Post-tetanic hyperpolarization and electrogenic Na pump in stretch receptor neurone of crayfish

1. Two types of after-potentials in the stretch receptor neurone of crayfish are described.2. A short-duration after-hyperpolarization associated with a single spike or a few spikes is diminished and reversed on applying hyperpolarizing currents. However, a much longer-lasting post-tetanic hyperpolarization (PTH) is enhanced by conditioning hyperpolarization; thus, no reversal potential can be obtained.3. No changes in membrane conductance occur during PTH.4. Reducing K concentration in the bathing fluid diminishes PTH, while it shifts the reversal potential of the short after-potential toward greater negativity.5. Replacement of Na with Li, or addition of 2,4-dinitrophenol in the bathing fluid suppresses PTH in a reversible manner.6. Electrophoretic injection of Na into the cell induces a long-lasting hyperpolarization.7. No change in K-equilibrium potential, as indicated by the reversal point of the short after-potential, is detected during PTH.8. It is concluded that the short after-potential is caused by a permeability increase for potassium ion, whereas PTH is produced by an electrogenic Na-pump.     

Block of receptor response in the stretch receptor neuron of the crayfish by gadolinium.

The trivalent lanthanide gadolinium was found to block the mechanotransducer response in the stretch receptor neuron of the crayfish. At normal calcium concentration (13.5 mM) a 50 per cent block of the receptor current was found at 395 +/- 59 (mean +/- SD) microM gadolinium. At a calcium concentration of 1.35 mM a 50 per cent block of the receptor current was obtained at 103 +/- 14 (mean +/- SD) microM gadolinium. The potential activated potassium current was also affected by gadolinium. At 200 microM the amplitude of the peak outward current as a result of a 90 mV positive potential step was decreased by about 40 per cent. The fast inward sodium current was decreased less than 10 per cent by gadolinium. It is concluded that in the crayfish stretch receptor gadolinium blocks the receptor current, reflecting block of stretch-activated channels, but at higher concentrations than have been described for other stretch-activated channels. In addition the outward rectifier potassium current is also blocked reflecting a block of potassium channels.     

The effects of Triton-detergents on the stretch receptor of the crayfish

The effects of the non-ionic detergents Triton X-45 and Triton X-100 on the action potential and the receptor potential of the stretch receptor neuron of the crayfish Astacus fluviatilis was studied with intracellular recording technique. Membrane currents were measured with voltage clamp technique. Both detergents blocked the action potential in 20–30 min at concentrations of 60–80 μM. Following blocking of spike electrogenesis the receptor potential evoked by stretch was obtained in isolation. With prolonged exposure of the neuron to the detergents there was a slowly developing reduction of the receptor potential and after 60–90 min no response to stretch could be obtained. These effects were produced without any significant change of the resting membrane potential. Following return to normal saline the responsiveness to stretch was completely restored in 80–100 min. Measurements with voltage clamp technique showed that the passive membrane properties were little affected by the two detergents. The stretch induced current on the other hand was severely depressed and almost abolished with prolonged exposure. The experimental results suggest that non-ionic detergents block the spike electrogenesis and the transducer action by a selective action on the sodium channels of the membrane of the receptor neuron.     

Block of receptor response in the stretch receptor neuron of the crayfish by gadolinium

The trivalent lanthanide gadolinium was found to block the mechanotransducer response in the stretch receptor neuron of the crayfish. At normal calcium concentration (13.5 mm) a 50 per cent block of the receptor current was found at 395 ± 59 (mean ± SD), μM gadolinium. At a calcium concentration of 1.35 mm a 50 per cent block of the receptor current was obtained at 103 ± 14 (mean ± SD) μM gadolinium. The potential activated potassium current was also affected by gadolinium. At 200 μM the amplitude of the peak outward current as a result of a 90 mV positive potential step was decreased by about 40 per cent. The fast inward sodium current was decreased less than 10 per cent by gadolinium. It is concluded that in the crayfish stretch receptor gadolinium blocks the receptor current, reflecting block of stretch-activated channels, but at higher concentrations than have been described for other stretch-activated channels. In addition the outward rectifier potassium current is also blocked reflecting a block of potassium channels.     

Potassium ion channels operated by receptor stimulation can be activated simply by raising temperature.

