NATURE OF THE ACTION CURRENT IN NITELLA : V. PARTIAL RESPONSE AND THE ALL-OR-NONE LAW

When a stimulus arrives before recovery is complete there may be no response or only a partial response. A typical response appears to involve an immediate loss of potential at the inner protoplasmic surface but not at the outer surface. As long as recovery is incomplete only a part of the total potential is located at the inner protoplasmic surface and the loss of this part of the total potential can cause only a partial response; i.e., one of smaller magnitude than the normal. Even after the action curve has returned to the base line recovery may be incomplete and the response only a partial one. The return of the action curve to the base line means a recovery of total potential but if part of this is located at the outer protoplasmic surface and if this part is not lost when stimulation occurs the response can be only a partial one. During recovery there is a shift of potential from the outer to the inner protoplasmic surface. Not until this shift is completed can recovery be called complete. The response to stimulation then becomes normal because the loss of potential reaches the normal amount. In many cases the partial responses appear to conform to the all-or-none law. In other cases this is doubtful.     

MECHANICAL RESTORATION OF IRRITABILITY AND OF THE POTASSIUM EFFECT

Treatment of Nitella with distilled water apparently removes from the cell something which is responsible for the normal irritability and the potassium effect, (i.e. the large P.D. between a spot in contact with 0.01 M KCl and one in contact with 0.01 M NaCl). Presumably this substance (called R) is partially removed from the protoplasm by the distilled water. When this has happened a pinch which forces sap out into the protoplasm can restore its normal behavior. The treatment with distilled water which removes the potassium effect from the outer protoplasmic surface does not seem to affect the inner protoplasmic surface in the same way since the latter retains the outwardly directed potential which is apparently due to the potassium in the sap. But the inner surface appears to be affected in such fashion as to prevent the increase in its permeability which is necessary for the production of an action current. The pinch restores its normal behavior, presumably by forcing R from the sap into the protoplasm.     

Mechanical stimulation-induced chilling tolerance in tobacco suspension cultured cells and its relation to proline

Mechanical stimulation (MS), widely existing but usually ignored in nature, is one of the major environmental stress factors. MS by increasing the rotational speed of shaker incubator could alleviate a decrease in vitality of tobacco (Nicotiana tabacum L.) suspension cultured cells and reduce the accumulation of MDA under chilling stress at 1°C, which in turn improved survival percentage under chilling stress and regrowth ability of tobacco suspension cells after chilling stress. In addition, MS could increase the activity of Δ¹-pyrroline-5-carboxylate synthetase (P5CS) and induce the accumulation of endogenous proline in tobacco cells; exogenously applied proline also could enhance its endogenous level under normal culture conditions and survival percent-age of the cells under chilling stress. These results suggest that MS could improve chilling tolerance of tobacco suspension cells and the acquisition of this chilling tolerance was related to proline.     

INCREASED IRRITABILITY IN NITELLA DUE TO GUANIDINE

Guanidine applied to Nitella may lower the threshold of E.M.F. required to produce electrical stimulation and may give rise to trains of action currents. Its effect thus appears to be somewhat similar to that observed in animals. Rapid action currents are produced as well as "square topped" action curves and transitional forms. These effects may be due in part to increased protoplasmic conductivity produced by the penetration of guanidine.     

ACTION CURVES WITH SINGLE PEAKS IN NITELLA IN RELATION TO THE MOVEMENT OF POTASSIUM

In Nitella the action curve has two peaks, apparently because both protoplasmic surfaces (inner and outer) are sensitive to K⁺. Leaching in distilled water makes the outer surface insensitive to K⁺. We may therefore expect the action curve to have only one peak. This expectation is realized. The action curve thus obtained resembles that of Chara which has an outer protoplasmic surface that is normally insensitive to K⁺. The facts indicate that the movement of K⁺ plays an important part in determining the shape of the action curve.     

