Membrane capacity of squid giant axon during hyper- and depolarizations

Summary The change in membrane capacitance and conductance of squid giant axons during hyper- and depolarizations was investigated. The measurements of capacitance and conductance were performed using an admittance bridge with resting, hyperpolarized and depolarized membranes. The duration of DC pulses is 20–40 msec and is long enough to permit the admittance measurements between 1 and 50 kHz. The amplitudes of DC pulses were varied between 0 and 40mV for both depolarization and hyperpolarization. Within these limited experimental conditions, we found a substantial increase in membrane capacitance with depolarization and a decrease with hyperpolarization. Our results indicate that the change in membrane capacitance will increase further if low frequencies are used with larger depolarizing pulses. The change in membrane capacitance is frequency dependent and it increases with decreasing frequencies. The analyses based on an equivalent circuit (vide infra) gives rise to a time constant of active membrane capacitance close to that of sodium currents. This result indicates that the observed capacitance changes may arise from sodium channels. A brief discussion is given on the nature of frequency-dependent membrane capacitance of nerve axons.     

Rapid evolution of sperm length in response to increased temperature in an ectothermic fish

The Intergovernmental Panel on Climate Change predicts an average global temperature increase of 1.8–4.0 °C by 2100. Tropical ectotherms are expected to be particularly sensitive to this temperature increase because they live close to their thermal limits. We investigated the phenotypic plasticity and evolutionary response of sperm traits in guppies (Poecilia reticulata) to increased temperatures after 6, 18, and 24 months. Guppies with evolution temperatures of 25 °C (control) or 28 °C were reared in either 25 or 28 °C in a 2 × 2 common garden design. The plastic response to increased temperature was a decreased sperm length, velocity, and path linearity. The evolutionary response was a subsequent increase in sperm length, resulting in complete compensation after just 6 months (at most four generations) in 28 °C water. Sperm velocity and linearity showed no sign of evolution even after 24 months. This study provides evidence that some reproductive traits can respond via rapid evolution to the temperature increase associated with climate change.     

The electrical properties of phospholipid bilayer langmuir films

Ordered phospholipid bilayers of the Langmuir type have been formed between aluminum electrodes. First electrical measurements are reported and a variety of phenomena observed. Current-voltage, capacitance-temperature, and conductivity-temperature characteristics are determined. Linear regions of log current vs. $\text{1}\text{T}$ are observed with slope changes near the known transition temperatures for the lamellar phase in aqueous solutions, and minima in the capacitance are also observed near these temperatures. Conductivity is essentially independent of temperature from ?100°C to +30°C. An activation energy of 0.65 eV is obtained for conduction above 60°C. Self-potentials are exhibited. The phenomena are discussed and various mechanisms considered.     

Axon Viability and Mitochondrial Function are Dependent on Local Protein Synthesis in Sympathetic Neurons

(1) Axons contain numerous mRNAs and a local protein synthetic system that can be regulated independently of the cell body. (2) In this study, cultured primary sympathetic neurons were employed, to assess the effect of local protein synthesis blockade on axon viability and mitochondrial function. (3) Inhibition of local protein synthesis reduced newly synthesized axonal proteins by 65% and resulted in axon retraction after 6 h. Acute inhibition of local protein synthesis also resulted in a significant decrease in the membrane potential of axonal mitochondria. Likewise, blockade of local protein transport into the mitochondria by transfection of the axons with Hsp90 C-terminal domain decreased the mitochondrial membrane potential by 65%. Moreover, inhibition of the local protein synthetic system also reduced the ability of mitochondria to restore axonal levels of ATP after KCl-induced depolarization. (4) Taken together, these results indicate that the local protein synthetic system plays an important role in mitochondrial function and the maintenance of the axon.     

Rapid activity-dependent delivery of the neurotrophic protein CPG15 to the axon surface of neurons in intact Xenopus tadpoles

CPG15 (aka neuritin) is an activity-induced GPI-anchored axonal protein that promotes dendritic and axonal growth, and accelerates synaptic maturation in vivo. Here we show that CPG15 is distributed inside axons and on the axon surface. CPG15 is trafficked to and from the axonal surface by membrane depolarization. To assess CPG15 trafficking in vivo, we expressed an ecliptic pHluorin (EP)-CPG15 fusion protein in optic tectal explants and in retinal ganglion cells of intact Xenopus tadpoles. Depolarization by KCl increased EP-CPG15 fluorescence on axons. Intraocular kainic acid (KA) injection rapidly increased cell-surface EP-CPG15 in retinotectal axons, but coinjection of TTX and KA did not. Consistent with this, we find that intracellular CPG15 is localized to vesicles and endosomes in presynaptic terminals and colocalizes with synaptic vesicle proteins. The results indicate that the delivery of the neurotrophic protein CPG15 to the axon surface can be regulated on a rapid time scale by activity-dependent mechanisms in vivo.     

