Freeze-fracture studies on the giant axon and ensheathing Schwann cells of the squid

Summary The giant axons and encompassing sheaths from the stellar nerves of the squidsSepioteuthis sepioidea andLoligo forbesi have been analysed by freeze-fracture. The axolemma exhibits many intramembranous particles (IMPs) that fracture onto the cytoplasmic membrane half-leaflet (P-face); the larger IMPs may be aggregated into clusters. Axoplasmic subsurface cisternae are found beneath this membrane. Clustered or aligned arrays of P-face IMPs are also found on the membranes of the Schwann cells that intimately encapsulate the giant axons as well as ‘capitate’ projections of Schwann cells into the axons. When adjacent Schwann cells abut directly against one another, aligned E-face IMPs are found along the fracture plane of the upturning membranes. These E-face alignments of IMPs have complementary furrows on the Schwann cell membranes which exhibit no complementary structure on the axolemma as they represent the clefts between adjacent glial cells. The other Schwann cell membranes exhibit P-face dimples and E-face (extracellular membrane half-leaflet) protuberances which may reflect endo- or exocytotic activity; alternatively they may represent caveolae. Comparable structures are occasionally observed at axo-glial interfaces. However, those in the Schwann cell membrane could be part of the transverse tubular lattice system which also exists in adaxonal glia. Beyond the Schwann cells, layers of endoneurial cells (fibrocytes) are interleaved by collagen-filled spaces. These cells exhibit extensive cross-fractured intracellular invaginations as well as inpushings of the extracellular matrix material. Their membranes exhibit a large number of IMPs.     

Transglial pathway of diffusion in the Schwann sheath of the squid giant axon

Summary In order to investigate the transglial pathways in the Schwann sheath of squid giant axons, an electron microscopic study of thin sections and freeze-fracture replicas was carried out. Hitherto the mesaxonal clefts between Schwann cells were regarded as the only pathway between the extracellular space and the periaxonal space which, like the clefts, is about 10 nm in width. The clefts were now found to be obstructed by a putative single-stranded tight junction between neighbouring Schwann cells along the entire border near the axon. The Schwann cells were found to be penetrated like a sponge by a three-dimensional tubular transglial lattice that is confluent with the periaxonal space, the mesaxonal clefts and the extracellular space. The transglial channel system (TGCS) would, therefore, serve as an alternative diffusional pathway, provided that the tubular lumen was permeable. The diameter of the tubules is about 40 nm. In freeze-fracture replicas the density of tubular openings towards the axon was estimated to be 3.3 ± 0.72 per µm². In relation to the periaxonal cell surface, this constitutes a relative opening area of 0.42% as compared to the 0.15% of the mesaxonal clefts (neglecting their tight junctions). Therefore, the TGCS would provide a ubiquitous access for ionic flow between axolemma and extracellular space. The fact that the TGCS has only recently been observed in squid, but has been described for some time in the giant nerve fibres of crayfish and lobster, can be explained by the use of different fixation methods. The TGCS system is preserved in aldehyde fixation as used in the present study, whereas osmium tetroxide was applied in earlier work on squid. The comparison with the results obtained in other species suggests strongly that the TGCS is permeable and constitutes a transglial pathway for rapid ionic flow.     

Temperature dependence of non-electrolyte and sodium permeability in giant axon of squid

1. The efflux of [(14)C]urea was measured in micro-injected axons at 18 degrees C. A permeability constant for urea of (0.55 +/- 0.18) x 10(-6) cm/sec was calculated from these experiments.2. The influxes of urea, thiourea, ethylene glycol, urethane and toluene were measured in perfused axons at 18 +/- 1 degrees C. The permeability constants obtained from these determinations increased in the order listed, from (0.76 +/- 0.19) x 10(-6) cm/sec for urea to 0.80 x 10(-4) cm/sec for toluene.3. The influxes of tritiated water and sodium ions at 18 degrees C were measured in perfused axons. An average permeability of (0.78 +/- 0.22) x 10(-4) cm/sec for titriated water and an average influx of 23 +/- 6 p-mole/cm(2) sec for sodium were obtained.4. Lowering the temperature of the external sea-water bathing the axon from 18 to 5 degrees C produced a decrease of 12% in the permeability of toluene, 30% for tritiated water and urethane, 55% for ethylene glycol and urea and 60% for thiourea. There was a 50% reduction in the influx of sodium for this same temperature change.5. The results obtained with the effect of temperature on permeabilities suggest that the axonal membrane has a non-homogeneous composition. A model based on the assumption of structured aqueous channels in the membrane is postulated.     

