Characterization of a delayed rectifier potassium channel in the slowly adapting stretch receptor neuron of crayfish

Prostaglandins, which are cyclooxygenase (COX) products, are pathologically up-regulated, and have been proven to be closely associated with neuronal death. In this study, we investigated a role of COX isoforms (COX-1 and COX-2) in kainic acid-induced neuronal death in cultured murine cortical or hippocampal neurons. In primary cortical neurons, both indomethacin (COX-1/-2 nonselective inhibitor) and aspirin (COX-1 preferential inhibitor) reduced basal and kainic acid-induced PGE(2) production significantly and prevented neuronal cell death after kainic acid treatment. In contrast, NS398 (COX-2 selective inhibitor) had no effect on kainic acid-induced neuronal cell death. In hippocampal neurons, however, COX-2 inhibitors prevented both kainic acid-induced neuronal death and PGE(2) production. COX-2 expression was remarkably up-regulated by kainic acid in hippocampal neurons; whereas in cortical neurons, COX-2 expression was comparatively less significant. Astrocytes were unresponsive to kainic acid in terms of PGE(2) production and cell death. In conclusion, we suggest that the release of PGE(2) induced by kainic acid occurred through COX-1 activity rather than COX-2 in cortical neurons. The inhibition of PGE(2) release by COX-1 inhibitors prevented kainic acid-induced cortical neuronal death, while in the hippocampal neurons, COX-2 inhibitors prevented kainic acid-induced PGE(2) release and hippocampal neuronal death.     

Comparison of GABA analogues at the crayfish stretch receptor neurone

The conductance increase induced by GABA and structurally related compounds has been measured in voltage clamped stretch receptor neurones of crayfish. GABA induced only at 10⁻³ M a rapid conductance increase. The response to lower concentrations between 10⁻⁶ and 10⁻⁴ M developed slowly (20–60 min). The postsynaptic conductance increase induced by repetitive application of the same GABA concentration was progressively enhanced in the speed and magnitude. In the presence of nipecotic acid or in Na⁺-free Ringer solutions, the response to all GABA concentrations was instantaneous and constant for each concentration. Muscimol between 10⁻⁶ and 10⁻³ M caused instantaneous dose-dependent conductance increases. These results suggest the presence of a saturable GABA uptake system limiting the access of bath applied GABA, but not of muscimol, to postsynaptic receptor sites.     

Distribution of sodium channels in chronically demyelinated spinal cord axons: immuno-ultrastructural localization and electrophysiological observations

The immuno-ultrastructural localization of voltage-sensitive sodium channels was demonstrated within a central demyelinating lesion induced in the rat spinal cord by ethidium bromide/irradiation using polyclonal antibody 7493. Antibody 7493 has previously been shown to immunostain intensely axon membrane at nodes of Ranvier, and also perinodal astrocyte processes. At 25–35 days post injection/irradiation, the central portion of the demyelinating lesion is populated with chronically demyelinated axons and there is an absence of glial processes. Sodum channel immunoreactivity was not observed on the chronically demyelinated axolemma within this central portion of the lesion. Within the peripheral portion of the lesion demyelinated axons were occasionally abutted by astrocyte and Schwann cell processes. At these focal sites of apposition, the axon membrane displayed intense sodium channel immunoreactivity, while the abutting astrocyte and Schwann cell processes did not exhibit immunostaining. Also in the periphery of the lesion, some axons become ensheathed and myelinated by oligodendrocytes and Schwann cells. The axon membrane of circumferentially ensheathed axons displayed antibody 7493 immunostaining, and this immunoreactivity persisted on the axolemma until the ensheathing cytoplasmic processes compacted into myelin. Internodal axon membrane beneath the myelin sheath did not display sodium channel immunoreactivity, though (putative) developing nodal axon membrane adjacent to terminal paranodal loops exhibited robust sodium channel staining. Electrophysiological recordings within the ethidium bromide/irradiation lesion demonstrated that at least some axons conducted action potentials within the lesion, while others exhibited conduction block. These results indicate that there is a reorganization of sodium channels within the axon membrane of chronically demyelinated central axons.     

