Decentralisation of neurones in the pelvic ganglion of the guinea-pig: Reinnervation by adrenergic nerves

An electron-microscopic study has been made of adrenergic and cholinergic nerve fibres and synapses in the pelvic ganglion of the guinea-pig at intervals of up to 60 days following section of the hypogastric and pelvic nerves. Transection of the hypogastric nerves led to degeneration of 80-90% of the cholinergic nerve profiles and synapses in the ganglion. The small number of adrenergic nerves and synapses did not change, but 30-60 days after section, this number increased 8-10 times. Transection of the pelvic nerves led to degeneration of about 15% of the cholinergic nerve terminals, but no change in adrenergic terminals. After transection of both hypogastric and pelvic nerves, only about 1% of cholinergic nerves survived, but after 30-60 days, the number of adrenergic nerves increased 8-10 times. It is concluded that following cholinergic nerve degeneration in the ganglion, adrenergic nerves, probably originating as collateral sprouts from postganglionic neurones and granule-containing cells, can replace them to some extent.     

Presence and distribution of cholinergic nerves in rat mediastinal brown adipose tissue.

Brown adipose tissue (BAT) is richly provided with sympathetic noradrenergic nerves but is believed to lack a parasympathetic nerve supply. Acetylcholine is the predominant transmitter of postganglionic parasympathetic nerves. The vesicular acetylcholine transporter (VAChT) resides in synaptic vesicles of cholinergic nerve terminals and is used as a marker for peripheral cholinergic nerves. We sought cholinergic nerves in rat BAT using VAChT immunohistochemistry (IHC) on cryosections of interscapular, cervical, mediastinal, and perirenal depots. Mediastinal BAT was the sole depot provided with putative parasympathetic perivascular and parenchymal cholinergic nerves. The absence of vasoactive intestinal peptide-positive nerves suggested their nature as pure cholinergic fibers. By confocal microscopy, both cholinergic and noradrenergic nerves were detected in mediastinal BAT. Cold exposure and fasting led to increased density of VAChT-positive fibers and of noradrenergic sympathetic nerves at morphometry. The unexpected double innervation of mediastinal BAT may explain the inhibitory influence on thermogenesis observed after systemic injection of muscarinic antagonists in rats, and raises questions about the physiological role of its cholinergic nerve supply.     

Localization of cholinergic innervation and neurturin receptors in adult mouse heart and expression of the neurturin gene

Neurturin (NRTN) is a neurotrophic factor required during development for normal cholinergic innervation of the heart, but whether NRTN continues to function in the adult heart is unknown. We have therefore evaluated NRTN expression in adult mouse heart and the association of NRTN receptors with intracardiac cholinergic neurons and nerve fibers. Mapping the regional distribution and density of cholinergic nerves in mouse heart was an integral part of this goal. Analysis of RNA from adult C57BL/6 mouse hearts demonstrated NRTN expression in atrial and ventricular tissue. Virtually all neurons in the cardiac parasympathetic ganglia exhibited the cholinergic phenotype, and over 90% of these cells contained both components of the NRTN receptor, Ret tyrosine kinase and GDNF family receptor α2 (GFRα2). Cholinergic nerve fibers, identified by labeling for the high affinity choline transporter, were abundant in the sinus and atrioventricular nodes, ventricular conducting system, interatrial septum, and much of the right atrium, but less abundant in the left atrium. The right ventricular myocardium contained a low density of cholinergic nerves, which were sparse in other regions of the working ventricular myocardium. Some cholinergic nerves were also associated with coronary vessels. GFRα2 was present in most cholinergic nerve fibers and in Schwann cells and their processes throughout the heart. Some cholinergic nerve fibers, such as those in the sinus node, also exhibited Ret immunoreactivity. These findings provide the first detailed mapping of cholinergic nerves in mouse heart and suggest that the neurotrophic influence of NRTN on cardiac cholinergic innervation continues in mature animals.This study was supported by the National Heart, Lung, and Blood Institute (grant HL-54633).     

Histochemical study of the postnatal development of autonomic nerves in the mouse iris,using a whole-mount preparation method

Summary Postnatal development of autonomic nerves in the mouse iris was studied histochemically from one day to five months of age. For the demonstration of aminergic nerves the glyoxylic acid method was used, while for cholinergic nerves, Karnovsky and Roots' method was utilized on the whole-mount preparations of irises. The results obtained were as follows. In one-day-old mice, many aminergic nerves could already be seen, while cholinergic nerves were scarcely observable. Both types of nerve increased rapidly in the first 2 weeks. Both completed development between 3 and 4 weeks, although aminergic nerves were observed to develop earlier than the cholinergic.     

