B. Rydqvist

Crayfish stretch receptor: an investigation with voltage‐clamp and ion‐sensitive electrodes.

1. The membrane characteristics of the slowly adapting stretch receptor from the crayfish, Astacus fluviatilis, were examined with electrophysiological techniques consisting of membrane potential recording, voltage clamp and ion‐sensitive microelectrodes. 2. The passive membrane current (Ip) following step changes of the membrane potential to levels above 0 mV required more than a minute to decay to a steady‐state level. 3. The stretch‐induced current (SIC, where SIC = Itotal‐‐Ipassive) was not fully developed until the Ip had decayed to a steady state. 4. With Ip at the steady state and the stretch‐induced current at the O‐current potential, a slow stretch‐induced inward current was isolated. The latter reaches a maximum after 1 sec of stretch and declines even more slowly after stretch. The I‐V relation of the slow current had a negative slope and reversed sign near the resting potential. It is suggested that this current is due to a Cl‐ conductance change. 5. The stretch‐induced current, consisting of a rapid transient phase and a steady component can be isolated from the slow stretch‐induced current at a holding potential corresponding to the resting potential. 6. The SIC‐Em relation is non‐linear and reverses sign at about +15 mV. 7. In a given cell, the reversal potential of the stretch‐induced potential change obtained with current clamp coincided with the 0‐current potential of the stretch‐induced current obtained by voltage clamp. The average value from twenty‐six cells was +13 +/‐ 6.5 mV; cell to cell variability seemed to be correlated with dendrite length. 8. Tris (mol. wt. 121) or arginine (mol. wt. 174) susbstituted for Na+ reduces but does not abolish the stretch‐induced current. 9. The permeability ratios of Tris:Na and arginine:Na were estimated from changes in the 0‐current potential as these cations replaced Na+ in the external medium. The PTris:PNa was somewhat higher (0.31) than the Parginine:PNa ratio (0.25). 10. Changes in the external Ca2+ concentration had no effect on the 0‐current potential in Na or Tris saline. However, reducing Ca2+ did augment the stretch‐induced current in either saline. A tenfold reduction of Ca2+ increased the conductance (at the 0‐current level) about twofold. 11. Intracellular K+ and Cl‐ activities were obtained with ion sensitive electrodes. The average values from six cells were aiK = 133 +/‐ 34 mM and aiCl = 15.2 +/‐ 1.8 mM S.D.). EK was about 20 mV more negative than Em and ECl was about 10 mV more positive than Em. 12. aik and resting Em undergo large changes in K+‐free solutions. After 60 min, ak was reduced eightfold and Em was reduced from ‐67 to ‐40 mV. Reduced Ca2+ in K+‐free augments the rate of these changes. Receptor potential amplitude was also reduced in K+‐free solution but could be restored upon polarizing the membrane to the pre‐existing resting level.

The permeability of the transducer membrane of the crayfish stretch receptor to calcium and other divalent cations.

Abstract The generator potential of the crayfish stretch receptor is produced by the movement of cations through a channel which is opened by stretch. Physiologically, Na + is the main ion moving through the channel; the channel is sufficiently large to admit arginine. The permeability to divalent cations has been measured in solutions in which all of the Na + was replaced by these ions. Ca 2+ , Mg 2+ , Sr 2+ and Ba 2+ were able to move through the channel, as shown by the membrane potential responses to stretch in solutions in which these ions were predominant. Ca 2+ (13.5 min) was found to be necessary in the Ba 2+ and Mg 2+ solutions to maintain membrane integrity; however, in the Sr 2+ solution Ca 2+ was not required. The results of voltage clamp studies showed the resting conductance to decrease in the divalent cation solutions. The reversal potential for the stretch induced current, measured with voltage clamp, was shifted in the negative direction by Ca 2+ , Sr 2+ and Mg 2+ . Two estimates of the value of the ratio of the permeability of Ca 2+ to that of Na + , calculated using the Goldman-Hodgkin-Katz equation, and either the change in reversal potential or the stretch induced current were 1.4 and 0.3 respectively.

The mechanism of action of ketamine of the myelinated nerve membrane

Ketamine is an intravenous anaesthetic that has been reported to react with a number of synaptic and non-synaptic receptors at both the spinal and supraspinal level. The present investigation was undertaken to analyse the effects of ketamine on the myelinated axon under voltage clamp conditions. Both sodium and potassium channels were affected. The effect may be described as mainly a reduction of the permeability constants. No effect on inactivation was observed. The effects were described by a first order binding to receptors within the ion channels that may be identical with the receptors for other anaesthetics. It was concluded from experiments with naloxone that no opiate receptors were involved in the axonal ketamine effects.

Transducer properties of the rapidly adapting stretch receptor neurone in the crayfish (Pacifastacus leniusculus).

