Electrical properties and transmitter output associated with growth and transformation in shrimp muscle fibers

Summary Comparisons were made of the passive electrical properties of closer muscle fibers in the dimorphic claws of snapping shrimp,Alpheus armillatus. During claw transformation the small fibers of pincer claws grow to become much larger snapper claw fibers. As muscle fibers grow, the relationship of fiber input resistance (R ₀) to fiber diameter (d) is predicted by the proportionality,R ₀d ^–3/2. Muscle fiber membrane resistance,R _m, is independent of fiber diameter, but membrane capacitance,C _m, grows with diameter. This results in a 40 to 50 fold reduction in fiber input impedance as fiber diameter enlarges during transformation. Reductions of muscle fiber impedance are partially compensated by 2–5 fold increases in quantal content at excitatory synapses on snapper muscle fibers. However, changes in quantal content during transformation apparently are independent of fiber diameter per se. Excitatory junction potentials in both pincer and snapper muscle fibers have equal amplitude. Because fiber input impedance decreases precipitously during transformation, and in view of the relatively small compensatory changes in quantal content at excitatory synapses, additional pre- or post-synaptic modifications must supplement increased quantal content to maintain synaptic efficacy in transformed muscle fibers.Abbreviations ejp excitatory junctional potential - epp endplate potential - mepp miniature endplate potential     

An analysis of the direct effect of chloridazepoxide on mammalian cardiac tissues and crayfish and squid giant axons: possible basis of antiarrhythmic activity.

Chlordiazepoxide has been found to be antiarrhythmic $\text{in vivo}$. The purpose of the present investigation is to identify the mechanism(s) of such antiarrhythmic activity. In canine heart, chlordiazepoxide effectively depressed the enhanced repetitive discharges in subendocardial Purkinje fibers surviving acute myocardial infarction. Chlordiazepoxide altered the action potential characteristics of Purkinje fiber by shortening the APD₅₀, APD₁₀₀ and effective refractory period with little effect on the resting membrane potential. The maximal rate of upstroke (dv/dt) was significantly reduced only at 1 × 10^?4M and above in Purkinje fibers and the membrane response curve was consistently shifted to the right by chlordiazepoxide. The ventricular muscle was little affected by chlordiazepoxide except for the shortened APD₅₀ and reduced dv/dt. Chlordiazepoxide exerted nerve blocking potency comparable to lidocaine in the crayfish giant axon. Voltage-clamp experiments in squid axon showed that chlordiazepoxide suppressed both components of membrane current, the transient inward sodium current being diminished far greater than the steady-state potassium current. These results demonstrate a direct action on cardiac and axonal membranes which may be partially responsible for the antiarrhythmic activity of this agent.     

The independence of membrane potential and potassium activation of the sodium pump in muscle

The membrane potential (E_m) of sartorius muscle fibers was made insensitive to [K⁺] by equilibration in a 95 mM K⁺, 120 mM Na⁺ Ringer solution. Under these conditions a potassium-activated, ouabain-sensitive sodium efflux was observed which had characteristics similar to those seen in muscles with E_m sensitive to [K⁺]. In addition, in the presence of 10 mM K⁺, these muscles were able to produce a net sodium extrusion against an electrochemical gradient which was also inhibited by 10^?4 M ouabain. This suggests that the membrane potential does not play a major role in the potassium activation of the sodium pump in muscles.     

Regulation of ClC-1 and KATP channels in action potential–firing fast-twitch muscle fibers

