AbstractÐThis study was performed using intracellular and multiunit extracellular recording techn... more AbstractÐThis study was performed using intracellular and multiunit extracellular recording techniques in order to characterize the role of nitric oxide in the afferent synaptic transmission of the vestibular system of the axolotl (Ambystoma tigrinum). Bath application of nitric oxide synthase inhibitors N G -nitro-l-arginine (0.01 mM to 10 mM) and N-nitro-larginine methyl ester hydrochloride (0.1 mM to 1000 mM) elicited a dose-dependent decrease in the basal discharge of the semicircular canal afferent ®bers. N G -Nitro-l-arginine also diminished the response to mechanical stimuli. Moreover, N G -nitro-l-arginine (1 mM) produced a hyperpolarization associated with a decrease in the spike discharge and diminished the frequency of the excitatory postsynaptic potentials on afferent ®bers recorded intracellularly. Nitric oxide donors were also tested: (i) S-nitroso-N-acetyl-dl-penicillamine (0.1 mM to 100 mM) increased the basal discharge and the response to mechanical stimuli. At the maximum effective concentration (100 mM) this drug affected neither the amplitude nor the frequency of the excitatory postsynaptic potentials. However, it slightly depolarized the afferent neurons and decreased their input resistance. (ii) 3-Morpholino-sydnonimine hydrochloride did not signi®cantly affect the basal discharge or the mechanically evoked peak response of afferent neurons at any of the concentrations used (1 mM to 1000 mM). However, after 10 min of perfusion in the bath, 1 mM and 10 mM 3-morpholino-sydnonimine hydrochloride signi®cantly modi®ed the baseline of the mechanically evoked response, producing an increase in the mean spike discharge of the afferent ®bers.
Conus californicus belongs to a genus of marine gastropods with more than 700 extant species. C. ... more Conus californicus belongs to a genus of marine gastropods with more than 700 extant species. C. californicus has been shown to be distantly related to all Conus species, but showing unusual biological features. We report a novel peptide isolated from C. californicus with a significant inhibitory action over neuronal voltage-gated calcium channels. The new toxin is formed by 13-amino acid residues with two disulfide bonds, whose sequence (NCPAGCRSQGCCM) is strikingly different from regular ω-conotoxins. In the HPLC purification procedure, the venom fraction eluted in the first 10–15 min produced a significant decrease (54% ± 3%) of the Ca2+ current in Xenopus laevis oocytes transfected with purified rat-brain mRNA. A specific peptide obtained from the elution at 13 min decreased the Ca2+ current in the adult rat dorsal-root ganglion neurons in a primary culture by 34% ± 2%. The cysteine pattern of this peptide corresponds to the framework XVI described for the M-superfamily of conopeptides and is unprecedented among Conus peptides acting on Ca2+ channels.
This study was undertaken to determine the possible role of GABA as an afferent transmitter in th... more This study was undertaken to determine the possible role of GABA as an afferent transmitter in the vestibular system of the axolotl. We studied the effects of GABA, muscimol, bicuculline and picrotoxin on the spontaneous spike discharge of the afferent fibers of the sac&i lagena and anterior semicircular canal. It was found that GABA and muscimol produce a very weak excitatory effect which does not mimic either the temporal course or the amplitude of the response of vestibular afferents to physiological stimuli. The GABA antagonist bicuculline has no significant effect on these fibers, and picrotoxin partially blocks the spontaneous activity in 33% of the fibers studied. These results indicate that GABA is probably not an afferent transmitter in the vestibular system as has previously been proposed.
By combining mathematical methods with the morphological analysis of the semicircular canals of t... more By combining mathematical methods with the morphological analysis of the semicircular canals of the axolotl (Ambystoma tigrinum), a system of differential equations describing the mechanical coupling in the semicircular canals was obtained. The coefficients of this system have an explicit physiological meaning that allows for the introduction of morphological and dynamical parameters directly into the differential equations. The cupula of the semicircular canals was modeled both as a piston and as a membrane (diaphragm like), and the duct canals as toroids with two main regions: i) the semicircular canal duct and, ii) a larger diameter region corresponding to the ampulla and the utricle. The endolymph motion was described by the Navier-Stokes equations. The analysis of the model demonstrated that cupular behavior dynamics under periodic stimulation is equivalent in both the piston and the membrane cupular models, thus a general model in which the detailed cupular structure is not relevant was derived.
