RESUMO
Neurons that release serotonin as a neurotransmitter project to most regions of the central and peripheral nervous system and mediate diverse neural functions. The physiological effects of serotonin are initiated by the activation of multiple, distinct receptor subtypes. Cloning in RNA expression vectors was combined with a sensitive electrophysiological assay in Xenopus oocytes in order to isolate a functional cDNA clone encoding the 5HTlc serotonin receptor. Injection of RNA transcribed in vitro from this clone into Xenopus oocytes elicits serotonin sensitivity. Mouse fibroblasts transformed with this clone bind serotonin agonists and antagonists and exhibit an increase in intracellular Ca2+ concentrations in response to serotonin. The sequence of the 5HTlc receptor reveals that it belongs to the family of G protein-coupled receptors, which are thought to traverse the cytoplasmic membrane seven times. Moreover, in situ hybridization and RNA blot analysis indicate that the 5HTlc receptor is expressed in neurons in many regions of the central nervous system and suggest that this subclass of receptor may mediate many of the central actions of serotonin.
Assuntos
Receptores de Serotonina/genética , Serotonina/fisiologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , DNA/genética , Fibroblastos/fisiologia , Regulação da Expressão Gênica , Glicoproteínas de Membrana/genética , Dados de Sequência Molecular , Oócitos/fisiologia , Fosfoproteínas/fisiologia , Ratos , Xenopus laevisRESUMO
Regulation of release of inhibitory neurotransmitter is a key element of plasticity in dorsal horn function. In this issue of Neuron, Kerchner et al. report that neurotransmitter release from inhibitory dorsal horn neurons is affected by activation of presynaptic kainate-type glutamate receptors.
Assuntos
Ácido Glutâmico/metabolismo , Dor/metabolismo , Ácido gama-Aminobutírico/metabolismo , Animais , Ácido Glutâmico/biossíntese , Células do Corno Posterior/metabolismo , Receptores de Ácido Caínico/metabolismo , Ácido gama-Aminobutírico/biossínteseRESUMO
Local inhibition within the spinal cord dorsal horn is mediated by the neurotransmitters GABA and glycine and strongly influences nociceptive and temperature signaling. Alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors are expressed by inhibitory interneurons and have been shown to modulate GABA release in other regions of the CNS. In the spinal cord, there is morphological evidence for presynaptic AMPA receptor subunits in GABAergic dorsal horn neurons, but functional data are lacking. To determine if AMPA receptors are indeed functional at presynaptic terminals of inhibitory neurons, we recorded evoked and miniature inhibitory postsynaptic currents (mIPSPs) in the superficial dorsal horn of the rat spinal cord. We show that AMPA receptor activation enhances spontaneous release of inhibitory amino acids in the presence of tetrodotoxin onto both lamina II neurons and NK1 receptor-expressing (NK1R+) lamina I neurons. This effect is sensitive to the concentration of extracellular Ca2+, yet is not fully blocked in most neurons in the presence of Cd2+, suggesting possible Ca2+ entry through AMPA receptors. Postsynaptic Ca2+ elevation is not required for these changes. AMPA-induced increases in mIPSP frequency are also seen in more mature dorsal horn neurons, indicating that these receptors may play a role in nociceptive processing in the adult. In addition, we have observed AMPA-induced depression of evoked release of GABA and glycine onto lamina I NK1R+ neurons. Taken together these data support a role for presynaptic AMPA receptors in modulating release of GABA and glycine in the superficial dorsal horn. Because inhibition in the dorsal horn is important for controlling pain signaling, presynaptic AMPA receptors acting to modulate the inhibitory inputs onto dorsal horn neurons would be expected to impact upon pain signaling in the spinal cord dorsal horn.