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

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

AMP‐activated protein kinase mediates activity‐dependent axon branching by recruiting mitochondria to axon

During development, axons are guided to their target areas and provide local branching. Spatiotemporal regulation of axon branching is crucial for the establishment of functional connections between appropriate pre‐ and postsynaptic neurons. Common understanding has been that neuronal activity contributes to the proper axon branching; however, intracellular mechanisms that underlie activity‐dependent axon branching remain elusive. Here, we show, using primary cultures of the dentate granule cells, that neuronal depolarization‐induced rebalance of mitochondrial motility between anterograde versus retrograde transport underlies the proper formation of axonal branches. We found that the depolarization‐induced branch formation was blocked by the uncoupler p‐trifluoromethoxyphenylhydrazone, which suggests that mitochondria‐derived ATP mediates the observed phenomena. Real‐time analysis of mitochondrial movement defined the molecular mechanisms by showing that the pharmacological activation of AMP‐activated protein kinase (AMPK) after depolarization increased anterograde transport of mitochondria into axons. Simultaneous imaging of axonal morphology and mitochondrial distribution revealed that mitochondrial localization preceded the emergence of axonal branches. Moreover, the higher probability of mitochondrial localization was correlated with the longer lifetime of axon branches. We qualitatively confirmed that neuronal ATP levels decreased immediately after depolarization and found that the phosphorylated form of AMPK was increased. Thus, this study identifies a novel role for AMPK in the transport of axonal mitochondria that underlie the neuronal activity‐dependent formation of axon branches. © 2013 Wiley Periodicals, Inc. Develop Neurobiol 74: 557–573, 2014     

Novel peripheral motor neurons in the posterior tentacles of the snail responsible for local tentacle movements

Three flexor muscles of the posterior tentacles of the snail Helix pomatia have recently been described. Here, we identify their local motor neurons by following the retrograde transport of neurobiotin injected into these muscles. The mostly unipolar motor neurons (15–35 µm) are confined to the tentacle digits and send motor axons to the M2 and M3 muscles. Electron microscopy revealed small dark neurons (5–7 µm diameter) and light neurons with 12–18 (T1 type) and 18–30 µm diameters (T2 type) in the digits. The diameters of the neurobiotin-labeled neurons corresponded to the T1 type light neurons. The neuronal processes of T1 type motor neurons arborize extensively in the neuropil area of the digits and receive synaptic inputs from local neuronal elements involved in peripheral olfactory information processing. These findings support the existence of a peripheral stimulus–response pathway, consisting of olfactory stimulus—local motor neuron—motor response components, to generate local lateral movements of the tentacle tip (“quiver”). In addition, physiological results showed that each flexor muscle receives distinct central motor commands via different peritentacular nerves and common central motor commands via tentacle digits, respectively. The distal axonal segments of the common pathway can receive inputs from local interneurons in the digits modulating the motor axon activity peripherally without soma excitation. These elements constitute a local microcircuit consisting of olfactory stimulus—distal segments of central motor axons—motor response components, to induce patterned contraction movements of the tentacle. The two local microcircuits described above provide a comprehensive neuroanatomical basis of tentacle movements without the involvement of the CNS.     

The Effects of Mechanical Stimulation on Some Electrical Properties of Axons

Rapid, short duration mechanical compression of lobster giant axons by a crystal-driven stylus produces a depolarization and an increase in membrane conductance which develop immediately with compression but take several seconds to recover. The conductance increase occurs even when the depolarization is prevented electrically. If sodium is removed from the external medium or if procaine is added to it, compression produces almost no depolarization. Small bundles of myelinated frog fibers are depolarized by rapid compression but recover very rapidly (milliseconds); "off" responses are occasionally seen. The results are discussed in terms of the mechanoelectric transducer behavior of an axon membrane.     

