Depletion of gastrin-releasing peptide (GRP) from nerves in the gastric body of rats with experimental ulcers. An immunohistochemical study

A predominant population of mucosal nerves in the mammalian gastric body has been known to contain large amounts of gastrin-releasing peptide (GRP). The present immunohistochemical study demonstrates the depletion of GRP immunoreactivity from nerves in the oxyntic mucosa of rats with ulcers induced by restraint plus water immersion. Depletion of GRP occurred in a major part of the nerves after 3 h exposure to the stress, and after 6 h exposure only a few nerve fibers could be recognized. In contrast, GRP fibers in the pyloric mucosa did not decrease significantly in number in any of the stressed rats. Since the depletion of GRP immunoreactivity preceded mucosal erosion in the gastric body, the possibility is proposed that GRP released from the nerves may be related to stress-related ulceration in the stomach.     

Immunoelectron microscopic observation of calcitonin gene-related peptide (CGRP)-positive nerves in the dental pulp of rat molars.

The ultrastructure of nerves containing immunoreactivity for calcitonin gene-related peptide (CGRP) was investigated in the dental pulp of rat molars. The immunoreactivity was recognized predominantly in unmyelinated nerve fibers, and sparsely in a few myelinated fibers. It was localized throughout the axoplasm, as well as in the large cored vesicles. Small clear vesicles and mitochondria were free of the immunoreaction. The CGRP-immunoreactive nerves were frequently observed to terminate, being devoid of Schwann cell investment, in the vicinity of blood vessels in the coronal pulp, suggesting that CGRP may be involved in the regulation of pulpal blood flow. Moreover, CGRP-immunoreactive axon terminals containing numerous small clear vesicles, a few large cored vesicles and mitochondria were recognized in contact with the cell bodies of odontoblasts and their processes in the dentinal tubules. Although specialized synaptic ultrastructures were not recognizable, a functional association of CGRP nerves and odontoblasts was suggested. Thus, CGRP in the dental pulp appears to have multiple functions, including vascular regulation and sensory transduction.     

Immunohistochemical localization of neurotensin in hamster adrenal medulla

Neurotensin-like immunoreactivity was localized in nerve fibers and terminals of hamster adrenal medulla at light and electron microscopy using the peroxidase-antiperoxidase method. Numerous varicose neurotensin-immunoreactive nerves and terminals were found among nonlabeled cell groups situated peripherally in the adrenal medulla. Combined formaldehyde-glutaraldehyde (Faglu) fluorescence and immunohistochemistry of the same vibratome section showed that only norepinephrine cells were innervated by neurotensin-immunoreactive nerves. All norepinephrine cells seemed to be innervated by neurotensin-immunoreactive nerves. Neurotensin-immunoreactive nerves disappeared after extrinsic denervation of the adrenal gland. By electron microscopy numerous neurotensin-immunoreactive terminals were seen to make synaptic contacts with norepinephrine cells and with autonomic ganglion cells present in small numbers among norepinephrine cells. In the terminals neurotensin-like immunoreactivity was localized mainly in large dense-cored vesicles, but some precipitates were also associated with small vesicles, diffusely scattered in the axoplasm. The present findings suggest that in the hamster adrenal medulla part of the nerve terminals arising from splanchnic nerves contain neurotensin-like peptide. The functional significance of these nerves in the hamster adrenal medulla remains to be elucidated.     

Morphological evidence for functional capsaicin receptor expression and calcitonin gene-related peptide exocytosis in isolated peripheral nerve axons of the mouse

Rat sciatic nerve axons express capsaicin, proton and heat sensitivity and respond to stimulation with a Ca2+-dependent and graded calcitonin gene-related peptide (CGRP) release. In this study we demonstrate that similar functions, including capsaicin-induced CGRP release, are to be found in the desheathed sciatic nerve of the mouse. We have morphologically investigated the mechanisms of this axonal release in regions away from the active zones of synapses. Capsaicin receptor 1 (TRPV1) and CGRP immunostaining was performed using electron microscopic visualization. TRPV1 was identified in the axoplasm and inside vesicles--presumably on axonal transport--as well as in considerable quantity in the axonal plasma membrane of unmyelinated nerve fibers. Most of the unmyelinated axons were immunopositive for CGRP and in unstimulated nerves CGRP-containing vesicles almost entirely filled the axoplasm. After capsaicin stimulation (10(-6) M for 5 min), the fibers appeared depleted of CGRP with only few vesicles remaining as well as some residual staining of the axoplasm. In addition a large number of vesicles were fused with the axonal membrane, forming classical exocytotic figures--the omega structures--lined with CGRP immunoreactive product. These results present morphological evidence for the distribution of TRPV1 along unmyelinated axons in peripheral nerve and also provide the first demonstration of vesicular neuropeptide exocytosis along unmyelinated axons in peripheral nerve.     