Adrenergic and cholinergic innervation of rat cerebral arteries

Summary A consecutive demonstration of both aminergic and cholinergic nerves of rat cerebral arteries was carried out on whole mount preparations. For demonstration of aminergic nerves the glyoxylic acid method was used, while for cholinergic nerves Karnovsky's technique was utilized consecutively. This procedure provided a highly sensitive and reproducible demonstration of the two systems on the same specimen. The results obtained were as follows: 1) Aminergic and cholinergic nerves were distributed densely in the proximal portions of the major cerebral arteries examined. They were dense in the arteries of the anterior circulation and sparse in the posterior circulation. Each nerve had two different patterns, i.e. circular and longitudinal. Both circular and longitudinal patterns were observed in the proximal portions of the arteries, while in the distal portions, longitudinal fibres were predominantly present. 2) Superior cervical ganglionectomy produced no change in cholinergic nerve distribution, while it caused decreased density of aminergic nerves on the ipsilateral side on the arteries of the anterior circulation. The contralateral anterior cerebral arteries were partially affected. Bilateral sympathectomies abolished aminergic nerves in all arteries except the vertebral artery.     

Partial degeneration of autonomic nerves of the heart of methotrexate-treated guinea pigs

In the present investigation partial degeneration of adrenergic and cholinergic nerves of the heart of guinea pigs following methotrexate treatment has been reported. No appreciable change was observed in the autonomic nerve fibres after 3 days of treatment. However, after prolonged treatment some nerve fibres showed signs of degeneration and a few adrenergic and cholinergic nerve fibres had disappeared.     

Effects of heterologous cross-suture between the postganglionic sympathetic and the preganglionic parasympathetic nerve trunks of submandibular glands in cats

Summary After sectioning the postganglionic adrenergic sympathetic nerve trunk for the submandibular gland, as close to the submandibular artery as practicable, its central end was sutured to the peripheral end of the preganglionic cholinergic parasympathetic nerve trunk for the gland, the chorda, which had been sectioned where it left the lingual nerve. The effects of this heterologous cross-sature were studied at different times, up to 1 year afterwards, by assessing the physiological and pharmacological responses of the glands and the neuro-histochemical changes in the nerve trunks and in the nerves within the glands.In all cases adrenergic sympathetic nerves grew across the site of suture and down the erstwhile cholinergic parasympathetic trunk, eventually to develop connections in the gland. In some cases the functional adrenergic reinnervation of the submandibular gland appeared to result exclusively or predominantly from the direct downgrowth of adrenergic axons to the gland, via the crossed nerves. In other cases however, in addition to a direct glandular reinnervation, there was some physiological and morphological evidence which suggested that possible heterogenous synaptic contacts may have been created between postganglionic sympathetic axons and cholinergic ganglion cells in the chorda nerve.This work was supported by a grant from the Joint Research Committee, King's College Hospital.     

Intrinsic innervation of the uterus in guinea pig and rat

The pattern of distribution of cholinergic and adrenergic nerves in the uterus of albino rats and guinea pigs was examined histochemically. In the albino rat, the uterus was found well-innervated by both adrenergic and cholinergic nerves with a clear regional variation. Dense innervation was demonstrated at the tubal and cervical ends of the uterus and in the cervix. Cholinergic nerves supplying the glands were more numerous than the adrenergic nerves which were relatively few. In the guinea-pigs, the uterus was richly innervated by adrenergic nerves with a clear regional variation. No cholinesterase-positive nerves or nerve cells were demonstrated.     

Immunoreactivity for high-affinity choline transporter colocalises with VAChT in human enteric nervous system

