The morphological basis of intracellular measurements in the cockroach tactile spine neuron

Summary The femoral tactile spine of the cockroach (Periplaneta americana) contains a single sensory neuron, which adapts rapidly and completely to step deformations of the spine. Techniques for stable intracellular recording from the tactile spine neuron have recently been established, allowing electrophysiological investigation of mechanotransduction and adaptation in this sensory neuron. However, intracellular recordings from the neuron produce a wide range of action potential heights and thresholds, raising the possibility that some penetrations are in adjacent, but closely coupled supporting glial cells. This problem is exacerbated because the cell cannot be visualized during penetration.Systematic measurements of action potential heights and thresholds were made in tactile spine cells, together with identification of some penetrated cells by intracellular injection of Lucifer Yellow. All stained cells were clearly sensory neurons, although their action potentials amplitudes varied from 9 mV to 80 mV. Smaller action potentials were broader than larger action potentials, and the changes in height and shape could be explained by a simple cable conduction model using measured morphological and electrical parameters. The model could also account for the observed relationship between action potential height and threshold.These results indicate that reliable recording from the tactile spine neuron is possible, but that variability in the positions of the penetration or the spike initiating zone cause an apparently wide range of electrophysiological measurements.     

Phentolamine selectively affects the fast sodium component of sensory adaptation in an insect mechanoreceptor

Phentolamine and related compounds have several different actions on nervous tissues in vertebrates and invertebrates, including a local anesthetic effect. However, recent work suggests that phentolamine can interfere with sensory transduction in insect mechanoreceptors at significantly lower concentrations than are required for conduction block. We tested the actions of phentolamine on sensory transduction and encoding in an insect mechanoreceptor, the cockroach tactile spine neuron and found that 500 microM phentolamine increased the action potential threshold by 50%. The passive membrane properties of the neuron were not affected, but one component of dynamic threshold change was strongly and selectively reduced. This component has previously been attributed to slowly inactivating sodium channels in the action potential initiating region, suggesting that these channels are the most phentolamine-sensitive sites.     

Selective oxidation of methionine residues in proteins.

Methionine residues in peptides and proteins were oxidized to methionine sulfoxides by mild oxidizing reagents such as chloramine-T and N-chlorosuccinimide at neutral and slightly alkaline pH. With chloramine-T cysteine was also oxidized to cystine but no other amino acid was modified; with N-chlorosuccinimide tryptophans were oxidized as well. In peptides and denaturated proteins all methionine residues were quantitatively oxidized, while in native proteins only exposed methionine residues could be modified. Extent of oxidation of methionine residues was determined by quantitative modification of the unoxidized methionine residues with cyanogen bromide (while methionine sulfoxide residues remained intact), followed by acid hydrolysis and amino acid analysis. Methionine was determined as homoserine and methionine sulfoxide was reduced back to methionine. Sites of oxidation were identified in a similar way by cleaving the unoxidized methionyl peptide bonds with cyanogen bromide, followed by quantitative end-group analysis of the new amino-terminal amino acids (by an automatic sequencer).     

A nonlinear model of step responses in the cockroach tactile spine neuron

Rapid sensory adaptation in the cockroach tactile spine neuron has previously been associated with a labile threshold for action potentials, which changes with the membrane potential by a process involving two time constants. A feed-forward, variable-threshold model has previously been used to account for the frequency response function of the neuron when stimulated with small-signal, white-noise currents. Here, we used a range of accurately controlled steps of extracellular current to stimulate the neuron. The same model was able to predict the individual step responses and could also fit the entire set of step responses from a single neuron if an initial, saturating, static nonlinearity was included. These results indicate that the two-time-constant, variable-threshold model can account for most of the rapidly adapting behavior of the tactile spine neuron.     

After-hyperpolarization and receptor potential attenuation following bursts of action potentials in an insect mechanoreceptor

The receptor potential in the sensory neuron of the cockroach femoral tactile spine was recently observed by raising the axon into an oil bath and measuring the decrementally conducted receptor current. Although action potential discharge in this receptor adapts rapidly, there was no evidence of adaptation in the receptor potential. In the present work we report that bursts of action potentials in the neuron produce a prolonged after-hyperpolarization and attenuate the receptor potential. Both of these effects could be important in receptor adaptation and we sought to identify their origin. It was impossible to control ionic concentrations in the fluid surrounding the sensory neuron because of an effective glial barrier, but it was possible to infuse the tissues with chemical agents which are known to block ionic membrane processes. Cobalt and cadmium, which inhibit calcium influx, eliminated the effects of action potentials, and ouabain had similar effects. These results suggest that both a calcium-activated potassium conductance and an electrogenic sodium pump are involved in these phenomena. However, it is argued that the former is probably more important.     

