Quantal potential fields around individual active zones of amphibian motor-nerve terminals

The release of a quantum from a nerve terminal is accompanied by the flow of extracellular current, which creates a field around the site of transmitter action. We provide a solution for the extent of this field for the case of a quantum released from a site on an amphibian motor-nerve terminal branch onto the receptor patch of a muscle fiber and compare this with measurements of the field using three extracellular electrodes. Numerical solution of the equations for the quantal potential field in cylindrical coordinates show that the density of the field at the peak of the quantal current gives rise to a peak extracellular potential, which declines approximately as the inverse of the distance from the source at distances greater than about 4 microm from the source along the length of the fiber. The peak extracellular potential declines to 20% of its initial value in a distance of about 6 microm, both along the length of the fiber and in the circumferential direction around the fiber. Simultaneous recordings of quantal potential fields, made with three electrodes placed in a line at right angles to an FM1-43 visualized branch, gave determinations of the field strengths in accord with the numerical solutions. In addition, the three electrodes were placed so as to straddle the visualized release sites of a branch. The positions of these sites were correctly predicted on the basis of the theory and independently ascertained by FM1-43 staining of the sites. It is concluded that quantal potential fields at the neuromuscular junction that can be measured with available recording techniques are restricted to regions within about 10 microm of the release site.     

Somatostatin-like immunoreactivity in human sympathetic ganglia

Summary The localization of somatostatin-like immunore-activity (SOM-LI) was examined in human lumbar sympathetic ganglia using the peroxidase-antiperoxidase method. Few of the principal neurons showed immunolabelling for somatostatin and sparse networks of nerve terminals were unevenly associated with ganglion cells. Using light microscopy, the most intense SOM-LI was seen in the perinuclear zone of the neurons. Electron-microscopically, the staining was localized on the membranes of the Golgi apparatuses. In the nerve terminals, SOM-LI was seen inside the small vesicles (40–60 nm diameter). All neurons with SOM-LI were also found to be tyrosine-hydroxylase immunoreactive when excamined with a double-staining technique. These results provide evidence that somatostatin and noradrenaline co-exist in human sympathetic neurons.     

Peptidergic synaptic transmission in sympathetic ganglia

Biologically active peptides have been localized in neuronal cell bodies, axons, and synaptic boutons of sympathetic ganglia; some of these peptides may be neurotransmitters. For example, substances immunologically similar to substance P and luteinizing hormone-releasing hormone appear to be released from nerve terminals in sympathetic ganglia. In each case, the postsynaptic action of the peptide lasts for several minutes and is accompanied by a combination of decreases and increases in the membrane conductance that are voltage dependent. These peripheral peptidergic synapses may be models for peptidergic transmission in the central nervous system where detailed analysis is more difficult.     

Amphibian sympathetic ganglia in tissue culture

1. A culture medium has been developed for amphibian sympathetic nervous tissue but it is suggested that the ionic values should be adjusted to correspond to the concentrations of salts in the plasma of particular species. 2. The morphology, monoamine fluorescence, growth and differentiation of sympathetic ganglia of the frog, Limnodynastes dumerili, have been studied in culture. 3. Two types of neuron could be distinguished largely according to size, namely small, 18 X 20 mum and large, 38 X 42 mum. The possibility that these represent one type at different stages in development or represent functionally distinct neurons is discussed. 4. The sympathetic neurons are extremely sensitive to nerve growth factor (NGF) which caused an increase in the size of the cell bodies, the number of nerve fibres regenerating, the rate of axonal growth and synthesis of catecholamines. 5. Various other cell types appearing in the cultures have been described, including chromaffin, satellite, Schwann, multipolar and epithelial cells as well as fibroblasts, melanocytes and macrophages. The epithelial cells show slow contractions and changes in shape.     

Synaptic transmission at visualized sympathetic boutons: stochastic interaction between acetylcholine and its receptors.

