Periaxonal K+ regulation in the small squid Alloteuthis. Studies on isolated and in situ axons.

A novel giant axon preparation from the squid Alloteuthis is described. Properties of in situ and isolated axons are similar. Periaxonal K+ accumulation is a function of the physiological state of the animal and of the axon and its sheathing layers. Carefully dissected isolated axons, and axons in situ in a healthy mantle, show much less K+ accumulation than previously reported in squid. It is suggested that the Schwann cells are involved in the observed K+ regulation.     

Significant potassium ion accumulation at the external surface of Myxicola giant axons

Potassium accumulation associated with outward membrane potassium current was investigated experimentally in Myxicola giant axon. During prolonged voltage-clamp pulses to positive transmembrane potentials, the K+ equilibrium potential may approach zero mV, suggesting massive K+ accumulation outside the axonal membrane to concentrations many-fold higher than those in the bathing medium. The potassium accumulation can be satisfactorily described by a three-compartment model, consisting of the nerve fiber, a restricted physiological periaxonal space and the bulk solution. The average thickness, theta, of the periaxonal space is calculated as 177 +/- 59 A, i.e., comparable to that in the squid, while the permeability coefficient of the external barrier, PKs, was calculated to be (1.4 +/- 0.4) X 10(-4) cm/s. These conclusions are well supported by morphological study.     

The periaxonal space of crayfish giant axons

The influence of the glial cell layer on effective external ion concentrations has been studied in crayfish giant axons. Excess K ions accumulate in the periaxonal space during outward K+ current flow, but at a rate far below that expected from the total ionic flux and the measured thickness of the space. At the conclusion of outward current flow, the external K+ concentration returns to normal in an exponential fashion, with a time constant of approximately 2 ms. This process is about 25 times faster than is the case in squid axons. K+ repolarization (tail) currents are generally biphasic at potentials below about -40 mV and pass through a maximum before approaching a final asymptotic level. The initial rapid phase may in part reflect depletion of excess K+. After block of inactivation and reversal of the Na+ concentration gradient, we could demonstrate accumulation and washout of excess Na ions in the periaxonal space. Characteristics of these processes appeared similar to those of K+. Crayfish glial cell ultrastructure has been examined both in thin sections and after freeze fracture. Layers of connective tissue and extracellular fluid alternate with thin layers of glial cytoplasm. A membranous tubular lattice, spanning the innermost glial layers, may provide a pathway allowing rapid diffusion of excess ions from the axon surface.     

Axonal contribution to subthreshold currents inaplysia bursting pacemaker neurons

The contribution of axonal activity to the ionic currents which generate bursting pacemaker activity was studied by using the two-electrode voltage-clamp technique in Aplysia bursting neuron somata in conjunction with intraaxonal voltage recordings. Depolarizing voltage-clamp pulses applied to bursting cell somata triggered axonal action potentials. The voltage-clamp current recording exhibited transient inward current "notches" corresponding to each of the axonal spikes. The addition of 50 microM tetrodotoxin (TTX) to the bathing medium blocked the fast axonal spikes and current notches, revealing a slower axonal spike which was blocked by the replacement of external Ca2+ with Co2+. The inward current evoked by applying a depolarizing voltage-clamp pulse in the soma is distorted by the occurrence of the axonal Ca2+ spike. Elimination of the axonal spike, by injecting hyperpolarizing current into the axon, changes both the time course and the magnitude of the inward current. The axonal Ca2+ spikes are followed by a series of Ca2+-dependent afterpotentials: a rapid postspike hyperpolarization, a depolarizing afterpotential (DAP) and, finally, a long-lasting postburst hyperpolarization. The long-lasting hyperpolarization is not blocked by 50 mM external tetraethyl ammonium, an effective blocker of Ca2+-activated K+ current [IK(Ca)], and does not appear to reverse at EK. Hence, the axonal long-lasting hyperpolarization may not be due to IK(Ca). Somatic voltage-clamp pulses in bursting neurons are followed by a slow inward tail current, which is sometimes coincident with a DAP in the axon. In some cells, the amplitude of the slow inward tail current is greatly reduced if axonal spikes and DAPs are prevented by hyperpolarization of the axon, while, in other cells, elimination of axonal activity has little effect. Therefore, the slow inward tail current is not necessarily an artifact of poor voltage-clamp control over the axonal membrane potential but probably results from the activation of an ionic conductance mechanism located partly in the axon and partly in the soma.     

