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.     

The sphincter of the efferent filament artery in teleost gills: II. Sympathetic innervation

In addition to the cholinergic innervation described in the sphincter of the efferent filament arteries (Bailly and Dunel-Erb, ′86), an aminergic component has been demonstrated by specific techniques. The Falck fluorescence technique reveals a network of nerve fibers displaying a green fluorescence characteristic of catecholamines. At the ultrastructural level two types of fibers are present, one with clear vesicles and another with densecored vesicles. Axo-axonal synaptic relationships exist between the two types. Results of 5- and 6-OHDA (hydroxydopamine) treatments confirm the presence of an aminergic component. These observations support the notion of a dual innervation: cholinergic and adrenergic of, respectively, parasympathetic and sympathetic origin. The presence of presynaptic modulation is suggested. The aminergic component could inhibit or reduce the release of acetylcholine from cholinergic nerve endings. These results suggest that the sympathetic innervation modulates the vasoconstriction effect of the parasympathetic component.     

Target influences on transmitter choice by sympathetic neurons developing in the anterior chamber of the eye

In contrast to the majority of sympathetic neurons which are noradrenergic, the sympathetic neurons which innervate sweat glands are cholinergic. Previous studies have demonstrated that during development the sweat gland innervation initially contains catecholamines which are lost as cholinergic function appears. The neurotransmitter phenotype of sweat gland neurons further differs from the majority in that they contain vasoactive intestinal peptide (VIP) rather than neuropeptide Y (NPY). In the experiments described here, we addressed the question of whether sympathetic targets influence the neurotransmitter-related properties of the neurons which innervate them; in particular, do sweat glands play a role in reducing the expression of noradrenergic properties and inducing the expression of cholinergic properties and VIP in sympathetic neurons? This was accomplished by cotransplanting to the anterior chamber of the eye of host rats the superior cervical ganglia (SCG) which contains neurons that normally innervate targets other than the sweat glands and differentiate noradrenergically and footpad tissue from neonatal rats. Sweat glands developed in the transplanted footpad tissue and became innervated by the cotransplanted SCG neurons. The transplanted neurons and sweat gland innervation initially exhibited catecholamine histofluorescence which declined with further development in the anterior chamber. After 4 weeks, choline acetyltransferase (ChAT) and VIP immunoreactivities were evident. These observations suggest that as in the neurons which innervate the glands in situ, noradrenergic properties were suppressed and cholinergic function was induced in the neurons which innervated the glands in oculo. To distinguish a specific influence of the sweat glands on transmitter choice, SCG were also cotransplanted with the pineal gland, a normal target of the ganglion. Neurons cotransplanted with the pineal gland continued to exhibit catecholamine histofluorescence and contained NPY immunoreactivity. At least some neurons in SCG/pineal cotransplants, however, developed ChAT immunoreactivity. The target-appropriate expression of catecholamines and peptides in these experiments is consistent with the hypothesis that some transmitter properties are influenced by target tissues. The indiscriminant expression of ChAT, however, suggests that at least in oculo, additional factors can influence transmitter choice.     

A Histochemical Study of the Gas Gland Innervation in the Atlantic God,Gadus morhua

A histochemical study was made of the distribution of catecholamines and cholinesterases in two autonomic ganglia closely associated with the swimbladder of the Atlantic cod, Gadus morhua. The “swimbladder nerve ganglion” comprised large (40 μm) neurones, the majority of which are positive for both catecholamines and acetylcholinesterase. It is argued that these neurones are mainly adrenergic postganglionic elements of sympathetic pathways which pass through the vago-sympathetic trunk. The “gas gland ganglion” comprised small (20 μm) neurones, positive for acetylcholinesterase but showing no catecholamine reaction. It is argued that these neurones are cholinergic postganglionic elements of the parasympathetic vagal innervation of gas gland cells.     

Ultrastructure of the nerve cells and fibres in the urinary bladder wall of the cat.

