Cytophotometric study of monoamines and energy exchange enzymes in paravertebral ganglia neurons during cold and emotional stress

Concentration of monoamines and energy metabolism mechanisms was investigated in neurons of the stellate ganglion (SG) and ganglia of the thoracic sympathetic trunk Th₆-Th₁₀ (GTST) using histochemical techniques and computer analysis. Intensification of monoamine fluorescence and energy metabolism enzyme activity was found in SG neurons during both cold and emotional stress. Such changes were only observed during development of hypothermia in GTST. Selective neuronal activation is thought to take place in paravertebral ganglia under different types of stressful action.Institute of Physiology, Academy of Sciences of the Belorussian SSR, Minsk. Translated from Neirofiziologiya, Vol. 22, No. 6, pp. 771–779, November–December, 1990.     

Cytophotometric and electrophysiological analysis of the role of the celiac plexus in maintenance of temperature homeostasis during cold stress in rats

The role of the celiac plexus in maintenance of temperature homeostasis in rats exposed to cold stress was studied by histochemical, ultrastructural, and electrophysiological methods. Inhibition of efferent impulsation was found in the preganglionic (splanchnic) nerves and potentiation in postganglionic (superior mesenteric) nerves under the influence of short-term cold stress, leading to a state of mild hypothermia. During cooling of the animals after decentralization of the celiac plexus an increase was observed in the intensity of fluorescence, in the activity of enzymes of energy metabolism, and in hyperplasia of the ultrastructural formations responsible for protein synthesis and the energy supply of the cell. It is suggested that during cold stress, when the flow of efferent impulses along preganglionic nerves is considerably reduced the celiac plexus becomes the center regulating autonomic functions that are involved in the maintenance of temperature homeostasis.Institute of Physiology, Belorussian Academy of Sciences, Minsk. Translated from Neirofiziologiya, Vol. 24, No. 6, pp. 659–667, November–December, 1992.     

Distribution and projections of neurons with immunoreactivity for both gastrin-releasing peptide and bombesin in the guinea-pig small intestine

Summary Bombesin-like and gastrin-releasing peptide (GRP)-like immunoreactivities were localized in nerves of the guinea-pig small intestine and celiac ganglion with the use of antibodies raised against the synthetic peptides. The anti-bombesin serum (preincubated to avoid cross reactivity with substance P) and the anti-GRP serum revealed the same population of neurons. Preincubation of the antibombesin serum with bombesin abolished the immunoreactivity in nerves while absorption of the anti-GRP serum with either bombesin or the 14–27 C-terminal of GRP only reduced the immunoreactivity. The immunoreactivity was abolished by incubation with GRP 1–27.Immunoreactive nerves were found in the myenteric plexus, circular muscle, submucous plexus and in the celiac ganglion. Faintly reactive nerve cell bodies were found in the myenteric ganglia (3.2% of all neurons) but not in submucous ganglia. After all ascending and descending pathways in the myenteric plexus had been cut, reactive terminals disappeared in the myenteric plexus, circular muscle (including the deep muscular plexus) and the submucous plexus on the anal side. After the mesenteric nerves were cut no changes were observed in the intestinal wall but the reactive fibres in celiac ganglia disappeared. It is deduced that GRP/bombesin-immunoreactive nerve cell bodies in myenteric ganglia project from the myenteric plexus to other myenteric ganglia situated further anally (average length 12 mm), anally to the circular muscle (average length 9 mm), anally to submucous ganglia (average length 13 mm) and external to the intestine to the celiac ganglia.It is concluded that the GRP/bombesin-reactive neurons in the intestinal wall represent a distinct population of enteric neurons likely to be involved in controlling motility and in the coordination of other intestinal functions.     

Structure and functional changes in the gastric intramural nervous plexus in emotionally stressed rats exposed to low doses of long-term ionizing irradiation and lead

Changes in the catecholamine content in adrenergic fibres, acetylcholinesterase activity, and in the energy metabolism enzymes lactate dehydrogenase and succinate dehydrogenase in neurons of the gastric intramural plexus during emotional stress in rats a day after combined exposure to prolonged (30 days) ionizing radiation in a total dose 1.0 Gy and 0.6 mg/kg lead were studied. A decrease in catecholamines in adrenergic fibres and acetylcholinesterase and lactate dehydrogenase activity in neurons was observed. An enhanced sensitivity of the gastric intramural plexus after the prolonged exposure to small doses of ionizing radiation and lead in conditions of emotional stress was suggested.     

