Autoradiographic localization of acetylcholine receptors in the Schwann cell membrane of the squid nerve fiber

Intact and slit nerve fibers of the squid Sepioteuthis sepioidea were incubated in a 50-nM solution of [125I] alpha-bungarotoxin in artificial seawater, in the absence and in the presence of D- tubocurarine (10(-4) M). The distribution of the radioactive label was then determined by electron microscope autoradiography. It was found that, in the fibers exposed solely to the radioactive toxin, the label was located mainly at the axon-Schwann cell boundary in the intact nerve fibers or at the axonal edge of the Schwann cell layer in the axon-free nerve fiber sheaths. Label was also present in those regions of the Schwann cell layer rich in intercellular channels. No signs of radioactivity were observed in the nerve fibers exposed to the labeled toxin in the presence of D-tubocurarine. These results indicate that the acetycholine receptors previously found in the Schwann cell plasma membrane are mainly located over the cell surfaces facing the neighboring axon and the adjacent Schwann cells. These findings represent a further advance in the understanding of the relationship between the axon and its satellite Schwann cell.     

Acetylcholine Synthesis in the Schwann Cell and Axon in the Giant Nerve Fiber of the Squid

Abstract: Acetyltransferase enzymatic activity was detected and measured in homogenates obtained from intact nerve fibers and their separate cellular components, in the tropical squid Sepioteuthis sepioidea. The levels of acetylcholine synthesis were determined in pooled samples of whole stellar nerve, intact giant nerve fiber, extruded axoplasm, axoplasm-free giant nerve fiber sheaths, and small nerve fibers. The values found per mg of protein for the axoplasm-free sheaths are about 3–9 times those of the extruded axoplasm, and comparable to those found for the intact giant nerve fiber. These experimental findings settle the question of whether the Schwann cells of the giant nerve fiber of S. sepioidea , under physiological conditions, contain acetyltransferase activity and are able to synthesize acetylcholine.     

ACETYLCHOLINE CONTENT OF THE SCHWANN CELL AND AXON IN THE GIANT NERVE FIBRE OF THE SQUID

Abstract— Acetylcholine and choline were identified and their concentrations measured, by means of gas chromatography/mass spectrometry, in extracts obtained from nerve fibers of the hindmost stellar nerve of the squid Sepioteuthis sepioidea. These compounds were quantitated in samples of stellar nerve devoid of giant fiber, intact giant nerve fiber, extruded axoplasm, and axoplasm-free giant nerve fiber sheaths. In 11 samples of stellar nerve devoid of giant fiber, weighing an average of 20.8 ± 2.3 mg ( s.e.m. ), 756 ± 91 pmol ACh and 8.65 ± 0.62 nmol of choline were found. The total ACh content of the largest fibre in this group (10 μ m in diameter), for a 5 cm length of nerve, is in the order of 0.16 pmol. The average wet weights of a single giant nerve fiber (270-420 μ m in diameter) and its separate components ( s.e.m .; in mg; number of fibers in parentheses) were: intact fiber, 4.58 ± 0.19 (25); extruded axoplasm, 3.38 ± 0.13 (20); sheaths, 1.21 ± 0.11 (16). The average ACh content per unit weight of sample was about 2-3 times higher in the sheaths (5-13 pmol-mg⁻¹) than in the axoplasm (2-4 pmol mg⁻¹), whereas the ACh concentrations estimated per unit volume of cellular water were about 40 times higher in the Schwann cell (107-222 μ m ) than in the axon (2-5 μ m ). These experimental findings establish the presence of ACh in the giant nerve fiber of S. sepioidea. They also indicate the Schwann cells themselves as the main source for the release of ACh, responsible for their long-lasting hyperpolarizations following the conduction of nerve impulse trains by the axon.     

