The innervation of the hepatic portal vein in the rabbit: Ultrastructural evidence against “purinergic” neurotransmission

Summary The relative density of adrenergic and non-adrenergic nerves in the hepatic portal vein of the rabbit has been determined ultrastructurally. Adrenergic nerves were visualised with the modified chromaffin procedure of Tranzer and Richards (1976). Nearly equal numbers of adrenergic and non-adrenergic nerve profiles were found, indicating a much greater density of innervation by non-adrenergic nerves than that described by Burnstock et al. (1979) using light microscopic histochemical methods. These results imply that part of the argument used by Burnstock et al. (1979) to support purinergic transmission in rabbit portal vein is probably invalid.     

Lack of correlation between ultrastructural and pharmacological types of non-adrenergic autonomic nerves

Summary In order to test the premise that non-adrenergic, non-cholinergic (NANC) autonomic nerves have a distinctive ultrastructural appearance, clearly different from that of cholinergic nerves, a detailed quantitative ultrastructural analysis has been made of the non-adrenergic innervation of 15 tissues thought from pharmacological evidence to be innervated by NANC nerves (rat and rabbit anococcygeus muscles; rabbit hepatic portal vein; extrinsically denervated toad lung); cholinergic nerves (atria of rat, rabbit, guinea-pig and toad); or both (guinea-pig cervical and thoracic trachealis muscle; rabbit rectococcygeus muscle; urinary bladder of rat, rabbit, guinea-pig and toad) in addition to their adrenergic supply. Following fixation with a modified chromaffin procedure allowing identification of adrenergic nerves, large, randomly selected samples of non-adrenergic nerve profiles from each tissue were analysed with respect to numbers, relative proportions, and size frequency distributions of different vesicle classes within the profiles. The neuromuscular relationships within each tissue were also analysed. On the basis of these analyses, it is clear that there are no consistent ultrastructural differences between cholinergic and NANC autonomic nerves: neither proportions nor sizes of the vesicles provide any clue as to the transmitter used by a particular nerve. The great majority of nerve profiles, whether cholinergic or NANC, contain predominantly small clear synaptic vesicles. Large filled peptidergic vesicles are no more common in most NANC nerves than in most cholinergic ones. It is concluded, on ultrastructural grounds, that the primary transmitter in these NANC autonomie nerves is most likely to be stored in and released from the small clear vesicles.     

Innervation of the large arteries and heart of the toad (Bufo marinus) by adrenergic and peptide-containing neurons

Summary The innervation of the major arteries and heart of the toad (Bufo marinus) was examined by use of glyoxylic acid-induced catecholamine fluorescence and peptide immunohistochemistry. All arteries possessed a moderate to dense plexus of adrenergic axons, which also showed neuropeptide Y-like immunoreactivity (NPY-LI). Some adrenergic axons in the intracardiac vagal trunks showed NPY-LI, but the varicose adrenergic axons innervating the cardiac muscle of the atria and ventricle, and the coronary blood vessels did not display NPY-LI. About half of the nerve cell bodies in the anterior sympathetic chain ganglia with dopamine--hydroxylase-LI (DBH-LI) also contained NPY-LI. The nerve cell bodies with DBH-LI alone were generally larger (median diameter 30 m) than those with both DBH-LI and NPY-LI (median diameter 20 m). Some cell bodies showing DBH-LI alone were surrounded by boutons with NPY-LI but not DBH-LI. Axons that displayed simultaneously both substance P-LI (SP-LI) and calcitonin gene-related peptide-LI (CGRP-LI) also formed a plexus around all arteries studied, being particularly dense around the mesenteric and pulmonary arteries. These axons are most likely sensory since SP-LI was reduced by capsaicin treatment, and nerve cell bodies with both SP-LI and CGRP-LI were found in dorsal root ganglia and the vagal ganglion. A dense plexus of axons showing somatostatin-LI was located around the pulmonary artery and its main intrapulmonary branches. A few nerves with vasoactive intestinal polypeptide-LI were found around the dorsal aorta and pulmonary artery. No perivascular nerves with enkephalin-LI were observed. Reversed-phase, high-pressure liquid chromatography of acid extracts of the large arteries showed that the major peaks of NPY-LI and SP-LI coeluted with porcine NPY (1–36) and synthetic SP (1–11), respectively. Thus, the location and structure of these peptides in perivascular nerves has been highly conserved during vertebrate evolution.     

