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The effect of hydrocortisone and adrenocorticotrophic hormone on monoamine oxidase and tyrosine hydroxylase in explant cultures of embryonic chick sympathetic ganglia.

Sympathetic ganglia from 13- to 15-day-old embryonic chicks were cultured for up to 2 days in Leighton tubes. The influence of hydrocortisone and ACTH added to the culture medium on the enzymes monoamine oxidase (MAO) and tyrosine hydroxylase was studied. Hydrocortisone (5 times 10(-5)M) had no effect on tyrosine hydroxylase but increased MAO activity by up to 46 percent over control values under conditions of low or zero nerve growth factor (NGF) concentration. ACTH also increased ganglionic MAO activity, the effect again depending on NGF concentration. This time the maximal response (an increase of 50 percent over controls) was seen at high NGF concentrations. This response was similar to the effect of 1 mM dibutyryl cyclic AMP, and was blocked by 1 times 10-5 M propranolol and 10 muM prostaglandin E(1). ACTH only slightly increased tyrosine hydroxylase activity and this effect was due to a small (18 percent) increase in sympathetic neurone number. Guanosine 5-diphosphate (0.5 mM) was found to increase tyrosine hydroxylase activity by 57 percent and this effect was blocked by the presence of ACTH.

Adrenocorticotropic Hormone

Small, intensely fluorescent cells of human sympathetic ganglia.

Human sympathetic ganglia obtained from sympathectomies were studied using the formaldehyde-induced fluorescence (FIF) method and electron microscopy to identify the small, intensely fluorescent (Sif) cells. Microspectrofluorimetry was used to study the intensity of the emitted FIF and the emission spectra. The SIF cells were found in every ganglion studied, but they were very sparce. They occurred alone or in small clusters and were mostly situated close to the blood vessels. The SIF cells contained high amounts of catecholamines, as indicated by microspectrofluorimetry. Electron microscopy revealed granule-containing cells (GC) enveloped by satellite cells; the former cells contained osmiophilic granules 300--400 nm in diameter and synapses of the cholinergic type were frequently found on their surface.

Dopamine

Ultrastructure and function of the granule-containing cells in the anuran sympathetic ganglia.

In the sympathetic ganglia of various anuran species clusters of small granule-containing cells (GC cells) appeared more often in the 5th to 7th paravertebral granglia rather than in the 9th and 10th ganglia of the sympathetic chain. The GC cells were classified into four types according to the granular vesicle morphology. Types I, II and III cells probably acted as endocrine cells which secrete catecholamines into blood capillaries or extracellular spaces. Type IV cells, however, resembled sympathetic ganglion cells. They seemed to be a special type of postganglionic adrenergic neuron rather than a type of interneuron. All these four cell types received cholinergic innervations. Ganglion cells in the anuran sympathetic ganglia formed no synapses with the adrenergic elements or the GC cells. Hence, it was unlikely that s-IPSP was mediated by catecholamines released from GC cells. Serial section studies revealed that both B- and C-neurons received only cholinergic innervation. A mechanism of s-IPSP production was discussed in correlation with the morphological findings.

Adrenergic Fibers

Identification of small intensely fluorescent (SIF) cells as chromaffin cells in bullfrog sympathetic ganglia.

Small intensely fluoresent (SIF) cells were investigated in the ninth and tenth paravertebral sympathetic ganglia of the bullfrog using histochemical and electron microscopic techniques. Fluorescence histochemistry revealed that clusters of SIF cells are sparsely distributed in the ninth and tenth ganglia; the clusters were usually located in the vicinity of blood vessels. Fluorescent processes were not observed emanating from SIF cells. The clusters stained positively for the chromaffin reaction indicating that SIF cells are chromaffin cells. Ultrastructurally, the SIF-chromaffin cells in the sympathetic ganglia appeared virtually identical to the chromaffin cells in the adrenal gland; this includes two cell types that appear morphologically the same as the epinephrine and norepinephrine containing chromaffin cells in the adrenal gland. Efferent synapses from the SIF-chromaffin cells in sympathetic ganglia to sympathetic ganglia to sympathetic neurons were not observed. The SIF-chromaffin cells in bullfrog sympathetic ganglia did not have the morphological characteristics of interneurons; it is suggested that they may function as extra-adrenal chromaffin tissue.

Adrenal Glands

On the occurrence of substance P-containing fibers in sympathetic ganglia: immunohistochemical evidence.