Catecholamine concentration in adrenergic plexuses of the spleen and intestine during cold-induced and emotional stress

Computer analysis and histochemical techniques were used to investigate catecholamine concentration in the adrenergic plexuses of the spleen and small intestine in rats. The concentration was found to increase during both emotional and short-term cold-induced stress. Injection of 10 mg/kg guanethidine i.p. does not counteract cold-induced accumulation of catecholamines in the adrenergic plexuses of the spleen and small intestine. During emotional stress, by contrast, an accumulation of this sort does take place. The mechanisms potentially underlying catecholamine accumulation in the adrenergic plexus of these organisms associated with different types of stress are discussed. Cold-induced stress is thought to produce catecholamine synthesis in the adrenergic nerve endings of the prevertebral ganglia, whereas emotional stress leads to catecholamine uptake by adrenergic terminals from the blood.Institute of Physiology, Academy of Sciences of the Byelorussian SSR, Minsk. Translated from Neirofiziologiya, Vol. 22, No. 3, pp. 347–354, May–June, 1990.     

Effect of dalargin on the activity of cytochrome oxidase and glutamate dehydrogenase in rat spinal cord dissociated cultured neurons

Dissociated cultured neurons from the rat embryo spinal cord were grown for six days in the presence of dalargin, the synthetic analog of leu-enkephalin. Then the activities of two enzymes of energy metabolism, cytochrome oxidase (CO) and glutamate dehydrogenase (GDH), were studied in these neurons using quantitative cytochemical technique. Dalargin, which possesses the properties of nerve growth factor, enhanced the nerve cell growth and increased the activity of the above enzymes, with GDH activity being increased more significantly. According to the classical standpoint, increased GDH activity under conditions of acute energy deflciency favors the invoivement of some amino acids in a citric acid cycle for subsequent reproduction. One can suggest, in this relation, that the increased energy production caused by the enhanced nerve cell growth in the presence of dalargin was partially compensated by the amino acid splitting. The results allow us to suggest that the effect of dalargin (growth factor) on the nerve cells is similar to the effects of the extremal factors, and requires additional energy to be supplied.Neirofiziologiya/Neurophysiology, Vol. 28, No. 2/3, pp. 95–99, March–June, 1996.     

Fluorescent properties of monoamine neurons following glyoxylic acid treatment of intact leech ganglia

Summary The SPG modification (de la Torre and Surgeon 1976) of the glyoxylic acid method for amine condensation is a straightforward procedure which can be used upon intact ganglia from the leech. Intense fluorescence of the neurosomata of identified neurons which contain either indoleamine (serotonin, 5-HT) or catecholamine (CA) is obtained in less than 30 min. The fluorescence of the 5-HT containing neurons is yellow (518–526 nm) and decays more rapidly than the dominant blue emission (478–480 nm) of the CA neurons. Most importantly, the SPG technique greatly enhances the visibility of the axonal processes of neurons which contain both classes of amines over the fluorescence produced by formaldehyde condensation techniques. Both blue and yellow fluorescent varicosities are easily distinguished in the longitudinal connectives and lateral roots of the leech C.N.S. Because of its simplicity and high fluorescence yields, the SPG method for histochemical fluorescence should contribute to investigations of amine functions in invertebrate nervous systems.Supported by NIH research grant No. NS14481-03 and NSF research grant BNS-7915/08     

Catecholamines and catecholamine-synthesizing enzymes in guinea-pig sensory ganglia