Action potential (AP) excitation requires a transient dominance of depolarizing membrane currents over the repolarizing membrane currents that stabilize the resting membrane potential. Such stabilizing currents, in turn, depend on passive membrane conductance (G_m), which in skeletal muscle fibers covers membrane conductances for K⁺ (G_K) and Cl⁻ (G_Cl). Myotonic disorders and studies with metabolically poisoned muscle have revealed capacities of G_K and G_Cl to inversely interfere with muscle excitability. However, whether regulation of G_K and G_Cl occur in AP-firing muscle under normal physiological conditions is unknown. This study establishes a technique that allows the determination of G_Cl and G_K with a temporal resolution of seconds in AP-firing muscle fibers. With this approach, we have identified and quantified a biphasic regulation of G_m in active fast-twitch extensor digitorum longus fibers of the rat. Thus, at the onset of AP firing, a reduction in G_Cl of ∼70% caused G_m to decline by ∼55% in a manner that is well described by a single exponential function characterized by a time constant of ∼200 APs (phase 1). When stimulation was continued beyond ∼1,800 APs, synchronized elevations in G_K (∼14-fold) and G_Cl (∼3-fold) caused G_m to rise sigmoidally to ∼400% of its level before AP firing (phase 2). Phase 2 was often associated with a failure to excite APs. When AP firing was ceased during phase 2, G_m recovered to its level before AP firing in ∼1 min. Experiments with glibenclamide (K_ATP channel inhibitor) and 9-anthracene carboxylic acid (ClC-1 Cl⁻ channel inhibitor) revealed that the decreased G_m during phase 1 reflected ClC-1 channel inhibition, whereas the massively elevated G_m during phase 2 reflected synchronized openings of ClC-1 and K_ATP channels. In conclusion, G_Cl and G_K are acutely regulated in AP-firing fast-twitch muscle fibers. Such regulation may contribute to the physiological control of excitability in active muscle.     

The equivalent circuit of single crab muscle fibers as determined by impedance measurements with intracellular electrodes

The input impedance of muscle fibers of the crab was determined with microelectrodes over the frequency range 1 cps to 10 kc/sec. Care was taken to analyze, reduce, and correct for capacitive artifact. One dimensional cable theory was used to determine the properties of the equivalent circuit of the membrane admittance, and the errors introduced by the neglect of the three dimensional spread of current are discussed. In seven fibers the equivalent circuit of an element of the membrane admittance must contain a DC path and two capacitances, each in series with a resistance. In two fibers, the element of membrane admittance could be described by one capacitance in parallel with a resistance. In several fibers there was evidence for a third very large capacitance. The values of the elements of the equivalent circuit depend on which of several equivalent circuits is chosen. The circuit (with a minimum number of elements) that was considered most reasonably consistent with the anatomy of the fiber has two branches in parallel: one branch having a resistance R_e in series with a capacitance C_e; the other branch having a resistance R_b in series with a parallel combination of a resistance R_m and a capacitance C_m. The average circuit values (seven fibers) for this model, treating the fiber as a cylinder of sarcolemma without infoldings or tubular invaginations, are R_e = 21 ohm cm²; C_e = 47 µf/cm²; R_b = 10.2 ohm cm²; R_m = 173 ohm cm²; C_m = 9.0 µf/cm². The relation of this equivalent circuit and another with a nonminimum number of circuit elements to the fine structure of crab muscle is discussed. In the above equivalent circuit R_m and C_m are attributed to the sarcolemma; R_e and C_e, to the sarcotubular system; and R_b, to the amorphous material found around crab fibers. Estimates of actual surface area of the sarcolemma and sarcotubular system permit the average circuit values to be expressed in terms of unit membrane area. The values so expressed are consistent with the dielectric properties of predominantly lipid membranes.     

Effects of temperature and thermal history on neuromuscular properties of two crustacean species

Summary The effect of temperature on the ability of neuromuscular junctions and muscle fibers to contract, release neurotransmitter, and maintein postsynaptic membrane properties, was measured in vivo and in vitro in claw closer muscles in stone crabsMenippe mercenaria (Say) and blues crabsCallinectes sapidus (Rathbun).In vivo muscle stress (defined as force generated per unit of muscle cross-sectional area) was measured in crabs of both species collected from northern populations (annual temperature range 2–30°C) and southern populations (annual temperature range 15–30°C). Muscle stress was compared between (1) crabs of both species maintained in the laboratory at 30°C (laboratory warmed); (2) crabs given a brief acclimation period (4 weeks for blue crabs; 7 weeks for stone crabs) at 8°C in the laboratory (laboratory colled), and (3) stone crabs that had been naturally acclimated from summer (30°C) to winter (8°C) temperatures over a 6 month period in the field (naturally cooled). No differences were found in the abilities of the northern and southern populations of either species to generate muscle stress when tested at summer temperatures (30°C) common to both populations. Northern and southern blue crabs produced similar levels of muscle stress whether laboratory warmed (30°C) or laboratory cooled (8°C). Conversely, northern and southern stone crabs showed significantly reduced muscle stress in laboratory cooled crabs compared with laboratory warmed crabs. Stone crabs from both populations generated the same amount of muscle stress after having been naturally cooled to 8°C as they had generated the previous summer (30°C).In vitro neuromuscular properties (i.e. (1) muscle stress as a measure of contractile ability; (2) excitatory junction potential (EJP) amplitude as a measure of neurotransmitter release; (3) specific membrane resistance (R_m) as a measure of postsynaptic membrane properties) were compared at 8, 20, and 30°C between northern cold acclimated (naturally cooled stone crabs and laboratory cooled blue crabs) and non-cold acclimated (laboratory cooled stone crabs. Muscle fibers in claws of stone crabs and blue crabs showing cold acclimation had higher R_m at 8°C than non-cold acclimated crabs. This higher R_m resulted in a broadening of EJP's which enhanced EJP summation, muscle fiber depolarization, and muscle stress.Abbreviations EJP excitatory junction potential - E _r resting membrane potential - F _e lacilitation - R _m specific membrane resistance     

