Splenic Nerve Research Articles

The ultrastructure of the plasma membrane and myelin sheath of peripheral nerve fibers after fixation by freeze-drying

The plasma membrane of frozen-dried unmyelinated splenic nerve fibers appears as a triple-layered asymmetric structure. This pattern is clearly seen after embedding in Vestopal W and section staining with either uranyl acetate or lead hydroxide. The cell boundaries between the axons and the Schwann cells usually have a five-layered appearance due to a close packing of the two adjacent plasma membranes and a fusion of the peripheral membrane components. Sometimes a light interspace of varying width and containing stainable material separates the plasma membranes, giving rise to a seven-layered membrane pattern. Also in the myelinated nerve fibers a triple-layered asymmetric pattern of the plasma membrane is seen after freeze-drying fixation, Vestopal embedding, and section staining. In the mesaxons, as well as in the cell boundary between the axon and the Schwann cell of the internode, a five-layered structure is usually observed. The layering of the myelin sheath appears after freeze-drying in principle similar to the one procured after chemical fixation.

Catecholamine Release and Uptake in Isolated Adrenergic Nerve Granules

AbstractIsolated storage granules from bovine splenic nerves, incubated in neutral isotonic K‐phosphate give off noradrenaline at rates rapidly increasing with temperature. Noradrenaline in concentrations of 10—20 μg/ml in the incubation fluid inhibits or prevents the release. The noradrenaline thus retained is released at the normal rate after removal of the amine in the incubation fluid. After depletion, the granules are able to take up noradrenaline and adrenaline to about the original content when exposed to catecholamines in concentrations of 10—20 μg/ml, suggesting a limited storage capacity of the granules. It is suggested that the noradrenaline in the adrenergic nerve terminals is partly present in a free pool available for immediate release during nerve stimulation and that a lowering of the concentration in this pool causes the granules to give off the stored transmitter until an equilibrium is reached. The following resynthesis is assumed to continue until the free pool has reached a concentration preventing further release from the granules. This concentration is about the same as that normally found in bovine splenic nerves.

Adrenergic neurone blockade and other acute effects caused by N-benzyl-N'N"-dimethylguanidine and its ortho-chloro derivative.

N-Benzyl-N'N"-dimethylguanidine sulphate (BW 467C60) and its ortho-chloro derivative (BW 392C60) had adrenergic neurone blocking and sympathomimetic effects resembling those of bretylium and guanethidine in cats, dogs and monkeys, but they were more potent in blocking adrenergic mechanisms in the cat. BW 467C60 was more active than its chloro derivative. Each compound inhibited release of noradrenaline during stimulation of the splenic nerve of cats, and increased smooth muscle responses to adrenaline and noradrenaline. Pressor responses to standard doses of tyramine were also increased except when large doses of BW 467C60 or BW 392C60 were given. The adrenergic neurone block by BW 467C60 was inhibited by dopamine, cocaine and amphetamine in situations in which these amines inhibit the effects of bretylium and guanethidine. In contrast to guanethidine, BW 467C60 and BW 392C60 did not lower the pressor amine content of the iris of cats 24 hr after administration of single doses of the compounds. BW 467C60 depressed the slope of curves relating the frequency of stimuli applied to the cervical sympathetic nerves and the resulting contraction of the nictitating membrane, but the effects of the lower rates of stimulation were preferentially inhibited. Large intravenous doses of BW 467C60 and BW 392C60 blocked autonomic cholinergic mechanisms and caused neuromuscular paralysis of voluntary muscle. These effects were brief, in contrast to the adrenergic neurone blockade. Both BW 467C60 and BW 392C60 were well absorbed from the alimentary tract. In contrast to guanethidine, BW 467C60 did not cause diarrhoea in guinea-pigs.

Electron microscopic studies on the effect of anisotonic solutions on the structure of unmyelinated splenic nerve fibers of the cat

In the first series of experiments unmyelinated splenic nerve fibers were fixed in highly hypotonic and hypertonic osmium tetroxide solutions. Fixation in a solution made up of distilled water produces only small changes in the fibers, observable as a slight swelling of the axons and of some of the axon mitochondria. Fixation in a solution made up of Tyrode's fluid containing 10 times its original content of sodium chloride brings about a shrinkage of axons and Schwann cells, along with compact axon mitochondria. In a second series of experiments nerve fibers were soaked in media with different ionic strengths before fixation with osmium tetroxide. Exposure to distilled water results in a swelling of the Schwann cells and a shrinkage of the axons. In Tyrode's fluid with reduced sodium chloride content, the Schwann cells swell and the axons either swell or shrink. In Tyrode's fluid with increased sodium chloride content, the Schwann cells, as well as many axons, shrink. The axon mitochondria swell in highly hypotonic media and appear compact after exposure to hypertonic media. The pattern of the filamentous material of the axoplasm is sensitive to treatment with anisotonic media. In contrast to the results reported by Robertson (15), the relationship and spacing of the axon and Schwann cell plasma membranes remains unchanged in all these experiments, with the exception of a possible widening of the cell boundaries in highly hypertonic media.