Summary Cranial and spinal sensory ganglia of the guinea-pig were investigated by means of histochemistry and biochemistry for the presence of catecholamines and catecholamine-synthesizing enzymes. Sensory neurons exhibiting immunoreactivity to the rate-limiting enzyme of catecholamine synthesis, tyrosine nydroxylase (TH), were detected by immunohistochemistry in lumbo-sacral dorsal root ganglia, the nodose ganglion and the petrosal/jugular ganglion complex. The carotid body was identified as a target of TH-like-immunoreactive (TH-LI) neurons by the use of combined retrograde tracing and immunohistochemistry. Double-labelling immunofluorescence revealed that most TH-LI neurons also contained somatostatin-LI, but TH-LI did not coexist with either calcitonin gene-related peptide- or substance P-LI. TH-LI neurons did not react with antibodies to other enzymes involved in catecholamine synthesis, i.e., aromatic amino acid decarboxylase (AADC), dopamine--hydroxylase (DH), and phenylethanolamine-N-methyltransferase (PNMT). Petrosal neurons as well as their endings in the carotid body lacked dopamine- and L-DOPA-LI. Sensory neurons did not display glyoxylic acid-induced catecholamine fluorescence. Ganglia containing TH-LI neurons were kept in short-term organ culture after crushing their roots and the exiting nerve in order to enrich intra-axonal transmitter content at the ganglionic side of the crush. However, even under these conditions, catecholamine fluorescence was not detected in axons projecting peripherally or centrally from the ganglia. Sympathetic noradrenergic nerves entered the ganglia and terminated within them. Accordingly, biochemical analyses of guinea-pig sensory ganglia revealed noradrenaline but no dopamine. In conclusion, catecholamines within guinea-pig sensory ganglia are confined to sympathetic nerves, which fulfill presently unknown functions. The TH-LI neurons themselves, however, lack any additional sign of catecholamine synthesis, and the presence of enzymatically active TH within these neurons is questionable.     

Conducting pathways of the rabbit lumbar sympathetic ganglia

Conducting pathways of ganglia from the lumbar portion (L₃–L₅) of the sympathetic trunk in rabbits were studied by recording action potentials from nerves of the ganglia evoked by stimulation of other nerves of these ganglia, and by intracellular recording from single neurons. Besides the well-known system of descending preganglionic fibers, which enter the trunk through white rami communicantes and, as they pass through the ganglia, form synapses on ganglionic neurons, some preganglionic fibers were shown to enter the sympathetic chain through gray rami communicantes and to run in both ascending and descending directions, forming synaptic connections with neurons of the lumbar ganglia.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 16, No. 2, pp. 247–254, March–April, 1984.     

Effects of the drug balis-2 on the growth of sympathetic ganglia from different strains of rat in culture

The growth-promoting properties of balis-2, produced by microbiological synthesis, were investigated in organotypic cultures of upper cervical ganglia from newborn Wistar, Wag, and August rats. Maximum size of the growth zone was calculated, as well as the density of neurite-glial bundles and numbers of catecholamine-containing fibers within this zone. It was found that ganglia cultures from Wistar and Wag strains grow at 2.0–2.1 times the rate of those from the August strain. Balis-2 exerts an activating action at concentrations of 0.001 and 0.0001%, mainly affecting neurite growth. Level of response was found to vary from one strain to the next: peak response, at 2.3–2.6 times control level was recorded in rats of the August strain, while growth in ganglia culture increased 1.8–2.0-fold against the controls in the remaining two strains under the effects of this substance. In this way it was first shown that balis-2 has neurotrophic properties when used on sympathetic ganglia in culture. In view of the fact that rats belonging to the August strain have a high level of plasma catecholamines and are classified as an emotional species according to their reaction to stress, the subject of how processes of nerve tissue regeneration connect up with the state of the sympathoadrenal system is discussed.Institute of Experimental Cardiology of the All-Union Cardiological Research Center, Academy of Medical Sciences of the USSR, Moscow. Institute of Physical and Organic Chemistry, Rostov University, Krasnodar. Translated from Neirofiziologiya, Vol. 20, No. 4, July–August, 1988, pp. 539–546.     

