of rat carotid body after fixation with permanganate or glutaraldehyde

Summary Glomus (Type I) cells of the carotid body of adult rats were studied electron microscopically after fixation with potassium permanganate or with glutaraldehyde and osmium tetroxide. Two permanganate fixation methods (using Krebs-Ringer-glucose, pH 7.0, or acetate buffer, pH 5.0) were compared. Numerous dense-cored vesicles were observed only in about one tenth of the glomus cells when neutral permanganate was used for fixation, although all glomus cells showed such vesicles after fixation with glutaraldehyde and osmium tetroxide. Numerous vesicles with a dense core were observed in about one third of the cells after fixation with acid potassium permanganate. With this fixation, small dense-cored vesicles similar to those in adrenergic nerve terminals were occasionally seen in the cytoplasm of glomus cells. It is tentatively concluded that the amine-storing vesicles of the carotid body are different from those in the small intensely fluorescent (SIF) cells and those in adrenergic nerve terminals.     

Interneuronal synapses in the local ganglia of the cat urinary bladder.

The interneuronal synapses of the urinary bladder in the cat were studied by electron microscopy. The great majority of the fibres containing vesicles are found within the ganglia occurring in the trigonum area. Morphologically differentiated synaptic contacts could be observed on the surface of the local neurons and between the different nerve processes. The presynaptic terminals can be divided into three types based on a combination of synaptic vesicles. Type I terminals, presumably cholinergic synaptic terminals, contain only small clear vesicles of 40-50 nm in diameter. Type II terminals, presumably adrenergic terminals, are characterized by small granulated vesicles of 40-60 nm in diameter. Type III terminals, probably of local origin, contain a variable number of large granulated vesicles of 80-140 nm in diameter. Occasionally, a single nerve fibre contacted several (two or four) other nerve processes forming a typical synapse. In other cases, on one nerve cell soma or on other nerve processes there are two or three different-type nerve terminals establishing synapses. It might be inferred from these observations that convergence and divergence can occur in the local ganglia and that cholinergic and adrenergic synaptic terminals can modulate the ganglionic activity. However, a local circuit also can play an important role in coordinating the function of the bladder.     

Catecholamine-rich cells and varicosities in bovine splenic nerve,vesicle contents and evidence for exocytosis

The bovine splenic nerve trunk contins mast cells, ganglion cells, small intensely flurescent (SIF) cells, and varicosities which exhibit a brilliant fluorescence characteristic for noradrenaline (NA) and dopamine (DA) after formaldehyde exposure. All these catecholamine-rich structure could contribute particles to isolated nerve vesicle fractions. Mast cells are recognized ultrastructurally by their large (300–800nm) dense granules. SIF cells may be represented by cells and processes containing dense cored vesicles (120–140 nm) which are larger than the typical vesicles in axons and terminals. Terminal-like areas with typical large dense cored vesicles (LDV, 75 nm) and small dense cored vesicles (SDV, 45–55 nm) probably correspond to the fluorescent varicosities. The LDV constitute about 40% of all vesicle in terminal-like areas and terminals. Their staining properties indicate the presence of protein, phospholipids, and ATP. Tyramine depletes NA without loss of matrix density. The LDV can fuse with the terminal membrane, and released material outside omega profiles is interpreted to depict exocytosis. Large and small vesicles are easily distinguished from the very large mast cell granules and the moderately dense Schwann cell vesicles. Neither appear to contaminate the LDV fractions but the latter may contain a small population of SIF cell vesicles. Golgi vesicles from the Schwann cells mainly occur in the lighter zones of the gradient.     

Vasoactive intestinal peptide (VIP)-like immunoreactivity in the human sympathetic ganglia

Vasoactive intestinal peptide immunoreactive (VIP-IR) nerve fibres and terminals, neurons and small granule containing cells were observed in human lumbal sympathetic ganglia. Electron-microscopically VIP-IR was localized in the large dense-cored vesicles in nerve terminals and on the membranes of the Golgi complexes in the neurons. A small population of principal ganglion cells was surrounded by VIP-IR nerve terminals. Most of these neurons contained acetylcholinesterase (AChE) enzyme but were not tyrosine hydroxylase-immunoreactive (TH-IR). All VIP-IR ganglion cells and most of the nerve fibres contained AChE but not TH-IR. It appears that in human sympathetic ganglia VIP is localized in the cholinergic neurons and nerve fibres and that the VIP-IR nerve terminals innervate mainly the cholinergic subpopulation of the sympathetic neurons.     

Vasoactive intestinal peptide (VIP)-like immunoreactivity in the human sympathetic ganglia

Summary Vasoactive intestinal peptide immunoreactive (VIP-IR) nerve fibres and terminals, neurons and small granule containing cells were observed in human lumbal sympathetic ganglia. Electron-microscopically VIP-IR was localized in the large dense-cored vesicles in nerve terminals and on the membranes of the Golgi complexes in the neurons. A small population of principal ganglion cells was surrounded by VIP-IR nerve terminals. Most of these neurons contained acetycholinesterase (AChE) enzyme but were not tyrosine hydroxylase-immnoreactive (TH-IR). All VIP-IR ganglion cells and most of the nerve fibres contained AChE but not TH-IR. It appears that in human sympathetic ganglia VIP is localized in the cholingergic neurons and nerve fibres and that the VIP-IR nerve terminals innervate mainly the cholinergic subpopulation of the sympathetic neurons.     

The innervation and fine structure of paraneuronic cells in an amphibian pulmonary artery

Summary The pulmonary artery of Bufo marinus contains large numbers of bipolar cells situated in the tunica adventitia and in the outer layers of the media. These cells show a bright green-yellow fluorescence (emission spectra 485 nm) after formaldehyde pre-treatment suggesting that they contain a primary monoamine. The most characteristic fine-structural feature of these cells is the presence of numerous dense-cored vesicles (80—300 nm diameter) in their cytoplasm. The cells are in close contact (20 nm gap) with both agranular and granular nerve fibres. Both EM-cytochemical and formaldehyde-induced fluorescence tests indicate that the granule-containing nerve fibres are adrenergic. The agranular nerve fibres form discrete synaptic contacts with pre-and post-synaptic membrane thickenings on the cells. This was never observed with respect to the adrenergic fibres. Each process of the cells is about 45 m long. The processes do not bear any special relationship to either vessels of the arterial vasa vasorum or medial smooth muscle cells. Their location in the wall of the artery suggests that they are functionally significant with respect to activity of the arterial media.