RESUMEN
Large conductance Ca(2+)-activated potassium (BK) channels play important roles in controlling neuronal excitability. We cloned the PISlo gene encoding BK channels from the spiny lobster, Panulirus interruptus. This gene shows 81-98% sequence identity to Slo genes previously found in other organisms. We isolated a number of splice variants of the PISlo cDNA within Panulirus interruptus nervous tissue. Sequence analysis indicated that there are at least seven alternative splice sites in PISlo, each with multiple alternative segments. Using immunohistochemistry, we found that the PISlo proteins are distributed in the synaptic neuropil, axon and soma of stomatogastric ganglion (STG) neurons.
Asunto(s)
Clonación Molecular/métodos , Canales de Potasio de Gran Conductancia Activados por el Calcio/genética , Palinuridae/genética , Empalme Alternativo/genética , Secuencia de Aminoácidos , Animales , Ganglios de Invertebrados/metabolismo , Canales de Potasio de Gran Conductancia Activados por el Calcio/metabolismo , Datos de Secuencia Molecular , Neurópilo/metabolismo , Palinuridae/metabolismo , Isoformas de Proteínas/genéticaRESUMEN
We studied the properties of currents generated in Xenopus oocytes by nine splice variants of the spiny lobster Shaker gene. These isoforms differ in their amino termini and in the P-loop region of the pore. Both the voltage dependence and kinetic properties of the currents varied significantly, depending on which amino terminus was present. A cluster of net positive charges at the N-terminus was not necessary for rapid inactivation: negatively charged N-termini also inactivated rapidly. There was no obvious correlation between N-terminus length and inactivation rate. These N-terminal effects were additive with a separate set of voltage and kinetic properties controlled by the two alternative P-loop exons.
Asunto(s)
Empalme Alternativo , Activación del Canal Iónico , Oocitos/metabolismo , Palinuridae/fisiología , Canales de Potasio/fisiología , Secuencia de Aminoácidos , Animales , Electrofisiología , Femenino , Cinética , Datos de Secuencia Molecular , Oocitos/citología , Canales de Potasio/genética , Isoformas de Proteínas , Eliminación de Secuencia , Canales de Potasio de la Superfamilia Shaker , Xenopus laevisRESUMEN
The motor pattern generated by the 14 neurons composing the pyloric circuit in the stomatogastric ganglion (STG) of the spiny lobster, Panulirus interruptus, is organized not only by the synaptic connections between neurons, but also by the characteristic intrinsic electrophysiological properties of the individual cells. These cellular properties result from the unique complement of ion channels that each cell expresses, and the distribution of those channels in the cell membranes. We have mapped the STG expression of shab and shaw, two genes in the Shaker superfamily of potassium channel genes that encode voltage-dependent, non-inactivating channels. Using antibodies developed against peptide sequences from the two channel proteins, we explored the localization and cell-specific expression of the channels. Anti-Shab and anti-Shaw antibodies both stain all the pyloric neurons in the somata, as well as their primary neurites and branch points of large neurites, but to varying degrees between cell types. Staining was weak and irregular (Shaw) or absent (Shab) in the fine neuropil of pyloric neurons, where most synaptic interactions occur. There is a high degree of variability in the staining intensity among neurons of a single cell class. This supports Golowasch et al.'s [J Neurosci 19 (1999) RC33; Neural Comput 11 (1999) 1079] hypothesis that individual cells can have similar firing properties with varying compositions of ionic currents. Both antibodies stain the axons of the peripheral nerves as they enter foregut muscles. We conclude that both Shab and Shaw channels are appropriately localized to contribute to the noninactivating potassium current in the stomatogastric nervous system.