Effects of anoxia and inhibitors of energy metabolism on potassium and sodium homeostasis in neurons of gastropod mollusk

The effect of anoxia and inhibitors of energy metabolism on intracellular concentrations of potassium and sodium, membrane potentials, permeability, active and passive ouabain-sensitive transport of potassium (determined with⁸⁶Rb) was studied in neurons of the freshwater planorbis mollusk (Planorbarius corneus). X-ray microanalysis showed that incubation of isolated ganglia in oxygen-free medium induced no change in intracellular concentrations of potassium and sodium. In the presence of cyanide, absorption of oxygen by the ganglia ceased, but accumulation of⁸⁶Rb decreased insignificantly. The membrane potential and permeability did not depend on addition of cyanide. Desoxyglucose, an inhibitor of glycolysis, decreased⁸⁶Rb accumulation more than cyanide did. In the presence of inhibitors of both glycolysis and respiration, which excluded the possibility of mutual compensation of oxidation and glycolytic sources of energy supply,⁸⁶Rb accumulation decreased to the highest degree. A hypothesis was formulated on the paramount importance of glycolytic ATP for maintaining ion homeostasis of the nerve cells. The problem of functionally facilitated compartmentation of intracellular energy sources is discussed.Institute of Evolutionary Physiology and Biochemistry, Academy of Sciences of the USSR, Leningrad. Translated from Neirofiziologiya, Vol. 23, No. 3, pp. 313–321, May–June, 1991.     

Changes in P2X<Subscript>3</Subscript> purinoceptors in sensory ganglia of the mouse during embryonic and postnatal development

The expression of the P2X₃ nucleotide receptor in embryonic day 14–18, postnatal day 1–14 and adult mouse sensory ganglia was examined using immunohistochemistry. Nearly all sensory neurons in dorsal root ganglia, trigeminal ganglia and nodose ganglia in embryos at embryonic day 14 expressed P2X₃ receptors, but after birth there was a gradual decline to about 50% of neurons showing positive immunostaining for P2X₃. In embryos there were only small neurons, while from postnatal day 7 both large and small neurons were present. Isolectin B₄ (IB₄)-positive neurons in dorsal, trigeminal and nodose ganglia did not appear until birth, but the numbers increased to about 50% by postnatal day 14 when a high proportion of IB₄-positive neurons were also positively labelled for the P2X₃ receptor. About 10% of neurons in dorsal, trigeminal and nodose ganglia were positive for calcitonin gene-related peptide in embryos, nearly all of which stained for P2X₃ receptors. This increased postnatally to about 35–40% in adults, although only a few colocalised with P2X₃ receptors. Neurofilament 200 was expressed in about 50% of neurons in trigeminal ganglia in the embryo, and this level persisted postnatally. All neurofilament 200-positive neurons stained for P2X₃ in embryonic dorsal root ganglia, trigeminal ganglia and nodose ganglia, but by adulthood this was significantly reduced. The neurons that were positive for calbindin in embryonic dorsal, trigeminal and nodose ganglia showed colocalisation with P2X₃ receptors, but few showed colocalisation postnatally.     

Microfluorimetric study of adrenergic structures in nerve plexuses of the cat pelvic cavity following decentralization and in the normal

Microfluorimetric techniques were used to investigate catecholamine concentration in small, intensely fluorescent cells and adrenergic fibers of the cat pelvic plexus ganglia and intramural ganglia of the urinary bladder and rectum in the control and following sympathetic and parasympathetic denervation. The cells examined could be divided between catecholamine- and serotonin-containing types. Parasympathetic denervation brought about an increase in the number of cells displaying serotinergic fluorescence and heightened fluorescence in the adrenergic fibers of the pelvic plexus ganglia and intramural ganglia of the urinary bladder, without affecting degree of fluorescence in those of the rectal intramural ganglia. Sympathetic denervation failed to change fluorescence level in the cells and adrenergic fibers in pelvic plexus and urinary bladder ganglia but caused the almost complete disappearance of the adrenergic fibers in the rectal intramural ganglia.Institute of Physiology, Academy of Sciences of the Belorussian SSR, Minsk. Translated from Neirofiziologiya, Vol. 18, No. 4, pp. 496–502, July–August, 1986.     

