Normal Artificial Sea Water Research Articles

Carbon dioxide sensitivity and its role in multifunctional neurons in the mollusk Onchidium

Intrinsically photoresponsive neurons in the abdominal ganglion of the amphibious mollusk Onchidium named Ip-1 and Ip-2 (Ip-1/2) react to several different stimuli. These neurons respond to light with slow hyperpolarization and to CO2 stimulation with slow depolarization. In this study, increasing the concentration of CO2 in the air caused hyperventilation and enlargement of the pneumostome in the intact animal. In a semi-intact preparation, pouring artificial seawater (ASW) with dissolved CO2 onto the central ganglia caused the previously closed pneumostome to open. In an ASW environment, Ip-1/2 neurons depolarized even under conditions of constant pH (alkaline ASW) and after dissolution of CO2. This depolarization prolonged the firing of action potentials in Ip-1/2 neurons. Adding protons (H+) to ASW caused Ip-1/2 depolarization only when the neurons' membranes were depolarized to a potential above the resting potential. Furthermore, in the presence of the carbonic anhydrase inhibitor acetazolamide (AZ), CO2-induced excitation in Ip-1/2 neurons was increased in both normal and alkaline ASW. These results suggest that when dissolved in ASW, CO2 directly induced the depolarizing response in Ip-1/2 neurons. Since Ip-1/2 neurons participate in pneumostome opening, these results suggest that increased CO2 levels in ASW directly stimulate CO2-sensitive central neurons, promoting ventilation.

A novel stiffening factor inducing the stiffest state of holothurian catch connective tissue

The dermis of sea cucumbers is a catch connective tissue or mutable collagenous tissue that shows large changes in stiffness. Extensive studies on the dermis revealed that it can adopt three different states having different mechanical properties that can be reversibly converted. These are the stiff, standard and soft states. The standard state is readily produced when a dermal piece is immersed in the sea water containing Ca²+, whereas the soft state can be produced by removal of Ca²+. A stiffening protein, tensilin, has been isolated from some sea cucumbers (Cucumaria frondosa and Holothuria leucospilota). Although tensilin converts the state of the dermis from soft to standard, it cannot convert from standard to stiff. In this study, we isolated and partially purified a novel stiffening factor from the dermis of Holothuria leucospilota. The factor stiffened the dermis in normal artificial sea water (ASW) but did not stiffen the soft dermis in Ca²+-free ASW. It also stiffened the dermis that had been converted to the standard state in Ca²+-free ASW by the action of tensilin. These results suggest that the factor produces the stiff dermis from the standard state but cannot work as a stiffener on the soft dermis. Its addition to longitudinal muscles of the sea cucumber produced no effects, suggesting that its effect is specific to the catch connective tissue. Its stiffening activity was susceptible to trypsin, meaning that it is a polypeptide, and its molecular mass estimated from gel filtration chromatography was 2.4 kDa.

Repetitive Firing Triggers Clustering of Kv2.1 Potassium Channels in Aplysia Neurons

The Kv2.1 gene encodes a highly conserved delayed rectifier potassium channel that is widely expressed in neurons of the central nervous system. In the bag cell neurons of Aplysia, Kv2.1 channels contribute to the repolarization of action potentials during a prolonged afterdischarge that triggers a series of reproductive behaviors. Partial inactivation of Aplysia Kv2.1 during repetitive firing produces frequency-dependent broadening of action potentials during the afterdischarge. We have now found that, as in mammalian neurons, Kv2.1 channels in bag cell neurons are localized to ring-like clusters in the plasma membrane of the soma and proximal dendrites. Either elevation of cyclic AMP levels or direct electrical stimulation of afterdischarge rapidly enhanced formation of these clusters on the somata of these neurons. In contrast, injection of a 13-amino acid peptide corresponding to a region in the C terminus that is required for clustering of Kv2.1 channels produced disassociation of the clusters, resulting in a more uniform distribution over the somata. Voltage clamp recordings demonstrated that peptide-induced dissociation of the Kv2.1 clusters is associated with an increase in the amplitude of delayed rectifier current and a shift of activation toward more negative potentials. In current clamp recording, injection of the unclustering peptide reduced the width of action potentials and reduced frequency-dependent broadening of action potentials. Our results suggest that rapid redistribution of Kv2.1 channels occurs during physiological changes in neuronal excitability.

