Some aspects of the physiological role of ion channels in the nervous system.

Recent analyses of the genomes of several animal species, including man, have revealed that a large number of ion channels are present in the nervous system. Our understanding of the physiological role of these channels in the nervous system has followed the evolution of biophysical techniques during the last century. The observation and the quantification of the electrical events associated with the operation of the ionic channels has been, and still is, one of the best tools to analyse the various aspects of their contribution to nerve function. For this reason, we have chosen to use electrophysiological recordings to illustrate some of the main functions of these channels. The properties and the roles of Na+ and K+ channels in neuronal resting and action potentials are illustrated in the case of the giant axons of the squid and the cockroach. The nature and role of the calcium currents in the bursting behaviour of the neurons are illustrated for Aplysia giant neurons. The relationship between presynaptic calcium currents and synaptic transmission is shown for the squid giant synapse. The involvement of calcium channels in survival and neurite outgrowth of cultured neurons is exemplified using embryonic cockroach brain neurons. This same neuronal preparation is used to illustrate ion channel noise and single-channel events associated with the binding of agonists to nicotinic receptors. Some features of the synaptic activity in the central nervous system are shown, with examples from the cercal nerve giant-axon preparation of the cockroach. The interplay of different ion conductances involved in the oscillatory behaviour of the Xenopus spinal motoneurons is illustrated and discussed. The last part of this review deals with ionic homeostasis in the brain and the function of glial cells, with examples from Necturus and squids.

Role of ligand-gated ion channels in the swimming behaviour of Xenopus tadpoles: experimental data and modelling experiments.

The swimming behaviour of lower vertebrates has been used as a model to study the function of simple neuronal circuits. Good examples are the lamprey and the Xenopus tadpole. In these two cases, glutamate-activated NMDA receptors are involved, and the relative importance of the NMDA and non-NMDA receptors as well as the involvement of other ion channels has been studied using a combination of electrophysiological recordings and modelling experiments, but little attention had been paid to their evolution during development. In the present experiments, which have been performed on Xenopus embryos from stages 31 to 42, we have probed the relative importance of the two categories of receptors using selective blockers [respectively dl-2-amino-5-phosphonovaleric acid (APV) and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX)]. The sensitivity of the swimming behaviour to APV was found to increase during development and that to CNQX to decrease. Furthermore, it has been observed that the spike activity recorded from the ventral roots is more complex in late embryonic stages that in early embryos. These modifications are associated with changes of the neuronal circuit, some of which correspond to a lengthening of the axon and an increased complexity of the dendritic tree of the motoneurons. We have incorporated these modifications in a simplified model of the central pattern generator built with Neuron software. The results indicate that at least part of the observed changes can be associated with changes in the length of the dendrites and axons.

Properties and development of calcium currents in embryonic cockroach neurons.

In freshly dissociated neurons from embryonic cockroach (Periplaneta americana L.) brains, voltage-dependent calcium currents appear early in development (E14). Their intensity increases progressively during embryonic life until eclosion (E35). Their time course and voltage dependency are characteristic of high voltage activated (HVA) currents although a 10 mV shift of the I/V curve towards more negative potentials was observed between E18 and E23. Their sensitivity to omega-AgaTx-IVA and omega-CgTx-GVIA and insensitivity to both amiloride and isradipine indicate that the corresponding channels are of the P/Q and N types. These channels, as well as a small proportion of toxin-resistant (R) channels (about 20%), are blocked by mibefradil and verapamil. The physiological significance of these currents and their modifications during embryonic life is discussed.

Quantitative morphological analysis of embryonic cockroach (Periplaneta americana) brain neurons developing in vitro.

Neurons dissociated from the brain of embryonic cockroaches (Periplaneta americana) can be maintained in culture for several weeks. The survival as well as the progressive organization of the neurons into a complex network was studied during a 5-week period under different culture conditions. About 10% of the dissociated cells adhered to the culture dish. This figure remained constant throughout the culture. The cell diameter ranged from 10 to 20 microns and did not change significantly over time in culture. Whereas only a few cells exhibited neurites at the start of the culture, the number of cells exhibiting neurites increased to reach about 99% after 2 weeks. The different cells were then connected to each other, forming a network, which became more and more complex. The number of cells per cluster as well as the length and the diameter of the "connectives" that linked the different clusters were found to increase with time. The morphology of individual neurons within the network was visualized after intracellular injection of biocytin. Labeling with antibodies raised against serotonin or GABA indicated that neurons were able to differentiate and to acquire specific neurotransmitter fates. The serotonergic phenotype was found to appear progressively throughout the culture, in parallel with the formation of the network. Cell density, addition of fetal calf serum, and ecdysone were shown to influence the development of the network.

omega-AgaIVA-sensitive (P/Q-type) and -resistant (R-type) high-voltage-activated Ba(2+) currents in embryonic cockroach brain neurons.