Application of either dopamine (DA), acetylcholine (ACh), or histamine (HA) to the identified ganglion cells of Aplysia elicits a K(+)-dependent slow hyperpolarization. When temperature of the bathing solution was raised from 22 to 32 degrees C, these cells were also hyperpolarized with a marked increase in K+ conductance. The warm- and transmitter-induced current responses recorded under voltage clamp were not blocked by either 1 mM Ba2+ or 10 mM TEA. Intracellularly injected guanosine 5'-O-(2-thiodiphosphate) (GDP beta S) depressed both warm- and transmitter-induced K+ responses immediately after the injection. Intracellular application of guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) caused a gradual, irreversible increase in K+ conductance of the plasma membrane and occluded both responses. Transmitter-induced response markedly decreased when the temperature was raised from 22 to 32 degrees C, suggesting that the response to transmitter was occluded during the warm-induced response. These results suggested that the G-protein regulating the receptor-operated K+ channels could be activated simply by raising temperature.     

Analysis of leak current properties in the lobster stretch receptor neurone

Experiments were performed to characterize the so-called leak current of the slowly adapting stretch receptor neurone of the European lobster with respect to its ionic basis, its kinetics and its pharmacology. Estimates of the leak current were obtained by subtraction of a Na-K pump current and of an unspecific impalement current from a non-dynamic (‘instantaneous’) current, recorded in a voltage range from ～-120 to ～-30 mV, after blockage of spike-generating currents and a hyperpolarization-activated inwardly rectifying current (Q-current). The leak current, estimated in this way, was seen to reverse direction at the cell's K⁺ equilibrium voltage, thus indicating that it is carried by K⁺ passing through channels which, also, proved to be permeable to Rb⁺ and NH₄⁺, but not permeable to Na⁺ or Cl^- to any significant extent. Kinetically, the leak current was found to be characterized by being enhanced by increases in extracellular K⁺ and by being subject to outward rectification, most distinctly at elevated extracellular [K⁺]. In quantitative terms, these kinetic properties could be accounted for by a mathematical model comprising (1) a one-site two-barrier Eyring formulation describing ion permeation through membrane channels and (2) an ordinary dose–response relationship describing the channel-opening effect of K⁺ at an extracellular regulatory site. Pharmacologically, the leak current proved to be distinguished by being reversibly blockable, in a non-voltage dependent manner, by Co²⁺ (K_d=0.9 mm , Hill coefficient 1.1) and procaine, but not by Ba²⁺, Gd³⁺, bupivacaine (a local anesthetic), or other K⁺ channel blockers such as TEA, 4-AP and Cs⁺. It is concluded that, in native unimpaled cells, the K⁺ carried leak current (1) is setting the resting voltage together with the (mainly) Na⁺-carried Q-current and the Na-K pump current, (2) is determining the cell's firing threshold, together with the spike generating currents, and (3) is also stabilizing the cell's membrane excitability in conditions of varying extracellular [K⁺], by virtue of its K⁺ sensitivity.     

Functional effects of a hyperpolarization-activated membrane current in the lobster stretch receptor neurone.

The functional effects of a hyperpolarization-activated membrane current (IQ) in the slowly adapting lobster stretch receptor neurone were investigated. From comparisons between changes in membrane excitability due to blockage of IQ by Cs+, in normally impaled and native unimpaled (Edman et al. 1987 b) cells, it could be concluded that the resting voltage of native cells is distinctly more negative than -65 mV (average membrane voltage of impaled cells) and, therefore, under the control of an activated IQ. Starting from this conclusion, impaled cells were polarized to holding (resting) voltages around -75 mV and their polarization and excitability properties studied after tetanic impulse activity and variation of various external influences (K+, pH, temperature), both in control conditions and after blockage of IQ by 2 mM Cs+. It was found that an unblocked IQ (a) greatly accelerates the initial (90%) decay of post-tetanic hyperpolarization, and (b) depresses distinctly any polarization and excitability alterations due to increases in extracellular K+ concentration (from 2.5 to 10 mM), variations in extracellular pH (between 6.4 and 8.6), and changes in temperature (between 14 and 24 degrees C). It was inferred that in well polarized cells, IQ plays a role as a stabilizer of membrane polarization and excitability in conditions of varying external influences. From a model study of IQ it could be concluded that, with its slow dynamic responses, the current is well adapted to its functional purposes and to the rather slow homeostatic effects of the cell's Na-K pump.     

