Microneurographic identification of spontaneous activity in C-nociceptors in neuropathic pain states in humans and rats.

C-nociceptors do not normally fire action potentials unless challenged by adequate noxious stimuli. However, in pathological states nociceptors may become hyperexcitable and may generate spontaneous ectopic discharges. The aim of this study was to compare rat neuropathic pain models and to assess their suitability to model the spontaneous C-nociceptor activity found in neuropathic pain patients. Studies were performed in normal rats (n=40), healthy human subjects (n=15), peripheral neuropathic pain patients (n=20), and in five rat neuropathic pain models: nerve crush (n=24), suture (n=14), chronic constriction injury (n=12), STZ-induced diabetic neuropathy (n=56), and ddC-induced neuropathy (n=15). Microneurographic recordings were combined with electrical stimulation to monitor activity in multiple C fibers. Stimulation at 0.25 Hz allowed spontaneous impulses to be identified by fluctuations in baseline latency. Abnormal latency fluctuations could be produced by several mechanisms, and spontaneous activity was most reliably identified by the presence of unexplained latency increases corresponding to two or more additional action potentials. Spontaneous activity was present in a proportion of mechano-insensitive C-nociceptors in the patients and all rat models. The three focal traumatic nerve injury models provided the highest proportion (59.5%), whereas the two polyneuropathy models had fewer (18.6%), and the patients had an intermediate proportion (33.3%). Spontaneously active mechano-sensitive C-nociceptors were not recorded. Microneurographic recordings of spontaneous activity in diseased C-nociceptors may be useful for both short- and long-term drug studies, both in animals and in humans.

Double and triple spikes in C-nociceptors in neuropathic pain states: an additional peripheral mechanism of hyperalgesia.

It was previously reported that in 5 patients with small-fiber neuropathy, neuropathic pain, and hyperalgesia, application of a single, brief electrical stimulus to the skin could give rise to 2 afferent impulses in a C-nociceptor fiber. These double spikes, which are attributed to unidirectional conduction failure at branch points in the terminal arborisation, provide a possible mechanism for hyperalgesia. We here report that similar multiple spikes are regularly observed in 3 rat models of neuropathic pain: nerve crush, nerve suture, and chronic constriction injury. The proportion of nociceptor fibers exhibiting multiple spikes was similar (10.1-18.5%) in the 3 models, and significantly greater than the proportion in control (unoperated) animals (1.2%). As in the human patients, multiple spikes in the rat models were often provoked by increasing the stimulation rate from 0.25 to 2Hz, but provocation by warming was less consistent. Multiple spiking was also directly dependent on stimulus intensity, consistent with a mechanism that depends on activation of multiple branches. Whereas only double spikes had previously been described in patients, in these more extensive recordings from rats we found that triple spikes could also be observed after a single electrical stimulus. The results strengthen the suggestion that multiple spiking, because of impaired conduction in the terminal branches of nociceptors, may contribute to hyperalgesia in patients with neuropathic pain. Double and triple spikes in c-nociceptors, caused by impaired conduction in terminal branches, may be an important cause of hyperalgesia in patients with neuropathic pain.

Purinergic and nitrergic junction potential in the human colon.

The aim of the present work is to investigate a putative junction transmission [nitric oxide (NO) and ATP] in the human colon and to characterize the electrophysiological and mechanical responses that might explain different functions from both neurotransmitters. Muscle bath and microelectrode techniques were performed on human colonic circular muscle strips. The NO donor sodium nitroprusside (10 microM), but not the P2Y receptor agonist adenosine 5'-O-2-thiodiphosphate (10 microM), was able to cause a sustained relaxation. NG-nitro-L-arginine (L-NNA) (1 mM), a NO synthase inhibitor, but not 2'-deoxy-N6-methyl adenosine 3',5'-diphosphate tetraammonium salt (MRS 2179) (10 microM), a P2Y antagonist, increased spontaneous motility. Electrical field stimulation (EFS) at 1 Hz caused fast inhibitory junction potentials (fIJPs) and a relaxation sensitive to MRS 2179 (10 microM). EFS at higher frequencies (5 Hz) showed biphasic IJP with fast hyperpolarization sensitive to MRS 2179 followed by sustained hyperpolarization sensitive to L-NNA; both drugs were needed to fully block the EFS relaxation at 2 and 5 Hz. Two consecutive single pulses induced MRS 2179-sensitive fIJPs that showed a rundown. The rundown mechanism was not dependent on the degree of hyperpolarization and was present after incubation with L-NNA (1 mM), hexamethonium (100 microM), MRS 2179 (1 microM), and NF023 (10 microM). We concluded that single pulses elicit ATP release from enteric motor neurons that cause a fIJP and a transient relaxation that is difficult to maintain over time; also, NO is released at higher frequencies causing a sustained hyperpolarization and relaxation. These differences might be responsible for complementary mechanisms of relaxation being phasic (ATP) and tonic (NO).