Molecular identity, ontogeny, and cAMP modulation of the hyperpolarization-activated current in v... more Molecular identity, ontogeny, and cAMP modulation of the hyperpolarization-activated current in vestibular ganglion neurons. ties, developmental regulation, and cAMP modulation of the hyperpolarization-activated current (I h ) were investigated by the whole cell patch-clamp technique in vestibular ganglion neurons of the rat at two postnatal stages (P7-10 and P25-28). In addition, by RT-PCR and immunohistochemistry the identity and distribution of hyperpolarization-activated and cyclic nucleotide-gated channel (HCN) isoforms that generate I h were investigated. I h current density was larger in P25-28 than P7-10 rats, increasing 410% for small cells (Ͻ30 pF) and 200% for larger cells (Ͼ30 pF). The half-maximum activation voltage (V 1/2 ) of I h was Ϫ102 mV in P7-10 rats and in P25-28 rats shifted 7 mV toward positive voltages. At both ages, intracellular cAMP increased I h current density, decreased its activation time constant (), and resulted in a rightward shift of V 1/2 by 9 mV. Perfusion of 8-BrcAMP increased I h amplitude and speed up its activation kinetics. I h was blocked by Cs ϩ , zatebradine, and ZD7288. As expected, these drugs also reduced the voltage sag caused with hyperpolarizing pulses and prevented the postpulse action potential generation without changes in the resting potential. RT-PCR analysis showed that HCN1 and HCN2 subunits were predominantly amplified in vestibular ganglia and end organs and HCN3 and HCN4 to a lesser extent. Immunohistochemistry showed that the four HCN subunits were differentially expressed (HCN1 Ͼ HCN2 Ͼ HCN3 Ն HCN4) in ganglion slices and in cultured neurons at both P7-10 and P25-28 stages. Developmental changes shifted V 1/2 of I h closer to the resting membrane potential, increasing its functional role. Modulation of I h by cAMP-mediated signaling pathway constitutes a potentially relevant control mechanism for the modulation of afferent neuron discharge.
In the isolated inner ear of the axolotl (Ambystoma tigrinum) acid pH decreased and basic pH incr... more In the isolated inner ear of the axolotl (Ambystoma tigrinum) acid pH decreased and basic pH increased the resting and mechanically evoked spike discharge of semicircular canal afferent neurons. Variations in pH also modified the afferent neuron response to N-methyl-D-aspartic acid and to (±)-α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid. Responses to both excitatory amino acid agonists increased at pH 7.8 (41% and 22%, respectively) and decreased by perfusion of the preparation with a saline solution, of pH 7.0 (28% in both cases). These results indicate that vestibular endorgans have a significant sensitivity to pH that could play a significant role in various pathological states, and may also contribute to the postransductional processing of sensory information.
Several studies have shown that type I hair cells present a large outward rectifying potassium cu... more Several studies have shown that type I hair cells present a large outward rectifying potassium current (g K;L ) that is substantially activated at the resting potential, greatly reducing cell input resistance and voltage gain. In fact, mechanoelectrical transducer currents seem not to be large enough to depolarize type I hair cells to produce neurotransmitter release. Also, the strongly nonlinear transducer currents and the limited voltage oscillations found in some hair cells did not account for the bidirectionality of response in hair cell systems. We developed a model based in the analysis of nonlinear Goldman^Hodgkin^Katz equations to calculate the hair cell receptor potential and ionic movements produced by transducer current activation. Type I hair cells displaying the large g K;L current were found to produce small receptor potentials (3^13.8 mV) in response to mechanoelectrical transducer current input. In contrast, type II cells that lack g K;L produced receptor potentials of about 30 mV. Properties of basolateral ionic conductances in type II hair cells will linearize hair bundle displacement to receptor potential relationship. The voltage to obtain the half maximal activation of g K;L significantly affects the resting membrane potential, the amplitude, and the linearity of the receptor potential. Electrodiffusion equations were also used to analyze ionic changes in the intercellular space between type I hair cell and calyx endings. Significant K þ accumulation could take place at the intercellular space depending on calyx structure. ß
International Journal of Bio-Medical Computing, 1990
A computer simulation of the extracellular field potential recording of nerve activity is present... more A computer simulation of the extracellular field potential recording of nerve activity is presented. An experimental setup composed of an oscilloscope, a nerve, a conductive surface, and a recording electrode is graphically simulated. The user may study the influence of the position of the recording electrode and of certain nerve properties (membrane potential, velocity of conduction and action potential duration), on the action potential shape. The different waves which constitute the action potential may be analyzed and their peak values plotted as a function of the independent variable (e.g. electrode distance from the nerve). Three-dimensional plots of a set of action potentials as a function of the independent variable may also be obtained. The simulation allows the user to study the basic factors which determine the configuration of an extracelluhuly recorded compound nerve action potential.
This work reviews the neuropharmacology of the vestibular system, with an emphasis on the mechani... more This work reviews the neuropharmacology of the vestibular system, with an emphasis on the mechanism of action of drugs used in the treatment of vestibular disorders. Otolaryngologists are confronted with a rapidly changing field in which advances in the knowledge of ionic channel function and synaptic transmission mechanisms have led to the development of new scientific models for the understanding of vestibular dysfunction and its management. In particular, there have been recent advances in our knowledge of the fundamental mechanisms of vestibular system function and drug mechanisms of action. In this work, drugs acting on vestibular system have been grouped into two main categories according to their primary mechanisms of action: those with effects on neurotransmitters and neuromodulator receptors and those that act on voltage-gated ion channels. Particular attention is given in this review to drugs that may provide additional insight into the pathophysiology of vestibular diseases. A critical review of the pharmacology and highlights of the major advances are discussed in each case. that probably accounts for the presence of catecholamines in the inner ear [47].