Several sympathetic ganglia of the guinea pig, cat and rat were studied with indirect immunofluorescence technique using antibodies to substance P (SP) and dopamine-beta-hydroxylase (DBH), the latter of which represents a marker for noradrenaline containing neurons. In all ganglia studied SP-like immunoreactivity could be observed in certain nerve fibers (SP-positive fibers). Their number, localization fluorescence intensity and 'fluorescence morphology' varied considerably between the different ganglia as well as between species. In the inferior mesenteric ganglion and the coeliac-superior mesenteric ganglion complex of all species, and in particular of the guinea pig, a dense plexus of varicose SP-positive fibers was observed around the mostly DBH-positive, principal ganglion cells. In contrast no such fibers were seen in relation to the DBH-positive SIF cells of the ganglia. No SP-positive ganglion cells were observed in any of the ganglia studied. Previous biochemical and immunohistochemical studies have indicated that peripheral SP-positive fibers may represent the branches of primary sensory neurons. The rich supply of varicose, SP-positive nerve terminals, often surrounding the noradrenergic ganglion cells in a basket-like manner, may indicate that these fibers may play a functional role within the ganglia. Instead of merely passing through the ganglion they may be involved in special types of reflex arches.

Animals

A peptide as a possible transmitter in sympathetic ganglia of the frog.

A search was made in sympathetic ganglia of the bullfrog for a noncholinergic, nonaminergic transmitter that is released by a distinct group of preganglionic axons. These initiate a late slow excitatory postsynaptic potential which lasts for many minutes. The most promising candidate for the role of transmitter is a peptide that resembles luteinizing hormone-releasing hormone (LHRH; luliberin). The reasons are: (i) LHRH (1 muM) and some of its analogs cause a slow depolarization of ganglion cells. (ii) Radioimmunoassays established that 100-800 pg of a LHRH-like substance is contained in the lumbar chain of sympathetic ganglia. (iii) The LHRH-like material is specifically distributed in those spinal nerves that contain axons that initiate the slow noncholinergic synaptic responses. (iv) Five days after ipsilateral preganglionic axons are cut, 95% of the LHRH-like substance disappears from ganglia, while the LHRH immunoreactivity triples in the spinal nerves proximal to the cut region. (v) About 0.6% of the LHRH-like material within ganglia can be collected from the perfusate after 30 min of incubation in isotonic KC1; this release is Ca dependent. (vi) The candidate for transmitter has several chemical characteristics of a peptide and has a Mr near 1000.

Animals

Monoamine-containing paraneurons in the sympathetic ganglia of mammals.

1. A combined method for glyoxylic acid induced catecholamine fluorescence and electron microscopy was introduced in this study of the small intensely fluorescent (SIF) or granule-containing (GC) cells of the sympathetic ganglia. 2. Adrenergic interneurons were demonstrated in the sympathetic ganglia of the rat and the rhesus monkey. 3. Existence of adrenergic paraneurons (neuro-endocrine cells) in the sympathetic ganglia of several mammalian species was suggesed by the following observations: a) catecholamines are contained in the granular vesicles of SIF cells; b) SIF cells are innervated by preganglionic cholinergic nerves and are often in close relation with fenestrated capillaries; c) SIF cells develop from the neuroblast which originated from the neural crest, and ; d) substantial surface areas of GC cells are covered by basal lamina alone. Figures indicating exocytosis of granular vesicles were often seen here. 4. There are remarkable species differences in quantity and quality of SIF cells and great discrepancies among the physiological, biochemical and the morphological observations on SIF cells.

Adrenergic Fibers

Discrepancies in the extracellular space of sympathetic ganglia measured using different isotopes of mannitol and sucrose.

The extracellular space of rat superior cervical ganglia in vitro was measured using mannitol and sucrose labelled with tritium and carbon-14. The volumes of distribution of the 3H-labelled derivatives, especially [3H]mannitol, exceeded those of the 14C-derivatives. The divergence increased with increasing lengths of incubation. Thus, after 30 min incubation, 'spaces' (ml . g-1) were: [14Cu]mannitol, 0.407; [3H]mannitol 0.447; [14C]mannitol, 0.458; [3H]mannitol, 0.645; [14C]sucrose, 0.430; [3H]sucrose, 0.497. Using thin layer chromatography, it was shown that an average of 22% of the label in ganglia incubated for 120 min with [3H]mannitol, but only 4% with [14C]mannitol, was not associated with the parent compound. Both [3H]- and [14C]sucrose appeared to be metabolized by 11%. It is concluded that mannitol and sucrose can be metabolized in isolated ganglia and that this may lead to substantial errors in estimating the extracellular space, particularly when [3H]markers are used.

