[Molecular pathogenesis of Duchenne muscular dystrophy-related fibrosis]. / Molekulare Pathogenese der Fibrose bei Muskeldystrophie vom Typ Duchenne.

Progressive myofibrosis plays a key role in Duchenne muscular dystrophy. The dystrophic loss of contractile cells triggers a relatively nonspecific restructuring of the surrounding mesenchyme. The increase in connective and fatty tissue leads to muscular weakness and is therefore of critical importance for the cellular pathogenesis of muscular dystrophy. The systematic biochemical analysis of fibrosis using comparative proteomics has identified a number of extracellular matrix proteins that are indirectly involved in muscular dystrophy. An increased concentration was established for collagen I, collagen IV, collagen VI, periostin, dermatopontin, fibronectin, biglycan, asporin, decorin, prolargin, mimecan and lumican. Based on these findings, the identified matrix proteins can now be characterized biochemically and their exact pathophysiological role in Duchenne muscular dystrophy determined.

Menthol suppresses nicotinic acetylcholine receptor functioning in sensory neurons via allosteric modulation.

In this study, we have investigated how the function of native and recombinant nicotinic acetylcholine receptors (nAChRs) is modulated by the monoterpenoid alcohol from peppermint (-) menthol. In trigeminal neurons (TG), we found that nicotine (75 µM)-activated whole-cell currents through nAChRs were reversibly reduced by menthol in a concentration-dependent manner with an IC50 of 111 µM. To analyze the mechanism underlying menthol's action in more detail, we used single channel and whole-cell recordings from recombinant human α4ß2 nAChR expressed in HEK tsA201 cells. Here, we found a shortening of channel open time and a prolongation of channel closed time, and an increase in single channel amplitude leading in summary to a reduction in single channel current. Furthermore, menthol did not affect nicotine's EC50 value for currents through recombinant human α4ß2 nAChRs but caused a significant reduction in nicotine's efficacy. Taken together, these findings indicate that menthol is a negative allosteric modulator of nAChRs.

Sphingosine-1-phosphate links glycosphingolipid metabolism to neurodegeneration via a calpain-mediated mechanism.

We have recently reported that the bioactive lipid sphingosine-1-phosphate (S1P), usually signaling proliferation and anti-apoptosis induces neuronal death when generated by sphingosine-kinase2 and when accumulation due to S1P-lyase deficiency occurs. In the present study, we identify the signaling cascade involved in the neurotoxic effect of sphingoid-base phosphates. We demonstrate that the calcium-dependent cysteine protease calpain mediates neurotoxicity by induction of the endoplasmic reticulum stress-specific caspase cascade and activation of cyclin-dependent kinase5 (CDK5). The latter is involved in an abortive reactivation of the cell cycle and also enhances tau phosphorylation. Neuroanatomical studies in the cerebellum document for the first time that indeed neurons with abundant S1P-lyase expression are those, which degenerate first in S1P-lyase-deficient mice. We therefore propose that an impaired metabolism of glycosphingolipids, which are prevalent in the central nervous system, might be linked via S1P, their common catabolic intermediate, to neuronal death.

Lysosulfatide regulates the motility of a neural precursor cell line via calcium-mediated process collapse.

Lysosulfatide is a derivative of the glycosphingolipid sulfatide. It is a major component of high density lipoproteins and was detected in the human brain. Here, we show that lysosulfatide acts as an extracellular signal regulating the migration of a neural precursor cell line (B35 neuroblastoma cells) by rapidly promoting process retraction and cell rounding. These cells express the lysosulfatide receptor S1P3 according to RT-PCR, western blotting and immunocytochemistry, but S1P3 does not mediate the effect since preincubation with three different compounds known to inhibit S1P3 did not block lysosulfatide-induced cell rounding. The signal transduction after stimulation with 3 microM lysosulfatide involves a rapid increase of [Ca2+]i which causes process retraction. This mechanism may be relevant under conditions where neural cells encounter elevated lysosulfatide levels as for example under pathological conditions after breakdown of the blood brain barrier or possibly in the lysosomal sulfatide storage disorder metachromatic leukodystrophy.