Opioid receptors are expressed in the vestibular endorgans (afferent neurons and hair cells) and ... more Opioid receptors are expressed in the vestibular endorgans (afferent neurons and hair cells) and are activated by the efferent system, which modulates the discharge of action potentials in vestibular afferent neurons (VANs). In mammals, VANs mainly express the μ opioid-receptor, but the function of this receptors activation and the cellular mechanisms by which they exert their actions in these neurons are poorly studied. To determine the actions of μ opioid receptor (MOR) and cell signaling mechanisms in VANs, we made perforated patch-clamp recordings of VANs that were obtained from postnatal days 7 to 10 (P7-10) rats and then maintained in primary culture. The MOR agonist [D-Ala 2 , N-Me-Phe 4 , Gly 5 -ol]-enkephalin (DAMGO) inhibited the total voltage-gated outward current; this effect was prevented by the perfusion of a Ca 2+ -free extracellular solution. We then studied the voltage-gated calcium current (I ca ) and found that DAMGO Met-enkephalin or endomorphin-1 inhibited the I Ca in a dose-response fashion. The effects of DAMGO were prevented by the MOR antagonist (CTAP) or by pertussis toxin (PTX). The use of specific calcium channel blockers showed that MOR activation inhibited T-, L-and N-type I Ca . The use of various enzyme activators and inhibitors and of cAMP analogs allowed us to demonstrate that the MOR acts through a cAMP dependent signaling mechanism. In current clamp experiments, MOR activation increased the duration and decreased the amplitude of the action potentials and modulated the discharge produced by current injection. Pre-incubation with PTX occluded MOR activation effect. We conclude that MOR activation inhibits the T-, L-and N-type I Ca through activation of a Gα i/o protein that involves a decrease in AC-cAMP-PKA activity. The modulation of I Ca may have an impact on the synaptic integration, excitability, and neurotransmitter release from VANs.
Aminoglycosides (AGs) modulate nociceptors and ionic channels expressed in sensory neurons. The A... more Aminoglycosides (AGs) modulate nociceptors and ionic channels expressed in sensory neurons. The AG applied in situ could be useful to alleviate hyperalgesia in animal models of inflammatory pain. We tested streptomycin (ST) and neomycin (NEO) as analgesic agents applied in situ in rat paw inflammation caused by formalin or carrageenan administration. The action of ST and NEO on the action potential discharge produced by acidic stimuli in isolated dorsal root ganglion neurons was also studied in current-clamp recordings. In the formalin test, ST and NEO significantly reduced the nociceptive behaviour. ST reduced the N-(4-methyl-2-quinazolinyl)-guanidine (GMQ)induced nociceptive behaviour, and NEO diminished the hyperalgesia to thermonociception and mechanonociception produced by CAR. In the current-clamp experiments, ST and NEO reduced the generation of action potentials when an acidic solution was applied. We conclude that ST and NEO produce analgesia to inflammatory pain, an effect that is due in part to the inhibition of ASIC activation in sensory neurons.
We present the study of the biocompatibility and surface properties of titanium dioxide (TiO 2 ) ... more We present the study of the biocompatibility and surface properties of titanium dioxide (TiO 2 ) thin films deposited by direct current magnetron sputtering. These films are deposited on a quartz substrate at room temperature and annealed with different temperatures (100, 300, 500, 800 and 1100 °C). The biocompatibility of the TiO 2 thin films is analyzed using primary cultures of dorsal root ganglion (DRG) of Wistar rats, whose neurons are incubated on the TiO 2 thin films and on a control substrate during 18 to 24 h. These neurons are activated by electrical stimuli and its ionic currents and action potential activity recorded. Through X-ray diffraction (XRD), the surface of TiO 2 thin films showed a good quality, homogeneity and roughness. The XRD results showed the anatase to rutile phase transition in TiO 2 thin films at temperatures between 500 and 1100 °C. This phase had a grain size from 15 to 38 nm, which allowed a suitable structural and crystal phase stability of the TiO 2 thin films for low and high temperature. The biocompatibility experiments of these films indicated that they were appropriated for culture of living neurons which displayed normal electrical behavior. OPEN ACCESS Materials 2014, 7 4106
Acknowledgements: The authors thank Professor Paul S. Guth for critical reading of the manuscript... more Acknowledgements: The authors thank Professor Paul S. Guth for critical reading of the manuscript. Work supported by MURST.
Extracellular protons have been shown to modulate voltage-activated ionic channels. It has been p... more Extracellular protons have been shown to modulate voltage-activated ionic channels. It has been proposed that synaptic modulation by exocytosed vesicular protons would be a characteristic feature of ribbon-type synapses. Type-I hair cells have a calyceal afferent junction with a diffusionally restricted synaptic cleft. These led us to study the action of extracellular pH changes on the voltage-activated Ca2+ and K+ currents evaluated using a whole-cell patch clamp in isolated cells. The amplitude of the Ca2+ and the K+ current were reduced by extracellular acidification, but without significant changes with extracellular alkalization. A shift in the voltage dependence to a more positive membrane potential was achieved at pH + and Ca2+ currents are modulated by protons, indicating that protons released along with an afferent neurotransmitter would participate as a feedback mechanism in type-I hair cells.