Some observations on the fine structure of the giant nerve fibers of the earthworm,Eisenia foetida

Sectioned dorsal giant fibers of the earthworm Eisenia foetida have been studied with the electron microscope. The giant axon is surrounded by a Schwannian sheath in which the lamellae are arranged spirally. They can be traced from the outer surface of the Schwann cell to the axon-Schwann membranes. Irregularities in the spiral arrangement are frequently observed. Desmosome-like attachment areas occur on the giant fiber nerve sheath. These structures appear to be arranged bilaterally in columns which are oriented slightly obliquely to the long axis of the giant fiber and aligned linearly from the axon to the periphery of the sheath. At these sites they bind together apposing portions of Schwann cell membrane comprising the sheath. Longitudinal or oblique sections of the nerve sheath attachment areas are reminiscent of the Schmidt-Lantermann clefts of vertebrate peripheral nerve. Septa of the giant fibers have been examined. They are symmetrical or non-polarized and consist of the two plasma membranes of adjacent nerve units. Characteristic vesicular and tubular structures are associated with both cytoplasmic surfaces of these septa.     

Some Observations on the Fine Structure of the Giant Nerve Fibers of the Earthworm, Eisenia foetida

Sectioned dorsal giant fibers of the earthworm Eisenia foetida have been studied with the electron microscope. The giant axon is surrounded by a Schwannian sheath in which the lamellae are arranged spirally. They can be traced from the outer surface of the Schwann cell to the axon-Schwann membranes. Irregularities in the spiral arrangement are frequently observed. Desmosome-like attachment areas occur on the giant fiber nerve sheath. These structures appear to be arranged bilaterally in columns which are oriented slightly obliquely to the long axis of the giant fiber and aligned linearly from the axon to the periphery of the sheath. At these sites they bind together apposing portions of Schwann cell membrane comprising the sheath. Longitudinal or oblique sections of the nerve sheath attachment areas are reminiscent of the Schmidt-Lantermann clefts of vertebrate peripheral nerve. Septa of the giant fibers have been examined. They are symmetrical or non-polarized and consist of the two plasma membranes of adjacent nerve units. Characteristic vesicular and tubular structures are associated with both cytoplasmic surfaces of these septa.     

Modulation of the membrane potential of the Schwann cell of the squid giant nerve fiber

1. The involvement of second messengers and of other chemical mediators, in the modulation of the membrane potential of the Schwann cell of the giant nerve fiber of the Tropical squid Sepioteuthis sepioidea is described. 2. The involvement of the cyclic nucleotide adenosine 3', 5' monophosphate (cAMP) in mediating the actions of the nicotinic Ach receptors of the Schwann cells is suggested. 3. The presence of octopaminergic receptors in the Schwann cells, mediating their actions through the activation of adenylate cyclase, is also described. 3. Receptors for vasoactive intestinal peptide (VIP) are also present on the Schwann cells, and their actions are mediated via a second messenger system that does not involve the activation of adenylate cyclase. 5. The three independent receptor systems referred above are able to interact in a complex way, which involves both their direct actions on the Schwann cell membrane potential and modulatory effects between the systems.     

The rate of action of tetrodotoxin on sodium conductance in the squid giant axon.

When tetrodotoxin is applied to or washed away from the squid giant axon, the rates at which the sodium conductatnce is blocked and unblocked are an order of magnitude smaller than those reported for the isolated node of Ranvier. This slowing is to be expected if in squid the tetrodotoxin binding sites act as a saturable sink in series with the barrier to free diffusion imposed by the presence of the Schwann cell. A comparison has been made between the rates observed experimentally and those calculated for a computer model of the system, in order to estimate the apparent density in the membrane of both specific and non-specific tetrodotoxin binding sites. The figure thus obtained for the number of sodium channels in the squid giant axon, several hundred per square micrometre, agrees well with those derived from other lines of argument.     

Grayanotoxin opens Na channels from inside the squid axonal membrane.