Statocyst hair cell activation of identified interneurons and foot contraction motor neurons in Hermissenda.

Pavlovian conditioning of Hermissenda produces both light-elicited inhibition of normal positive phototactic behavior and conditioned stimulus (CS)-elicited foot-shortening. Rotation, the unconditioned stimulus (US) elicits foot-shortening and reduced forward ciliary locomotion. The neural circuit supporting ciliary locomotion and its modulation by light is known in some detail. However, the neural circuits responsible for rotation-elicited foot-shortening and reduced forward ciliary locomotion are not known. Here we describe components of the neural circuit in Hermissenda that produce anterior foot contraction and ciliary activation mediated by statocyst hair cells. We have characterized in semi-intact preparations newly identified pedal ventral contraction motor neurons (VCMNs) and interneurons (I(b)). Type I(b) interneurons receive polysynaptic input from statocyst hair cells and project directly to VCMNs and cilia-activating motor neurons. Depolarization of VCMNs with extrinsic current in normal artificial seawater (ASW) and high-divalent cation ASW, and under conditions where central synaptic transmission was suppressed with 5 mM Ni(2+) ASW, elicited a contraction of the ipsilateral anterior foot measured from videotape recordings. Mechanical displacement of the statocyst or depolarization of identified statocyst hair cells with extrinsic current elicited spikes and complex excitatory postsynaptic potentials (EPSPs) in type I(b) interneurons and complex EPSPs and spikes recorded in VCMNs. Type I(b) interneurons are electrically coupled and project to VCMNs and VP1 cilia-activating motor neurons located in the contralateral pedal ganglia. The results indicate that statocyst hair-cell-mediated anterior foot contraction and graviceptive ciliary locomotion involve different interneuronal circuit components from the circuit previously identified as supporting light modulated ciliary locomotion.

Competence of the animal cap to react with the inductive signal from micromere descendants in the hatching blastula stage of echinoid embryos

Micromere signalling is a key to understanding the developmental mechanisms underlying endomesoderm differentiation along the A-V axis in sea urchin embryos. A recent study has shown that micromere activity in inducing endo-mesoderm differentiation of mesomere descendants is, unexpectedly, maximal at the hatching blastula (HB) stage in the echinoids Scaphechinus mirabilis and Hemicentrotus pulcherrimus. This study focused mainly on the timing of emission of the inductive signal by the micromere descendants. The timing of animal cap competent to react to the inductive signal from micromeres was not specifically investigated.In the present study, we examined the competence of mesomere descendants at the HB stage to react to the inductive signal of micromere descendants by producing recombinant embryos of the descendants of mesomeres and micromeres.Adults of the sand dollar Scaphechinus mirabilis and the sea urchin Hemicentrotus pulcherrimus were collected along the shore of Shiraishi Island, Okayama Prefecture and in the vicinity of the Misaki Marine Biological Station, Kanagawa Prefecture, respectively. The fertilised eggs were separated into two groups immediately after removal of the fertilisation membranes. One group was cultured in normal artificial seawater (ASW), and the other in ASW containing 50 mg/ml of rhodamine B isothiocyanate (RITC). At the early 16-cell stage unlabelled embryos were transferred to calcium-free seawater (CFSW) and dissected in the equatorial plane with a glass needle to isolate animal caps consisting of eight mesomeres. Embryos labeled with RITC were also transferred to CFSW at the same stage and dissected to isolate four micromeres. An isolated animal cap and a quartet of micromeres were cultured separately in ASW and recombined at the stage corresponding to the HB of control (undisturbed) embryos.

