Specificity of capsaicin treatment in the cerebral vasculature

Capsaicin has been shown to specifically deplete substance P from primary sensory afferents, including sensory nerves innervating blood vessels of the cerebral circulation as well as other vascular beds. In order to further document the specificity of this treatment, we examined the effect of capsaicin treatment on 3 other types of nerves in the guinea pig. Four tissues were examined: cerebral arteries, the mesenteric artery, the heart and iris. Norepinephrine content was not altered after capsaicin treatment, confirming that adrenergic nerves are unaffected. As indices of cholinergic nerves, activities of choline acetyltransferase and acetylcholinesterase were also unchanged after capsaicin treatment. In addition, no significant differences in levels of vasoactive intestinal peptide in cerebral arteries and the heart were found in animals treated with capsaicin. These findings underscore the specificity of capsaicin treatment for substance P containing nerves.     

Reduction in noradrenergic perivascular nerve density in the left and right cerebral arteries of old rabbits

With the use of fluorescence and acetylcholinesterase histochemistry, marked reductions have been shown in the noradrenergic and acetylcholinesterase-positive innervation of the right ( RMC ) and left (LMC) middle cerebral arteries of old compared with young adult rabbits. The decrease in noradrenergic nerve density tended to be greater in LMC than in RMC : Nerve density fell by approximately 45% in LMC and by approximately 30% in RMC . The reductions in acetylcholinesterase-positive nerves were similar in both LMC and RMC (29 and 33%, respectively). Vessel circumference and cross-sectional wall area appeared to increase in old age in LMC and RMC .     

Origin of cholinergic nerves to the rat major cerebral arteries: Coexistence with vasoactive intestinal polypeptide

The distribution and density of vasoactive intestinal polypeptide (VIP) immunoreactive and acetylcholinesterase (AChE)-containing nerves around the cerebral arteries was studied by using whole mounts with or without lesioning the sphenopalatine ganglia. Abundant VIP immunoreactive and AChE-containing nerves were observed around the cerebral blood vessels in normal rats especially in the anterior circulation of the cerebral arteries. VIP-immunoreactivity and AChE-staining was also demonstrated in neurons within the sphenopalatine ganglia. Lesions of the sphenopalatine ganglia resulted in a marked reduction of both VIP-immunoreactivity and AChE activity. In many neurons, coexistence of both VIP and AChE was revealed. These results demonstrate that cholinergic neurons from the sphenopalatine ganglia innervate the cerebral vasculature at the base of the brain, and that VIP and AChE coexists within the same fibers.     

Cytophotometric study of catecholamines and acetylcholinesterase in neurons of the intramural plexus of the urinary bladder of the cat in controls and animals exposed to experimental conditions

The intramural plexus of the cat urinary bladder was studied with histochemical methods for catecholamines and acetylcholinesterase. Ganglia of the plexus showed differences in the number of neurons (5 to 150 cells). They were adrenergic, cholinergic or mixed. Section of pelvic nerves increased the amount of catecholamines in adrenergic neurons and fibres and decreased the acetylcholinesterase activity. Section of hypogastric nerves did not affect the catecholamine level and acetylcholinesterase activity.     

The adrenergic innervation of pulmonary vasculature in the normal and pulmonary hypertensive rat

It would appear that susceptibility to chronic proliferative pulmonary hypertension in response to chronic alveolar hypoxia is most severe in species in which adrenergic innervation of pulmonary arteries is reduced or lacking. Intrapulmonary arteries of the rat have been reported to lack adrenergic innervation by some workers but not others. Since the rat develops severe proliferative pulmonary hypertension in response to prolonged alveolar hypoxia, the different divisions of the lung vasculature of Sprague-Dawley rats were thoroughly examined to determine the presence or absence of an adrenergic innervation. The degree of innervation in normal rats was compared with that of rats developing pulmonary hypertension. Both in normal and experimental pulmonary hypertensive rats the pulmonary arteries, all their branches and the small pulmonary veins with a smooth muscle media were found to be devoid of adrenergic innervation. In contrast, the cardiac-like muscle in the media of large pulmonary veins, the bronchial arteries and the vasa vasorum of larger vessels were richly innervated by adrenergic nerves. Thus the increase in medial smooth muscle which occurs in pulmonary arteries during chronic alveolar hypoxia is independent of a pre-existing adrenergic innervation or of such an innervation newly derived from that of adjacent vessels or structures. This is in contrast to systemic vessels where it has been suggested that increased adrenergic activity and density of innervation may augment hypertrophy of the media in hypertensive animals. Adrenergic nerves are suggested to have a protective action on pulmonary vessels.     