Animals

Physical properties of the detergent-extracted nerve growth factor receptor of sympathetic ganglia.

The nerve growth factor (NGF) receptor from microsomes of adult rabbit superior cervical ganglia has been solubilized with Triton X-100 and sodium deoxycholate. The physical properties of the detergent-extracted NGF receptor were assessed by Sepharose 6B chromatography and sucrose density gradient ultracentrifugation studies in H2O and D2O. The predominant form of the NGF receptor has a Stokes radius of 71 A, a partial specific volume of 0.74 ml/g, a sedimentation coefficient of 4.3 S, and a frictional ratio of 1.8. From these parameters, it can be calculated that the NGF receptor in Triton X-100 is a minimally hydrophobic, highly asymmetric, intrinsic membrane protein with a molecular weight of approximately 135,000. A form of the receptor with a sedimentation coefficient of 10.4 S was occasionally seen which appears to represent an aggregated form of the 4.3 S moiety.

Animals

Evaluation of cytotoxic responses caused by selected organophosphorus esters in chick sympathetic ganglia cultures.

Ten day old chick sympathetic ganglia cultured in a microslide assembly were treated with a selected group of organophosphate pesticides to evaluate their cytotoxicity ranges, and the usefulness of such a model for screening pesticides. Examination by phase contrast and light microscopy for chemically-induced morphological alteration of nerve fibers, glial cells and neurons provided the criteria for quantitation and assessment of the toxic effects. Concentrations that produced half-maximal effects ranged from 1 x 10(-6)M (severely toxic) for methylparathian, diazinon, paraoxon, mevinphos, diisopropylfluorophosphate, tri-o-tolyl phosphate and its mixed isomers to a 1 x 10(-3)M (intermediate) for malathion, leptophos, coumaphos, mono- and dicrotophos. Some or no effects were evident at 1 x 10(2-)M for O'ethyl-O-p-nitrophenyl phenyl phosphonothioate, tri-m-tolylphosphate, chlorpyriphos and triphenyl phosphate. In all instances, nerve fibers were more sensitive than neurons or glial cells to insecticides. All cellular growth was inhibited at 1 x 10(-2)M (except triphenyl phosphate). Below 1 x 10(-7)M, no inhibitory effects were evident. The secondary abnormalities included decreased cellular migration, diffuse cellular growth pattern, increased vacuolization, nerve fiber swelling and cellular degeneration. The cytotoxic effects of these chemicals do not appear to be related to in vivo toxicity or cholinesterase inhibition potential.

Animals

Ultrastructure and biochemistry of sympathetic ganglia in idiopathic orthostatic hypotension.

The role of transsynaptic dysfunction in the pathogenesis of idiopathic orthostatic hypotension (IOH, or idiopathic autonomic insufficiency) was examined microscopically and biochemically in autopsy specimens. Light microscopy of the sympathetic ganglia showed abnormalities in all 4 IOH patients, including focal phagocytosis of neurons, increased numbers of satellite cells, and perivascular lymphocytic infiltrates. Electron microscopy revealed proliferation and hypertrophy of satellite cells and abnormalities in the unmyelinated axons. In contrast, the spinal cord intermediolateral columns, containing the presynaptic neurons, were unremarkable in 1 patient, exhibited only mild gliosis in another, and showed neuron loss and fibrillary gliosis in 2 patients. Postsynaptic dopamine-beta-hydroxylase (DBH) activity was decreased at least fourfold (p less than 0.02) in sympathetic ganglia of patients with IOH, while tyrosine hydroxylase (T-OH) was normal. Ganglion choline acetyltransferase (ChAc) activity, an index of presynaptic function and integrity, was normal in the IOH group. A number of our observations suggest that presynaptic disease is not an absolute requirement for adrenergic abnormalities in IOH. The intermediolateral columns of the spinal cord were histologically normal in 2 of the patients with IOH, and ultrastructural abnormalities in sympathetic ganglia were consistent with primary adrenergic degeneration. In addition, presynaptic ChAc activity was normal in IOH ganglia, whereas postsynaptic DBH activity was depressed. Finally, postsynaptic T-OH activity, which is regulated by transsynaptic mechanisms, was normal in IOH ganglia.

Adrenergic Fibers

Developmental variations of tyrosine hydroxylase and acetylcholinesterase in embryonic and post-hatching chicken sympathetic ganglia.