External application of alpha-dihydro-grayanotoxin II (alpha-H2-GTX II) to squid giant axon under nonperfused condition caused substantial membrane depolarization. Intracellular perfusion of the fibers retarded this depolarization appreciably. Tritium-labeled alpha-dihydro-grayanotoxin II ([3H]alpha-H2-GTX II) in the external medium can permeate through the cell membrane, but permeation of alpha-H2-GTX II does not occur either with the carrier-mediated system or through the pores of the Na channel. The finding that the most hydrophilic grayanotoxin analogue, desacyl asebotoxin VII, is effective only when applied internally, strongly suggests that the receptor for grayanotoxin does not exist on the external surface of the membrane. The linear relationship between the concentration of [3H]alpha-H2-GTX II in the external medium and the count in the effluent from the perfused axon indicates that GTX II diffuses through the cell membrane's lipid phase and reaches the site of action only approached from the internal medium.     

Transport of electrolytes in muscle

Summary The muscle fiber stands alongside the red blood cell and the giant axon as one of the three classical cell types that have had major application in investigating ion transport processes in cell membranes. Of these three cell types, the muscle fiber was the first to provide definite evidence for a sodium pump. The ability of the sodium pump to produce an electrical potential difference across the cell membrane was also first demonstrated in muscle fibers. This important property of the sodium pump is now known to have physiological significance in many other types of cells.In this review, electrolyte transport investigations in skeletal muscle are traced from their inception to the current state of the field. Applications of major research techniques are discussed and key results are summarized. An overview of electrolyte transport in muscle, this article emphasizes relationships between the muscle fiber membrane potential and ionic transport processes.     

A study of the crustacean axon repetitive response. 3. A comparison of the effect of veratrine sulfate solution and potassium-rich solutions

The crustacean single nerve fiber gives rise to trains of impulses during a prolonged depolarizing stimulus. It is well known that the alkaloid veratrine itself causes a prolonged depolarization; and consequently it was of interest to investigate the effect of this chemically produced depolarization on repetitive firing in the single axon and compare it with the effect of depolarization by an applied stimulating current or by a potassium-rich solution. It was found that veratrine depolarization, though similar in some respects to a potassium-rich depolarization of depolarizing current effect, was in many respects quite different. (1) At low veratrine concentration, less than 1 Mg%, the negative after potential following a spike action potential was prolonged and augmented. At higher concentrations or after a long period of time, veratrine caused a prolonged steady state depolarization of the membrane, the “veratrine response”. The prolonged plateau depolarization response could be elicited with or without an action potential spike by a short or long duration stimulating pulse, but only if the veratrine depolarization was prevented or offset by an applied conditioning hyperpolarizing inward current. (2) The “veratrine response” resembled the potassium-rich solution response in the plateau-like contour of the depolarization and the very low membrane resistance during this plateau phase. Like the potassium response, it was possible to obtain a typical hyperpolarizing response with an inwardly directed current pulse if applied during the plateau phase. During the negative after potential augmented with veratrine, however, this hyperpolarizing response was not observed. (3) In contrast to the potassium response, however, the “veratrine response” is intimately associated with the sodium concentration in the external medium. The depolarization in millivolts is linearly related to the log of the concentration of external sodium. Moreover, during veratrine action there is a continuous and progressive inactivation of the sodium mechanism which ultimately terminates repetitive firing and abolishes the spike action potential. Then even with conditioning hyperpolarization only the slow response may be elicited in veratrine, occasionally with a spike superimposed if sodium is present, but without repetitive firing. (4) It is concluded that veratrine action is the result of a chemical or metabolic reaction by the alkaloid in the membrane. It is suggested that veratrine may inhibit the sodium extrusion mechanism, or may itself compete for sites in the membrane with calcium and/or sodium. This explains the inhibiting effect of high calcium, the abolition of the “veratrine response” with low temperature and high calcium combined and the progressive inactivation of the sodium system.     