Spinal inhibitory synaptic transmission in the glycine receptor mouse mutant spastic.

Inhibitory glycine receptor (GlyR) and GABA(A) receptor (GABA(A)R)-mediated synaptic transmission was examined in two strains of the GlyR mutant mouse spastic and the respective wild types. The mutants display a mild and a severe neurological phenotype. Electrically evoked postsynaptic whole-cell currents were recorded from alpha-motoneurons in lumbar spinal cord slices. Amplitudes of GlyR-mediated IPSCs were significantly reduced in the severe phenotype in comparison to the respective wild type and the mild phenotype mutants. Surprisingly, amplitudes of GABA(A)R-mediated IPSCs were also significantly reduced in both mutants. Fast time constants of the decay phase of IPSCs were slightly reduced for the GlyR-mediated IPSCs and significantly larger for the GABA(A)R-mediated IPSCs in both mutant strains.

The use of control theory for the design of voltage clamp systems: a simple and standardized procedure for evaluating system parameters.

Voltage clamp (VC) instruments are closed-loop control systems based on electronic feedback. Such feedback systems can be described in the framework of control theory. We used a mathematical approach based on control theory to improve the performance of VC systems. This approach considerably simplifies the design and optimal tuning of these systems, as is demonstrated for a standard two electrode and a time-sharing single electrode clamp system. The major advantage of this approach and the consequent optimization procedure is that only proportional-integral controllers for VC systems must be used. As a consequence, the design of such VC systems is solely based on the time constants of the clamp circuit. In our approach, the 'symmetrical optimum' rule was applied for the first time to VC systems. This yields optimized systems with respect to speed of response and clamp accuracy. An empirical procedure has been derived from this theoretical approach which allows the optimal tuning of VC instruments based on PI controllers while running an experiment.

Calcium-dependent inactivation of neuronal calcium channel currents is independent of calcineurin.

Dephosphorylation by the Ca2+/calmodulin-dependent phosphatase calcineurin has been suggested as an important mechanism of Ca2+-dependent inactivation of voltage-gated Ca2+ channels. We have tested whether calcineurin plays a role in the inactivation process of two types of high-voltage-activated Ca2+ channels (L and N type) widely expressed in the central nervous system, using the immunosuppressive drug FK506 (tacrolimus), which inhibits calcineurin after binding to intracellular FK506 binding proteins. Inactivation of L- and N-type Ca2+ channels was studied in a rat pituitary tumor cell line (GH3) and chicken dorsal root ganglion neurons, respectively. With the use of antisera directed against the calcineurin subunit B and the 12,000 mol. wt binding protein, we show that both proteins are present in the cytoplasm of GH3 cells and chicken dorsal root ganglion neurons. Ionic currents through voltage-gated Ca2+ channels were investigated in the perforated-patch and whole-cell configurations of the patch-clamp technique. The inactivation of L- as well as N-type Ca2+ currents could be well fitted with a bi-exponential function. Inactivation was largely reduced when Ba2+ substituted for extracellular Ca2+ or when the Ca2+ chelator EGTA was present intracellularly, indicating that both types of Ca2+ currents exhibited Ca2+-dependent inactivation. Extracellular (perforated-patch configuration) or intracellular (whole-cell configuration) application of FK506 to inactivate calcineurin had no effect on the amplitude and time-course of Ca2+ channel current inactivation of either L- or N-type Ca2+ channels. In addition, we found that recovery from inactivation and rundown of N-type Ca2+ channel currents were not affected by FK506. Our results provide direct evidence that the calcium-dependent enzyme calcineurin is not involved in the inactivation process of the two Ca2+ channel types which are important for neuronal functioning, such as gene expression and transmitter release.