Sensory receptor organs of the human nasal respiratory mucosa

Human nasal respiratory mucosa has a limited capacity for localization of stimuli and discrimination of sensory modalities. In order to obtain morphological data on its receptor organs, histochmeical and electron microscopical studies were undertaken in six individuals ranging in age from 18 to 39 years. It was found that the nasal respiratory mucosa was supplied by non-myelinated nerves which approached the mucosa in fascicles containing up to 200 axons. These fascicles were devoid of perineurial sheaths. They ramified repeatedly producing only one type of receptor organ — a simple terminal arborization. The finest terminal fascicles of the plexus ended either in the cell free zone of the lamina propria or in the spaces between the epithelial cells next to the basement membrane. The axons in the fascicles and in the endings were only partly insulated from one another by Schwann cell folds. The axoplasm of the terminal and preterminal nerve fibers contained accumulations of fine vesicles, simple granules and clusters of mitochondria. All nerve fibers gave a positive acetyl- and a negative butyro-cholinesterase reaction. The plexiform endings of the nasal respiratory mucosa are different from any receptor organ of the adult human skin. They are reminiscent of the transitory, yet functional plexiform endings of the fetal skin which are found prior to the formation of the definitive receptor organs.     

Two populations or granular vesicles in constricted post-ganglionic sympathetic nerves.

1. A comparative study has been made of the effects of different fixatives on the ultrastructural appearance of granular vesicles accumulated against a constriction in cat hypogastric nerves. 2. After fixation in either osmium tetroxide or in glutaraldehyde followed by post-osmication accumulations of large granular vesicles (65-90 nm in diameter) were observed in profiles of swollen axons. 3. Fixation in acrolein and sodium dichromate revealed a second population of small granular vesicles (mostly 30-50 nm in diameter) in addition to the large vesicles seen after the other fixatives. 4. In reserpinized cats the small granular vesicles were absent. The large granular vesicles were much less numerous, irrespective of the fixative used. 5. It is suggested that either the small vesicles occur in normal noradrenergic axons but are only revealed by catecholamine-sensitive fixatives or they occur as a result of constricting the nerve trunk. Evidence for and against these possibilities is discussed.     

Does substance P comediate with acetylcholine in nerves of opossum esophageal muscularis mucosa?

In opossum esophagus the highest density of varicose substance P-like immunoreactive deposits was in the muscularis mucosa, and this muscle layer, which is thick and prominent, was the most sensitive in contracting to exogenous substance P. On field stimulation, a tetrodotoxin-sensitive biphasic contraction was produced; an initial phasic component was followed after cessation of stimulation by a prolonged tonic component. This contractile response was unaffected by guanethidine or antagonists to several nerve mediators and to histamine and serotonin antagonists. Each component showed a different frequency dependence. The phasic component was abolished by atropine and potentiated by physostigmine; the tonic component was reduced or abolished after carbachol or physostigmine and potentiated by atropine. The tonic component was inhibited after substance P tachyphylaxis or by the substance P antagonist [D-Pro2, D-Trp7,9]substance P. On electron microscopic examination, the majority of nerve varicosities contained a mixture of small agranular and large granular vesicles. We postulate that acetylcholine and substance P may coexist in and be coreleased from nerves of this muscle and that the acetylcholine release occurs first, causing an initial phasic response and delaying and diminishing the release of substance P. The two mediators cause muscle contraction by actions on independent receptors.     

Cytoplasmic constriction and vesiculation after axotomy in the squid giant axon

Summary The squid giant axon responded to a transection injury by producing a gradient of cytoplasmic and vesicular changes at the cut end. At the immediate opening of the cut axon the cytoplasm was fragmented and dispersed and the vesicles in this region were in rapid Brownian movement. Approximately 0.1 mm further in, at the site of maximal axonal constriction, the axoplasm was condensed into a compact, constricted mass containing many large vesicles. The axoplasm was normal a few millimetres beyond this constricted, vesiculated end. It appears that transection triggered the transformation of normal axoplasm into a tightly constricted, highly vesiculated structure. This modified axoplasm at the cut end may slow the spread of damage and degeneration by preventing the bulk outflow of axoplasm, by slowing down the loss of intracellular molecules and by slowing down the influx of destructive extracellular ions (like calcium and chloride).     