Asunto(s)
Ganglios de Invertebrados/metabolismo , Canales de Potasio con Entrada de Voltaje , Canales de Potasio/metabolismo , Estómago/inervación , Análisis de Varianza , Animales , Western Blotting/métodos , Canales de Potasio de Tipo Rectificador Tardío , Inmunohistoquímica/métodos , Microscopía Confocal/métodos , Red Nerviosa/metabolismo , Unión Neuromuscular/metabolismo , Palinuridae , Péptidos/inmunología , Nervios Periféricos/metabolismo , Canales de Potasio/química , Píloro/inervación , Canales de Potasio Shab , Canales de Potasio ShawRESUMEN
The shal gene encoding the transient potassium current, I(A), plays important roles in shaping the firing properties of neurons in the pyloric network in the stomatogastric ganglion (STG) of the spiny lobster, Panulirus interruptus. However, when we overexpressed the shal protein in pyloric dilator (PD) neurons, the effect of increased I(A )was compensated by a parallel upregulation of the hyperpolarization activated inward current ( I(h)). In an attempt to temporally separate the overexpression of shal from the compensatory up-regulation of I(h) channels, we inserted an endoplasmic reticulum (ER) export signal sequence, FCYENE, into the shal gene. This signal sequence accelerated the surface expression of shal protein in Xenopus oocytes and PD neurons. However, the accelerated expression of shal still did not alter the firing properties of the injected neuron, suggesting that the compensatory upregulation of I(h) occurs simultaneously with the upregulation of I(A).
Asunto(s)
Retículo Endoplásmico/metabolismo , Ganglios de Invertebrados/metabolismo , Neuronas/metabolismo , Palinuridae , Canales de Potasio con Entrada de Voltaje , Canales de Potasio/metabolismo , Señales de Clasificación de Proteína , Secuencia de Aminoácidos , Animales , Ganglios de Invertebrados/citología , Neuronas/citología , Oocitos/fisiología , Palinuridae/anatomía & histología , Palinuridae/metabolismo , Canales de Potasio/química , Canales de Potasio/genética , Estructura Secundaria de Proteína , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Canales de Potasio Shal , Xenopus laevisRESUMEN
The transient potassium current (I(A)) plays an important role in shaping the firing properties of pyloric neurons in the stomatogastric ganglion (STG) of the spiny lobster, Panulirus interruptus. The shal gene encodes I(A) in pyloric neurons. However, when we over-expressed the lobster Shal protein by shal RNA injection into the pyloric dilator (PD) neuron, the increased I(A) had somewhat different properties from the endogenous I(A). The recently cloned K-channel interacting proteins (KChIPs) can modify vertebrate Kv4 channels in cloned cell lines. When we co-expressed hKChIP1 with lobster shal in Xenopus oocytes or lobster PD neurons, they produced A-currents resembling the endogenous I(A) in PD neurons; compared with currents evoked by shal alone, their voltage for half inactivation was depolarized, their kinetics of inactivation were slowed, and their recovery from inactivation was accelerated. We also co-expressed shal in PD neurons with lobster frequenin, which encodes a protein belonging to the same EF-hand family of Ca(2+) sensing proteins as hKChIP. Frequenin also restored most of properties of the shal-evoked currents to those of the endogenous A-currents, but the time course of recovery from inactivation was not corrected. These results suggest that lobster shal proteins normally interact with proteins in the KChIP/frequenin family to produce the transient potassium current in pyloric neurons.
