Search

Crayfish is a common model animal for different experimental purposes. However, the lack of information about the genetic properties of the animal limits its use in comparison to other model animals. In the present study, a putative crayfish sodium/calcium exchanger gene has firstly been cloned in ganglia cDNA samples by conducting a series of PCR experiments, where a set of degenerate and specific primers and RACE method were used. The complete sequence is 2955 bp, and the ORF is 2718 bp in length. Molecular properties of the calculated peptide were similar to the sodium/calcium exchangers reported in the other species. Analysis of the qPCR data indicated that the putative gene has the highest expression level in the ganglia. However, an apparently elevated level of expression is observed in highly active tissues like heart, muscle and intestine, while the least expression level was observed in the stomach samples. It was proposed that the cloned gene may code the sodium/calcium exchanger protein in the crayfish.

In the present study, cytosolic calcium concentration changes were recorded in response to various forms of excitations, using the fluorescent calcium indicator dye OG-BAPTA1 together with the current or voltage clamp methods in stretch receptor neurons of crayfish. A single action potential evoked a rise in the resting calcium level in the axon and axonal hillock, whereas an impulse train or a large saturating current injection would be required to evoke an equivalent response in the dendrite region. Under voltage clamp conditions, amplitude differences between axon and dendrite responses vanished completely. The fast activation time and the modulation of the response by extracellular calcium concentration changes indicated that the evoked calcium transients might be mediated by calcium entry into the cytosol through a voltage-gated calcium channel. The decay of the responses was slow and sensitive to extracellular sodium and calcium concentrations as well as exposure to 1–10 mM NiCl2 and 10–500 µM lanthanum. Thus, a sodium calcium exchanger and a calcium ATPase might be responsible for calcium extrusion from the cytosol. Present results indicate that the calcium indicator OG-BAPTA1 might be an efficient but indirect way of monitoring regional membrane potential differences in a single neuron.

Voltage-gated sodium channel genes and associated proteins have been cloned and studied in many mammalian and invertebrate species. However, there is no data available about the sodium channel gene(s) in the crayfish, although the animal has frequently been used as a model to investigate various aspects of neural cellular and circuit function. In the present work, by using RNA extracts from crayfish abdominal ganglia samples, the complete open reading frame of a putative sodium channel gene has firstly been cloned and molecular properties of the associated peptide have been analyzed. The open reading frame of the gene has a length of 5793 bp that encodes for the synthesis of a peptide, with 1930 amino acids, that is 82 % similar to the a-peptide of a sodium channel in a neighboring species, Cancer borealis. The transmembrane topology analysis of the crayfish peptide indicated a pattern of four folding domains with several transmembrane segments, as observed in other known voltage-gated sodium channels. Upon analysis of the obtained sequence, functional regions of the putative sodium channel responsible for the selectivity filter, inactivation gate, voltage sensor, and phosphorylation have been predicted. The expression level of the putative sodium channel gene, as defined by a qPCR method, was measured and found to be the highest in nervous tissue.

The effects of antidromic potential spread were investigated in the stretch receptor neurons of the crayfish. Current and potential responses to conductance changes were recorded in the dynamic clamp condition and compared to those obtained by using some conventional clamp methods and a compartmental neuron model. An analogue circuit was used for dynamic calculation of the injected receptor current as a function of the membrane potential and the given conductance change. Alternatively, receptor current responses to a mechanical stimulus were recorded and compared when the cell was voltage clamped to a previously recorded impulse wave form and the resting potential, respectively. Under dynamic clamp, the receptor current had an oscillating waveform which contrasts with the conventional recordings. Frequency, amplitude and sign of the oscillations were dependent on the applied conductance level, reversal potential and electrotonic attenuation. Mean current amplitude and frequency of the evoked impulse responses were smaller under dynamic clamp, especially for large conductance increases. However, firing frequency was larger if plotted against the mean current response. Recorded responses were similar to those calculated in the model. It was not possible to evoke any adaptation in the slowly adapting neuron by using the dynamic clamp. Evoked potential change served as a self limiting response, preventing the depolarization block. However, impulse duration was significantly shorter in the rapidly adapting neuron when the dynamic clamp was used. It was concluded that, in the stretch receptor neurons during a conductance increase, antidromic potential spread modulates the receptor responses and contributes to adaptation.