Opioidergic modulation of voltage-activated K+ currents in magnocellular neurons of the supraoptic nucleus in rat.

Opioidergic modulation plays an important role in the control of oxytocin and vasopressin release by magnocellular neurons (MCNs) in the supraoptic and paraventricular nuclei of the hypothalamus. We have used whole cell patch-clamp recording in acute slices of the supraoptic nucleus (SON) of the hypothalamus to study opioidergic modulation of voltage-dependent K+ currents in MCNs that are involved in release activity. The mu-receptor agonist D-Ala2, N-Me-Phe4, Gly5-ol-enkephalin (DAMGO, 2 microM) affected K+ currents in 55% of magnocellular neurons recorded from. In these putative oxytocinergic cells, DAMGO increased the delayed rectifier current (IK(V)) amplitude by approximately 50% without significant effects on its activation kinetics. The transient A current (IA) was enhanced by DAMGO by approximately 36%. Its inactivation kinetic was accelerated slightly while the voltage dependence of steady-state inactivation was shifted by -6 mV to more negative potentials. All DAMGO effects were blocked by the preferential non-kappa-opioid antagonist naloxone (10 microM). The kappa-opioid agonist trans-(+/-)-3, 4-dichloro-N-methyl-N(2-[1-pyrrolidinyl]cyclohexyl)benzeneacetamide (U50,488; 10 microM) strongly suppressed IK(V) by approximately 57% and evoked a 20-mV hyperpolarizing shift and an acceleration of activation in both, DAMGO-sensitive and -insensitive putative vasopressinergic MCNs. U50,488 reduced IA by approximately 29% and tau of inactivation by -20% in DAMGO-sensitive cells. In contrast, in DAMGO-insensitive cells U50,488 increased IA by approximately 23% and strongly accelerated inactivation (tau -44%). The effects of U50,488 were suppressed by the selective kappa-receptor antagonist nor-binaltorphimine (5 microM). We conclude that mu- and kappa-opioidergic inputs decrease and increase excitability of oxytocinergic MCNs, respectively, through modulation of voltage-dependent K+ currents. In vasopressinergic MCNs, kappa-opioidergic inputs differentially modulate these K+ currents. The modulation of K+ currents is assumed to significantly contribute to opioidergic control of hormone release by MCNs within the supraoptic nucleus and from the axon terminals in the neural lobe.

Mechanisms of IL-8-induced Ca2+ signaling in human neutrophil granulocytes.

Interleukin-8 (IL-8) plays an important role in the activation of neutrophil granulocytes. Although intracellular Ca2+ signals are essential in this process, they have not been studied in great detail so far. Here, we have measured IL-8-induced Ca2+ signals in single human neutrophil granulocytes using the Ca2+ indicator dye FURA-2 AM and we have investigated the signal transduction that leads to these Ca2+ signals with various pharmacological tools. Our results indicate that IL-8-induced Ca2+ signals consist of at least two components. An initial fast component was followed by a smaller and more persistent one. The initial Ca2+ signal was independent of extracellular Ca2+. It required the activation of phospholipase C via a pertussis toxin sensitive G-protein and was due to activation of IP3 receptor-coupled Ca2+ release channels. The late phase of the Ca2+ signal was suppressed when extracellular Ca2+ was removed suggesting that it was generated by Ca2+ influx through Ca2+ release-activated Ca2+ (CRAC) channels. This Ca2+ influx may prolong IL-8-induced Ca2+ signals during granulocyte activation.

Expression of early hippocampal CA1 LTP does not lead to changes in AMPA-EPSC kinetics or sensitivity to cyclothiazide.

