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.     

A histochemical study of the innervation of cerebral blood vessels in the snake

Summary Dual innervation of snake cerebral blood vessels by adrenergic and cholinergic fibres was demonstrated with the use of histochemical methods. Although the nerve plexuses are somewhat less dense, the essential features of innervation of the blood vessels are similar to those of mammals with the exception that the adrenergic plexuses are more prominent than the cholinergic plexuses. The major arteries of the cerebral carotid system have a rich nerve supply. However, the innervation is less rich in the basilar and poor in the spinal (vertebral) arteries. Although the arteries supplying the right side of head are poorly developed, three pairs of arteries, cerebral carotids, ophthalmics and spinals, supply the snake brain. The carotids and ophthalmics are densely innervated and are accompanied by thick nerve bundles, suggesting that the nerves preferentially enter the skull along those arteries. Some parenchymal arterioles are also dually innervated. Connection between the brain parenchyma and intracerebral capillaries via both cholinergic and adrenergic fibres was observed. In addition cholinergic nerve fibres, connecting capillaries and the intramedullary nerve fibre bundles, were noticed. Capillary blood flow may be influenced by both adrenergic and cholinergic central neurons. The walls of capillaries also exhibit heavy acetylcholinesterase activity. This may indicate an important role for the capillary in the regulation of intracerebral blood flow.     

Ultrastructure and innervation of the swimbladder of Tetractenos glaber (Tetraodontidae)

Summary The general structure, ultrastructure and innervation of the swimbladder of the smooth toadfish, Tetractenos glaber, were examined with light-microscopic, fluorescence-histochemical, and transmission electron-microscopic techniques. The structure of the swimbladder is similar to that of other euphysoclists. Fluorescence histochemistry showed adrenergic fibres in both the secretory and resorptive areas of the swimbladder. Transmission electron microscopy revealed two morphologically distinct axon profiles type-I profiles containing many small, flattened vesicles; type-II profiles containing both large, granular vesicles and rounded, small clear vesicles in varying proportions.The gas-gland cells and surrounding muscularis mucosae are innervated by both type-I and type-II fibres. Type-I fibres also innervate pre-rete arteries. The rete- and gas-gland capillaries do not appear to be innervated. Arteries running to the resorptive area are innervated by type-I fibres. Both type-I and type-II profiles make contact with the muscularis mucosae in the resorptive area. Only type-I fibres innervate the radial dilator muscle in the oval sphincter region, whereas only type II fibres innervate the circular muscle of the oval sphincter.Type-I fibres took up -methyl-noradrenaline, and could not be found after pre-treatment with 6-hydroxydopamine. They are, therefore, assumed to be adrenergic. Type-II fibres were tentatively identified, by exclusion, as cholinergic.     

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.     

Adrenergic innervation of the umbilical vessels

Summary The intra- and extrafetal portions of the umbilical vessels in the guinea-pig and the umbilical cord of man, mouse and rabbit have been investigated by means of the Falck-Hillarp method for the fluorescence microscopical demonstration of catecholamines.The umbilical cord was found to be devoid of nerves in all species investigated. Adrenergic nerves are present only in the immediate vicinity of the umbilicus.The intrafetal portions of the umbilical artery and umbilical vein receive adrenergic nerves, the distribution pattern of which is different for each vessel. In the guinea-pig the ductus venosus is an intrahepatic branch of the vena umbilicalis. No adrenergically innervated sphincter has been detected in the initial segment of the ductus venosus. Regional variations in the pattern of innervation of the intrafetal portion of the umbilical vein are paralleled by regional differences in the construction pattern of the vessel's wall. Regional differences in the noradrenaline concentration (measured by fluorometry) which correspond to the fluorescence microscopical findings have been detected in umbilical vessels: low noradrenaline content of the umbilical cord, high concentrations in the intrafetal sections of the umbilical vessels. The noradrenaline concentration of the guinea-pig umbilical artery is three times that of the umbilical vein.Supported by the Joachim Jungius-Gesellschaft der Wissenschaften, Hamburg.For continuous advice and constructive criticism I am indebted to Prof. Dr. Dr. E. Horstmann.     

The adrenergic innervation of the pulmonary vasculature,the lung and the thoracic aorta,and on the presence of aortic bodies in the domestic fowl (Gallus gallus domesticus L.)

