Autonomic innervation of the arteriovenous anastomoses in the dog tongue

Summary The innervation of the arteriovenous anastomoses in the dog tongue has been investigated. At the lightmicroscopic level, the vessels were found to be densely supplied with adrenergic and AChE-positive nerve plexuses and less densely with the quinacrine-binding nerve plexus. At the electron-microscopic level, at least two apparently different types of axon profiles were identified: 1) Small vesicle-containing axons, characterized by many small granular vesicles, variable numbers of small clear vesicles and large granular vesicles. Storage of endogenous amines and uptake of exogenous amines into most small granular vesicles and many large granular vesicles was demonstrated. These axons stained only lightly with reaction products for AChE activity and thus seemed to be adrenergic in nature. Some axons contained numerous large granular vesicles, whose cores occasionally stained with uranyl ions; this suggests a co-localization of ATP or peptides as neurotransmitters. 2) Small granular vesicle-free axons, containing small clear vesicles and large granular vesicles in variable ratio. Most cores of these large granular vesicles were heavily stained with uranyl ions. No storage or uptake of amine into the synaptic vesicles was detected. Some axons appeared to be typically cholinergic, some, typically non-adrenergic, noncholinergic, and the rest, intermediate between the two. All axons stained heavily with reaction products for AChE activity, suggesting their cholinergic nature.     

Morphological and biochemical studies on the development of cholinergic properties in cultured sympathetic neurons. I. Correlative changes in choline acetyltransferase and synaptic vesicle cytochemistry

Under certain culture conditions, neonatal rat superior cervical ganglion neurons display not only a number of expected adrenergic characteristics but, paradoxically, also certain cholinergic functions such as the development of hexamethonium-sensitive synaptic contacts and accumulation of choline acetyltransferase (ChAc). The purpose of this study was to determine whether the entire population of cultured neurons was aquiring cholinergic capabilities, or whether this phenomenon was restricted to a subpopulation. After 1--6 and 8 wk in culture, neurons were fixed in KMnO4 after incubation in norepinephrine and prepared for electron microscopy analysis of synaptic vesicle content to determine whether vesicles were dense cored or clear. ChAc, acetylcholinesterase (AChE), and DOPA-decarboxylase (DDC) activities were assayed in sister cultures. In the period from 1 to 8 wk in culture, the average ChAc activity per neuron increased 1,100-fold, and the DDC and AChE activities increased 20- and 30-fold, respectively. After 1 wk in culture, 48 of 50 synaptic boutons contained predominantly dense-cored vesicles, but by 8 wk the synaptic vesicle population was predominantly of the clear type. At intermediate times, the vesicle population in many boutons was mixed. The morphology of the synaptic contacts on neuronal surfaces was that characteristic of autonomic systems, with no definite clustering of the vesicles adjacent to the area of contact. Increased vesicle size correlated with increasing age in culture and the presence of a dense core. Considering these data along with available physiological studies, we conclude that these cultures contain one population of neurons that is initially adrenergic. Over time, under conditions of this culture system, this population develops cholinergic mechanisms. That a neuron may, at a given time, express both cholinergic and adrenergic mechanisms is suggested by the approximately equal numbers of clear and dense-cored vesicles in the boutons found at the intermediate times.     

Electron microscopic analysis of tyrosine hydroxylase, dopamine-beta-hydroxylase and phenylethanolamine-N-methyltransferase immunoreactive innervation of the hypothalamic paraventricular nucleus in the rat