Solvent-Solute Interactions within the Nexal Membrane

Several methionine-reactive reagents, including N-bromoacetamide, N-bromosuccinimide, chloramine-T, and N-chlorosuccinimide, irreversibly slowed and prevented Na channel inactivation in single myelinated nerve fibers, whereas sulfhydryl- or tyrosine-modifying reagents had little effect. The activation process was not modified by the reagents that altered inactivation and could be modulated normally by Ca++ ions and Centruroides scorpion toxin II alpha. These results suggest that externally applied N-bromoacetamide and its related compounds specifically affect Na channel inactivation by modifying methionine residues of the channel.     

Intracellular nonlinear frequency response measurements in the cockroach tactile spine neuron

The threshold of the cockroach tactile neuron increases strongly with depolarization by a process involving at least two time constants. This effect is probably responsible for the rapid and complete adaptation of the neuron's response to step inputs. A technique for intracellular recording and stimulation of the neuron has recently been established and this allows direct observation of the dynamic response of the neuronal encoder. A white noise stimulus was used to modulate the membrane potential of the neuron. The first-order frequency response function between membrane potential and action potential discharge could be explained by a variable threshold model with two time constants. Second-order frequency response functions could be accounted for by a Wiener cascade model. The dynamic nonlinear behavior of the encoder can therefore be explained by a unidirectional threshold which increases linearly and dynamically with membrane potential.     

Removal of sodium inactivation and block of sodium channels by chloramine-T in crayfish and squid giant axons.

Modification of sodium channels by chloramine-T was examined in voltage clamped internally perfused crayfish and squid giant axons using the double sucrose gap and axial wire technique, respectively. Freshly prepared chloramine-T solution exerted two major actions on sodium channels: (a) an irreversible removal of the fast Na inactivation, and (b) a reversible block of the Na current. Both effects were observed when chloramine-T was applied internally or externally (5-10 mM) to axons. The first effect was studied in crayfish axons. We found that the removal of the fast Na inactivation did not depend on the states of the channel since the channel could be modified by chloramine-T at holding potential (from -80 to -100 mV) or at depolarized potential of -30 mV. After removal of fast Na inactivation, the slow inactivation mechanism was still present, and more channels could undergo slow inactivation. This result indicates that in crayfish axons the transition through the fast inactivated state is not a prerequisite for the slow inactivation to occur. During chloramine-T treatment, a distinct blocking phase occurred, which recovered upon washing out the drug. This second effect of chloramine-T was studied in detail in squid axons. After 24 h, chloramine-T solution lost its ability to remove fast inactivation but retained its blocking action. After removal of the fast Na inactivation, both fresh and aged chloramine-T solutions blocked the Na currents with a similar potency and in a voltage-dependent manner, being more pronounced at lower depolarizing potentials.(ABSTRACT TRUNCATED AT 250 WORDS)     

Involvement of Methionine Residues in the Fast Inactivation Mechanism of the Sodium Current from Toad Skeletal Muscle Fibers

The role of methionine residues on the fast inactivation of the sodium channel from toad skeletal muscle fibers was studied with the mild oxidant chloramine-T (CT). Isolated segments of fibers were voltage clamped in a triple Vaseline? gap chamber. Sodium current was isolated by replacing potassium ions by tetramethylammonium ions in the external and internal solutions. Externally applied chloramine-CT was found to render noninactivating a large fraction of sodium channels and to slow down the fast inactivation mechanism of the remainder fraction of inactivatable channels. The action of CT appeared to proceed first by slowing and then removing the fast inactivation mechanism. The voltage dependence of the steady-state inactivation of the inactivatable CT-treated currents was shifted +10 mV. CT also had a blocking effect on the sodium current, but was without effect on the activation mechanism. The effects of CT were time and concentration dependent and irreversible. The use of high CT concentrations and/or long exposure times was found to be deleterious to the fiber. This side effect precluded the complete removal of fast inactivation. The effects of CT on the fast inactivation of the sodium current can be explained assuming that at least two methionine residues are critically involved in the mechanism underlying this process. Received: 10 November 1998/Revised: 4 January 1999     