Asunto(s)
Proteínas de Unión al Calcio/metabolismo , Neuronas/fisiología , Canales de Potasio con Entrada de Voltaje , Canales de Potasio/metabolismo , Proteínas de Xenopus , Animales , Proteínas de Unión al Calcio/genética , Ganglios de Invertebrados/citología , Ganglios de Invertebrados/fisiología , Expresión Génica/fisiología , Proteínas de Interacción con los Canales Kv , Potenciales de la Membrana/fisiología , Microinyecciones , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Proteínas Sensoras del Calcio Neuronal , Neuropéptidos , Oocitos/fisiología , Palinuridae , Técnicas de Placa-Clamp , Potasio/metabolismo , Canales de Potasio/genética , Píloro/inervación , ARN/farmacología , Canales de Potasio Shal , XenopusRESUMEN
Voltage-gated calcium channels are critical to all aspects of nervous system function, with differing roles within the neuronal somata, at synaptic terminals, and at the neuromuscular junction. We have developed antibodies against two voltage-gated Ca(2+) channel genes from the spiny lobster, Panulirus interruptus, which are homologous to the Drosophila Ca1A (a P/Q-type channel) and Ca1D (an L-type channel) genes. Using these antibodies, we have found that each channel shows unique patterns of localization within the stomatogastric nervous system. Both antibodies stain somata of most of the neurons in the pyloric network to varying degrees. The high degree of variability in staining intensity within individual pyloric cell classes supports the hypothesis of Golowasch et al. (1999a,b) that individual cells can vary in their composition of ionic currents and still have similar firing properties. Anti-Ca1A stains structures in the neuropil, some of which are terminals of axons descending from higher ganglia; however, the majority of these are neither neurites nor blood vessels, but may instead be glial cells or other support elements. Anti-Ca1A labeling was also prominent in the peripheral axons of pyloric motoneurons as they enter muscles, indicating that this channel may be involved in regulation of synaptic transmission onto the foregut muscles. Anti-Ca1D does not label neurites in the neuropil of the stomatogastric ganglion. It stains glial cells in the stomatogastric ganglion in the region of their nuclei, presumably from protein being produced in the perinuclear rough endoplasmic reticulum, en route to the glial cell periphery. While anti-Ca1D labeling is seen in a patchy distribution along peripheral pyloric axons, it was never seen near the muscle. We conclude that the localization of these two calcium channels is tightly controlled within the stomatogastric nervous system, but we cannot conclusively demonstrate that Ca1A and/or Ca1D channels play roles in synaptic integration within the stomatogastric ganglion.
Asunto(s)
Canales de Calcio Tipo L/metabolismo , Nephropidae/metabolismo , Red Nerviosa/metabolismo , Estómago/inervación , Secuencia de Aminoácidos/genética , Animales , Western Blotting , Canales de Calcio Tipo L/genética , Canales de Calcio Tipo P/genética , Canales de Calcio Tipo P/metabolismo , Inmunohistoquímica/métodos , Datos de Secuencia Molecular , Neurópilo/metabolismo , Píloro/inervación , Coloración y Etiquetado , Distribución TisularRESUMEN
The pyloric network of the stomatogastric ganglion of the lobster Panulirus interruptus is a model system used to understand how motor networks change their output to produce a variety of behaviors. The transient potassium current (I(A)) shapes the activity of individual pyloric neurons by affecting their rate of postinhibitory rebound and spike frequency. We used two electrode voltage clamp to study the modulatory effects of dopamine (DA), octopamine (OCT), and serotonin (5-HT) on I(A) in the anterior burster (AB), inferior cardiac (IC), and ventricular dilator (VD) neurons of the pyloric circuit. DA significantly reduced I(A) in the AB and IC neurons and shifted their voltages of activation (V(act)) and inactivation (V(inact)) in a depolarized direction. These ionic changes contribute to the depolarization and increased firing rate of the AB and IC neurons produced by DA. Likewise, 5-HT significantly reduced I(A) and shifted V(inact) in the depolarized direction in the IC neuron, consistent with 5-HT's enhancement of IC firing. None of the amines evoked significant changes in I(A) in the VD neuron, suggesting that other currents mediate the amine effects on this neuron.
Asunto(s)
Monoaminas Biogénicas/fisiología , Ganglios de Invertebrados/fisiología , Nephropidae/fisiología , Canales de Potasio/fisiología , Estómago/inervación , Algoritmos , Animales , Separación Celular , Dopamina/fisiología , Electrofisiología , Ganglios de Invertebrados/citología , Técnicas In Vitro , Neuronas Motoras/fisiología , Octopamina/fisiología , Técnicas de Placa-Clamp , Píloro , Serotonina/fisiologíaRESUMEN
The pyloric motor system, in the crustacean stomatogastric ganglion, produces a continuously adaptive behavior. Each cell type in the neural circuit possesses a distinct yet dynamic electrical phenotype that is essential for normal network function. We previously demonstrated that the transient potassium current (I(A)) in the different component neurons is unique and modulatable, despite the fact that the shal gene encodes the alpha-subunits that mediate I(A) in every cell. We now examine the hypothesis that alternate splicing of shal is responsible for pyloric I(A) diversity. We found that alternate splicing generates at least 14 isoforms. Nine of the isoforms were expressed in Xenopus oocytes and each produced a transient potassium current with highly variable properties. While the voltage dependence and inactivation kinetics of I(A) vary significantly between pyloric cell types, there are few significant differences between different shal isoforms expressed in oocytes. Pyloric I(A) diversity cannot be reproduced in oocytes by any combination of shal splice variants. While the function of alternate splicing of shal is not yet understood, our studies show that it does not by itself explain the biophysical diversity of I(A) seen in pyloric neurons.