Effect of growth temperature,surface type and incubation time on the resistance of Staphylococcus aureus biofilms to disinfectants

The goal of this study was to investigate the effect of the environmental conditions such as the temperature change, incubation time and surface type on the resistance of Staphylococcus aureus biofilms to disinfectants. The antibiofilm assays were performed against biofilms grown at 20 °C, 30 °C and 37 °C, on the stainless steel and polycarbonate, during 24 and 48 h. The involvement of the biofilm matrix and the bacterial membrane fluidity in the resistance of sessile cells were investigated. Our results show that the efficiency of disinfectants was dependent on the growth temperature, the surface type and the disinfectant product. The increase of growth temperature from 20 °C to 37 °C, with an incubation time of 24 h, increased the resistance of biofilms to cationic antimicrobials. This change of growth temperature did not affect the major content of the biofilm matrix, but it decreased the membrane fluidity of sessile cells through the increase of the anteiso-C19 relative amount. The increase of the biofilm resistance to disinfectants, with the rise of the incubation time, was dependent on both growth temperature and disinfectant product. The increase of the biofilm age also promoted increases in the matrix production and the membrane fluidity of sessile cells. The resistance of S. aureus biofilm seems to depend on the environment of the biofilm formation and involves both extracellular matrix and membrane fluidity of sessile cells. Our study represents the first report describing the impact of environmental conditions on the matrix production, sessile cells membrane fluidity and resistance of S. aureus biofilms to disinfectants.     

Analysis of nerve conduction block induced by direct current

The mechanisms of nerve conduction block induced by direct current (DC) were investigated using a lumped circuit model of the myelinated axon based on Frankenhaeuser–Huxley (FH) model. Four types of nerve conduction block were observed including anodal DC block, cathodal DC block, virtual anodal DC block, and virtual cathodal DC block. The concept of activating function was used to explain the blocking locations and relation between these different types of nerve block. Anodal/cathodal DC blocks occurred at the axonal nodes under the block electrode, while virtual anodal/cathodal DC blocks occurred at the nodes several millimeters away from the block electrode. Anodal or virtual anodal DC block was caused by hyperpolarization of the axon membrane resulting in the failure of activating sodium channels by the arriving action potential. Cathodal or virtual cathodal DC block was caused by depolarization of the axon membrane resulting in inactivation of the sodium channel. The threshold of cathodal DC block was lower than anodal DC block in most conditions. The threshold of virtual anodal/cathodal blocks was about three to five times higher than the threshold of anodal/cathodal blocks. The blocking threshold was decreased with an increase of axonal diameter, a decrease of electrode distance to axon, or an increase of temperature. This simulation study, which revealed four possible mechanisms of nerve conduction block in myelinated axons induced by DC current, can guide future animal experiments as well as optimize the design of electrodes to block nerve conduction in neuroprosthetic applications.     

Modelled temperature-dependent excitability behaviour of a single ranvier node for a human peripheral sensory nerve fibre

The objective of this study was to determine whether the Hodgkin–Huxley model for unmyelinated nerve fibres could be modified to predict excitability behaviour at Ranvier nodes. Only the model parameters were modified to those of human, with the equations left unaltered. A model of a single Ranvier node has been developed as part of a larger model to describe excitation behaviour in a generalised human peripheral sensory nerve fibre. Parameter values describing the ionic and leakage conductances, corresponding equilibrium potentials, resting membrane potential and membrane capacitance of the original Hodgkin–Huxley model were modified to reflect the corresponding parameter values for human. Parameter temperature dependence was included. The fast activating potassium current kinetics were slowed down to represent those of a slow activating and deactivating potassium current, which do not inactivate. All calculations were performed in MATLAB^TM. Action potential shape and amplitude were satisfactorily predicted at 20, 25 and 37°C, and were not influenced by activation or deactivation of the slow potassium current. The calculated chronaxie time constant was 65.5 μs at 37°C. However, chronaxie times were overestimated at temperatures lower than body temperature.     

The Neuroprotective Ability of Polyethylene Glycol is Affected by Temperature in Ex Vivo Spinal Cord Injury Model

Immediate membrane sealing after spinal cord injury (SCI) can prevent further degradation and result in ultimate functional recovery. It has been reported that polyethylene glycol (PEG) can repair membrane damage caused by mechanical insults to the spinal cord. Furthermore, membrane fluidity and its sealing process vary at different temperatures. Here, we have assessed the possible synergistic effects of PEG and temperature on the repair of neural membranes in an SCI model. The effects of PEGs (400, 1,000 and 2,000 Da) were studied at different temperatures (25, 37 and 40 °C) by means of compound action potential (CAP) recovery and a lactate dehydrogenase (LDH) assay. Isolated spinal cords were mounted in a double sucrose gap chamber, where the amplitude and area of CAPs were recorded after implementing injury, in the presence and absence of PEG. Moreover, the LDH assay was used to assess the effects of PEG on membrane resealing. Data showed that the least CAP recovery occurred at 25 °C, followed by 37 and 40 °C, in all treated groups. Moreover, maximum CAP amplitude recovery, 65.46 ± 5.04 %, was monitored in the presence of PEG400 at 40 °C, followed by 41.49 ± 2.41 % in PEG1000 and 37.36 ± 1.62 % in PEG2000. Furthermore, raising the temperature from 37 to 40 °C significantly increased CAP recovery in the PEG2000 group. Similar recovery patterns were obtained by CAP area measurements and LDH assay. The results suggest that application of low-molecular weight PEG (PEG400) in mild hyperthermia conditions (40 °C) provides the optimum condition for membrane sealing in SCI model.     