Effects of acetylcholine and carbamylcholine on the axon and Schwann cell electrical potentials in the squid nerve fibre

1. The effect of acetylcholine and carbamylcholine on the axon and Schwann cell membrane potential have been studied in the giant nerve fibre of the squid. The addition of carbamylcholine (10(-6)M) to the external sea-water medium has no appreciable effects on the resting and action potentials of the axon. However, it induces a long-lasting hyperpolarization in the surrounding Schwann cells of the unstimulated intact or slit nerve fibres which is completely blocked by D-tubocurarine (10(-9)M). Eserine (10(-9)M) prolongs the Schwann cell hyperpolarizations induced by a 1 min exposure of the unstimulated nerve fibres to acetylcholine (10(-7)M).2. The addition of carbamylcholine (10(-6)M) to the external medium increases the relative permeability of the Schwann cell membrane to the potassium ion in slit nerve fibres. Yet, a hundredfold reduction in external sodium concentration has no appreciable effect on the hyperpolarization of the Schwann cells of the slit nerve fibre under similar conditions.3. Tetrodotoxin at a concentration of 5 x 10(-8)M has no appreciable effects on either the Schwann cell electrical potential or on the hyperpolarizing action of carbamylcholine on the Schwann cells of the unstimulated intact nerve fibres.4. These findings indicate the presence of acetylcholine receptors in the plasma membrane of the Schwann cell in these nerve fibres and give further support to the hypothesis on the role of the cholinergic system in the genesis of the long-lasting Schwann cell hyperpolarizations caused by the conduction of nerve impulse trains by the axon.     

Neurofilament phosphorylation and axon diameter in the squid giant fibre system

Electron energy loss spectroscopic analysis of squid giant axons in a phosphorus energy window yielded bright signals, which were shown to originate from highly phosphorylated neurofilaments. The frequency and distribution of these signals were analysed at defined intervals in cross-sections of the giant axon, starting from its origin in the stellate ganglion and extending distally along the stellar nerve. The analysis revealed a proximodistal gradient of increasing neurofilament phosphorylation. Within the stellate ganglion and for some distance beyond, the increase in frequency of signals correlated with the widening of the neurofilament meshwork and the radial growth of the axon. This agrees with the hypothesis that neurofilament phosphorylation regulates axon calibre by affecting interfilament spacing. In distal axon domains where the axon diameter diminished, contrary to expectations, the spacing of signals increased and the signals were significantly larger. Hyperphosphorylation apparently compensated for a diminishing supply of neurofilament protein. Contrary to predictions, the presynaptic terminal of the giant synapse contained a distinct and highly phosphorylated neurofilament meshwork. We conclude that the growth of the axon diameter is a function of neurofilament phosphorylation, interfilament spacing and neurofilament density. A mature and highly phosphorylated neurofilament cytoskeleton completely filled the presynaptic terminal of the giant synapse.     

Cytoplasmic constriction and vesiculation after axotomy in the squid giant axon

Summary The squid giant axon responded to a transection injury by producing a gradient of cytoplasmic and vesicular changes at the cut end. At the immediate opening of the cut axon the cytoplasm was fragmented and dispersed and the vesicles in this region were in rapid Brownian movement. Approximately 0.1 mm further in, at the site of maximal axonal constriction, the axoplasm was condensed into a compact, constricted mass containing many large vesicles. The axoplasm was normal a few millimetres beyond this constricted, vesiculated end. It appears that transection triggered the transformation of normal axoplasm into a tightly constricted, highly vesiculated structure. This modified axoplasm at the cut end may slow the spread of damage and degeneration by preventing the bulk outflow of axoplasm, by slowing down the loss of intracellular molecules and by slowing down the influx of destructive extracellular ions (like calcium and chloride).     

