An Analysis of the Membrane Potential along a Clamped Squid Axon

A partially depolarized squid axon membrane is assumed to have a quasi-steady state negative resistance, the membrane potential is clamped at one point, and a distribution of potential along the axon is obtained from the cable equation. Nominal experimental values of -2 ohm cm² for the membrane and 6 ohm cm² for the internal and external current electrodes and the axoplasm and sea water between them are used for illustration. The potential and current may be uniform for an axon and electrode length less than 1.2 mm. For a long axon the potential varies as the cosine of the distance within 0.8 mm of the control point. Beyond this the potential variation is exponential and the entire pattern is about 5 mm long. The average current density out to 0.3 mm from the control point is within 10 per cent of the potential clamp value. These distributions are stable for control amplifications of about unity and more.     

TRANSVERSE ELECTRIC IMPEDANCE OF THE SQUID GIANT AXON

The impedance of the excised giant axon from hindmost stellar nerve of Loligo pealii has been measured over the frequency range from 1 to 2500 kilocycles per second. The measurements have been made with the current flow perpendicular to the axis of the axon to permit a relatively simple analysis of the data. It has been found that the axon membrane has a polarization impedance with an average phase angle of 76° and an average capacity of 1.1µf./cm² at 1 kilocycle. The direct current resistance of the membrane could not be measured, but was greater than 3 ohm cm.² and the average internal specific resistance was four times that of sea water. There was no detectable change in the membrane impedance when the axon lost excitability, but some time later it decreased to zero.     

Analysis of Certain Errors in Squid Axon Voltage Clamp Measurements

Localized membrane current and potential measurements were made on the squid giant axon in voltage clamp experiments. Spatial control of potential was impaired by the use of axial current supplying electrodes with surface resistance greater than 20 ohms for a centimeter length of axon. No region of membrane which was indeed subjected to a potential step showed more than one inward current peak. Other patterns were results of space clamp failure. Membrane current and potential patterns during space clamp failure were approximately reproduced in computations on a model containing two membrane patches obeying the equations of Hodgkin and Huxley. Non-uniformities in the axon or electrodes are not necessary for non-uniform electrical behavior. An extension of the core conductor model which includes the axial wire and external solution has been analyzed. The space constant of electrotonic spread is less than 0.5 mm with a usable electrode. Errors of about 5 per cent are introduced by ignoring the external solution. Resistance between the membrane and the control electrodes reduces the control and a few ohm cm² could lead to serious errors in interpretation.     

Microbial Degradation of Crude Oil: Factors Affecting the Dispersion in Sea Water by Mixed and Pure Cultures

By means of the enrichment culture technique, a mixed population of microorganisms was obtained which catalyzed the dispersion of crude oil in supplemented sea water. From this enrichment culture, eight pure cultures were isolated and studied. Only one of the isolates (RAG-1) brought about a significant dispersion of crude oil. RAG-1 has been tentatively characterized as a member of the genus Arthrobacter. The other seven isolates gave rise to colonies on supplemented oil agar, but were neither able to disperse oil nor to stimulate the dispersion catalyzed by RAG-1. The dispersion of crude oil by either RAG-1 or the enrichment culture was absolutely dependent on exogenous sources of nitrogen and phosphorous and completely inhibited by 10⁻²m azide. The increase in cell number of RAG-1 was directly proportional to the concentration of crude oil added to the medium over the range 0.05 to 1.0 mg/ml. Within this linear region, 1.0 mg of crude oil yielded 9 × 10⁷ cells and approximately 65% of the oil was converted into a nonbenzene extractable form. Accompanying the emulsification was a decrease in the pH from 7.6 to 5.0. Acidic conditions, however, were neither necessary nor sufficient for oil dispersion. When sea water was supplemented with 0.029 mm K₂HPO₄ and 3.8 mm (NH₄)₂SO₄ and inoculated with RAG-1, oil dispersion occurred within 1 day. This dispersion could also be brought about by the supernatant following separation of the cells from the medium. Similarly, the supernatant obtained following growth of RAG-1 on hexadecane was capable of emulsifying crude oil in 60 min.     