The nervous system of Microstomum lineare (Turbellaria,Macrostomida)

Summary The nervous system (NS) of Microstomum lineare (Turbellaria, Macrostomida) was studied by electron and light microscopy, combined with fluorescence histochemistry (Falck-Hillarp method for biogenic monoamines). The NS is primitively organized, with a bilobed brain, two lateral nerve cords lacking commissures, and peripheral nerve cells scattered along the nerve cords. The stomatogastric NS, with a pharyngeal nerve ring, is joined to the central NS by a pair of connective ganglia. A green fluorescence in all parts of the NS indicates catecholaminergic neurons as the dominant neuron type.Ultrastructurally, two types of neurons were identified on the basis of their vesicle content: 1. Aminergic (catecholaminergic) neurons containing densecore vesicles of varying electron-density and size, i.e., small dense-core vesicles (diameter 50–100 nm), vesicles with a highly electron-dense core (60–140 nm), and vesicles with an eccentric dense-core. 2. Presumed peptidergic neuro-secretory neurons containing large granular vesicles (diameter about 200 nm) in the stomatogastric NS and peripheral parts of the central NS. In light microscopy, paraldehyde-thionin stained neurons were observed in the same areas.     

Location of efferent sympathetic neurons and afferent neurons in spinal ganglia innervating the heart

Location and numbers of neurons associated with sympathetic innervation of the heart within the right stellate and accessory cervical ganglia, the spinal cord, and spinal ganglia were investigated using horseradish peroxidase retrograde axonal transport techniques in cats. The enzyme was applied to central sections of the anastomosis of the stellate ganglion with the vagus nerve, the inferior cardiac nerve, and the vagosympathetic trunk caudal to the anastomosis. Labeled neurons within the stellate ganglion were located close to the point of departure of the nerves and more thinly distributed in the accessory cervical ganglion. A group of labeled cells was found in the anastomosis itself. Preganglionic neurons associated with sympathetic innervation of the heat were detected at segmental levels T₁–T₅ in the spinal cord. Labeled neurons were diffusely located in the spinal ganglia, concentrated mainly at levels T₂–T₄.Medical Institute, Ministry of Public Health of the RSFSR, Yaroslavl'. Translated from Neirofiziologiya, Vol. 21, No. 1, pp. 106–111, January–February, 1989.     

Histochemical correlates of cold-induced trans-synaptic induction in the rat superior cervical ganglion

Synopsis The effect of prolonged pre-ganglionic activity induced by exposure to cold (5°C) was studied in intact and decentralized superior cervical ganglia of rats. Intact and decentralized ganglia of rats kept at room temperature served as controls. catecholamines were demonstrated histochemically using the formaldehyde-induced fluorescence method. The intensity of the fluorescence of the nerve cell bodies was estimated both visually and photometrically.Decentralization in itself had no effect on the intensity of the cell bodies for up to 16 days. Exposure to cold had no effect on the decentralized ganglia but caused a marked increase in the fluorescence intensity of some nerve cells of the intact ganglia, indicating that the increased fluorescence was mediated by the pre-ganglionic nerves. The increase lasted for the whole 16-day-length of exposure to cold.It is suggested that the observed change in the fluorescence intensity reflects an increase of the enzymes tyrosine hydroxylase and dopamine--hydroxylase, and thus represents a histochemical correlate of trans-synaptic induction.Sigrid Jusélius Foundation Research Fellow in the Department of Anatomy, University of Helsinki, April–July 1973.     

Experimental cytophotometric investigation of the effects of denervation on catecholamines and acetylcholinesterase in neurons of the intramural plexus of the cat urinary bladder

Catecholamines and acetylcholinesterase were determined by histochemical methods in neurons of the intramural plexus of the cat urinary bladder. The ganglia of this plexus were found to differ in the number of neurons they contained (from five to 150 cells). Adrenergic, cholinergic, and mixed ganglia exist. Division of the pelvic nerves increased the catecholamine content in adrenergic neurons and fibers and reduced acetylcholinesterase activity. Division of the hypogastric nerves has no effect on the catecholamine level and acetylcholinesterase activity in ganglia of the intramural plexus of the urinary bladder.Institute of Physiology, Academy of Sciences of the Belorussian SSR, Minsk. Translated from Neirofiziologiya, Vol. 17, No. 2, pp. 263–270, March–April, 1985.     

