Potentiation of capsaicin receptor activity by metabotropic ATP receptors as a possible mechanism for ATP-evoked pain and hyperalgesia

The capsaicin (vanilloid) receptor, VR1, is a sensory neuron-specific ion channel that serves as a polymodal detector of pain-producing chemical and physical stimuli. It has been proposed that ATP, released from different cell types, initiates the sensation of pain by acting predominantly on nociceptive ionotropic purinoceptors located on sensory nerve terminals. In this study, we examined the effects of extracellular ATP on VR1. In cells expressing VR1, ATP increased the currents evoked by capsaicin or protons through activation of metabotropic P2Y1 receptors in a protein kinase C-dependent pathway. The involvement of Gq/11-coupled metabotropic receptors in the potentiation of VR1 response was confirmed in cells expressing both VR1 and M1 muscarinic acetylcholine receptors. In the presence of ATP, the temperature threshold for VR1 activation was reduced from 42°C to 35°C, such that normally nonpainful thermal stimuli (i.e., normal body temperature) were capable of activating VR1. This represents a novel mechanism through which the large amounts of ATP released from damaged cells in response to tissue trauma might trigger the sensation of pain.

Role of Rab3 GDP/GTP Exchange Protein in Synaptic Vesicle Trafficking at the Mouse Neuromuscular Junction

The Rab3 small G protein family consists of four members, Rab3A, -3B, -3C, and -3D. Of these members, Rab3A regulates Ca2+-dependent neurotransmitter release. These small G proteins are activated by Rab3 GDP/GTP exchange protein (Rab3 GEP). To determine the function of Rab3 GEP during neurotransmitter release, we have knocked out Rab3 GEP in mice. Rab3 GEP−/− mice developed normally but died immediately after birth. Embryos at E18.5 showed no evoked action potentials of the diaphragm and gastrocnemius muscles in response to electrical stimulation of the phrenic and sciatic nerves, respectively. In contrast, axonal conduction of the spinal cord and the phrenic nerve was not impaired. Total numbers of synaptic vesicles, especially those docked at the presynaptic plasma membrane, were reduced at the neuromuscular junction ∼10-fold compared with controls, whereas postsynaptic structures and functions appeared normal. Thus, Rab3 GEP is essential for neurotransmitter release and probably for formation and trafficking of the synaptic vesicles.

Computation of Surface Electrical Potentials of Plant Cell Membranes : Correspondence to Published Zeta Potentials from Diverse Plant Sources

A Gouy-Chapman-Stern model has been developed for the computation of surface electrical potential (ψ0) of plant cell membranes in response to ionic solutes. The present model is a modification of an earlier version developed to compute the sorption of ions by wheat (Triticum aestivum L. cv Scout 66) root plasma membranes. A single set of model parameters generates values for ψ0 that correlate highly with published ζ potentials of protoplasts and plasma membrane vesicles from diverse plant sources. The model assumes ion binding to a negatively charged site (R− = 0.3074 μmol m−2) and to a neutral site (P0 = 2.4 μmol m−2) according to the reactions R− + IΖ ⇌ RIΖ−1 and P0 + IΖ ⇌ PIΖ, where IΖ represents an ion of charge Ζ. Binding constants for the negative site are 21,500 m−1 for H+, 20,000 m−1 for Al3+, 2,200 m−1 for La3+, 30 m−1 for Ca2+ and Mg2+, and 1 m−1 for Na+ and K+. Binding constants for the neutral site are 1/180 the value for binding to the negative site. Ion activities at the membrane surface, computed on the basis of ψ0, appear to determine many aspects of plant-mineral interactions, including mineral nutrition and the induction and alleviation of mineral toxicities, according to previous and ongoing studies. A computer program with instructions for the computation of ψ0, ion binding, ion concentrations, and ion activities at membrane surfaces may be requested from the authors.