The distribution,growth, and reproduction of the Antarctic limpet Nacella (Patinigera) concinna (Strebel, 1908)

Twenty-one monthly collections of the Antarctic limpet Nacella (Patinigera) concinna (Strebel, 1908) were obtained by divers at Signy Island, South Orkney Islands. A mean monthly population density of 123.7 ± 21.2 · m^?2, mean biomass of 13.7 ± 2.7 g dry tissue wt · m^?2, and annual production of 2.9 g · m^?2 were recorded in the depth range 2–12 m below mean low water.Shell growth was slow with a maximum growth rate, in the first 3–5 yr of life, of 3 4 mm per year. Maturity was attained at 7–8 yr (21 mm length), and maximum size (41 mm length) at about 21 yr. Unique spawning behaviour was observed in two Austral springs, and data relating spawning to the spring increase in sea temperature were obtained.     

ELECTRIC IMPEDANCE OF ARBACIA EGGS

The alternating current resistance and capacity of suspensions of unfertilized and fertilized eggs of Arbacia punctulata have been measured at frequencies from 10³ to 1.64 x 10⁷ cycles per second. The unfertilized egg has a static plasma membrane capacity of 0.73 µf./cm.² which is practically independent of frequency. The fertilized egg has a static membrane capacity of 3.1 µf./cm.² at low frequencies which decreases to a value of 0.55 µf./cm.² at high frequencies. The decrease follows closely the relaxation dispersion of the dielectric constant if the dissipation of such a system is ignored. It is considered more probable that the effect is due to a fertilization membrane of 3.1 µf./cm.² capacity lifted 1.5 µ. from the plasma membrane, the interspace having the conductivity of sea water. The suspensions show a frequency-dependent capacity at low frequencies which may be attributable to surface conductance. The equivalent low frequency internal specific resistance of both the unfertilized and fertilized egg is about 186 ohm cm. or about 6 times that of sea water, while the high frequency data extrapolate to a value of about 4 times sea water. There is evidence at the highest frequencies that the current is penetrating the nucleus and other materials in the cytoplasm. If this effect were entirely due to the nucleus it would lead to a very approximate value of 0.1 µf./cm.² for the capacity of the nuclear membrane. The measurements do not indicate any change in this effect on fertilization.     

BIOELECTRIC POTENTIALS IN VALONIA : THE EFFECT OF SUBSTITUTING KCl FOR NaCl IN ARTIFICIAL SEA WATER

The P.D. across the protoplasm of Valonia macrophysa has been studied while the cells were exposed to artificial solutions resembling sea water in which the concentration of KCl was varied from 0 to 0.500 mol per liter. The P.D. across the protoplasm is decreased by lowering and increased by raising the concentration of KCl in the external solution. Changes in P.D. with time when the cell is treated with KCl-rich sea water resemble those observed with cells exposed to Valonia sap. Varying the reaction of natural sea water from pH 5 to pH 10 has no appreciable effect on the P.D. across Valonia protoplasm. Similarly, varying the pH of KCl-rich sea water within these limits does not alter the height of the first maximum in the P.D.-time curve. The subsequent behavior of the P.D., however, is considerably affected by the pH of the KCl-rich sea water. These changes in the shape of the P.D.-time curve have been interpreted as indicating that potassium enters Valonia protoplasm more rapidly from alkaline than from acidified KCl-rich sea water. This conclusion is discussed in relation to certain theories which have been proposed to explain the accumulation of KCl in Valonia sap. The initial rise in P.D. when a Valonia cell is transferred from natural sea water to KCl-rich sea water has been correlated with the concentrations of KCl in the sea waters. It is assumed that the observed P.D. change represents a diffusion potential in the external surface layer of the protoplasm, where the relative mobilities of ions may be supposed to differ greatly from their values in water. Starting with either Planck''s or Henderson''s formula, an equation has been derived which expresses satisfactorily the observed relationship between P.D. change and concentration of KCl. The constants of this equation are interpreted as the relative mobilities of K⁺, Na⁺, and Cl^- in the outer surface layer of the protoplasm. The apparent relative mobility of K⁺ has been calculated by inserting in this equation the values for the relative mobilities of Na⁺ (0.20) and Cl^- (1.00) determined from earlier measurements of concentration effect with natural sea water. The average value for the relative mobility of K⁺ is found to be about 20. The relative mobility may vary considerably among different individual cells, and sometimes also in the same individual under different conditions. Calculation of the observed P.D. changes as phase-boundary potentials proved unsatisfactory.     