Cellular Analog of Differential Classical Conditioning inAplysia: Disruption by the NMDA Receptor Antagonistdl-2-Amino-5-Phosphonovalerate

We previously showed that the associative enhancement of Aplysia siphon sensorimotor synapses in a cellular analog of classical conditioning is disrupted by infusing the Ca(2+) chelator 1, 2-bis(2-aminophenoxy)ethane-N,N-N',N'-tetraacetic acid into the postsynaptic motor neuron before training or by training in the presence of the NMDA receptor antagonist DL-2-amino-5-phosphonovalerate (APV). Our earlier experiments with APV used a nondifferential training protocol, in which different preparations were used for associative and nonassociative training. In the present experiments we extended our investigation of the role of NMDA receptor type potentiation in learning in Aplysia to differential conditioning. A cellular analog of differential conditioning was performed with a reduced preparation that consisted of the CNS plus two pedal nerves. A siphon motor neuron and two siphon sensory neurons, both of which were presynaptically connected to the motor neuron, were impaled with sharp microelectrodes. One sensorimotor synapse received paired stimulation with a conditioned stimulus (brief activation of a single sensory neuron) and an unconditioned stimulus (pedal nerve shock), whereas the other sensorimotor synapse received unpaired stimulation. Training in normal artificial seawater (ASW) resulted in significant differential enhancement of synapses that received the paired stimulation. Training in APV blocked this differential synaptic enhancement. A comparison of the present data with the data from earlier experiments that used nondifferential training is consistent with the possibility that differential training comprises competition between the presynaptic sensory neurons. Synaptic competition may contribute significantly to the associative effect of paired stimulation in the differential training paradigm.

Dye coupling in the muscles controlling squid chromatophore expansion

Dye coupling between the cone-shaped radial muscle fibres, which control the expansion and closing of a squid chromatophore organ, was investigated in the squid Loligo vulgaris. Particular attention was paid to the role of the myomuscular junctions located between the muscle fibres. Lucifer Yellow was injected ionophoretically into single muscle fibres under normal artificial sea water (ASW) and under various concentrations of calcium in ASW. Under ASW, 44% of muscle fibres examined were dye-coupled, 82% were coupled under calcium-free sea water and 67% were coupled under sea water containing high concentrations of calcium. Dye transfer was blocked by octanol. Muscle fibres were never seen to link adjacent chromatophore organs. Results are discussed in terms of the role of the myomuscular junctions in the regulation of chromatophore expansion in the living animal.

Calcium and Cyclic AMP Mediate Sperm Activation, but Ca2+ Alone Contributes Sperm Chemotaxis in the Ascidian, Ciona savignyi: (ascidian/sperm motility/chemotaxis/calcium/cAMP).

Sperm-activating and -attracting factor (SAAF) released from the ascidian, Ciona savignyi, was partially purified from egg seawater with ethanol extraction and separation with the two-phase system of chloroform and water. SAAF did not activate sperm motility and cAMP synthesis in calcium-free seawater (CaFSW), but activated the both in the presence of Ca2+ . Sperm activation by SAAF in Ca2+ -containing medium was inhibited by flunarizine, a T-type Ca2+ channel antagonist, but L-type Ca2+ channel specific antagonists had no effect. Theophylline, a phosphodiesterase inhibitor, induced the increase of cAMP level and sperm activation in CaFSW without SAAF. On the other hand, the theophylline-activated sperm in CaFSW did not exhibit chemotaxis toward the tip of glass capillary containing SAAF, but upon the addition of Ca2+ they were attracted toward SAAF in the same manner as chemotaxis in normal artificial seawater. These results suggest that sperm activation is induced by the increased cAMP level caused by Ca2+ influx through T-type Ca2+ channel, and that Ca2+ alone mediates the sperm chemotaxis in Ciona.