The structure of the neurofilament cytoskeleton in the squid giant axon and synapse

Summary Stereo views of thick sections (<0.5m), or freeze etch replicas taken from selected regions of the squid giant axon and giant synapse, using an electron energy loss spectroscopic ultrastructural method, revealed a meshwork of anastomosing neurofilaments intersecting at a wide range of angles. This type of architectural organisation differs significantly from the ladder-like construction of the mammalian neurofilament cytoskeleton, in which longitudinally oriented core filaments are interconnected by lateral crossbridges. The deviant organization of squid neurofilaments is consistent with recent evidence that squid neurofilament proteins more closely resemble nuclear lamin rather than mammalian neurofilament proteins. A proximo-distal gradient of increasing width of the neurofilament meshes along the giant axon correlated well with the previously described gradient of increasing neurofilament phosphorylation. In these thick sections the presynaptic terminal of the giant synapse exhibited a mature neurofilament cytoskeleton that extended to the active zones without detectable signs of degradation. The observations are discussed in the context of current hypotheses concerned with the function of phosphorylation of neurofilaments, and with the steady state maintenance of the cytoskeleton in the squid axon and presynaptic terminal.     

Temperature and impulse velocity in giant axon of squid Loligo pealei.

Impulse propagation velocity as a function of temperature in the range 5--20degreesC was obtained by external recording from the giant axon of Loligo pealei. The stellar nerve was set into a chamber allowing continuous superfusion, temperature control, and double recording of the impulse. Velocity was calculated from the interval between the spike peaks. The Q10 of velocity was about 1.8. At all temperatures, the velocity increased with time so that only data obtained during the 1st h or 2 could be generally considered to be comparable. Impulse block occurred below --3.4degreesC, in contrast to the giant axon of L. vulgaris, which blocks at about 0degreeC, but at the higher range of temperatures, the velocity in the L. pealei axons was not as well sustained as in those of L. vulgaris. The expected impulse velocity was calculated from Huxley's stability function f(beta) by approximating that function to a fourth-order polynominal and by substituting into it suitable ratios of available Q10 values relating to membrane conductance, ionic current, capacitance, and axoplasmic resistance. The calculation provided an improved fit to published experimental data on L. vulgaris. The difference in slope of the log velocity versus temperature plots, between the presumably warm acclimatized L. vulgaris and the cold-acclimatized L. pealei, was present in both experimental and calculated curves.     

Interaction of DDT with sodium channels in squid giant axon membranes

The effects of DDT on the sodium channel gating mechanism were studied with internally perfused squid giant axons under voltage clamp conditions. In contrast to earlier studies, DDT has pronounced effects on the sodium conductance system of squid axons. Internal application of 1 × 10^?4M DDT had no effect on the activation time constant (τ_m) or on the peak current, but the falling phase of the sodium current and the tail current upon repolarization were slowed dramatically (τ_tail= 3–13 ms). The sodium inactivation process became second order after DDT treatment. The time constant of the fast phase (τ₁) was identical to τ_h recorded in the control axon, while the time constant of the slow phase (τ₂) was more than 10 times greater. The voltage dependence of τ₂ was different from τ_h, increasing with depolarization. The steady-state inactivation curve was shifted in the direction of hyperpolarization by 6–8 mV, but even after prolonged depolarizing pulses, the sodium conductance did not inactivate completely.These data are consistent with the hypothesis that a population of sodium channels modified by DDT activates normally but is then retained in a second open state which inactivates slowly.     

Analysis of the potential-dependent changes in optical retardation in the squid giant axon