The nerve elements in the urinary bladder of the cat were studied by electron microscopy. According to their ultrastructure, nerve cell somata can be classified into three types: the large cells with a cytoplasm rich in organelles, several processes and numerous synaptic contacts on their surface; the cytoplasm contained 80- 120-nm granulated vesicles. The second type is poor in cytoplasmic organelles and has very few processes and virtually no synaptic contacts on the soma. The third type contains numerous large 160- to 220-nm 'neurosecretory' vesicles in the cytoplasm. According to the morphology of the vesicle population, four types of nerve processes could be distinguished: Type a, with a dominant population of small (40-60 nm) agranular vesicles. These are thought to be sacral parasympathetic fibres. Type b, with small (40-60 nm) granular vesicles, which may be the noradrenergic sympathetic fibres. Type c, with 80- to 120-nm granulated vesicles, probably of local origin. Typed d, with large 160- to 220-nm 'neurosecretory' vesicles also of local origin. Different types of nerve fibres are converging on the local nerve cells. This suggests that the local circuits can play an important role in coordinating the function of the bladder. Therefore, ganglia may be considered as an elementary functional unit.     

Fine-structural study of the pineal body of the monkey (Macaca fuscata) with special reference to synaptic formations

Summary Various types of synaptic formations on pinealocytes and pineal neurons were found in the pineal body of Macaca fuscata. Axo-somatic synapses of the Gray type-II category were detected on the pinealocyte cell body. Gap junctions and ribbon synapses were observed between adjacent pinealocytes. About 70 nerve-cell bodies were detected in one half of the whole pineal body bisected midsagittally. They were localized exclusively deep in the central part. When examined electron-microscopically, they were found to receive ribbon-synapse-like contacts from pinealocytic processes. They also received synaptic contacts of the Gray type-I category on their dendrites, and those of the Gray type-II category on their cell bodies from nerve terminals of unknown origin. All these synapse-forming axon terminals contained small clear vesicles. Thus, the pineal neurons of the monkey, at least in part, are suggested to be derived from the pineal ganglion cells in the lower vertebrates and not from the postganglionic parasympathetic neurons. The functional significance of these observations is discussed in relation to the innervation of the pineal body of the monkey.     

Development of the cholinergic neurotransmitter phenotype in postganglionic sympathetic neurons

Sympathetic ganglia are composed of noradrenergic neurons and cholinergic neurons that differ in the expression of neurotransmitter-synthesizing enzymes, neurotransmitter transporters and neuropeptides. The analysis of the cholinergic differentiation during development revealed important principles involved in the generation of neuronal diversity, in particular the importance of signals from the innervated target. Some peripheral targets, such as the sweat glands in the mammalian footpads, are purely cholinergically innervated in the adult, whereas skeletal muscle arteries receive both noradrenergic and cholinergic innervation. For sympathetic neurons innervating sweat glands there is convincing evidence that these neurons are initially noradrenergic and that the interaction of innervating fibers and target tissue induces a shift in the neurotransmitter phenotype from noradrenergic to cholinergic. In addition to this target-dependent differentiation, an earlier expression of cholinergic characters was observed in sympathetic ganglia that occurs before target contact. These data raise the possibility that different subpopulations of cholinergic sympathetic neurons, innervating distinct peripheral targets, may develop along distinct schedules. In vitro studies suggest that growth factors of the family of neuropoietic cytokines are involved in the specification of the cholinergic sympathetic phenotype. Recent in vivo studies that interfered with cytokine receptor expression in developing avian sympathetic ganglia indicate that only the late, target-dependent differentiation depends on cytokine signaling. The signals involved in the early, target-independent expression of cholinergic properties remain to be determined, as well as the identity of the target-derived cytokine. Thus, cholinergic sympathetic differentiation seems to be more complex than expected, involving either both target-independent and target-dependent control or only target-induced differentiation, according to the specific neuronal subpopulation and target.     