Preface

The physiological function of the axon is to conduct short all-or-none action potentials from their site of initiation (usually the cell body) to the synapse. To ensure this function, both passive and active biophysical properties of the axons are tuned very precisely, especially the voltage-dependent ionic conductances to sodium and potassium. Under normal conditions, axons are not spontaneously active. Minor modifications of their ionic micro-environment or slight changes in the membrane properties are however sufficient to induce rhythmical activity and modify the time course of the action potentials. These modifications can be induced by a variety of pharmacological agents. Some typical examples taken from original studies on invertebrate preparations are illustrated. The experiments were carried out on two axonal preparations: the giant axon of the squid Loligo forbesi and the giant axon of the cockroach Periplaneta americana. The axons were ‘space-clamped’ and studied under both current-clamp and voltage-clamp conditions. Voltage-clamp experiments were used to dissect out the mechanisms underlying repetitive activity and to extract the relevant parameters. These parameters were then used to rebuild the observed effects using an extended version of the Hodgkin and Huxley (1952, J Physiol (Lond) 117, 500–544) formulation. One easy way to get repetitive firing in both preparations is to reduce potassium conductance. The effect of 4-aminopyridine on squid axon is illustrated here. The experimental results, including the occurrence of bursts of activity, can be described by adding a time- and voltage-dependent block of the potassium channels to the original Hodgkin and Huxley (1952, J Physiol (Lond) 117, 500–544) model. Repetitive spike activity and plateau action potentials are also produced when the depolarising effect of the voltage-dependent potassium current is counterbalanced by a maintained inward sodium current. This maintained sodium current can be due to several different mechanisms. This will be illustrated by five structurally unrelated molecules: two scorpion toxins, two insecticide molecules and one sea anemone toxin. One toxin purified from the venom of the scorpion Buthotus judaïcus (insect toxin 1) exerts its effects by shifting the sodium activation curve towards more hyperpolarized potentials. Another toxin purified from the venom of another scorption Androctonus australis (mammal toxin 1) modifies a significant proportion of normal (fast) sodium channels into slowly activating and inactivating sodium channels. The main effect of the insecticide DDT is to maintain sodium channels in the ‘open’ configuration. Another insecticide molecule known to induce repetitive activity, S-bioallethrin, activates voltage-dependent sodium channels with slow activation and inactivation kinetics. The sea anemone toxin anthopleurin A, purified from the venom of Anthopleura xanthogrammica, delays inactivation of the sodium current without changing its activation kinetics. These examples show that minor modifications of the properties of the nerve membrane are sufficient to alter nerve function. These deleterious effects will be amplified at the synapse through dramatic changes in transmitter release and will lead eventually to disastrous alterations of brain function.     

Control of the spatial distribution of sodium channels in giant fiber lobe neurons of the squid

Na+ channels are present at high density in squid giant axon but are absent from its somata in the giant fiber lobe (GFL) of the stellate ganglion. GFL cells dispersed in vitro maintain growing axons and develop a Na+ channel distribution similar to that in vivo. Tunicamycin, a glycosylation inhibitor, selectively disrupts the spatially appropriate, high level expression of Na+ channels in axonal membrane but has no effect on expression in cell bodies, which show low level, inappropriate expression in vitro. This effect does not appear to involve alteration in Na+ channel turnover or axon viability. K+ channel distribution is unaffected. Thus, glycosylation appears to be involved in controlling Na+ channel localization in squid neurons.     

Delayed kinetics of squid axon potassium channels do not always superpose after time translation.

The activation of potassium ion conductance in squid axons by voltage-clamp depolarization is delayed when the depolarizing step is preceded by a conditioning hyperpolarization of the axonal membrane. Moreover, the control conductance kinetics superpose with the delayed kinetics when they are translated along the time axis by an amount equal to the delay. We have found that the degree of superposition with internally perfused axons depends upon voltage-clamp protocol. The kinetics superpose almost exactly for modest test depolarizations, whereas they clearly fail to superpose completely for more positive levels of membrane depolarization. We have modeled these results by incorporating a time dependence into the rate constant of activation of potassium channel gates in the Hodgkin and Huxley model of potassium ionic conductance.     

Protein release from the internal surface of the squid giant axon membrane during excitation and potassium depolarization

The proteins in the perfusate collected from intracellularly perfused squid giant axons were analyzed after being labeled with radioactive ¹²⁵I-labeled Bolton-Hunter reagent. The rate of protein release into the perfusate was found to be increased by the following electrophysiological manipulations of the axons: (1) repetitive electrical stimulation at 60 Hz in axons perfused with normal potassium fluoride-containing solution or at 0.125 Hz in axons perfused with tetraethylammonium containing solution, (2) perfusion with 4-amino-pyridine solution which induces spontaneous electrical activity in the axon, and (3) depolarization of the axon induced by raising the external potassium concentration. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of the proteins released under these conditions yielded molecular weight profiles different from those of the extruded axoplasmic proteins. These observations indicate that there exists, in close association with the axonal membrane, a particular group of proteins, the solubility of which is readily affected by changes in the state of the membrane.     