Root Growth and Oxygen Relations at Low Water Potentials. Impact of Oxygen Availability in Polyethylene Glycol Solutions1

Polyethylene glycol (PEG), which is often used to impose low water potentials (ψw) in solution culture, decreases O2 movement by increasing solution viscosity. We investigated whether this property causes O2 deficiency that affects the elongation or metabolism of maize (Zea mays L.) primary roots. Seedlings grown in vigorously aerated PEG solutions at ambient solution O2 partial pressure (pO2) had decreased steady-state root elongation rates, increased root-tip alanine concentrations, and decreased root-tip proline concentrations relative to seedlings grown in PEG solutions of above-ambient pO2 (alanine and proline accumulation are responses to hypoxia and low ψw, respectively). Measurements of root pO2 were made using an O2 microsensor to ensure that increased solution pO2 did not increase root pO2 above physiological levels. In oxygenated PEG solutions that gave maximal root elongation rates, root pO2 was similar to or less than (depending on depth in the tissue) pO2 of roots growing in vermiculite at the same ψw. Even without PEG, high solution pO2 was necessary to raise root pO2 to the levels found in vermiculite-grown roots. Vermiculite was used for comparison because it has large air spaces that allow free movement of O2 to the root surface. The results show that supplemental oxygenation is required to avoid hypoxia in PEG solutions. Also, the data suggest that the O2 demand of the root elongation zone may be greater at low relative to high ψw, compounding the effect of PEG on O2 supply. Under O2-sufficient conditions root elongation was substantially less sensitive to the low ψw imposed by PEG than that imposed by dry vermiculite.

Cooperative interactions in the induction of long-term potentiation and depression of synaptic excitation between hippocampal CA3-CA1 cell pairs in vitro.

The requirement for cooperative interactions between multiple synaptic inputs in the induction of long-term potentiation (LTP) and long-term depression (LTD) has been tested at Schaffer collateral synapses with paired recordings from monosynaptically coupled CA3-CA1 cell pairs in rat hippocampal slice cultures. Tetanization of single presynaptic neurons at 50 Hz (repeated 5-7 times for 300-500 ms each) induced only a transient potentiation (< 3 min) of excitatory postsynaptic potentials (EPSPs). Persistent potentiation (> 15 min) was induced only when single presynaptic action potentials were synchronously paired with directly induced postsynaptic depolarizing pulses (repeated 50-100 times). Tetanus-induced potentiation of extracellularly evoked EPSPs lasting > 4 min could only be obtained if the EPSP was > 4 mV. Because unitary EPSP amplitudes average approximately 1 mV, we conclude that high-frequency discharge must occur synchronously] in 4-5 CA3 cells for LTP to be induced in a common postsynaptic CA1 cell. Asynchronous pairing of presynaptic action potentials with postsynaptic depolarizing current pulses (preceding each EPSP by 800 ms) depressed both naive and previously potentiated unitary EPSPs. Likewise, homosynaptic LTD of unitary EPSPs was induced when the presynaptic cell was tetanized at 3 Hz for 3 min, regardless of their amplitude (0.3-3.2 mV). Homosynaptic LTD of extracellularly evoked Schaffer collateral EPSPs < 4 mV could be induced if no inhibitory postsynaptic potential was apparent, but was prevented by eliciting a large inhibitory postsynaptic potential or by injection of hyperpolarizing current in the postsynaptic cell. We conclude that cooperative interactions among multiple excitatory inputs are not required for induction of homosynaptic LTD of unitary EPSPs.

NO hyperpolarizes pulmonary artery smooth muscle cells and decreases the intracellular Ca2+ concentration by activating voltage-gated K+ channels.

NO causes pulmonary vasodilation in patients with pulmonary hypertension. In pulmonary arterial smooth muscle cells, the activity of voltage-gated K+ (Kv) channels controls resting membrane potential. In turn, membrane potential is an important regulator of the intracellular free calcium concentration ([Ca2+]i) and pulmonary vascular tone. We used patch clamp methods to determine whether the NO-induced pulmonary vasodilation is mediated by activation of Kv channels. Quantitative fluorescence microscopy was employed to test the effect of NO on the depolarization-induced rise in [Ca2+]i. Blockade of Kv channels by 4-aminopyridine (5 mM) depolarized pulmonary artery myocytes to threshold for initiation of Ca2+ action potentials, and thereby increased [Ca2+]i. NO (approximately 3 microM) and the NO-generating compound sodium nitroprusside (5-10 microM) opened Kv channels in rat pulmonary artery smooth muscle cells. The enhanced K+ currents then hyperpolarized the cells, and blocked Ca(2+)-dependent action potentials, thereby preventing the evoked increases in [Ca2+]i. Nitroprusside also increased the probability of Kv channel opening in excised, outside-out membrane patches. This raises the possibility that NO may act either directly on the channel protein or on a closely associated molecule rather than via soluble guanylate cyclase. In isolated pulmonary arteries, 4-aminopyridine significantly inhibited NO-induced relaxation. We conclude that NO promotes the opening of Kv channels in pulmonary arterial smooth muscle cells. The resulting membrane hyperpolarization, which lowers [Ca2+]i, is apparently one of the mechanisms by which NO induces pulmonary vasodilation.