Effect of carbon dioxide on the activity of slowly and rapidly adapting pulmonary stretch receptors in cats

Single fiber activity from slowly and rapidly adapting pulmonary stretch receptors (PSR) was recorded from the left cervical vagus of anesthetized, open-chested and artificially ventilated cats. Reducing the end-tidal CO2 to low values did not affect the frequency of occurrence of higher threshold (HT) PSR. Occlusion of the left pulmonary artery (LPA) had no significant effect on the resting discharge of both HT and low threshold (LT) PSR. Cyclic ventilation with 8% CO2 in O2 reduced the activity of LT and HT receptors by similar amounts, irrespective of their anatomical location. After LPA occlusion, CO2 ventilation reduced markedly the activity of both type of PSR but to the greatest extent that of the HT receptors, the majority of which were located in the intrapulmonary airways. The CO2 depressant effect may not be due solely to changes in H+ concentration at the receptor level, since acetazolamide did not totally abolish the effect even though it significantly reduced it. Sustained inflation with 8% CO2 in O2 significantly reduced the activity of HT receptors in both the dynamic and static phases of inflation, but had no effect on the activity of LT receptors. Direct localization showed that the receptors which were more accessible to CO2 (all HT and one LT) were located in the lung parenchyma. In the case of rapidly adapting receptors, sustained inflations with CO2 gave inconsistent results. The results show clearly that, as in other mammalian species, the PSR activity in cats is also reduced by hypercapnia. The present study stresses the importance of localizing the PSR and making the observations separately on the two types of PSR, for there may be qualitative and quantitative differences.     

Differential effects of the pyrethroid tetramethrin on tetrodotoxin-sensitive and tetrodotoxin-resistant single sodium channels

The differential effects of the pyrethroid tetramethrin on tetrodotoxin-sensitive (TTX-S) and tetrodotoxin-resistant (TTX-R) single sodium channel currents in rat dorsal root ganglion (DRG) neurons were investigated using the outside-out configuration of patch-clamp technique. Channel conductances were 10.7 and 6.3 pS for TTX-S and TTX-R sodium channels, respectively, at a room temperature of 24–26°C. The single-channel current of TTX-S sodium channels at the test potential of −30 mV was −1.27 ± 0.25 pA, and was not changed after exposure to 10 μM tetramethrin (−1.28 ± 0.23 pA). The open time histogram of TTX-S single-channel currents could be fitted by a single exponential function with a time constant of 1.27 ms. After exposure to 10 μM tetramethrin, the open time histogram could be fitted by the sum of two exponential functions with time constants of 1.36 ms (τ_fast) and 5.73 ms (τ_low). The percentage of contribution of each component to the population was 62% for the fast component representing the normal channels and 38% for the slow component representing the tetramethrin modified channels. The amplitudc of TTX-R single-channel currents was slightly changed from −0.72 ± 0.14 to −0.83 ± 0.07 pA by 10 μM tetramethrin. The open time histogram of TTX-R single-channel currents could be fitted by a single exponential function with a time constant of 1.92 ms. In the presence of 10 μM tetramethrin, the open time histogram could be fitted by the sum of two exponential functions with time constants of 2.07 ms (τ_fast) and 9.75 ms (τ_slow). The percentage of contribution of each component was 15% for the fast, unmodified component and 85% for the slow, modified component. Differential effects of tetramethrin on the open time distribution of single sodium channel currents explains the differential sensitivity of TTX-S and TTX-R sodium channels.     

Structure and function relationship in the abdominal stretch receptor organs of the crayfish

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.     

Effects of 5-hydroxytryptamine on rapidly adapting pulmonary stretch receptor activity in the rabbit

In anesthetized rabbits, intravenous administration of 5-hydroxytryptamine (5-HT, 10, 20, 40 and 80 micrograms/kg) led to an increase of rapidly adapting pulmonary stretch receptor activity. The magnitude of the increase was dose-dependent. The excitatory responses to any doses of 5-HT were not inhibited by prior treatment with isoprenaline (100-150 micrograms). The results indicate that the stimulation of rapidly adapting pulmonary stretch receptors by 5-HT is not caused by contraction of the airway smooth muscle.     