Long Term Survival of Severed Distal Axonal Stumps in Vertebrates and Invertebrates

SYNOPSIS. Severed distal stumps of nerve axons have now beenreported to survive for months to years in both vertebrate andinvertebrate nervous systems While low (>15°C) temperaturesmay increase survival times in some preparations such as unmyelinatedgarfish olfactory axons, temperature between 15 and 25°Cis not the only significant factor determining the time courseof survival in goldfish Mauthner axons and for many invertebrateaxons For example when different axons in a crayfish are allstudied at the same temperature, long term survival differsin different axons In some cases these differences appear tobe due to differences in the nature of the ghal reaction orthe presence of synaptic contacts. The possible cellular mechanisms for long term survival fallinto three general cate gories slow degradation of axonal proteinsde novo axoplasmic protein synthesis, and transfer of proteinsfrom adjacent cells to severed axonal stumps In crayfish andsquid giant axons, there is evidence that proteins are indeedtransferred intact from glia to axons or from axon to axon,possibly via exocytotic/endocytotic processes However cellularmechanisms for long term survival may well differ in differentaxons of the same organism, much less between axons in organismsfrom different phyla In particular the ghal sheaths of myehnatedvertebrate axons which demonstrate long term survival mightbe expected to impede ghal/axonal or axonal/axonal protein transfer. The study of long term survival of severed distal stumps isimportant for studies of axonal regeneration because axons inorganisms having long survival times often show functional reconnectionmuch more rapidly and with higher specificity than do axonsin organisms lacking long survival times The study of long termsurvival is also important to cell biologists for an understandingof the molecular mechanisms which allow a piece of cytoplasmseparated from direct cytoplasmic contact with any nucleus toremain morphologically intact and functionally competent formonths to years.     

Methylmercury: Effects on electrical properties of squid axon membranes

At low concentrations (25–100 μM) methylmercury chloride caused a steady increase in the threshold for excitation and on eventual block of action potentials without changing the resting membrane potential in squid giant axons. In the axons exposed to 25 μM methylmercury chloride, peak transient and steady-state conductances were decreased by 58.8 ± 5.1% and 35.9 ± 4.3% (mean ± SEM, 4 axons), respectively and leakage conductance increased to about five times of the control value. Higher concentrations of methylmercury chloride decreased the resting membrane potential. A concentration of 0.5 mM depolarizing the nerve membrane by 16 ± 2 mV (mean ± SEM, 3 axons) in 40 minutes. These changes in ionic conductances and membrane potential were irreversible on washing the axon with drug-free sea water.     

Local anaesthetic block protects against electrically-induced damage in peripheral nerve

This study is one of a series addressing the mechanisms involved in the production of neural damage caused by continuous, prolonged electrical stimulation of peripheral nerve. It has been previously shown that sustained, high frequency electrical stimulation of the cat's peroneal nerve may cause irreversible neural damage in the form of axonal degeneration of the large myelinated fibres. In this study we demonstrate that blocking the action potentials on most of the nerve fibres with local anaesthetics (10% procaine or 2% lidocaine) almost completely prevents the axonal degeneration. The abolition of axonal injury by local anaesthetic block strongly suggests that the electrically-induced damage is due to prolonged electrical excitation of axons. Furthermore, since less than complete suppression of the induced neural activity by local anaesthetic engenders essentially complete sparing of all axons, our results suggest that the damage to individual axons derives, at least in part, from stimulation-induced global changes in the nerve.     

Anatomical basis for an apparent paradox concerning conduction velocities of two identified axons in Aplysia

Larger axons usually have faster conduction velocities, lower thresholds, and larger extracellular action potentials than smaller axons. However, it has been shown that the largest fiber, R2, in the right pleurovisceral connective of the marine mollusc, Aplysia, has a higher threshold and a slower conduction velocity than does the smaller axon of cell R1, even though the amplitude of R2's spike is larger than R1's spike. One explanation of this apparent paradox is that the two axons have different “intrinsic membrane and axoplasmic constants” (Goldman, L. (1961), J. Cell Comp. Physiol. 57: 185–191). However, the deep infolding of R2's axonal membrane suggested that differences in the shape of the two axons might also account for the paradox. Accordingly, we measured the conduction velocities of the two axons and then examined the same axons in the electron microscope in order to measure their volumes and surface areas. Our morphological observations indicate that the extensive infolding of surface membrane causes R2 to have a smaller volume to surface area ratio than R1. Thus, since conduction velocity is proportional to the square root of the volume to surface area ratio (Hodgkin, A. L. (1954), J. Physiol. 125: 221–224), it is predictable that the smaller axon would have a faster conduction velocity. The results suggest that the paradoxical conduction velocities can be explained largely as resulting from differences in the shapes of the two axons. However, certain discrepancies between the measured and the predicted values suggest that other factors are contributing as well.     