Effects of tetrodotoxin on the slowly adapting stretch receptor neurone of lobster

1. A study has been made of the effects of tetrodotoxin on the impulse activity, resting membrane potential, input resistance, and the generator potential and its after-hyperpolarization of the slowly adapting stretch receptor neurone of the lobster.2. Tetrodotoxin was able to abolish completely within about 2 min the impulse activity in most cells, when given in a dose of 2 x 10(-8) g/ml., but in all cells, when administered in a dose of 4 x 10(-8) g/ml. After blockage by the toxin in concentrations as high as 4 x 10(-6) g/ml. for periods of up to 30 min the action potential was restored by washing the preparation in physiological solution for 1 hr.3. In a concentration of 4 x 10(-8) g/ml. tetrodotoxin produced within 1-2 min an average increase of 4.8 mV of the resting membrane potential and a simultaneous 47% reduction of the resting input resistance. These effects were reversed by washing the preparation in physiological solution for 1 hr.4. Tetrodotoxin administered in doses as high as 4 x 10(-6) g/ml. for periods of up to 30 min had no effect on the amplitude of the steady phase of the generator potential.5. In a concentration of 4 x 10(-8) g/ml. tetrodotoxin produced within 5 min a 65% reduction of the amplitude of the hyperpolarization following the generator potential. This effect was reversed by washing the preparation in physiological solution for 1 hr.6. The simultaneous increase in resting membrane potential and decrease in membrane resistance is suggested to be due to an elevated potassium permeability besides a reduced sodium conductance. The constancy in height of the generator potential in the presence of a decreased membrane resistance makes necessary the assumption of an augmented generator current. The decrease in amplitude of the hyperpolarization following the generator potential may be the result of an increase in potassium conductance and/or a reduction in acceleration of an electrogenic pump in consequence of a diminished sodium influx during the generator potential.     

Viscoelastic properties of the slowly adapting stretch receptor muscle of the crayfish

The viscoelastic properties of the muscle associated with the slowly adapting stretch receptor organ of the crayfish (Astacus astacus) were studied by recording the tension response to various length changes. When steady-state length changes were applied to the muscle, the tension developed in a non-linear way, increasing slowly for small extensions and rapidly when extension increased. Muscle tension responses to ramp-and-hold extensions were characterized by a transient peak followed by a gradual decline in tension. At the onset of the ramp the tension increased rapidly, similar to the response seen in resting skeletal muscle. The relation between peak dynamic tension and extension was non-linear. In a log-log plot the relation was linear with a mean slope of 1.4. At small extensions (less than 5%) the slope seemed to be lower. The experimental results have been analysed in relation to a viscoelastic model consisting of a Voigt element in series with a non-linear spring. The model could describe both the static length-tension relation and the dynamic response, but different parameters for the springs had to be used for the two cases. When the measured tension response was transformed by an exponential function of the squared tension, in accord with recent findings on stretch-activated channels, a good agreement was obtained with the time course of the receptor currents. Adaptation is thus likely to be caused by both the mechanical properties of the receptor muscle and the characteristics of stretch-activated channels of the neuron.     

Stimulus-response properties of the slowly adapting stretch receptor neuron of the crayfish

The receptor potential and receptor current in response to ramp-and-hold extensions were measured in the slowly adapting stretch receptor of the crayfish, using potential clamp technique. The stimulus-response relationship for the peak amplitude of the receptor current showed a linear behaviour for extensions less than 2% and a nonlinear behaviour for extensions larger than 5%. Using the Stevens power law, R = k(S--S0)n, where R is response, S is stimulus, S0 is threshold stimulus and n the power coefficient, n was found to be 3 for extensions between 5 and 15%. The receptor current saturated at extensions above 20-25% of the zero length of the muscle, resulting in a lower n value. However, the n value is difficult to define in this region due to the saturation. The stimulus-response relation for the receptor current can be explained by the properties of the stretch-activated channels for which the open probability is exponentially dependent on the square of the membrane tension, as suggested by recent findings. The receptor potential, using tetrodotoxin, in response to identical ramp-and-hold extensions as those used to record current responses showed a more complex time-course, indicating involvement of potential-dependent channels, potassium channels being the most probable candidate. This was supported by a mathematical model which takes into account the viscoelastic properties of the receptor muscle, the properties of the stretch-activated channels and a potential dependent K+ current.     

Transfer properties of the slowly adapting stretch receptor of the crayfish abdomen

The slowly adapting stretch receptor in the abdomen of freshwater crayfish (Astacus fluviatilis) was investigated to determine its properties under dynamic conditions. An in situ preparation was used; the necessary dissection did not involve the receptor organ or its immediate surroundings. Sinusoidal variations in the angle of flexion in the joint to which the receptor organ was connected, were generated by a feed-back controlled stretcher. Nerve spikes recorded from the axon of the receptor neurone and information about angle of flexion in the joint obtained by position transducers, were fed into a computer. Fourier transforms were performed on both input and output data to determine the amplitude of the 0. and I. harmonic together with the phase of the 1. harmonic. The receptor organ was investigated for linearity up to 1.5 degrees input amplitude, and proved to be surprisingly linear within this range. In addition, the transfer function of the receptor organ was determined by stimulating it with small-amplitude sinusoidals with different frequencies. With a steady flexion of 35–40° in the joint, the gain of the receptor organ increased 5–6 times when the modulation frequency of the input signal was increased from 0.1 to 5 cycles/s. A maximum in gain was constantly found at about 5 cycles/s, with a rapid fall towards 0 when the modulation frequency was increased further. A change in phase lead from positive (leading output) to negative with change in sign about 1 cycle/s was also found. These results resemble the results found by investigators of isolated preparations. A “hold” property is probably a part of the overall property of the receptor organ together with an element of Maxwell type. An element of the form h(s)=ksⁿ with n=0.45 is also a part of the transfer function of the receptor organ, although the physiological parallel to this element is uncertain.     