Effect of Temperature on the Potential and Current Thresholds of Axon Membrane

The effect of temperature on the potential and current thresholds of the squid giant axon membrane was measured with gross external electrodes. A central segment of the axon, 0.8 mm long and in sea water, was isolated by flowing low conductance, isoosmotic sucrose solution on each side; both ends were depolarized in isoosmotic KCl. Measured biphasic square wave currents at five cycles per second were applied between one end of the nerve and the membrane of the central segment. The membrane potential was recorded between the central sea water and the other depolarized end. The recorded potentials are developed only across the membrane impedance. Threshold current values ranged from 3.2 µa at 267deg;C to 1 µa at 7.5°C. Threshold potential values ranged from 50 mv at 26°C to 6 mv at 7.5°C. The mean Q₁₀ of threshold current was 2.3 (SD = 0.2), while the Q₁₀ for threshold potentials was 2.0 (SD = 0.1).     

A New Species of Rhaponticoides (Asteraceae) from Southern Italy

A new diploid (2n?=?30) species, Rhaponticoides calabrica, is described from Calabria and Basilicata (southern Italy). This species differs from the closely related R. centaurium – an endemic of Apulia and northern Basilicata – by different flower colour (white in the former, purplish in the latter), width of membranous margin of phyllaries (1.8?±?0.4 mm vs 0.8?±?0.3 mm) and pappus length (6.8?±?0.9 mm vs 9.1?±?1.0 mm). The two species are completely allopatric and seem to have also different chromosome numbers.     

Sodium and Potassium Ion Effluxes from Squid Axons under Voltage Clamp Conditions

Squid giant axons loaded with Na²⁴ were subjected to short duration (0.5 msec.) clamped depolarizations of about 100 mv at frequencies of 20/sec. and 60/sec. while in choline sea water. Under such conditions the early outward current was just about maximal at the time of termination of the clamping pulse. An integration of the early current versus time record gave 1.2 μcoulomb/cm² pulse, while a measurement of the extra Na²⁴ efflux resulting from repetitive pulsing gave a charge transfer of 1.4 μcoulomb/cm² pulse. In sodium-containing sea water and with pulses 50-75 mv more positive than E_Na the Na²⁴ efflux is about 3 times the measured charge transfer. The efflux of K⁴² from a previously loaded axon into normal sea water is only 50 per cent of the measured charge transfer when the membrane is held for about 5 msec. at a potential such that there is no early current, and such pulses are at 10-20/sec. The experiments appear to confirm the suggestion that the early current during bioelectric activity is sodium but provide unsatisfactory support for the identification of the delayed but sustained current solely with potassium ions. Resting Na⁺ efflux is 0.6 pmole/cm² sec. mmole [Na]₁, while the apparent K⁺ efflux is about 250 pmole/cm² sec. and is little affected by hyperpolarization.     