Developmental interactions between sweat glands and the sympathetic neurons which innervate them: effects of delayed innervation on neurotransmitter plasticity and gland maturation

The neurotransmitter properties of the sympathetic innervation of sweat glands in rat footpads have previously been shown to undergo a striking change during development. When axons first reach the developing glands, they contain catecholamine histofluorescence and immunoreactivity for catecholamine synthetic enzymes. As the glands and their innervation mature, catecholamines disappear and cholinergic and peptidergic properties appear. Final maturation of the sweat glands, assayed by secretory competence, is correlated temporally with the development of cholinergic function in the innervation. To determine if the neurotransmitter phenotype of sympathetic neurons developing in vivo is plastic, if sympathetic targets can play a role in determining neurotransmitter properties of the neurons which innervate them, and if gland maturation is dependent upon its innervation, the normal developmental interaction between sweat glands and their innervation was disrupted. This was accomplished by a single injection of 6-hydroxy-dopamine (6-OHDA) on Postnatal Day 2. Following this treatment, the arrival of noradrenergic sympathetic axons at the developing glands was delayed 7 to 10 days. Like the gland innervation of normal rats, the axons which innervated the sweat glands of 6-OHDA-treated animals acquired cholinergic function and their expression of endogenous catecholamines declined. The change in neurotransmitter properties, however, occurred later in development than in untreated animals and was not always complete. Even in adult animals, some fibers continued to express endogenous catecholamines and many nerve terminals contained a small proportion of small granular vesicles after permanganate fixation. The gland innervation in the 6-OHDA-treated animals also differed from that of normal rats in that immunoreactivity for VIP was not expressed in the majority of glands. It seems likely that following treatment with 6-OHDA sweat glands were innervated both by neurons that would normally have done so and by neurons that would normally have innervated other, noradrenergic targets in the footpads, such as blood vessels. Contact with sweat glands, therefore, appears to suppress noradrenergic function and induce cholinergic function not only in the neurons which normally innervate the glands but also in neurons which ordinarily innervate other targets. Effects of delayed innervation were also observed on target development. The appearance of sensitivity to cholinergic agonists by the sweat glands was coupled with the onset of cholinergic transmission.(ABSTRACT TRUNCATED AT 400 WORDS)     

Intramural ganglia of the human gallbladder: morphological and functional observations]

The purpose of this study was the reinvestigation of the intrinsic innervation of human gall bladder with an immunohistochemical technique named peroxidase anti-peroxidase (PAP). The antigen demonstrated was the S100 protein normally present in the surface of glial cells, Schwann cells and satellite cells in ganglia. The tissues used were taken from 20 human gall bladders, fixed after surgery. This technique is not specific to demonstrate adrenergic or cholinergic innervation but it reveals just myelinated fibers. The current study was undertaken in order to study the organization and the function of plexus of nerves and ganglia present in the wall of the gall bladder. The neck of the gall bladder was the region in which the higher number of nerve cells and nervous fibers was present. The technique used has demonstrated ganglionated plexus and nerves in submucosal layer, fibromuscular and adventitial layer according to the enteric nervous system. All ganglia are postganglionic stations related with preganglionic cholinergic fibers. These results confirm that the intramural ganglia of the gall bladder are analogous to those of the enteric nervous system according to their common origin.     

The sphincter of the efferent filament artery in teleost gills: I. Structure and parasympathetic innervation

Previous studies have shown the existence of a sphincter in the efferent filament artery of the teleost gill and its constrictory response to acetylcholine (ACH) and vagal stimulation. This study deals with the muscular organization of this sphincter and the distribution of its innervation as elucidated by degeneration methods and cytochemistry. The sphincter innervation is supplied by the protrematic vagus nerves. Nerve endings filled with cholinergic-type vesicles are located in close association with the adventitial smooth muscle cells and display a strong acetylcholinesterase (ACHE) activity. Section of the protrematic vagus nerve induces a nearly complete degeneration of the sphincter innervation. ACHE-positive nerve cell bodies are present both in the sphincter area and in the protrematic vagus nerve. These results suggest that innervation of the sphincter in the efferent filament artery is cholinergic through the activity of postganglionic axons of the parasympathetic system.     