1. The transducer properties of the rapidly adapting stretch receptor neurone of the crayfish (Pacifastacus leniusculus) were studied using a two‐microelectrode voltage clamp technique. 2. The impulse response to ramp‐and‐hold extensions of the receptor muscle typically consisted of a high frequency burst followed by cessation of impulses within a relatively short time depending on the amplitude of extension. The type of adaptation was consistent with earlier studies. The stimulus‐response relationship for the impulse frequency was non‐linear and had a slope in a log‐log plot of 2.9. 3. When impulse generation was blocked by tetrodotoxin (TTX), (block of Na+ channels) the receptor potential was extension dependent and similar to that found in the slowly adapting receptor. For small extensions there was an initial peak followed by a fall to a steady potential level. For large extensions the potential response during the ramp phase consisted of a peak followed by a constant potential level lasting to the end of the ramp. When the extension changed to the hold phase the potential fell towards a steady state. The relation between extension and amplitude of receptor potential was non‐linear and saturated at ‐40 to ‐30 mV (extensions > 15% of zero length, lo). 4. When potassium channels were blocked by TEA (50 mM) and 4‐aminopyridine (4‐AP, 5 mM) (and Na+ channels blocked by TTX) the shape of the generator potential become less complex with an increased amplitude for large extensions. 5. When the receptor neurone was voltage clamped at the resting potential, extension of the receptor muscle produced an inwardly directed receptor current, the stretch‐induced current (SIC). The response consisted of a fast transient phase which decayed towards a steady state. The SIC peak amplitude was dependent on extension in a sigmoidal fashion and saturated at 190 nA (extensions > 25% of lo). The slope of the steepest part of the stimulus‐response relation (between 10 and 20% extension) was 4.7 +/‐ 0.25 (mean +/‐ S.E.M.) in a log‐log plot. 6. The peak amplitude of the SIC increased with increasing extension speed (ramp steepness), the relation between the slope of the ramp and current amplitude being a first order (hyperbolic) function. The amplitude of the receptor current was voltage dependent and had a reversal potential of +16.2 +/‐ 1.8 mV (mean +/‐ S.E.M., 32 cells). From the reversal potential the permeability ratio, PNa/PK, of the transducer permeability system was calculated to be 1.5. The I‐V curve of SIC was non‐linear.(ABSTRACT TRUNCATED AT 400 WORDS)

Potential-dependent potassium currents in the slowly adapting stretch receptor neuron of the crayfish

The outward current was analysed in the rapidly adapting stretch receptor neuron of the crayfish Pacifastacus leniusculus with a two-micropipette potential clamp technique and K(+)-selective microelectrodes in an attempt to establish if the properties of this current could explain the difference in adaptive behaviour compared to the slowly adapting receptor. A fast activating outward current carried by K+ was revealed. The time constant of activation(tau n) was dependent on potential and had a mean value of 0.5 ms at potential steps to 0 mV. Activation followed a second-order process according to the Hodgkin-Huxley model. The potential dependence of activation (n infinity) followed by a sigmoid curve n infinity = 1/(1 + exp/[(E - En)/a]) with a half maximal activation potential En = -44 mV and a = -13 mV. When long pulses were applied the outward potassium current decreased with two time constants, one that was potential independent (0.2 s) and one that was potential dependent (2-8 s). The latter could be explained by accumulation of K+ in the extracellular space of the neuron. The potential dependence of inactivation followed a sigmoid function infinity = 1/(1 + exp[(E - Ek)/+a]) with Ek = -36 mV and a = 13 mV. The inactivation properties are different from those of the classical fast transient (IA) current. The transport system for the outward potassium current during depolarizing potential steps in the rapidly adapting stretch receptor is similar to the current found in the slowly adapting receptor neuron. However, the activation is faster and seems to occur at potentials more negative than in the slowly adapting receptor. These differences can contribute to but not entirely explain the difference in adaptive behaviour between the slowly and rapidly adapting receptor.

Step-like shifts of membrane potential in the stretch receptor neuron of the crayfish (Astacus fluviatilis) at high temperatures

Summary1.The effects of temperatures between 10–32 °C on the slowly adapting stretch receptor neuron of the crayfish were studied with intracellular recording and potential clamp techniques.2.The membrane potential was found to decrease about 0.3 mV/ °C with increasing temperature in the range of 10° to 30 °C.3.At temperatures in the range of 25° to 32 °C stretch produced a brief intense impulse discharge followed by a maintained depolarization. A similar response could be elicited by intracellular cathodal pulses or appeared sometimes spontaneously.4.Tetrodotoxin (TTX) at a concentration of 10−7–10−8 mg/ml completely abolished the plateau, the receptor potential remaining unaffected.5.Lowering of Ca+ + concentration facilitated the development of the plateau while increase of Ca+ + concentration had the opposite effect.6.At a resting potential of −68 mV and at +30 °C the pNa/pK was 0.03 using the Goldman equation, the PNa/PK at a plateau level of −35 mV being 0.18. —The time constant for the inactivation of the plateau response was in the order of 1 s.7.The passive membrane conductance as measured with the potential clamp technique increased with increasing temperature.8.The stretch induced current (SIC) decreased and the reversal potential (Erev) was shifted towards more positive values when temperature was raised.9.It is suggested that the steplike shift of membrane potential at high temperatures may be attributed to an increase in PNa/PK of the regenerative portion of the membrane and that this increase as well as the increase in passive membrane conductance are accounted for by changes in the physical properties of the lipid phase of the membrane at high temperatures.