Acetylcholine and vasoactive intestinal peptide in cerebral blood vessels: effect of extirpation of the sphenopalatine ganglion

The innervation of cerebral blood vessels by nerve fibers containing acetylcholinesterase (AChE) and vasoactive intestinal peptide (VIP) and the vasomotor effects of the two neurotransmitters have been analyzed in the rat following the uni- or bilateral removal of the sphenopalatine ganglion (SPG), which is thought to be the major origin of this innervation. Histochemistry of AChE-positive nerve fibers and the immunoreactivity toward VIP revealed only a 30% reduction in the innervation pattern of the rostral part of the cerebral circulation following the operation. At approximately 4 weeks postoperatively, the original nerve network was restored. Quantitative measurements of cholineacetyltransferase activity and VIP revealed similar reductions in the levels of collected large cerebral arteries at the base of the brain and in small pial vessels overlying the cerebral cortex at the various postoperative times following uni- or bilateral removal of the SPG. The two techniques thus complemented each other. Vasomotor reactivity to acetylcholine (ACh) and VIP was examined in proximal segments of the middle cerebral artery at the various postoperative times. Generally, the removal of the SPG had no effect on the responses to ACh or VIP. The evidence indicates that only approximately one-third of the cholinergic/VIP innervation of the rostral part of the cerebral circulation originates in the SPG.     

The distribution of autonomic nerves in the musculature of the rat vas deferens. A light and electron microscope investigation

Light microscope histochemical techniques and electron microscopy were used to study the distribution of autonomic nerves in the muscle coat of the rat vas deferens. Numerous fluorescent catecholamine containing nerves were demonstrated throughout the length of the vas; muscle cells on its inner aspect were particularly well innervated. This distribution differed markedly from that of acetylcholinesterase containing fibres, the majority of which occurred at the testicular end. Few similar nerves were observed in the muscle coat further towards the urethral end of the vas. The distribution of fluorescent and acetylcholinesterase-positive nerves correlated well with the arrangement of adrenergic and non-adrenergic terminal regions identified on permanganate fixed sections using the electron microscope. Their distribution was endorsed using 6-hydroxydopamine to selectively damage adrenergic terminals. Adrenergic nerve terminals were frequently observed in isolation amongst smooth muscle cells while the non-adrenergic type were almost invariably associated with other axons. The significance of these observations is discussed.     

Extrinsic pathways of the adrenergic innervation of the guinea-pig trachealis muscle

Origins and extrinsic pathways of the adrenergic innervation of the guinea-pig trachealis muscle were studied using fluorescence histochemical techniques. Bilateral superior cervical ganglionectomy caused a marked reduction in the adrenergic innervation of the extra-thoracic region, which suggests that these ganglia are a major source of adrenergic innervation to this muscle. Combined anterior and posterior transection of the recurrent laryngeal nerves also caused a marked reduction in the density of adrenergic fibres in the extra-thoracic trachealis muscle. Crushing of these nerves revealed adrenergic fibres running both anteriorly and posteriorly. The majority of these adrenergic nerves were lost after superior cervical ganglionectomy and thus the fibres running in both directions originate in the superior cervical ganglion. Antero-posteriorly directed fibres entered the recurrent laryngeal nerve from the superior cervical ganglion via an anastomosis at the level of the cricoid cartilage, while those running postero-anteriorly entered the recurrent laryngeal nerve posteriorly from the vagus nerve and these adrenergic fibres were lost after cervical vagotomy.     