Excitatory postsynaptic currents (EPSCs) were recorded with loose patch electrodes placed over visualized boutons on the surface of rat pelvic ganglion cells. At 34 degrees C the time to peak of the EPSC was about 0.7 ms, and a single exponential described the declining phase with a time constant of about 4.0 ms; these times were not correlated with changes in the amplitude of the EPSC. The amplitude-frequency histogram of the EPSC at individual boutons was well described by a single Gaussian-distribution that possessed a variance similar to that of the electrical noise. Nonstationary fluctuation analysis of the EPSCs at a bouton indicated that about 120 ACh receptor channels were available beneath boutons for interaction with a quantum of ACh. The characteristics of these EPSCs were compared with the results of Monte Carlo simulations of the quantal release of 9000 acetylcholine (ACh) molecules onto receptor patches of density 1400 microns-2 and 0.41 micron diameter, using a kinetic scheme of interaction between ACh and the receptors similar to that observed at the neuromuscular junction. The simulated EPSC generated in this way had temporal characteristics similar to those of the experimental EPSC when either the diffusion of the ACh is slowed or allowance is made for a finite period of transmitter release from the bouton. The amplitude of the simulated EPSC then exhibited stochastic fluctuations similar to those of the experimental EPSC.     

Membrane responses of neurons in human sympathetic ganglia

Electrophysiological studies were performed on in vitro slice preparations of sympathetic ganglia excised from peripherally perfused, brain-dead human donors. The intracellular recordings in 16 neurons showed resting potentials and input resistances mostly in the ranges reported for sympathetic neurons in other mammals. The high input resistances (approximately 29 M omega) can account for the long membrane time constants measured in three neurons (means = 13.9 ms). Spikes that were part of anodal break responses as well as those evoked by current pulse injections were tetrodotoxin sensitive and were more prolonged in duration by tetraethylammonium than by 4-aminopyridine applications. Administrations of isoflurane (0.5-2 minimum alveolar concentrations) by perfusion did not greatly affect the membrane properties, but produced a marked reduction in repetitive spike firing evoked by current pulse injections as well as in the postspike afterhyperpolarizations, suggesting that a sympathetic neurogenic mechanism may contribute to the hypotension observed clinically during isoflurane anaesthesia. These investigations demonstrate for the first time that human sympathetic ganglion neurons can be studied successfully in in vitro preparations, and hence are valuable for direct relevance to the human condition.     

Analysis of hyposmolarity-induced taurine efflux pathways in the bullfrog sympathetic ganglia.

Hyposmolarity-induced taurine release was dependent on the decrease in medium osmolarity (5-50%) in the satellite glial cells of the bullfrog sympathetic ganglia. Release of GABA induced by hyposmolarity was much less than that of taurine. Omission of external Cl- replaced with gluconate totally suppressed taurine release, but only slightly suppressed GABA release. Bumetanide and furosemide, blockers of the Na+/K+/2Cl- cotransport system, inhibited taurine release by about 40%. Removal of external Na+ by replacement with choline, or omission of K+, suppressed taurine release by 40%. Antagonists of the Cl-/HCO3 exchange system, SITS, DIDS and niflumic acid, significantly reduced taurine release. The carbonic anhydrase inhibitor, acetazolamide, reduced the taurine release by 34%. Omission of external HCO3 by replacement with HEPES caused a 40% increase in the hyposmolarity-induced taurine release. Hyposmolarity-induced GABA release was not affected by bumetanide or SITS. Chloride channel blockers, 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB) and N-phenylanthranilic acid (DPC), practically abolished taurine release. Blockers of K+ channels, clofilium and quinidine, had no effect on the taurine release. The hyposmolarity-induced taurine release was considerably enhanced by a simultaneous increase in external K+. GABA was not mediated by the same transport pathway as that of taurine. These results indicate that Cl- channels may be responsible for the hyposmolarity-induced taurine release, and that Na+/K+/2Cl- cotransporter and Cl-/HCO3 exchanger may contribute to maintain the intracellular Cl- levels higher than those predicted for a passive thermodynamic distribution in the hyposmolarity-induced taurine release.     