Summary The Falck-Hillarp technique for the localisation of biogenic amines has been used to examine the adrenergic innervation of the thoracic vasculature and lung, and to demonstrate the occurrence of aortic bodies in the domestic fowl. The proximal pulmonary vein is very densely innervated but distally the innervation becomes sparse. The pulmonary artery is sparsely innervated over its whole length. The bronchial muscle of the lung has little adrenergic innervation and fluorescent cell bodies are absent from the lung. The thoracic aorta receives a moderate adrenergic innervation. In the region of the aortic arch and pulmonary arteries groups of fluorescent cells are common. Extramedullary chromaffin cells and small, intensely fluorescent cells occur within these groups. In the media of the aorta and pulmonary artery other types of fluorescent cells are found. These results are discussed in the light of previous observations.Part of this work was performed while the author was a postdoctoral research fellow of the National Heart Foundation of Australia. His thanks are due to Prof. G. Burnstock for use of laboratory facilities.     

Reinnervation of regenerating smooth muscle cells in minced vas deferens of the guinea-pig

Summary In adult guinea-pigs, a portion of the wall of the vas deferens was removed, minced and replaced. This caused muscle cells to dedifferentiate, divide and redifferentiate. Reinnervation of redifferentiating cells was followed using electron microscopy and histochemistry. Adrenergic nerves were first observed to re-enter the regenerating area 5 days after operation, and close contacts (within 20 nm) with muscle cells were first seen at 10 days. The total number of adrenergic nerves per 100 muscle cells reached control values by 5 weeks, and by 15 weeks was higher than control levels. Cholinergic nerves first appeared in the regenerating area about 3–4 weeks after the operation. The total number of cholinergic nerves present had not reached control values even at 15 weeks, and no nerve muscle contacts within 20 nm were observed. The ratio of adrenergic to cholinergic nerves in the regenerating area was higher at 15 weeks than in control tissue.This work was supported by grants from the Wellcome Trust and the Medial Research Council     

Adrenergic innervation of the tunica media in the saphenous artery of the fetal and newborn guinea-pig

Summary Previous studies have demonstrated that adrenergic nerves are located in the medial-adventitial border of the muscular arteries. Observations made in this study have revealed that adrenergic nerves penetrate into the outer medial layer of the saphenous artery in fetal and newborn guinea-pigs, while in the adult these nerves are located in the medial-adventitial border. It is proposed that the adrenergic nerves located in the tunica media may have a trophic effect on the medial smooth muscle. It is further suggested that the final refinement of the dual control system of arterial walls, by nerves and circulating catecholamines, involves exclusion of adrenergic nerves from the tunica media.     

Comparative study of the autonomic innervation of the mammalian ovary,with particular regard to the follicular system

Summary The autonomic innervation of the ovary was studied in 12 mammalian species utilizing the cholinesterase method in combination with pseudocholinesterase inhibition for the cholinergic component, and glyoxylic acid histochemistry together with fluorometric determination of noradrenaline for the adrenergic component. Ovaries from cow, sheep, cat, and guinea pig were very richly supplied with adrenergic nerves in the cortical stroma, particularly enclosing follicles in various stages of development. In the follicular wall the nerve terminals were located in the theca externa, where they ran parallel to the follicular surface. Numerous adrenergic terminals also surrounded ovarian blood vessels. The adrenergic innervation was of intermediary density in the human ovary and in the pig, dog, cat, and opossum. Ovaries from rabbit, mouse and hamster had a sparse adrenergic nerve supply. The amount of intraovarian adrenergic nerves agreed well with the tissue concentration of noradrenaline in the various species. The cholinergic innervation was generally less well developed, but had the same distribution as the adrenergic system around blood vessels and in the ovarian stroma, including follicular walls.     

Distribution and vasomotor effects of peptide HI (PHI) in feline cerebral blood vessels in vitro and in situ

Peptide HI (PHI)-immunoreactive nerve fibres were numerous around cerebral blood vessels of the cat. The number and distribution resemble that previously found for vasoactive intestinal polypeptide (VIP), a peptide with which PHI co-exists in pial arteries, at least in some segments. PHI and VIP elicit dilatation in a concentration-dependent manner in isolated middle cerebral arteries; the maximum effects were similar but VIP was considerably more potent. Neither effect was blocked by atropine, cimetidine or propranolol, confirming an action at a non-adrenergic, non-cholinergic site. In chloralose-anaesthetized cats PHI and VIP elicited concentration-dependent dilatations; the magnitude of responses was similar, however, considerably more PHI was necessary to elicit the same response as that of VIP. The results suggest that though both peptides are co-localized and may act at the same receptor, VIP is a more likely candidate for eliciting dilatation during physiological conditions.     