Intracellular calcium changes in Balanus photoreceptor. A study with calcium ion-selective electrodes and Arsenazo III

Intracellular Ca2+ concentration (Cai) in the dark and during light stimulation, was measured in Balanus photoreceptors with Ca2+ ion-selective electrodes (Ca-ISE) and Arsenazo III absorbance changes (AIII). The average basal Cai of 17 photoreceptors in darkness was 300 +/- 160 nM determined with liquid ion-exchanger (t-HDOPP) Ca-ISE. Ca-ISE measurements indicated that light increased Cai by 700 nM (average), whereas AIII indicated an average change of 450 nM. The time course of AIII absorbance changes matched the time course of changes in the receptor potential more closely than did the Ca-ISE. Changes in Cai were graded with light intensity but the change in Cai was much greater for a decade change in intensity at high light intensity than at low intensity. The peak light induced conductance change of voltage clamped cells had a relationship to light intensity similar to that of the change in Cai. The peak Cai level measured with Ca-ISE was in good agreement with the free Ca2+ concentration of injected buffer solutions. Control Cai levels were usually restored within 5 min following injection of Ca2+ buffers. Injection of Ca2+ buffers with free Ca2+ of 0.6 microM produced a membrane depolarization. Larger increases in Cai (greater than microM) produced by injection of CaCl2 or release of Ca2+ from injected buffers by acidifying the cell, produced a pronounced membrane hyperpolarization. Increasing Cai with all of these techniques reduced the amplitude of the receptor potential. The time course of the receptor potential recovery was usually similar to that of Cai recovery.

Local anaesthetics potentiate GABA-mediated Cl- currents by inhibiting GABA uptake.

A two-electrode voltage clamp was used to examine the effects of the local anaesthetics (LAs) lidocaine, tetra- caine and bupivacaine, and the meta-isomer of lidocaine (LL33), on the γ-aminobutyric acid (GABA)-gated Cl− channel in the crayfish stretch receptor neurone. The voltage-induced current changes were recorded while exposing the neurone to GABA in saline containing different LAs. All LAs enhanced the voltage-induced Cl− current equivalent to a shift of the conductance vs GABA curve towards lower concentrations. The mechanism of the facilitation of GABA-induced conductance was found to be mainly a block of the GABA uptake in the stretch receptor neurone. Tetracaine, and to a lesser extent LL33, also seem to affect directly the GABA/receptor Cl−/channel complex resulting in an increased conductance of the channel.

Effect of a highly purified phospholipase C on some electrophysiological properties of the frog muscle fibre membrane

Abstract Isolated sartorius frog muscles were treated with a highly purified phospholipase C (from Clostridium perfringens ) which was shown to be devoid of other biological and enzymatic activities. The resting membrane potential, action potential and input resistance were seriously affected. It is concluded that polar groups of the phospholipids are accessible to phospholipase C in the absence of other hydrolytic enzymes and that intact phospholipids are implicated in the ionic selectivity of the resting muscle cell membrane.

Mechanotransduction and the crayfish stretch receptor.

Mechanotransduction or mechanosensitivity is found in almost every cell in all organisms from bacteria to vertebrates. Mechanosensitivity covers a wide spectrum of functions from osmosensing, cell attachment, classical sensory mechanisms like tactile senses in the skin, detection of sound in hair cells of the hearing apparatus, proprioceptive functions like recording of muscle length and tension in the muscle spindle and tendon organ, respectively, and pressure detection in the circulation etc. Since most development regarding the molecular aspects of the mechanosensitive channel has been made in nonsensory systems it is important to focus on mechanosensitivity of sensory organs where the functional importance is undisputed. The stretch receptor organ of the crustaceans is a suitable preparation for such studies. The receptor organ is experimentally accessible to mechanical manipulation and electrophysiological recordings from the sensory neuron using intracellular microelectrode or patch clamp techniques. It is also relatively easy to inject substances into the neuron, which also makes the neuron accessible to measurements with fluorescent techniques. The aim of the present paper is to give an up to date summary of observations made on the transducer properties of the crayfish stretch receptor (Astacus astacus and Pacifastacus leniusculus) including some recent unpublished findings. Finally some aspects on future line of research will be presented.