Asunto(s)
Empalme Alternativo/genética , Ganglios de Invertebrados/metabolismo , Movimiento/fisiología , Red Nerviosa/metabolismo , Neuronas/metabolismo , Canales de Potasio con Entrada de Voltaje , Canales de Potasio/genética , Animales , ADN Complementario/análisis , Femenino , Ganglios de Invertebrados/citología , Potenciales de la Membrana/genética , Datos de Secuencia Molecular , Nephropidae/citología , Nephropidae/genética , Nephropidae/metabolismo , Red Nerviosa/citología , Neuronas/citología , Oocitos/citología , Oocitos/metabolismo , Sistemas de Lectura Abierta/genética , Canales de Potasio/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Píloro/citología , Píloro/inervación , Píloro/fisiología , ARN Mensajero/aislamiento & purificación , Homología de Secuencia de Aminoácido , Canales de Potasio Shal , Xenopus/genética , Xenopus/metabolismoRESUMEN
Organotypic cultures of the lobster (Homarus gammarus) stomatogastric nervous system (STNS) were used to assess changes in membrane properties of neurons of the pyloric motor pattern-generating network in the long-term absence of neuromodulatory inputs to the stomatogastric ganglion (STG). Specifically, we investigated decentralization-induced changes in the distribution and density of the transient outward current, I(A), which is encoded within the STG by the shal gene and plays an important role in shaping rhythmic bursting of pyloric neurons. Using an antibody against lobster shal K(+) channels, we found shal immunoreactivity in the membranes of neuritic processes, but not somata, of STG neurons in 5 d cultured STNS with intact modulatory inputs. However, in 5 d decentralized STG, shal immunoreactivity was still seen in primary neurites but was likewise present in a subset of STG somata. Among the neurons displaying this altered shal localization was the pyloric dilator (PD) neuron, which remained rhythmically active in 5 d decentralized STG. Two-electrode voltage clamp was used to compare I(A) in synaptically isolated PD neurons in long-term decentralized STG and nondecentralized controls. Although the voltage dependence and kinetics of I(A) changed little with decentralization, the maximal conductance of I(A) in PD neurons increased by 43.4%. This increase was consistent with the decentralization-induced increase in shal protein expression, indicating an alteration in the density and distribution of functional A-channels. Our results suggest that, in addition to the short-term regulation of network function, modulatory inputs may also play a role, either directly or indirectly, in controlling channel number and distribution, thereby maintaining the biophysical character of neuronal targets on a long-term basis.
Asunto(s)
Relojes Biológicos/fisiología , Canales Iónicos/metabolismo , Neuronas Motoras/metabolismo , Neurotransmisores/metabolismo , Canales de Potasio con Entrada de Voltaje , Animales , Anticuerpos/farmacología , Relojes Biológicos/efectos de los fármacos , Membrana Celular/metabolismo , Sistema Digestivo/inervación , Ganglios de Invertebrados/citología , Ganglios de Invertebrados/efectos de los fármacos , Ganglios de Invertebrados/metabolismo , Técnicas In Vitro , Canales Iónicos/antagonistas & inhibidores , Neuronas Motoras/efectos de los fármacos , Nephropidae , Red Nerviosa/citología , Red Nerviosa/efectos de los fármacos , Red Nerviosa/metabolismo , Neuritas/metabolismo , Técnicas de Placa-Clamp , Periodicidad , Potasio/metabolismo , Bloqueadores de los Canales de Potasio , Canales de Potasio/metabolismo , Canales de Potasio ShalRESUMEN
Ion channels and receptors play critical roles in shaping neuronal activity, and thus are appropriate targets for evolutionary change to generate new behaviors. In this review, the evolution and differentiation of the many voltage-gated ion channels and transmitter-activated receptors is summarized; these channels and receptors evolved very early, and with some exceptions all species with nervous systems use similar sets of channels and receptors. Several examples are given of mechanisms for species-specific behavioral evolution that arise from mutations involving the structure, alternative splicing, level of expression, targeting and modulation of these important neural proteins.