The catecholaminergic innervation of the hypothalamic paraventricular nucleus (PVN) of the rat was studied by preembedding immunocytochemical methods utilizing specific antibodies which were generated against catecholamine synthesizing enzymes. Phenylethanolamine-N-methyltransferase (PNMT)-immunoreactive terminals contained 80-120 nm dense core granules and 30-50 nm clear synaptic vesicles. The labeled boutons terminated on cell bodies and dendrites of both parvo- and magnocellular neurons of PVN via asymmetric synapses. The parvocellular subnuclei received a more intense adrenergic innervation than did the magnocellular regions of the nucleus. Dopamine-beta-hydroxylase (DBH)-immunopositive axons were most numerous in the periventricular zone and the medial parvocellular subnucleus of PVN. Labeled terminal boutons contained 70-100 nm dense granules and clusters of spherical, electron lucent vesicles. Dendrites, perikarya and spinous structures of paraventricular neurons were observed to be the postsynaptic targets of DBH axon terminals. These asymmetric synapses frequently exhibited subsynaptic dense bodies. Paraventricular neurons did not demonstrate either PNMT or DBH immunoreactivity. The fibers present within the nucleus which contained these enzymes are considered to represent extrinsic afferent connections to neurons of the PVN. Tyrosine hydroxylase (TH)-immunoreactivity was found both in neurons and neuronal processes within the PVN. In TH-cells, the immunolabel was associated with rough endoplasmic reticulum, free ribosomes and 70-120 nm dense granules. Occasionally, nematosome-like bodies and cilia were observed in the TH-perikarya. Unlabeled axons established en passant and bouton terminaux type synapses with these TH-immunopositive cells. TH-immunoreactive axons terminated on cell bodies as well as somatic and dendritic spines of paraventricular parvocellular neurons. TH-containing axons were observed to deeply invaginate into both dendrites and perikarya of magnocellular neurons. These observations provide ultrastructural evidence for the participation of central catecholaminergic neuronal systems in the regulation of the different neuronal and neuroendocrine functions which have been related to hypothalamic paraventricular neurons.     

Membrane Composition of Adrenergic Large and Small Dense Core Vesicles and of Synaptic Vesicles: Consequences for Their Biogenesis

The membrane proteins of adrenergic large dense core vesicles, in particular those of chromaffin granules, have been characterized in detail. With the exception of the nucleotide carrier all major peptides have been cloned. There has been a controversy whether these vesicles contain antigens like synaptophysin, synaptotagmin and VAMP or synaptobrevin found in high concentration in synaptic vesicles. One can now conclude that large dense core vesicles also contain these peptides although in lower concentrations. The biosynthesis of large dense core vesicles is analogous to that of other peptide secreting vesicles of the regulated pathway. One cannot yet definitely define the biosynthesis of small dense core vesicles which apparently have a very similar membrane composition to that of large dense core vesicles. They may form directly from large dense core vesicles when their membranes have been retrieved after exocytosis. These membranes may become sorted in an endosomal compartment where peptides may be deleted or added. Such an addition could be derived from synaptophysin-rich vesicles present in adrenergic axons. However small dense core vesicle peptides may also be transported axonally independent of large dense core vesicles. For proving one of these possibilities some crucial experiments have been suggested.     

A CHOLINERGIC COMPONENT IN THE INNERVATION OF THE LONGITUDINAL SMOOTH MUSCLE OF THE GUINEA PIG VAS DEFERENS : The Fine Structural Localization of Acetylcholinesterase

Acetylcholinesterase (AChE) has been detected on the plasma membrane of about 25% of the axons in the longitudinal smooth muscle tissue of guinea pig vas deferens. These axons are presumably cholinergic. No enzyme was detected in the remaining 75% of axons. These axons are presumably adrenergic. The plasma membrane of the Schwann cells associated with the cholinergic axons also stained for AChE. Some axon bundles contained only cholinergic or adrenergic axons while others contained both types of axon. When a cholinergic axon approached within 1100 A of a smooth muscle cell, there was a patch of AChE activity on the muscle membrane adjacent to the axon. It is suggested that these approaches are the points of effective transmission from cholinergic axons to smooth muscle cells. Butyrylcholinesterase activity was detected on the plasma membranes of all axons and smooth muscle cells in this tissue.     

Electron microscopic histochemistry of acetylcholinesterase of rat brain by Karnovsky's method

Summary Karnovsky's electron microscopic acetylcholinesterase method was successfully applied to rat brain fixed by vascular perfusion with either 2% glutataldehyde or 4% formaldehyde. 2% glutaraldehyde showed better fine structure but worse preservation of the enzyme than 4% formaldehyde.In the neuropil of the caudate nucleus, locus coeruleus and dorsal nucleus of the vagus, AChE activity was most intensely demonstrated on the plasma membranes of preterminal axons and somewhat less strongly on those of axon terminals and contacting dendritic branches. The axoplasm and synaptic vesicles were usually negative, while the cytoplasm and neurotubules of the dendritic branches showed some activity. In the nodule and uvula of the cerebellum moderate activity was exhibited on the synaptic contacts between the mossy fiber endings and granule cell dendrites. In the hypothalamus and other autonomic regions the characteristic coexistence of AChE and granulated vesicles of axon terminals could be demonstrated.In the perikaryon of positive nerve cells, AChE was observed strongly in the cytoplasm, disseminated irregularly or attached to the endoplasmic reticulum, while it was absent in the mitochondria and lysosomal dense bodies.     