Opposite actions of nitric oxide on cholinergic synapses: which pathways?

Nitric oxide (NO) produced opposite effects on acetylcholine (ACh) release in identified neuroneuronal Aplysia synapses depending on the excitatory or the inhibitory nature of the synapse. Extracellular application of the NO donor, SIN-1, depressed the inhibitory postsynaptic currents (IPSCs) and enhanced the excitatory postsynaptic currents (EPSCs) evoked by presynaptic action potentials (1/60 Hz). Application of a membrane-permeant cGMP analog mimicked the effect of SIN-1 suggesting the participation of guanylate cyclase in the NO pathway. The guanylate cyclase inhibitor, methylene blue, blocked the NO-induced enhancement of EPSCs but only reduced the inhibition of IPSCs indicating that an additional mechanism participates to the depression of synaptic transmission by NO. Using nicotinamide, an inhibitor of ADP-ribosylation, we found that the NO-induced depression of ACh release on the inhibitory synapse also involves ADP-ribosylation mechanism(s). Furthermore, application of SIN-1 paired with cGMP-dependent protein kinase (cGMP-PK) inhibitors showed that cGMP-PK could play a role in the potentiating but not in the depressing effect of NO on ACh release. Increasing the frequency of stimulation of the presynaptic neuron from 1/60 Hz to 0.25 or 1 Hz potentiated the EPSCs and reduced the IPSCs. In these conditions, the potentiating effect of NO on the excitatory synapse was reduced, whereas its depressing effect on the inhibitory synapse was unaffected. Moreover the frequency-dependent enhancement of ACh release in the excitatory synapse was greatly reduced by the inhibition of NO synthase. Our results indicate that NO may be involved in different ways of modulation of synaptic transmission depending on the type of the synapse including synaptic plasticity.

Long-term modifications of synaptic efficacy in the human inferior and middle temporal cortex.

The primate temporal cortex has been demonstrated to play an important role in visual memory and pattern recognition. It is of particular interest to investigate whether activity-dependent modification of synaptic efficacy, a presumptive mechanism for learning and memory, is present in this cortical region. Here we address this issue by examining the induction of synaptic plasticity in surgically resected human inferior and middle temporal cortex. The results show that synaptic strength in the human temporal cortex could undergo bidirectional modifications, depending on the pattern of conditioning stimulation. High frequency stimulation (100 or 40 Hz) in layer IV induced long-term potentiation (LTP) of both intracellular excitatory postsynaptic potentials and evoked field potentials in layers II/III. The LTP induced by 100 Hz tetanus was blocked by 50-100 microM DL-2-amino-5-phosphonovaleric acid, suggesting that N-methyl-D-aspartate receptors were responsible for its induction. Long-term depression (LTD) was elicited by prolonged low frequency stimulation (1 Hz, 15 min). It was reduced, but not completely blocked, by DL-2-amino-5-phosphonovaleric acid, implying that some other mechanisms in addition to N-methyl-DL-aspartate receptors were involved in LTD induction. LTD was input-specific, i.e., low frequency stimulation of one pathway produced LTD of synaptic transmission in that pathway only. Finally, the LTP and LTD could reverse each other, suggesting that they can act cooperatively to modify the functional state of cortical network. These results suggest that LTP and LTD are possible mechanisms for the visual memory and pattern recognition functions performed in the human temporal cortex.

Inhibition of sustained gamma oscillations (35-80 Hz) by fast transient responses in cat visual cortex.

Interactions between stimulus-induced oscillations (35-80 Hz) and stimulus-locked nonoscillatory responses were investigated in the visual cortex areas 17 and 18 of anaesthetized cats. A single square-wave luminance grating was used as a visual stimulus during simultaneous recordings from up to seven electrodes. The stimulus movement consisted of a superposition of a smooth movement with a sequence of dynamically changing accelerations. Responses of local groups of neurons at each electrode were studied on the basis of multiple unit activity and local slow field potentials (13-120 Hz). Oscillatory and stimulus-locked components were extracted from multiple unit activity and local slow field potentials and quantified by a combination of temporal and spectral correlation methods. We found fast stimulus-locked components primarily evoked by sudden stimulus accelerations, whereas oscillatory components (35-80 Hz) were induced during slow smooth movements. Oscillations were gradually reduced in amplitude and finally fully suppressed with increasing amplitudes of fast stimulus-locked components. It is argued that suppression of oscillations is necessary to prevent confusion during sequential processing of stationary and fast changing retinal images.

