Effects of NMDA receptor antagonists on nociceptive responsesin vivo: Comparison of antagonists acting at the glycine site with uncompetitive antagonists

Summary We have shown that members of a new series of tricyclic pyridophthalazine diones, defined as glycine_B site NMDA antagonistsin vitro, are selective and systemically active NMDA antagonistsin vivo. In electrophysiological tests in -chloralose anaesthetised rats, these compounds reduced nociceptive reflex responses. In conscious rats they displayed analgesic properties. These glycine_B antagonists were compared electrophysiologically with several uncompetitive NMDA channel blockers. The degree of voltage dependence previously reportedin vitro related to the effectiveness of the agents against different amplitude nociceptive responses of spinal cord neuronesin vivo.     

非洲爪蟾卵细胞膜去极化后由超极化激活的瞬间内向电流

以双微电极电压钳制技术研究了未成熟非洲爪蟾卵细胞膜的离子流,发现在较长时间的去极化（－３０ｍｖ,５秒）前脉冲后由超极化引出的一个内向电流,其潜伏期约为０．５秒,经过０．４秒左右到达高峰,随近经０．８秒左右完全回复。其幅值随超极化程度增强而增大,其翻转电位接近氯平衡电位,并随胞外ＣＬ－浓度改变而变化。降低胞外ＣＬ－浓度使其幅值增大。降低胞外Ｎａ＾＋浓度对其无明显影响,提示此内几电流可能是氯离子流。以     

Voltage clamp calculations for myelinated and demyelinated axons

Exact cable theory is used to calculate voltage distributions along fully myelinated axons and those with various patterns of demyelination. The model employed uses an R-C circuit for the soma, an equivalent cable for the dendrites, a myelinated axon with n internodes and a cable representing telodendria. For the case of a voltage clamp at the soma, a system of 2n + 1 equations must be solved to obtain the potential distribution and this is done for arbitrary n. An explicit calculation is performed for one internode whereas computer-generated solutions are obtained for several internodes. The relative importance of the position of a single demyelinated internode is determined. An approximate expression is given for the critical internodal length necessary for action potential generation.     

The neural basis of catalepsy in the stick insect

The known nonlinearities of the femur-tibia control loop of the stick insect Carausius morosus (enabling the system to produce catalepsy) are already present in the nonspiking interneuron E4: (1) The decay of depolarizations in interneuron E4 following slow elongation movements of the femoral chordotonal organ apodeme could be described by a single exponential function, whereas the decay following faster movements had to be characterized by a double exponential function. (2) Each of the two corresponding time constants was independent of stimulus velocity. (3) The relative contribution of each function to the total amount of depolarization changed with stimulus velocity. (4) The characteristics described in (1)–(3) were also found in the slow extensor tibiae motoneuron. (5) Single electrode voltage clamp studies on interneuron E4 indicated that no voltage dependent membrane properties were involved in the generation of the observed time course of decay. Thus, we can trace back a certain behavior (catalepsy) to the properties of an identified, nonspiking interneuron.Abbrevations FETi fast extensor tibiae motor neuron - FT-joint femur-tibia joint - FT-control loop femur-tibia control loop - SETi slow extensor tibiae motor neuron - R regression coefficient     

Neuron–transistor coupling: interpretation of individual extracellular recorded signals

The electrical coupling of randomly migrating neurons from rat explant brain-stem slice cultures to the gates of non-metallized field-effect transistors (FETs) has been investigated. The objective of our work is the precise interpretation of extracellular recorded signal shapes in comparison to the usual patch-clamp protocols to evaluate the possible use of the extracellular recording technique in electrophysiology. The neurons from our explant cultures exhibited strong voltage-gated potassium currents through the plasma membrane. With an improved noise level of the FET set-up, it was possible to record individual extracellular responses without any signal averaging. Cells were attached by patch-clamp pipettes in voltage-clamp mode and stimulated by voltage step pulses. The point contact model, which is the basic model used to describe electrical contact between cell and transistor, has been implemented in the electrical simulation program PSpice. Voltage and current recordings and compensation values from the patch-clamp measurement have been used as input data for the simulation circuit. Extracellular responses were identified as composed of capacitive current and active potassium current inputs into the adhesion region between the cell and transistor gate. We evaluated the extracellular signal shapes by comparing the capacitive and the slower potassium signal amplitudes. Differences in amplitudes were found, which were interpreted in previous work as enhanced conductance of the attached membrane compared to the average value of the cellular membrane. Our results suggest rather that additional effects like electrodiffusion, ion sensitivity of the sensors or more detailed electronic models for the small cleft between the cell and transistor should be included in the coupling model.     

Interactions Between Charged Residues in the Transmembrane Segments of the Voltage-sensing Domain in the hERG Channel

Studies on voltage-gated K channels such as Shaker have shown that positive charges in the voltage-sensor (S4) can form salt bridges with negative charges in the surrounding transmembrane segments in a state-dependent manner, and different charge pairings can stabilize the channels in closed or open states. The goal of this study is to identify such charge interactions in the hERG channel. This knowledge can provide constraints on the spatial relationship among transmembrane segments in the channel’s voltage-sensing domain, which are necessary for modeling its structure. We first study the effects of reversing S4’s positive charges on channel activation. Reversing positive charges at the outer (K525D) and inner (K538D) ends of S4 markedly accelerates hERG activation, whereas reversing the 4 positive charges in between either has no effect or slows activation. We then use the ‘mutant cycle analysis’ to test whether D456 (outer end of S2) and D411 (inner end of S1) can pair with K525 and K538, respectively. Other positive charges predicted to be able, or unable, to interact with D456 or D411 are also included in the analysis. The results are consistent with predictions based on the distribution of these charged residues, and confirm that there is functional coupling between D456 and K525 and between D411 and K538.     