Intraepithelial blood vessels in the vomeronasal neuroepithelium of the rat

Summary Epithelial-vascular relationships are established during the development of the vomeronasal neuroepithelium of the rat. Special attention is given to the fine structure of the endothelial wall of intra-epithelial vessels, to ultrastructural aspects of the neuronal-vascular relationships, and to the appearance of inclusion bodies in the neuronal cells adjacent to these vessels. The neuronal perikarya surrounding the blood vessels are filled with highly developed smooth endoplasmic reticulum. Possible functional implications of the vascularization of the neuroepithelium of the vomeronasal organ in mediating olfacto-endocrine relationships are discussed. It is suggested that the intra-epithelial blood vessels are at least supportive and nutritive in nature, while their implication in an olfacto-endocrine connection remains obscure.On sabattical leave: Visiting Professor, Department of Anatomy, University of Kentucky, Lexington, Ky., USA     

A comparative study of incisor procumbency and mandibular morphology in vampire bats

The three species of vampire bats (Phyllostomidae: Desmodontinae), Desmodus rotundus, Diaemus youngi, and Diphylla ecaudata, are the only mammals that obtain all nutrition from vertebrate blood (sanguinivory). Because of the unique challenges of this dietary niche, vampire bats possess a suite of behavioral, physiological, and morphological specializations. Morphological specializations include a dentition characterized by small, bladelike, non‐occlusive cheek teeth, large canines, and extremely large, procumbent, sickle‐shaped upper central incisors. The tips of these incisors rest in cuplike pits in the mandible behind the lower incisors (mandibular pits). Here, we use microCT scanning and high‐resolution radiography to describe the morphology of the mandible and anterior dentition in vampire bats, focusing on the relationship between symphyseal fusion, mandibular pit size, incisor size, and procumbency. In Desmodus and Diaemus, highly procumbent upper incisors are associated with relatively small mandibular pits, an unfused mandibular symphysis with substantial bony interdigitations linking the dentaries, and a diastema between the lower central incisors that helps to facilitate the lapping of blood from a wound. In Diphylla, less procumbent upper incisors are associated with relatively large mandibular pits, a completely fused mandibular symphysis, and a continuous lower toothrow lacking a central diastema. We hypothesize that symphyseal morphology and the presence or absence of the diastema are associated with the angle of upper incisor procumbency and mandibular pit development, and that spatial constraints influence the morphology of the symphysis. Finally, this morphological variation suggests that Diphylla utilizes a different feeding strategy as compared to Desmodus and Diaemus, possibly resulting from the functional demands of specialization on avian, rather than mammalian, blood. J. Morphol., 2010. © 2010 Wiley‐Liss, Inc.     

Body and brain temperature coupling: the critical role of cerebral blood flow

Direct measurements of deep-brain and body-core temperature were performed on rats to determine the influence of cerebral blood flow (CBF) on brain temperature regulation under static and dynamic conditions. Static changes of CBF were achieved using different anesthetics (chloral hydrate, CH; α-chloralose, αCS; and isoflurane, IF) with αCS causing larger decreases in CBF than CH and IF; dynamic changes were achieved by inducing transient hypercapnia (5% CO₂ in 40% O₂ and 55% N₂). Initial deep-brain/body-core temperature differentials were anesthetic-type dependent with the largest differential observed with rats under αCS anesthesia (ca. 2°C). Hypercapnia induction raised rat brain temperature under all three anesthesia regimes, but by different anesthetic-dependent amounts correlated with the initial differentials—αCS anesthesia resulted in the largest brain temperature increase (0.32 ± 0.08°C), while CH and IF anesthesia lead to smaller increases (0.12 ± 0.03 and 0.16 ± 0.05°C, respectively). The characteristic temperature transition time for the hypercapnia-induced temperature increase was 2–3 min under CH and IF anesthesia and ~4 min under αCS anesthesia. We conclude that both, the deep-brain/body-core temperature differential and the characteristic temperature transition time correlate with CBF: a lower CBF promotes higher deep-brain/body-core temperature differentials and, upon hypercapnia challenge, longer characteristic transition times to increased temperatures.     