Fine structure and innervation of the avian adrenal gland

Summary Apart from cholinergic nerve fibers, which make up the main part of efferent fibers to the avian adrenal gland (Unsicker, 1973b), adrenergic, purinergic and afferent nerve fibers occur. Adrenergic nerve fibers are much more rare than cholinergic fibers. With the Falck-Hillarp fluorescence method they can be demonstrated in the capsule of the gland, in the pericapsular tissue and near blood vessels. By their green fluorescent varicosities they may be distinguished characteristically from undulating yellow fluorescent ramifications of small nerve cells which are found in the ganglia of the adrenal gland and below the capsule. The varicosities of adrenergic axons exhibit small (450 to 700 Å in diameter) and large (900 to 1300 Å in diameter) granular vesicles with a dense core which is usually situated excentrically. After the application of 6-hydroxydopamine degenerative changes appear in the varicosities. Adrenergic axons are not confined to blood vessels but can be found as well in close proximity of chromaffin cells. Probably adrenergic fibers are the axons of large ganglion cells which are situated mainly within the ganglia of the adrenal gland and in the periphery of the organ and whose dendritic endings show small granular vesicles after treatment with 6-OHDA.A third type of nerve fiber is characterized by varicosities containing dense-cored vesicles with a thin light halo, the mean diameter (1250 Å) of which exceeds that of the morphologically similar granular vesicles in cholinergic synapses. Those fibers resemble neurosecretory and purinergic axons and are therefore called p-type fibers. They cannot be stained with chromalum-hematoxyline-phloxine. Axon dilations showing aggregates of mitochondria, myelin bodies and dense-cored vesicles of different shape and diameter are considered to be afferent nerve endings. Blood vessels in the capsule of the gland are innervated by both cholinergic and adrenergic fibers.Supported by a grant from the Deutsche Forschungsgemeinschaft (Un 34/1).     

Ultrastructure of catecholamine-containing axons in the intestine of the domestic fowl

Axons in the duodenum, ileum and rectum of the domestic fowl were identified as catecholamine-containing (CA) on the basis of positive reactivity following chromaffin fixation for electron microscopy. CA-axons in association with blood vessels in all regions of the intestine and in non-vascular sites in the small intestine had a 'typical' adrenergic appearance, in that they contained many small granular vesicles (SGV) and variable numbers of large granular vesicles (LGV). In the rectum the non-vascular CA-axon profiles were atypical, in that there were many elongated LGV and few SGV, and the chromaffin reactivity was weak. The nerve profiles in the rectum were dramatically reduced following 6-hydroxydopamine and reserpine treatment and were absent in rectum cultured in the absence of extrinsic ganglia. It was concluded that the profiles, in spite of their low chromaffin reactivity, truely represent CA-axons. The possibility was raised that the atypical morphology and reduced chromaffin reactivity is due to the presence of adrenaline.     

Observations on the fine structure of the baroreceptors and adrenergic innervation of the guinea-pig carotid sinus

We examined the fine structure of the baroreceptors and the adrenergic innervation of the guinea-pig carotid sinus. The tunica adventitia contained many nerve bundles whose perineuria enclosed unmyelinated nerve fibers, alone or together with myelinated nerve fibers. Baroreceptors, which lay close to elastic and collagen fibers in the adventitia and media, were surrounded by “terminal” cells with ultrastructural features characteristic of Schwann cells and contained inclusions of various types. Morphologic features of the baroreceptors included densely packed mitochondria, osmiophilic lamellated and homogeneous bodies, clear and granular vesicles, lamellar systems, glycogen granules, neurofilaments, neurotubuli, and vacuolated mitochondria. In animals that had been treated with 6-hydroxydopamine, occasional electrondense endings (or fibers) were observed in the adventitial layer. The baroreceptors in the guinea-pig carotid sinus appear to have most of the morphologic features reported for other species.     