Antagonism of glutamate receptor by a chromatographic fraction from the exudate of sea anemone Ph... more Antagonism of glutamate receptor by a chromatographic fraction from the exudate of sea anemone Phyllactis flosculifera. Toxicon 34, [443][444][445][446][447][448][449][450] 1996. -In the search for new glutamate antagonists it seems promising to characterize the effects of venom from invertebrates that prey mainly on crustaceans. In this work, the exudate of the sea anemone P. flosculifera was used as a source of this kind of compounds. The action of chromatographic fraction D from P. flosculifera was tested upon microiontophoretically evoked glutamate responses in intracellular recordings from central neurons of the land snail Zachrysia guanensis. Bath application of fraction D (2-8 mg/ml, n = 13) diminished both the excitatory and the inhibitory components of glutamate agonists in Z. guanensis neurons; this action was dose-dependent and partially reversible. Fraction D actions were also tested in the multiunit spontaneous and mechanically evoked responses of the glutamatergic junction between hair cells and afferent neurons of the axolotl Ambystoma tigrinum. Pressure ejection of fraction D in concentrations ranging from 0.5 to 2 mg/ml (n = 9) decreased the spontaneous and mechanically evoked activity of semicircular canal afferent neurons and the responses evoked by kainic acid and α-amino-3-hydroxy-5-methylisoxasole-4-propionic acid. This action was also dose-dependent and partially reversible. These results indicate that fraction D acts as a glutamate receptor antagonist in snail and amphibian neurons. Further studies are required to characterize the active compounds responsible for this action and its specificity upon the subtypes of glutamate receptors.
Conus californicus belongs to a genus of marine gastropods with more than 700 extant species. C. ... more Conus californicus belongs to a genus of marine gastropods with more than 700 extant species. C. californicus has been shown to be distantly related to all Conus species, but showing unusual biological features. We report a novel peptide isolated from C. californicus with a significant inhibitory action over neuronal voltage-gated calcium channels. The new toxin is formed by 13-amino acid residues with two disulfide bonds, whose sequence (NCPAGCRSQGCCM) is strikingly different from regular u-conotoxins. In the HPLC purification procedure, the venom fraction eluted in the first 10-15 min produced a significant decrease (54% AE 3%) of the Ca 2þ current in Xenopus laevis oocytes transfected with purified rat-brain mRNA. A specific peptide obtained from the elution at 13 min decreased the Ca 2þ current in the adult rat dorsal-root ganglion neurons in a primary culture by 34% AE 2%. The cysteine pattern of this peptide corresponds to the framework XVI described for the M-superfamily of conopeptides and is unprecedented among Conus peptides acting on Ca 2þ channels.
Nicotinamide adenine dinucleotide phosphate reduced-diaphorase (NADPH-d) histochemistry was inves... more Nicotinamide adenine dinucleotide phosphate reduced-diaphorase (NADPH-d) histochemistry was investigated in the axolotl (Ambystoma tigrinum ) inner ear. Hair cells showed a intense NADPH-d reaction; afferent neurones also stained but less intensely than hair cells. Effects of N G -nitro-L-arginine (L-NOARG) on the basal discharge and mechanical responses of semicircular canal afferent neurones recorded extracellularly were also studied.
In the vestibular system, the electrical discharge of the afferent neurons has been found to be h... more In the vestibular system, the electrical discharge of the afferent neurons has been found to be highly sensitive to external pH changes, and acid-sensing ionic-channels (ASIC) have been found to be functionally expressed in afferent neurons. No previous attempt to assay the ASIC function in vestibular afferent neurons has been done. In our work we studied the electrical discharge of the afferent neuron of the isolated inner ear of the axolotl (Ambystoma tigrinum) to determine the participation of proton-gated currents in the postransductional information processing in the vestibular system. Microperfusion of FMRF-amide significantly increased the resting activity of the afferent neurons of the semicircular canal indicating that ASIC currents are tonically active in the resting condition. The use of ASIC antagonists, amiloride and acetylsalicylic acid (ASA), significantly reduced the vestibular-nerve discharge, corroborating the idea that the afferent neurons of the vestibular system express ASICs that are sensitive to amiloride, ASA, and to FMRF-amide. The sensitivity of the vestibular afferent-resting discharge to the microperfusion of ASIC acting agents indicates the participation of these currents in the establishment of the afferent-resting discharge.
Nicotinamide adenine dinucleotide phosphate reduced-diaphorase (NADPH-d) histochemistry was inves... more Nicotinamide adenine dinucleotide phosphate reduced-diaphorase (NADPH-d) histochemistry was investigated in the axolotl (Ambystoma tigrinum) lateral line. Hair cells of neuromast organs of the head skin and neurons of the postotic ganglia showed a significant NADPH-d reaction.