We have analysed whether the expression of long-term potentiation (LTP) in rat hippocampal CA1 neurons involves a change in the kinetics of (S)-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor-mediated excitatory postsynaptic currents (EPSCs) (AMPA-EPSCs) or their susceptibility to the AMPA receptor modulator cyclothiazide. AMPA-EPSCs in the CA1 region were evoked by alternate stimulation of two independent Schaffer collateral-commissural inputs of slices of adult rat hippocampus. In the current-clamp mode a strong tetanus (100 Hz, 1 s) applied to one input (input I) induced stable LTP of AMPA-EPSCs in this input, while the control input (input II) remained unaffected. For neither input were EPSC rise time and decay kinetics significantly changed. The application of cyclothiazide prolonged the rise time and the decay time constants of the AMPA-EPSCs in both control and potentiated inputs to the same extent (Input I-rise time: 198+/-8%, decay: 148+/-12%; input II-rise time: 212+/-14%, decay: 144+/-19%; n=8). Furthermore, when present during tetanization cyclothiazide did not occlude LTP, suggesting that cyclothiazide and tetanic stimulation enhance AMPA-EPSCs via independent mechanisms. Our findings argue against changes in (de-)activation or desensitization of AMPA receptors as the molecular basis for the expression of LTP.

Interactions of GYKI 52466 and NBQX with cyclothiazide at AMPA receptors: experiments with outside-out patches and EPSCs in hippocampal neurones.

In outside-out patches from cultured hippocampal neurones, glutamate (1 mM) applied for 1 ms evoked currents which rose rapidly (tau(on) 451 +/- 31 micros) to a peak and then deactivated with slower kinetics (1.95 +/- 0.13 ms). Offset time constants were significantly slower with longer application durations (tau(off) 3.10 +/- 0.19, 3.82 +/- 0.25, 4.80 +/- 0.65 and 7.56 +/- 0.65 ms with 10, 20, 100 and 500 ms applications respectively). Desensitization was complete within 100 ms with a similar rate for all application durations (4.74 +/- 0.34 ms with 100 ms applications). GYKI 52466 reduced inward peak currents with an IC50 of 11.7 +/- 0.6 microM and had similar potency on steady-state currents to longer glutamate applications. GYKI 52466 had no significant effect on desensitization or deactivation time constants but caused a modest and significant prolongation of onset kinetics at higher concentrations. Cyclothiazide (100 microM) potentiated steady-state currents 25-fold at 100 ms and caused a modest but significant slowing in onset kinetics (601 +/- 49 micros with 1 ms applications) but a more pronounced prolongation of deactivation time constants (5.55 +/- 0.66 ms with 1 ms applications). In 50% of neuronal patches cyclothiazide completely eliminated desensitization. In those patches with residual desensitization, the rate was not significantly different to control (5.36 +/- 0.43 ms with 100 ms applications). Following 100 ms applications of glutamate, GYKI 52466 had IC50s of 11.7 +/- 1.1 microM and 75.1 +/- 7.0 microM in the absence and presence of cyclothiazide (100 microM) respectively. Onset kinetics were slowed from 400 +/- 20 micros to 490 +/- 30 micros by cyclothiazide (100 microM) and then further prolonged by GYKI 52466 (100 microM) to a double exponential function (tau(on1) 1.12 +/- 0.13 ms and tau(on2) 171.5 +/- 36.5 ms). GYKI 52466 did not re-introduce desensitization but concentration-dependently weakened cyclothiazide's prolongation of deactivation time constants (1 ms applications: 5.01 +/- 0.71, 4.47 +/- 0.80 and 2.28 +/- 0.64 ms with GYKI 52466 30, 100 and 300 microM respectively). NBQX reduced peak current responses with an IC50 of 28.2 +/- 1.3 nM. Paradoxically, steady-state currents with 500 ms applications of glutamate were potentiated from 3.3 +/- 1.2 pA to 29.4 +/- 6.4 pA by NBQX (1 nM). Higher concentrations of NBQX then antagonized this potentiated response. The potency of NBQX in antagonizing steady-state currents to 500 ms applications of glutamate (IC50 120.9 +/- 30.2 nM) was 2-fold less than following 100 ms applications (IC50 67.7 +/- 2.6 nM). NBQX had no effect on rapid onset, desensitization or deactivation time constants. However, a slow relaxation of inhibition was seen with longer applications. NBQX was 2-5-fold less potent against inward currents in the presence of cyclothiazide (100 microM) depending on the application duration but had no effect on the rapid onset, desensitization or deactivation time constants. The same relaxation of inhibition was seen as with NBQX alone. NBQX (1 microM) reduced AMPA receptor-mediated EPSC amplitude to 7 +/- 1% of control with no effect on kinetics. Cyclothiazide (330 microM) caused a 2.8-fold prolongation of the decay time constant (control 26.6 +/- 2.2 ms, cyclothiazide 74.2 +/- 7.6 ms, n = 9). Additional application of NBQX (1 microM) partly reversed this prolongation to 1.9 fold (47.7 +/- 2.5 ms, n = 5). These results support previous findings that cyclothiazide also allosterically influences AMPA receptor agonist/antagonist recognition sites. There were no interactions between NBQX and cyclothiazide on desensitization or deactivation time constants of glutamate-induced currents but clear interactions on EPSC deactivation kinetics. This raises the possibility that the interactions of NBQX, GYKI 52466 and cyclothiazide on AMPA-receptor-mediated EPSC kinetics observed are due to modulation of glutamate-release at presynaptic AMPA receptors.