Cholinergic nerves in mouse uterus

Summary The topographical distribution of cholinergic nerve fibres in the uterus was studied in normal as well as chemically sympathectomized mice.Our analysis was performed using both transverse sections and whole mounts, in order to analyse the distribution of cholinergic nerves within the various layers of the uterus and to study the morphology of nerve plexuses that supply the organ.Cholinergic nerve fibres were found chiefly around the uterine artery and its primary ramifications. Nerve fibres coming from arterial plexuses are distributed to the myometrium and more infrequently to the endometrium.Groups of acetylcholinesterase positive ganglion cells were located in the cervix.The distribution pattern of the cholinergic innervation of mouse uterus shows important topographical differences. In the tubal end of uterine horn the innervation is poor and cholinergic nerve fibres are organized in a sparse plexus. The number and the density of uterine nerves increase gradually proceeding toward the cervix. The cervix shows the richest innervation.The findings that observed nerve fibres are unaltered by chemical sympathectomy and are revealed using short incubation times suggest their parasympathetic nature.     

Innervation of the human spleen: A complete hilum-embedding approach

IntroductionThe spleen is hypothesized to play a role in the autonomic nervous system (ANS)-mediated control of host defence, but the neuroanatomical evidence for this assumption rests on a sparse number of studies, which mutually disagree with respect to the existence of cholinergic or vagal innervation.MethodsWe conducted an immuno- and enzyme-histochemical study of the innervation of the human spleen using a complete hilum-embedding approach to ensure that only nerves that entered or left the spleen were studied, and that all splenic nerves were included in the sampled area. Furthermore, a complete embedded spleen was serially sectioned to prepare a 3D reconstruction of the hilar nerve plexus.ResultsAll detected nerves entering the spleen arise from the nerve plexus that surrounds branches of the splenic artery and are catecholaminergic. Inside the spleen these nerves continue within the adventitia of the white pulpal central arteries and red pulpal arterioles. Staining for either choline acetyltransferase or acetylcholinesterase did not reveal any evidence for cholinergic innervation of the human spleen, irrespective of the type of fixation (regularly fixed, fresh-frozen post-fixed or fresh-frozen cryoslides). Furthermore, no positive VIP staining was observed (VIP is often co-expressed in postganglionic parasympathetic nerves).ConclusionOur comprehensive approach did not produce any evidence for a direct cholinergic (or VIP-ergic) innervation of the spleen. This finding does not rule out (indirect) vagal innervation via postganglionic non-cholinergic periarterial fibres.     

Increased density of perivascular nerves to the major cerebral vessels of the spontaneously hypertensive rat: differential changes in noradrenaline and neuropeptide Y during development

Fluorescence and immunohistochemical techniques were used to study the pattern and density of perivascular nerves containing noradrenaline (NA) and neuropeptide Y (NPY) supplying the major cerebral arteries of 4-, 6-, 8- and 12-week-old spontaneously hypertensive rats (SHR) and normotensive Wistar (WIS) controls. Levels of NA and NPY in the superior cervical ganglia were measured. The density of nerves containing NA and NPY was greater in the hypertensive animals at all ages studied. However, the developmental changes in the density of innervation showed similar trends in both SHR and WIS groups. With few exceptions, there was a significant increase in the density of nerves containing NA from 4 to 6 weeks and from 8 to 12 weeks of age. This was in contrast to a low expression, and in some vessels a significant decrease in the number of NPY-containing nerves from 4 to 6 weeks. The density of nerve fibres containing NPY increased significantly in almost all vessels between 6 and 8 weeks of age and then stabilized. Thus there is a differential time course for the appearance of NA and NPY during development. Furthermore, the hyperinnervation of cerebral vessels in SHR by nerves containing NA and NPY precedes the onset of hypertension and associated medial hypertrophy. High-performance liquid chromatography and enzyme-linked immunosorbant assays show that the NA and NPY contents of the superior cervical ganglion do not reflect the changes in innervation pattern seen in the terminal fibres in the cerebral arteries. This tends to support the view that a local neurovascular mechanism is involved in the maintenance of hypertension. The possibility that increase in NPY as well as NA in cerebral perivascular nerves of hypertensive animals is involved in the protection of the blood-brain barrier against oedema and cerebral haemorrhage is raised.     