We report the existence and anatomical distribution to nociceptin/orphanin-FQ (N/O FQ)-like immun... more We report the existence and anatomical distribution to nociceptin/orphanin-FQ (N/O FQ)-like immunoreactivity in neurons and fibers in the perioesophageal ganglia of the snail (Helix aspersa). Intracellular recordings from perioesophageal ganglion neurons showed that the application of 10 mM N/O FQ produced an excitatory action in 22% of the neurons studied and an inhibitory action in 33% of the neurons regardless of their origin (cerebral or parietal ganglion). Our result provides evidence that N/O FQ-like peptide is located in whole perioesophageal ganglia (mainly in the cerebral one), and that it may serve as a neuromodulator of the neuronal spike discharge. These data support the idea that the N/ O FQ opioid system has an early phylogenetic origin and a functional continuity during the course of evolution. q
AbstractÐThis study was performed using intracellular and multiunit extracellular recording techn... more AbstractÐThis study was performed using intracellular and multiunit extracellular recording techniques in order to characterize the role of nitric oxide in the afferent synaptic transmission of the vestibular system of the axolotl (Ambystoma tigrinum). Bath application of nitric oxide synthase inhibitors N G -nitro-l-arginine (0.01 mM to 10 mM) and N-nitro-larginine methyl ester hydrochloride (0.1 mM to 1000 mM) elicited a dose-dependent decrease in the basal discharge of the semicircular canal afferent ®bers. N G -Nitro-l-arginine also diminished the response to mechanical stimuli. Moreover, N G -nitro-l-arginine (1 mM) produced a hyperpolarization associated with a decrease in the spike discharge and diminished the frequency of the excitatory postsynaptic potentials on afferent ®bers recorded intracellularly. Nitric oxide donors were also tested: (i) S-nitroso-N-acetyl-dl-penicillamine (0.1 mM to 100 mM) increased the basal discharge and the response to mechanical stimuli. At the maximum effective concentration (100 mM) this drug affected neither the amplitude nor the frequency of the excitatory postsynaptic potentials. However, it slightly depolarized the afferent neurons and decreased their input resistance. (ii) 3-Morpholino-sydnonimine hydrochloride did not signi®cantly affect the basal discharge or the mechanically evoked peak response of afferent neurons at any of the concentrations used (1 mM to 1000 mM). However, after 10 min of perfusion in the bath, 1 mM and 10 mM 3-morpholino-sydnonimine hydrochloride signi®cantly modi®ed the baseline of the mechanically evoked response, producing an increase in the mean spike discharge of the afferent ®bers.
Conus californicus belongs to a genus of marine gastropods with more than 700 extant species. C. ... more Conus californicus belongs to a genus of marine gastropods with more than 700 extant species. C. californicus has been shown to be distantly related to all Conus species, but showing unusual biological features. We report a novel peptide isolated from C. californicus with a significant inhibitory action over neuronal voltage-gated calcium channels. The new toxin is formed by 13-amino acid residues with two disulfide bonds, whose sequence (NCPAGCRSQGCCM) is strikingly different from regular ω-conotoxins. In the HPLC purification procedure, the venom fraction eluted in the first 10–15 min produced a significant decrease (54% ± 3%) of the Ca2+ current in Xenopus laevis oocytes transfected with purified rat-brain mRNA. A specific peptide obtained from the elution at 13 min decreased the Ca2+ current in the adult rat dorsal-root ganglion neurons in a primary culture by 34% ± 2%. The cysteine pattern of this peptide corresponds to the framework XVI described for the M-superfamily of conopeptides and is unprecedented among Conus peptides acting on Ca2+ channels.
This study was undertaken to determine the possible role of GABA as an afferent transmitter in th... more This study was undertaken to determine the possible role of GABA as an afferent transmitter in the vestibular system of the axolotl. We studied the effects of GABA, muscimol, bicuculline and picrotoxin on the spontaneous spike discharge of the afferent fibers of the sac&i lagena and anterior semicircular canal. It was found that GABA and muscimol produce a very weak excitatory effect which does not mimic either the temporal course or the amplitude of the response of vestibular afferents to physiological stimuli. The GABA antagonist bicuculline has no significant effect on these fibers, and picrotoxin partially blocks the spontaneous activity in 33% of the fibers studied. These results indicate that GABA is probably not an afferent transmitter in the vestibular system as has previously been proposed.
By combining mathematical methods with the morphological analysis of the semicircular canals of t... more By combining mathematical methods with the morphological analysis of the semicircular canals of the axolotl (Ambystoma tigrinum), a system of differential equations describing the mechanical coupling in the semicircular canals was obtained. The coefficients of this system have an explicit physiological meaning that allows for the introduction of morphological and dynamical parameters directly into the differential equations. The cupula of the semicircular canals was modeled both as a piston and as a membrane (diaphragm like), and the duct canals as toroids with two main regions: i) the semicircular canal duct and, ii) a larger diameter region corresponding to the ampulla and the utricle. The endolymph motion was described by the Navier-Stokes equations. The analysis of the model demonstrated that cupular behavior dynamics under periodic stimulation is equivalent in both the piston and the membrane cupular models, thus a general model in which the detailed cupular structure is not relevant was derived.