An analysis of conductance changes in squid axon

The membrane of the squid axon is considered on the basis of a pore model in which the distribution of the pore sizes strongly favors K⁺ transfer when there is no potential. Electrical asymmetry causes non-penetrating ions on the membrane capacitor to exert a mechanical force on both membrane surfaces and this force results in a deformation of the membrane pore system such that it assumes a distribution of sizes favoring the ions exerting mechanical force. The ions involved appear to be Ca⁺⁺ on the outside of the membrane and isethionate^-, (i^-) on the inside; as Ca⁺⁺ is equivalent in size to Na⁺, the charged membrane is potentially able to transfer Na⁺, when the ions deforming the membrane pore distribution are removed. A depolarization of the membrane leads to an opening of pores that will allow Na⁺ penetration and a release of the membrane from deformation. The pores revert to the zero-potential pore size distribution hence the Na permeability change is a transient. Calculation shows that the potassium conductance vs. displacement of membrane potential curve for the squid axon and the "inactivation" function, h, can be obtained directly from the assumed membrane distortion without the introduction of arbitrary parameters. The sodium conductance, because it is a transient, requires assumptions about the time constants with which ions unblock pores at the outside and the inside of the membrane.     

MEMBRANE POTENTIAL OF THE SQUID GIANT AXON DURING CURRENT FLOW

The squid giant axon was placed in a shallow narrow trough and current was sent in at two electrodes in opposite sides of the trough and out at a third electrode several centimeters away. The potential difference across the membrane was measured between an inside fine capillary electrode with its tip in the axoplasm between the pair of polarizing electrodes, and an outside capillary electrode with its tip flush with the surface of one polarizing electrode. The initial transient was roughly exponential at the anode make and damped oscillatory at the sub-threshold cathode make with the action potential arising from the first maximum when threshold was reached. The constant change of membrane potential, after the initial transient, was measured as a function of the total polarizing current and from these data the membrane potential is obtained as a function of the membrane current density. The absolute value of the resting membrane resistance approached at low polarizing currents is about 23 ohm cm.². This low value is considered to be a result of the puncture of the axon. The membrane was found to be an excellent rectifier with a ratio of about one hundred between the high resistance at the anode and the low resistance at the cathode for the current range investigated. On the assumption that the membrane conductance is a measure of its ion permeability, these experiments show an increase of ion permeability under a cathode and a decrease under an anode.     

Distribution, standing stock, growth, mortality and production of Strongylocentrotus pallidus (Echinodermata: Echinoidea) in the northern Barents Sea

The regular sea urchin, Strongylocentrotus pallidus (G.O. Sars, 1871), is a widespread epibenthic species in high-Arctic waters. However, little is known about its distribution, standing stock, population dynamics and production. In the northern Barents Sea, S. pallidus was recorded on seabed still photographs at 10 out of 11 stations in water depths of 80–360?m. Mean abundances along photographic transects of 150–300 m length ranged between <0.1 and 14.7?ind. m^?2 yielding a grand average of 3.6?ind.?m^?2. The small-scale distribution along the transects was patchy, with densities varying from nil to an overall maximum of 25.5 ind. m^?2, and exhibited a significant relation to the number of stones present. Sea urchin test diameters, measured on scaled photographs, extended from 7 to 90?mm. Median values at single stations varied from 14 to 46?mm, showing a significant inverse relationship to water depth. Biomass, estimated by combining photographic abundances, size frequencies and a size-mass function established with trawled specimens, ranged between <0.1 and 3.0?g ash-free dry mass m^?2, averaging about 1.0?g ash free dry mass m^?2. An analysis of skeletal growth bands in genital plates was carried out with 143 trawled individuals ranging in test diameter (D) from 4 to 48?mm. Assuming these bands to represent annual growth marks, the ages of the specimens analysed ranged between 3 and 42 years. A von Bertalanffy function was fitted to size-at-age data to model individual growth pattern (D_∞?=?102.3?mm, k?=?0.011 year^?1, t₀?=?0.633?year). The annual mortality rate Z of the population in the northern Barents Sea was estimated from a size-converted catch curve to be 0.08 year^?1. Applying the weight-specific growth rate method, the average P/B ratio and the mean annual production of this population were estimated as 0.07 year^?1 and 0.076?g AFDM m^?2 year^?1, respectively. In conclusion, S. pallidus is characterized by slow growth, low mortality, high longevity and low productivity. Because of its relatively high biomass, it is considered to contribute significantly to total benthic standing stock and carbon flux in the study area.     