Evidence for exclusive adrenergic innervation of feather muscles (mm. pennati) in the chicken

Summary Feather follicles in the avian skin are interconnected by well-defined bundles of smooth muscle cells, which are responsible for the erection and depression of feathers and thus play an important role in thermoregulation. The depressing and erecting muscle bundles were found to receive a very dense supply of unmyelinated nerve fibres that displayed ultrastructural and histochemical characteristics of noradrenergic axons (formaldehyde- and glyoxylic acid-induced catecholamine fluorescence; uptake to 5-hydroxydopamine). No nerve fibres were encountered showing histochemical acetylcholinesterase activity. There was no indication of the presence of peptidergic or purinergic nerve endings.The neuromuscular space usually ranged from 40–60 nm in width and contained a basal lamina. Occasionally, this space was reduced to approximately 20 nm. At such close neuromuscular contacts a basal lamina was lacking, and focal densities beneath the pre- and postsynaptic plasma membrane were observed. Since no gap junctions between muscle cells were detected, the dense supply with noradrenergic nerve fibres indicates a high amount of directly innervated smooth muscle cells.An additional finding of the present study was the observation that high local concentrations of 5-hydroxydopamine led to degeneration of noradrenergic nerve endings.Supported by a grant from the Deutsche Forschungsgemeinschaft (Dr. 91)     

Intralobular lymphatic vessels and their relationship to blood vessels in the mouse thymus

Summary The spatial distribution and fine structure of the lymphatic vessels within the thymic lobules of normal and hydrocortisone-injected mice were studied by light- and electron microscopy. The lymphatic vessels of the cortex and medulla of normal thymus are irregularly shaped spaces closely associated with branches of the intralobular artery and vein. The overall distribution of these vessels in the greatly involuted thymus of hydrocortisone-treated mice is essentially the same as in the normal thymus. The wall of the lymphatic vessels consists of only a layer of endothelial cells supported by underlying reticular cells. The luminal surface of the endothelial cell is smooth, but trabecular processes are often seen. There are three morphological types of intercellular contacts between contiguous cells, namely, end-to-end, overlapping and interdigitating. The lymphatic vessel has anchoring filaments and collagen fibrils, but a basal lamina is either absent, or if present, is discontinuous. This is in contrast to the continuous basal lamina of the venule. The perivascular space surrounding the postcapillary venule opens into a terminal lymphatic vessel at the cortico-medullary junction and in the medulla. Lymphocytes are seen penetrating the lymphatic endothelium, particularly in acutely involuted thymuses. These findings suggest that the intralobular lymphatic vessels may originate from the vacuities that surround the postcapillary venules, and the lymphatic system may function as a pathway for the migration of lymphocytes into or out of the lymphatic circulation.     

Studies on the fine structure of invertebrate blood vessels

Summary The position, structure and function of the valves within the lateral sinus of the medical leech, Hirudo medicinalis, are described on the basis of vital, light- and electron microscopy. In this species the valvular apparatus consists of multiple elongated fir cone-shaped fibrous villi surrounding the orifices of the latero-lateral and latero-dorsal vessel like a tentacular crest. Each villus is covered by a thin sheet of a continuous endothelium. The valves prevent the backflow of hemolymph during systolic contraction of the lateral sinus.The endothelium contains many small mitochondria and polyribosomes in the perinuclear cytoplasm and it develops deep projections into the underlying connective tissue. Each of these consists of a multilayered system of closely interwoven thin endothelial membranes. The endothelium is anchored to its basement membrane by means of a great number of poorly defined hemidesmosomes. The fibrous tissue of the villi consists mainly of a homogeneous vitreous matrix in which few cellular components and very fine filaments are dispersed. Close to the endothelium this matrix appears to be condensed to form a multilayered framework made out of a basement membrane-like material.Though the valves themselves are devoid of muscle cells, those situated at their base and thus belonging to the vascular wall proper, display some specific morphological features: in particular the nuclei of these cells show a distinct fibrous lamina. Moreover, these muscle cells seem to be innervated only by one type of axon, containing both small, lucent synaptic vesicles as well as some of the dense-core variety.These findings are compared with data from earlier works and are discussed in relation to the hemodynamic functions of this valvular apparatus.     