Endogenous hemichannels play a role in the release of ATP from Xenopus oocytes.

ATP is an electrically charged molecule that functions both in the supply of energy necessary for cellular activity and as an intercellular signaling molecule. Although controlled ATP secretion occurs via exocytosis of granules and vesicles, in some cells, and under certain conditions, other mechanisms control ATP release. Gap junctions, intercellular channels formed by connexins that link the cytoplasm of two adjacent cells, control the passage of ions and molecules up to 1 kDa. The channel is formed by two moieties called hemichannels, or connexons, and it has been suggested that these may represent an alternative pathway for ATP release. We have investigated the release of ATP through hemichannels from Xenopus oocytes that are formed by Connexin 38 (Cx38), an endogenous, specific type of connexin. These hemichannels generate an inward current that is reversibly activated by calcium-free solution and inhibited by octanol and flufenamic acid. This calcium-sensitive current depends on Cx38 expression: it is decreased in oocytes injected with an antisense oligonucleotide against Cx38 mRNA (ASCx38) and is increased in oocytes overexpressing Cx38. Moreover, the activation of these endogenous connexons also allows transfer of Lucifer Yellow. We have found that the release of ATP is coincident with the opening of hemichannels: it is calcium-sensitive, is inhibited by octanol and flufenamic acid, is inhibited in ASCx38 injected oocytes, and is increased by overexpression of Cx38. Taken together, our results suggest that ATP is released through activated hemichannels in Xenopus oocytes.

Cholinergic currents in Xenopus oocytes transplanted with human brain membranes

Corrientes colinérgicas de cerebro humano fueron registradas en oocitos de Xenopus laevis trasplantados con membranas de cerebro humano procedentes de dos zonas diferentes, la corteza frontal y el hipocampo. Las corrientes registradas fueron activadas por el receptor nicotínico o por el receptor nicotínico o muscarínico de la acetilcolina. Se probaron los efectos de diferentes agonistas nicotínicos como acetilcolina, nicotina y yoduro de 1,1-dimetil-4-fenil-piperazinio (DMPP), y antagonistas del receptor nicotínico como a-bungarotoxina y d-tubocurarina en los oocitos transplantados. Detectamos cuatro clases de cinéticas de corrientes nicotínicas. Las diferencias en la amplitud y en la carga eléctrica total de las corrientes provocadas por varios agonistas en el rango de potencial mantenido no fueron significativas, excepto en el caso del DMPP a un potencial mantenido de -90 mV. Nuestros resultados indican que las formas alfa4beta2, alfa3beta4 y alfa7 son los principales receptores nicotínicos en el cerebro humano

Cholinergic human brain currents were recorded in Xenopus laevis oocytes transplanted with human cerebral membranes from two different zones, the frontal cortex and the hippocampus. The recorded currents were supported by the nicotinic or the muscarinic acetylcholine receptor. We tested the effects of a number of several nicotinic agonists acetylcholine, nicotine and 1,1-dimethyl-4-phenylpiperazinium iodide (DMPP), and the nicotinic receptor antagonists a-bungarotoxin and d-tubocurarine on the transplanted oocytes. We detected four kinds of nicotinic current kinetics. The differences in the amplitude and in the total electric charge of the currents elicited by various agonists at a range of holding potentials were not significant, except in the case of DMPP at a holding potential of -90 mV. Our results indicate that alpha4beta2, alpha3beta4 and alpha7 are the main nicotinic receptors in human brain

Effect of galantamine on the human alpha7 neuronal nicotinic acetylcholine receptor, the Torpedo nicotinic acetylcholine receptor and spontaneous cholinergic synaptic activity.

