Ladislav Vyklický

Temperature coefficient of membrane currents induced by noxious heat in sensory neurones in the rat

1 Membrane currents induced by noxious heat (Iheat) were studied in cultured dorsal root ganglion (DRG) neurones from newborn rats using ramps of increasing temperature of superfusing solutions. 2 I heat was observed in about 70 % of small (< 25 μm) DRG neurones. At ‐60 mV, Iheat exhibited a threshold at about 43 °C and reached its maximum, sometimes exceeding 1 nA, at 52 °C (716 ± 121 pA; n= 39). 3 I heat exhibited a strong temperature sensitivity (temperature coefficient over a 10 °C temperature range (Q10) = 17·8 ± 2·1, mean ± s.d., in the range 47‐51 °C; n= 41), distinguishing it from the currents induced by capsaicin (1 μM), bradykinin (5 μM) and weak acid (pH 6·1 or 6·3), which exhibited Q10 values of 1·6‐2·8 over the whole temperature range (23‐52 °C). Repeated heat ramps resulted in a decrease of the maximum Iheat and the current was evoked at lower temperatures. 4 A single ramp exceeding 57 °C resulted in an irreversible change in Iheat. In a subsequent trial, maximum Iheat was decreased to less than 50 %, its threshold was lowered to a temperature just above that in the bath and its maximum Q10 was markedly lower (5·6 ± 0·8; n= 8). 5 DRG neurones that exhibited Iheat were sensitive to capsaicin. However, four capsaicin‐sensitive neurones out of 41 were insensitive to noxious heat. There was no correlation between the amplitude of capsaicin‐induced responses and Iheat. 6 In the absence of extracellular Ca2+, Q10 for Iheat was lowered from 25·3 ± 7·5 to 4·2 ± 0·4 (n= 7) in the range 41‐50 °C. The tachyphylaxis, however, was still observed. 7 A high Q10 of Iheat suggests a profound, rapid and reversible change in a protein structure in the plasma membrane of heat‐sensitive nociceptors. It is hypothesized that this protein complex possesses a high net free energy of stabilization (possibly due to ionic bonds) and undergoes disassembly when exposed to noxious heat. The liberated components activate distinct cationic channels to generate Iheat. Their affinity to form the complex at low temperatures irreversibly decreases after one exposure to excessive heat.

Improved superfusion technique for rapid cooling or heating of cultured cells under patch-clamp conditions

We have developed an improved technique for fast cooling and heating of solutions superfusing isolated cells under patch-clamp or calcium imaging conditions. The system meets the requirements for studying temperature dependency of all kinds of ion channels, in particular temperature-gated ion channels. It allows the application of temperature changes within a range of 5-60 degrees C at maximum rates of -40 degrees C/s to 60 degrees C/s. Barrels filled with different solutions are connected to a manifold consisting of seven silica capillaries (320 microm inner diameter, i.d.). A common outlet consists of a glass capillary through which the solutions are applied onto the cell surface. The upper part of this capillary is embedded in a temperature exchanger driven by a miniature Peltier device which preconditions the temperature of the passing solution. The lower part of the capillary carries an insulated copper wire, densely coiled over a length of 7 mm, and connected to a dc current source for resistive heating. The Peltier device and the heating element are electrically connected to the headstage probe which is fixed on to a micromanipulator for positioning of the manifold. The temperature of the flowing solution is measured by a miniature thermocouple inserted into the common outlet capillary near to its orifice which is placed at a distance of less than 100 microm from the surface of the examined cell. The temperature is either manually controlled by voltage commands or adjusted via the digital-to-analog converter of a conventional data acquisition interface. Examples are given of using the device in patch-clamp studies on heterologously expressed TRPV1, TRPM8, and on cultured rat sensory neurons.