Assuntos
Glicina/genética , Células do Corno Posterior/metabolismo , Receptores de AMPA/fisiologia , Receptores Pré-Sinápticos/fisiologia , Medula Espinal/citologia , Ácido gama-Aminobutírico/metabolismo , Fatores Etários , Animais , Animais Recém-Nascidos , Benzodiazepinas/farmacologia , Cloreto de Cádmio/farmacologia , Cálcio/farmacologia , Bloqueadores dos Canais de Cálcio/farmacologia , Relação Dose-Resposta a Droga , Interações Medicamentosas , Estimulação Elétrica/métodos , Técnicas In Vitro , Ácido Caínico/análogos & derivados , Ácido Caínico/farmacologia , Inibição Neural/fisiologia , Inibição Neural/efeitos da radiação , Técnicas de Patch-Clamp/métodos , Ratos , Receptores de AMPA/agonistas , Receptores de AMPA/antagonistas & inibidores , Ácido alfa-Amino-3-hidroxi-5-metil-4-isoxazol Propiônico/farmacologiaRESUMO
Dorsal root ganglion (DRG) neurons first express kainate receptor subunits, predominantly GluR5, during embryonic development. In the DRG and throughout the nervous system, substantial editing of GluR5 mRNA occurs with developmental maturation (Bernard et al., 1999). The accompanying change in Ca(2+) permeability of functional kainate receptors that is the predicted outcome of this developmental regulation of mRNA editing has not been investigated. Here we report that kainate receptors on DRG neurons from late embryonic and newborn rats are predominantly Ca(2+) permeable but then become fully Ca(2+) impermeable later in the first postnatal week. Using multiple markers for nociceptor subpopulations, we show that this switch in Ca(2+) permeability is not caused by the appearance of a new subpopulation of nociceptors with different receptor properties. Instead, the change in Ca(2+) permeability matches the time course of post-transcriptional RNA editing of GluR5 at the Q/R site within the pore of the channel, indicating that the change is probably caused by developmentally regulated RNA editing. We also report that, on the basis of the strong correlation of receptor expression with expression of the surface markers LA4, isolectin B4, and LD2, kainate receptors are present on C-fiber-type neurons projecting to lamina II of spinal cord dorsal horn. These results raise the possibility that kainate receptors in their Ca(2+)-permeable form serve a developmental role in synapse formation between this population of C-fibers and their targets in the spinal cord dorsal horn. Thereafter, the receptors may serve a new function that does not require Ca(2+) permeability.
Assuntos
Cálcio/metabolismo , Nociceptores/metabolismo , Receptores de Ácido Caínico/biossíntese , Animais , Antígenos de Diferenciação/biossíntese , Diferenciação Celular , Permeabilidade da Membrana Celular/fisiologia , Células Cultivadas , Gânglios Espinais/citologia , Gânglios Espinais/efeitos dos fármacos , Gânglios Espinais/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Ácido Caínico/farmacologia , Fibras Nervosas/metabolismo , Neurônios/citologia , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Nociceptores/citologia , Células do Corno Posterior/citologia , Edição de RNA , Ratos , Receptores de Ácido Caínico/genéticaRESUMO
Excitatory postsynaptic currents (EPSCs) have been studied in voltage-clamped bullfrog sympathetic ganglion B cells. The EPSC was small, rose to a peak within 1-3 ms, and then decayed exponentially over most of its time-course. For 36 cells at --50 mV (21-23 degrees C), peak EPSC size was --6.5 +/- 3.5 nA (mean +/- SD), and the mean decay time constant tau was 5.3 +/- 0.9 ms. tau showed a small negative voltage dependence, which appeared independent of temperature, over the range --90 to --30 mV; the coefficient of voltage dependence was --0.0039 +/-0.0014 mV-1 (n = 29). The peak current-voltage relationship was linear between --120 and --30 mV but often deviated from linearity at more positive potentials. The reversal potential determined by interpolation was approximately --5 mV. EPSC decay tau had a Q10 = 3. The commonly used cholinesterase inhibitors, neostigmine and physostigmine, exhibited complex actions at the ganglia. Neostigmine (1 X 10(-5)M) produced a time-dependent slowing of EPSC decay without consistent change in EPSC size. In addition, the decay phase often deviated from a single exponential function, although it retained its negative voltage dependence. With 1 x 10(-6) M physostigmine, EPSC decay was slowed by the decay phase remained exponential. At higher concentrations of physostigmine, EPSC decay was markedly prolonged and was composed of at least two decay components. High concentrations of atropine (10(-5) to 10(-4) M) produced complex alterations in EPSC decay, creating two or more exponential components; one decay component was faster and the other was slower than that observed in untreated cells. These results suggest that the time-course of ganglionic EPSC decay is primarily determined by the kinetics of the receptor-channel complex rather than hydrolysis or diffusion of transmitter away from the postsynaptic receptors.