THE EXPERIMENTAL PRODUCTION OF DOUBLE PEAKS IN CHARA ACTION CURVES AND THEIR RELATION TO THE MOVEMENT OF POTASSIUM

The action curve in Chara seems to depend (as in Nitella) on the outward movement of K⁺ from the sap. Presumably the increase in permeability in the inner protoplasmic surface and the outward movement of K⁺ destroy the concentration gradient of K⁺ across the inner protoplasmic surface. Hence the outwardly directed P.D. disappears, causing the rise (spike) of the action curve. The outer protoplasmic surface is normally insensitive to K⁺. But when it is made sensitive to K⁺ by treatment with guanidine the outwardly moving K⁺ sets up a positive P.D. on reaching the outer surface and this causes the action curve to fall, producing a peak. Then the curve has 2 peaks, the second being due to the process of recovery. The action curve thus comes to resemble that of Nitella in which the outer protoplasmic surface is normally sensitive to K⁺.     

DELAYED POTASSIUM EFFECT IN NITELLA

In normal cells of Nitella replacement of NaCl by KCl makes the P.D. much less positive: this is called the potassium effect. Cells which have lost the potassium effect usually show little or no change of P.D. when NaCl is replaced by KCl but an occasional cell responds after a delay. It seems possible that the delay may be largely due to the time required for potassium to combine with an organic substance, thus forming a compound which sensitizes the protoplasmic surface to the action of potassium.     

An adaptive and robust biological network based on the vacant-particle transportation model

A living system reveals local computing by referring to a whole system beyond the exploration-exploitation dilemma. The slime mold, Physarum polycephalum, uses protoplasmic flow to change its own outer shape, which yields the boundary condition and forms an adaptive and robust network. This observation suggests that the whole Physarum can be represented as a local protoplasmic flow system. Here, we show that a system composed of particles, which move and are modified based upon the particle transformation that contains the relationship between the parts and the whole, can emulate the network formed by Physarum. This system balances the exploration-exploitation trade-off and shows a scale-free sub-domain. By decreasing the number of particles, our model, VP-S, can emulate the Physarum adaptive network as it is attracted to a food stimulus. By increasing the number of particles, our model, VP-D, can emulate the pattern of a growing Physarum. The patterns produced by our model were compared with those of the Physarum pattern quantitatively, which showed that both patterns balance exploration with exploitation. This model should have a wide applicability to study biological collective phenomena in general.     

Studies on the electrical properties of a single plant cell; internodal cell of Nitella flexilis

Impedance changes of single plant cells of Nitella flexilis were studied under different environmental conditions. With the analysis presented changes in resistance of the protoplasmic membrane and of cell sap can be studied independently and simultaneously. Under "transcellular osmosis," the resistance of the protoplasmic membrane and of the cell sap increase at the part of the cell where water enters, while they decrease where water goes out. Ethanol of low concentration (below 8 per cent) first decreases and later increases the resistance of the protoplasmic membrane. Concentrated ethanol (over 10 per cent), however, brings about a large decrease in resistance of the protoplasmic membrane. Its time course is not simple, but undulatory changes occur. When ethanol is applied to one part of the cell, the resistance of the protoplasmic membrane shows a different type of change, which may be attributed to the local osmotic effect of ethanol; injury generally occurs with comparatively low concentration. Methanol, ethanol, and propanol have almost the same effect upon the cell, while butanol is toxic at the same concentration. When the cell dies, the resistance of the protoplasmic membrane decreases greatly, while the resistance of the cell sap increases to a level (several hundred kilo ohms or more), expected when external solution and cell sap are freely mixed with each other.     

THE RELATION OF THE STABILITY OF PROTOPLASMIC FILMS IN NOCTILUCA TO THE DURATION AND INTENSITY OF AN APPLIED ELECTRIC POTENTIAL