Synaptic modulation of oscillatory activity of hypothalamic neuronal networks in vitro.

1. Rhythmic bursts of action potentials in neurosecretory cells are a key factor in hypothalamic neurosecretion. Rhythmicity and synchronization may be accomplished by pacemaker cells synaptically driving follower cells or by a network oscillator. 2. In this review we describe a hypothalamic cell culture which may serve as a model for a hypothalamic network oscillator. An overview is given of neurochemical phenotypes, synaptic mechanisms and their development, properties of receptors for fast synaptic transmission, and membrane properties of cells in dissociated rat embryonic hypothalamic culture. 3. Rhythmic activity spreads in the cultured network through synapses that release glutamate, activating a heteromultimeric AMPA-type receptor containing a GluR2 subunit which is associated with a high-conductance channel for Na+ and K+. Rhythmic activity is controlled by synapses that release GABA to activate GABAA receptors. The presumed function of the two receptor types is facilitated by their respective location, GABAA receptors predominating near the soma and AMPA receptors being abundant in dendrites. 4. Network oscillators may be more reliable for the presumed function than single-cell oscillators. They are controlled through synaptic modulation, which may prove to represent a process important for the release of hormones.

Fractional Ca2+ currents through capsaicin- and proton-activated ion channels in rat dorsal root ganglion neurones.

1. Capsaicin and protons cause excitation and sensitization of primary nociceptive afferents. In a subset of dorsal root ganglion (DRG) neurones, which probably represent nociceptive neurones, both capsaicin and protons induce slowly inactivating non-selective cation currents. Whole-cell as well as single channel currents activated by these two stimuli share many biophysical and physiological properties in these neurones. This has lead to the suggestion that protons and capsaicin might activate the same ion channels. 2. In this study we simultaneously measured fluorescence signals and whole-cell currents activated by capsaicin or protons in acutely isolated DRG neurones filled with a high concentration (1 mM) of the Ca2+ indicator dye fura-2. From these measurements the fractional contribution of Ca2+ (Pf; the portion of the whole-cell current carried by Ca2+) to capsaicin- and two types of proton-induced (fast and slowly inactivating) membrane currents was determined. 3. Capsaicin- and slowly inactivating proton-induced currents were accompanied by a change in fluorescence that was dependent on the presence of extracellular Ca2+. With 1.6 mM extracellular Ca2+ and at a holding potential of -80 mV Pf of capsaicin-induced currents (at pH 7.3) was 4.30 +/- 0.17% (mean +/- S.E.M.; no. of experiments, n = 16) and of slowly inactivating proton-induced currents (at pH 5.1) was 1.65 +/- 0.11% (n = 17). Pf of fast inactivating proton-induced currents was negligible. 4. Pf of capsaicin- and slowly inactivating proton-induced currents increased with increasing extracellular Ca2+ concentration (0.5-4.8 mM). 5. Pf of both current types decreased linearly with decreasing extracellular pH by about 0.7% per pH unit over the pH range investigated. When determined at the same extracellular pH Pf values were significantly different for the two current types at all pH values tested. 6. In summary, our results provide evidence that capsaicin and protons activate ion channels which are markedly permeable to Ca2+. The fractional contribution of Ca2+, however, was significantly different for capsaicin- and slowly inactivating proton-induced currents. This strongly suggests that the two stimuli activate different populations of ion channels and supports the possibility that Ca2+ influx through these channels may be important for Ca(2+)-dependent sensitization of primary nociceptive neurones.