Stimulation-depletion of serotonin and noradrenaline from vesicles of sympathetic nerves in the pineal gland of the rat

Summary The pineal gland of the rat receives a rich nervous supply originating from the superior cervical ganglia. These fibers contain serotonin in addition to their neurotransmitter, noradrenaline. Cytochemical studies at the ultrastructural level have shown that both amines are present in the cores of the granular vesicles that are characteristic of these nerves. It is presently shown that the bilateral electrical stimulation of the preganglionic fibers innervating the ganglia markedly reduces the number of small sites reacting cytochemically for both noradrenaline and serotonin, these sites corresponding to the cores of small granular vesicles, while the larger reactive sites (cores of large vesicles) remain unaltered. The vesicles are retained in nerve terminals after stimulation, as observed in conventionally processed tissues, although with altered sizes and shapes. Apart from these cytochemical and structural changes, nerve stimulation also reduces the endogenous noradrenaline content of the pineal gland. Thus, both noradrenaline and serotonin are released from their storage sites in pineal sympathetic nerves after electrical stimulation in vivo. This suggests the possibility that several substances with presumed transmitter or modulatory functions might be simultaneously released by nerve impulses from a given nerve terminal.     

Vasoactive intestinal peptide (VIP)-like immunoreactivity in the human sympathetic ganglia

Vasoactive intestinal peptide immunoreactive (VIP-IR) nerve fibres and terminals, neurons and small granule containing cells were observed in human lumbal sympathetic ganglia. Electron-microscopically VIP-IR was localized in the large dense-cored vesicles in nerve terminals and on the membranes of the Golgi complexes in the neurons. A small population of principal ganglion cells was surrounded by VIP-IR nerve terminals. Most of these neurons contained acetylcholinesterase (AChE) enzyme but were not tyrosine hydroxylase-immunoreactive (TH-IR). All VIP-IR ganglion cells and most of the nerve fibres contained AChE but not TH-IR. It appears that in human sympathetic ganglia VIP is localized in the cholinergic neurons and nerve fibres and that the VIP-IR nerve terminals innervate mainly the cholinergic subpopulation of the sympathetic neurons.     

Immunohistochemical properties and spinal connections of pelvic autonomic neurons that innervate the rat prostate gland

Autonomic innervation of the prostate gland supplies the acini, and non-vascular and vascular smooth muscle. The activity of each of these tissues is enhanced by sympathetic outflow, whereas the role of the parasympathetic nervous system in this organ is unclear. In the present study, a range of methods was applied in rats to determine the location of autonomic neurons supplying this gland, the immunohistochemical properties of these neurons, the spinal connections made with the postganglionic pathways and the distribution of various axon types within the gland. Injection of the retrograde tracer, FluoroGold, into the ventral gland visualised neurons within the major pelvic ganglion and sympathetic chain. Fluorescence immunohistochemical studies on the labelled pelvic neurons showed that most were noradrenergic (also containing neuropeptide Y, NPY), the others being non-noradrenergic and containing either vasoactive intestinal peptide (VIP) or NPY. Sympathetic dyelabelled neurons were identified by the presence of varicose nerve terminals stained for synaptophysin on their somata following lesion of sacral inputs. Parasympathetic innervation of dye-labelled neurons was identified by continued innervation after hypogastric nerve lesion. Most noradrenergic prostate-projecting neurons were sympathetic, as were many of the non-noradrenergic VIP neurons. Parasympathetic prostate-projecting neurons were largely non-noradrenergic and contained either VIP or NPY. All substances found in retrogradely labelled somata were located in axons within the prostate gland but had slightly different patterns of distribution. The studies have shown that there are a significant number of non-noradrenergic sympathetic prostate-projecting neurons, which contain VIP.     

Neonatal treatment with nerve growth factor antiserum eliminates cholinergic sympathetic innervation of rat sweat glands

Most mammalian sympathetic neurons are noradrenergic, and their dependence upon nerve growth factor (NGF) for survival during development is well established. A minor population of sympathetic neurons, including those that innervate sweat glands, is cholinergic. To determine whether cholinergic sympathetic neurons, like their noradrenergic counterparts, require NGF during development, neonatal rats were treated with NGF-antiserum and 3 weeks later their sweat glands were examined for the presence of innervation. Acetylcholinesterase (AChE) staining and vasoactive intestinal polypeptide-like immunoreactivity (VIP-IR) which mark the mature sweat gland innervation were absent from the sweat glands of the anti-NGF treated animals. Further, when the glands were examined with the electron microscope, no axons or nerve terminals were evident. These observations indicate that the elaboration of the sweat gland plexus is NGF-dependent and suggest that at least one population of cholinergic sympathetic neurons in the rat requires NGF for survival. Our findings are consistent with the idea that during development NGF is a required trophic factor not only for noradrenergic sympathetic but also for cholinergic sympathetic neurons.     