20-Oxo-5β-Pregnan-3α-yl Sulfate Is a Use-Dependent NMDA Receptor Inhibitor

NMDA receptors are ligand-gated ion channels permeable to calcium and play a critical role in excitatory synaptic transmission, synaptic plasticity, and excitotoxicity. They are heteromeric complexes of NR1 combined with NR2A-D and/or NR3A-B subunits that are activated by glutamate and glycine and whose activity is modulated by allosteric modulators. In this study, patch-clamp recordings from human embryonic kidney 293 cells expressing NR1/NR2 receptors were used to study the molecular mechanism of the endogenous neurosteroid 20-oxo-5β-pregnan-3α-yl sulfate (3α5βS) action at NMDA receptors. 3α5βS was a twofold more potent inhibitor of responses mediated by NR1/NR2C-D receptors than those mediated by NR1/NR2A-B receptors. The structure of the extracellular loop between the third and fourth transmembrane domains of the NR2 subunit was found to be critical for the neurosteroid inhibitory effect. The degree of 3α5βS-induced inhibition of responses to glutamate was voltage independent, with recovery lasting several seconds. In contrast, application of 3α5βS in the absence of agonist had no effect on the subsequent response to glutamate made in the absence of the neurosteroid. A kinetic model was developed to explain the use-dependent action of 3α5βS at NMDA receptors. In accordance with the model, 3α5βS was a less potent inhibitor of NMDA receptor-mediated EPSCs and responses induced by a short application of 1 mm glutamate than of those induced by a long application of glutamate. These results suggest that 3α5βS is a use-dependent but voltage-independent inhibitor of NMDA receptors, with more potent action at tonically than at phasically activated receptors. This may be important in the treatment of excitotoxicity-induced neurodegeneration.

PROPERTIES OF NMDA RECEPTORS IN RAT SPINAL CORD MOTONEURONS

Postnatal development and properties of N‐methyl‐d‐aspartate (NMDA) receptors were studied with whole‐cell and outside‐out patch‐clamp techniques in interneurons and fluorescence‐labelled motoneurons in rat spinal cord slices. Both the absolute amplitude of NMDA‐induced currents and currents normalized with respect to the motoneuron capacitance increased significantly at postnatal days 10–13 when compared to the responses evoked at postnatal days 2–3. The mean amplitude of the responses to kainate also increased in motoneurons of postnatal days 10–13. Single‐channel currents induced by low concentrations of glutamate, exhibited four distinct amplitude levels corresponding to 19.2 ± 2.4 pS, 38.4 ± 3.5 pS, 56.3 ± 2.4 pS and 69.6 ± 3.7 pS. In contrast, the conductance of single channels, recorded under identical conditions, in rat spinal cord interneurons was less, 15.3 ± 3.2 pS, 29.9 ± 5.4 pS, 46.7 ± 4.8 pS and 62.4 ± 3.9 pS. The high (56/70 pS) conductance single‐channel openings in motoneuron patches were sensitive to NMDA receptor inhibitors d‐2‐amino‐5‐phosphonovalerate, 7‐chlorokynurenic acid and ifenprodil. Whole‐cell NMDA‐evoked currents were blocked in a voltage‐dependent manner by extracellular Mg2+ with an apparent dissociation constant for Mg2+ binding at 0 mV of 1.8 ± 0.5 mm. The conductance and relative distribution of NMDA receptor channel openings induced by 1 μm glutamate in patches isolated from the motoneurons were independent of age from postnatal day 4 to 14.

Reducing agent dithiothreitol facilitates activity of the capsaicin receptor VR-1

The vanilloid receptor subtype 1 (VR1) is expressed in a sub-population of small dorsal root ganglion (DRG) neurones in mammals and serves as the common transducer of the pain-producing signals, such as noxious heat, acids and capsaicin [Caterina et al., Nature 389 (1997) 816-824; Tominaga et al., Neuron 21 (1998) 531-543]. On the extracellular side, VR1 has three cysteine residues at positions 616, 621 and 634. Here we report that dithiothreitol (DTT) (2-60 mM), an agent that maintains -SH groups of cysteines in a reduced state, greatly facilitates membrane currents induced by noxious heat or capsaicin (1 microM) in cultured DRG neurones from the rat and in VR1-transfected HEK293 cells. The effects of DTT are concentration-dependent and fully reversible. We suggest that the ratio between free sulfhydryl groups and disulfide bonds of the cysteine residues of VR1 pre-sets sensitivity of primary nociceptors to algogens and may represent a new target for treating some pain states in humans.