Effects of batrachotoxin on membrane potential and conductance of squid giant axons

The effects of batrachotoxin (BTX) on the membrane potential and conductances of squid giant axons have been studied by means of intracellular microelectrode recording, internal perfusion, and voltage clamp techniques. BTX (550–1100 nM) caused a marked and irreversible depolarization of the nerve membrane, the membrane potential being eventually reversed in polarity by as much as 15 mv. The depolarization progressed more rapidly with internal application than with external application of BTX to the axon. External application of tetrodotoxin (1000 nM) completely restored the BTX depolarization. Removal or drastic reduction of external sodium caused a hyperpolarization of the BTX-poisoned membrane. However, no change in the resting membrane potential occurred when BTX was applied in the absence of sodium ions in both external and internal phases. These observations demonstrate that BTX specifically increases the resting sodium permeability of the squid axon membrane. Despite such an increase in resting sodium permeability, the BTX-poisoned membrane was still capable of undergoing a large sodium permeability increase of normal magnitude upon depolarizing stimulation provided that the membrane potential was brought back to the original or higher level. The possibility that a single sodium channel is operative for both the resting sodium, permeability and the sodium permeability increase upon stimulation is discussed.     

The coupling of Na,K-ATPase with the sodium channels in the plasma membranes of the brain synaptosomes of rats]

Effect of neurotoxins veratrine (100 micrograms/ml) and tetrodotoxin (1 microM) on the binding of 3H-ouabain (10(-8) M) with Na,K-ATPase of intact synaptosomes and isolated synaptic membranes was studied. The persistent opening of sodium channels in synaptosomes by veratrine results in an increase of specific binding of the labeled ligand by 20%. A similar effect was caused by Na/H exchanger monensin. Destruction of microtubules with vinblastine and colchicine has no influence on veratrine action, while depolymerization of microfilaments with cytochalasin B reverses the neurotoxin effect. In isolated synaptic membranes veratrine and tetrodotoxin stimulate ouabain binding, the absolute veratrine-induced increment being several times higher in the presence of ATP than in its absence. Since the closed vesicles of any type are not permeable to ATP and ouabain, it means that in the isolated membranes an interaction between sodium channels and Na,K-ATPase molecules takes place. In intact nerve endings such a mechanism may be operative along with the known ways of control of sodium pump and its ouabain-binding site.     

Diffusion Models for the Squid Axon Schwann Cell Layer

The Schwann cell, basement membrane, and connective tissue layers that surround the squid giant axon and constitute barriers to diffusion, were modeled in a number of ways to analyze various experimental results. The experiments considered are (a) the time-course of the potassium concentration in the space between the Schwann cell and the axon membrane (from now on referred to as the F-H space) after an initial loading, (b) the time-course of sodium concentration in the F-H space after a sudden change in the sodium concentration in the external fluid; (c) the time-course of the concentration of tetrodotoxin (TTX) or saxitoxin (STX) in the F-H space after a sudden change in external concentration, including (or not) the effects of specific binding of TTX or STX to sites on the axon membrane and nonsaturable binding to sites in the F-H space or in the spaces (clefts) between Schwann cells; (d) the effects of the F-H space, clefts, and diffusion into the clefts from the outside (from now on referred to as convergence into the clefts) on the measured series resistance.

The analysis shows that (1) in no case is it necessary to include the effects of the convergence into the clefts from the outside; (2) in case a, the basement membrane, connective tissue layers, and the unstirred layer may be neglected, i.e., the clefts are rate limiting; (3) in case b the clefts may be neglected, i.e., the unstirred layer is rate limiting; (4) in most cases the clefts may be replaced by an equivalent thin diffusion barrier.