Cholinergic nerves are identified by labelling molecules in the ACh synthesis, release and destruction pathway. Recently, antibodies against another molecule in this pathway have been developed. Choline reuptake at the synapse occurs via the high-affinity choline transporter (CHT1). CHT1 immunoreactivity is present in cholinergic nerve fibres containing vesicular acetylcholine transporter (VAChT) in the human and rat central nervous system and rat enteric nervous system. We have examined whether CHT1 immunoreactivity is present in nerve fibres in human intestine and whether it is colocalised with markers of cholinergic, tachykinergic or nitrergic circuitry. Human ileum and colon were fixed, sectioned and processed for fluorescence immunohistochemistry with antibodies against CHT1, class III beta-tubulin (TUJ1), synaptophysin, common choline acetyl-transferase (cChAT), VAChT, nitric oxide synthase (NOS), substance P (SP) and vasoactive intestinal peptide (VIP). CHT1 immunoreactivity was present in many nerve fibres in the circular and longitudinal muscle, myenteric and submucosal ganglia, submucosa and mucosa in human colon and ileum and colocalised with immunoreactivity for TUJ1 and synaptophysin confirming its presence in nerve fibres. In nerve fibres in myenteric ganglia and muscle, CHT1 immunoreactivity colocalised with immunoreactivity for VAChT and cChAT. Some colocalisation occurred with SP immunoreactivity, but little with immunoreactivity for VIP or NOS. In the mucosa, CHT1 immunoreactivity colocalised with that for VIP and SP in nerve fibres and was also present in vascular nerve fibres in the submucosa and on epithelial cells on the luminal border of crypts. The colocalisation of CHT1 immunoreactivity with VAChT immunoreactivity in cholinergic enteric nerves in the human bowel thus suggests that CHT1 represents another marker of cholinergic nerves.     

Adhesion-dependent interactions between eosinophils and cholinergic nerves

Eosinophils adhere to airway cholinergic nerves and influence nerve cell function by releasing granule proteins onto inhibitory neuronal M(2) muscarinic receptors. This study investigated the mechanism of eosinophil degranulation by cholinergic nerves. Eosinophils were cocultured with IMR32 cholinergic nerve cells, and eosinophil peroxidase (EPO) or leukotriene C(4) (LTC(4)) release was measured. Coculture of eosinophils with nerves significantly increased EPO and LTC(4) release compared with eosinophils alone. IMR32 cells, like parasympathetic nerves, express the adhesion molecules vascular cell adhesion molecule-1 and intercellular adhesion molecule-1 (ICAM-1). Inhibition of these adhesion molecules alone or in combination significantly inhibited eosinophil degranulation. IMR32 cells also significantly augmented the eosinophil degranulation produced by formyl-Met-Leu-Phe. Eosinophil adhesion to IMR32 cells resulted in an ICAM-1-mediated production of reactive oxygen species via a neuronal NADPH oxidase, inhibition of which significantly inhibited eosinophil degranulation. Additionally, eosinophil adhesion increased the release of ACh from IMR32 cells. These neuroinflammatory cell interactions may be relevant in a variety of inflammatory and neurological conditions.     

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.     

Peripheral choline acetyltransferase in rat skin demonstrated by immunohistochemistry

Conventional choline acetyltransferase immunohistochemistry has been used widely for visualizing central cholinergic neurons and fibers but not often for labeling peripheral structures, probably because of their poor staining. The recent identification of the peripheral type of choline acetyltransferase (pChAT) has enabled the clear immunohistochemical detection of many known peripheral cholinergic elements. Here, we report the presence of pChAT-immunoreactive nerve fibers in rat skin. Intensely stained nerve fibers were distributed in association with eccrine sweat glands, blood vessels, hair follicles and portions just beneath the epidermis. These results suggest that pChAT-positive nerves participate in the sympathetic cholinergic innervation of eccrine sweat glands. Moreover, pChAT also appears to play a role in cutaneous sensory nerve endings. These findings are supported by the presence of many pChAT-positive neuronal cells in the sympathetic ganglion and dorsal root ganglion. Thus, pChAT immunohistochemistry should provide a novel and unique tool for studying cholinergic nerves in the skin.     

Effects of infantile/prepubertal chronic estrogen treatment and chemical sympathectomy with guanethidine on developing cholinergic nerves of the rat uterus.