Removal of sodium channel inactivation in squid axon by the oxidant chloramine-T

We have investigated the effects of a mild oxidant, chloramine-T(CT), on the sodium and potassium currents of squid axons under voltage-clamp conditions. Sodium channel inactivation of squid giant axons can be completely removed by CT at neutral pH. Internal and external CT treatment are both effective. CT apparently removes inactivation in an irreversible, all-or-none manner. The activation process of sodium channels is little affected, as judged from the voltage dependence of peak sodium currents, the rising phase of sodium currents, and the time course of tail currents following the repolarization. The removal of inactivation by CT is pH-dependent; higher pH decreases the removal rate, whereas lower pH increases it. Internal metabisulfite, a strong reductant, does not protect inactivation from the action of external CT, nor does external metabisulfite protect from internal CT application. CT slightly depresses the peak potassium currents at comparable concentrations but has no apparent effects on their kinetics. Our results suggest that the neutral form of CT modifies an embedded methionine residue that is involved in sodium channel inactivation.     

Nonlinear neuronal mode analysis of action potential encoding in the cockroach tactile spine neuron

 Neuronal mode analysis is a recently developed technique for modelling the behavior of nonlinear systems whose outputs consist of action potentials. The system is modelled as a set of parallel linear filters, or modes, which feed into a multi-input threshold. The characteristics of the principal modes and the multi-input threshold device can be derived from Laguerre function expansions of the computed first- and second-order Volterra kernels when the system is stimulated with a randomly varying input. Neuronal mode analysis was used to model the encoder properties of the cockroach tactile spine neuron, a nonlinear, rapidly adapting, sensory neuron with reliable behavior. The analysis found two principal modes, one rapid and excitatory, the other slower and inhibitory. The two modes have analogies to two of the pathways in a block-structured model of the encoder that was developed from previous physiological investigations of the neuron. These results support the block-structured model and offer a new approach to identifying the components responsible for the nonlinear dynamic properties of this neuronal encoder. Received: 30 December 1994/Accepted in revised form: 25 April 1995     

A simple Markov model of sodium channels with a dynamic threshold

Characteristics of action potential generation are important to understanding brain functioning and, thus, must be understood and modeled. It is still an open question what model can describe concurrently the phenomena of sharp spike shape, the spike threshold variability, and the divisive effect of shunting on the gain of frequency-current dependence. We reproduced these three effects experimentally by patch-clamp recordings in cortical slices, but we failed to simulate them by any of 11 known neuron models, including one- and multi-compartment, with Hodgkin-Huxley and Markov equation-based sodium channel approximations, and those taking into account sodium channel subtype heterogeneity. Basing on our voltage-clamp data characterizing the dependence of sodium channel activation threshold on history of depolarization, we propose a 3-state Markov model with a closed-to-open state transition threshold dependent on slow inactivation. This model reproduces the all three phenomena. As a reduction of this model, a leaky integrate-and-fire model with a dynamic threshold also shows the effect of gain reduction by shunt. These results argue for the mechanism of gain reduction through threshold dynamics determined by the slow inactivation of sodium channels.     

Radioiodination of naturally occurring phospholipids

A simple and rapid nonenzymatic method for radioiodination of phospholipids is described. It involves oxidation of Na125I with TlCl3 (or chloramine-T) in an aqueous medium, with subsequent exposure of the phospholipids, dissolved in chloroform/methanol, to the action of the oxidizing mixture. Purification of the radiolabelled phospholipids was effected by washing with sodium thiosulphate followed by thin-layer chromatography on silica gel. Specific radioactivity of 125I-labelled phosphatidylcholine was estimated to be about 10 muCi/mg phospholipid. The method is designed for radioiodination of various naturally occurring phospholipids.     

Properties of chemically oxidized kininogens

Kininogens are multifunctional proteins involved in a variety of regulatory processes including the kinin-formation cascade, blood coagulation, fibrynolysis, inhibition of cysteine proteinases etc. A working hypothesis of this work was that the properties of kininogens may be altered by oxidation of their methionine residues by reactive oxygen species that are released at the inflammatory foci during phagocytosis of pathogen particles by recruited neutrophil cells. Two methionine-specific oxidizing reagents, N-chlorosuccinimide (NCS) and chloramine-T (CT), were used to oxidize the high molecular mass (HK) and low molecular mass (LK) forms of human kininogen. A nearly complete conversion of methionine residues to methionine sulfoxide residues in the modified proteins was determined by amino acid analysis. Production of kinins from oxidized kininogens by plasma and tissue kallikreins was significantly lower (by at least 70%) than that from native kininogens. This quenching effect on kinin release could primarily be assigned to the modification of the critical Met-361 residue adjacent to the internal kinin sequence in kininogen. However, virtually no kinin could be formed by human plasma kallikrein from NCS-modified HK. This observation suggests involvement of other structural effects detrimental for kinin production. Indeed, NCS-oxidized HK was unable to bind (pre)kallikrein, probably due to the modification of methionine and/or tryptophan residues at the region on the kininogen molecule responsible for the (pro)enzyme binding. Tests on papain inhibition by native and oxidized kininogens indicated that the inhibitory activity of kininogens against cysteine proteinases is essentially insensitive to oxidation.     