Effect of ouabain on the afterhyperpolarization of slowly adapting pulmonary stretch receptors in the rat lung

In anesthetized, artificially ventilated rats with one vagus nerve section, the purposes of the present study were to investigate whether release from phasic consecutive hyperinflations (inflation volume=3 tidal volumes) results in the afterhyperpolarization (AHP) of the slowly adapting pulmonary stretch receptor (SAR) activity and whether the effect of ouabain, a Na+-K+ ATPase inhibitor, alters AHP of the SAR activity seen after release from maintained inflations. Release from 10 consecutive phasic hyperinflations did not cause any significant inhibition of SAR activity. Release from maintained inflations (for approximately 10 and 15 cmH2O) for 5 s produced the induction of disappearance of SAR activity, corresponding with the AHP. Intravenous administration of ouabain (20 and 40 microg/kg) had no significant effects on the responses of SAR activity and SAR adaptation index (AI) to maintained inflations, but ouabain treatment with at 40 microg/kg resulted in a significant increase in the SAR activity after stopping the respirator and significantly attenuated the AHP of the SAR activity. In the immunohistochemical study, we found Na+-K+ ATPase alpha3-subunit-isoforms-like immunoreactivity in SAR terminals, forming leaflike extensions in the intrapulmonary bronchioles at different diameters, and those terminals were buried in the smooth muscle. In the same sections, the alpha1 subunit immunoreactivity of SAR terminals was not found. These results suggest that the mechanism of generating the AHP of SARs is mainly mediated by the activation of Na+-K+ ATPase alpha3 subunit isoform.     

A novel stretch receptor in the jaw of the rat

We report here an unusual type of stretch receptor found on each side of the rat jaw. This receptor has unique morphological featurs: it is quite long (24–28 mm), lies in connective tissue in between masticatooy muscle, and extends between the medial pterygoid muscle-tendon on the maxilla and the masseter-tendon on the mandible through a zigzag course, forming a Greek capital letter sigma when viewed from the side. The receptor is neither in parallel nor in series with any masticatory muscles and receives multiple innervation. The receptor increases its length when the jaw closes and shortens when the jaw opens. Electron microscopy revealed axial structures composed of a central cellular core surrounded by tightly packed collagen bundles which are separated from the capsule by a wide capsular space. Most of the sensory endings are found among axial collagen bundles, some in between core cells. The core cells have many finger-like processes on their surface, being coupled by desmosomes. The origin and nature of these cells are unclear. The wide capsular space is filled with Alcian blue positive substrate, probably acid glycosaminoglycans. The structures of outer and inner capsules are similar to those of muscle spindles, the former being composed of three to ten layers of thin flattened cells. The response of the receptor was examined with in vivo as well as in vitro preparations. In in vivo experiments, impulse discharges from this receptor increased with the increase in jaw closing. When the jaw was fully opened the impulse discharge from this receptor disappeared. It is clear from these experiments that this receptor is a slowly adapting stretch receptor, being sensitive to jaw position and movement. With in vitro preparations, a linear relation was obtained between the frequency of impulse discharge measured during the static phase of stretch and the amplitude of stretch. Beyond 20% of stretch the response tended to saturate. The dynamic component of discharge was less marked, and comparable to the response of Golgi tendon organs or secondary endings of muscle spindles. The conduction velocity of innervating axons was 23–31 m/s at 35°C.     

Two modes of activity of nicotinic acetylcholine receptor channels in sympathetic neurons

Spectral analysis of acetylcholine (ACh) noise was performed in voltage-clamped neurons of the isolated rabbit superior cervical ganglion at 34–37°C and at membrane potential −80 mV. Two modes of activity were found in the ionic channels of nicotinic ACh receptors, with mean channel life-times of τ_f = 1.1 ± 0.1 ms for fast-operating channels and τ_s = 5.6 ± 0.6 ms for slow-operating channels. Excitatory postsynaptic current (EPSC) decays exponentially with a time constant which is very close to τ_s, indicating that the slow-operating channel activity determines the duration of EPSC. The mean value of conductance of single nicotinic ACh-receptor channel is 36 ± 3 pS.     

Distribution and location of slowly adapting pulmonary stretch receptors in the airways of cats

The distribution and location of slowly adapting pulmonary stretch receptors (PSRs) that affect respiratory and cardiovascular functions were investigated in anaesthetized, artificially ventilated and thoracotomized cats. The location of the receptors was done by punctate stimulation and local mechanical stimulation after occlusion of the trachea at end-expiration (Exp). 84% of the slowly adapting PSRs were found to be located in the lung parenchyma. The occlusion technique alone was found to be of help only for a limited population of stretch receptors. The intrapulmonary distribution of PSRs revealed a greater percentage of receptors in the diaphragmatic lobe. No correlation was found between conduction velocity and receptor location. Both the slowly and rapidly conducting receptors were found to be scattered throughout the entire lung parenchyma. However, it was observed that while the majority of low threshold (LT) PSRs were located closer to the hilum of the lung, many of the higher threshold (HT) receptors were located farther away. In addition, when veratrine was administered into the pulmonary circulation, 83% of HT PSRs studied were stimulated by the drug, while only 25% of LT PSRs under study could be stimulated this way. The significance of the above findings is discussed.     