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)

The effects of 4-aminopyridine (4-AP) and tetraethylammonium (TEA) on the outward potassium currents in the rapidly and slowly adapting stretch receptor neurons (SRNs) of the crayfish (Pacifastacus leniusculus) were studied using a two micro-electrode voltage-clamp technique. The leakage current was not affected by either 4-AP or TEA. External 4-AP blocked the peak outward current in a dose-dependent manner (1:1 stoichiometry) with an apparent dissociation constant (Kd) of 2.3 +/- 0.2 mM (mean +/- SEM) in the slowly and 1.4 +/- 0.2 mM in the rapidly adapting SRN, the block being voltage dependent. External application of TEA resulted in a block of the steady state current enhancing the transient characteristics of the current response. The block appeared to deviate from a 1:1 stoichiometry and the apparent Kd for TEA was 9.6 +/- 3.4 mM with a cooperativity factor n = 0.43 +/- 0.03 in the slowly adapting SRN and 34.5 +/- 9.2 mM and 0.37 +/- 0.03 respectively in the rapidly adapting SRN. Low Ca2+, apamin and charybdotoxin, which are known to block Ca(2+)-dependent K-currents, had no effects on the outward current as was also the case with catechol. It is concluded that the different effects of TEA and 4-AP on the outward current in the two types of SRNs can be explained by the presence of at least two, probably heteromultimeric, channel populations having similar sensitivity to 4-AP but different sensitivity to TEA. One channel has a high affinity (Kd = 0.8-1.6 mM) for TEA and the other a low affinity (Kd = 173-213 mM) for TEA. The low-affinity channel seems to dominate in the slowly adapting SRN while both channels are equally common in the rapidly adapting SRN. Further, the present results do not support the existence of a macroscopic Ca(2+)-dependent K+ current in the SRNs.

The structure/function relationship in the rapidly and slowly adapting stretch receptor organs of the crayfish (Astacus leptodactylus) was investigated using confocal microscopy and neuronal modeling methods. Both receptor muscles were single muscle fibers with structural properties closely related to the function of the receptors. Dendrites of the rapidly adapting neuron terminated in a common pile of nerve endings going in all directions. Dendrites of the slowly adapting neuron terminated in a characteristic T shape in multiple regions of the receptor muscle. The slowly adapting main dendrite, which was on average 2.1 times longer and 21% thinner than the rapidly adapting main dendrite, induced larger voltage attenuation. The somal surface area of the slowly adapting neuron was on average 51% larger than that of the rapidly adapting neuron. Variation in the neuronal geometry was greatest among the slowly adapting neurons. A computational model of a neuron pair demonstrated that the rapidly and the slowly adapting neurons attenuated the dendritic receptor potential like low-pass filters with cut-off frequencies at 100 and 20 Hz, respectively. Recurrent dendrites were observed mostly in the slowly adapting neurons. Voltage signals were calculated to be propagated 23% faster in the rapidly adapting axon, which is 51% thicker than the slowly adapting axon. The present findings support the idea that the morphology of the rapidly and the slowly adapting neurons evolved to optimally sense the dynamic and the static features of the mechanical stimulus, respectively. J. Comp. Neurol. 488:369–383, 2005. © 2005 Wiley-Liss, Inc.