Electron microscopic analysis of tyrosine hydroxylase,dopamine-β-hydroxylase and phenylethanolamine-N-methyltransferase immunoreactive innervation of the hypothalamic paraventricular nucleus in the rat

Summary The catecholaminergic innervation of the hypothalamic paraventricular nucleus (PVN) of the rat was studred by preembedding immunocytochemical methods utilizing specific antibodies which were generated against catecholamine synthesizing enzymes. Phenylethanolamine-N-methyltransferase (PNMT)-immunoreactive terminals contained 80–120 nm dense core granules and 30–50 nm clear synaptic vesicles. The labeled boutons terminated on cell bodies and dendrites of both parvo- and magnocellular neurons of PVN via asymmetric synapses. The parvocellular subnuclei received a more intense adrenergic innervation than did the magnocellular regions of the nucleus. Dopamine--hydroxylase (DBH)-immunopositive axons were most numerous in the periventricular zone and the medial paryocellular subnucleus of PVN. Labeled terminal boutens contained 70–100 nm dense granules and clusters of spherical, electron lucent vesicles. Dendrites, perikarya and spinous structures of paraventricular neurons were observed to be the postsynaptic targets of DBH axon terminals. These asymmetric synapses frequently exhibited subsynaptic dense bodies. Paraventricular neurons did not demonstrate either PNMT or DBH immunoreactivity. The fibers present within the nucleus which contained these enzymes are considered to represent extrinsic afferent connections to neurons of the PVN.Tyrosine hydroxylase (TH)-immunoreactivity was found both in neurons and neuronal processes within the PVN In TH-cells, the immunolabel was associated with rough endoplasmic reticulum, free ribosomes and 70–120 nm dense granules. Occasionally, nematosome-like bodies and cilia were observed in the TH-perikarya. Unlabeled axons established en passant and bouton terminaux type synapses with these TH-immunopositive cells. TH-immunoreactive axons terminated on cell bodies as well as somatic and dendritic spines of paraventricular parvocellular neurons. TH-containing axons were observed to deeply invaginate into both dendrites and perikarya of magnocellular neurons.These observations provide ultrastructural evidence for the participation of central catecholaminergic neuronal systems in the regulation of the different neuronal and neuroendocrine functions which have been related to hypothalamic paraventricular neurons.Supported by NIH Grant NS 19266 to W.K. Paull     

Acetylcholinesterase activity at the synapses of presynaptic boutons with presumed alpha-motoneurons in chicken ventral horn. Light- and electron-microscopic studies

Acetylcholinesterase (AChE) activity at the synapses of presynaptic boutons on presumed alpha-motoneurons in the chicken ventral horn was studied histochemically at the light- and electron-microscope levels. At the light-microscope level, many dot-like AChE-active sites were observed on the soma and dendrites of presumed alpha-motoneurons. On electron microscopy, reaction products for AChE activity were observed mainly in the synaptic clefts of the four kinds of presynaptic boutons: (1) S type boutons, (2) boutons containing small, spherical, dense cored vesicles (diameter range, 60-105 nm) and spherical, clear vesicles, (3) boutons containing medium-sized, spherical, dense cored vesicles (65-115 nm) and spherical, clear vesicles, and (4) boutons containing large, spherical, dense cored vesicles (80-130 nm) and spherical, clear vesicles. In the light of previous physiological and biochemical studies, the present results suggest the possibility that each of these presynaptic boutons which are AChE-active in their synaptic clefts may contain acetylcholine, substance P, or enkephalins which acts as a neurotransmitter or modulator.     

Ultrastructure of calcitonin gene-related peptide-immunoreactive nerve fibres in guinea-pig peribronchial ganglia.