Heterogeneous histochemical reaction pattern of the lectin Bandeiraea (Griffonia) simplicifolia with blood vessels of human full-term placenta

Bandeiraea simplicifolia lectin (BS-I) stains vascular endothelium in various species. In humans, less than 10% of the specimens studied exhibit a reaction with BS-I. In the present histochemical study, the reactivity of BS-I with placental blood vessels and its correlation with the blood group from mother and newborn child was investigated. Acetone-fixed cryosections of representative tissue segments of human full-term placenta and umbilical cord were stained with BS-I. The staining pattern of tissues from patients with different blood groups was identical, although the reaction of BS-I in the placenta was heterogeneous. BS-I did not react with the umbilical cord. Vascular smooth muscle cells at the insertion site of the umbilical cord into the chorionic plate, and endothelium deeper in the chorionic plate, became progressively stained. The endothelial cells and tunica muscularis of smaller arteries and veins in stem villi lost their reactivity in parallel with decreasing vessel size. Arterioles and venules reacted heterogeneously. Capillaries, trophoblastic basement membranes, especially epithelial plates, and sometimes the syncytiotrophoblast were labelled in several terminal villi. The data indicate that 1) the placenta binds BS-I to fetal endothelium independent of the blood group, 2) cell-surface antigens on placental endothelial cells are expressed heterogeneously and 3) cell-surface glycans are constituted in an organ-specific manner on human endothelial cells.     

Distribution and origin of vasoactive intestinal polypeptide (VIP)-immunoreactive,acetylcholinesterase-positive and adrenergic nerves of the cerebral arteries in the bent-winged bat (Mammalia: Chiroptera)

Summary The overall distribution and origins of vasoactive intestinal polypeptide (VIP)-immunoreactive (IR), acetylcholinesterase (AChE)-positive and adrenergic nerves in the walls of the cerebral arteries were investigated in the bent-winged bat. VIP-IR and AChE-positive nerves innervating the bat cerebral vasculature appear to arise mainly from VIP-IR and AChE-positive cell bodies within microganglia found in the nerve bundle accompanying the sympathetic nerve bundle within the tympanic cavity. These microganglia, as well as the nerve bundle containing them, do not emit catecholamine fluorescence, suggesting that they are of the cranial parasympathetic outflow, probably the facial or glossopharyngeal one. The axons from VIP-IR and AChE-positive microganglia run intermingled with sympathetic adrenergic nerves in the same thick fiber bundles, and reach the cranial cavity through the carotid canal. In addition, some of the VIP-IR fibers innervating the vertebro-basilar system, at least the basilar artery, originate from VIP-IR nerve cells located in the wall of this artery.The supply of VIP-IR fibers to the bat major cerebral arteries is the richest among mammals that have been studied, and differs from other mammals in that it is much greater in the vertebro-basilar system than in the internal carotid system: plexuses of VIP-IR nerves are particularly dense along the walls from the posterior ramus to posterior cerebral and basilar arteries. Small pial and intracerebral arteries of the vertebro-basilar system, especially those of the posterior cerebral artery which supply most parts of the diencephalon and cerebrum, are also richly innervated by peripheral VIP-IR fibers. This pattern corresponds well with the innervation pattern of adrenergic and AChE-positive nerves.     

Fine structural and cytochemical study of the innervation of smooth muscle in an amphibian (Bufo marinus) lung before and after denervation

Summary The innervation of the toad (Bufo marinus) lung was studied with transmission electron microscopy and fluorescence techniques, both before and after 12 or 20 days close vagosympathetic denervation. Four cytologically distinct types of neuronal processes were recognised, in relation to the visceral muscles of the lung. These were described as cholinergic, adrenergic, nonadrenergic/non-cholinergic (NANC) and sensory on the basis of the characteristics of their vesicular content and cytochemical reactions. An apparent efferent innervation of visceral smooth muscle was achieved by NANC (50%), cholinergic (25%) and adrenergic (25%) fibres. A few sensory fibres were also present. After denervation only NANC fibres persisted, showing that the cell bodies of these fibres were intrapulmonary. The vascular smooth muscle was supplied by cholinergic, adrenergic and sensory fibres. In the walls of the proximal branches of the pulmonary artery were fibres containing large dense-cored vesicles. These profiles, which were associated with the vasa vasorum, were similar to neurosecretory fibres. After denervation all neural profiles associated with the vasculature had degenerated. The observations suggest that vagal vasodepressor effects in the toad lung are mediated indirectly through relaxation of visceral muscle strands which in their contracted state compress vascular channels.The authors would like to thank Dr. J.R. McLean for technical advice on fluorescence microscopy. This work was supported by a grant from the Australian Research Grants Committee