Specific blockage of squid axon resting potassium permeability by Haliclona viridis (Porifera: Haliclonidae) toxin (HvTX)

C. Sevcik, A. I. García-Rodriguez, G. D'Suze and A. J. Mijares. Specific blockage of squid axon resting potassium permeability by Haliclona viridis (Porifera: Haliclonidae) toxin (HvTX). Toxicon 32, 773–788, 1994.—The action of partially purified HvTX, toxin of the marine sponge H. viridis, was explored on the giant axon of the tropical squids Doryteuthis plei and Sepioteuthis sepioidea. HvTX depolarizes the nerves dose dependently. The effect occurs after blocking sodium channels with tetrodotoxin (1 μM), removing external Na +, blocking electrically excitable K + channels with 3,4-diaminopyridine (10 mM) or internal and external application of tetraethylammonium (40 mM). Ouabain (up to 10 mM) does not modify HvTX effect. The action of HvTX occurs only when it is applied to the outer phase of the nerve membrane; microinjection of the toxin into the axons lacks depolarizing effects. HvTX reduces the dependence of membrane potential on external potassium concentration. The apparent 86Rb + permeability ( π′) was measured in axons of S. sepioidea. The value of π′ in normal artificial sea water was 80 (61, 96) nm/sec (median and its 95% confidence interval, n = 8) and raised to 1030 (588, 2113) nm/sec ( n = 7) when the axons were depolarized to 0 mV raising external K + to 300 mM. In axons depolarized with HvTX (10 mM external K +) to 0 mV, π′ was 88 (55, 97) nm/sec ( n = 8). HvTX could not prevent ( P ⪢ 0.05) the increase in π′ induced by 300 mM K + when the ion concentration was raised before toxin application [ π′ = 660 (354, 1876) nm/sec, n = 7]. Most of the 86Rb + permeability increase in high K + was prevented if HvTX was added before external K + was raised [ π′ = 298 (264, 337) nm/sec, n = 8]. All the measures of π′ were carried out in solutions containing 1 μM tetrodotoxin, 1 mM 3,4-diaminopyridine and 2 mM ouabain.

Spatiotemporal distribution of Ca2+ following axotomy and throughout the recovery process of cultured Aplysia neurons.

This study investigates the alterations in the spatiotemporal distribution pattern of the free intracellular Ca2+ concentration ([Ca2+]i) during axotomy and throughout the recovery process of cultured Aplysia neurons, and correlates these alterations with changes in the neurons input resistance and trans-membrane potential. For the experiments, the axons were transected while imaging the changes in [Ca2+]i with fura-2, and monitoring the neurons' resting potential and input resistance (Ri) with an intracellular microelectrode inserted into the cell body. The alterations in the spatiotemporal distribution pattern of [Ca2+]i were essentially the same in the proximal and the distal segments, and occurred in two distinct steps: concomitantly with the rupturing of the axolemma, as evidenced by membrane depolarization and a decrease in the input resistance, [Ca2+]i increased from resting levels of 0.05-0.1 microM to 1-1.5 microM along the entire axon. This is followed by a slower process in which a [Ca2+]i front propagates at a rate of 11-16 microns/s from the point of transection towards the intact ends, elevating [Ca2+]i to 3-18 microM. Following the resealing of the cut end 0.5-2 min post-axotomy, [Ca2+]i recovers in a typical pattern of a retreating front, travelling from the intact ends towards the cut regions. The [Ca2+]i recovers to the control level 7-10 min post-axotomy. In Ca(2+)-free artificial sea water (2.5 mM EGTA) axotomy does not lead to increased [Ca2+]i and a membrane seal is not formed over the cut end. Upon reperfusion with normal artificial sea water, [Ca2+]i is elevated at the tip of the cut axon and a membrane seal is formed. This experiment, together with the observations that injections of Ca2+, Mg2+ and Na+ into intact axons do not induce the release of Ca2+ from intracellular stores, indicates that Ca2+ influx through voltage gated Ca2+ channels and through the cut end are the primary sources of [Ca2+]i following axotomy. However, examination of the spatiotemporal distribution pattern of [Ca2+]i following axotomy and during the recovery process indicates that diffusion is not the dominating process in shaping the [Ca2+]i gradients. Other Ca2+ regulatory mechanisms seem to be very effective in limiting these gradients, thus enabling the neuron to survive the injury.

Integumental taurine transport in Mytilus gill: short-term adaptation to reduced salinity.