1. The experiments demonstrate that when a constant electric potential of sufficient intensity is applied to Noctiluca, the protoplasmic films which represent a part of the visible continuous phase of the cytoplasm and plasma membrane at the surface of the cell, become unstable and break down, thus releasing the acid contents of one of the internal discontinuous phases present in the cytoplasm of Noctiluca. This process which occurs first at anode then at the cathode side of the cell, appears to be a selective deemulsification or coalescence similar to that at the surface of an emulsion having a viscous continuous phase. 2. The experiments demonstrate that Nernst''s equation See PDF for Equation which expresses approximately the relation of duration and intensity of a constant electric current to threshold stimulation of striated muscle, applies equally well to the process of anodal coalescence in Noctiluca. 3. Anodal and cathodal coalescence have different thresholds, due to the fact that the semipermeable plasma film at the surface of the cell is asymmetric with respect to the direction of the applied current. Attention is called to the possible relation between this phenomenon and the conditions occurring at the synapse between neurons. 4. The stability of the protoplasmic films in relation to the applied electric potential is greater in young cells than in old cells, or in other words the threshold intensity of the stimulus is higher for young than for old cells. 5. Attention is called to the occurrence in the same cell of different receptor-affector mechanisms having a corresponding difference in intensity threshold when an electric current is acting as a stimulus.     

A chilling sensitive mutant of Arabidopsis with altered steryl-ester metabolism

A chilling-sensitive mutant of Arabidopsis thaliana was isolated and subjected to genetic, physiological, and biochemical analysis. The chilling-sensitive nature of the mutant line is due to a single recessive nuclear mutation at a locus designated chs1. In contrast to wild-type plants, which are not adversely affected by low temperatures, the chs1 mutant is killed by several days of exposure to temperatures below 18°C. Following exposure to chilling temperatures, the mutant displays two common symptoms of chilling injury—leaf chlorosis and electrolyte leakage. In these respects, the physiological response of the mutant to low temperatures mimics the response observed in some naturally occurring chilling sensitive species. The biochemical basis of chilling sensitivity was explored by examining the pattern of incorporation of ¹⁴CO₂ into soluble metabolites and lipids in wild-type and mutant plants. The only difference observed between the mutant and wild type was that following low temperature treatment, the mutant accumulated 10-fold more radioactivity in a specific class of neutral lipids which were identified by a variety of criteria to be steryl-esters. The accumulation of radioactivity in the steryl-ester fraction occurs 24 hours before there is any visible evidence of chilling injury. These results suggest one of two possible explanations: either the mutation directly affects sterol metabolism, which in turn leads to chilling sensitivity, or the mutation affects another unidentified function and the accumulation of radioactivity in steryl-esters is a secondary consequence of chilling injury.     

CHANGES OF APPARENT IONIC MOBILITIES IN PROTOPLASM : IV. INFLUENCE OF GUAIACOL ON THE EFFECTS OF SODIUM AND POTASSIUM IN NITELLA

In Nitella, as in Halicystis, guaiacol increases the mobility of Na⁺ in the outer protoplasmic surface but leaves the mobility of K⁺ unaffected. This differs from the situation in Valonia where the mobility of Na⁺ is increased and that of K⁺ is decreased. The partition coefficient of Na⁺ in the outer protoplasmic surface is increased and that of K⁺ left unchanged. Recovery after the action current is delayed in the presence of guaiacol and the action curves are "square topped."     

Protoplasmic streaming of an internodal cell of Nitella flexilis; its correlation with electric stimulus

The sudden cessation or sudden decrease in velocity of the protoplasmic streaming of Nitella flexilis is observed whenever an action potential is elicited. The action potential can be generated by an electric stimulus after its refractory period, whether the flow is at a complete standstill or on the way to recovery. The membrane potential is generally decreased more or less when the rate of flow is decreased on application of salts or other agents. There is, however, no parallelism between these two. The membrane potential decreases proportionally with applied voltage of subthreshold intensity, while the rate of flow does not change appreciably. Only on application of a superthreshold voltage does the flow stop suddenly. In one case the rate of flow decreased to half without appreciable decrease in membrane potential. In another case it continued flowing at about one-half rate, although the membrane potential was almost zero. The Q₁₀ of the rate of flow is about 2, while it is 1.1 to 1.5 for the membrane potential. The sudden cessation of the protoplasmic streaming is supposed to be caused by the temporary formation of certain interlinkages among contractile protein networks in the endoplasm during excitation at the cathodal half of Nitella.     