Asunto(s)
Conducta Animal/fisiología , Evolución Biológica , Canales Iónicos/fisiología , Receptores de Superficie Celular/fisiología , Animales , Humanos , Canales Iónicos/genética , Receptores de Superficie Celular/genéticaRESUMEN
The patterned activity generated by the pyloric circuit in the stomatogastric ganglion of the spiny lobster, Panulirus interruptus, results not only from the synaptic connectivity between the 14 component neurons but also from differences in the intrinsic properties of the neurons. Presumably, differences in the complement and distribution of expressed ion channels endow these neurons with many of their distinct attributes. Each pyloric cell type possesses a unique, modulatable transient potassium current, or A-current (I(A)), that is instrumental in determining the output of the network. Two genes encode A-channels in this system, shaker and shal. We examined the hypothesis that cell-specific differences in shaker and shal channel distribution contribute to diversity among pyloric neurons. We found a stereotypic distribution of channels in the cells, such that each channel type could contribute to different aspects of the firing properties of a cell. Shal is predominantly found in the somatodendritic compartment in which it influences oscillatory behavior and spike frequency. Shaker channels are exclusively localized to the membranes of the distal axonal compartments and most likely affect distal spike propagation. Neither channel is detectably inserted into the preaxonal or proximal portions of the axonal membrane. Both channel types are targeted to synaptic contacts at the neuromuscular junction. We conclude that the differential targeting of shaker and shal to different compartments is conserved among all the pyloric neurons and that the channels most likely subserve different functions in the neuron.
Asunto(s)
Ganglios de Invertebrados/fisiología , Neuronas/fisiología , Canales de Potasio/fisiología , Secuencia de Aminoácidos , Animales , Axones/fisiología , Axones/ultraestructura , Membrana Celular/fisiología , Membrana Celular/ultraestructura , Datos de Secuencia Molecular , Nephropidae , Neuritas/fisiología , Neuritas/ultraestructura , Neuronas/ultraestructura , Canales de Potasio/análisis , Canales de Potasio/genética , Canales de Potasio de la Superfamilia Shaker , Sinapsis/fisiologíaRESUMEN
The patterns of expression of voltage gated potassium channel genes of the Shaker family have been mapped in identified neurons of the lobster (Homarus americanus) ventral nerve cord using a single cell reverse transcriptase polymerase chain reaction procedure. Using specific oligonucleotides derived from the sequences of the shaker, shab, and shaw genes of the spiny lobster, Panulirus interruptus, we detected the corresponding potassium channel DNA fragments from Homarus americanus. The Homarus DNA fragments are 87-98% identical at the nucleotide level to the Panulirus DNA fragments. We used the Panulirus primers to measure the complement of RNAs for shaker, shab, and shaw in single identified cells that use GABA, glutamate, octopamine or serotonin as chemical messengers. Shaker and shaw RNAs were found in all four identified neuron types but shab RNA was not detected in serotonin cells under the present experimental conditions. All cells expressed alpha-tubulin RNA, which serves as an internal control suggesting that cells are intact after dissection. In glial cells that surround the neuronal cell bodies, the potassium channel genes are expressed at low to non-detectable levels.