The distribution of P2X receptor clusters on individual neurons in sympathetic ganglia and their redistribution on agonist activation

The distribution of P2X receptors on neurons in rat superior cervical ganglia and lability of P2X receptors on exposure to agonists were determined. Antibody labeling of each P2X subtype P2X(1)-P2X(7) showed neurons isolated into culture possessed primarily P2X(2) subunits with others occurring in order P2X(7) > P2X(6) > P2X(3) > P2X(1) > P2X(5) > P2X(4). Application of ATP and alpha,beta-meATP to neurons showed they possessed a predominantly nondesensitizing P2X receptor type insensitive to alpha,beta-meATP, consistent with immunohistochemical observations. P2X(1)-green fluorescent protein (GFP) was used to study the time course of P2X(1) receptor clustering in plasma membranes of neurons and internalization of receptors following prolonged exposure to ATP. At 12-24 h after adenoviral infection, P2X(1)-GFP formed clusters about 1 microm diameter in the neuron membrane. Application of ATP and alpha,beta-meATP showed these neurons possessed a predominantly desensitizing P2X receptor type sensitive to alpha,beta-meATP. Infection converted the major functional P2X receptor type in the membrane to P2X(1). Exposure of infected neurons to alpha,beta-meATP for less than 60 s led to the disappearance of P2X(1)-GFP fluorescence from the cell surface that was blocked by monensin, indicating the chimera is normally endocytosed into these organelles on exposure to agonist.     

A model of selective synapse formation in sympathetic ganglia

In the sympathetic system, neurons from several spinal segments are mapped onto targets in the periphery in a topographically ordered way by means of selective synaptic connections in the superior cervical ganglion. Experimental evidence points to a crucial role for chemoaffinity in establishing this topographic map. Furthermore, rearrangements of synapses after surgical manipulations indicate that this chemoaffinity is not based on rigid “key-and-lock” markers. Our model is used to study how such nonrigid markers may interact with other regulatory factors, including growth-regulating signals and the growth potential of individual nerons. In the model, these latter factors are limiting, so that an increasing number of synaptic contacts decreases the likelihood of further synapse formation. These factors are combined with chemoaffinity using a linear threshold model. The model is robust to parameter changes and reproduces experimental observations with reasonable detail. Simulation results are used to discuss characteristic experimental results, such as the substantial plasticity of the connections seen after partial denervation. A surprisingly small effect of transient hyperinnervation in the model may help explain why final connectivities are similar in two real situations with high and low degrees of transient hyperinnervation (development and adult reinnervation). It is shown that spatial restrictions on post-synaptic neurons (dendrites) may contribute significantly to the segmentally broad innervation of each ganglion cell. Finally, we discuss potential effects of presynaptic neuronal death in systems with a high degree of plasticity. © 1993 John Wiley & Sons, Inc.     

Acetyl- and pseudocholinesterase activities in sympathetic ganglia of rats

—The quantitative method of Ellman , Courtney , Andres and Featherstone (1961) was adapted to a differential assay for the determination of acetyl- and pseudocholinesterase activities of sympathetic ganglia of rats. The activities of the cholinesterases of superior cervical, stellate and thoracic chain ganglia and of the abdominal ganglionic complexes in apposition to the superior mesenteric and coeliac arteries (superior mesenteric, coeliac and cardiac ganglia) were measured. B.W.284C51 dibromide, 5 × 10^?5m , and ethopropazine hydrochloride, 3·15 × 10^?5m , were employed to inhibit selectively acetyl- and pseudocholinesterases, respectively. Linearity was shown to be maintained with enzyme concentrations corresponding to 0·12-0·5 mg of ganglion (wet wt.)/incubation. Under the experimental conditions of this assay, the rates of the reaction of ganglionic acetyl- and pseudocholinesterases were linear for time periods greater than those employed for calculating the rates of hydrolysis in the homogenates of sympathetic ganglia. Several experimental approaches were used to ascertain the specificity of the inhibitors and of the reaction. Of the total cholinesterase activity of sympathetic ganglia of rats, 55-63 per cent was due to acetylcholinesterase and 31-39 per cent to pseudocholinesterase. On the basis of the specific enzyme activity, superior cervical, stellate and superior mesenteric ganglia contained higher acetyl- and pseudocholinesterase activities than did thoracic chain, coeliac and cardiac (abdominal) ganglia. The specific activity of acetylcholinesterase was similar in rat and cat superior cervical ganglia and sympathetic cervical trunks while the pseudocholinesterase activity of these two tissues was somewhat lower in cats than in rats.