Visco-elastic properties of the rapidly adapting stretch receptor muscle of the crayfish

The visco-elastic properties of the receptor muscle associated with the rapidly adapting stretch receptor organ of crayfish (Pacifastacus Leniusculus) were studied by recording the tension responses to various length changes. Steady-state length changes resulted in a non-linear tension development in the receptor muscle. The tension increased slowly for small extensions and more rapidly when extension increased. Muscle tension responses to ramp-and-hold extension were characterized by a transient peak followed by a gradual non-exponentional decline in tension. At the onset of the ramp the tension increased rapidly, similar to what has been observed in the muscle of the slowly adapting receptor (SM). The steeper rise in tension during the first part of the ramp indicating higher initial stiffness, resulted in a ‘hump’ when large extensions (> 15%) were applied. The results show that the rapidly adapting receptor muscle has a more pronounced dynamic component; the ratio between the amplitude of the peak and the steady state response was larger in the rapidly than in the slowly adapting receptor muscle. Accordingly, different values for the elements of a visco-elastic model of the muscle had to be set for the two types of receptors. The different properties of the rapidly and slowly adapting receptor muscles are in line with the differences in the overall adaptive behaviour of the organ and give further support to the idea that mechanical factors contribute to the adaptive properties.     

Functional effects of a hyperpolarization-activated membrane current in the lobster stretch receptor neurone

The functional effects of a hyperpolarization-activated membrane current (I_Q) in the slowly adapting lobster stretch receptor neurone were investigated. From comparisons between changes in membrane excitability due to blockage of I_Q by Cs⁺, in normally impaled and native unimpaled (Edman et al. 1987 b) cells, it could be concluded that the resting voltage of native cells is distinctly more negative than –65 mV (average membrane voltage of impaled cells) and, therefore, under the control of an activated I_Q. Starting from this conclusion, impaled cells were polarized to holding (resting) voltages around –75 mV and their polarization and excitability properties studied after tetanic impulse activity and variation of various external influences (K⁺, pH, temperature), both in control conditions and after blockage of IQ by 2 mm Cs⁺. It was found that an unblocked I_Q (a) greatly accelerates the initial (90%) decay of post-tetanic hyperpolarization, and (b) depresses distinctly any polarization and excitability alterations due to increases in extracellular K⁺ concentration (from 2.5 to 10 mM), variations in extracellular pH (between 6.4 and 8.6), and changes in temperature (between 14 and 24 °C). It was inferred that in well polarized cells, I_Q plays a role as a stabilizer of membrane polarization and excitability in conditions of varying external influences. From a model study of I_Q it could be concluded that, with its slow dynamic responses, the current is well adapted to its functional purposes and to the rather slow homeostatic effects of the cell's Na-K pump.     

Ultrastructure of the phasic stretch receptor in the crayfish abdominal nerve cord

Summary The ultrastructure of the abdominal ganglionic cord stretch receptor of the crayfishPacifastacus leniusculus is described. This bilaterally-paired, segmentally-repeating phasic receptor monitors stretch applied to the central nervous system itself. It consists of a connective tissue mass closely applied to the medial margin of each medial giant fibre, into which ramifies a collection of specialized terminal dendrites originating from branches (primary dendrites) of a single axon. The connective tissue consists of an electron-opaque matrix in which are embedded many short, electron-lucent, tubular structures whose lumens are continuous with the matrix. Some filamentous material penetrates the connective tissue from its boundaries, and glial cells are present. The primary dendrites are irregular in size and orientation, and contain many microtubules and much filamentous material. The terminal dendrites are of consistent diameter and longitudinal orientation, containing very regularly-spaced microtubules with no microfilaments. The terminal dendrites contain a well-defined cytoskeletal tube or lamina 6 nm thick, evenly spaced about 25 nm below the plasma membrane and connected to it by filamentous material 5 nm in diameter, which is deposited in rings or helices. This lamina arises just at the point where the primary dendrites gave rise to the terminal dendrites. Its function is not known, but it shows some similarities to the subaxolemmal lamina found in some regions of spike initiation.