Assuntos
Gânglios Simpáticos/fisiologia , Sinapses/fisiologia , Animais , Anuros , Atropina/farmacologia , Inibidores da Colinesterase/farmacologia , Gânglios Simpáticos/efeitos dos fármacos , Técnicas In Vitro , Potenciais da Membrana/efeitos dos fármacos , Neostigmina/farmacologia , Fisostigmina/farmacologia , Rana catesbeiana , Sinapses/efeitos dos fármacosRESUMO
Molecular, biochemical and physiological evidence for the existence of releasable Ca2+ stores in neurons is strong. There are two separate molecules that function as release channels from those Ca2+ stores, the RyanR and InsP3R, and both have multiple regulatory sites for positive and negative control. Perhaps most intriguing is the biphasic, concentration-dependent action of cytosolic Ca2+ on both channels, first to stimulate release then, at higher concentration, to depress release. Whether the InsP3R and RyanR channels regulate Ca2+ release from different or identical functional compartments will need to be defined for each neuron type and perhaps even for each intracellular region within neurons since the evidence for functional separation of stores is mixed. The identification of Ca2+ storage and releasing capacity throughout all subcellular regions of neurons and the increasing evidence for a role for Ca2+ stores in neuronal plasticity suggests that the further characterization of the functional properties of Ca2+ stores will be an increasingly important and expanding area of interest in neurobiology.
Assuntos
Cálcio/metabolismo , Inosina Trifosfato/metabolismo , Neurônios/metabolismo , Animais , Cálcio/fisiologia , Canais de Cálcio/efeitos dos fármacos , Canais de Cálcio/metabolismo , HumanosRESUMO
Sensory neurons express purinergic P2X receptors on their central and peripheral terminals as well as their cell bodies. ATP activation of these receptors drives action potential firing and glutamate release with potentially important consequences for sensory function. Here we show ATP-gated currents activated in cultured embryonic dorsal root ganglion neurons have heterogeneity of time-courses comparable to those observed in different subpopulations of acutely dissociated adult dorsal root ganglion neurons. The distribution of time-courses across the population of cultured neurons is strongly influenced by culture conditions. Heterogeneity in ATP current kinetics occurs even though immunocytochemical staining reveals a relatively homogeneous and widespread expression of the P2X2 and P2X3 subunits. We show that the time-courses of ATP-gated currents recorded at the cell bodies are mirrored by the time-courses of transmitter release from the dorsal root ganglion nerve terminals, indicating similar P2X receptor properties on the soma and their associated terminals. Our results illustrate a functional heterogeneity of P2X receptor-mediated currents that is strongly influenced by external factors. This heterogeneity in current kinetics may have implications for neuronal function as it constrains the time-course of ATP-mediated modulation of neurotransmitter release at sensory nerve terminals.
Assuntos
Trifosfato de Adenosina/farmacologia , Gânglios Espinais/efeitos dos fármacos , Gânglios Espinais/fisiologia , Neurônios/efeitos dos fármacos , Neurônios/fisiologia , Animais , Células Cultivadas , Resistência a Medicamentos , Condutividade Elétrica , Embrião de Mamíferos , Gânglios Espinais/citologia , Ácido Glutâmico/metabolismo , Imuno-Histoquímica , Cinética , Ratos , Receptores Purinérgicos P2/metabolismo , Receptores Purinérgicos P2X2 , Receptores Purinérgicos P2X3 , Fatores de TempoRESUMO
Dorsal root ganglion neurons express functional AMPA and kainate receptors near their central terminals. Activation of these receptors causes a decrease in glutamate release during action potential evoked synaptic transmission. Due to differences in kinetic properties and expression patterns of these two families of glutamate receptors in subpopulations of sensory neurons, AMPA and kainate receptors are expected to function differently. We used embryonic dorsal root ganglion (DRG) neurons maintained in culture to compare functional properties of kainate and AMPA receptors. Most DRG neurons in culture expressed kainate receptors and about half also expressed AMPA receptors. Most AMPA and kainate receptor-expressing DRG neurons were sensitive to capsaicin, suggesting involvement of these glutamate receptors in nociception. When activated by kainate, AMPA receptors were capable of driving a sustained train of action potentials while kainate receptors tended to activate action potential firing more transiently. Glutamate elicited more action potentials and a larger steady-state depolarization in neurons expressing both AMPA and kainate receptors than in neurons expressing only kainate receptors. Adding to their more potent activation properties, AMPA receptors recovered from desensitization much more quickly than kainate receptors. Activation of presynaptic receptors by low concentrations of kainate, but not ATPA, caused a tetrodotoxin-sensitive increase in the frequency of spontaneous EPSCs recorded in dorsal horn neurons. By recording synaptic pairs of DRG and dorsal horn neurons, we found that activation of presynaptic kainate and AMPA receptors decreased evoked glutamate release from terminals of DRG neurons in culture. Our data suggest that the endogenous ligand, glutamate, will cause a different physiological impact when activating these two types of non-NMDA glutamate receptors at central or peripheral nerve endings of sensory neurons.