Intrinsic choroidal neurons in the duck eye receive sympathetic input: anatomical evidence for adrenergic modulation of nitrergic functions in the choroid

Intrinsic choroidal neurons (ICN) in the duck eye form an intramural ganglionic plexus that may subserve complex integrative functions. A key feature of such ganglia is an innervation by sympathetic postganglionic neurons. The present study was thus aimed at determining the sympathetic postganglionic innervation of ICN. Choroids were processed for double immunofluorescence labelling with the following markers: tyrosine-hydroxylase (TH)/nitric oxide synthase (nNOS), TH/galanin (GAL), dopamine-beta-hydroxylase (DBH)/vasoactive intestinal polypeptide (VIP), TH/DBH and DBH/alpha-smooth-muscle actin (alphaSMA), and for triple immunofluorescence labelling with VIP/DBH/TH. Epifluorescence and confocal laser scanning microscopy were used for evaluation. Immunoperoxidase staining for TH or DBH in combination with NADPH-diaphorase histochemistry was applied for electron microscopy. ICN spread over the entire choroid but were concentrated in an equatorial zone passing obliquely from naso-cranial to temporocaudal. More than 80% of nNOS-positive ICN showed close appositions of TH/DBH-immunoreactive varicose nerve fibres at the light-microscopic level, as could be confirmed by confocal laser scanning microscopy. Ultrastructurally, these appositions could be defined as both synapses or close contacts without synaptic specialisation. Vascular and non-vascular smooth muscle fibres also received TH/DBH-immunopositive innervation. Our findings suggest that most ICN receive a sympathetic input that might modulate their nitrergic effects upon vascular and non-vascular smooth muscle fibres in the choroid and that they may have more complex functions than merely being a simple parasympathetic relay.     

Light and electron microscopic histochemical observations on cholinesterase-containing sympathetic nerve fibres in the pineal body of the rat

Synopsis Pineal glands of adult albino rats were examined histochemically using, first, formaldehyde-induced fluorescence to study monoamines and, second, copper thiocholine or copper ferrocyanide methods to study acetylcholinesterase and non-specific cholinesterase by light and electron microscopy. Cholinesterase was determined quantitatively by a constant pH titration assay.Fluorescent and acetylcholinesterase-positive nerve nets formed identical patterns. Nonspecific cholinesterase was observed only in nerve trunks outside the pineal. Bilateral removal of superior cervical ganglia resulted in complete disappearance of fluorescence and acetylcholinesterase from nerve fibres. Electron microscopically, acetylcholinesterase was found on sympathetic axons containing small granular vesicles. Quantitative cholinesterase determinations suggested that the pineal activity was mainly due to acetylcholinesterase. Comparison of the incubation times required for equal histochemical acetylcholinesterase reactions suggested that the activity of the sympathetic nerve fibres in the pineal is of the same order of magnitude as that in the nerve fibres of the iris.     

Norepinephrine transporter expression in cholinergic sympathetic neurons: differential regulation of membrane and vesicular transporters