The viscosity of mammalian nerve axoplasm measured by electron spin resonance.

1. The microviscosity of the axoplasm of can sciatic nerve was determined by an in vitro electron spin resonance (e.s.r.) method using the spin label tempone. To identify the spin label signal as one arising only from within the axoplasm, Ni2+ was used as a line broadening agent. In one series of experiments in nerves with sheath intact the Ni2+ ion was shown to eliminate the tempone signal arising from the surface water, and in another series of experiments, with the sheath slit, to eliminate the signal from the extracellular space as well. 2. A microviscosity of less than 5 centipoise (cP), i.e. 5x that of water, was determined for the axoplasm. Changes in the viscosity of the nerve axoplasm as a function of temperature over a range of 38 degrees down to 2 degrees C were seen to follow closely the viscosity change found for a water solution. 3. The microviscosity of nerve axoplasm and its change with temperature were related to axoplasmic transport of material in nerve fibres. The results were used to exclude a large increase in viscosity at low temperatures as the cause for the cold-block of fast axoplasmic transport.     

Turnover of adenine nucleotides in cholinergic synaptic vesicles of the Torpedo electric organ

Synaptic vesicles were isolated from perfused blocks of electric tissue on sucrose density gradients in a zonal rotor. In vesicles from control tissue the composition was ATP (83%), ADP (15%) and AMP (2%): the corresponding figures for stimulated tissue were 69, 22 and 6% respectively: thus ATP is the predominant vesicular adenine nucleotide in both types of vesicle. Stimulation of the nerves to the tissue at a frequency (0.1 Hz) which does not cause a fall in vesicle numbers induces an approx. 50% loss of total vesicular nucleotides, the same as the degree of loss of acetylcholine in previous experiments.When tissue blocks are perfused with a solution containing [2-³H]adenosine for several hours, 85% of the radiolabel recovered in the isolated vesicle fraction is in the form of ATP. Besides some radiolabelled ADP and a reproducible but small contribution of inosinemonophosphate, traces of radiolabelled AMP, adenosine, adenine, hypoxanthine and inosine were detected. On stimulation of nerves to tissue blocks at 0.1 Hz two populations of synaptic vesicles can be isolated, the denser one of which contains the bulk (70%) of the newly synthesized vesicular ATP as well as acetylcholine. Vesicles sedimenting at the original sucrose density lose both ATP and acetylcholine. The specific radioactivity of ATP in the denser vesicles after a simulation of 1280–1800 impulses was about four times higher than that of vesicles equilibrating at the original sucrose density.The results suggest that adenosine is an effective precursor of vesicular adenine nucleotides. On stimulation nucleotides are lost from synaptic vesicles together with the neurotransmitter. The new population of vesicles appearing on stimulation has a high turnover rate for both ATP and acetylcholine.     

Ultrastructural localization of VIP-like immunoreactivity in large dense-core vesicles of ‘cholinergic-type’ nerve terminals in cat exocrine glands

Exocrine glands of the cat were analysed with the peroxidase-antiperoxidase method and routine electron microscopy. Vasoactive intestinal polypeptide (VIP)-like immunoreactivity was observed in certain nerve endings in the submandibular salivary gland, lacrimal gland and Harderian gland. The distribution of the VIP immunoreactive nerve fibres agreed well with earlier light microscopic findings. At the electron microscopic level electron-dense precipitates representing VIP-like immunoreactivity were seen in so-called large dense-core vesicles (median diameter about 990A?) in nerve fibres and varicosities also containing many small (‘immunonegative’) agranular vesicles. In conventional electron microscopy, the small agranular vesicles outnumbered the large dense-core vesicles by about 9 to 1. Immunoreactive fibres and varicosities could be seen close to the secretory acini (distance less than 400A?) and more distant (1500A?or more) to e.g. demilunes, ducts and blood vessels of the glands. The number and distribution of immunoreactive nerve fibres were not affected by sympathectomy. Furthermore, no typical ‘p-type’ bouton profiles, which are dominated by large opaque vesicles (dia. 800–2000A?), could be seen in the ultrastructural analysis of conventional preparations of the glands.The morphological features of the VIP immunoreactive nerve endings could not be distinguished from those often described as representing cholinergic fibres. These findings are in agreement with earlier suggestions of a possible coexistence of acetylcholine and VIP in neurons innervating exocrine glands and indicate possible functions for VIP in the roles of these nerves in evoking vasodilation and exocrine secretion.     

Close association of peptidergic nerves with lymphocytes in canine and monkey ileal villi.