Catecholaminergic nerves in the embryonic chick ovary: co-localization with β2-adrenoceptor-bearing steroidogenic cells

Summary The present study investigates the innervation of the embryonic chick ovary with regard to (i) development and compartmentalization of catecholaminergic nerves, and (ii) presence of adrenoceptors on steroidogenic target cells of catecholaminergic nerve terminals. Catecholaminergic nerve fibers visualized by glyoxylic acid-induced histofluorescence first appeared at embryonic day (E) 13. From E15 through E21 the density of fluorescent aminergic nerves increased markedly in parallel with the concentration of catecholamines and numbers of nerve bundles and single axons seen at the electron-microscopic level. Catecholaminergic nerves were confined to the ovarian medulla and closely associated with interstitial cells. Nerve terminals approached interstitial cells up to a distance of 20 nm and, in their majority, exhibited uptake of the false adrenergic transmitter 5-hydroxydopamine. Although adrenaline amounted to 14% of the total catecholamine content at E21, adrenaline immunoreactivity was only detected in adrenal chromaffin cells, but not in nerve fibers or cell bodies within the ovary. Interstitial cells structurally matured between E15 and E21 as documented by an increase of smooth endoplasmic reticulum and tubular mitochondria. Monoclonal antibodies mAB 120 and BRK 2 raised against avian ₁ and mammalian ₂-adrenergic receptors revealed the presence of ₂-adrenoceptor-like immunoreactivity on the surface of interstitial cells, but not on any other cell type.The results are consistent with the notion of a dense adrenergic innervation of the embryonic chick ovarian medulla and its steroidogenic interstitial cells, and suggest the chick ovary as an excellent model for elucidating the functional role of a neural input to steroidogenic cells during development.     

Ultrastructural identification of non-adrenergic,non-cholinergic nerves in the rat anococcygeus muscle

The innervation of the rat anococcygeus muscle has been investigated ultrastructurally following fixation with a modified chromaffin reaction for the demonstration of biogenic amines (Tranzer and Richards, 1976). Three types of nerve profiles were revealed: (1) 60-70% of the profiles are adrenergic; (2) less than 5% of the profiles appear to be cholinergic; (3) up to 40% of the profiles are distinguished by the presence of a characteristically high proportion of electron-opaque, chromaffin-negative vesicles, 85-110 nm in diameter. This third type of profile was not affected by 6-OHDA, and is considered to represent the non-adrenergic, non-cholinergic inhibitory innervation of this tissue. Because of the morphological similarity of this nerve type, apart from the smaller vesicle size, to classical peptidergic nerve endings, they have been termed "small p-type" (sp-type). These results are discussed in relation to a previous report describing only two types of nerve profiles in this tissue (Gillespie and Lüllmann-Rauch, 1974).     

Partial Isolation of Two Classes of Dopamine β-Hydroxylase-Containing Particles Undergoing Rapid Axonal Transport in Rat Sciatic Nerve

The rapid bidirectional transport of dopamine beta-hydroxylase (DBH) in adrenergic axons provides a means of analyzing the life cycle of adrenergic storage vesicles. We compared the physical characteristics of DBH-containing particles traveling to or returning from the terminal varicosities of ligated rat sciatic nerves. Density gradient centrifugation and Sephacryl S1000 gel-permeation chromatography were used to fractionate extracts from nerve segments proximal or distal to the ligatures. A series of experiments indicated the existence of at least two populations of rapidly transported DBH-containing particles, a "light" 85-nm particle and a larger "dense" 120-nm particle. The 85-nm particles were prevalent in unligated nerve, but accounted for only one-third of the total anterogradely transported DBH activity accumulated after 18 h. The 120-nm particles were barely detectable in the unligated nerve, but they accumulated at twice the rate of the 85-nm particles and accounted for the rest of the anterogradely transported particulate DBH activity. These two populations of particles were readily isolated from proximal nerve extracts by sucrose density gradient centrifugation. Similar-appearing dense and light peaks of particulate DBH activity were obtained from distal nerve extracts. Much of the retrogradely transported DBH of the extracts, however, was associated with large particles (greater than 300 nm) not resolved by Sephacryl S1000. Retrogradely transported exogenous NGF was found only in the dense sucrose gradient peak. We propose that the 85-nm DBH-containing particles correspond to "large dense-cored vesicles," and that the 120-nm particles are derived from the dense tubules visualized in adrenergic nerves by the chromaffin reaction.     