Interaction of Bacillus subtilis spores with sodium hypochlorite, sodium dichloroisocyanurate and chloramine-T

S.F. BLOOMFIELD AND M. ARTHUR. 1992. Solutions of chlorine-releasing agents (CRAs) show varying activity against Bacillus subtilis spores; sodium hypochlorite (NaOCl) shows higher activity than sodium dichloroisocyanurate (NaDCC) which is more active than chloramine-T. Investigations with coat- and cortex-extracted spores indicate that resistance to CRAs depends not only on the spore coat but also the cortex. Whereas extraction of alkali-soluble coat protein increased sensitivity to NaOCl and NaDCC, degradation of coat and cortex material was required to achieve significant activity with chloramine-T. NaOCl (in the presence and absence of NaOH) and NaDCC (in the presence of NaOH only) produced degradation of spore coat and cortes material which may be related to their rapid sporicidal action at low concentrations under these conditions. By contrast, chloramine-T produced no degradation of cortex peptidoglycan and was only effective against normal and alkali-treated spores at high concentrations, requiring extraction of peptidoglycan with urea/dithiothreitol/sodium lauryl sulphate (UDS) or UDS/lysozyme to achieve significant activity at low concentrations. Results suggest that the sporicidal action of CRAs is associated with spore coat and cortex degradation causing rehydration of the protoplast allowing diffusion to the site of action on the underlying protoplast.     

Interaction of Bacillus subtilis spores with sodium hypochlorite, sodium dichloroisocyanurate and chloramine-T.

Solutions of chlorine-releasing agents (CRAs) show varying activity against Bacillus subtilis spores; sodium hypochlorite (NaOCl) shows higher activity than sodium dichloroisocyanurate (NaDCC) which is more active than chloramine-T. Investigations with coat- and cortex-extracted spores indicate that resistance to CRAs depends not only on the spore coat but also the cortex. Whereas extraction of alkali-soluble coat protein increased sensitivity to NaOCl and NaDCC, degradation of coat and cortex material was required to achieve significant activity with chloramine-T. NaOCl (in the presence and absence of NaOH) and NaDCC (in the presence of NaOH only) produced degradation of spore coat and cortex material which may be related to their rapid sporicidal action at low concentrations under these conditions. By contrast, chloramine-T produced no degradation of cortex peptidoglycan and was only effective against normal and alkali-treated spores at high concentrations, requiring extraction of peptidoglycan with urea/dithiothreitol/sodium lauryl sulphate (UDS) or UDS/lysozyme to achieve significant activity at low concentrations. Results suggest that the sporicidal action of CRAs is associated with spore coat and cortex degradation causing rehydration of the protoplast allowing diffusion to the site of action on the underlying protoplast.     

Effects of chloramine-T on activation and inactivation of sodium channels in neuroblastoma cells

The effects of chloramine-T, a reagent specific to methionine residues, on sodium channel gating mechanisms was investigated in neuroblastoma cell membrane. Treating the membrane with chloramine was found to retard inactivation kinetics and considerably reduce the slope of the inactivation curve, while pushing the activation curve toward hyperpolarization ranges without changing the slope of the central portion perceptibly. Effective activation charge, as determined from the limiting logarithmic slope of activation, was reduced by a factor of 1.17. Possible reasons for the changes observed in sodium channel gating mechanisms are discussed.Institute of Cytology, Academy of Sciences of the USSR, Leningrad. Translated from Neirofiziologiya, Vol. 19, No. 6, pp. 789–795, November–December, 1987.     