Intimate association of peptidergic nerves with lymphocytes of canine and monkey ileal villi was demonstrated by immunohistochemistry and transmission electron microscopy. A swollen, presumably terminal, portion of nerves containing large cored vesicles and small clear vesicles was in direct contact with a lymphocyte. The apposing membranes of the nerve and lymphocyte were thickened and darkened, being separated by a narrow uniform space. The lymphocyte-associated nerves contained immunoreactivity for substance P(SP), calcitonin gene-related peptide (CGRP) or vasoactive intestinal polypeptide (VIP), localized in large-cored vessels. These result support the hypothesis that peptidergic nerves may play a regulatory role in mucosal immune responses.     

Fine structure of nerves in the regenerating limb of the newt Triturus

The structural integrity of some tissues and the regeneration of extremities in some vertebrates depends upon the nervous system. To investigate the structure of nerves exhibiting trophic function, nerves in regenerating forelimbs of adult newts, Triturus viridescens, were studied with the electron microscope. Nerve fibers sprout from the transected axons, 2–3 days after amputation of the limb, and invade all portions of the blastema and epidermis in large numbers. Regenerating nerve fibers contain a greatly increased amount of smooth-surfaced channels of endoplasmic reticulum containing moderately dense material, an increased number of microtubules, and large (1000 Å) membrane-bounded dense granules. The latter were not observed normally and could be distinguished from synaptic vesicles and dense-core vesicles thought to contain catechol amines. In larger nerves, the organelles are distributed in the peripheral axoplasm around a central zone containing neurofilaments. The relationship of the fine structural changes occurring during regeneration to the trophic action of nerves and the regeneration of nerve fibers is discussed. The tortuous membranous tubules of endoplasmic reticulum could serve as channels for the transport of substances, either trophic material or materials necessary for growth of the nerve, down the axon. Microtubules may play a role in the regulation of form of the growing axon and also be related to axoplasmic flow or migration of particles (e.g., granules) along their length. The large membrane-bounded dense granules appearing during regeneration resemble neurosecretory granules, which have been associated with regeneration in some invertebrates. These structures could, therefore, contain a trophic substance or hormone that is transported down the axon, released into the intercellular spaces, and controls subsequent regeneration of the limb.     

Subcellular fractionation of cat submandibular gland: Comparative studies on the distribution of acetylcholine and vasoactive intestinal polypeptide (VIP)

In previous studies, evidence has been obtained for the presence of vasoactive intestinal polypeptide (VIP) in presumptive cholinergic neurons innervating the exocrine glands in the cat. In the present study, an attempt was made to define the storage sites of these two compounds in the cat submandibular gland using subcellular fractionation techniques. Particulate VIP was preferentially found in dense fractions (0.78–0.97 M sucrose) of a density gradient. Particle-bound acetylcholine showed a bimodal distribution in the gradient with relative enrichment in a dense fraction (0.87 M sucrose) but mainly in lighter fractions (0.28–0.44 M sucrose). More than 50% of all acetylcholine was recovered in a soluble form. For comparison, noradrenaline was also analysed and found in paniculate form in fractions ranging between 0.44–0.78 M sucrose. The electron microscopic analysis revealed presence of i.a. small clear vesicles in the lighter fractions, whereas the fractions rich in VIP contained i.a. many larger vesicles, sometimes with an electron-dense core. In agreement with earlier ultrastructural immunocytochemical studies on intact tissue, the present results support the view that VIP is present in large dense-cored vesicles. Most of the particulate acetylcholine seems to be localized in small clear vesicles, although a small proportion could be associated with large vesicles. Whether acetylcholine and VIP coexist in such large vesicles or whether separate populations of large vesicles exist, remains to be elucidated.     

Light and electron microscopic immunocytochemical localization of vasoactive intestinal polypeptide VIP-like activity in the rat small intestine

Vasoactive intestinal polypeptide nerve processes and cell bodies were identified by electron microscopic immunocytochemistry in the rat small intestine. Labeled nerve processes were numerous in the inner circular smooth muscle coat and mainly in the mucosa, but were absent in the longitudinal muscle layer. Submucosal blood vessels were often surrounded by immunoreactive vasoactive intestinal polypeptide positive nerves, in close associations (distance less than 40 mn) to blood vessel basement membranes and to smooth muscle cells. In the ganglia of the myenteric and submucous plexuses, labeled fibers surrounded unstained neural cell bodies. The synaptic vesicles of vasoactive intestinal polypeptide positive terminals were 35-40 nm in diameter and some dense core vesicles (80-120 nm in diameter) were also observed in the same profiles. These observations suggest that vasoactive intestinal polypeptide nerves may participate in regulating smooth muscle activity and local blood flow in the small intestine.     