Autonomic nervous control of the circulatory system in the air-breathing fish Channa argus

The control of the cardiovascular system with particular emphasis on the regulation of blood distribution in the gills and air-breathing organ was studied in the air-breathing teleost Channa argus. Perfused head preparations were used in addition to experiments with isolated strip preparations of arteries and heart chambers. The distribution of adrenergic nerves was investigated using Falck-Hillarp fluorescence histochemistry. This preliminary study shows an adrenergic control system composed of chromaffin cells and adrenergic nerves similar to that found in other teleosts investigated, although the systemic arteries (coeliac artery, dorsal aorta and the vasculature of the air-breathing organ) appear to lack an adrenergic innervation. The reactions of isolated artery strip preparations to acetylcholine and adrenaline resemble those seen in other teleosts, and there is a prominent inhibitory effect of L-isoprenaline suggestive of arterial beta-adrenoceptors. The general vascular resistance of the gill apparatus-air-breathing organ increases in response to acetylcholine or adrenaline, and there is a redistribution of perfusion flow from the air-breathing organ circuit (anterior venous outflow from the first and second pair of gills and the air-breathing organ) to the general systemic circuit (dorsal aortic outflow from the third and fourth pair of gills). Stimulation of the vagal branch entering the air-breathing organ mimics the effects of acetylcholine or adrenaline. This innervation is probably non-adrenergic since no adrenergic nerve fibres could be demonstrated in the vasculature of the air-breathing organ using the histochemical technique. An adrenergic control of the vasculature of the air-breathing organ is not likely, since the concentration of adrenaline needed to affect the vasculature is not reached in the plasma even during "stress".     

Synaptic organisation of the pelvic ganglion in the guinea-pig

Summary A semi-quantitative electron-microscopic study of neuronal cell bodies, nerve profiles and synapses in the anterior pelvic ganglia of the guinea-pig has been carried out following in vivo labelling of adrenergic nerve endings with 5-hydroxydopamine. Ganglion cells of three main types have been distinguished: 1) the majority (about 70%) not containing granular vesicles, probably cholinergic elements; 2) those containing large granular vesicles of uniform size (80–110 nm), with granules of medium density and prominent halos; and 3) those containing vesicles of variable size (60–150 nm), with very dense eccentrically placed granular cores. Some non-neuronal granule-containing cells were present, mainly near small blood vessels. Some 95% of the total axon profiles within the ganglia were cholinergic, the remaining 5% were adrenergic. However, 99% of synapses (i.e. axons within 50 nm of nerve cell membrane with pre-and post-synaptic specialisations) were cholinergic, and 1 % were adrenergic. Only three examples of nerve cell bodies exhibiting both cholinergic and adrenergic synapses were observed. Unlike the para-and prevertebral ganglia, the pelvic ganglia contained large numbers of axo-somatic synapses. As many as 20% of the nucleated neuronal cell profiles displayed two distinct nuclei.     

Innervation of the renal vasculature of the toad (Bufo marinus)