1. An analysis has been made of the change in optical retradation of the membrane elicited by the application of voltage-clamp pulses in squid giant axons.2. The retardation response consists of three separate voltage-dependent components. For freshly mounted axons, defined as being in state 1, hyperpolarizing pulses give a rapid increase in the light intensity measured with crossed polarizers which has been termed the fast phase. This is followed by a rather slow return towards the base line termed the rebound. On treatment of the axon with certain agents that include tetrodotoxin, high calcium and terbium, the rebound disappears and the fast phase slows down, increases in size, and has a new slow component added to it. This transition from state 1 to a second state, 2, appears to be irreversible.3. In state 1, the time constant of the fast phase is 20-40 musec at 13 degrees C; it has a very large negative temperature coefficient (Q(10) = Ca.(1/8)). The size of the retardation change is independent of temperature and varies as the square of the applied voltage, but the voltage-retardation curve is symmetrical about a point well beyond zero membrane potential, at an internal potential of around + 70 mV. In state 2, the time constant is about five times larger, and varies much less markedly with temperature; the apex of the voltage-retardation curve is shifted to + 200 mV.4. The rebound has a time constant of the order of 20 msec at 13 degrees C. A 10 degrees rise in temperature more than halves the time constant and roughly doubles the amplitude of the rebound. The voltage dependence of the rebound differed from that of the fast phase.5. The slow component of state 2 has a time constant of about 2 msec which does not change noticeably between 10 and 25 degrees C. The size of this component seems to be linearly dependent on the applied voltage, rather than obeying a square law.6. A tenfold increase in external calcium concentration had no discernible effect on the fast and slow phases, but reversibly reduced the amplitude of the rebound nearly to half.7. In experiments on perfused axons, the retardation response was not measurably altered by any of the modifications made to the composition of the perfusing fluid.8. There was some indication of the possible existence of a small current- or conductance-dependent component of the retardation response.9. These phenomena seem likely to originate either from molecular relaxation processes analogous with the Kerr effect, or from changes in membrane thickness under the influence of the pressure exerted by the electric field. However, the specific molecules involved in the retardation response cannot yet be identified.     

Trypargine blocks the sodium channels only from the inside of squid giant axon

A new tetrahydro-beta-carboline, trypargine (TRG), specifically suppresses the Na current (INa) when applied to the internal surface of the squid axon membrane without affecting the K current (IK). The binding of TRG to its receptor is potential-dependent and occurs at a site about halfway through the membrane electric field from the outside; the dissociation constant is 11 microM at 0 mV.     

Sodium influxes in internally perfused squid giant axon during voltage clamp

1. An experimental method for measuring ionic influxes during voltage clamp in the giant axon of Dosidicus is described; the technique combines intracellular perfusion with a method for controlling membrane potential.

2. Sodium influx determinations were carried out while applying rectangular pulses of membrane depolarization. The ratio `measured sodium influx/computed ionic flux during the early current' is 0·92 ± 0·12.

3. Plots of measured sodium influx and computed ionic flux during the early current against membrane potential are very similar. There was evidence that the membrane potential at which the sodium influx vanishes is the potential at which the early current reverses.

     

The mechanisms of sodium current inhibition by benzocaine in the squid giant axon

(1) The effects of benzocaine on the ionic currents in the voltage-clamped squid giant axon have been examined under various conditions; intact axons internally perfused with CsF and axons dialysed with tetraethyl-ammonium ions were used. (2) Both the steady state outward (potassium) current and the early transient (sodium) current were reduced by ca. 50% by benzocaine (1 mM). (3) Plots of the changes produced by benzocaine (1 mM) in the Hodgkin-Huxley parameters for the steady state activation (m), the steady state inactivation (h) and the time constants (_m and _h) for activation and inactivation of the sodium current are shown. Them andh curves are shifted in positive and negative directions respectively on the voltage axis. The time constants are not greatly affected. (4) In axons in which the sodium current inactivation had been largely removed by treatment with chloramine T, the sodium current was still reduced by ca. 50% by 1 mM benzocaine and the positive shift in activation remained unchanged. (5) The dependence on benzocaine concentration (for2mM) of the peak sodium current reduction and the shift in steady state inactivation have been determined. (6) It is concluded that in the squid axon the effects on inactivation are not the main reason for the reduction of the sodium current by benzocaine and that, in common with many other neutral anaesthetics, there are at least two sites at which benzocaine acts.     

Potassium and sodium ion current noise in the membrane of the squid giant axon.

1. The spectral density of current noise power from 20 mm segments of giant axons of the squid Loligo vulgaris has been measured under space-clamp and voltage-clamp conditions. From 4 to 1000 Hz the measured noise is larger by several orders of magnitude than the theoretical thermal noise. The amplifier's noise, which may yield appreciable contributions above 200 Hz, could be evaluated and subtracted from the total noise using direct measurements of the membrane impedance...