Expression and characterization of chimeric spidroins from flagelliform-aciniform repetitive modules

Spiders can produce up to seven different types of silks or glues with different mechanical properties. Of these, flagelliform (Flag) silk is the most elastic, and aciniform (AcSp1) silk is the toughest. To produce a chimeric spider silk (spidroin) Flag_R-AcSp1_R, we fused one repetitive module of flagelliform silk from Araneus ventricosus and one repetitive module of aciniform silk from Argiope trifasciata. The recombinant protein expressed in E. coli formed silk-like fibers by manual-drawing. CD analysis showed that the secondary structure of Flag_R-AcSp1_R spidroin remained stable during the gradual reduction of pH from 7.0 to 5.5. The spectrum of FTIR indicated that the secondary structure of Flag_R-AcSp1_R changed from α-helix to β-sheet. The conformation change of Flag_R-AcSp1_R was similar to other spidroins in the fiber formation process. SEM analysis revealed that the mean diameter of the fibers was around 1 ~ 2 μm, and the surface was smooth and uniform. The chimeric fibers exhibited superior toughness (~33.1 MJ/m³) and tensile strength (~261.4 MPa). This study provides new insight into design of chimeric spider silks with high mechanical properties.     

Correlated Electrophysiological and Ultrastructural Studies of a Crustacean Motor Unit

Structural and functional interrelationships between the pre- and postsynaptic elements of a singly motor innervated crab muscle (stretcher of Hyas araneus L.) were examined using electrophysiological and electron microscopic techniques. Excitatory postsynaptic potential (EPSP) amplitude at 1 Hz was found to be inversely related to the extent of facilitation, and directly related both to the amount of transmitter released at 1 Hz and the muscle fiber input resistance (R_in). The extent of facilitation (F_e), taken as the ratio of the EPSP amplitude at 10 Hz to that 1 Hz, was inversely related to muscle fiber R_in, τ_m, and sarcomere length. Sarcomere length was directly related to R_in and τ_m. The excitatory nerve terminals of low F_e muscle fibers had larger neuromuscular synapses than did those of high F_e fibers. Inhibitory axo-axonal synapses were more often found in low F_e muscle fibers. These structural features may account for the greater release of transmitter at low frequencies from the low F_e nerve terminals as well as provide for a greater amount of presynaptic inhibition of low F_e muscle fibers. The implications of these findings for the development and physiological performance of the crustacean motor unit are discussed. It is proposed that both nerve and muscle fiber properties may be determined by the developmental pattern of nerve growth.     

The Structure of Mytilus Smooth Muscle and the Electrical Constants of the Resting Muscle

The individual muscle fibers of the anterior byssus retractor muscle (ABRM) of Mytilus edulis L. are uninucleate, 1.2–1.8 mm in length, 5 µm in diameter, and organized into bundles 100–200 µm in diameter, surrounded by connective tissue. Some bundles run the length of the whole muscle. Adjacent muscle cell membranes are interconnected by nexuses at frequent intervals. Specialized attachments exist between muscle fibers and connective tissue. Electrical constants of the resting muscle membrane were measured with intracellular recording electrodes and both extracellular and intracellular current-passing electrodes. With an intracellular current-passing electrode, the time constant τ, was 4.3 ± 1.5 ms. With current delivered via an extracellular electrode τ was 68.3 ± 15 ms. The space constant, λ, was 1.8 mm ± 0.4. The membrane input resistance, R_eff, ranged from 23 to 51 MΩ. The observations that values of τ depend on the method of passing current, and that the value of λ is large relative to fiber length and diameter are considered evidence that the individual muscle fibers are electrically interconnected within bundles in a three-dimensional network. Estimations are made of the membrane resistance, R_m, to compare the values to fast and slow striated muscle fibers and mammalian smooth muscles. The implications of this study in reinterpreting previous mechanical and electrical studies are discussed.     