Taurine, a principal osmolyte in molluscan integument, is actively transported from sea water by Mytilus gill by means of a Na(+)-dependent process. In this study we examined the response of this transport to reductions in external salinity, i.e. the response to reductions in osmotic concentration as well as Na+ concentration. Acute exposure of isolated gill tissue to 60% artificial sea water (ASW) resulted in a greater than 85% inhibition of taurine uptake, substantially more than the 45% inhibition predicted on the basis of the acute reduction in external [Na+]. Within 60 min, however, taurine transport recovered to the level predicted by the Na+ concentration in dilute sea water. Isolated gills acutely exposed to 60% ASW made isosmotic to normal (100%) ASW with mannitol had rates of taurine uptake comparable to gills acclimated for 60 min. Taurine uptake by gill tissue exposed to 60% ASW for 60 min and then returned to 100% ASW for 90 min was not significantly different from that of control gills held in 100% ASW. Glucose uptake by the gill during acute exposure to reduced salinity responded in a pattern similar to that of taurine. Gill tissue increased by 20% in wet mass within 2 min of exposure to 60% ASW, but returned to control mass within 30-60 min, presumably reflecting cell volume regulation. Long-term (12 days) exposure to reduced salinities was not accompanied by increases in taurine transport over that of gills observed following the 60 min 'short-term' acclimation. These results suggest that Mytilus gill undergoes a rapid (albeit incomplete) recovery from the extreme inhibition of transport associated with abrupt changes in salinity, and the extent of recovery is defined by the availability of Na+ in the external medium. The extreme sensitivity of taurine uptake observed after acute exposure of gills to reduced salinity is related to the osmotic concentration of the medium, and is possibly linked to a change in cell volume.

Larval metamorphosis of the opisthobranch molluscAdalaria proxima(Gastropoda: Nudibranchia): the effects of choline and elevated potassium ion concentration

Veliger larvae of the nudibranch molluscAdalaria proximaare triggered to metamorphose to the benthic form by the adult prey bryozoan,Electro pilosa.Ion substitution and supplementation experiments with artificial sea water (ASW) have, however, shown that metamorphosis can be induced by elevation of the potassium ion concentration alone. Approximately 19 mM K+ASW (10 mM ‘excess’) was found to elicit maximal metamorphic responses: 29 and 39 mM K+ASW had no inductive effect. Choline chloride was also found to induce metamorphosis in a dose-dependent manner, with lO M ineffective, 10 M approximately threshold, and 5×10 M to 10 M optimal. Concentrations of choline >10 M were sub-lethally toxic. That the absence of larval metamorphosis on exposure to 29 and 39 mM K ASW was due to inhibition is inferred from interaction experiments with choline: at these concentrations of K, metamorphosis in response to choline could be abolished. Timed-exposure experiments indicated that artificial induction elicited by elevated K and choline involve either separate mechanisms, or different parts of the same pathway. Thus, whilst larvae required to be continuously exposed to 19 mM K ASW in order to complete metamorphosis, those exposed to 10 M choline would subsequently complete development in normal ASW following only 1–2 h exposure to the inducer. Preliminary experiments failed to specify further the nature of the natural inducer, beyond the confirmation that live intact colonies of the bryozoanElectro pilosawill trigger larvae to metamorphose.

Characterization of Vegetal Plate Cells Separated from Cytochalasin B-Treated Blastulae of the Sea Urchin, Clypeaster japonicus: (cytochalasin B/cell isolation/presumptive primary mesenchyme cell/vegetal plate cell/sea urchin).