Neurochemical coupled actions of transmitters in the microvasculature of the brain

The discovery that monoamine nerves end on the central microvessels of the choroid plexus, pia-arachnoid and parenchyma has prompted an intense investigation as to their physiological and neuropathological roles. The source of the monoamine fibers to the pial vessels and choroid plexus was shown to be the superior cervical ganglion. Ganglionic stimulation causes vasoconstriction or vasodilation of pial vessels, an event depending upon the functional ratio of alpha to beta adrenergic receptors. Moreover, stimulation of the superior cervical ganglion evokes an inhibition of cerebrospinal fluid formation in choroid plexus. The locus coeruleus is the site of adrenergic nerve supply to the parenchymal capillaries and stimulation of this nucleus increases capillary permeability to small molecules and water. Neurotransmitter receptors (adrenergic, histamine, adenosine, dopamine, prostacyclin, prostaglandins and specific amino acids or neuropeptides) have been identified on microvessels and in many instances these transmitter actions are coupled to cyclic AMP synthesis. Moreover, cyclic AMP has been shown to increase the rate of capillary endothelial pinocytosis and produce brain edema. In small vessels containing smooth muscle cells cyclic AMP production improves cerebral blood flow via an initiation of vasodilatory processes. The presence of receptors for serotonin and acetylcholine have likewise been demonstrated to occur on cerebral microvessels. Limited information is available as to the receptor coupled actions of these two transmitters, but cholinergic mechanisms may act to restrict catecholamine-induced formation of cyclic AMP. Altered sensitivity of microvessels to neurotransmitters has been demonstrated following conditions of stroke, hypertension, aging, diabetes and X-irradiation.     

Perinatal development of adrenergic, cholinergic and non-adrenergic, non-cholinergic nerves and sif cells in the rabbit urinary bladder

The development of adrenergic (indicated by catecholamine fluorescence), acetylcholine-sterase-positive (possibly cholinergic), non-adrenergic, non-cholinergic (indicated by quinacrine fluorescence) nerves and small intensely fluorescent (SIF) cells in the rabbit urinary bladder was examined in foetal (from 23 days of gestation), newborn and adult animals. Acetylcholinesterase-positive nerve fibres and ganglion cells and quinacrine-positive ganglion cells were both present on day 23 of gestation, while quinacrine-positive varicose nerve fibres were first seen on day 24. At foetal age 26 days, 25–38 ganglia containing quinacrine-positive cells were seen in whole-mount preparations of detrusor muscle of the bladder. Each ganglion contained 30–40 quinacrine-positive cells (diameter 20–40 μm). In contrast, only 5–12 ganglia contained acetylcholinesterase-positive nerve cell bodies at the same foetal age with only 3–20 cells in each ganglion; these figures remained at about the same level from foetal age 23 days to maturity. No catecholamine-containing nerve cell bodies were seen at any foetal age or in the adult. Adrenergic nerve fibres were not detected until day 28 of gestation, although small intensely fluorescent cells were first observed on day 26 of gestation. In the adult bladder there was a reduction of approximately 25–35% in the number of quinacrine-positive nerve cell bodies within the ganglia when compared with the ganglia in 1-day-old bladders and an increase in nerve fibre density of about 50% when compared with bladders of earlier ages. A reduction of approximately 90% in small intensely fluorescent cells and a 2-fold increase of adrenergic nerves was also characteristic of the adult bladder, although no changes were observed in the density of the acetylcholinesterase-positive cell bodies and nerve fibres. It is concluded that catecholamine-containing, acetylcholinesterase-positive and non-adrenergic, non-cholinergic nerves follow very different developmental patterns in the bladder.     