Molecular identity, ontogeny, and cAMP modulation of the hyperpolarization-activated current in v... more Molecular identity, ontogeny, and cAMP modulation of the hyperpolarization-activated current in vestibular ganglion neurons. ties, developmental regulation, and cAMP modulation of the hyperpolarization-activated current (I h ) were investigated by the whole cell patch-clamp technique in vestibular ganglion neurons of the rat at two postnatal stages (P7-10 and P25-28). In addition, by RT-PCR and immunohistochemistry the identity and distribution of hyperpolarization-activated and cyclic nucleotide-gated channel (HCN) isoforms that generate I h were investigated. I h current density was larger in P25-28 than P7-10 rats, increasing 410% for small cells (Ͻ30 pF) and 200% for larger cells (Ͼ30 pF). The half-maximum activation voltage (V 1/2 ) of I h was Ϫ102 mV in P7-10 rats and in P25-28 rats shifted 7 mV toward positive voltages. At both ages, intracellular cAMP increased I h current density, decreased its activation time constant (), and resulted in a rightward shift of V 1/2 by 9 mV. Perfusion of 8-BrcAMP increased I h amplitude and speed up its activation kinetics. I h was blocked by Cs ϩ , zatebradine, and ZD7288. As expected, these drugs also reduced the voltage sag caused with hyperpolarizing pulses and prevented the postpulse action potential generation without changes in the resting potential. RT-PCR analysis showed that HCN1 and HCN2 subunits were predominantly amplified in vestibular ganglia and end organs and HCN3 and HCN4 to a lesser extent. Immunohistochemistry showed that the four HCN subunits were differentially expressed (HCN1 Ͼ HCN2 Ͼ HCN3 Ն HCN4) in ganglion slices and in cultured neurons at both P7-10 and P25-28 stages. Developmental changes shifted V 1/2 of I h closer to the resting membrane potential, increasing its functional role. Modulation of I h by cAMP-mediated signaling pathway constitutes a potentially relevant control mechanism for the modulation of afferent neuron discharge.
In the isolated inner ear of the axolotl (Ambystoma tigrinum) acid pH decreased and basic pH incr... more In the isolated inner ear of the axolotl (Ambystoma tigrinum) acid pH decreased and basic pH increased the resting and mechanically evoked spike discharge of semicircular canal afferent neurons. Variations in pH also modified the afferent neuron response to N-methyl-D-aspartic acid and to (±)-α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid. Responses to both excitatory amino acid agonists increased at pH 7.8 (41% and 22%, respectively) and decreased by perfusion of the preparation with a saline solution, of pH 7.0 (28% in both cases). These results indicate that vestibular endorgans have a significant sensitivity to pH that could play a significant role in various pathological states, and may also contribute to the postransductional processing of sensory information.
Several studies have shown that type I hair cells present a large outward rectifying potassium cu... more Several studies have shown that type I hair cells present a large outward rectifying potassium current (g K;L ) that is substantially activated at the resting potential, greatly reducing cell input resistance and voltage gain. In fact, mechanoelectrical transducer currents seem not to be large enough to depolarize type I hair cells to produce neurotransmitter release. Also, the strongly nonlinear transducer currents and the limited voltage oscillations found in some hair cells did not account for the bidirectionality of response in hair cell systems. We developed a model based in the analysis of nonlinear Goldman^Hodgkin^Katz equations to calculate the hair cell receptor potential and ionic movements produced by transducer current activation. Type I hair cells displaying the large g K;L current were found to produce small receptor potentials (3^13.8 mV) in response to mechanoelectrical transducer current input. In contrast, type II cells that lack g K;L produced receptor potentials of about 30 mV. Properties of basolateral ionic conductances in type II hair cells will linearize hair bundle displacement to receptor potential relationship. The voltage to obtain the half maximal activation of g K;L significantly affects the resting membrane potential, the amplitude, and the linearity of the receptor potential. Electrodiffusion equations were also used to analyze ionic changes in the intercellular space between type I hair cell and calyx endings. Significant K þ accumulation could take place at the intercellular space depending on calyx structure. ß
International Journal of Bio-Medical Computing, 1990
A computer simulation of the extracellular field potential recording of nerve activity is present... more A computer simulation of the extracellular field potential recording of nerve activity is presented. An experimental setup composed of an oscilloscope, a nerve, a conductive surface, and a recording electrode is graphically simulated. The user may study the influence of the position of the recording electrode and of certain nerve properties (membrane potential, velocity of conduction and action potential duration), on the action potential shape. The different waves which constitute the action potential may be analyzed and their peak values plotted as a function of the independent variable (e.g. electrode distance from the nerve). Three-dimensional plots of a set of action potentials as a function of the independent variable may also be obtained. The simulation allows the user to study the basic factors which determine the configuration of an extracelluhuly recorded compound nerve action potential.
This work reviews the neuropharmacology of the vestibular system, with an emphasis on the mechani... more This work reviews the neuropharmacology of the vestibular system, with an emphasis on the mechanism of action of drugs used in the treatment of vestibular disorders. Otolaryngologists are confronted with a rapidly changing field in which advances in the knowledge of ionic channel function and synaptic transmission mechanisms have led to the development of new scientific models for the understanding of vestibular dysfunction and its management. In particular, there have been recent advances in our knowledge of the fundamental mechanisms of vestibular system function and drug mechanisms of action. In this work, drugs acting on vestibular system have been grouped into two main categories according to their primary mechanisms of action: those with effects on neurotransmitters and neuromodulator receptors and those that act on voltage-gated ion channels. Particular attention is given in this review to drugs that may provide additional insight into the pathophysiology of vestibular diseases. A critical review of the pharmacology and highlights of the major advances are discussed in each case. that probably accounts for the presence of catecholamines in the inner ear [47].