Comparison of regulated passive membrane conductance in action potential–firing fast- and slow-twitch muscle

In several pathological and experimental conditions, the passive membrane conductance of muscle fibers (G_m) and their excitability are inversely related. Despite this capacity of G_m to determine muscle excitability, its regulation in active muscle fibers is largely unexplored. In this issue, our previous study (Pedersen et al. 2009. J. Gen. Physiol. doi:10.1085/jgp.200910291) established a technique with which biphasic regulation of G_m in action potential (AP)-firing fast-twitch fibers of rat extensor digitorum longus muscles was identified and characterized with temporal resolution of seconds. This showed that AP firing initially reduced G_m via ClC-1 channel inhibition but after ∼1,800 APs, G_m rose substantially, causing AP excitation failure. This late increase of G_m reflected activation of ClC-1 and K_ATP channels. The present study has explored regulation of G_m in AP-firing slow-twitch fibers of soleus muscle and compared it to G_m dynamics in fast-twitch fibers. It further explored aspects of the cellular signaling that conveyed regulation of G_m in AP-firing fibers. Thus, in both fiber types, AP firing first triggered protein kinase C (PKC)-dependent ClC-1 channel inhibition that reduced G_m by ∼50%. Experiments with dantrolene showed that AP-triggered SR Ca²⁺ release activated this PKC-mediated ClC-1 channel inhibition that was associated with reduced rheobase current and improved function of depolarized muscles, indicating that the reduced G_m enhanced muscle fiber excitability. In fast-twitch fibers, the late rise in G_m was accelerated by glucose-free conditions, whereas it was postponed when intermittent resting periods were introduced during AP firing. Remarkably, elevation of G_m was never encountered in AP-firing slow-twitch fibers, even after 15,000 APs. These observations implicate metabolic depression in the elevation of G_m in AP-firing fast-twitch fibers. It is concluded that regulation of G_m is a general phenomenon in AP-firing muscle, and that differences in G_m regulation may contribute to the different phenotypes of fast- and slow-twitch muscle.     

Influence of the State of TTX-Resistant Sodium Channels on Electrical Activity of a Nociceptive Sensory Fiber: A Model Study

On a model of a thin (C-type) primary afferent fiber, we examined one of the hypotheses related to the phenomenon of initiation of long-lasting tonic discharges in nociceptive afferents. In the membrane of a region corresponding to the free peripheral terminal of the modeled nociceptive C fiber, there were sodium channels of three types (channels of rapidly inactivating TTX-sensitive current and TTX-resistant channels of two types, Na_V1.8/SNS/PN3 and Na_V1.9/NaN/SNS2). As is known, TTX-resistant sodium currents promote the development of long-lasting trains of action potentials, APs, where the duration of tonic discharges exceeds by orders of magnitude the duration of short stimuli inducing such discharges. Such trains, when transmitted to the spinal cord, are interpreted as pain signals. Using the model, we obtained the time course of changes in the membrane potential in the distal and proximal segments of the nerve fiber and values of the densities of inward and outward TTX-resistant sodium currents through channels Na_V1.9/NaN/SNS2 and Na_V1.8/SNS/PN3 in the norm and in a state mimicking the action of inflammation factors. Results of modeling demonstrated that TTX-resistant sodium currents provide intensification of slow components in the generated APs (plateau afterdepolarization). Having a higher inactivation threshold, these currents are inactivated more slowly and recover more rapidly after inactivation, as compared with the currents through TTX-sensitive sodium channels. Such behavior presupposes a considerable role of the TTX-resistant currents in facilitation of transmission of nociceptive signals under conditions of neuropathic pain characterized by excessive “upregulation” of the respective channels. It can be concluded that expression of TTX-resistant sodium channels in nociceptive sensory neurons possessing primary afferent C fibers, the presence of these channels in the membranes of peripheral terminals of the above fibers, and modification of biophysical properties of such channels under conditions of action of inflammation mediators, when taken together, create substantial prerequisites for initiation of anomalous long-lasting AP trains in the above peripheral terminals and, therefore, for transmission of such signals to the CNS. Such a situation appears to be a key electrophysiological phenomenon responsible for generation of neuropathic and inflammation-related pain.     