Synaptic connectivity in cultured hypothalamic neuronal networks.

We have developed a novel approach to analyze the synaptic connectivity of spontaneously active networks of hypothalamic neurons in culture. Synaptic connections were identified by recording simultaneously from pairs of neurons using the whole cell configuration of the patch-clamp technique and testing for evoked postsynaptic current responses to electrical stimulation of one of the neurons. Excitatory and inhibitory responses were distinguished on the basis of their voltage and time dependence. The distribution of latencies between presynaptic stimulation and postsynaptic response showed multiple peaks at regular intervals, suggesting that responses via both monosynaptic and polysynaptic paths were recorded. The probability that an excitatory event is transmitted to another excitatory neuron and results in an above-threshold stimulation was found to be only one in three to four. This low value indicates that in addition to evoked synaptic responses other sources of excitatory drive must contribute to the spontaneous activity observed in these networks. The various types of synaptic connections (excitatory and inhibitory, monosynaptic, and polysynaptic) were counted, and the observations analyzed using a probabilistic model of the network structure. This analysis provides estimates for the ratio of inhibitory to excitatory neurons in the network (1:1.5) and for the ratio of postsynaptic cells receiving input from a single GABAergic or glutamatergic neuron (3:1). The total number of inhibitory synaptic connections was twice that of excitatory connections. Cell pairs mutually connected by an excitatory and an inhibitory synapse occurred significantly more often than predicted by a random process. These results suggests that the formation of neuronal networks in vitro is controlled by cellular mechanisms that favor inhibitory connections in general and specifically enhance the formation of reciprocal connections between pairs of excitatory and inhibitory neurons. These mechanisms may contribute to network formation and function in vivo.

Modulation of excitatory synaptic transmission by nociceptin in superficial dorsal horn neurones of the neonatal rat spinal cord.