Calcitonin gene-related peptide-immunoreactive (CGRP-IR) nerves within guinea-pig peribronchial ganglia were studied at ultrastructural level using pre-embedding immunohistochemistry. Preterminal CGRP-IR axons were unmyelinated and contained singular immunoreactive dense core vesicles. CGRP-IR axon terminals were filled with numerous non-reactive small clear vesicles and few immunoreactive dense core vesicles. Some of these terminals were presynaptic to large neuronal processes emerging from local ganglion cells. Another population of presynaptic varicosities lack CGRP-IR. Within CGRP-IR terminals, non-reactive clear vesicles were clustered at the presynaptic membrane whereas CGRP-IR large vesicles remained in some distance from the synaptic cleft. The present observations indicate that: (1) at least two neurochemically different types of synaptic input exist to guinea-pig peribronchial ganglia. (2) CGRP-IR presynaptic terminals probably utilize a non-peptide transmitter for fast synaptic transmission, whilst the peptides are likely to be released parasynaptically and may act in a modulatory fashion.     

Effect of sympathomimetic substances and DOPA on the ultrastructure of the nervous apparatus of the heart]

After a single administration of norepinephrine or DOPA to albino rats there occurred an incorporation of norepinephrine into the adrenergic axons of the heart and its deposition in the form of granules in small synaptic vesicles, about 300 A in diameter. The adrenergic and cholinergic axons can be thus differentiated. The amount of cholinergic axons in the auricles is greater than that of the adrenergic ones. The adrenergic terminals came into the most intimate contact with the cholinergic terminals and with the endothelial cells of the blood capillaries and the myocardial muscle cells. It is supposed that adrenergic fibers can act upon the heart muscle in three ways: by means of presynaptic inhibition through the cholinergic axons, by humoral route, and directly on the myocardial muscle cells.     

Innervation of the rabbit cornea. A histochemical and electron-microscopic study

The innervation of the rabbit cornea was investigated histochemically and electron-microscopically with special reference to the autonomic nerves. Both formaldehyde- and glyoxylic-acid-induced fluorescence methods revealed adrenergic nerves in the stroma; a few fibres were also observed between the basal epithelial cells near the limbus. Acetylcholinesterase- (AChE-) positive nerves were found both in the stroma and in the epithelium, whereas nonspecific cholinesterase (NsChE) activity appeared only in the stromal nerves. Under the electron microscope, both AChE and NsChE activities were observed to be located in the axon membranes. A weak NsChE reaction also appeared in the Schwann cells. When the specimens fixed with KMnO4 were examined under the electron microscope, most nerve fibres did not contain any special axoplasmic structures, although several axons contained mitochondria. Moreover, two vesicle-containing axon types were found in the stromal nerves; axons with small granular vesicles and axons containing small agranular vesicles. In the epithelium, two types of fibres were observed; one type containing only mitochondria while the other showed both agranular vesicles and mitochondria.     

Occurrence of uranaffin-positive synaptic vesicles in both adrenergic and non-adrenergic nerves of the rat anococcygeus muscle

Summary The uranaffin reaction in rat anococcygeus muscle, which receives a dual innervation of both adrenergic and non-cholinergic, non-adrenergic nerves was examined. Dense reaction product was observed in the vesicular membranes and/or the cores of some synaptic vesicles in the adrenergic nerve terminals. Occasional vesicles were filled up with dense reaction product. In the prominent population of small clear vesicles, however, no dense reaction product was observed. The number of small granular vesicles in the adrenergic nerve terminals was markedly increased after the administration of 5-hydroxydopamine (5-OHDA). These granular vesicles were moderately stained with uranaffin deposit on the cores but their limiting membranes possessed no uranaffin deposit at all.In the non-adrenergic nerve terminals, on the other hand, uranaffin deposit of variable density was observed on the cores of large granular vesicles but never on their limiting membranes or on the small clear vesicles. There was no change in the axon profiles after the administration of 5-OHDA.The possible occurrence of purines in the cores of large granular vesicles in the non-adrenergic nerves is discussed.     