Changes in resting potential due to a shift of electrolytes in the cell produced by non-electrolytes

Experiments on Nitella indicate that the resting potential is due chiefly to the outwardly directed diffusion potential of electrolytes which is set up at the inner, non-aqueous, protoplasmic surface surrounding the vacuole. We might therefore expect that any change in the concentration of these electrolytes would affect the resting potential. The experiments described here indicate that this expectation is justified. When a sucrose solution is applied at one end of the cell and water is placed at another spot, water enters at the latter, passes along inside the cell, and escapes into the sucrose solution, but the electrolytes are unable to escape into the sucrose solution (except very slowly) so that the concentration of electrolytes increases in the region in contact with the sucrose solution. Hence the potential at this spot increases. At the other spot where the water enters, the concentration of electrolytes decreases and the potential at this spot falls off. The changes can be carried out reversibly without injury to the cell.     

Distribution of Chlorophyll-Protein Complexes during Chilling in the Light Compared with Heat-Induced Modifications

The effects of chilling in the light (4 days at 5°C and 100-200 micromoles of photons per square meter per second) on the distribution of chlorophyll (Chl) protein complexes between appressed and nonappressed thylakoid regions of pumpkin (Cucurbita pepo L.) chloroplasts were studied and compared with the changes occurring during in vitro heat treatment (5 minutes at 40°C) of isolated thylakoids. Both treatments induced an increase (18 and 65%, respectively) in the relative amount of the antenna Chl a protein complexes (CP47 + CP43) of photosystem II (PSII) in stroma lamellae vesicles. Freeze-fracture replicas of light-chilled material revealed an increase in the particle density on the exoplasmic fracture face of unstacked membrane regions. These two treatments differed markedly, however, in respect to comigration of the light-harvesting Chl a/b protein complex (LHCII) of PSII. The LHCII/PSII ratio in stroma lamellae vesicles remained fairly constant during chilling in the light, whereas it dropped during the heat treatment. Moreover, it was a minor light-harvesting Chl a/b protein complex of PSII, CP29, that increased most in stroma lamellae vesicles during light-chilling. Changes in the organization of LHCII during chilling were suggested by a shift to particles of smaller sizes on the protoplasmic fracture face of stacked membrane regions and a decrease in the amount of trans-Δ³-hexadecenoic acid in the phosphatidyldiacylglycerol fraction.     

Diapause development in Bombyx eggs in relation to ‘esterase A’ activity

Diapause is broken by hydrochloric acid treatment and also terminated by long chilling of eggs in the silkworm, Bombyx mori. One of esterases in silkworm eggs named ‘esterase A’ is closely related to diapause of this insect.Hydrochloric acid treatment of diapause eggs induced a prompt elevation of esterase A activity. The elevation was observed within 30 min after treatment. This HCl treatment effectively stimulated the eggs to hatch. This indicates that the increase of esterase A activity is correlated with an active resumption of morphogenesis.The question was examined of whether chilling also increases esterase A activity or not. It was found that chilling also caused an increase of esterase A activity. This increase occurred before the re-appearance of glycogen in eggs, which indicates the termination of diapause in this insect. In fact, the establishment of hatchability in chilled eggs was observed after esterase A activity has reached the maximum level. Thus the increase of esterase A activity could be regarded as associated with the termination of diapause per se but not with the subsequent process of post-diapause development. This change in esterase A activity was observed only in chilled diapause eggs and was not observed in diapause eggs without chilling and non-diapause eggs.These results suggest that the increase of esterase A activity during chilling may be a kind of activity that occurs during the diapause stage in preparation for resumption of morphogenesis or diapause development.     

Application of a NMDA Receptor Conductance in Rat Midbrain Dopaminergic Neurons Using the Dynamic Clamp Technique