Opioid receptors are expressed in the vestibular endorgans (afferent neurons and hair cells) and ... more Opioid receptors are expressed in the vestibular endorgans (afferent neurons and hair cells) and are activated by the efferent system, which modulates the discharge of action potentials in vestibular afferent neurons (VANs). In mammals, VANs mainly express the μ opioid-receptor, but the function of this receptors activation and the cellular mechanisms by which they exert their actions in these neurons are poorly studied. To determine the actions of μ opioid receptor (MOR) and cell signaling mechanisms in VANs, we made perforated patch-clamp recordings of VANs that were obtained from postnatal days 7 to 10 (P7-10) rats and then maintained in primary culture. The MOR agonist [D-Ala 2 , N-Me-Phe 4 , Gly 5 -ol]-enkephalin (DAMGO) inhibited the total voltage-gated outward current; this effect was prevented by the perfusion of a Ca 2+ -free extracellular solution. We then studied the voltage-gated calcium current (I ca ) and found that DAMGO Met-enkephalin or endomorphin-1 inhibited the I Ca in a dose-response fashion. The effects of DAMGO were prevented by the MOR antagonist (CTAP) or by pertussis toxin (PTX). The use of specific calcium channel blockers showed that MOR activation inhibited T-, L-and N-type I Ca . The use of various enzyme activators and inhibitors and of cAMP analogs allowed us to demonstrate that the MOR acts through a cAMP dependent signaling mechanism. In current clamp experiments, MOR activation increased the duration and decreased the amplitude of the action potentials and modulated the discharge produced by current injection. Pre-incubation with PTX occluded MOR activation effect. We conclude that MOR activation inhibits the T-, L-and N-type I Ca through activation of a Gα i/o protein that involves a decrease in AC-cAMP-PKA activity. The modulation of I Ca may have an impact on the synaptic integration, excitability, and neurotransmitter release from VANs.
Aminoglycosides (AGs) modulate nociceptors and ionic channels expressed in sensory neurons. The A... more Aminoglycosides (AGs) modulate nociceptors and ionic channels expressed in sensory neurons. The AG applied in situ could be useful to alleviate hyperalgesia in animal models of inflammatory pain. We tested streptomycin (ST) and neomycin (NEO) as analgesic agents applied in situ in rat paw inflammation caused by formalin or carrageenan administration. The action of ST and NEO on the action potential discharge produced by acidic stimuli in isolated dorsal root ganglion neurons was also studied in current-clamp recordings. In the formalin test, ST and NEO significantly reduced the nociceptive behaviour. ST reduced the N-(4-methyl-2-quinazolinyl)-guanidine (GMQ)induced nociceptive behaviour, and NEO diminished the hyperalgesia to thermonociception and mechanonociception produced by CAR. In the current-clamp experiments, ST and NEO reduced the generation of action potentials when an acidic solution was applied. We conclude that ST and NEO produce analgesia to inflammatory pain, an effect that is due in part to the inhibition of ASIC activation in sensory neurons.
We present the study of the biocompatibility and surface properties of titanium dioxide (TiO 2 ) ... more We present the study of the biocompatibility and surface properties of titanium dioxide (TiO 2 ) thin films deposited by direct current magnetron sputtering. These films are deposited on a quartz substrate at room temperature and annealed with different temperatures (100, 300, 500, 800 and 1100 °C). The biocompatibility of the TiO 2 thin films is analyzed using primary cultures of dorsal root ganglion (DRG) of Wistar rats, whose neurons are incubated on the TiO 2 thin films and on a control substrate during 18 to 24 h. These neurons are activated by electrical stimuli and its ionic currents and action potential activity recorded. Through X-ray diffraction (XRD), the surface of TiO 2 thin films showed a good quality, homogeneity and roughness. The XRD results showed the anatase to rutile phase transition in TiO 2 thin films at temperatures between 500 and 1100 °C. This phase had a grain size from 15 to 38 nm, which allowed a suitable structural and crystal phase stability of the TiO 2 thin films for low and high temperature. The biocompatibility experiments of these films indicated that they were appropriated for culture of living neurons which displayed normal electrical behavior. OPEN ACCESS Materials 2014, 7 4106
Acknowledgements: The authors thank Professor Paul S. Guth for critical reading of the manuscript... more Acknowledgements: The authors thank Professor Paul S. Guth for critical reading of the manuscript. Work supported by MURST.
Extracellular protons have been shown to modulate voltage-activated ionic channels. It has been p... more Extracellular protons have been shown to modulate voltage-activated ionic channels. It has been proposed that synaptic modulation by exocytosed vesicular protons would be a characteristic feature of ribbon-type synapses. Type-I hair cells have a calyceal afferent junction with a diffusionally restricted synaptic cleft. These led us to study the action of extracellular pH changes on the voltage-activated Ca2+ and K+ currents evaluated using a whole-cell patch clamp in isolated cells. The amplitude of the Ca2+ and the K+ current were reduced by extracellular acidification, but without significant changes with extracellular alkalization. A shift in the voltage dependence to a more positive membrane potential was achieved at pH + and Ca2+ currents are modulated by protons, indicating that protons released along with an afferent neurotransmitter would participate as a feedback mechanism in type-I hair cells.