Innervation of iridic melanophores

Summary The nerve supply to the iridic melanophores of the rat was studied with the electron microscope. The adrenergic and cholinergic terminals were identified with the aid of 5-hydroxydopamine, which produces dense-cored 400–800 Å synaptic vesicles in adrenergic axon varicosities, whereas the synaptic vesicles of cholinergic axons remain empty. It was found that both adrenergic and cholinergic terminal axons come in close apposition (200–250 Å) with the melanophores. The appositions have the same appearance as synapses in peripheral tissues. It seems likely that the murine iridic melanophores have a double innervation, although its functional significance is obscure.This work has been supported by grants from Lunds Läkarsällskap, the Swedish Medical Research Council (Project no. B69-14X-2321-02 and B69-14X-712-04C) and NIH (06701-02).     

Differential expression of acetylcholinesterase in the brainstem, ventrobasal thalamus and primary somatosensory cortex of perinatal rats, mice, and hamsters

Acetylcholinesterase (AChE) is transiently expressed by thalamocortical axons in the rat, and staining for this enzyme has been used extensively to study the development of thalamocortical projections. In the present study, patterns of AChE staining were compared in the trigeminal brainstem, thalami and primary somatosensory cortices of perinatal rats, mice, and hamsters. As previously reported, the ventral posteromedial nucleus (VPM) of rats showed dense AChE staining from P-0 at least through P-8. The ventral posterolateral nucleus (VPL) contained heavy AChE staining at least through P-60. In the cortex, there was also dense AChE staining which was organized somatotopically in patches similar to those observed with other methods such as cytochrome oxidase (CO) staining. However, by adulthood, AChE staining revealed a negative image of the CO staining pattern in lamina IV. In the mouse and hamster, there was dense AChE staining inVPL from P-0 through adulthood, but VPM was much less heavily stained for this enzyme. Moreover, the staining in VPL of mice was markedly reduced after transection of axons that travel to the thalamus in the medial lemniscus, suggesting that much of it was contained in these afferent fibers. In the cortices of both perinatal and adult mice and hamsters, AChE staining yielded a negative image of the somatotopically organized patches demonstrable with CO staining. This negative image was apparent by P-2 in the mouse and P-4 in the hamster. These results document a dramatic species difference with respect to the expression of AChE in the thalami and cortices of developing rodents. The differences between the patterns observed in rats vs mice and hamsters probably reflect the fact that cortical AChE in the latter species is not contained in thalamocortical afferents arising from either VPM or VPL.     

Ultrastructural changes in the nerves innervating the cerebral artery after sympathectomy

Summary The ultrastructure of the innervation of the anterior cerebral artery of the rat was studied in control animals and in animals after superior cervical ganglionectomy.Fluorescence histochemistry shows a periarterial network of intensely fluorescent fibers which are divided into two groups, adventitial and periadventitial. The fluorescence begins to decrease 26 hours after, and completely disappears about 32 hours after, ganglionectomy.Fine structural changes are first observed 18 hours after ganglionectomy, when the axoplasm of degenerating axons becomes electron dense. This density gradually increases up to about 32 hours. By 32 hours most axons with disintegrating axolemmas become inclusion bodies of the Schwann cells. At this stage, synaptic vesicles can still be distinguished as less dense areas, but the membrane structures of synaptic vesicles and mitochondria are difficult to recognize. The degenerating axons are gradually absorbed and by 38 hours dense, residual bodies are observed in the Schwann cells. Generally speaking, the degeneration occurs first in the adventitial fibers and then in the periadventitial fibers. The transient appearance of small, granular vesicles is noticed in axon terminals about 18 hours after denervation, although very few small, granular vesicles are seen in control tissue or at later stages of degeneration.     

Innervation of somatostatin synthesizing neurons by adrenergic,phenylethanolamine-N-methyltransferase (PNMT)-immunoreactive axons in the anterior periventricular nucleus of the rat hypothalamus

Summary The adrenergic innervation of somatostatin synthesizing neurons located in the anterior region of the rat hypothalamic periventricular nucleus was studied by means of a light and electron microscopic immunocytochemical double labelling technique. This region which is the source of hypophysiotrophic somatostatin immunoreactive (IR) neurons also receives a dense plexus of adrenergic axons as determined by immunocytochemistry of phenylethanolamine-N-methyltransferase (PNMT), the marker enzyme for the central adrenergic system. The simultaneous detection of PNMT and somatostatin antigens in hypothalamic sections of colchicine pretreated animals revealed a congruency in the distribution of the labelled elements and also close juxtaposition of PNMT-IR axons to somatostatin producing neurons. At the ultrastructural level, axo-somatic and axo-dendritic synaptic connections were found between PNMT-containing axons and somatostatin expressing neurons. These morphological findings support the view that the central adrenergic system might influence the production and secretion of growth hormone in the pituitary gland by a direct monosynaptic interaction with somatostatin synthesizing neurons.     