Electrical properties of skeletal muscle fibres of the flour moth larva Ephestia kuehniella

The electrical properties of the ventral longitudinal muscle fibres in the flour moth larva Ephestia kuehniella were investigated at rest and during electrical activity. The membrane resting potential was only partially dependent on the K-concentration gradient across the muscle membrane. The electrical constants λ, τ, R_m, R_i, and C_m were determined according to the equations for ‘short cables’ (Table 1). Current-voltage relationships of the muscle membrane were measured: they revealed anomalous as well as delayed rectification of the membrane. Stimulation of the muscle fibres with intracellular current pulses elicited graded action potentials in most fibres; in some fibres ‘all-or-none’ action potentials were generated. In contrast to graded action potentials these ‘all-or-none’ action potentials were propagated without decrement along the muscle fibre. Indirect stimulation of the muscle fibres resulted in large excitatory junction potentials which generally gave rise to action potentials.     

Graded and All-or-None Electrogenesis in Arthropod Muscle : I. The effects of alkali-earth cations on the neuromuscular system of Romalea microptera

Graded electrically excited responsiveness of Romalea muscle fibers is converted to all-or-none activity by Ba⁺⁺, Sr⁺⁺, or Ca⁺⁺, the two former being much the more effective in this action. The change occurs with as little as 7 to 10 per cent of Na⁺ substituted by Ba⁺⁺. The spikes now produced have overshoots and may be extremely prolonged, lasting many seconds. During the spike the membrane resistance is lower than in the resting fiber, but the resting resistance and time constant are considerably increased by the alkali-earth ions. The excitability is also increased, spikes arising neurogenically from spontaneous repetitive discharges in the axon as well as myogenically from spontaneous activity in the muscle fibers. Repetitive responses frequently occur on intracellular stimulation with a brief pulse. The data indicate that the alkali-earth ions exert a complex of effects on the different action components of electrically excitable membrane. They may be described in terms of the ionic theory as follows: The resting K⁺ conductance is diminished. The sodium inactivation process is also diminished, and sodium activation may be increased. Together these changes can act to convert graded responsiveness to the all-or-none variety. The alkali-earth ions can also to some degree carry inward positive charge during activity, since spikes are produced when Na⁺ is fully replaced with the divalent ions.     

Maximum Rate of Depolarization of Single Muscle Fiber in Normal and Low Sodium Solutions

The values of membrane action potentials and maximum depolarization rates of single muscle fibers in normal Tyrode solution and in low sodium solutions containing as little as 20 per cent of the sodium chloride were measured with intracellular microelectrodes. Under these conditions the membrane potential remains unchanged up to 36 per cent of [Na⁺]_out concentration, whereas the overshoot of the action potential varies linearly with the logarithm of the external sodium concentration. The maximum depolarization rate is a linear function of the external sodium concentration. The results obtained support the ionic theory for sodium and the independence principle for sodium current related to the external sodium concentration.     

Denervation causes fiber atrophy and myosin heavy chain co-expression in senescent skeletal muscle