1. The modulatory actions of nociceptin/orphanin FQ on excitatory synaptic transmission were studied in superficial dorsal horn neurones in transverse slices from 7 to 14 day old rats. 2. Glutamatergic excitatory postsynaptic currents (e.p.s.cs) were recorded from the somata of the neurones in the whole-cell patch-clamp configuration. E.p.s.cs were evoked by extracellular electrical stimulation (100 microns, 3-10 V) of the ipsilateral dorsal root entry zone by use of a glass electrode. E.p.s.cs with constant short latency (< 2.3 ms) and with no failures upon stimulation were assumed to be monosynaptic. These e.p.s.cs occurred with an average latency of 1.72 +/- 0.098 ms and exhibited a fast decay with a time constant, tau, of 4.8 +/- 0.53 ms (n = 30). 3. Nociceptin reversibly reduced the amplitudes of e.p.s.cs in a concentration-dependent manner in 25 out of 27 cells tested. Average maximum inhibition was 51.6 +/- 5.7% (mean +/- s.e.mean; n = 9), at concentrations > 3 microM. EC30 was 485 +/- 47 nM and the Hill coefficient was 1.29 +/- 0.09. 4. Inhibition of synaptic transmission by nociceptin (10 microM) was insensitive to the non-specific opioid receptor antagonist naloxone (10 microM) indicating that nociceptin did not act via classical opioid receptors. 5. In order to determine the site of action of nociceptin spontaneous miniature e.p.s.cs (m-e.p.s.cs) were recorded. Nociceptin reduced the frequency of m-e.p.s.cs in 6 out of 7 cells but had no effect on their amplitude distribution or on their time course. These findings suggest a pre- rather than a postsynaptic modulatory site of action. This is in line with the finding that current responses elicited by extracellular application of L-glutamate (10 microM) were not affected by nociceptin (10 microM; n = 7). 6. No positive correlation was found between the degree of inhibition by nociceptin (10 microM) and by the mixed delta- and mu-receptor agonist methionine-enkephalin (10 microM). This suggests that both neuropeptides acted on different but perhaps overlapping populations of synaptic connections. 7. Our results indicate that nociceptin inhibits excitatory synaptic transmission in the superficial layers of the rat dorsal horn by acting on presynaptic, presumably ORL1 receptors. This may be an important mechanism for spinal sensory information processing including nociception.

A simple computer model to simulate ECG based on ionic channels.

The presented computer model reconstructs the ECG with 500 myocardium cells based on seven ionic channels. The low hardware-requirement and the exact demonstration of physiological and pathophysiological coherence make this simulation model very useful for medical scientific education.

Ca2+ permeability of the sustained proton-induced cation current in adult rat dorsal root ganglion neurons.

1. Microfluorometric Ca2+ measurements using FURA-2 and whole cell patch-clamp recordings were performed to investigate the Ca2+ permeability of ion channels underlying the proton-induced sustained cation current in adult rat dorsal root ganglion neurons. 2. In a subpopulation of these neurons, extracellular application of acidic solutions (pH 5.1) elicited a sustained cation current and a concomitant reversible rise in the intracellular free Ca2+ concentration ([Ca2+]i), which depended on the presence of external Ca2+. Ruthenium red (10 microM) reduced both the current and the rise in [Ca2+]i to about the same extent. 3. In the presence of 2 mM external Ca2+, sustained proton-induced currents reversed sign at -4.6 +/- 1.2 (SE) mV, with external Na+ and internal Cs+ as the major charge carriers. Increasing the external Ca2+ concentration to 30 mM shifted the reversal potential (Erev) by 3.0 +/- 0.9 mV toward more positive values, suggesting a permeability ratio of Ca2+/Cs+ of 0.41. A similar value (0.35) could be obtained from Erev (-21 mV) under bi-ionic conditions with 100 mM external Ca2+ and 154 mM internal Cs+. 4. These results demonstrate that the proton-activated cation channels investigated here are moderately permeable to Ca2+. This may lead to pathophysiologically relevant increases in [Ca2+]i on prolonged exposure of the cells to an acidic environment in inflamed or ischemic tissue.

Calcium channel types contributing to excitatory and inhibitory synaptic transmission between individual hypothalamic neurons.

The contribution of L-, N-, P- and Q-type Ca2+ channels to excitatory and inhibitory synaptic transmission and to whole-cell Ba2+ currents through Ca2+ channels (Ba2+ currents) was investigated in rat hypothalamic neurons grown in dissociated cell culture. Excitatory and inhibitory postsynaptic currents (EPSCs and IPSCs) were evoked by stimulating individual neurons under whole-cell patch-clamp conditions. The different types of high-voltage-activated (HVA) Ca2+ channels were identified using nifedipine, omega-Conus geographus toxin VIA (omega-CTx GVIA), omega-Agelenopsis aperta toxin IVA (omega-Aga IVA), and omega-Conus magus toxin VIIC (omega-CTx MVIIC). N-, but not P- or Q-type Ca2+ channels contributed to excitatory as well as inhibitory synaptic transmission together with Ca2+ channels resistant to the aforementioned Ca2+ channel blockers (resistant Ca2+ channels). Reduction of postsynaptic current (PSC) amplitudes by N-type Ca2+ channel blockers was significantly stronger for IPSCs than for EPSCs. In most neurons whole-cell Ba2+ currents were carried by L-type Ca2+ channels and by at least two other Ca2+ channel types, one of which is probably of the Q-type and the others are resistant Ca2+ channels. These results indicate a different contribution of the various Ca2+ channel types to excitatory and inhibitory synaptic transmission and to whole-cell currents in these neurons and suggest different functional roles for the distinct Ca2+ channel types.