Neuroscientists study the function of the brain by investigating how neurons in the brain communicate. Many investigators look at changes in the electrical activity of one or more neurons in response to an experimentally-controlled input. The electrical activity of neurons can be recorded in isolated brain slices using patch clamp techniques with glass micropipettes. Traditionally, experimenters can mimic neuronal input by direct injection of current through the pipette, electrical stimulation of the other cells or remaining axonal connections in the slice, or pharmacological manipulation by receptors located on the neuronal membrane of the recorded cell.Direct current injection has the advantages of passing a predetermined current waveform with high temporal precision at the site of the recording (usually the soma). However, it does not change the resistance of the neuronal membrane as no ion channels are physically opened. Current injection usually employs rectangular pulses and thus does not model the kinetics of ion channels. Finally, current injection cannot mimic the chemical changes in the cell that occurs with the opening of ion channels.Receptors can be physically activated by electrical or pharmacological stimulation. The experimenter has good temporal precision of receptor activation with electrical stimulation of the slice. However, there is limited spatial precision of receptor activation and the exact nature of what is activated upon stimulation is unknown. This latter problem can be partially alleviated by specific pharmacological agents. Unfortunately, the time course of activation of pharmacological agents is typically slow and the spatial precision of inputs onto the recorded cell is unknown.The dynamic clamp technique allows an experimenter to change the current passed directly into the cell based on real-time feedback of the membrane potential of the cell (Robinson and Kawai 1993, Sharp et al., 1993a,b; for review, see Prinz et al. 2004). This allows an experimenter to mimic the electrical changes that occur at the site of the recording in response to activation of a receptor. Real-time changes in applied current are determined by a mathematical equation implemented in hardware.We have recently used the dynamic clamp technique to investigate the generation of bursts of action potentials by phasic activation of NMDA receptors in dopaminergic neurons of the substantia nigra pars compacta (Deister et al., 2009; Lobb et al., 2010). In this video, we demonstrate the procedures needed to apply a NMDA receptor conductance into a dopaminergic neuron.     

DIFFERING RATES OF DEATH AT INNER AND OUTER SURFACES OF THE PROTOPLASM : I. EFFECTS OF FORMALDEHYDE ON NITELLA

When protoplasm dies it becomes completely and irreversibly permeable and this may be used as a criterion of death. On this basis we may say that when 0.2 M formaldehyde plus 0.001 M NaCl is applied to Nitella death arrives sooner at the inner protoplasmic surface than at the outer. If, however, we apply 0.17 M formaldehyde plus 0.01 M KCl death arrives sooner at the outer protoplasmic surface. The difference appears to be due largely to the conditions at the two surfaces. With 0.2 M formaldehyde plus 0.001 M NaCl the inner surface is subject to a greater electrical pressure than the outer and is in contact with a higher concentration of KCl. In the other case these conditions are more nearly equal so that the layer first reached by the reagent is the first to become permeable. The outer protoplasmic surface has the ability to distinguish electrically between K⁺ and Na⁺ (potassium effect). Under the influence of formaldehyde this ability is lost. This is chiefly due to a falling off in the partition coefficient of KCl in the outer protoplasmic surface. At about the same time the inner protoplasmic surface becomes completely permeable. But the outer protoplasmic surface retains its ability to distinguish electrically between different concentrations of the same salt, showing that it has not become completely permeable. After the potential has disappeared the turgidity (hydrostatic pressure inside the cell) persists for some time, probably because the outer protoplasmic surface has not become completely permeable.     

Different photosynthetic responses to night chilling among twelve populations of Jatropha curcas

Jatropha curcas, one of the most important energy plant resources, is vulnerable to chilling. To evaluate the effects of chilling on photosynthesis of J. curcas and intraspecific differences in chilling tolerance, seedlings of twelve populations were treated with the temperature of 4–6°C for five consecutive nights with normal environmental temperature during the day. Night chilling treatment decreased light-saturated photosynthetic rate (P _max) significantly for all populations. Stomatal limitation could not explain the decreased P _max because intracellular CO₂ concentration was not significantly reduced by night chilling in all populations (with only one exception). The decreased soluble-protein content, which may be related to the increased malondialdehyde (MDA) content, contributed to the decreased P _max. The increased MDA content indicated that oxidative stress occurred after night chilling, which was associated with the larger decrease in P _max compared with the decrease in actual photochemical efficiency of photosystem II, and the slight increase in thermal dissipation of excessive energy. After five-day recovery, MDA (with two exceptions) and P _max still did not recover to the levels as those before night chilling treatment for all populations, indicating that J. curcas was vulnerable to chilling. Chilling tolerance was significantly different among populations. Populations originating from high elevations had greater chilling-tolerant abilities than populations originating from low elevations, showing a local adaptation to environmental temperatures of origins. Our study shed light on the possibility to find or breed chilling-tolerant genotypes of J. curcas.