Asunto(s)
Expresión Génica , Nephropidae/metabolismo , Neuronas/metabolismo , Canales de Potasio/genética , Animales , Secuencia de Bases , Clonación Molecular , ADN Complementario , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , ARN Mensajero/genética , Homología de Secuencia de Ácido Nucleico , Canales de Potasio de la Superfamilia ShakerRESUMEN
Calcium is essential for synaptic transmission and the control of the intrinsic firing properties of neurons; this makes Ca(2+) channels a prime target for neuromodulators. A combination of multiphoton microscopy and voltage-clamp recording was used to determine the localization of voltage-dependent Ca(2+) accumulation in the two pyloric dilator (PD) neurons of the pyloric network in the spiny lobster, Panulirus interruptus, and its modulation by dopamine. We monitored [Ca(2+)](i) in fine distal branches in the neuropil >350 microm below the surface of the ganglion during controlled voltage steps in voltage clamp. Ca(2+) accumulation originated mostly from small, fairly rare, spatially restricted varicosities on distal neuritic arborizations. Ca(2+) diffused from these point sources into adjacent regions. Varicosities with similar morphology in the PD neuron have been shown previously to be sites of synaptic contacts. We have demonstrated in earlier studies that dopamine inhibits activity and greatly reduces synaptic transmission from the PD neuron. In approximately 60% of the varicosities, the voltage-activated Ca(2+) accumulation was reduced by exogenous dopamine (DA) (10(-4) M). DA decreased the peak amplitude of Ca(2+) accumulation but had no effect on the rise and decay time. We conclude that DA reduces chemical synaptic transmission from the PD neurons at least in part by decreasing Ca(2+) entry at neurotransmitter release sites.
Asunto(s)
Calcio/metabolismo , Dopamina/farmacología , Neuronas Motoras/metabolismo , Animales , Microscopía , Neuronas Motoras/efectos de los fármacos , Nephropidae , Neurópilo/metabolismo , Transmisión SinápticaRESUMEN
Technological advances in electrophysiology and molecular biology in the last two decades have led to great progress in ion channel research. The invention of the patch-clamp recording technique has enabled the characterization of the biophysical and pharmacological properties of single channels. Rapid progress in the development of molecular biology techniques and their application to ion channel research led to the cloning, in the 1980s, of genes encoding all major classes of voltage- and ligand-gated ionic channels. It has become clear that operationally defined channel types represent extended families of ionic channels. Several experimental approaches have been developed to test whether there is a correlation between the detection of particular ion channel subunit mRNAs and the electrophysiological response to a pharmacological or electrical stimulus in a cell. In one method, whole-cell patch-clamp recording is performed on a cell in culture or tissue-slice preparation. The biophysical and pharmacological properties of the ionic channels of interest are characterized and the cytoplasmic contents of the recorded cell are then harvested into the patch pipette. In a variant of this method, the physiological properties of a cell are characterized with a two-electrode voltage clamp and, following the recording, the entire cell is harvested for its RNA. In both methods, the RNA from a single cell is reverse-transcribed into cDNA by a reverse transcriptase and subsequently amplified by the polymerase chain reaction, i.e. by the so-called single-cell/reverse transcription/polymerase chain reaction method (SC-RT-PCR). This review presents an analysis of the results of work obtained by using a combination of whole-cell patch-clamp recording or two-electrode voltage clamp and SC-RT-PCR with emphasis on its potential and limitations for quantitative analysis.
Asunto(s)
Canales Iónicos/genética , Técnicas de Placa-Clamp/métodos , Reacción en Cadena de la Polimerasa/métodos , Activación Transcripcional , Animales , Cartilla de ADN , Canales Iónicos/metabolismo , Neuronas/metabolismo , ARN/biosíntesis , ARN/aislamiento & purificación , Procesamiento Postranscripcional del ARN , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Motor neurons are endowed with intrinsic and conditional membrane properties that may shape the final motor output. In the first half of this paper we present data on the contribution of I(h), a hyperpolarization-activated inward cation current, to phase-transition in motor neurons during rhythmic firing. Motor neurons were recorded intracellularly during locomotion induced with a mixture of N-methyl-D-aspartate (NMDA) and serotonin, after pharmacological blockade of I(h). I(h) was then replaced by using dynamic clamp, a computer program that allows artificial conductances to be inserted into real neurons. I(h) was simulated with biophysical parameters determined in voltage clamp experiments. The data showed that electronic replacement of the native I(h) caused a depolarization of the average membrane potential, a phase-advance of the locomotor drive potential, and increased motor neuron spiking. Introducing an artificial leak conductance could mimic all of these effects. The observed effects on phase-advance and firing, therefore, seem to be secondary to the tonic depolarization; i.e., I(h) acts as a tonic leak conductance during locomotion. In the second half of this paper we discuss recent data showing that the neonatal rat spinal cord can produce a stable motor rhythm in the absence of spike activity in premotor interneuronal networks. These coordinated motor neuron oscillations are dependent on NMDA-evoked pacemaker properties, which are synchronized across gap junctions. We discuss the functional relevance for such coordinated oscillations in immature and mature spinal motor systems.