By means of the whole cell patch-clamp technique, the biophysical and pharmacological properties of voltage-dependent Ba(2+) currents (I(Ba)) were characterized in embryonic cockroach brain neurons in primary culture. I(Ba) was characterized by a threshold of approximately -30 mV, a maximum at approximately 0 mV, and a reversal potential near +40 mV. Varying the holding potential from -100 to -40 mV did not modify these properties. The steady-state, voltage-dependent activation and inactivation properties of the current were determined by fitting the corresponding curves with the Boltzmann equation and yielded V(0.5) of -10 +/- 2 (SE) mV and -30 +/- 1 mV, respectively. I(Ba) was insensitive to the dihydropyridine (DHP) agonist BayK8644 (1 microM) and antagonist isradipine (10 microM) but was efficiently and reversibly blocked by the phenylalkylamine verapamil in a dose-dependent manner (IC(50) = 170 microM). The toxin omega-CgTxGVIA (1 microM) had no significant effect on I(Ba). Micromolar doses of omega-CmTxMVIIC were needed to reduce the current amplitude significantly, and the effect was slow. At 1 microM, 38% of the peak current was blocked after 1 h. In contrast, I(Ba) was potently and irreversibly blocked by nanomolar concentrations of omega-AgaTxIVA in approximately 81% of the neurons. Approximately 20% of the current was unaffected after treatment of the neurons with high concentrations of the toxin (0. 4-1 microM). The steady-state dose-response relationship was fitted with a Hill equation and yielded an IC(50) of 17 nM and a Hill coefficient (n) of 0.6. A better fit was obtained with a combination of two Hill equations corresponding to specific (IC(50) = 9 nM; n = 1) and nonspecific (IC(50) = 900 nM; n = 1) omega-AgaTxIVA-sensitive components. In the remaining 19% of the neurons, concentrations >/=100 nM omega-AgaTxIVA had no visible effect on I(Ba). On the basis of these results, it is concluded that embryonic cockroach brain neurons in primary culture express at least two types of voltage-dependent, high-voltage-activated (HVA) calcium channels: a specific omega-AgaTxIVA-sensitive component and DHP-, omega-CgTxGVIA-, and omega-AgaTxIVA-resistant component related respectively to the P/Q- and R-type voltage-dependent calcium channels.

N-Methyl-D-aspartate-induced oscillations in whole cell clamped neurons from the isolated spinal cord of Xenopus laevis embryos.

The patch-clamp technique was used to measure the effect of N-methyl-D-aspartate (NMDA) on Xenopus embryonic neurons in an isolated, but intact spinal cord. Whole cell recordings were done at external calcium concentrations of 1 mM. NMDA alone (50-200 microM) or in association with 10 microM serotonin or glycine induced oscillatory activity in most presumed motoneurons, which were therefore considered part of rhythm generating networks. In the presence of TTX, one-half of these neurons maintained this activity. The oscillations fell into two main categories: voltage-dependent, low-frequency (0.3-0.5 Hz) and voltage-independent, high-frequency (3-8 Hz) oscillations. NMDA alone induced TTX-insensitive oscillations in one-third of the neurons; however, the percentage of neurons showing oscillations was greater in the presence of exogenous 5-hydroxytryptamine (5-HT) or glycine. Because these observations were made at embryonic stages where little or no serotonergic innervation exists, it is likely that NMDA-induced intrinsic oscillatory activity in Xenopus embryonic neurons does not require 5-HT.

Effects of nicotinic and muscarinic ligands on embryonic neurones of Periplaneta americana in primary culture: a whole cell clamp study.

The pharmacological properties of acetylcholine (ACh) receptors of cultured neurones from embryonic cockroach brains were studied using the whole-cell configuration of the patch-clamp technique. More than 90% of the studied neurones responded to ACh by a monophasic inward current, the intensity of which varied from cell to cell. The sequence of potency of the five tested agonists was ACh > nicotine=carbamylcholine > suberyldicholine=oxotremorine. The dose-response relationship was complex, suggesting the existence of two populations of receptors: high-affinity receptors (extrapolated K(d) around 10(-7) M) and low-affinity receptors (extrapolated K(d) around 5x10(-5) M). The current-voltage relationship of the induced current was linear between -80 and -40 mV and the extrapolated reversal potential was not significantly different from 0 mV. The sequence of decreasing potency of the antagonists of the ACh response was: methyllycaconitine > alpha-bungarotoxin > mecamylamine > curare > strychnine > bicuculline > atropine > picrotoxin. These results show: (1) that, in embryonic brain neurones, the response to ACh corresponds to the opening of non-selective cationic channels; and (2) that the pharmacology of the ACh receptors is mainly but not solely nicotinic. The nature of the single events which underlie this response, as well as the structure of the channels (homo or hetero-oligomeric) remain to be investigated.