A possible role for phosphate in complexing the arginines of S4 in voltage gated channels

Phosphate ions are known to complex guanidinium groups, which are the side chains of arginine. Voltage gated channels that allow passage of ions through cell membranes, producing, for example the nerve impulse, are in many cases composed of four domains, each with six transmembrane segments. The S4 transmembrane segments of these channels have arginines placed in such a way that they would be expected to complex phosphate. Known phosphate-arginine complexes are reasonably strong. Here, an ab initio calculation reinforces the expectation that a strong complex could form. As a consequence, if the S4 moved, it would carry either no charge, or at most half of what is expected from fully charged arginines. This suggests that it may be necessary to rethink voltage gating models in which the gating current is produced by physical motion of the S4 transmembrane segments.     

Effects of the Enantiomers of BayK 8644 on the Charge Movement of L-type Ca Channels in Guinea-pig Ventricular Myocytes

The effects of the agonist enantiomer S(-)Bay K 8644 on gating charge of L-type Ca channels were studied in single ventricular myocytes. From a holding potential (Vh) of -40 mV, saturating (250 nm) S(-)Bay K shifted the half-distribution voltage of the activation charge (Q1) vs. V curve -7.5 +/- 0.8 mV, almost identical to the shift produced in the Ba conductance vs. V curve (-7.7 +/- 2 mV). The maximum Q1 was reduced by 1.7 +/- 0.2 nC/microF, whereas Q2 (charge moved in inactivated channels) was increased in a similar amount (1.4 +/- 0.4 nC/microF). The steady-state availability curves for Q1, Q2, and Ba current showed almost identical negative shifts of -14.8 +/- 1.7 mV, -18.6 +/- 5.8 mV, and -15.2 +/- 2.7 mV, respectively. The effects of the antagonist enantiomer R(+)BayK 8644 were also studied, the Q1 vs. V curve was not significantly shifted, but Q1max (Vh = -40 mV) was reduced and the Q1 availability curve shifted by -24.6 +/- 1.2 mV. We concluded that: a) the left shift in the Q1 vs. V activation curve produced by S(-)BayK is a purely agonistic effect; b) S(-)BayK induced a significantly larger negative shift in the availability curve than in the Q1 vs. V relation, consistent with a direct promotion of inactivation; c) as expected for a more potent antagonist, R(+)Bay K induced a significantly larger negative shift in the availability curve than did S(-)Bay K.     

Nefopam,an analogue of orphenadrine,protects against both NMDA receptor-dependent and independent veratridine-induced neurotoxicity

Summary.  Nefopam hyghochloride is a potent analgesic compound commercialized in most Western Europe for 20 years, which possesses a profile distinct from that of opioids or anti-inflammatory drugs. Previous evidence suggested a central action of nefopam but the detailed mechanisms remain unclear. While, nefopam structure resembles that of orphenadrine, an uncompetitive NMDA receptor antagonist, here we report that differently from orphenadrine, nefopam (100 μM) failed to protect cultured cerebellar neurons from excitotoxicity following direct exposure of neurons to glutamate. Moreover, nefopam failed to displace MK-801 binding to hippocampal membranes. Nefopam effectively prevented NMDA receptor-mediated early appearance (30 min) of toxicity signs induced by the voltage sensitive sodium channel (VSSC) activator veratridine. The later phase (24 h) of neurotoxicity by veratridine occurring independently from NMDA receptor activation, was also prevented by nefopam. Nefopam effect was not mimicked by the GABA receptor agonist muscimol. Received June 29, 2001 Accepted August 6, 2001 Published online June 3, 2002     

CO<Subscript>2</Subscript> Sensitivity of Voltage Gating and Gating Polarity of GapJunction Channels—Connexin40 and its COOH-Terminus-Truncated Mutant

The CO₂ sensitivity of transjunctional voltage (V _j) gating was studied by dual voltage clamp in oocytes expressing mouse Cx40 or its COOH terminus (CT)-truncated mutant (Cx40-TR). V _j sensitivity, determined by a standard V _j protocol (20 mV V _j steps, 120 mV maximal), decreased significantly with exposure to 30% CO₂. The Boltzmann values of control versus CO₂-treated oocytes were: V ₀ = 36.3 and 48.7 mV, n = 5.4 and 3.7, and G _{j min} = 0.21 and 0.31, respectively. CO₂ also affected the kinetics of V _j-dependent inactivation of junctional current (I _j); the time constants of two-term exponential I _j decay, measured at V _j = 60 mV, increased significantly with CO₂ application. Similar results were obtained with Cx40-TR, suggesting that CT does not play a role in this phenomenon. The sensitivity of Cx40 channels to 100% CO₂ was also unaffected by CT truncation. There is evidence that CO₂ decreases the V _j sensitivity of Cx26, Cx50 and Cx37 as well, whereas it increases that of Cx45 and Cx32 channels. Since Cx40, Cx26, Cx50 and Cx37 gate at the positive side of V _j, whereas Cx45 and Cx32 gate at negative V _j, it is likely that V _j behavior with respect to CO₂-induced acidification varies depending on gating polarity, possibly involving the function of the postulated V _j sensor (NH₂-terminus).This revised version was published online in June 2005 with a corrected cover date.