Biochemical evidence for cholinergic innervation of intracerebral blood vessels

Muscarinic cholinergic receptor sites were detected with [3H]quinuclidinylbenzilate (QNB) binding techniques in two fractions of bovine intracerebral vessels; one of the fractions contained primarily small arteries and veins with some attached capillaries, and the other one was highly enriched in capillaries. The amounts of binding were similar in equivalent vascular fractions isolated from cerebral cortex, caudate nucleus and cerebellar cortex in spite of large differences among the 3 regions in [3H]QNB binding to brain tissue. The different distribution of muscarinic receptors in brain tissue and blood vessels argues against the possibility that the receptors represent a contamination of the vascular fractions by brain parenchyma. Cerebral endothelial cells, which were isolated by treating capillaries with collagenase, bound [3H]QNB to the same extent as did cerebral capillaries. This is consistent with an endothelial localization of capillary muscarinic receptors. Choline acetyltransferase (ChAT) activity, a marker for cholinergic neurons, also was present in the vascular preparations. Within each brain region, ChAT activities in capillaries and larger vessels were similar, but significant regional differences were found for vascular ChAT activity, with the highest values in the caudate. Isolated endothelial cells contained significantly lower levels of ChAT activity than intact capillaries, suggesting a periendothelial location of the enzyme, as would also be the case for attached nerve terminals. The presence of [3H]QNB binding sites and ChAT activity in intracerebral blood vessels is consistent with an innervation of the cerebral vasculature by a cholinergic system that may regulate cerebral blood flow and capillary permeability.     

Pathway of acetylcholinesterase containing nerves to the major cerebral arteries in rats

The pathway of nerves containing acetylcholinesterase (AChE) to the major cerebral arteries was investigated in the rat. In this species, the internal ethmoidal artery (IEA) arises from the anterior cerebral artery (ACA) and anastomoses with the external ethmoidal artery (EEA), forming the ethmoidal rete on the cribriform plate. The ethmoidal nerve (EN) and EEA enter the cranial cavity through the ethmoidal foramen. Densely distributed adventitial nerve plexi were present around the IEA, ethmoidal rete, and EEA. Many thick nerve bundles were found in the periadventitial layers in association with these vessels. Around the EN, just before it enters the ethmoid foramen, intensely staining nerve bundles were present that entered the cranial cavity with the EN. After unilateral section of the EN and EEA, a marked decrease of the nerve fibers was observed around the arteries of the anterior part of the circle of Willis on the operated side, whereas the basilar artery (BA) showed a moderate decrease in the AChE activity. After bilateral section of the EN and EEA, nerves disappeared from around all the major cerebral vessels including the BA. Section of the EEA alone did not produce any visible change of the cerebral perivascular innervation. The present study suggests that AChE-containing nerves on the cerebral arteries arise from the AChE-positive nerve bundles, which enter the cranial cavity with the EN through the ethmoid foramen; The anterior part of the circle of Willis is innervated unilaterally by the AChE-positive nerve bundles from the ethmoidal foramen, whereas the BA receives bilateral innervation.     

Postnatal development of adrenergic innervation in the tibial and vagus nerves of Fischer-344 rats

Adrenergic innervation of rat tibial and vagus nerves was studied in male Fischer-344 rats between 1 and 84 days of age, using sucrose-phosphate-glyoxylic acid (SPG) histochemistry and the formaldehyde-induced fluorescence (FIF) method. Adrenergic nerve fibers were found in epi-perineurial blood vessels of the vagus nerve at one day of age, whereas blood vessels in the tibial nerve received the first adrenergic nerve fibers at 3 days. A few adrenergic nerve fibers were seen in the endoneurium of both tibial and vagus nerves at 7 days. The densities of adrenergic innervation increased gradually during the first 4 postnatal weeks, and at 21 days the distributions of adrenergic innervation in both nerves resembled those in adult animals. The results suggest that development of adult adrenergic innervation in rat peripheral nerves occurs during the first postnatal month and that sympathetic innervation becomes available to regulate nerve blood flow within this period.     