The innervation of the dorsal aorta and renal vasculature in the toad (Bufo marinus) has been studied with both fluorescence and ultrastructural histochemistry. The innervation consists primarily of a dense plexus of adrenergic nerves associated with all levels of the preglomerular vasculature. Non-adrenergic nerves are occasionally found in the renal artery, and even more rarely near the afferent arterioles. Many of the adrenergic nerve profiles in the dorsal aorta and renal vasculature are distinguished by high proportions of chromaffin-negative, large, filled vesicles. Close neuromuscular contacts are common in both the renal arteries and afferent arterioles. Possibly every smooth muscle cell in the afferent arterioles is multiply innervated. The glomerular capillaries and peritubular vessels are not innervated, and only 3-5% of efferent arterioles are accompanied by single adrenergic nerve fibres. Thus, nervous control of glomerular blood flow must be exerted primarily by adrenergic nerves acting on the preglomerular vasculature. The adrenergic innervation of the renal portal veins and efferent renal veins may play a role in regulating peritubular blood flow. In addition, glomerular and postglomerular control of renal blood flow could be achieved by circulating agents acting via contractile elements in the glomerular mesangial cells, and in the endothelial cells and pericytes of the efferent arterioles. Some adrenergic nerve profiles near afferent arterioles are as close as 70 nm to distal tubule cells, indicating that tubular function may be directly controlled by adrenergic nerves.     

A histochemical study of the innervation of the cerebral blood vessels in the domestic fowl

Summary Adrenergic and cholinergic nerves innervating the cerebral arteries of the domestic fowl were examined by specific histochemical techniques.The adrenergic nerve plexuses of the cerebral carotid system are markedly denser than those of other vertebrates observed by similar techniques. They form longitudinally elongated meshworks of fine fibres in the vascular wall of the arterial branches. Those innervating the vertebro-basilar system are less dense and more elongated, and, as the size of the artery diminishes, the fibres of the plexus become coarser. In the small pial and parenchymal arteries they are reduced to a few fibres running parallel to, or spiralling around the vascular axis.The cholinergic nerve plexuses are not as dense as the adrenergic system. The acetylcholinesterase activity is very weak, except in the plexuses innervating the cerebral carotid artery and the proximal portion of the anterior and posterior rami. In the vertebro-basilar system, a few thick nerve bundles run alongside the blood vessels of the vertebral and basilar arteries. Cholinergic nerves enter the cranial cavity along the internal carotid, the vertebral and possibly the cerebro-ethmoidal arteries.Intracerebral capillaries and some arterioles are not innervated with cholinergic and adrenergic fibres of peripheral origin, but with ones arising from parenchymal nerve cells.     

Auerbach's plexus of mammals and man: Electron microscopic identification of three different types of neuronal processes in myenteric ganglia of the large intestine from rhesus monkeys,guinea-pigs and man

Summary Ganglia from Auerbach's plexus of the large intestine (caecum, appendix vermiformis, colon transversum and rectum) in man, rhesus monkey and guinea-pig are composed of nerve cells and their processes, typical Schwann cells and a vast neuropil. The neuropil consists of dendrites and axons of intrinsic nerve cell perikarya and axons of extrinsic neurons. Axonal profiles in large nerve fibre bundles are of uniform size and appearance, embedded in infoldings of Schwann cell cytoplasm and contain occasional large granular vesicles, mitochondria and neurotubules. Preterminal axons widen into vesicle filled varicosities, some of which establish synaptic contact with intrinsic nerve cell bodies.At least three different types of neuronal processes can be distinguished in the myenteric neuropil according to the size, appearance and commutual proportion of vesicles present in axonal varicosities, and their ability to accumulate exogenous 5- and 6-hydroxydopamine and 5-hydroxydopa: 1. Axonal enlargements containing a major population of small electron lucent synaptic vesicles (350–600 Å in diameter) together with a small number of membrane-bound, opaque granules (800–1,100 Å). These profiles have been identified as cholinergic axons. The boutons establish synaptic contacts with dendritic processes of intrinsic nerve cell bodies; membrane specializations are found at the preand postsynaptic sites. 2. Axonal beads of sometimes very large diameter, containing an approximately equal amount of large granular vesicles (850–1,600 Å) and small, electron lucent or faintly opaque vesicles (400–600 Å). The granular core of the large vesicles is of medium electron density and may either fill the entire vesicle or is separated from the limiting membrane by a more or less clear interspace. The fibres probably belong to intrinsic neurons, and because of the similarity of the large, membrane-bound vesicles with neurosecretory elementary granules, they have been designated p-type fibres (polypeptide fibres). The granular core of the vesicles in these fibres becomes more electron dense after treatment with 5-OH-dopa. The accumulation of an amine precursor analogue in combination with a possible storage of a polypeptide substance (or an ATP-like substance) resembles the situation in several diffusely distributed endocrine cell systems. 3. Varicosities of axons equipped with small (400–600 Å) empty or sometimes granular vesicles, medium sized (500–900 Å) vesicles with highly electron dense cores and occasional large (900–1,300 Å) granular vesicles. Pretreatment with 5-OH-dopamine increases the electron density in almost all medium-sized granular vesicles and some of the large granular vesicles; an osmiophilic core develops in some small vesicles. 6-hydroxydopamine results in degenerative changes in the varicosities of this type of neurons. Concomitantly, both catecholamine analogues markedly reduce neuronal noradrenaline in the large intestine, as demonstrated by fluorescence histochemistry and in fluorimetric determinations. The ultrastructural features of these varicosities and their reaction to 5- and 6-OH-dopamine indicate that they belong to adrenergic, sympathetic nerves. No membrane specializations could be detected at sites of close contact of the adrenergic boutons with dendrites and cell bodies of intrinsic nerve cells.Supported by grants from the Deutsche Forschungsgemeinschaft.Supported by a grant from Albert Pahlsson's Foundation, Sweden. The work was carried out within a research organization sponsored by the Swedish Medical Research Council (projects No. B70-14X-1007-05B, B70-14X-712-05, and B70-14X-56-06).     