Effect of temperature and ripening stages on membrane integrity of fresh-cut tomatoes

The shelf-life of fresh-cut tomatoes mainly depends on loss of tissue integrity and firmness that occurs also in intact fruits after long-term cold storage due to chilling injury. Round-fruit tomatoes (Solanum lycopersicum L.) cv. Jama were stored in 1.1-L plastic (polyethylene) fresh-cut produce containers as 10.0-mm-thick tomato slices and as intact tomatoes at 4 ± 0.5 °C. The aim of this work was to study the loss of membrane integrity and biochemical processes involved in membrane disruption. Electrolyte leakage and lipid peroxidation were studied at different stages of maturity: mature green, pink (PK), fully ripe and two different storage temperatures: 4 and 15 °C. The tomato slices of PK stage stored at 4 °C did not show changes for both parameters, while significant increase in membrane leakage and lipid peroxidation was observed at 15 °C, especially after 24 h of storage. The enzymes showed a simultaneous increase in their activities with a rise in electrolyte leakage and lipid peroxidation after 7 days of storage. Finally, phospholipase C (PLC) and phospholipase D (PLD) were investigated for intact fruit and tomato slices stored at 4 °C. The PLC had higher activity compared with PLD. In conclusion, the loss of membrane integrity in fresh-cut tomatoes is mainly affected by ripening stages, storage temperature and duration. The wounds enhance the PLC and PLD activities and they play a role late during storage.     

Stochastic simulation of neurofilament transport in axons: the "stop-and-go" hypothesis

According to the "stop-and-go" hypothesis of slow axonal transport, cytoskeletal and cytosolic proteins are transported along axons at fast rates but the average velocity is slow because the movements are infrequent and bidirectional. To test whether this hypothesis can explain the kinetics of slow axonal transport in vivo, we have developed a stochastic model of neurofilament transport in axons. We propose that neurofilaments move in both anterograde and retrograde directions along cytoskeletal tracks, alternating between short bouts of rapid movement and short "on-track" pauses, and that they can also temporarily disengage from these tracks, resulting in more prolonged "off-track" pauses. We derive the kinetic parameters of the model from a detailed analysis of the moving and pausing behavior of single neurofilaments in axons of cultured neurons. We show that the model can match the shape, velocity, and spreading of the neurofilament transport waves obtained by radioisotopic pulse labeling in vivo. The model predicts that axonal neurofilaments spend approximately 8% of their time on track and approximately 97% of their time pausing during their journey along the axon.     

Low temperature alters plasma membrane lipid composition and ATPase activity of pineapple fruit during blackheart development

Plasma membrane (PM) plays central role in triggering primary responses to chilling injury and sustaining cellular homeostasis. Characterising response of membrane lipids to low temperature can provide important information for identifying early causal factors contributing to chilling injury. To this end, PM lipid composition and ATPase activity were assessed in pineapple fruit (Ananas comosus) in relation to the effect of low temperature on the development of blackheart, a form of chilling injury. Chilling temperature at 10 °C induced blackheart development in concurrence with increase in electrolyte leakage. PM ATPase activity was decreased after 1 week at low temperature, followed by a further decrease after 2 weeks. The enzyme activity was not changed during 25 °C storage. Loss of total PM phospholipids was found during postharvest senescence, but more reduction was shown from storage at 10 °C. Phosphatidylcholine and phosphatidylethanolamine were the predominant PM phospholipid species. Low temperature increased the level of phosphatidic acid but decreased the level of phosphatidylinositol. Both phospholipid species were not changed during storage at 25 °C. Postharvest storage at both temperatures decreased the levels of C18:3 and C16:1, and increased level of C18:1. Low temperature decreased the level of C18:2 and increased the level of C14:0. Exogenous application of phosphatidic acid was found to inhibit the PM ATPase activity of pineapple fruit in vitro. Modification of membrane lipid composition and its effect on the functional property of plasma membrane at low temperature were discussed in correlation with their roles in blackheart development of pineapple fruit.