Complete Spinal Cord Injury and Brain Dissection Protocol for Subsequent Wholemount In Situ Hybridization in Larval Sea Lamprey

After a complete spinal cord injury, sea lampreys at first are paralyzed below the level of transection. However, they recover locomotion after several weeks, and this is accompanied by short distance regeneration (a few mm) of propriospinal axons and spinal-projecting axons from the brainstem. Among the 36 large identifiable spinal-projecting neurons, some are good regenerators and others are bad regenerators. These neurons can most easily be identified in wholemount CNS preparations. In order to understand the neuron-intrinsic mechanisms that favor or inhibit axon regeneration after injury in the vertebrates CNS, we determine differences in gene expression between the good and bad regenerators, and how expression is influenced by spinal cord transection. This paper illustrates the techniques for housing larval and recently transformed adult sea lampreys in fresh water tanks, producing complete spinal cord transections under microscopic vision, and preparing brain and spinal cord wholemounts for in situ hybridization. Briefly, animals are kept at 16 °C and anesthetized in 1% Benzocaine in lamprey Ringer. The spinal cord is transected with iridectomy scissors via a dorsal approach and the animal is allowed to recover in fresh water tanks at 23 °C. For in situ hybridization, animals are reanesthetized and the brain and cord removed via a dorsal approach.     

Determination of ketotifen fumarate by capillary electrophoresis with tris(2,2′‐bipyridyl) ruthenium(II) electrochemiluminescence detection

On the basis of an europium (III)‐doped Prussian blue analog film modifying platinum electrode as the working electrode, a Ru(bpy)₃²⁺‐based electrochemiluminescence (ECL) assay coupled with capillary electrophoresis has been first established for the determination of ketotifen fumarate (KTF). Analytes were injected onto a separation capillary of 50 cm length (50 μm i.d., 360 μm o.d.) by electrokinetic injection for 10 s at 10 kV. Parameters related to the separation and detection were discussed and optimized. It was proved that 15 mm phosphate buffer at pH 8.0 could achieve the most favorable resolution, and the highest sensitivity of detection was obtained using the detection potential at 1.25 V and 5 mm Ru(bpy)₃²⁺ in 100 mm phosphate buffer at pH 8.0 in the detection reservoir. Under the optimized conditions, the ECL intensity was in proportion to KTF concentration over the range from 3.0 × 10^?8 to 5.0 × 10^?6 g mL^?1 with a detection limit of 2.1 × 10^?8 g mL^?1 (3σ). The relative standard deviations of the ECL intensity and the migration time were 0.95 and 0.26%, respectively. The developed method was successfully applied to determine KTF contents in pharmaceuticals and human urine with recoveries between 99.5 and 107.0%. Copyright © 2010 John Wiley & Sons, Ltd.     

Effects of Replacement of External Sodium Chloride with Sucrose on Membrane Currents of the Squid Giant Axon

It was observed that a reduction of the sodium chloride concentration in the external solution bathing a squid giant axon by replacement with sucrose resulted in marked decreases in the peak inward and steady-state outward currents through the axon membrane following a step decrease in membrane potential. These effects are quantitatively acounted for by the increase in series resistance resulting from the decreased conductivity of the sea water and the assumption that the sodium current obeys a relation of the form I = k₁C₁ - k₂C₂ where C₁, C₂ are internal and external ion activities and k₁, k₂ are independent of concentration. It is concluded that the potassium ion current is independent of the sodium concentration. That the inward current is carried by sodium ions has been confirmed. The electrical potential (or barrier height) profile in the membrane which drives sodium ions appears to be independent of sodium ion concentration or current. A specific effect of the sucrose on hyperpolarizing currents was observed and noted but not investigated in detail.     