Asunto(s)
Células del Asta Anterior/crecimiento & desarrollo , Potenciales de la Membrana/fisiología , Movimiento/fisiología , Animales , Células del Asta Anterior/efectos de los fármacos , Células del Asta Anterior/fisiología , Uniones Comunicantes/efectos de los fármacos , Uniones Comunicantes/metabolismo , Canales Iónicos/efectos de los fármacos , Canales Iónicos/metabolismo , Potenciales de la Membrana/efectos de los fármacos , Inhibición Neural/efectos de los fármacos , Inhibición Neural/fisiología , Periodicidad , RatasRESUMEN
The nervous system is evolutionarily conservative compared to the peripheral appendages that it controls. However, species-specific behaviors may have arisen from very small changes in neuronal circuits. In particular, changes in neuromodulatory systems may allow multifunctional circuits to produce different sets of behaviors in closely related species. Recently, it was demonstrated that even species differences in complex social behavior may be attributed to a change in the promoter region of a single gene regulating a neuromodulatory action.
Asunto(s)
Conducta Animal/fisiología , Evolución Biológica , Vías Nerviosas/fisiología , Neuronas/fisiología , Animales , Especificidad de la EspecieRESUMEN
The monoamines dopamine (DA), serotonin (5HT), and octopamine (Oct) can each sculpt a unique motor pattern from the pyloric network in the stomatogastric ganglion (STG) of the spiny lobster Panulirus interruptus. In this paper we investigate the contribution of individual network components in determining the specific amine-induced cycle frequency. We used photoinactivation of identified neurons and pharmacological blockade of synapses to isolate the anterior burster (AB) and pyloric dilator (PD) neurons. Bath application of DA, 5HT, or Oct enhanced cycle frequency in an isolated AB neuron, with DA generating the most rapid oscillations and Oct the slowest. When an AB-PD or AB-2xPD subnetworks were tested, DA often reduced the ongoing cycle frequency, whereas 5HT and Oct both evoked similar accelerations in cycle frequency. However, in the intact pyloric network, both DA and Oct either reduced or did not alter the cycle frequency, whereas 5HT continued to enhance the cycle frequency as before. Our results show that the major target of 5HT in altering the pyloric cycle frequency is the AB neuron, whereas DA's effects on the AB-2xPD subnetwork are critical in understanding its modulation of the cycle frequency. Octopamine's effects on cycle frequency require an understanding of its modulation of the feedback inhibition to the AB-PD group from the lateral pyloric neuron, which constrains the pacemaker group to oscillate more slowly than it would alone. We have thus demonstrated that the relative importance of the different network components in determining the final cycle frequency is not fixed but can vary under different modulatory conditions.
Asunto(s)
Monoaminas Biogénicas/fisiología , Ganglios de Invertebrados/fisiología , Nephropidae/fisiología , Red Nerviosa/fisiología , Píloro/inervación , Animales , Dopamina/farmacología , Dopamina/fisiología , Vías Eferentes/fisiología , Femenino , Masculino , Neuronas/efectos de los fármacos , Neuronas/fisiología , Octopamina/fisiología , Serotonina/fisiologíaRESUMEN
To understand the physiology of Schwann cells and myelinated nerve, we have been engaged in identifying K+ channels in sciatic nerve and determining their subcellular localization. In the present study, we examined the slo family of Ca(2+)-activated K+ channels, a class of channel that had not previously been identified in myelinated nerve. We have determined that these channels are indeed expressed in peripheral nerve, and have cloned rat homologues of slo that are more than 95% identical to the murine slo. We found that sciatic nerve RNA contained numerous alternatively spliced variants of the slo homologue, as has been seen in other tissues. We raised a polyclonal antibody against a peptide from the carboxyl terminal of the channels. Immunocytochemistry revealed that the channel proteins are in Schwann cells and are associated with canaliculi that run along the outer surface of the cells. They are also relatively concentrated near the node of Ranvier in the Schwann cell outer membrane. This staining pattern is quite similar to what we previously reported for the voltage-dependent K+ channel Kv 1.5. We did not observe staining of axons or connective tissue in the nerve and so it seems likely that most or all of the splicing variants are located in the Schwann cells. The localization of these channels also suggests that they may participate in maintaining the resting potential of the Schwann cells during K+ buffering.