Treatment with oxidizing agents damages the inner membrane of spores of Bacillus subtilis and sensitizes spores to subsequent stress

AIMS: To determine if treatment of Bacillus subtilis spores with a variety of oxidizing agents causes damage to the spore's inner membrane. METHODS AND RESULTS: Spores of B. subtilis were killed 80-99% with wet heat or a variety of oxidizing agents, including betadine, chlorine dioxide, cumene hydroperoxide, hydrogen peroxide, Oxone, ozone, sodium hypochlorite and t-butylhydroperoxide, and the agents neutralized and/or removed. Survivors of spores pretreated with oxidizing agents exhibited increased sensitivity to killing by a normally minimal lethal heat treatment, while spores pretreated with wet heat did not. In addition, spores treated with wet heat or the oxidizing agents, except sodium hypochlorite, were more sensitive to high NaCl in plating media than were untreated spores. The core region of spores treated with at least two oxidizing agents was also penetrated much more readily by methylamine than was the core of untreated spores, and spores treated with oxidizing agents but not wet heat germinated faster with dodecylamine than did untreated spores. Spores of strains with very different levels of unsaturated fatty acids in their inner membrane exhibited essentially identical resistance to oxidizing agents. CONCLUSIONS: Treatment of spores with oxidizing agents has been suggested to cause damage to the spore's inner membrane, a membrane whose integrity is essential for spore viability. The sensitization of spores to killing by heat and to high salt after pretreatment with oxidizing agents is consistent with and supports this suggestion. Presumably mild pretreatment with oxidizing agents causes some damage to the spore's inner membrane. While this damage may not be lethal under normal conditions, the damaged inner membrane may be less able to maintain its integrity, when dormant spores are exposed to high temperature or when germinated spores are faced with osmotic stress. Triggering of spore germination by dodecylamine likely involves action by this agent on the spore's inner membrane allowing release of the spore core's depot of dipicolinic acid. Presumably dodecylamine more readily alters the permeability of a damaged inner membrane and thus more readily triggers germination of spores pretreated with oxidizing agents. Damage to the inner spore membrane by oxidizing agents is also consistent with the more rapid penetration of methylamine into the core of treated spores, as the inner membrane is likely the crucial permeability barrier to methylamine entry into the spore core. As spores of strains with very different levels of unsaturated fatty acids in their inner membrane exhibited essentially identical resistance to oxidizing agents, it is not through oxidation of unsaturated fatty acids that oxidizing agents kill and/or damage spores. Perhaps these agents work by causing oxidative damage to key proteins in the spore's inner membrane. SIGNIFICANCE AND IMPACT OF THE STUDY: The more rapid heat killing and germination with dodecylamine, the greater permeability of the spore core and the osmotic stress sensitivity in outgrowth of spores pretreated with oxidizing agents is consistent with such agents causing damage to the spore's inner membrane, even if this damage is not lethal under normal conditions. It may be possible to take advantage of this phenomenon to devise improved, less costly regimens for spore inactivation.     

White-noise analysis of nonlinear behavior in an insect sensory neuron: Kernel and cascade approaches

A functional expansion was used to model the relationship between a Gaussian white noise stimulus current and the resulting action potential output in the single sensory neuron of the cockroach femoral tactile spine. A new precise procedure was used to measure the kernels of the functional expansion. Very similar kernel estimates were obtained from separate sections of the data produced by the same neuron with the same input noise power level, although some small time-varying effects were detectable in moving through the data. Similar kernel estimates were measured using different input noise power levels for a given cell, or when comparing different cells under similar stimulus conditions. The kernels were used to identify a model for sensory encoding in the neuron, comprising a cascade of dynamic linear, static nonlinear, and dynamic linear elements. Only a single slice of the estimated experimental second-order kernel was used in identifying the cascade model. However, the complete second-order kernel of the cascade model closely resembled the estimated experimental kernel. Moreover, the model could closely predict the experimental action potential train obtained with novel white noise inputs.     

Na activation delays and their relation to inactivation in frog skeletal muscle

Summary Delays in the development of activation of Na currents were studied using voltage-clamped frog skeletal muscle fibers. Na currents elicited by a depolarizing voltage step from a hyperpolarized membrane potential were delayed in their activation when compared to Na currents elicited from the resting potential. The magnitude of the delay increased with larger hyperpolarizing potentials and decreased with larger depolarizing test potentials. Delays in activation observed following chloramine-T treatment that partially removes inactivation did not differ from delays observed before treatment. Longer exposures of the muscle fiber to chloramine-T led to a complete loss of inactivation, coincident with an elimination of the hyperpolarization-induced delays in activation. Steady-state slow inactivation was virtually unaffected by prolonged exposures of the fibers to chloramine-T that eliminated fast inactivation. The results show that chloramine-T acts at a number of sites to alter both activation and inactivation. Markov model simulations of the results show that chloramine-T alters fundamental time constants of the system by altering both activation and inactivation rate constants.