A large proportion of afferent neurons innervating the uterine cervix of the cat contain VIP and other neuropeptides

Summary Axonal tracing techniques were used in combination with immunohistochemistry to examine the distribution of neuropeptides in afferent pathways from the uterine cervix of the cat. Primary afferent neurons innervating the uterine cervix were identified by axonal transport of the dye, fast blue, injected into the cervix. Fifteen to twenty-five days after the injection, dorsal root ganglia (L1–S3) were removed and incubated for 48–72 h in culture medium containing colchicine to increase the levels of peptides. Calcitonin gene-related peptide (CGRP), cholecystokinin (CCK), leucine-enkephalin (LENK), somatostatin, substance P and vasoactive intenstinal polypeptide (VIP) were identified by use of indirect immunohistochemical techniques. Eighty-four percent of uterine cervix afferent neurons were identified in the sacral dorsal root ganglia (S1–S3), and 16% in the middle lumbar dorsal root ganglia (L3–L4). In sacral dorsal root ganglia, VIP was present in the highest percentage of dye-labeled cells (71%), CGRP in 42%, and substance P in 18% of the cells. CCK and LENK were present in 13% of the cells. In lumbar dorsal root ganglia, CGRP (51%) was most prominent peptide followed by VIP (34%), substance P (28%), LENK (17%) and CCK (13%). Somatostatin was present in the ganglia but did not occur in dye-labeled neurons. In conclusion, the uterine cervix of the cat receives a prominent VIP-and CGRP-containing afferent innervation. The percentage of neurons containing VIP is three to five times higher than the percentage of these neurons in afferent pathways to other pelvic organs. These observations coupled with the results of physiological studies suggest that VIP is an important transmitter in afferent pathways from the cervix.     

Lobula plate and ocellar interneurons converge onto a cluster of descending neurons leading to neck and leg motor neuropil in Calliphora erythrocephala

Summary In the fly, Calliphora erythrocephala, a cluster of three Y-shaped descending neurons (DNOVS 1–3) receives ocellar interneuron and vertical cell (VS4–9) terminals. Synaptic connections to one of them (DNOVS 1) are described. In addition, three types of small lobula plate vertical cell (sVS) and one type of contralateral horizontal neuron (Hc) terminate at DNOVS 1, as do two forms of ascending neurons derived from thoracic ganglia. A contralateral neuron, with terminals in the opposite lobula plate, arises at the DNOVS cluster and is thought to provide heterolateral interaction between the VS4–9 output of one side to the VS4–9 dendrites of the other. DNOVS 2 and 3 extend through pro-, meso-, and metathoracic ganglia, branching ipsilaterally within their tract and into the inner margin of leg motor neuropil of each ganglion. DNOVS 1 terminates as a stubby ending in the dorsal prothoracic ganglion onto the main dendritic trunks of neck muscle motor neurons. Convergence of VS and ocellar interneurons to DNOVS 1 comprises a second pathway from the visual system to the neck motor, the other being carried by motor neurons arising in the brain. Their significance for saccadic head movement and the stabilization of the retinal image is discussed.     

Quantitative fluorescence studies of the effects of catecholamines and hydrocortisone on endogenous amine levels in neurones and small intensely fluorescent cells of embryonic chick sympathetic ganglia in vivo and in vitro

Summary Chick embryo lumbar sympathetic ganglia (11 day) cultured for three days and uncultured (in vivo) ganglia of comparable age were freeze-dried and processed by the formaldehyde-induced fluorescence technique for the demonstration of biogenic monoamines. The catecholamine levels within principal neurone cell bodies and small intensely fluorescent (SIF) cells were then examined in plastic sections of the in vivo and in vitro ganglia by a quantitative fluorescence method under various experimental conditions. Culture of ganglia for three days in the presence of hydrocortisone acetate (10g/ml) resulted in an increased SIF cell fluorescence (P<0.001 compared to control) and a green to yellow colour shift in the fluorophore of SIF cells. No detectable alteration in the fluorescence level of neurones was observed. When neurones after three days in culture were incubated for 1 h in exogenous catecholamines, a significant increase in fluorescence levels (interpreted as an increase in catecholamine content) occurred with noradrenaline (2×10^–6 M; 2×10^–5 M). SIF cells in ganglia removed directly from 14-day old chicks similarly took up noradrenaline and dopamine, and also adrenaline (2×10^–5 M). Morphological results are presented which indicate that the cellular appearances and architecture of cultured ganglion explants are very similar to those in comparable ganglia in vivo.This work was supported by a grant from the Medical Research Council. We thank Mrs. G. O'Shea, Mr. T.T. Lee and Mr. P.F. Hire for their valuable technical assistance