     

Histoenzymological and ultrastructural changes in lateral muscle fibers of Oreochromis niloticus (Teleostei: Cichlidae) after local injection of veratrine

The effects of veratrine have been investigated in mammalian, amphibian, and crustacean muscle, but not in fish. In this work, the action of veratrine was studied in the lateral muscle of the freshwater teleost Oreochromis niloticus after intramuscular injection. Histoenzymological typing and electron microscopy of muscle fibers before and 15, 30, and 60 min after veratrine injection (10 ng/kg fish) were used to indirectly assess the morphological changes and the oxidative and m-ATPase activities. In some cases, muscles were pretreated with tetrodotoxin to determine whether the ultrastructural changes were the result of Na(+) channel activation by veratrine. Veratrine altered the metabolism of fibers mainly after 30 min. Oxidative fibers showed decreased NADH-TR activity, whereas that of glycolytic and oxidative-glycolytic type fibers increased. There was no change in the m-ATPase activity of the three fiber types, except at 60 min postveratrine, when a novel fiber type, which showed no reversal after acidic and alkaline preincubations, appeared. Ultrastructural damage involved sarcomeres, myofibrils, and mitochondria, but the T-tubules remained intact. Pretreatment with tetrodotoxin (1 ng/ml) prevented the ultrastructural changes caused by veratrine. These results show that in fish skeletal muscle veratrine produces some effects that are not seen in mammalian muscle.     

Transport of electrolytes in muscle

The muscle fiber stands alongside the red blood cell and the giant axon as one of the three classical cell types that have had major application in investigating ion transport processes in cell membranes. Of these three cell types, the muscle fiber was the first to provide definite evidence for a sodium pump. The ability of the sodium pump to produce an electrical potential difference across the cell membrane was also first demonstrated in muscle fibers. This important property of the sodium pump is now known to have physiological significance in many other types of cells. In this review, electrolyte transport investigations in skeletal muscle are traced from their inception to the current state of the field. Applications of major research techniques are discussed and key results are summarized. An overview of electrolyte transport in muscle, this article emphasizes relationships between the muscle fiber membrane potential and ionic transport processes.     

The pH Dependency of Relative Ion Permeabilities in the Crayfish Giant Axon

The dependence of the membrane potential on potassium, chloride, and sodium ions, was determined at the pH's of 6.0, 7.5, and 9.0 for the resting and depolarized crayfish ventral nerve cord giant axon. In normal saline (external potassium = 5.4 mM), the dependence of the membrane potential on the external potassium ions decreased with lowered pH while that for chloride increased. In contrast, in the potassium depolarized axon (external potassium = 25 mM), the dependence of the membrane potential on external potassium was minimum around pH 7.5 and increased in either more acidic or basic pH. In addition, the dependence of the membrane potential on external chloride in the depolarized axon was maximum at pH 7.5 and decreased in either more acidic or basic pH. The sodium dependency of the membrane potential was small and relatively unaffected by pH or depolarization. The data are interpreted as indicating a reversible surface membrane protein-phospholipid conformation change which occurs in the transition from the resting to the depolarized axon.     

Rate of Movement and Composition of Rapidly Transported Proteins in Regenerating Olfactory Nerve

In a previous study, three successive groups of regenerative fibers, growing initially at 5.8, 2.1, and 0.8 mm/day, were observed in the regenerating garfish olfactory nerve. In the present study, fast axonal transport in the most rapidly regenerating axons (phase I and II) has been examined. Rapid transport in phase I fibers occurs at a velocity of 208 +/- 9 mm/day at 23 degrees, a rate identical to that measured in intact nerves. This first phase of regenerating fibers represents only 3 to 5% of the original axonal population, but each fiber appears to contain 6 to 16 times more transported radioactivity than an axon in an intact nerve. Subcellular distribution of rapidly moving material in phase I and II fibers was closely related to the distribution obtained in intact nerves. Small but significant differences indicate a shift of the transported radioactivity from a heavier to a light axonal membranous fraction. This shift might be characteristic of the immature membrane of a growing axon. The polypeptide distribution of transported radioactivity was also very similar to that of a normal nerve, with most of the radioactivity associated with high-molecular-weight polypeptides.     