Vanilloid receptor TRPV1 is not activated by vanilloids applied intracellularly.

&NA; The vanilloid receptor TRPV1 is a ligand‐gated cation channel that can be activated by capsaicin, acids and noxious heat. For vanilloids, a stretch of ˜8 amino acids in the vicinity of the TM3 region on the cytosolic side of TRPV1 and R114 and E761 in the N‐ and C‐cytosolic tails, respectively, has been shown to be critical for capsaicin binding and channel activation. Here, we report that intracellular application of vanilloids is insufficient for activating TRPV1 channels in HEK293T cells. Pipette solution (ICS) for recording membrane currents was supplemented with 50 &mgr;M capsaicin (n=14) or 1 &mgr;M resiniferatoxin (RTX) (n=39) and the responses induced by extracellular capsaicin (1 &mgr;M) or RTX (100 nM) were recorded at intervals >50% of that needed for diffusion of Lucifer yellow from the pipette to reach maximum fluorescence (n=7). We found that all cells with expressed TRPV1 exhibited a similar sensitivity to vanilloids irrespective of whether the membrane currents were recorded with electrodes filled with ICS containing capsaicin or RTX or only with control ICS. We suggest that, in addition to intracellularly located agonist recognition sites of TRPV1, there is at least one resides on the extracellular side, which needs to be occupied to activate the channel.

Molecular and functional properties of synaptically activated NMDA receptors in neonatal motoneurons in rat spinal cord slices

The functional properties of N‐methyl‐ d‐aspartate (NMDA) receptor‐mediated excitatory postsynaptic currents (EPSC) were studied in fluorescence‐labelled motoneurons in thin spinal cord slices. The deactivation of NMDA receptor EPSCs in motoneurons voltage‐clamped at +40 mV was independent of intensity or location of stimulation and of postnatal age [τfast = 28.5 ± 4.6 ms (63.6 ± 8.8%) and τslow = 165.6 ± 49.6 ms]. In the presence of 1 mm Mg2+ the amplitude of NMDA receptor EPSCs was voltage‐dependent. Boltzmann analysis of the relationship between peak NMDA receptor EPSC amplitude and membrane potential suggested an apparent Kd of Mg2+ (at 0 mV) of 0.87 mm. Nonstationary variance analysis of NMDA receptor EPSCs gave an estimated single‐channel conductance of 59 ± 14 pS. Direct measurement of the NMDA receptor channel openings in outside‐out patches isolated from motoneurons indicated the presence of single‐channel conductance levels of 21.8 ± 2.8 pS, 37.1 ± 3.2 pS, 49.6 ± 5.1 pS and 69.6 ± 4.2 pS. Single‐cell RT‐PCR analysis of mRNA revealed that NR1, NR2A–D and NR3A transcripts were expressed in motoneurons. These results suggest that specific assembly of NMDA receptor subunits in motoneurons determines the functional and pharmacological properties of the receptors in these cells.

The effects of capsaicin and acidity on currents generated by noxious heat in cultured neonatal rat dorsal root ganglion neurones.