Propagation of action potentials in squid giant axons. Repetitive firing at regions of membrane inhomogeneities

Effects of reduction in potassium conductance on impulse conduction were studied in squid giant axons. Internal perfusion of axons with tetraethylammonium (TEA) ions reduces G K and causes the duration of action potential to be increased up to 300 ms. This prolongation of action potentials does not change their conduction velocity. The shape of these propagating action potentials is similar to membrane action potentials in TEA. Axons with regions of differing membrane potassium conductances are obtained by perfusing the axon trunk and one of its two main branches with TEA after the second branch has been filled with normal perfusing solution. Although the latter is initially free of TEA, this ion diffuses in slowly. Up until a large amount of TEA has diffused into the second branch, action potentials in the two branches have very different durations. During this period, membrane regions with prolonged action potentials are a source of depolarizing current for the other, and repetitive activity may be initiated at transitional regions. After a single stimulus in either axon region, interactions between action potentials of different durations usually led to rebound, or a short burst, of action potentials. Complex interactions between two axon regions whose action potentials have different durations resembles electric activity recorded during some cardiac arrhythmias.     

Potassium currents in the giant axon of the crab Carcinus maenas

Measurements were made of the kinetic and steady-state characteristics of the potassium conductance in the giant axon of the crab Carcinus maenas. These measurements were made in the presence of tetrodotoxin, using the feedback amplifier concept introduced by Dodge and Frankenhaeuser (J. Physiol, (London) 143:76-90). The conductance increase during depolarizing voltage-clamp pulses was analyzed assuming that two separate potassium channels exist in these axons. The first potassium channel exhibited activation and fast inactivation gating which could be fitted using the m3h, Hodgkin-Huxley formalism. The second potassium channel exhibited the standard n4 Hodgkin-Huxley kinetics. These two postulated channels are blocked by internal application of caesium, tetraethylammonium and sodium ions. External application of 4 amino-pyridine also blocks these channels.     

K+ accumulation in the space between giant axon and Schwann cell in the squid Alloteuthis. Effects of changes in osmolarity.

In a train of impulses in squid giant axon, accumulation of extracellular potassium causes successive afterhyperpolarizations to be progressively less negative. In Loligo, Frankenhaeuser and Hodgkin had satisfactorily accounted for the characteristics of this effect with a model in which the axon is surrounded by a space, width theta, and a barrier of permeability P. In axons isolated from Alloteuthis, we found that the model fitted the observations quite well. Superfusing the axon with hypotonic artificial seawater (ASW) caused theta and P to decrease, and, conversely, hypertonic ASW caused them to increase: this would be the case if both the space and the pathway through the barrier were extracellular. In some cases, in normal ASW, the afterhyperpolarizations in a train decreased very little, less than 0.7 mV. In these extreme cases, theta was estimated to be 190 nm and P to be 7 x 10(-4) cm s-1, both several times the values of 30 nm and 6 x 10(-5) cm s-1 estimated by Frankenhaeuser and Hodgkin. We suggest that in vivo the periaxonal space may be considerably wider than that seen in conventionally fixed squid tissue.     

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

Transmembrane potentials in the crayfish giant axon have been investigated as a function of the concentration of normally occurring external cations. Results have been compared with data already available for the lobster and squid giant axons. The magnitude of the action potential was shown to be a linear function of the log of the external sodium concentration, as would be predicted for an ideal sodium electrode. The resting potential is an inverse function of the external potassium concentration, but behaves as an ideal potassium electrode only at the higher external concentrations of potassium. Decrease in external calcium results in a decrease in both resting potential and action potential; an increase in external calcium above normal has no effect on magnitude of transmembrane potentials. Magnesium can partially substitute for calcium in the maintenance of normal action potential magnitude, but appears to have very little effect on resting potential. All ionic effects studied are completely reversible. The results are in generally good agreement with data presently available for the lobster giant axon and for the squid giant axon.     