Cholinergic and H1-receptor influences of histamine on slowly adapting pulmonary stretch receptor activity in the rabbit.

Afferent impulses of slowly adapting pulmonary stretch receptors (SARs) were obtained by dissecting fine slips from the left vagus nerve (LVN) and by leaving the rest of the nerve intact. In the same SAR preparation, changes of the receptor activity in response to right atrial injections of histamine (10 and 60 micrograms/kg) were successively examined before and after atropine (1 mg/kg), partial vagal efferent ablation, and mequitazine (1 mg/kg) in 10 rabbits. Administration of histamine led to an increase in the SAR activity, and this effect became more pronounced by increasing the dose of histamine. Atropine treatment diminished the responses of SARs to histamine at different doses. Partial vagal efferent ablation produced by denervation of the rest of the intact LVN slightly reduced the response of SARs to histamine at 10 micrograms/kg but had no significant effect on the SAR response to 60 micrograms/kg histamine. In the absence of vagal afferent and efferent activities on the left side, mequitazine, a potent H1-receptor blocker, completely blocked low- and high-dose effects of histamine on SARs. We compared the responses of the receptor activity to aerosol histamine (1 and 4%) and to topical application of histamine (0.1 ml, 0.025% and 0.1%) in six SAR preparations. The magnitude and duration of increased SAR activity became more prominent by increasing the concentration of histamine. The firing pattern and discharge rate of SARs following aerosol or intratracheal administration of histamine were similar to those after intra-atrial histamine. In addition, we also examined the excitatory responses of SAR activity to right atrial injections of histamine at 10 and 60 micrograms/kg before and after topical administration of atropine (0.1 ml, 1%, n = 6) or mequitazine (0.1 ml, 1%, n = 6) in 12 SAR preparations. Intratracheal atropine diminished the response of SARs to 10 micrograms/kg of histamine but had no significant effect on the response of SARs to histamine at 60 micrograms/kg. All the responses of SARs to histamine were completely blocked by topical application of mequitazine. These results suggest that the change of SAR activity produced by histamine at 10 micrograms/kg occurs mainly as a result of the release of acethylcholine (ACh) via the vagovagal reflex and that the activation of H1-receptors of the airway smooth muscle contributes importantly to the response of SARs to histamine at 60 micrograms/kg.     

Dose-conductance relationships for GABA agonists and the effect of uptake inhibitors in crayfish stretch receptor neurons

The interaction of GABA (γ-aminobutyric acid) and structurally-related compounds with postsynaptic GABA receptors was studied quantitatively by measuring receptor-mediated increases in membrane input conductance in isolated crayfish stretch receptor neurons (SRN). The following compounds, in order of decreasing potency, were effective agonists:muscimol>GABA>isoguvacine> (−)γ-amino-β-hydroxybutyric acid> β-guanidinopropionic acid> 3-aminopropanesulfonic acid> (+)γ-amino-β-hydroxybutyric acid>isonipecotic acid>THIP. A highly significant correlation was found between the log potencies for GABA agonists that were obtained in the SRN and those obtained in our laboratory using mammalian GABA receptor binding assays. Hill plot analyses of the log concentration-conductance data from the SRN indicated a Hill slope (n_H) of approximately 2 for all agonists except GABA and guanidinopropionic acid(n_H > 2), two compounds known to be actively accumulated by cellular GABA uptake processes, Nipecotic acid, guvacine, andl-α, β-diaminopropionic acid, blockers of GABA uptake processes, had essentially no effect by themselves on the SRN membrane input conductance at concentrations up to 5 mM, however, they potentiated the effects of sub-maximal concentrations of GABA and decreased the steepness of the log concentration-conductance curve, and consequently n_H, for GABA. The effects of muscimol, however, were not affected. When the influence of uptake processes was considered, it appeared that all agonists tested acted by the same cooperative mechanism which required at least two molecules of agonist to activate a receptor-ionophore unit.     