1. Various types of anticholinesterasic agents have been used to improve the daily activities of Alzheimer's disease patients. It was recently demonstrated that Galantamine, described as a molecule with anticholinesterasic properties, is also an allosteric enhancer of human alpha4beta2 neuronal nicotinic receptor activity. We explored its effect on the human alpha7 neuronal nicotinic acetylcholine receptor (nAChR) expressed in Xenopus oocytes. 2. Galantamine, at a concentration of 0.1 microM, increased the amplitude of acetylcholine (ACh)-induced ion currents in the human alpha7 nAChR expressed in Xenopus oocytes, but caused inhibition at higher concentrations. The maximum effect of galantamine, an increase of 22% in the amplitude of ACh-induced currents, was observed at a concentration of 250 microM Ach. 3. The same enhancing effect was obtained in oocytes transplanted with Torpedo nicotinic acetylcholine receptor (AChR) isolated from the electric organ, but in this case the optimal concentration of galantamine was 1 microM. In this case, the maximum effect of galantamine, an increase of 35% in the amplitude of ACh-induced currents, occurred at a concentration of 50 microM ACh. 4. Galantamine affects not only the activity of post-synaptic receptors but also the activity of nerve terminals. At a concentration of 1 microM, quantal spontaneous events, recorded in a cholinergic synapse, increased their amplitude, an effect which was independent of the anticholinesterasic activity associated with this compound. The anticholinesterasic effect was recorded in preparations treated with a galantamine concentration of 10 microM. 5. In conclusion, our results show that galantamine enhances human alpha7 neuronal nicotinic ACh receptor activity. It also enhances muscular AChRs and the size of spontaneous cholinergic synaptic events. However, only a very narrow range of galantamine concentrations can be used for enhancing effects.

Gadolinium inhibition of ecto-nucleoside triphosphate diphosphohydrolase activity in Torpedo electric organ.

Ecto-nucleoside triphosphate diphosphohydrolases (E-NTPDases) are widely expressed enzymes implicated in the modulation of nucleotide cell signaling. They dephosphorylate either ATP or ADP in the presence of divalent cations, and efforts have been made to identify efficient inhibitors. E-NTPDase activity has been described in Torpedo electric organ electrocytes. We show here that gadolinium, an established blocker of stretch-activated channels, efficiently inhibits E-NTPDase activity of Torpedo electric organ (Ki = 3 microM for ATPase) as well as apyrase from potato tuber, frequently used in inhibition experiments. To our knowledge, gadolinium is the most potent inhibitor described to date for both membrane-bound and soluble E-NTPDases.

Kainate-triggered currents in Xenopus oocytes injected with chick retinal membrane fragments: effect of guanine nucleotides.

PURPOSE: To electrophysiologically characterize alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA)/kainate receptors in chick retinal membrane fragments, incorporated into Xenopus oocytes by direct microinjection. METHODS: A 6-day retinal membrane suspension was injected into Xenopus oocytes by use of an electronic nanoliter injector. Fifteen to 40 hours after injection, the oocytes were assayed for kainate-elicited inward currents, under voltage-clamp conditions (membrane potential held at -70 mV). The structural incorporation of the retinal membrane fragments into the oocyte membrane was verified by specific immunofluorescent staining. RESULTS: Chick retinal membrane fragments were efficiently grafted onto Xenopus oocytes after microinjection, with 22.9% +/- 7.6% of the oocyte membrane being stained with anti-chick retina antibody. Part of the retinal material was seen as patches of relatively uniform size (292.1 +/- 72.3 microm(2)). Bath-applied kainate induced dose-dependent (EC(50): 64 +/- 7 microM), nondesensitizing inward currents (15-90 nA) in the chimeric Xenopus oocytes. Sham-injected oocytes did not respond to kainate. Kainate-driven currents were blocked by 6,7-dinitroquinoxaline-2,3-dione (DNQX) and 1-(4-aminopropyl)-4-methyl-7,8-methylenedioxy-5H-2,3-benzodiazepine hydrochloride (GYKI 52466), but not by gamma-D-glutamylaminomethyl sulfonic acid (GAMS) or aminophosphonoheptanoate (AP7), suggesting the involvement of AMPA receptors in the observed responses. Guanine nucleotides (GNs) also blocked kainate currents in a concentration-dependent manner. CONCLUSIONS: An alternative oocyte microinjection technique to analyze the electrophysiological properties of glutamate receptors in chick retinal membranes is described. The results show the functional activity of putative AMPA-preferring receptors from chick retina and confirm, in the chick retinal model, the antagonistic behavior of guanine nucleotides toward glutamate receptors and their potential role as neuroprotective agents under excitotoxic conditions.