Asunto(s)
Calcio/fisiología , Canales de Potasio Calcio-Activados , Canales de Potasio/metabolismo , Nervio Ciático/metabolismo , Secuencia de Aminoácidos , Animales , Formación de Anticuerpos/fisiología , ADN Recombinante , Técnica del Anticuerpo Fluorescente , Técnicas In Vitro , Subunidades alfa de los Canales de Potasio de Gran Conductancia Activados por Calcio , Canales de Potasio de Gran Conductancia Activados por el Calcio , Datos de Secuencia Molecular , Canales de Potasio/genética , Canales de Potasio/inmunología , Isoformas de Proteínas/genética , Ratas , Ratas Sprague-Dawley , Distribución Tisular/fisiologíaRESUMEN
We consider whole-cell voltage-clamp data of isolated currents characterized by the Hodgkin-Huxley paradigm. We examine the errors associated with the typical parameter estimation method for these data and show them to be unsatisfactorally large especially if the time constants of activation and inactivation are not sufficiently separated. The size of these errors is due to the fact that the steady-state and kinetic properties of the current are estimated disjointly. We present an improved parameter estimation method that utilizes all of the information in the voltage-clamp conductance data to estimate steady-state and kinetic properties simultaneously and illustrate its success compared to the standard method using simulated data and data from P. interruptus shal channels expressed in oocytes.
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Modelos Neurológicos , Neuronas/fisiología , Técnicas de Placa-Clamp/normas , Animales , ADN Complementario/farmacología , Conductividad Eléctrica , Canales Iónicos/genética , Canales Iónicos/metabolismo , Análisis de los Mínimos Cuadrados , Potenciales de la Membrana/fisiología , Oocitos/fisiologíaRESUMEN
The two pyloric dilator (PD) neurons are components [along with the anterior burster (AB) neuron] of the pacemaker group of the pyloric network in the stomatogastric ganglion of the spiny lobster Panulirus interruptus. Dopamine (DA) modifies the motor pattern generated by the pyloric network, in part by exciting or inhibiting different neurons. DA inhibits the PD neuron by hyperpolarizing it and reducing its rate of firing action potentials, which leads to a phase delay of PD relative to the electrically coupled AB and a reduction in the pyloric cycle frequency. In synaptically isolated PD neurons, DA slows the rate of recovery to spike after hyperpolarization. The latency from a hyperpolarizing prestep to the first action potential is increased, and the action potential frequency as well as the total number of action potentials are decreased. When a brief (1 s) puff of DA is applied to a synaptically isolated, voltage-clamped PD neuron, a small voltage-dependent outward current is evoked, accompanied by an increase in membrane conductance. These responses are occluded by the combined presence of the potassium channel blockers 4-aminopyridine and tetraethylammonium. In voltage-clamped PD neurons, DA enhances the maximal conductance of a voltage-sensitive transient potassium current (IA) and shifts its Vact to more negative potentials without affecting its Vinact. This enlarges the "window current" between the voltage activation and inactivation curves, increasing the tonically active IA near the resting potential and causing the cell to hyperpolarize. Thus DA's effect is to enhance both the transient and resting K+ currents by modulating the same channels. In addition, DA enhances the amplitude of a calcium-dependent potassium current (IO(Ca)), but has no effect on a sustained potassium current (IK(V)). These results suggest that DA hyperpolarizes and phase delays the activity of the PD neurons at least in part by modulating their intrinsic postinhibitory recovery properties. This modulation appears to be mediated in part by an increase of IA and IO(Ca). IA appears to be a common target of DA action in the pyloric network, but it can be enhanced or decreased in different ways by DA in different neurons.