Effects of surgical denervations on the autonomic nerves in parotid glands of dogs

Summary The innervation of the dog's parotid has been studied by cholinesterase staining and catecholamine fluorescence. In normal glands cholinergic and adrenergic nerves are plentiful around acini, muscular blood vessels, and to a lesser extent striated ducts. The main ducts, although surrounded by many cholinesterase-positive nerves, are associated with few adrenergic nerves. Severance of the classical parasympathetic post-ganglionic nerve to the gland, the auriculo-temporal, caused a moderate loss of cholinesterase-positive nerves. When this procedure was combined with section of the nerves on the internal maxillary artery there was a greater loss. Fewest cholinesterase-positive nerves remained when, in addition to these two procedures, the facial nerve was cut. These findings support the concept that all three sets of nerves contain some post-ganglionic parasympathetic fibres for the dog's parotid. The source of the remaining nerves is unknown. Preganglionic parasympathetic denervation by section of the tympanic branch of the glossopharyngeal nerve did not reduce the number of cholinesterase-positive nerves. None of these parasympathetic denervations caused reduction of adrenergic nerves, indicating that they do not travel to the gland with the parasympathetic nerves. After superior cervical ganglionectomy a few scattered fluorescent nerves remained in the gland; their origin is unknown.     

The innervation of the trachea and extrapulmonary bronchi of the mouse

Summary In the mouse, nerves were located throughout the trachea and extrapulmonary bronchi in both the smooth muscle and the connective tissue. However, no nerves were found within the epithelium. In the smooth muscle there were large numbers of nonmyelinated nerves. These were usually en passant elements but varicosities containing small mitochondria and vesicles were also seen; these axons sometimes appeared to be efferent to the muscle.Unilateral cervical vagotomy reduced the numbers of nerves in the muscle of the trachea and ipsilateral primary bronchus, suggesting that they were afferent. The intramuscular nerves were characterized in terms of their complement of cytoplasmic organelles; in particular nerves containing many mitochondria disappeared following vagotomy.Pretreatment of mice with 5-hydroxydopamine to accentuate the electron-opacity of catecholamine-containing granules resulted in 3.5% of the nerves within tracheal muscle showing such granules.The afferent nerves of the smooth muscle may be complex branching structures with many varicosities. The absence of epithelial nerves may be related to the absence of the cough reflex in the mouse.     