Although denervation has long been implicated in aging muscle, the degree to which it is causes the fiber atrophy seen in aging muscle is unknown. To address this question, we quantified motoneuron soma counts in the lumbar spinal cord using choline acetyl transferase immunhistochemistry and quantified the size of denervated versus innervated muscle fibers in the gastrocnemius muscle using the in situ expression of the denervation-specific sodium channel, Nav_1.5, in young adult (YA) and senescent (SEN) rats. To gain insights into the mechanisms driving myofiber atrophy, we also examined the myofiber expression of the two primary ubiquitin ligases necessary for muscle atrophy (MAFbx, MuRF1). MN soma number in lumbar spinal cord declined 27% between YA (638±34 MNs×mm⁻¹) and SEN (469±13 MNs×mm⁻¹). Nav_1.5 positive fibers (1548±70 μm²) were 35% smaller than Nav_1.5 negative fibers (2367±78 μm²; P<0.05) in SEN muscle, whereas Nav_1.5 negative fibers in SEN were only 7% smaller than fibers in YA (2553±33 μm²; P<0.05) where no Nav_1.5 labeling was seen, suggesting denervation is the primary cause of aging myofiber atrophy. Nav_1.5 positive fibers had higher levels of MAFbx and MuRF1 (P<0.05), consistent with involvement of the proteasome proteolytic pathway in the atrophy of denervated muscle fibers in aging muscle. In summary, our study provides the first quantitative assessment of the contribution of denervation to myofiber atrophy in aging muscle, suggesting it explains the majority of the atrophy we observed. This striking result suggests a renewed focus should be placed on denervation in seeking understanding of the causes of and treatments for aging muscle atrophy.     

A fully coupled transient excited state model for the sodium channel

Summary The axon membrane is simulated by standard Hodgkin-Huxley leakage and potassium channels plus a coupled transient excited state kinetic scheme for the sodium channel. This scheme for the sodium channel is as proposed previously by the author. Simultations are presented showing the form of the action potential, threshold behavior, accommodation, and repetitive firing. It is seen that the form of the individual action potential, its all-or-none nature, and its refractory period are well simulated by this model, as they are by the standard Hodgkin-Huxley model. However, the model differs markedly from the Hodgkin-Huxley model with respect to repetitive firing and accommodation to stimulating currents of slowly rising intensity, in ways that are anomn to be related to those features of the sodium inactivation which are anomalous to the H-H model. The tendency for repetitive firing is highly dependent on that parameter which primarily determintes the existence of the inactivation shift in voltage clamp experiments, in such a way that the more pronounced the inactivation shift, the less the tendency for repetitive firing,. The tendency for accommodation is highly dependent on that parameter which primarily determines the “τ_c − τ_h” separation, in such a way that the greater the separation the greater the tendency for the membrane to accommodate without firing action potentials to a slowly rising current.     

Neuromuscular transmission in cultures of adult human and rodent skeletal muscle after innervation in vitro by fetal rodent spinal cord

Electrophysiologic analyses have been carried out on in vitro-coupled explants of fetal rodent spinal cord and adult skeletal muscle of human as well as rodent origin. The studies demonstrate that characteristic neuromuscular transmission can develop and be maintained in these unusual tissue combinations during long-term culture. After coupling periods of 2–7 weeks in vitro, selective stimulation of spinal cord evokes widespread coordinated contractions in the muscle tissue. Simultaneous microelectrode recordings of cord and muscle responses to local cord, or ventral root, stimuli show that muscle action potentials (and contractions) generally occur with latencies of several msec after onset of cord discharges. Similar temporal relations are often seen during spontaneous rhythmic discharges of the coupled cord and muscle tissues. Long series of repetitive discharges, at 2–5 sec intervals, may occur synchronously between these cord and muscle explants, in response to single cord (or dorsal-root ganglion) stimuli, and they may also appear spontaneously. d-Tubocurarine (1–10 μg/ml) selectively and reversibly blocks neuromuscular transmission in these cultures. Eserine accelerates recovery of normal function. Spontaneous repetitive fibrillations of many of the cultured muscle fibers are observed sporadically, and these contractions often continue unabated after block of neuromusclar transmission by d-tubocurarine. Many of the fibers which show asynchronous fibrillations are probably not innervated (as in denervated muscle in situ). In some cases, however, extracellular as well as intracellular recordings indicate that similar fibrillations may also occur in fibers which are clearly innervated. Repetitive cord and muscle discharges are greatly augmented after introduction of strychnine. Complex rhythmic oscillatory (ca. 10/sec) afterdischarges generated in strychninized cord explants lead to similarly patterned muscle discharges (and contractions), which may also occur, at, times, in normal medium.     