Pharmacological induction of rhythmical activity and plateau action potentials in unmyelinated axons.

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 judaicus (insect toxin 1) exerts its effects by shifting the sodium activation curve towards more hyperpolarized potentials. Another toxin purified from the venom of another scorpion 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.

Patch-clamp analysis of the effects of the insecticide deltamethrin on insect neurones.

1. The mode of action of the pyrethroid insecticide deltamethrin on inexcitable embryonic cultured cockroach neurones has been investigated using the patch-clamp technique. 2. Whole-cell recordings of the current induced by step depolarizations of the cell membrane showed that concentrations of deltamethrin ranging from 10(-8) to 5 x 10(-6) mol l-1 induced a small tetrodotoxin (TTX)-sensitive inward current that peaked at around +10 mV and reversed at around +60 mV. The activation and inactivation kinetics of this current were much slower than those of the axonal sodium current in this same species and were relatively insensitive to membrane potential. Steady-state inactivation was almost absent. 3. Single-channel activity associated with the action of the insecticide was analyzed using the cell-attached configuration. Three distinct patterns of activity were found: (1) discrete single-channel events of relatively short duration, (2) long events of comparatively small amplitude and (3) complex bursts made up of a succession of openings and closings to several levels. These three patterns were analyzed quantitatively using specially designed programs. 4. The first pattern of activity could be seen in most patches. It consisted of short (1-10 ms) rectangular events of comparatively small amplitude (1.5 pA at rest) and very low open time probability (around 0.001). The current-voltage relationship of these small events was linear over the voltage range studied and the (extrapolated) reversal potential approximated ENa. 5. The second pattern of activity was observed less frequently. The channels could stay open for very long periods (up to several seconds) and occasionally flickered between two or more levels. 6. The third pattern of activity was observed in many patches. During the burst, which could last from a few milliseconds to a few hundred milliseconds, the single-channel current jumped almost continuously between several levels (up to 7 or 8).

Micromolar concentrations of veratridine activate sodium channels in embryonic cockroach neurones in culture.

The mode of action of the alkaloid veratridine has been reinvestigated on cultured cockroach neurones, which are normally inexcitable and do not have a detectable fast sodium current. The whole-cell and cell-attached configurations of the patch-clamp technique were used to record the macroscopic and single channel currents, respectively. Concentrations of veratridine ranging from 10(-8) to 10(-5) M were found to induce a small tetrodotoxin (TTX)-sensitive inward current, which peaked around +10 mV and reversed around +55 mV. This current exhibited a pronounced plateau and was insensitive to changes in the holding potential. Bath application of veratridine induced typical TTX-sensitive inwardly-directed single-channel activity, falling into two (apparently coupled) categories of events: first, relatively large events (1 pA at a hyperpolarized potential of -125 mV relative to rest) of short duration and, second, small bursting events (0.4 pA under similar conditions) of slightly longer duration. Pipette application of similar concentrations of veratridine had similar effects in that two categories of events were observed: first, bursts of large events with multiple conductance states and, second, small events of very long duration. The current/voltage relationship of these events was linear for the voltage range studied and the (extrapolated) reversal potential approximated +110 mV. These results support the hypothesis that veratridine, in small concentrations, induces a slow voltage-dependent activation of TTX-sensitive sodium channels, independent of the fast activating and inactivating sodium channels involved in action potential generation.

Quantitative analysis of sodium and potassium activation delays in fresh axons of the squid: Loligo forbesi.

Activation kinetics of the sodium and potassium conductances were re-examined in fresh axons of Loligo forbesi exhibiting very little if any potassium accumulation and a very small leak conductance, special attention being paid to the initial lag phase which precedes the turning-on of the conductances. The axons were kept intact and voltage-clamped at 2-3 degrees C. In all cases, the rising phase of the currents could be fitted with very good accuracy using the Hodgkin-Huxley (1952) equations although, in most cases, the turning-on of the conductance did not coincide with the beginning of the depolarizing test pulse. The delay which separates the change in potential and the turning-on of current (the activation delay) was analyzed quantitatively for different prepulse and pulse potentials. The measured activation delay differed significantly from the delay predicted by the original HH equations. This difference (the 'non-HH delay') varied with prepulse and pulse potentials. For the potassium current, the relationship between the non-HH delay and pulse potential for a constant prepulse was bell shaped, the maximum value (0.7 ms for a prepulse to -80 mV) being reached for about 0 mV. For this same current, the relationship between the non-HH delay and the prepulse potential for a constant pulse potential was sigmoidal, starting from a minimum value of around 0.5 ms at -100 mV and rising to 5 ms at -15 mV. Essentially similar results were obtained for the sodium current although the non-HH delay was three to five times smaller and the dependency upon prepulse potential not significant.(ABSTRACT TRUNCATED AT 250 WORDS)

Ionic currents in neurones cultured from embryonic cockroach (Periplaneta americana) brains.