The innervation of the uterus is remarkable in that it exhibits physiological changes in response to altered levels in the circulating levels of sex hormones. Previous studies by our group showed that chronic administration of estrogen to rats during the infantile/prepubertal period provoked, at 28 days of age, an almost complete loss of norepinephrine-labeled sympathetic nerves, similar to that observed in late pregnancy. It is not known, however, whether early exposure to estrogen affects uterine cholinergic nerves. Similarly, it is not known to what extent development and estrogen-induced responses in the uterine cholinergic innervation are affected by the absence of sympathetic nerves. To address this question, in this study we analyzed the effects of infantile/prepubertal chronic estrogen treatment, chronic chemical sympathectomy with guanethidine, and combined sympathectomy and chronic estrogen treatment on developing cholinergic nerves of the rat uterus. Cholinergic nerves were visualized using a combination of acetylcholinesterase histochemistry and the immunohistochemical demonstration of the vesicular acetylcholine transporter (VAChT). After chronic estrogen treatment, a well-developed plexus of cholinergic nerves was observed in the uterus. Quantitative studies showed that chronic exposure to estrogen induced contrasting responses in uterine cholinergic nerves, increasing the density of large and medium-sized nerve bundles and reducing the intercept density of fine fibers providing myometrial and perivascular innervation. Estrogen-induced changes in the uterine cholinergic innervation did not appear to result from the absence/impairment of sympathetic nerves, because sympathectomy did not mimic the effects produced by estrogen. Estrogen-induced responses in parasympathetic nerves are discussed, considering the direct effects of estrogen on neurons and on changes in neuron-target interactions.     

The ligament of Marshall as a parasympathetic conduit

The objective of the study was to investigate the morphology, distribution, and electrophysiological profile of the autonomic fibers that innervate the ligament of Marshall (LOM). Gross anatomical dissections were performed in 10 dogs. Sections of the left vagus nerve, left stellate ganglion, and the LOM were immunostained to identify adrenergic and cholinergic nerves. Hearts were also stained for acetylcholinesterase to identify epicardial cholinergic nerves. In vivo electrophysiological studies were performed in another 10 dogs before and after LOM ablation. The anatomical examination revealed that the LOM is innervated by a branch of the left vagus. Immunohistochemistry confirmed that these nerve bundles are predominantly cholinergic (cholinergic-to-adrenergic ratio of 12.6 +/- 3.9:1). Cholinergic nerves originating in the LOM were found to innervate surrounding left atrial structures, including the pulmonary veins, left atrial appendage, coronary sinus, and posterior left atrial fat pad. Ablation of the LOM significantly attenuated effective refractory period shortening at distant sites, such as pulmonary veins and left atrial appendage, in response to vagal stimulation (vagal-induced ERP decrease in the left atrium: baseline vs. postablation = 17 vs. 4%; P = 0.0056). In conclusion, the LOM contains a predominance of cholinergic nerve fibers. Cholinergic fibers arising from the LOM innervate surrounding structures and contribute to the electrophysiological profile of the left atrium. These findings may provide a basis for the role of the LOM in the genesis and maintenance of atrial fibrillation.     

Structure and innervation of the pineal gland of the rabbit,Oryctolagus cuniculus (L.)

In the rabbit pineal gland two types of postganglionic nerve endings were found which are characterized by the presence of small dense-core vesicles or small clear vesicles. Pharmacological and cytochemical experiments showed then to be noradrenergic and cholinergic, respectively. Both types were often present in the same nerve bundle, occasionally in close opposition. Intrapineal neurons were only rarely observed. They showed cholinergic synapses on their perikaryon and dendrites as well as noradrenergic axo-dendritic close contacts. Bilateral extirpation of the superior cervical ganglia revealed the postganglionic sympathetic origin of the pineal noradrenergic nerve fibres. Moreover, it appeared that these ganglia are hardly, if at all, involved in the pathway of pineal cholinergic innervation. The results obtained from lesions of both facial nerves, taken together with the results reported in the literature, led to the conclusion that the postganglionic cholinergic nerve fibers in the pineal are of parasympathetic origin. A model for the sympathetic and parasympathetic pineal innervation is proposed.     

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.     

Histoarchitectonics of the cholinergic innervation of the frog tongue

Acetylcholinesterase (AChE) activity was studied in dorsal tongue surface structures and in both tongue nerves (hypoglossal and glossopharyngeal) of frogs (Rana temporaria). AChE was found in nerve fibers of fungiform and filiform papillae, blood vessels, glandular ducts of tongue mucosa, both nerve fibers and also in the bodies of cholinergic neurons in subepithelial connective tissue and along the glossopharyngeal nerve. Their parasympathetic origin was suggested. The experiments with butirilthiocholin have revealed no activity of non-specific cholinesterases in the above structures. Possible role of cholinergic system in the regulation of tongue receptor function is discussed.     