Interactions of 2,3-benzodiazepines and cyclothiazide at AMPA receptors: patch clamp recordings in cultured neurones and area CA1 in hippocampal slices.

1. The 2,3-benzodiazepines GYKI 52466, GYKI 53405 and GYKI 53655 antagonized AMPA-induced currents in cultured superior colliculus neurones in a non use-dependent manner (steady state IC50s: GYKI 52466 9.8 +/- 0.6 microM; GYKI 53405 3.1 +/- 0.6 microM; GYKI 53655 0.8 +/- 0.1 microM). 2. Higher concentrations of all three antagonists slowed the onset kinetics and quickened the offset kinetics of AMPA-induced currents indicative of an allosteric interaction with the AMPA recognition site. 3. Cyclothiazide (3-300 microM) dramatically slowed desensitization of AMPA-induced currents and potentiated steady state currents (EC50 10.0 +/- 2.5 microM) to a much greater degree than peak currents. Both tau on and tau off were also increased by cyclothiazide in a concentration-dependent manner (EC50: tau on 42.1 +/- 4.5 microM; tau off 31.6 +/- 6.6 microM). 4. Cyclothiazide (10-100 microM) shifted the concentration-response curves of the 2,3-benzodiazepines to the right. For example, with 10 microM cyclothiazide the IC50s of GYKI 52466 and GYKI 53405 on steady-state AMPA-induced currents were 57.9 +/- 9.5 and 41.6 +/- 1.5 microM, respectively. 5. GYKI 53405 and GYKI 52466 concentration-dependently reversed the effects of cyclothiazide (100 microM) on offset kinetics (GYKI 53405 IC50 16.6 +/- 4.2 microM). However, the 2,3-benzodiazepines were unable to reintroduce desensitization in the presence of cyclothiazide and even concentration-dependently slowed the onset kinetics of AMPA responses further (GYKI 53405 EC50 8.0 +/- 2.8 microM). 6. GYKI 52466 decreased the peak amplitude of hippocampal area CA1 AMPA receptor-mediated excitatory postsynaptic currents (e.p.s.cs) (IC50 10.8 +/- 0.8 microM) with no apparent effect on response kinetics. Cyclothiazide prolonged the decay time constant of AMPA receptor-mediated e.p.s.cs (EC50 35.7 +/- 6.5 microM) with less pronounced effects in slowing e.p.s.c. onset kinetics and increasing e.p.s.c. amplitude. 7. Cyclothiazide (330 microM) shifted the concentration-response curve for the effects of GYKI 52466 on AMPA receptor-mediated e.p.s.c. peak amplitude to the right (GYKI 52466 IC50 26.9 +/- 9.4 microM). Likewise, GYKI 52466 (30-100 microM)) shifted the concentration-response curve for the effects of cyclothiazide on AMPA receptor-mediated e.p.s.c. decay time constants to the right. 8. In conclusion, cyclothiazide and the 2,3-benzodiazepines seem to bind to different sites on AMPA receptors but exert strong allosteric interactions with one another and with other domains such as the agonist recognition site. The interactions of GYKI 52466 and cyclothiazide on AMPA receptor-mediated e.p.s.cs in area CA1 of hippocampal slices provide evidence that the decay time constant of these synaptic events are not governed by desensitization.