The glial blood-brain barrier of crustacea and cephalopods: a review

1. The glial blood-brain barrier of invertebrates is an accessible, polarised glial layer that permits study of glial cells in their normal relations with neurons. Crayfish 2. The glial "perineurium" forms the blood-brain interface in crayfish, and acts as a barrier to horseradish peroxidase (HRP) and ionic lanthanum. By contrast, the perineurium of the peripheral nervous system is relatively permeable. 3. The ionic permeability of the blood-brain interface can be studied in a sucrose gap chamber, using an extra-cellular microelectrode to monitor the potential across the perineurium following changes in the bathing medium. Subtraction of the microelectrode trace from the sucrose gap records gives the change in the axonal membrane potential. 4. Raised [K+] in the bath causes a complex change in perineurial potential, with the initial transient indicating that the outer (basal) glial membrane is highly K+ selective. The axonal response shows that the time constant for K+ uptake (tau u) and efflux (tau E) across the perineurium of the order of 3-4 min, but the interstitial [K+] in the steady state, [K+] infinity is always less than in the bathing medium. The results are explained by a model incorporating a K+ sink, which may be glial. 5. Strophanthidin and ethacrynic acid have little effect on tau u or K infinity, but cause a rise of tau E. Cold temperature pulses causes changes in the perineurial potential compatible with depolarisation of the inner (apical) membrane. A model is proposed with a Na+-K+-2 Cl co-transporter on the perineurial basal membrane, and an electrogenic Na+-K+-ATPase on the apical.membrane, consistent with results from vertebrate glial/ependymal epithelia. Cephalopods 6. The brain of the cuttlefish Sepia has an extensive system of microvessels. In the vertical and optic lobes studied, a perivascular glial layer forms a barrier to HRP. The occluding structure appears not to be a classical tight junction but may involve condensation of extracellular material. There is no barrier between retinal axons and blood. 7. Studies with radiolabelled polyethylene glycol (PEG4000) and EDTA show that the Sepia blood-brain barrier is as tight as the endothelial barrier of mammals. 8. A modification of the Oldendorf arterial injection technique is used to show that glucose transport at the Sepia barrier is mediated by a Na+-independent hexose carrier resembling that of mammalian red cells and blood-brain barrier. 9. The blood-axon interface fo mantle nerves in the squid Alloteuthis is relatively impermeable to small ions.(ABSTRACT TRUNCATED AT 400 WORDS)     

Axonal excitability revisited

The original papers of Hodgkin and Huxley (J. Physiol. 116 (1952a) 449, J. Physiol. 116 (1952b) 473, J. Physiol. 116 (1952c) 497, J. Physiol. 117 (1952d) 500) have provided a benchmark in our understanding of cellular excitability. Not surprisingly, their model of the membrane action potential (AP) requires revisions even for the squid giant axon, the preparation for which it was originally formulated. The mechanisms they proposed for the voltage-gated potassium and sodium ion currents, IK, and INa, respectively, have been superceded by more recent formulations that more accurately describe voltage-clamp measurements of these components. Moreover, the current-voltage relation for IK has a non-linear dependence upon driving force that is well described by the Goldman-Hodgkin-Katz (GHK) relation, rather than the linear dependence on driving force found by Hodgkin and Huxley. Furthermore, accumulation of potassium ions in the extracellular space adjacent to the axolemma appears to be significant even during a single AP. This paper describes the influence of these various modifications in their model on the mathematically reconstructed AP. The GHK and K+ accumulation results alter the shape of the AP, whereas the modifications in IK and INa gating have surprisingly little effect. Perhaps the most significant change in their model concerns the amplitude of INa, which they appear to have overestimated by a factor of two. This modification together with the GHK and the K+ accumulation results largely remove the discrepancies between membrane excitability of the squid giant axon and the Hodgkin and Huxley (J. Physiol. 117 (1952d) 500) model previously described (Clay, J. Neurophysiol. 80 (1998) 903).     

Protein Transport in Intact and Severed (Anucleate) Crayfish Giant Axons

Abstract: Using video-enhanced microscopy and a pulse-radiolabeling paradigm, we show that proteins synthesized in the medial giant axon cell body of the crayfish ( Procambarus clarkii ) are delivered to the axon via fast (∼62 mm/day) and slow (∼0.8 mm/day) transport components. These data confirm that the medial giant axon cell body provides protein to the axon in a manner similar to that reported for mammalian axons. Unlike mammalian axons, the distal (anucleate) portion of a medial giant axon remains intact and functional for >7 months after severance. This axonal viability persists long after fast transport has ceased and after the slow wave front of radiolabeled protein has reached the terminals. These data are consistent with the hypothesis that another source (i.e., local glial cells) provides a significant amount of protein to supplement that delivered to the medial giant axon by its cell body.     

Extracellular [K+] fluctuations in voltage-clamped canine cardiac Purkinje fibers.