Eosinophil adhesion to cholinergic nerves via ICAM-1 and VCAM-1 and associated eosinophil degranulation

In vivo, eosinophils localize to airway cholinergic nerves in antigen-challenged animals, and inhibition of this localization prevents antigen-induced hyperreactivity. In this study, the mechanism of eosinophil localization to nerves was investigated by examining adhesion molecule expression by cholinergic nerves. Immunohistochemical and functional studies demonstrated that primary cultures of parasympathetic nerves express vascular cell adhesion molecule-1 (VCAM-1) and after cytokine pretreatment with tumor necrosis factor-alpha and interferon-gamma intercellular adhesion molecule-1 (ICAM-1). Eosinophils adhere to these parasympathetic neurones after cytokine pretreatment via a CD11/18-dependent pathway. Immunohistochemistry and Western blotting showed that a human cholinergic nerve cell line (IMR-32) expressed VCAM-1 and ICAM-1. Inhibitory experiments using monoclonal blocking antibodies to ICAM-1, VCAM-1, or CD11/18 and with the very late antigen-4 peptide inhibitor ZD-7349 showed that eosinophils adhered to IMR-32 cells via these adhesion molecules. The protein kinase C signaling pathway is involved in this process as a specific inhibitor-attenuated adhesion. Eosinophil adhesion to IMR-32 cells was associated with the release of eosinophil peroxidase and leukotriene C(4). Thus eosinophils adhere to cholinergic nerves via specific adhesion molecules, and this leads to eosinophil activation and degranulation; this may be part of the mechanism of eosinophil-induced vagal hyperreactivity.     

Comparative studies of purinergic nerves.

Purinergic nerves supply the gastrointestinal tract of vertebrates, including fish, amphibians, reptiles and birds, as well as mammals. Their cell bodies are located in Auerbach's plexus and their axons extend in an anal direction before innervating mainly the circular muscle coat. In the stomach they are controlled by preganglionic cholinergic fibres of parasympathetic origin. They are involved in "receptive relaxation" of the stomach, "descending inhibition" in peristalsis and reflex relaxation of oesophageal and internal anal sphincters. The terminal varicosities of purinergic nerves are characterised by a predominance of "large opaque vesicles," which can be distinguished from the "large granular vesicles" found in small numbers in both adrenergic and cholinergic nerves. Stimulation of purinergic nerves with single pulses produces hyperpolarisations of up to 25 mV (inhibitory junction potentials) in smooth muscle cells. These potentials are unaffected by atropine, adrenergic neuron blocking agents or sympathetic denervation, but are abolished by tetrodotoxin. The "rebound contraction" which characteristically follows cessation of purinergic nerve stimulation is probably due to prostaglandin. Evidence that ATP is the transmitter released from purinergic nerves includes: (1) synthesis and storage of ATP in nerves; (2) release of ATP from the nerves when they are stimulated; (3) exogenously applied ATP mimicking the action of nerve-released transmitter, both producing a specific increase in K+ conductance; (4) the presence of Mg-activated ATPase, 5'-nucleotidase and adenosine deaminase, enzymes which inactivate ATP; (5) drugs (including quinidine, some 2-substituted imidazolines, 2-2'pyridylisatogen and dipyridamole) which produce similar blocking or potentiating effects on the response to exogenously applied ATP and nerve stimulation. Speculations are made about the evolution and development of the nervous system, including the possibility that purinergic nerves are a primitive nerve type.     

A histochemical study on the innervation of the cerebral blood vessels in bats

Summary The adrenergic and cholinergic nerves innervating the cerebral blood vessels of four species of Japanese chiropterids (Rhinolophus ferrumequinum, Murina leucogaster, Vespertilio superans and Miniopterus Schreibersi) have been investigated using specific histochemical techniques. In all these species of bats arteries of the internal carotid system are poorly developed, whereas those of the vertebro-basilar system are well developed. The adrenergic and cholinergic nerves innervating these cerebral arteries, however, all originate from the stem nerve bundles entering the cranial cavity along the internal carotid artery. Both nerve plexuses are among the densest of any vertebrate species so far investigated. Adrenergic nerve plexuses are usually composed of complicated meshworks of fine fibres, while cholinergic ones are composed of rather longitudinally arranging meshworks of both thick and thin fibres, exhibiting a very high acetylcholinesterase activity. Small parenchymal arteries and arterioles are also dually innervated by adrenergic and cholinergic nerve fibres of peripheral origin. Intracerebral capillaries, on the other hand, are in several places directly connected with both adrenergic and cholinergic fibres of parenchymal origin. Capillaries in the cerebral and cerebellar cortices, diencephalon and cochlear nucleus in V. superans exhibit a heavy non-nervous acetylcholinesterase activity in their walls, but in R.ferrumequinum and M. schreibersi, the response is weak or negative, except for that in the cochlear nucleus.