Fluorescence histochemical observations on the intrinsic adrenergic innervation and monoamine-containing cells of the feline pancreas,with special reference to the terminal main pancreatic duct of wirsung

Summary Intrinsic adrenergic innervation of the pancreas of the cat was studied using two fluorescence histochemical methods. Special emphasis was focused on the monoaminergic regulation of the mechanisms responsible for dynamically active suction-pressure pumping at the choledocho-pancreatico-duodenal junction, consisting of a labyrinthine reservoir chamber surrounded by a contractile mantle of smooth muscle, described recently (Kyösola, 1976). In that junction area, a rich distribution of single varicose fluorescing nerve fibers and small nerve fascicles, both free (i.e., unrelated to the blood vessels) and forming typical perivascular nerve plexuses, as well as large ganglia of nonfluorescing nerve cells surrounded by fluorescing baskets of varicose nerves were observed in the connective, fat, and pancreatic tissues, as well as between the smooth muscle bundles of the contractile smooth muscle mantle surrounding the reservoir chamber and its minor compartments. The epithelial lining of the labyrinthine duct system contained some solitary brightly yellow fluorescing enterochromaffin cells. In addition, two other categories of fluorescing cells were observed: (1) Small rounded cells with a relatively large rounded nucleus, and exhibiting a clearly bluish, usually granular, fluorescence varying from weak to intense; (2) Larger cells exhibiting quite a weak, clearly greenish to greenish-yellow fluorescence, either gathered in homogeneous clusters or mingled with the cells of the former type into heterogeneous cells clusters. These two cell types were clearly distinguishable from each other, but intermediate cell types were also seen. Thus, a continuous scale of fluorescing cells was observed, the color of the fluorescence varying from clearly bluish to greenish-yellow, the intensity of the fluorescence varying from intense to weak (respectively, in general), and the size ranging from small to large (respectively). These cells, probably heterotopic alpha cells of the islets of Langerhans, were frequently in quite close proximity to the smooth muscle surrounding the contractile labyrinthine system or its minor compartments. On the other hand, they were frequently in close proximity to quite large thin-walled sinusoid-type blood vessels. At least some of these fluorescing cells were surrounded by fluorescing varicose nerves, suggesting the existence of a neuroendocrine link. Scattered among these fluorescing cells, and also gathered into small clusters, were nonfluorescing nerve cells interconnected with fluorescing varicose nerves. The fluorescing cells of the endocrine pancreas, similarly, could be arbitrarily classified into two main categories, although intermediate cell types were observed as well: (1) Small rounded cells with a large, ovoid to rounded, centrally placed nucleus, and exhibiting a moderate to intense granular cytoplasmic fluorescence. The color of the fluorescence varied from yellow-green to blue-green, and there were considerable differences also in the fluorescence intensity. (2) Mixed irregularly with the type (1) cells were cells with more or less the same cytologic characteristics, but slightly larger, and exhibiting a rather homogeneous weak yellow fluorescence. Sometimes the fluorescence intensity was so weak that it was hardly discernible. These cells were packed densely and irregularly with the type (1) cells within the clusters, and no clear-cut spatial organization between these two cell types was observed. In the wall of the main pancreatic duct of Wirsung, both free and perivascular fluorescing nerves were seen within the different layers, including the (distally incomplete) smooth muscle layer. The epithelial lining of the duct contained enterochromaffin cells, sometimes located so deeply in the epithelial invaginations that they were in close proximity to the smooth muscle. The distribution of fluorescing nerves in the exocrine pancreas was sparse. Typical enterochromaffin cells exhibiting a bright yellow granular cytoplasmic fluorescence were scattered either solitarily or in small groups in the epithelial lining of the acini and of the small excretory ducts. Blue-green fluorescing varicose adrenergic axons were some-times seen to connect enterochromaffin cells or groups of them located in different acini. Scattered within the parenchyme of the exocrine pancreas were clusters of nonfluorescing nerve cells surrounded by typical baskets of fluorescing varicose terminal ramifications of adrenergic axons. In the endocrine pancreas, similarly, only a relatively sparse distribution of fluorescing nerves was observed among the islet cells and surrounding clusters of them. However, groups of islet cells were observed to be interconnected by fluorescing varicose nerves coursing through or near nonfluorescing ganglia, suggesting an integrative neural connection between pancreatic islets and nonadrenergic ganglia via adrenergic nerves.     