Low ethanol concentrations enhance GABAergic inhibitory postsynaptic potentials in hippocampal pyramidal neurons only after block of GABAB receptors.

Despite considerable evidence that ethanol can enhance chloride flux through the gamma-aminobutyric acid type A (GABA/A/) receptor-channel complex in several central neuron types, the effect of ethanol on hippocampal GABAergic systems is still controversial. Therefore, we have reevaluated this interaction in hippocampal pyramidal neurons subjected to local monosynaptic activation combined with pharmacological isolation of the various components of excitatory and inhibitory synaptic potentials, using intracellular current- and voltage-clamp recording methods in the hippocampal slice. In accord with our previous findings, we found that ethanol had little effect on compound inhibitory postsynaptic potentials/currents (IPSP/Cs) containing both GABA/A/ and GABA/B/ components. However, after selective pharmacological blockade of the GABA/B/ component of the IPSP (GABA/B/-IPSP/C) by CGP-35348, low concentrations of ethanol (22-66 mM) markedly enhanced the peak amplitude, and especially the area, of the GABA/A/ component (GABA/A/-IPSP/C) in most CA1 pyramidal neurons. Ethanol had no significant effect on the peak amplitude or area of the pharmacologically isolated GABA/B/-inhibitory postsynaptic current (IPSC). These results provide new data showing that activation of GABAB receptors can obscure ethanol enhancement of GABA/A/ receptor function in hippocampus and suggest that similar methods of pharmacological isolation might be applied to other brain regions showing negative or mixed ethanol-GABA interactions.

Rapid local synchronization of action potentials: toward computation with coupled integrate-and-fire neurons.

The collective behavior of interconnected spiking nerve cells is investigated. It is shown that a variety of model systems exhibit the same short-time behavior and rapidly converge to (approximately) periodic firing patterns with locally synchronized action potentials. The dynamics of one model can be described by a downhill motion on an abstract energy landscape. Since an energy landscape makes it possible to understand and program computation done by an attractor network, the results will extend our understanding of collective computation from models based on a firing-rate description to biologically more realistic systems with integrate-and-fire neurons.

Neurosteroids, via sigma receptors, modulate the [3H]norepinephrine release evoked by N-methyl-D-aspartate in the rat hippocampus.

N-Methyl-D-aspartate (NMDA, 200 microM) evokes the release of [3H]norepinephrine ([3H]NE) from preloaded hippocampal slices. This effect is potentiated by dehydroepiandrosterone sulfate (DHEA S), whereas it is inhibited by pregnenolone sulfate (PREG S) and the high-affinity sigma inverse agonist 1,3-di(2-tolyl)guanidine, at concentrations of > or = 100 nM. Neither 3 alpha-hydroxy-5 alpha-pregnan-20-one nor its sulfate ester modified NMDA-evoked [3H]NE overflow. The sigma antagonists haloperidol and 1-[2-(3,4-dichlorophenyl)-ethyl]-4-methylpiperazine, although inactive by themselves, completely prevented the effects of DHEA S, PREG S, and 1,3-di(2-tolyl)guanidine on NMDA-evoked [3H]NE release. Progesterone (100 nM) mimicked the antagonistic effect of haloperidol and 1-[2-(3,4-dichlorophenyl)ethyl]-4-methyl-piperazine. These results indicate that the tested steroid sulfate esters differentially affected the NMDA response in vitro and suggest that DHEA S acts as a sigma agonist, that PREG S acts as a sigma inverse agonist, and that progesterone may act as a sigma antagonist. Pertussis toxin, which inactivates the Gi/o types of guanine nucleotide-binding protein (Gi/o protein) function, suppresses both effects of DHEA S and PREG S. Since sigma 1 but not sigma 2 receptors are coupled to Gi/o proteins, the present results suggest that DHEA S and PREG S control the NMDA response via sigma 1 receptors.

Rapid endocytosis of a G protein-coupled receptor: substance P evoked internalization of its receptor in the rat striatum in vivo.