Electrical activity and structural correlates of giant nerve fibers in Kuruma shrimp (Penaeus japonicus)

Morphology and recordings of electrical activity of Kuruma shrimp (Penaeus japonicus) giant medullated nerve fibers were carried out. A pair of giant fibers with external diameter of about 120 μ and 10 μ in myelin thickness were found in the ventral nerve cord. The diameter of the axon is about 10 μ. Thus there is a wide gap between the axon and the external myelin sheath. Each axon is doubly coated directly by Schwann cells and indirectly by the myelin sheath layer which is produced by those Schwann cells. Impulse conduction velocities of these giant fibers showed a range between 90–210 m/sec at about 22°C. Large action potentials (up to 113 mV, rise time of 0.16–0.3 msec, maximum rate of rise of 650–1250 V/sec, half decay time of 0.2–0.3 msec, maximum rate of fall of 250–450 V/sec and total duration of less than 1.5 msec) could be obtained by inserting microelectrodes or by longitudinal insertion of 25 μ diameter capillary electrodes into the gap but no DC-potential difference was observed across the myelin sheath. Transmyelin electrical parameters were very favorable for fast impulse conduction: myelin resistance of 3 × 10⁴ Ω cm²; time constant of 0.38 msec; myelin capacitance of 1.35 × 10^?8 F/cm²; gap fluid resistivity of 23 Ω cm. The existence of nodes of Ranvier could not be demonstrated morphologically, but electrophysiological evidence suggests that a type of saltatory conduction occurs in these giant fibers.     

FINE STRUCTURAL ALTERATIONS ASSOCIATED WITH VENOM ACTION ON SQUID GIANT NERVE FIBERS

(1) Block of conduction and marked increase in permeability of the squid giant axon, when surrounded by adhering small nerve fibers, is caused by the venoms of cottonmouth, ringhals, and cobra snakes and by phospholipase A (PhA). This phenomenon is associated with a marked breakdown of the substructure of the Schwann sheath into masses of cytoplasmic globules. Low concentrations of these agents which render the axons sensitive to curare cause less marked changes in the structure of the sheath. (2) Rattlesnake venom, the direct lytic factor obtained from ringhals venom, and hyaluronidase caused few observable changes in structure, correlating with the inability of these agents to increase permeability. (3) Cottonmouth venom did not alter the structure of giant axons freed of all adhering small nerve fibers. This is in agreement with previous evidence that the venom effects are due to an action of lysophosphatides liberated as a result of PhA action. Cetyltrimethylammonium chloride, a cationic detergent, produces effects that resemble those of venom and PhA. (4) The results provide evidence that PhA is the component of the venoms that is responsible for their effects. It also appears that the Schwann cell and possibly the axonal membrane are the major permeability barriers in the squid giant axon.     

Effects of external ions on membrane potentials of a lobster giant axon

The effects of varying external concentrations of normally occurring cations on membrane potentials in the lobster giant axon have been studied and compared with data presently available from the squid giant axon. A decrease in the external concentration of sodium ions causes a reversible reduction in the amplitude of the action potential and its rate of rise. No effect on the resting potential was detected. The changes are of the same order of magnitude, but greater than would be predicted for an ideal sodium electrode. Increase in external potassium causes a decrease in resting potential, and a decrease in potassium causes an increase in potential. The data so obtained are similar to those which have been reported for the squid giant axon, and cannot be exactly fitted to the Goldman constant field equation. Lowering external calcium below 25 mM causes a reduction in resting and action potentials, and the occasional occurrence of repetitive activity. The decrease in action potential is not solely attributable to a decrease in resting potential. Increase of external calcium from 25 to 50 mM causes no change in transmembrane potentials. Variations of external magnesium concentration between zero and 50 mM had no measurable effect on membrane potentials. These studies on membrane potentials do not indicate a clear choice between the use of sea water and Cole's perfusion solution as the better external medium for studies on lobster nerve.