Antagonism of glutamate receptor by a chromatographic fraction from the exudate of sea anemone Ph... more Antagonism of glutamate receptor by a chromatographic fraction from the exudate of sea anemone Phyllactis flosculifera. Toxicon 34, [443][444][445][446][447][448][449][450] 1996. -In the search for new glutamate antagonists it seems promising to characterize the effects of venom from invertebrates that prey mainly on crustaceans. In this work, the exudate of the sea anemone P. flosculifera was used as a source of this kind of compounds. The action of chromatographic fraction D from P. flosculifera was tested upon microiontophoretically evoked glutamate responses in intracellular recordings from central neurons of the land snail Zachrysia guanensis. Bath application of fraction D (2-8 mg/ml, n = 13) diminished both the excitatory and the inhibitory components of glutamate agonists in Z. guanensis neurons; this action was dose-dependent and partially reversible. Fraction D actions were also tested in the multiunit spontaneous and mechanically evoked responses of the glutamatergic junction between hair cells and afferent neurons of the axolotl Ambystoma tigrinum. Pressure ejection of fraction D in concentrations ranging from 0.5 to 2 mg/ml (n = 9) decreased the spontaneous and mechanically evoked activity of semicircular canal afferent neurons and the responses evoked by kainic acid and α-amino-3-hydroxy-5-methylisoxasole-4-propionic acid. This action was also dose-dependent and partially reversible. These results indicate that fraction D acts as a glutamate receptor antagonist in snail and amphibian neurons. Further studies are required to characterize the active compounds responsible for this action and its specificity upon the subtypes of glutamate receptors.
Conus californicus belongs to a genus of marine gastropods with more than 700 extant species. C. ... more Conus californicus belongs to a genus of marine gastropods with more than 700 extant species. C. californicus has been shown to be distantly related to all Conus species, but showing unusual biological features. We report a novel peptide isolated from C. californicus with a significant inhibitory action over neuronal voltage-gated calcium channels. The new toxin is formed by 13-amino acid residues with two disulfide bonds, whose sequence (NCPAGCRSQGCCM) is strikingly different from regular u-conotoxins. In the HPLC purification procedure, the venom fraction eluted in the first 10-15 min produced a significant decrease (54% AE 3%) of the Ca 2þ current in Xenopus laevis oocytes transfected with purified rat-brain mRNA. A specific peptide obtained from the elution at 13 min decreased the Ca 2þ current in the adult rat dorsal-root ganglion neurons in a primary culture by 34% AE 2%. The cysteine pattern of this peptide corresponds to the framework XVI described for the M-superfamily of conopeptides and is unprecedented among Conus peptides acting on Ca 2þ channels.
Nicotinamide adenine dinucleotide phosphate reduced-diaphorase (NADPH-d) histochemistry was inves... more Nicotinamide adenine dinucleotide phosphate reduced-diaphorase (NADPH-d) histochemistry was investigated in the axolotl (Ambystoma tigrinum ) inner ear. Hair cells showed a intense NADPH-d reaction; afferent neurones also stained but less intensely than hair cells. Effects of N G -nitro-L-arginine (L-NOARG) on the basal discharge and mechanical responses of semicircular canal afferent neurones recorded extracellularly were also studied.
In the vestibular system, the electrical discharge of the afferent neurons has been found to be h... more In the vestibular system, the electrical discharge of the afferent neurons has been found to be highly sensitive to external pH changes, and acid-sensing ionic-channels (ASIC) have been found to be functionally expressed in afferent neurons. No previous attempt to assay the ASIC function in vestibular afferent neurons has been done. In our work we studied the electrical discharge of the afferent neuron of the isolated inner ear of the axolotl (Ambystoma tigrinum) to determine the participation of proton-gated currents in the postransductional information processing in the vestibular system. Microperfusion of FMRF-amide significantly increased the resting activity of the afferent neurons of the semicircular canal indicating that ASIC currents are tonically active in the resting condition. The use of ASIC antagonists, amiloride and acetylsalicylic acid (ASA), significantly reduced the vestibular-nerve discharge, corroborating the idea that the afferent neurons of the vestibular system express ASICs that are sensitive to amiloride, ASA, and to FMRF-amide. The sensitivity of the vestibular afferent-resting discharge to the microperfusion of ASIC acting agents indicates the participation of these currents in the establishment of the afferent-resting discharge.
Nicotinamide adenine dinucleotide phosphate reduced-diaphorase (NADPH-d) histochemistry was inves... more Nicotinamide adenine dinucleotide phosphate reduced-diaphorase (NADPH-d) histochemistry was investigated in the axolotl (Ambystoma tigrinum) lateral line. Hair cells of neuromast organs of the head skin and neurons of the postotic ganglia showed a significant NADPH-d reaction.
We report the existence and anatomical distribution to nociceptin/orphanin-FQ (N/O FQ)-like immun... more We report the existence and anatomical distribution to nociceptin/orphanin-FQ (N/O FQ)-like immunoreactivity in neurons and fibers in the perioesophageal ganglia of the snail (Helix aspersa). Intracellular recordings from perioesophageal ganglion neurons showed that the application of 10 mM N/O FQ produced an excitatory action in 22% of the neurons studied and an inhibitory action in 33% of the neurons regardless of their origin (cerebral or parietal ganglion). Our result provides evidence that N/O FQ-like peptide is located in whole perioesophageal ganglia (mainly in the cerebral one), and that it may serve as a neuromodulator of the neuronal spike discharge. These data support the idea that the N/ O FQ opioid system has an early phylogenetic origin and a functional continuity during the course of evolution. q
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