Actions of bretylium and guanethidine on the uptake and release of [3H]-noradrenaline.

The effect of bretylium and guanethidine has been studied on the uptake and the spontaneous and reserpine-induced release of [(3)H]-noradrenaline in the rat heart and in the splenic nerve endings of the cat. Bretylium and guanethidine inhibited the uptake by the heart of circulating [(3)H]-noradrenaline. Bretylium blocked spontaneous and reserpine-induced release of [(3)H]-noradrenaline, while guanethidine caused release and partially antagonized the reserpine-induced release. Both compounds produced a transient liberation of [(3)H]-noradrenaline from splenic nerves, but blocked the release of the [(3)H]-catechol amine following stimulation of the splenic nerve.

Effect of morphine on some sympathetically innervated effectors.

Morphine, in doses of 0.5 to 55 mg/kg, inhibited contraction of the nictitating membrane of the cat following stimulation of the postganglionic sympathetic nerve fibres. Morphine was more effective at low than at high frequencies of stimulation, independently of the size of contraction of the membrane; the speed of contraction was reduced at all frequencies. Cocaine potentiated the contraction of the nictitating membrane following nerve stimulation more at low than at high frequencies, and antagonized the action of morphine. These findings, and the absence of an effect of morphine on the action of injected noradrenaline, make it likely that morphine interferes with the release of noradrenaline from the postganglionic nerve endings in the nictitating membrane. Morphine had no effect on the cardioaccelerator action of the cardiac nerves and inconsistent results were obtained on the emptying of the spleen after stimulation of the splenic nerves.

Electron microscopic investigation of the plasma membrane and myelin sheath of autonomic nerve fibers in the cat

In unmyelinated splenic nerve fibers the plasma membranes of both the axon and the Schwann cell contain an osmiophilic layer which is approximately 50–60 Å thick. It appears more dense in the axolemma than in the plasma membrane of the Schwann cell. After-staining of the sections with uranyl acetate reveals an asymmetric triple-layered pattern in connection with this opaque plasma membrane layer. In myelinated fibers of the sympathetic trunk the axolemma has an osmiophilic layer about 45 Å thick, which is more dense than the corresponding layer of the Schwann cell, which is about 30 Å thick. The opaque layers sometimes exhibit an asymmetric triple-layered structure in uranyl acetate-stained material. Two about 30-Å thick opaque layers of the Schwann cell plasma membrane merge into the major dense layer of the myelin sheath. The light interspace of mesaxons is continuous with the light intermediate space of the myelin sheath. After-staining of the myelin sheath with uranyl acetate reveals intraperiod layers which frequently have a triple-layered substructure.

Effect of reserpine on the release of catecholamines from isolated nerve and chromaffin cell granules.

AbstractIn a suspension of bovine splenic nerve granules, reserpine in concentrations of 10‐8‐10‐5 M strongly inhibits the spontaneous release of noradrenaline. The catecholamine releasing action of tyramine on nerve granules is unaffected by low concentrations of reserpine, but is almost completely blocked by concentrations of 10‐5 M. Reserpine is less active as inhibitor on suprarenal medullary granules from the rabbit and still less on cats' medullary granules. The spontaneous release occurs at a lower rate in rabbit's medullary granules than in those from the cat. No difference in the rate of release was found between adrenaline and noradrenaline from the cat's medullary granules. After administration of dopa 100 mg/kg i.v. in the rabbit the rate of release from the medullary granules was greatly enhanced. The possible action of reserpine as enzyme inhibitor is briefly discussed.

Electron-microscopic investigation of filament structures in unmyelinated fibers of cat splenic nerve

The ultrastructural pattern of the axoplasm in unmyelinated splenic nerve fibers is characterized in great part by morphologically well-differentiated neurofilaments. Some of the filaments are approximately 100 A thick and have a homogeneously opaque structure; others are about 300 A in diameter and of irregular density. Even the Schwann cell contains filaments around 100 A thick and usually disposed in groups contiguous to the plasma membrane. The filament structures appear distinctly in osmium-fixed, Araldite-embedded material that has been after-stained with phosphotungstic acid. The filaments also stain with uranyl acetate.

Improved technique for the fluorimetric estimation of catecholamines.