Evapotranspiration over the Grassland Field in the Liudaogou Basin of the Loess Plateau,China

Severe desertification on the Loess Plateau of China since the 17th century as a result of improper land use has caused critical soil erosion and water shortages in the lower reaches of the Yellow River. To prevent further soil erosion, it is very important to cover the bare land surface with natural vegetation. Evapotranspiration (ET) over the grassland (Stipa bungeana) in Shenmu County of the Loess Plateau during the growing season is estimated to be about 1 mm day^–1, and the ratio of ET to reference Evapotranspiration (ET/ET₀) is below 0.3. Evaporation from the bare soil surface simulated by a three-layer soil model is less than ET; that is, ET is 1.5 times greater than evaporation from the soil surface during the growing season.This study examines the relationships among the surface resistance r_s in the Penman–Monteith equation, solar radiation, vapor-pressure deficit, temperature, wind speed, and soil water content. Surface resistance (r_s) is strongly affected by the vapor-pressure deficit and soil water content.     

Kinetics of ion movement in the squid giant axon

The loss of Na²², K⁴², and Cl³⁶ from single giant axons of the squid, Loligo pealii, following exposure to an artificial sea water containing these radioisotopes, occurs in two stages, an initial rapid one followed by an exponential decline. The time constants of the latter stage for the 3 ion species are, respectively, 290, 200, and 175 minutes. The outflux of sodium is depressed while that of potassium is accelerated in the absence of oxygen; the emergence of potassium is slowed by cocaine, while that of sodium is unaffected. One cm. ends of the axons take up about twice as much radiosodium as the central segment; this difference in activity is largely preserved during exposure to inactive solution. Such marked differences are not observed with radiopotassium. From the experimental data estimates are given of the influxes and outfluxes of the individual ions. The kinetics of outflux suggests a cortical layer of measureable thickness which contains the ions in different proportions from those in the medium and which governs the rate of emergence of these ions from the axon as though it contained very few but large (relative to ion dimensions) pores.     

Sea water chlorophyll-a estimation using hyperspectral images and supervised Artificial Neural Network

The use of satellite hyperspectral images has improved the extraction of information compared to multispectral images. Although designed as a technical demonstration for land applications, Hyperion satellite hyperspectral images are used to estimate sea water parameters in the coastal area. A combination of turbid river inputs, as well as the open sea flushing, determines the quality of the sea water in the coastal area and the status of its environment. In addition, the existence of different source of pollution adds to the complexity of the coastal sea water analysis. The field campaigns to retrieve sea water parameters provided by the past completed projects were coincident with acquisition of the Hyperion image covering the pilot area. A robust method based on a supervised Feed-Forward Back-Propagation Artificial Neural Network (ANN-BP) algorithm is applied to retrieve the concentration of chlorophyll-a from hyperspectral image. In addition, Hyperion images are used to show the variation of chlorophyll-a during two different periods of time. The variation is due to many manmade environmental disasters such as oil spill and continuous discharge of chemical and solid wastes. The research proves that the new method based on ANN has improved the mathematical regression methods to a coefficient of determination almost equal 1 compared to about 0.4 for the methods not based on ANN-BP.     

ELECTRIC IMPEDANCE OF SUSPENSIONS OF ARBACIA EGGS

Apparatus has been designed and constructed for the measurement of the electric impedance of suspensions of Arbacia eggs in sea water to alternating currents of frequencies from one thousand to fifteen million cycles per second. This apparatus is simple, rugged, compact, accurate, and rapid. The data lead to the conclusions that the specific resistance of the interior of the egg is about 90 ohm cm. or 3.6 times that of sea water, and that the impedance of the surface of the egg is probably similar to that of a "polarization capacity". The characteristics of this surface impedance can best be determined by measurements of the capacity and resistance of suspensions of eggs. No specific change has been found in the interior resistance or the surface impedance which can be related either to membrane formation or to cell division.