1 The effects of capsaicin, acidic pH, ATP, kainate and GABA on currents generated by noxious heat were studied in cultured dorsal root ganglion (DRG) neurones (< 20 μm in diameter) isolated from neonatal rats. The patch clamp technique was used to record membrane currents or changes of membrane potential. 2 In agreement with previous results, inward membrane currents (Iheat) induced by a 3 s ramp of increasing temperature from room temperature (≈23 °C) to over 42 °C varied greatly between cells (‐100 pA to −2.4 nA at 48 °C) and had a temperature coefficient (Q10) > 10 over the range of 43‐52 °C. 3 Capsaicin potentiated the heat‐induced current even when capsaicin, at room temperature, had little or no effect on its own. In cells in which capsaicin induced no or very small membrane current at room temperature (< 50 pA), Iheat exhibited detectable activation above 40 °C and increased 5.1 ± 1.1 (n= 37) and 6.3 ± 2.0 (n= 18) times at 0.3 and 1 μM capsaicin, respectively. 4 A rapid decrease in extracellular pH from 7.3 to 6.8, 6.3 or 6.1 produced an inward current which inactivated in ≈5 s either completely (pH 6.8 or 6.3) or leaving a small current (≈50 pA) for more than 2 min (pH 6.1). After inactivation of the initial low pH‐induced current, Iheat at 48 °C increased 2.3 ± 0.4 times at pH 6.8, 4.0 ± 0.6 times at pH 6.3 and 4.8 ± 0.8 times at pH 6.1 with a Q10 > 10 (n= 16). 5 ATP (n= 22), kainate (n= 7) and GABA (n= 8) at 100 μM, produced an inactivating inward current in all heat‐sensitive DRG neurones tested. During inactivation and in the presence of the drug, Iheat was increased slightly with ATP and unaffected with kainate and GABA. These agents apparently do not directly affect the noxious heat receptor. 6 The results indicate a novel class of capsaicin‐sensitive cells, in which capsaicin evokes no or very small inward current but nevertheless increases sensitivity to noxious heat.

Spontaneous openings of NMDA receptor channels in cultured rat hippocampal neurons.

Spontaneous and N‐methyl‐D‐aspartate (NMDA)‐evoked single‐channel currents were studied in outside‐out patches isolated from cultured rat hippocampal neurons. Both spontaneous and NMDA‐evoked single‐channel currents reversed at potentials close to 0 mV and exhibited multiple amplitude levels of similar amplitude. Both spontaneous and NMDA‐evoked single‐channel currents were inhibited by Mg2+ in a voltage‐dependent manner and by 7‐chlorokynurenic acid. The activity of spontaneous single‐channel currents was reduced by the competitive NMDA receptor antagonists, but by one to three orders of magnitude less than expected assuming that the spontaneous activity is due to an ambient NMDA receptor agonist present in the extracellular solution. Our results suggest that, similar to other ligand‐gated ion channels, NMDA receptor channels have a dual mode of activation ‐ spontaneous and agonist induced.

Effects of steroids on NMDA receptors and excitatory synaptic transmission in neonatal motoneurons in rat spinal cord slices.

The effect of steroids on NMDA receptors and excitatory postsynaptic transmission was studied in fluorescence‐labelled motoneurons in thin spinal cord slices. In outside‐out patches, NMDA‐induced responses were potentiated by 79% in the presence of 20‐oxopregn‐5‐en‐3β‐yl sulfate (PS), while in the presence of 20‐oxo‐5α‐pregnan‐3α‐yl sulfate (3α5αS) and 20‐oxo‐5β‐pregnan‐3α‐yl sulfate (3α5βS) they were diminished by 57% and 66%, respectively. PS and 3α5βS had no effect on the amplitude of single NMDA receptor channel openings, however, both compounds altered relative distribution of the openings to individual conductance levels. In control cases, the most frequent openings of the NMDA receptor channels were at the 70 pS conductance level, while in the presence of PS or 3α5βS, the most frequent openings were at the 55 pS conductance level. Analysis of the mean current transferred by NMDA receptor channel openings at individual conductance levels indicated that in the presence of PS, the mean current induced by 55 pS conductance openings was significantly increased. In the presence of 3α5βS, the mean currents induced by 55 pS and 70 pS conductance openings were significantly decreased. The amplitude of NMDA receptor‐mediated EPSCs was potentiated by 54% in the presence of PS and the deactivation kinetics slowed. Neither the amplitude nor the kinetics of NMDA receptor‐mediated EPSCs was significantly changed in the presence of 3α5βS. The results of our experiments indicate that neurosteroids affect NMDA receptors in motoneurons. The effect appears to be influenced by the receptor subunit composition.