Vegetal plate forming cells (VPCs) of the vegetal plate blastulae of the sea urchin, Clypeaster japonicus, had a layer of microfilaments on the basal side. The VPCs specifically protruded from the embryos after a treatment with 1 μg/ml of cytochalasin B (CB). Based on scanning electron microscopy, unlike other epithelial cells the protruded VPCs possessed neither cilium nor microvilli on their surface. The protruded VPCs were easily separated from the embryos by stirring the embryo suspension with pipette. An in vitro immunohistochemistry using a primary mesenchyme cell (PMC) surface-specific monoclonal antibody (MAb) raised against PMCs of Strongylocentrotus purpuratus showed that the MAb also specifically bound to the PMCs in mesenchyme blastulae of C. japonicus. The MAb bound in 81% of the separated VPCs in C. japonicus vegetal plate blastulae examined. However, the MAb binding occurred only after the separated VPCs were incubated in artificial sea water (ASW) for at least 1 hr. In the VPC-deprived embryos, gastrulation occurred after they were transferred to normal ASW. However, the PMCs and the spicules were not formed in these embryos. In conclusion, a majority of the VPCs separated from the CB-treated blastulae were presumptive PMCs. These VPCs provide an excellent source of presumptive PMCs.

The isotopic effects of D2O in developing sea urchin eggs.

When developing sea urchin eggs were treated with sea water containing 40% D2O (D2O-SW) at the 8-cell stage, the micromere formation was delayed and micromeres were larger than those seen in the control. But eggs returned to normal artificial sea water (NASW) at the 16- to 32-cell stage did not form abnormal spicules in larvae of Pseudocentrotus depressus. Little effect on the spicule formation of Hemicentrotus pulcherrimus was also noted. If the culture period in D2O-SW was extended until the hatching stage, the number of plutei with abnormal spicules increased. Primary mesenchyme cells of Pseudocentrotus depressus larvae failed to make two aggregated spicule rudiments on the ventral side of the larva and developed a ring-like spicule. This ring-like spicule, however, only occasionally occurred in the larvae of Hemicentrotus pulcherrimus. The cell cycle was longer in the presence of D2O. However, blastomeres managed to divide throughout the development. Larvae reared in 20% D2O-SW after the hatching stage developed into quasi-normal plutei but smaller than control. We found no exogastrulation in these larvae. Exogastrulation was found only in larvae continuously cultured in 40% D2O-SW from the early development. These results are inconsistent with previous reports made by other authors.

The Acrosome Reaction Induced by Dimethylsulfoxide in Sea Urchin Sperm: (sea urchin/sperm/acrosome reaction/dimethyl sulfoxide/nifedipine).

The acrosome reaction in sea urchin sperm, as judged by disappearance of the acrosomal vesicles in Nomarski optics, was induced by dimethylsulfoxide (DMSO) at concentration above 0.1% in normal artificial sea water. The number of the acrosome-reacted spermatozoa increased in proportion to DMSO concentration. The DMSO-induced acrosome reaction, as well as the jelly water- or A23187-induced one, was inhibited by nifedipine and hardly occurred in Ca2+ -free artificial sea water. However, the DMSO-induced acrosome reaction was found in a few number of spermatozoa in the presence of Ca2+ at above 0.5 mM, though the jelly water- or A23187-induced acrosome reaction did not occur at external Ca2+ levels lower than 1 mM. Dependency of the acrosome reaction by DMSO on external Ca2+ is somewhat lower than that of the reaction by jelly water. In Ca2+ -free artificial sea water, the acrosomal regions of DMSO-treated spermatozoa attached to their own tails. In some cases, spermatozoa thus treated with DMSO in Ca2+ free artificial sea water caused formation of fertilization membrane in a few number of eggs kept in Ca2+ -free artificial sea water. Even in the absence of extermal Ca2+ , preliminary step of the acrosome reaction seems to be completed probably by DMSO-induced weak Ca2+ -mobilization in spermatozoa.

Activation of Sea Urchin Eggs by Halothane and Its Inhibition by Dantrolene: (sea urchin eggs/fertilization/halothane/dantrolene/nifedipine).