Effects of various types of scorpion venom on gamma-aminobutyric acid (GABA) release were studied in an isolated synapse in the crayfish. Post-synaptic GABA-induced currents were recorded to monitor the GABA release from the pre-synaptic site. In 20 mM tetraethylammonium (TEA) chloride solution the GABA-induced currents increased 71%. Exposing the preparations to Leiurus quinquestriatus hebraeus , Leiurus quinquestriatus quinquestriatus or Tityus serrulatus venom (0.1 mg/ml) increased GABA-induced currents 4–5 fold. The effect was present in the presence of tetrodotoxin (TTX) but diminished significantly when verapamil was applied. Exposing the preparations Androctonus australis or Buthus tamulus venom did not affect the GABA-induced currents. The results indicate that stimulation of the GABA release by some of the scorpion venoms may partly be due to a possible block of pre-synaptic potassium channels, but not due to an abnormal increase in sodium channel activation.

Purali, Nuhan and Bo Rydqvist. Action potential and sodium current in the slowly and rapidly adapting stretch receptor neurons of the crayfish ( Astacus astacus). J. Neurophysiol. 80: 2121–2132, 1998. Action potentials (APs) and sodium current from the slowly and the rapidly adapting stretch receptor neurons in the crayfish ( Astacus astacus) were recorded with a two microelectrode voltage- and current-clamp technique. In the rapidly adapting neuron the APs had a duration of 3.2 ± 0.2 ms (means ± SE) and an amplitude of 55.2 ± 1.5 mV. In the slowly adapting receptor neuron APs had a duration of 4.1 ± 0.2 ms and an amplitude 79.9 ± 2.0 mV. APs in the rapidly adapting neuron had a larger amplitude if they were recorded from the axon. In the rapidly adapting neuron adaptation of the impulse response was prolonged by hyperpolarization or by exposure to scorpion venom. Also, sinusoidal current stimulation added to the current steps prevented impulse adaptation. Block of the potassium currents in the slowly adapting neuron resulted in a rapid adaptation of the impulse response. The maximum sodium current amplitude was 313 ± 15 nA in slowly adapting neuron and 267 ± 11 nA in the rapidly adapting neuron. The current-voltage relationship showed a hump most marked in the slowly adapting neuron and abolished when a depolarizing prepulse was given. In the rapidly adapting neuron the inactivation starts at a more negative potential ( Eh = −45 mV) and is faster compared with the slowly adapting neuron ( Eh = −41 mV). The crude scorpion venom of Leiurus quinquestriatus (ScVLq) shifted h ∞ curve toward more positive potentials and slowed down the rate of inactivation. The results indicate the possible presence of more than one Na+ channel population and that the relative density and the spatial distribution is different in the slowly and rapidly adapting neuron. The difference contributes to the adaptive properties of the two receptor neurons.

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.

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 outward current in the stretch receptor neuron of the crayfish Pacifastacus leniusculus was analysed using a two-micropipette potential-clamp technique. The outward current was shown to be carried by K+. When the sodium-dependent inward current was blocked by tetrodotoxin a fast-activating potassium current was revealed, resembling the delayed rectifier. The time-course of activation (Tau n) was dependent on potential and had a mean value of I ms at potential steps of to mV. The activation followed a second-order process according to the Hodgkin-Huxley model. The potential dependence of activation (n infinity) followed a sigmoid curve, n infinity = I/(I + exp [(E-En)/a]) with half-maximal activation potential En = -31 mV and a = -13 mV. When long pulses were applied, the potassium current showed marked inactivation with a fast time constant of 0.5 s that was potential independent and a slow component that was slightly potential dependent. The minimum value for the slow time constant was 4 s for steps to about 0 mV. The potential dependence of inactivation followed a sigmoid function k infinity = I/(I + exp [(E-Ek)/a]) with Ek = -39 mV and a = II mV. No transient potassium outward current (IA) was found in the crayfish stretch receptor neuron. In experiments on tail currents after depolarizing potential steps of different duration, it was found that the reversal potential changed in the positive direction when the duration of the pre-pulse increased. This could be due to K- accumulation in a space close to the neuronal membrane. The potassium current during depolarizing potential steps in the crayfish stretch receptor is similar to the delayed current found in other cells, for example the frog myelinated nerve, but different from many other invertebrate neurons.