AbstractEuler U. S. V. and F. Lishajko. Noradrenaline release from isolated nerve granules.Some properties of isolated transmitter granules obtained by high g centrifugation of press juice from bovine splenic nerves have been studied. Of the total noradrenaline present in the press juice 20–35 per cent was recovered in the sediment. The granules are stable for several hours at + 4° C but rapidly lose their noradrenaline on incubation in isotonic neutral media at 37° C. Noradrenaline is rapidly released even at low temperature at pH 4 and below, and by detergents. Hypo‐ and hyperosmotic solutions and freezing and thawing, had a moderate releasing effect. Acetylcholine, nicotine, serotonin and GABA had no detectable effect on the noradrenaline release from the granules. Nerve granules are more sensitive to temperature than adrenal medullary granules but show higher resistance to freezing and thawing and osmotic changes.

Conditions affecting rate of development of anoxic block in C fibers.

The isolated splenic nerve of sheep conducted impulses for an average of 10 minutes in the complete absence of oxygen at 37.5°C. If, immediately following conduction failure, the nerve was allowed to recuperate for 2 hours in oxygen it became able to conduct impulses as long during a repeated period of anoxia as it had initially. If the initial period of anoxia was prolonged beyond 28 minutes the nerve sustained irreversible damage and its ability to conduct impulses during subsequent periods of anoxia was shortened proportionally. For recuperation periods shorter than 2 hours the conduction block during the course of the second period of anoxia appeared earlier. Temperatures below 37.5°C or CO2 concentrations above 10% prolonged the time to conduction block during anoxia; temperatures above 37.5°C or frequencies of stimulation greater than 1/sec. shortened the time to conduction block during anoxia.

Improved Chlorophyll Extraction Method

Direct evidence is given that reserpine, in concentrations of 0.125 to 0.625 mM, effects a release of noradrenaline from a suspension of transmitter granules isolated from bovine splenic nerves.

Effect of Reserpine on Release of Noradrenaline from Transmitter Granules in Adrenergic Nerves

Direct evidence is given that reserpine, in concentrations of 0.125 to 0.625 m M , effects a release of noradrenaline from a suspension of transmitter granules isolated from bovine splenic nerves.

SYMPATHETIC POSTGANGLIONIC CHOLINERGIC FIBRES

When the postganglionic fibres to the nictitating membrane are stimulated in a cat treated with reserpine, the membrane contracts. The contraction is increased after the injection of eserine and is abolished with atropine. Thus the fibres stimulated appear to be cholinergic. When the splenic nerve is stimulated in a cat treated with reserpine there is similar evidence of the presence of cholinergic fibres; the spleen dilates, the dilatation is greater in the presence of eserine and is abolished by atropine. The hypogastric nerves when stimulated cause inhibition of the uterus of the virgin cat. When the cat is treated with reserpine, stimulation then causes contraction which is increased in the presence of eserine and abolished by atropine. There is also evidence of cholinergic fibres in the postganglionic supply to the vessels of the rabbit's ear. From this and other evidence it is suggested that a form of adrenergic mechanism may exist such that cholinergic fibres in the postganglionic sympathetic supply liberate acetylcholine; this in turn liberates noradrenaline from a store around the sympathetic nerve ending.

Autonomic stimulation and blockade on canine splenic inflow, outflow and weight.

Blood flow (autoperfusion-electromagnetic meters), pressures and splenic weight (strain gauges) were recorded in barbitalized dogs. Splenic nerve stimulation and epinephrine decreased inflow, increased outflow and pressure and diminished splenic weight; during recovery inflow exceeded outflow, and splenic weight returned to control. Progressively increasing adrenergic blockade partially reversed the inflow reduction (vasoconstriction) but only decreased the outflow surge and weight reduction. Cholinergic substances increased inflow, outflow and splenic weight; atropine abolished these without significant effect on adrenergic or splenic nerve responses. No reactive vasodilation followed interruption of inflow. Conclusions: a) metabolic dilator receptors are absent; b) afferent arterioles possess innervated α adrenergic constrictor receptors and noninnervated ß adrenergic and γ cholinergic dilator receptors; c) an innervated ‘emptying mechanism’ exists which may: 1) involve efferent venular relaxation and/or contraction of smooth muscle in the capsule or substance of the spleen, 2) have neither α receptors (arterenol is minimally effective and the response is resistant to adrenergic blockage) nor ß receptors (isoproterenol is ineffective) and 3) have epinephrine as its neuromediator.

Mechanism of splenic contraction produced by severe hypercapnia.