The ontogeny and fine structure of calcitonin gene-related peptide (CGRP)-immunoreactive nerve fibers in the celiac ganglion of rats

Calcitonin gene-related peptide (CGRP)-like immunoreactivity has been reported to occur in both varicose and non-varicose nerve fibers among and around principal ganglion cells in the celiac ganglion of rats. The present electron microscopic immunohistochemistry revealed a small number of myelinated nerve fibers immunoreactive for CGRP, although most of the immunoreactive fibers were unmyelinated. In the fetal development, CGRP-immunoreactive nerve fibers were first revealed at day 18 of gestation and thereafter gradually increased in number. Both the density and distribution of the immunoreactive fibers within the ganglion similar to those in adult rats were attained by postnatal day 14. This developmental change was compared with that of enkephalin-immunoreactive nerve fibers in the same ganglion. In electron microscopy, the immunoreactive fibers with varicosities were characterized by abundant small clear vesicles mixed with large granular vesicles. These formed numerous axo-dendritic and several axo-somatic synapses with the principal ganglion cells, whereby the immunoreactive fibers were presynaptic. The immunoreactive material was localized in the core of the large granular vesicles as well as in the axoplasm. On the other hand, the immunoreactive fibers without varicosities were characterized by neurofilaments, neurotubules and small mitochondria; small clear or large granular vesicles were rarely found in the immunoreactive fibers. They lay in no direct apposition to adjacent neuronal elements and were, therefore, regarded either as fibers passing through the ganglion or as preterminal portions of the CGRP-immunoreactive varicose terminals.     

Pharmacological and ultrastructural studies on the electron dense cores of the vesicles that accumulate in noradrenergic axons constricted in vitro.

The noradrenaline storage vesicles that accumulate in axons proximal to ligatures applied to cat hypogastric nerves incubatedin vitro have been examined in the electron microscope with different fixation and staining procedures after exposure of the nerves to a variety of drugs which alter their noradrenaline content. After glutaraldehyde/osmium tetroxide fixation and lead citrate staining the granules possess electron-dense cores irrespective of their noradrenaline content. After fixation with Palade's osmium tetroxide or glutaraldehyde/dichromate followed by lead citrate staining, dense cores are not seen after treatment with noradrenaline-depleting agents but reappear after staining additionally with uranyl acetate.It is concluded that the matrix of axonal (large) noradrenaline storage vesicles contains some material in addition to catecholamines and this possibility is discussed in connection with the relationship between large and small dense-cored vesicles seen in sympathetic nerve endings.     

Axoplasm architecture and physical properties as seen in the Myxicola giant axon.

A technique is described for extracting axoplasm from the giant axon of a marine worm, Myxicola infundibulum. The operation can be completed in 10 sec. 2. Axoplasm is pulled from the axon of a living worm as a long, clear cylinder, up to 35 cm long and 70 mg wet weight. The worm regenerates a new giant axon in about 4 months. 3. Myxicola axoplasm is a gel, 87% water, held together by protein neurofilaments. It contains small amounts of mitochondria and vesicles, but no detectable microtubules. 4. The internal structure of the gel is superficially similar to that of yarn. Closer inspection with light and electron microscopy, and X-ray diffraction, show it to be organized in a hierarchy of helical forms. Squid giant axons have a similar structure. 5. Initial estimates of the bulk physical properties of extracted Myxicola axoplasm give: breaking strength, 1400 g/cm2; specific gravity, 1-05 g/cm3; birefringence, 1-6 X 10(-4); index of refraction, 1-351; resistivity, 57 omega cm. These average values are shown to be compatible with the observed structure and composition. 6. Despite its mechanical strength, the axoplasm gel is so hydrated that Na+, K+ and homarine diffuse through it at rates approaching those in free solution. Fewer than about 5% of each of these ions are tightly bound to the gel. 7. It is argued that (a) the structure and physical properties of Myxicola axoplasm are representative of those in other axons, (b) the compound helix architecture results from twist of parallel, cross-linked fibrous proteins, and (c) this sturcture serves as a flexible internal skeleton for nerve cell processes.     

Suggestive evidence for a direct innervation of mucosal mast cells

Mast cells are often observed near nerves and functional evidence suggests an innervation of these cells. In the present ultrastructural study, nerve terminals containing many small clear vesicles and a few large vesicles with dense matrix were observed in direct contact with the plasma membrane of mucosal mast cells in the rat ileum, strongly suggestive of a direct innervation.