Evidence for the presence of cholinergic nerves in cerebral arteries: an immunohistochemical demonstration of choline acetyltransferase

The presence of cholinergic nerves in cerebral arteries of several species was investigated by an immunohistochemical method using antibodies against choline acetyltransferase (ChAT). In cats, pigs, rats, and dogs, ChAT immunoreactivities were found to be associated with large bundles and single fibers in the circle of Willis and anterior cerebral, middle cerebral, and basilar arteries. In the rabbit, the ChAT-immunoreactive (ChAT-I) nerves were also observed in the circle of Willis and anterior and middle cerebral arteries, but only few or none were found in the basilar and vertebral arteries. The ChAT-I nerves were found only in the adventitial layer of vessels examined. Superior cervical ganglionectomy did not appreciably affect the distribution of ChAT-I nerves. These results indicate the presence of cholinergic nerves in cerebral arteries. The distribution pattern of ChAT-I nerves was different from that of vasoactive intestinal polypeptide (VIP)-like-immunoreactive nerves and acetylcholinesterase-positive nerves. The possible coexistence of ChAT and VIP-like substance in the same neuron is discussed.     

Autonomic nerves, mast cells, and amine receptors in human brain vessels. A histochemical and pharmacological study.

The studies were performed on operation material from 17- to 63-year-old patients and on fetuses at 19-23 weeks gestational age. Formaldehyde histofluorescence showed the presence of numerous perivascular adrenergic nerves around pial and intracerebral vessels, the carotid system being better supplied than the vertebral system. Cholinergic nerves, visualized by the cholinesterase technique, followed the adrenergic fibers in the plexus formations of the pial arterial system. Histamine-containing mast cells, often with a perivascular distribution, were located with the o-phthaldiadehyde method. Transmural electrical stimulation of the perivascular nerves contracted isolated pieces of pial arteries in a frequency-dependent manner, and the response was inhibited by the adrenergic nerve blocking agent, guanethidine. On the basis of the relative potency of various amines and related compounds in producing a motor response of isolated pial arteries, and the mode of inhibition caused by specific antagonists, various amine receptors could be demonstrated: adrenergic alpha-receptors (mediating contraction) and beta-receptors (dilation), cholinergic muscarinic receptors (dilation) and histamine H2-receptors (mediating dilation). Thus, the amine mechanisms demonstrated in human brain vessels appear to be principally the same of those shown in more extensive studies on laboratory animals.     

Evidence that vasoactive intestinal polypeptide (VIP) mediates neurogenic vasodilation of feline cerebral arteries

In this study the magnitude of non-sympathetic, non-cholinergic neurogenic vasodilation of feline cerebral arteries in vitro was correlated with the extent of innervation by VIP-immunoreactive nerves. Well-innervated arteries underwent nerve-mediated relaxation whereas those that are not supplied with VIP-containing axons did not relax to transmural nerve stimulation. The relaxation of cerebral arteries that are well endowed with VIP-immunoreactive nerves was selectively and reversibly inhibited by VIP-specific antiserum. Substance P-specific antiserum did not affect the dilator responses. We conclude that VIP is a functional neurodilator transmitter in the cerebral circulation.     

Autonomic innervation of the mammalian penis: A histochemical and physiological study

Summary The distribution of adrenergic and cholinergic nerves was studied in penile tissue from rat, rabbit, cat, monkey and man. Glyoxylic acid histofluorescence and acetylcholinesterase-positive fibers were found in all tissues examined, but histofluorescent, presumably adrenergic fibers predominated. In general, except in the rabbit, more nerve fibers of both types were found in the corpus cavernosum (CC) than in the corpus spongiosum/penile urethra (CS). The rabbit penis showed slightly more adrenergic fibers in the CC than in the CS and more cholinergic nerves in the CS than in the CC. The CC of the monkey demonstrated an alpha receptor mediated contractile response to norepinephrine (NE) stimulation and no response to acetylcholine (ACh) in anin vitro muscle bath.