Adrenergic innervation of the large arteries and veins of the semiarboreal rat snake Elaphe obsoleta

Fluorescence histochemistry was used to study the adrenergic innervation of the large arteries and veins at six points along the body of the semiarboreal rat snake Elaphe obsoleta. Apart from the vessels adjacent to the heart, there was a marked contrast in the density of adrenergic innervation of anterior and posterior systemic arteries and veins. The anterior arteries and veins have little adrenergic innervation in contrast to the extremely dense innervation of the arteries and veins posterior to the heart. The innervation pattern is consistent with known physiological adjustments to gravity and suggests a mechanism for regulating dependent blood flow via sympathetic nerves. In comparison to the posterior systemic arteries, parallel segments of pulmonary artery taken from the same body position of Elaphe contained a much sparser innervation by adrenergic nerves. The sparser innervation can be correlated with less gravitational disturbance in the pulmonary artery, which is relatively short in this and in other arboreal snakes.     

The adrenergic innervation of the renal artery and vein of the rat

Summary The adrenergic innervation of the extrarenal blood vessels of the rat left kidney was investigated by fluorescence histochemistry and by electron microscopy. The trunk of the renal artery proximal to the aorta is elastic and appears to be very sparsely innervated. In contrast, near the kidney the renal artery—which divides into 3 to 4 large branches of the muscular type possesses a dense adrenergic innervation. The adrenergic terminal axons are situated in the adventitia close to the external elastic lamella, but only rarely in close contact with smooth muscle cells. In most instances several terminal axons are grouped and enclosed by a Schwann cell, single axons being rare. All terminal axons are able to take up and to store 5-hydroxydopamine which strongly suggests that they are adrenergic. The innervation of the renal vein is more sparse than that of the muscular arteries but somewhat denser than that of the elastic artery. In addition, close to the origin of the renal artery the presence of small intensively fluorescent (SIF) cells as well as of some adrenergic ganglion cells is noted. The latter are situated in the adrenergic nonterminal axon bundles, which run parallel to the blood vessels.It is concluded that the uneven adrenergic innervation along the artery as well as individual variations in the branching of the artery are the main causes of the unusually high individual variations of the NA content of this organ such as used in pharmacological experiments.     

Cholinergic mechanisms in pial vessels

Summary Plexuses of cholinergic nerve terminals were demonstrated (acetylcholinesterase staining) in pial arteries (down to a diameter of about 15) at the base of the brain and on the brain convexities of mice, rats, rabbits, hamsters, guinea-pigs, and cats. The pial veins were less well supplied than the arteries. Consecutive formaldehyde gas treatment (to visualize adrenergic nerves) and acetylcholinesterase staining revealed that the adrenergic and cholinergic plexuses followed each other closely, the axon terminals running together in the same Schwann cell strands. This was confirmed by electron microscopy after KMnO₄ fixation or 5-hydroxydopamine treatment. The varicosities of cholinergic and adrenergic axons were sometimes seen as close as 250 Å. In the neuro-effector area, the terminals of both nerve types (naked or surrounded by an incomplete Schwann cell covering) approached the smooth muscle cells as close as 800–1100 Å, and they were separated from the latter only by the fused neuronal and muscular basement membranes. In this area axo-axonal contacts were observed. The adrenergic, but not the cholinergic, nerves disappeared after bilateral removal of the superior cervical sympathetic ganglia. Isolated cat middle cerebral artery contracted strongly with acetylcholine, and the effect was inhibited by atropine.With regard to the cholinergic neural control of the intracranial arteries, it may have particular functional implications: (1) that these vessels do have a cholinergic parasympathetic innervation in contrast to most other vascular systems, for example, in the mesenterium, (2) that this cholinergic nerve supply was found to be about equally prominent as the adrenergic (sympathetic) innervation which, in some pial vessels, is even better developed than in the mesenteric arteries, and (3) that the adrenergic and cholinergic systems in the intracranial arteries may interact, even at the level of the neuro-muscular contacts, a complex situation which may be partly responsible for the previous difficulties in defining the autonomic neural influence on the brain circulation.Part of the findings were reported at Journées Internationales de Circulation Cérébrale, Toulouse, April 21–22, 1972.