Sympathetic neurons that undergo a noradrenergic to cholinergic change in phenotype provide a useful model system to examine the developmental regulation of proteins required to synthesize, store, or remove a particular neurotransmitter. This type of change occurs in the sympathetic sweat gland innervation during development and can be induced in cultured sympathetic neurons by extracts of sweat gland-containing footpads or by leukemia inhibitory factor. Sympathetic neurons initially produce norepinephrine (NE) and contain the vesicular monoamine transporter 2 (VMAT2), which packages NE into vesicles, and the norepinephrine transporter (NET), which removes NE from the synaptic cleft to terminate signaling. We have used a variety of biochemical and molecular techniques to test whether VMAT2 and NET levels decrease in sympathetic neurons which stop producing NE and make acetylcholine. In cultured sympathetic neurons, NET protein and mRNA decreased during the switch to a cholinergic phenotype but VMAT2 mRNA and protein did not decline. NET immunoreactivity disappeared from the developing sweat gland innervation in vivo as it acquired cholinergic properties. Surprisingly, NET simultaneously appeared in sweat gland myoepithelial cells. The presence of NET in myoepithelial cells did not require sympathetic innervation. VMAT2 levels did not decrease as the sweat gland innervation became cholinergic, indicating that NE synthesis and vesicular packaging are not coupled in this system. Thus, production of NE and the transporters required for noradrenergic transmission are not coordinately regulated during cholinergic development.     

Vasoactive intestinal peptide (VIP)-like immunoreactivity in the human sympathetic ganglia

Summary Vasoactive intestinal peptide immunoreactive (VIP-IR) nerve fibres and terminals, neurons and small granule containing cells were observed in human lumbal sympathetic ganglia. Electron-microscopically VIP-IR was localized in the large dense-cored vesicles in nerve terminals and on the membranes of the Golgi complexes in the neurons. A small population of principal ganglion cells was surrounded by VIP-IR nerve terminals. Most of these neurons contained acetycholinesterase (AChE) enzyme but were not tyrosine hydroxylase-immnoreactive (TH-IR). All VIP-IR ganglion cells and most of the nerve fibres contained AChE but not TH-IR. It appears that in human sympathetic ganglia VIP is localized in the cholingergic neurons and nerve fibres and that the VIP-IR nerve terminals innervate mainly the cholinergic subpopulation of the sympathetic neurons.     

Interneuronal synapses in the local ganglia of the cat urinary bladder.

The interneuronal synapses of the urinary bladder in the cat were studied by electron microscopy. The great majority of the fibres containing vesicles are found within the ganglia occurring in the trigonum area. Morphologically differentiated synaptic contacts could be observed on the surface of the local neurons and between the different nerve processes. The presynaptic terminals can be divided into three types based on a combination of synaptic vesicles. Type I terminals, presumably cholinergic synaptic terminals, contain only small clear vesicles of 40-50 nm in diameter. Type II terminals, presumably adrenergic terminals, are characterized by small granulated vesicles of 40-60 nm in diameter. Type III terminals, probably of local origin, contain a variable number of large granulated vesicles of 80-140 nm in diameter. Occasionally, a single nerve fibre contacted several (two or four) other nerve processes forming a typical synapse. In other cases, on one nerve cell soma or on other nerve processes there are two or three different-type nerve terminals establishing synapses. It might be inferred from these observations that convergence and divergence can occur in the local ganglia and that cholinergic and adrenergic synaptic terminals can modulate the ganglionic activity. However, a local circuit also can play an important role in coordinating the function of the bladder.     

Localization of neuropeptide Y in human sympathetic ganglia: Correlation with met-enkephalin, tyrosine hydroxylase and acetylcholinesterase

Summary The relationships of immunoreactive neuropeptide Y, enkephalin and tyrosine hydroxylase, on the one hand, and acetylcholinesterase histochemical activity, on the other, were studied in human lumbar sympathetic ganglia. Two thirds of the ganglion cells contained immunoreactive neuropeptide Y. Electron microscopically the immunoreaction was localized in the Golgi apparatus and in large dense-cored vesicles in the nerve endings. Most of the neuropeptide-containing neurons and nerve fibres were also reactive for tyrosine hydroxylase. Nerve fibres reactive for neuropeptide Y were found around ganglion cells regardless of their transmitter contents, whereas enkephalin-reactive nerve terminals surrounded only acetylcholinesterase-containing neurons. The results demonstrate that neuropeptide Y is colocalized with noradrenaline in most of the human sympathetic neurons and that the nerve fibres may innervate selectively the noradrenergic and cholinergic subpopulations of ganglion cells depending on the transmitters of the nerves.