Chromogranin A processing in sympathetic neurons and release of chromogranin A fragments from sheep spleen.

Chromogranin A (CGA) has been localized to the large dense cored vesicles (LDV) of sympathetic neurons. SDS-PAGE and immunoblotting of soluble LDV proteins from ox and dog adrenergic neuronal cell bodies, axons and nerve terminals, revealed an increasing number of CGA-immunoreactive forms, consistent with proteolytic processing during axonal transport. Splenic nerve electrical stimulation (10 Hz, 2 min) revealed that, apart from CGA, these CGA-processing products are released from the sheep spleen. The secretion of CGA-derived fragments from sympathetic neurons might suggest a role in the regulation of synaptic transmission.     

Innervation of the mucosa of the small intestine of laboratory animals. I. Architecture, light microscopic structure and histochemical differentiation

1. The small intestine mucosa has a dense and differentiated innervation. The most dense nerve plexuses are contained in the villi. Each tubular gland is surrounded by a rope-ladder-like plexus, mostly developed in the lower and upper third. 2. According to our observations most of the axons in the mucosa are cholinergic. Cholinergic and adrenergic axons take part in the innervation of the glands. Moreover enterochromaffine cells can be innervated for instance sympathetically. Intraepithelial axons could not be found.     

Observations on the ultrastructure of nerve cells in the brain of the planarian,Dugesia gonocephala

Summary The submicroscopic structure of the nerve cells in the planarian brain was studied. Close similarities with neurons of other invertebrates were noted. In the cytoplasm of the planarian nerve cells there are at least three types of vesicular inclusions: 1) Clear vesicles (200–800 Å in epon embedded tissue) similar in morphological appearance to classical synaptic vesicles. These have generally some content of extremely low density but occasionally a dense core. 2) Dense vesicles (400–1,200 Å in epon embedded tissue) containing highly osmiophilic granules. Between the limiting membrane of the vesicle and the granule there is always a clear rim of variable width. These vesicles closely resemble synaptic vesicles described in vertebrate adrenergic endings. 3) Neurosecretory vesicles (600–1,300 Å in Vestopal embedded tissue) similar to elementary granules observed in neurosecretory systems in vertebrates and invertebrates. All three vesicle types have the same mode of origin from the Golgi membranes. All are present in the nerve cell processes of the neuropil as well as in the perikarya. Any given perikaryon or axon contains only one of the three vesicle types. All of these vesicles are considered to be discharged into the axons from their site of origin within the perikaryon.     

Fine structure of the autonomic nerves of the rabbit myometrium

Summary The fine structure of the preterminal nerve fibers of the rabbit myometrial smooth muscle was studied using potassium permanganate fixation or glutaraldehyde fixation with postosmification. The preterminal fibers were mostly formed by 2–10 axons enveloped by Schwann cells. Two kinds of axons and axon terminals were found. (1) Adrenergic axons, which contained many small, granular vesicles (diameter 300–600 Å) and large granular vesicles (diameter 700–1200 Å) which represented ca. 2% of the total count of the vesicles. (2) Nonadrenergic axons, which contained small agranular vesicles (diameter 300–600 Å) and large granular vesicles (diameter 700–1200 Å). Both types of axons formed preterminal varicosities along their course. The real terminal varicosities, representing the anatomical end of the axons, were usually larger than the preterminal ones and showed close contact to the plasma membranes of the smooth muscle cells. Both adrenergic and nonadrenergic terminals were found close to the smooth muscle cells, but a gap of at least 2000 Å was always present between the two cell membranes. The axons and preterminal varicosities of both types of nerves were in intimate contact with each other within the preterminal nerve fiber. Axo-axonal interactions between the two types of axons are possible in the rabbit myometrium. The relative proportion of the nonadrenergic axons from the total was about one fourth.