Neurones isolated from embryonic cockroach brains were maintained in culture for up to 8 weeks. A single patch electrode was used to record voltage changes in response to injected current, membrane ionic currents under whole-cell voltage-clamp conditions or single-channel currents from isolated membrane patches. The voltage changes in response to injected current that depolarized the cell indicated increases in membrane permeability to calcium and potassium. These observations were confirmed using a voltage clamp. The potassium current observed in the youngest cultures turned on with a delay and was blocked by tetraethylammonium (TEA) and 4-aminopyridine (4-AP). Two kinds of decrease in the outward potassium current were observed. One may be associated with extracellular potassium accumulation, inactivation of the potassium channel or inactivation of a calcium channel. The other appears to be a voltage-dependent inactivation. The magnitude of the calcium permeability appeared to increase as the cultures developed, being most prominent in cultures more than 2 weeks old. Single-channel conductance measured from an analysis of records from six isolated membrane patches ranged from 15 to 110 pS. Except for one channel, the probability of the channels being open did not change appreciably with membrane potential. Our results suggest that much of the increase in potassium permeability may be due an increase in intracellular calcium level.

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.

Potassium homeostasis in the nervous system of cephalopods and crustacea.

1. Previous work has shown that nerve activity is associated with a significant release of potassium in the vicinity of the axonal membrane. Several mechanisms are normally present which reduce K+ accumulation in the extra-axonal space. 2. In intact connectives of the crayfish, Procambarus clarkii, repetitive stimulation of the giant axons was associated with an apparent hyperpolarization measured by an interstitial microelectrode, which most probably corresponds to depolarization of the inner face of the perineurial cells by K+ ions leaving the axons. 3. In desheathed connectives of the crayfish, potassium accumulated during long depolarizing voltage-clamp pulses but cleared away very quickly at the end of the pulse. 4. In the small squid, Alloteuthis subulata, repetitive stimulation of giant axons in situ in fresh and well-perfused animals did not result in a large decrease in the positive after potential (undershoot), reflecting the absence of potassium accumulation. A similar absence of accumulation was observed in vitro for carefully and freshly dissected isolated axons from live squids. 5. In both cases, deterioration of the physiological state of the axon was accompanied by a significant potassium accumulation. Potassium accumulation could also be reversibly enhanced by decreasing the osmotic pressure of the bathing medium, whereas hyperosmotic solutions had the opposite effect. These results are compatible with the idea that Schwann cells around the axon play a key role in K+ homeostasis. 6. Experiments on giant axons of the large squid species, Loligo forbesi confirmed the observations made on Alloteuthis in that fresh preparations exhibited little potassium accumulation. Under voltage-clamp conditions, 10 ms depolarizing pulses to various potential levels did not induce any accumulation in these preparations as reflected by the outward tail current. Large accumulation was observed in older axons under similar experimental conditions. 7. A large peri-axonal space associated with healthy glial cells appears to be a prerequisite for efficient K+ homeostasis in both crayfish and squid. Other mechanisms involving specific transport mechanisms across axonal and glial membranes are also likely to be involved.

Effects of N-alcohols on potassium conductance in squid giant axons.

The effect of bath application of several short chain N-alcohols on voltage-dependent potassium conductance has been studied in intact giant axons of Loligo forbesi under voltage-clamp conditions. All tested alcohols (methanol, ethanol, propanol, butanol, heptanol and octanol) were found to depress potassium conductance only at concentrations much larger than those necessary to reduce sodium conductance. The efficacy of the different molecules was correlated with the carbon-chain length. In all cases the effects were found to be at least partly reversible. Low concentrations of propanol (100 mM) or heptanol (1 mM) were found to increase potassium conductance whereas higher concentrations had the usual depressing effect. The two alcohols were found to induce a slow inactivation of the potassium conductance. A detailed analysis of the time course of the turning-on of the potassium current for various pulse potentials in the presence of TTX revealed that, for membrane potential values more positive than -20 mV, the time constant of activation was reduced in the presence of propanol or heptanol. The delay which separates the change in potential and the turning-on of the potassium current, which was systematically analysed for different pulse and prepulse potential values, was increased by the two alcohols, the curve relating this delay to prepulse potential being shifted towards larger (positive) delays. This high degree of complexity in the effects on potassium conductance suggests that the alcohol molecules modify several more or less independent mechanisms associated with the turning-on of the potassium current.