Eggs of the sea urchin, Hemicentrotus pulcherrimus, were stimulated by halothane, known to induce Ca2+ release from sarcosome, to cause fertilization membrane formation in normal and Ca2+ free artificial sea water. In the absence of external Ca2+ , halothane-induced formation of fertilization membrane was inhibited by dantrolene, an inhibitor of Ca2+ release from sarcosome, but was not blocked by nifedipine, a Ca2+ antagonist specific to Ca2+ channels in plasma membrane. Ca2+ release from sedimentable fraction isolated from eggs was induced by halothane and was inhibited by dantrolene, but was not blocked by nifedipine. In normal artificial sea water, halothane-caused egg activation was not inhibited either by dantrolene or by nifedipine, but was blocked in the presence of both compounds. 45 Ca2+ influx was substantially stimulated by halothane in eggs exposed to 45 CaCl2 . Halothane-induced 45 Ca2+ influx into eggs was inhibited by nifedipine but was not blocked by dantrolene. When Ca2+ release from intracellular organellae is blocked, Ca2+ transport through Ca2+ channels in plasma membrane probably acts as a "fail-safe" system to induce an increase in cytosolic Ca2+ level, resulting in egg activation.

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.

D2O and the sodium pump in squid nerve membrane.

In 10 K artificial seawater (ASW), D2O replacement reduced the Na efflux of squid axons by about one third. In 0 K ASW, D2O replacement had little effect. D2O reduced the K+ sensitivity of the efflux but increased the affinity for K+. A 4 degrees decrease in temperature mimicked the effects of D2O. When axons were injected with arginine, to decrease the ATP/ADP ratio, they lost K+ sensitivity in normal ASW, as expected. Their efflux into 0 K ASW became D2O sensitive. The results are discussed in terms of conformational changes in the Na pump molecular complex.

Barium spikes are generated in the spines of the sea urchin Diadema antillarum

1. 1. In the presence of calcium, Ba 2+ ions, at concentrations as low as 1–2 mM, block the action potentials (a.p.'s) elicited by the electrical stimulation of the primary spines of Diadema antillarum. 2. 2. Lower concentrations of barium (0.1 mM) potentiate the a.p.'s recorded from spines equilibrated with Ca-Free artificial sea water (ASW). 3. 3. Exposure of the spines to a saturated solution of EDTA in Ca-free, unbuffered ASW reversibly blocks their electrical activity. 4. 4. Spines blocked by EDTA continue to generate a.p.'s following their equilibration with Ca-free ASW containing 1 m of Ba 2+ ions. 5. 5. The time course of the a.p.'s recorded from spines equilibrated with normal ASW is only slightly affected by the combined action of 15 mM of tetraethylammonium (TEA) and 5 mM of 4-aminopyridine (4-AP). 6. 6. In contrast, the duration of the a.p.'s recorded from spines blocked by EDTA and placed in 1 mM barium is markedly increased by the combined actions of TEA and 4-AP at the above concentrations. 7. 7. We conclude that the Ca-channels of the neurites present in Diadema spines are not, at least qualitatively, an exception with regard to their permeation by Ba 2+. The blocking action of low concentrations of these ions, particularly in the presence of calcium, may be explained by the extremely high surface to volume ratio prevailing in neurites with a diameter of only < 0.1–0.3 μm.

Light-evoked depolarizations in the retina of Strombus: role of calcium and other divalent cations.

Previous studies indicate that overlapping inward sodium and outward potassium currents play a role in generating the waveform of light-evoked depolarizations (LEDs) in one type of retinal neuron in Strombus luhuanus, a marine gastropod [Chinn, K. S., and Gillary, H. L. (1985). Comp. Biochem. Physiol. 80A:233-245]. This paper concerns the effects of divalent cations on the LED. The LED can exhibit a distinct early phase of depolarization (DE). Increasing the [Ca2+] in the artificial seawater (ASW) bathing medium reduced the amplitude of the entire LED, and omitting Ca2+ increased it. Adding 10 mM Sr2+ or 10 mM Mn2+ to either normal ASW or 0-Ca2+ ASW decreased the LED amplitude. Adding 10 mM Ba2+ to 0-Ca2+ ASW also decreased the LED amplitude, but adding Ba2+ to normal ASW selectively increased DE. Cd2+ (100 microM) selectively reduced DE when added to normal ASW but not when added to 0-Ca2+ ASW. The results show that a variety of divalent cations can alter the currents that underlie the LED. They also suggest that an inward Ca2+ current occurs during DE.