Studies on cultured cells have shown that agonists induce several types of G protein-coupled receptors to undergo internalization. We have investigated this phenomenon in rat striatum, using substance P (SP)-induced internalization of the SP receptor (SPR) as our model system. Within 1 min of a unilateral striatal injection of SP in the anesthetized rat, nearly 60% of the SPR-immunoreactive neurons within the injection zone display massive internalization of the SPR--i.e., 20-200 SPR+ endosomes per cell body. Within the dendrites the SPR undergoes a striking translocation from the plasma membrane to endosomes, and these dendrites also undergo a morphological reorganization, changing from a structure of rather uniform diameter to one characterized by large, swollen varicosities connected by thin fibers. In both cell bodies and dendrites the number of SPR+ endosomes returns to baseline within 60 min of SP injection. The number of neurons displaying substantial endosomal SPR internalization is dependent on the concentration of injected SP, and the SP-induced SPR internalization is inhibited by the nonpeptide neurokinin 1 receptor antagonist RP-67,580. These data demonstrate that in the central nervous system in vivo, SP induces a rapid and widespread SPR internalization in the cell bodies and dendrites and a structural reorganization of the dendrites. These results suggest that many of the observations that have been made on the internalization and recycling of G protein-coupled receptors in in vitro transfected cell systems are applicable to similar events that occur in the mammalian central nervous system in vivo.

Mechanistic Importance of Redox Potentials and Conformational Flexibility in Electron-Transferring Flavoproteins

The mitochondrial matrix flavoproteins electron transfer flavoprotein (ETF) and electron transfer flavoprotein-ubiquinone oxidoreductase (ETF-QO) are responsible for linking fatty acid β-oxidation with the main mitochondrial respiratory chain. Electrons derived from flavoprotein dehydrogenases are transferred sequentially through ETF and ETF-QO to ubiquinone and then into the respiratory chain via complex III. In this study, the effects of changes in ETF-QO redox potentials on its activity and the conformational flexibility of ETF were investigated. ETF-QO contains one [4Fe-4S]2+,1+ and one flavin adenine dinucleotide (FAD). In the porcine protein, threonine 367 is hydrogen bonded to N1 and O2 of the flavin ring of the FAD. The analogous site in Rhodobacter sphaeroides ETF-QO is asparagine 338. Mutations N338T and N338A were introduced into the R. sphaeroides protein by site-directed mutagenesis to determine the impact of hydrogen bonding at this site on redox potentials and activity. FAD redox potentials were measured by potentiometric titration probed by electron paramagnetic resonance (EPR) spectroscopy. The N338T and N338A mutations lowered the midpoint potentials, which resulted in a decrease in the quinone reductase activity and negligible impact on disproportionation of ETF1e- catalyzed by ETF-QO. These observations indicate that the FAD is involved in electron transfer to ubiquinone, but not in electron transfer from ETF to ETF-QO. Therefore it is proposed that the iron-sulfur cluster is the immediate acceptor from ETF. It has been proposed that the αII domain of ETF is mobile, allowing promiscuous interactions with structurally different partners. Double electron-electron resonance (DEER) was used to measure the distance between spin labels at various sites and an enzymatically reduced FAD cofactor in Paracoccus denitrificans ETF. Two or three interspin distance distributions were observed for spin-labels in the αI (A43C) and βIII (A111C) domains, but only one is observed for a label in the βII (A210C) domain. This suggests that the αII domain adopts several stable conformations which may correspond to a closed/inactive conformation and an open/active conformation. An additional mutation, E162A, was introduced to increase the mobility of the αII domain. The E162A mutation doubled the activity compared to wild-type and caused the distance distributions to become wider. The DEER method has the potential to characterize conformational changes in ETF that occur when it interacts with various redox partners.

Detection of concealed information using event-related potentials

Existe un interés considerable en hallar métodos que nos ayuden a saber cuándo una persona miente y cuándo dice la verdad desde un punto de vista forense. Actualmente, una de las líneas de investigación se inclina hacia el uso de potenciales relacionados con eventos. Se pretende hacer una revisión de los artículos que estudian estos procedimientos mediante distintos métodos: propiedades, fiabilidad, validez y limitaciones. Los resultados indican tasas de acierto en la discriminación de culpables en un rango de 7 al 100 por ciento, y en la de inocentes de 31 a 100 por ciento. La gran variabilidad y la posibilidad de “falsear” las respuestas llevan a cuestionar la inexactitud utilizada en algunos círculos mediáticos respecto a las cualidades y finalidades de dicha prueba. Se concluye la necesidad de profundizar más la posibilidad de que esta prueba sea utilizada con fines forenses.