The developmental variations of tyrosine hydroxylase (TH) and of acetylcholinesterase (AChE) were studied in embryonic and post-hatching chicken sympathetic ganglia. Different levels of TH activity were found in two different flocks of White Leghorn chicken, which are probably dependent on genetic differences. These enzymatic differences, however, do not become apparent before hatching and may indicate a combined effect of genetic variation and functional demands. During the period of incubation, TH activity is characterized by a pronounced and steady increase from the twelfth day of incubation up to day 2 after hatching. This corresponds to a period of intense maturation of the sympathetic neuron. In the period following hatching, the 'fourth day fall phenomenon' previously described by us for DOPA decarboxylase (DDC), dopamine-beta-hydroxylase (DBH), and monoamine oxidase (MAO) is not seen in the TH curve. Instead, TH activity tends to remain constant between days 2 and 14 after hatching (ah). Both ganglionic protein and weight remain constant in this period, indicating a phase of general pause in protein synthesis. AChE activity increases steadily from the eighth until the twenty-first day of incubation. A sudden and significant drop in AChE activity was found at day 2 ah followed by a period of rapid increase at day 3 ah and a levelling of activity up to day 30 ah. Comparing the present variations to those observed in our previous studies on DBH, a temporal relationship between TH and DBH activity is observed during the phases of synaptogenesis and maturation but not during the phase of intense functional activity. Our results strongly suggest that before hatching in chick embryo sympathetic ganglia, the cholinergic presynaptic terminals play a role in regulating the development of the adrenergic neurons. In the period following hatching, however, the DBH and TH levels in cell bodies seem to be principally regulated by the functional activity. This results in depletion of DBH, but not TH, through liberation along with the neurotransmitter at the periphery. Depletion of DBH at the terminals may result in increased transport and thereby depletion in the cell body. This mechanism is probably responsible for the difference in the profiles of activity of DBH and TH in the cell bodies observed in the first week after hatching.

Acetylcholinesterase

Evidence for autonomic paraneurons in sympathetic ganglia of a shrew (Tupaia glis).

The morphological relationships between blood vessels and the small monoamine-storing cells have been studied in the superior sympathetic ganglia of Tupaia. Substantial clusters of these cells were found to be associated anatomically with vascular loops or glomeruli. Blood from these glomeruli enters the capillary bed that supplies principal ganglionic neurons. Thus the vascular path for catecholamine transportation is rather clearly defined in Tupaia, providing new morphological evidence for the existence of a portal system for conveying catecholamine from clusters of paraneurons to principal ganglionic neurons. On the basis of vesicle criteria, two populations of small, granule-containing cell were distinguished, and both were encountered in the same cluster. On the basis of fluorescence microscopy, the relatively small number (14.3%) of solitary SIF cells may be true interneurons because, although there has been no electron microscopic identification of efferent synapses, their processes are much longer than those of the paraneurons.

Animals

Selective induction of tyrosine hydroxylase and dopamine beta-hydroxylase by nerve growth factor: comparison between adrenal medulla and sympathetic ganglia of adult and newborn rats.

Administration of NGF to newborn and adult rats elicits a selective increase in TH and DBH both in sympathetic ganglia and adrenal medulla. This effect does not depend on intact preganglionic cholinergic fibers. The augmented enzyme activity results from enhanced enzyme synthesis since it can be abolished by cycloheximide and NGF has been shown to enhance the incorporation of [3H]leucine into DBH molecules. The responsiveness of the adrenal medulla to NGF is also supported by light and electron microscopic autoradiograms which show that intravenously injected 125I-NGF is accumulated with high selectivity in adrenal chromaffin as compared to adjacent adrenal cortical cells. In spite of the many similarities between the response of the adrenergic neurons and adrenal chromaffin cells to NGF, there are also two distinct differences. (a) In newborn rats the ratio between the TH increase effected by a single and 10 subsequent daily injections of NGF is 1:2 in the adrenal medulla and 1:7 in the superior cervical ganglia. (b) If adrenal medullae are transferred to organ culture after intravenous injection of NGF, maximal TH response is initiated 60-90 min after NGF administration. In superior cervical ganglia only a half-maximal response is initiated at that time. After a stationary phase a second increase starts after about 6 h to reach the maximum after 12 h. The biphasic time course of the initiation of TH induction by NGF in sympathetic ganglia is in agreement with the time course of 125I-NGF accumulation after intravenous injection27 reflecting the moiety of NGF reaching the cell bodies of the adrenergic neurons directly by the blood stream (initial accumulation) and by retrograde axonal transport (second phase).

Adrenal Medulla