Potassium and sodium channels in human malignant glioma cells

Human malignant glioma cells from 5 different cell lines were voltage clamped and examined for the presence of depolarization-activated ion channels. Outward K-currents were elicited at membrane potentials > 40 mV, which had two main components, one which was delayed and blocked by externally applied tetraethylammonium (TEA, 10 mM), and another which was instantaneous and insensitive to TEA in the outside solution. The proportion of the two K-current components varied between cell lines. An increase in [Ca²⁺] in the range 0–4 mM, decreased the leak conductance and shifted the activation of the instantaneous outward K-current towards more positive potenttials. Mg²⁺, Zn²⁺ and Co²⁺ had qualitatively similar effects. Patch recordings with 150–160 mM K⁺-solution on both sides of the membrane revealed that the delayed outward K-current was carried through large conductance (250–300 pS) channels. Changes in free [Ca²⁺]_i from 0 to 2 × 10⁻⁸ M increased the activation of the large conductance K-channel. Small Na-currents were identified in cells from one cell line (Tp-378MG). The Na-conductance rangedfrom 0.5 to 7.5 nS in 25% of the cells, and was less than 0.5 nS in 75%. The Na-channels were activated and inactivated at 30–40 mV more positive potentials than in the mammalian peripheral nerve. Tetrodotoxin (100 mM) blocked g_Na almost completely.     

Single-channel analysis of tetrodotoxin-sensitive and tetrodotoxin-resistant sodium channels in rat dorsal root ganglion neurons

Tetrodotoxin-sensitive and tetrodotoxin-resistant single sodium channel currents were recorded from rat dorsal root ganglion neurons. The two types of sodium channel currents could be distinguished by the effects of predepolarization, 10 nM tetrodotoxin, and the inactivation during depolarization. Single-channel conductances were calculated to be 6.3 and 3.4 pS in the tetrodotoxin-sensitive and tetrodotoxin-resistant channels, respectively.     

Axonal coding of action potentials in demyelinated nerve fibers

Conduction in individual axons ofXenopus has been measured optically in response to short trains of stimuli, following demyelination of the sciatic nerve. In many cases theinitial action potential in a burst is absent. Failure may also occur later in the train, resulting in a profound alteration of signal coding by the axon. Integration leading to delayed transmission occurred at the heminode forming the proximal border of the demyelinated zone, as well as at new nodes of Ranvier forming in remyelinating axons. This process appeared to involve a depolarizing afterpotential and seemed to be analogous to the threshold changes involved in superexcitability. Axonal coding was very sensitive to small changes in the stimulus pattern. Neither 1 mM tetraethylammonium ion, which blocks nodal and Ca²⁺ activated K⁺ channels, nor 1 mM 4-aminopyridine, which blocks fast internodal K⁺ channels, prevented loss of the initial spike in a burst. Similarly, neither block of Ca²⁺ channels by Cd²⁺ nor lowering of Cl⁻ had a notable effect. Ouabain, on the other hand, had small but possibly significant effects on responses to repetitive stimuli. A computational model was used to test mechanisms involving passive cable properties. Lowering the myelin resistance and the nodal leakage conductance, in accord with recent evidence from intracellular recordings, reproduced many of the results and was accurate with respect to stimulus frequency, temperature and sensitivity to average potential. The coding of action potentials observed here may have clinical consequences in demyelinating diseases such as multiple sclerosis.     

Macrocurrents of voltage gated Na+ and K+ channels from the crayfish stretch receptor neuronal soma

Currents from the slowly adapting stretch receptor neuron of the crayfish (Pacifastacus leniusculus) were studied in a cell attached configuration using patch pipettes with an opening diameter of 2-10 microm. The neuronal membrane was enzymatically freed from the glial layer. The voltage gated Na+ and K+ channels seemed to be more concentrated in the lower part of soma close to the axon hillock. The Na+ and K+ currents could be analysed by fitting the currents to a fourth-order exponential function for Na+ current and a second-order exponential function for the K+ current. The macropatch recordings of enzymatically treated neurons are superior to two electrode voltage clamp recordings when analyzing voltage gated Na+ and K+ currents.     

Development of inflammatory hypersensitivity and augmentation of sodium channels in rat dorsal root ganglia

The development of thermal allodynia in relationship to sodium channel augmentation in dorsal root ganglia (DRGs) was studied in albino rats. Paw withdrawal latencies were measured hourly following complete Freund's adjuvant (CFA) injections. Sodium channels were demonstrated with immunocytochemistry. Sustained minimum latencies were attained between 10 and 12 h post-injection. Sodium channel labeling began to increase at 23 h post-injection and reached maximum levels by 24 h. Thermal hypersensitivity is thus established 12 h before sodium channel augmentation can be demonstrated.