Unilaterally adrenalectomized and bilaterally adrenalectomized mongrel dogs with both normal and denervated spleen were made hypercapnic by inhalation of 30% CO2 in oxygen in order to determine the mechanism of the resulting splenic contraction. The results obtained indicate that splenic contraction in dogs breathing 30% CO2 is mediated by both the splenic nerves and by the adrenal glands. The nerves appear to act more rapidly but contraction still occurs in the absence of all innervation. Neither bilateral adrenalectomy nor splenic denervation alone completely blocked the splenic response in all animals, but of the two procedures the former appeared to be much more effective in this respect. In order to completely block splenic response to inhalation of 30% CO2, both denervation of the spleen and bilateral adrenalectomy were necessary.

The ultrastructure of unmyelinated fibers in the splenic nerve of the cat

The unmyelinated fibers in the splenic nerve of the cat are usually enveloped by the Schwann cell plasma membrane. Sometimes they have a small surface in contact with the basement membrane. The axolemma and plasma membrane of the Schwann cell contain an opaque layer with a thickness of about 60 Å. Here and there the membrane is triple-layered and has a total thickness of approximately 75 Å. Between the membranes of the axon and of the cell there is a lighter interspace around 90 Å thick. The axoplasm contains neurofibrils approximately 60 Å in diameter, long slender mitochondria about 0.1 μ thick and vesicular structures about 450 Å in diameter. Intra-axonal granular structures are present. These granules have a diameter of about 0.28 μ and are of somewhat varying appearance. The Schwann sheath consists of cells linked in series. The sheath is separated from the connective tissue space by a basement membrane about 450 Å thick. The nucleus of the Schwann cell is enclosed by a double membrane. There appears to be no specific nucleolar structure. The ground cytoplasm exhibits no structures specific for the Schwann cell. No equivalents of the Golgi apparatus were observed. Mitochondria about 0.18 μ thick and of varying length are relatively sparse in the cell. The outer and inner membranes are triple-layered and show the conventional arrangement. The cytoplasm contains granular structures varying in diameter between 0.2 and 1.1 μ.

�ber den Noradrenalin- und ATP-Gehalt sympathischer Nerven

1. Splenic nerves of cattle have been found to contain 3–5 γ of noradrenaline and about 40 γ of ATP per 1 g. of fresh tissue. The molar ratio noradrenaline/ATP was 0,38. 2. Homogenates of splenic nerves were centrifuged at 80 × g. for 15 min. The supernatant contained 1,85 γ of noradrenaline per 1 g. of original tissue. 3. When the supernatant was centrifuged at high speed (12 000 × g.) for 15 min. a sediment of submicroscopical granules was obtained which contained nearly 30% of the noradrenaline. 4. The amounts of 0,43 γ noradrenaline and 0,25 γ of ATP found in these submicroscopical granules (mean of 4 experiments) give a molar ratio noradrenaline/ATP of 5,2. This ratio agrees with the ratio of 5,0 found in chromaffine granules of the adrenal medulla of cattle by Falck et al. and of chicken and rats in my own earlier experiments. 5. The role of ATP in the storage of catechol aminesj in granular cell constituents is discussed.

The blocking action of choline 2:6-xylyl ether bromide on adrenergic nerves.

Choline 2:6-xylyl ether bromide (TM 10), given systemically to cats in doses of 5 to 15 mg./kg., abolishes the effects of adrenergic nerve stimulation whilst leaving the reactions of the effector organs to adrenaline unimpaired. The effects of a single dose may take up to one hour to become fully established and last for more than twenty-four hours. Apart from transitory ganglionic blockade, cholinergic autonomic nerves are unaffected even by large doses of TM 10. Doses of TM 10 which produce effective blockade do not impair conduction along adrenergic nerve trunks; the drug must, therefore, act at, or close to, the nerve terminals. TM 10 prevents the output of noradrenaline from the spleen on stimulating the splenic nerves; but, in acute experiments, it does not influence the liberation of pressor amines from the stimulated suprarenals. Examination of some ethers related to TM 10 revealed no correlation between TM 10-like adrenergic blocking activity and local anaesthetic activity. The action of TM 10 on adrenergic nerves does not, therefore, seem to be accounted for by axonal block.

The reflex production of antibodies caused by the injection of antigens into the isolated spleen

Various antigens were injected into a spleen which had been isolated from circulation. Isolation was effected by separating the splenic nerve and tying all the underlying blood vessels and tissues. The degree of splenic isolation was tested by introducing P32 into the spleen and subsequently determining its presence in the blood. When the isolated spleen was removed five minutes after being injected with the antigen, the antigen injection brought about the formation of specific antibodies, although no resorption occurred. When no antigen was injected into the spleen, surgical intervention did not change the antibody titer.