Control of neurotransmitter release by an internal gel matrix in synaptic vesicles.

Neurotransmitters are stored in synaptic vesicles, where they have been assumed to be in free solution. Here we report that in Torpedo synaptic vesicles, only 5% of the total acetylcholine (ACh) or ATP content is free, and that the rest is adsorbed to an intravesicular proteoglycan matrix. This matrix, which controls ACh and ATP release by an ion-exchange mechanism, behaves like a smart gel. That is, it releases neurotransmitter and changes its volume when challenged with small ionic concentration change. Immunodetection analysis revealed that the synaptic vesicle proteoglycan SV2 is the core of the intravesicular matrix and is responsible for immobilization and release of ACh and ATP. We suggest that in the early steps of vesicle fusion, this internal matrix regulates the availability of free diffusible ACh and ATP, and thus serves to modulate the quantity of transmitter released.

Release of ATP induced by hypertonic solutions in Xenopus oocytes.

ATP mediates intercellular communication. Mechanical stress and changes in cell volume induce ATP release from various cell types, both secretory and non-secretory. In the present study, we stressed Xenopus oocytes with a hypertonic solution enriched in mannitol (300 mM). We measured simultaneously ATP release and ionic currents from a single oocyte. A decrease in cell volume, the activation of an inward current and ATP release were coincident. We found two components of ATP release: the first was associated with granule or vesicle exocytosis, because it was inhibited by tetanus neurotoxin, and the second was related to the inward current. A single exponential described the correlation between ATP release and the hypertonic-activated current. Gadolinium ions, which block mechanically activated ionic channels, inhibited the ATP release and the inward current but did not affect the decrease in volume. Oocytes expressing CFTR (cystic fibrosis transmembrane regulator) released ATP under hypertonic shock, but ATP release was significantly inhibited in the first component: that related to granule exocytosis. Since the ATP measured is the balance between ATP release and ATP degradation by ecto-enzymes, we measured the nucleoside triphosphate diphosphohydrolase (NTPDase) activity of the oocyte surface during osmotic stress, as the calcium-dependent hydrolysis of ATP, which was inhibited by more than 50 % in hypertonic conditions. The best-characterized membrane protein showing NTPDase activity is CD39. Oocytes injected with an antisense oligonucleotide complementary to CD39 mRNA released less ATP and showed a lower amplitude in the inward current than those oocytes injected with water.

Calcium-dependent acetylcholine release from Xenopus oocytes: simultaneous ionic currents and acetylcholine release recordings.

The fusion of synaptic vesicles with presynaptic membranes is controlled by a complex network of protein-protein and protein-lipid interactions. SNAP-25, syntaxin and synaptobrevin (SNARE complex) are thought to participate in the formation of the core of the membrane fusion machine but the molecular basis of SNARE interactions is not completely understood. Thus, it would be interesting to design experiments to test those relationships in a new model. Xenopus laevis oocytes are valuable tools for studying the molecular structure and function of ionic channels and neurotransmitter receptors. Here we show that SNARE proteins are present in native Xenopus oocytes and that those oocytes injected with acetylcholine and presynaptic plasma membranes extracted from the electric organ of Torpedo marmorata assume some of the functions of a cholinergic nerve terminal. Neurotransmitter release and macroscopic currents were recorded and analysed simultaneously in a single oocyte electrically depolarized: acetylcholine release was detected using a chemiluminiscent method and calcium entry was measured by exploiting the endogenous Ca2+-activated chloride current of the oocyte with a two-electrode voltage-clamp system. Neurotransmitter release was calcium- and voltage-dependent and partially reduced in the presence of several calcium channel blockers. Clostridial neurotoxins, both holotoxin and injected light-chain forms, also inhibited acetylcholine release. We also studied the role of the SNARE complex in synaptic transmission and membrane currents by using monoclonal antibodies against SNAP-25, syntaxin or VAMP/synaptobrevin. The use of antibodies against VAMP/synaptobrevin, SNAP-25 and syntaxin inhibited acetylcholine release, as did clostridial toxins. However, macroscopic currents were only modified either by syntaxin antibody or by Botulinium-C1 neurotoxin. This model constitutes a new approach for understanding the vesicle exocytosis processes.