Membrane currents and extracellular [K+] were measured in canine Purkinje strands during voltage-clamp steps to plateau or diastolic potentials. Extracellular [K+] increased during step depolarizations and decreased during step hyperpolarizations. On hyperpolarization, the largest fraction of the K+ depletion occurred during the initial 500 ms of the voltage-clamp step and was correlated with a potassium depletion current, the id. A slower component of the depletion also occurred on hyperpolarization and had a time constant consistent with cylindrical diffusion of potassium within the Purkinje strands. On depolarization, there is an accumulation of K+ that is correlated with the plateau current ix. On termination of depolarizing test pulses, the K+ accumulation decays with a time course similar to the ix tail current. Surprisingly, no accumulation of K+ occurred during the arrhythmogenic transient inward current, TI, suggesting that the selectivity of this current should be reevaluated.     

Calcium entry in squid axons during voltage clamp pulses

Squid giant axons were injected with aequorin and tetraethylammonium and were impaled with sodium ion sensitive, current and voltage electrodes. The axons were usually bathed in a solution of varying Ca2+ concentration ([Ca2+]o) containing 150mM each of Na+, K+ and an inert cation such as Li+, Tris or N-methylglucamine and had ionic currents pharmacologically blocked. Voltage clamp pulses were repeatedly delivered to the extent necessary to induce a change in the aequorin light emission, a measure of axoplasmic Ca2+ level, [Ca2+]i. The effect of membrane voltage on [Ca2+]i was found to depend on the concentration of internal Na+ ([Na+]i). Voltage clamp hyperpolarizing pulses were found to cause a reduction of [Ca2+]i. For depolarizing pulses a relationship between [Ca2+]i gain and [Na+]i indicates that Ca2+ entry is sigmoid with a half maximal response at 22 mM Na+. This Ca2+ entry is a steep function of [Na+]i suggesting that 4 Na+ ions are required to promote the influx of 1 Ca2+. There was little change in Ca2+ entry with depolarizing pulses when [Ca2+]o is varied from 1 to 10mM, while at 50mM [Ca2+]o calcium entry clearly increases suggesting an alternate pathway from that of Na+/Ca2+ exchange. This entry of Ca2+ at high [Ca2+]o, however, was not blocked by Cs+o. The results obtained lend further support to the notion that Na+/Ca2+ exchange in squid giant axon is sensitive to membrane voltage no matter whether this is applied as a constant change in membrane potential or as an intermittent one.     

Axonal and presynaptic RNAs are locally transcribed in glial cells.

In the last few years, the long-standing opinion that axonal and presynaptic proteins are exclusively derived from the neuron cell body has been substantially modified by the demonstration that active systems of protein synthesis are present in axons and nerve terminals. These observations have raised the issue of the cellular origin of the involved RNAs, which has been generally attributed to the neuron soma. However, data gathered in a number of model systems indicated that axonal RNAs are synthesized in the surrounding glial cells. More recent experiments on the perfused squid giant axon have definitively proved that axoplasmic RNAs are transcribed in periaxonal glia. Their delivery to the axon occurs by a modulatory mechanism based on the release of neurotransmitters from the stimulated axon and on their binding to glial receptors. In additional experiments on squid optic lobe synaptosomes, presynaptic RNA has been also shown to be synthesized locally, presumably in nearby glia. Together with a wealth of literature data, these observations indicate that axons and nerve terminals are endowed with a local system of gene expression that supports the maintenance and plasticity of these neuronal domains.     

Differential blockage of two types of potassium channels in the crab giant axon

Summary Measurements were made of the kinetic and steady-state characteristics of the potassium conductance in the giant axon of the crabsCarcinus maenas andCancer pagirus. The conductance increase during depolarizing voltage-clamp pulses was analyzed assuming that two separate types of potassium channels exist in these axons (M. E. Quinta-Ferreira, E. Rojas and N. Arispe,J. Membrane Biol. 66:171–181, 1982). It is shown here that, with small concentrations of conventional K⁺-channel blockers, it is possible to differentially inhibit these channels. The potassium channels with activation and fast inactivation gating (m³h, Hodgkin-Huxley kinetics) were blocked by external application of 4 amino-pyridine (4-AP). The potassium channels with standard gating (n⁴, Hodgkin-Huxley kinetics) were preferentially inhibited by externally applied tetraethylammonium (TEA). The differential blockage of the two types of potassium conductance changes suggests that they represent two different populations of potassium channels.It is further shown here that blocking the early transient conductance increase leads to the inhibition of the repetitive electrical activity induced by constant depolarizing current injection in fibers fromCardisoma guanhumi.