The ionic requirements for the initiation of action potentials in insect muscle fibers

Electrical properties of locust leg muscle fibers were studied by means of intracellular electrodes. In most fibers, a depolarizing current pulse initiated a local response. A delayed decrease in membrane resistance appeared with more than about 10 mv depolarization. In some fibers a regenerative response also was found. Membrane constants were measured, applying the short cable model. The value of the space constant λ was 1.6 mm and the calculated value of R_m was about 1750 ohm cm². Action potentials could be elicited when the bathing fluid contained more than 2–5 mM Ba or Sr. Similar responses were seen with 2 mM Ca in the presence of tetraethylammonium (TEA). The overshoot of these action potentials increased with increasing [Ca⁺⁺]_o, [Sr⁺⁺]_o, or [Ba⁺⁺]_o, the increment for a 10-fold increase being about 29 mv for Ca and Sr and between 40 and 50 mv for Ba. These action potentials were inhibited by Mn ions but were not affected by tetrodotoxin or procaine. In solutions containing Ba or Sr, action potentials generated were suppressed by addition of Ca. The removal of Na ions did not change the configuration of the action potential. The results suggest that an increase in permeability to Ca, Ba, or Sr ions makes a major contribution to the initiation of action potentials in this tissue.     

Analysis of the transformation effect in cytochrome b559 of photosystem II in terms of the model of the heme-quinone redox interaction

Transformation of three-component redox pattern of cytochrome (Cyt) b559 in PS II membrane fragments upon various treatments is manifested in decrease of the relative content (R) of the high potential (HP) redox form of Cyt b559 and concomitant increase in the fractions of the two lower potential forms. Redox titration of Cyt b559 in different types of PS II membrane preparations was performed and revealed that (1) alteration of redox titration curve of Cyt b559 upon treatment of a sample is not specific to the type of treatment; (2) each value of R_HP defines the individual shape of the redox titration curve; (3) population of Cyt b559 may exist in several stable forms with multicomponent redox pattern: three types of three-component redox pattern and one type of two-component redox pattern as well as in the form with a single E_m; (4) transformation of Cyt b559 proceeds as successive conversion between the stable forms with multicomponent redox pattern; (5) upon harsh treatments, Cyt b559 abruptly converts into the state with a single E_m which value is intermediate between the E_m values of the two lower potential forms. Analysis of the data using the model of Cyt b559-quinone redox interaction revealed that diminution of R_HP in a range from 80 to 10% reflects a shift in redox equilibrium between the heme group of Cyt b559 and the interacting quinone, due to a gradual decrease of 90?mV in E_m of the heme group at the virtually unchanged E_m of the quinone component.     

Electric membrane properties of adult mouse DRG neurons and the effect of culture duration

The electrical membrane properties (EMP) of adult mouse dorsal root ganglion (DRG) neurons were characterized by an extensive electrophysiological investigation of 450 cells. The neurons were divided into two types: an M-type having an action potential with monophasic falling phase and a B-type with a more complex biphasic or triphasic falling phase. Compared to M-type, B-type were “slow” neurons with a higher specific membrane resistance (R_m), and a longer time constant (τ), duration of action potential (Δt), and absolute refractory period (ARP). B-type also had a larger amplitude action potential, afterhyperpolarization and positive overshoot. The action potential of the M-type neuron had only a Na⁺ component while that of the B-type had both a Na⁺ and a Ca²⁺ component. After two days in culture, M-type neurons exhibited phase bright cytoplasmic granules, which were seldom observed for B-type neurons. Although neuron survival remained constant during the first six days in culture (DIV), the relative frequency of occurrence of the M-type decreased from 82 to 50%. Thereafter, it decreased more gradually to a final value of approximately 20% after 40 DIV. It was concluded that at least during the first 6 DIV and possibly through to 40 DIV, M-type neurons transformed into B-type. Both M- and B-type neurons showed significant and similar changes in their EMP with increasing DIV (up to 40 DIV). For M- and B-types combined, R_m increased approximately 142%, τ by 204%, and no significant change in specific membrane capacitance was observed. Rheobasic threshold depolarization decreased 58%, while the resting membrane potential decreased by only 19%. These changes in the EMP of adult neurons are strikingly similar to changes in EMP observed in adult denervated muscle and in cultures of either embryonic nerve or muscle. This similarity suggested that the adult DRG neurons in cell culture undergo progressive dedifferentiation because of isolation from their usual trophic interactions. Determination of neuronal membrane electrical characteristics provides a new method for evaluating the effects of various possible trophic agents, e.g., hormones and tissue extracts, on the state of differentiation of neurons in cell culture.