Assuntos
Neurônios Aferentes/fisiologia , Receptores de AMPA/metabolismo , Receptores de Ácido Caínico/metabolismo , Potenciais de Ação/efeitos dos fármacos , Potenciais de Ação/fisiologia , Animais , Capsaicina/farmacologia , Células Cultivadas , Embrião de Mamíferos , Potenciais Pós-Sinápticos Excitadores/efeitos dos fármacos , Potenciais Pós-Sinápticos Excitadores/fisiologia , Gânglios Espinais/efeitos dos fármacos , Gânglios Espinais/fisiologia , Ácido Glutâmico/metabolismo , Ácido Glutâmico/farmacologia , Neurônios Aferentes/efeitos dos fármacos , Técnicas de Patch-Clamp , Ratos , Receptores de AMPA/efeitos dos fármacos , Receptores de Ácido Caínico/efeitos dos fármacosRESUMO
Excitatory amino acid receptor distribution was mapped on acutely dissociated neurons from postnatal rat spinal cord dorsal horn. N-methyl D-aspartate, quisqualate and kainate were applied to multiple locations along the somal and dendritic surfaces of voltage-clamped neurons by means of a pressure application system. To partially compensate for the decrement of response amplitude due to current loss between the site of activation on the dendrite and the recording electrode at the soma, a solution containing 0.15 M KCl was applied on the cell bodies and dendrites of some cells to estimate an empirical length constant. In the majority of the cells tested, the dendritic membrane had regions of higher sensitivity to excitatory amino acid agonists than the somatic membrane, with dendritic response amplitudes reaching more than seven times those at the cell body. A comparison of the relative changes in sensitivity between each combination of two of the three excitatory amino acid agonists along the same dendrite showed different patterns of agonist sensitivity along the dendrite in the majority of the cells. These data were obtained from dorsal horn neurons that had developed and formed synaptic connections in vivo. They demonstrate that in contrast to observations made on ventral horn neurons, receptor density for all the excitatory amino acid receptors on dorsal horn neurons, including the N-methyl-D-aspartate receptor, are generally higher on the dendrites than on the soma. Further, these results are similar to those obtained from dorsal horn neurons grown in culture.
Assuntos
Animais Recém-Nascidos/metabolismo , Neurônios/metabolismo , Receptores de Aminoácido/metabolismo , Medula Espinal/metabolismo , Animais , Dendritos/efeitos dos fármacos , Dendritos/ultraestrutura , Eletrofisiologia , Técnicas In Vitro , Ácido Caínico/farmacologia , N-Metilaspartato/farmacologia , Neurônios/ultraestrutura , Potássio/farmacologia , Ácido Quisquálico/farmacologia , Ratos , Ratos Sprague-Dawley , Receptores de Aminoácido/efeitos dos fármacos , Medula Espinal/ultraestruturaRESUMO
We have investigated the role of NMDA receptors in mediating synaptic transmission in spinal cord lamina II over the first 2 weeks of postnatal development. High intensity root stimulation evoked D-APV-sensitive slow synaptic activity in lamina II neurons that drove action potential firing. This NMDA receptor-mediated activity was enhanced when bicuculline and strychnine were used to block synaptic inhibition. When activated by repetitive focal stimulation, synaptic activity mediated by NMDA receptors alone drove action potential firing. NMDA receptors were also able to drive action potential firing at synapses where AMPA receptors were present but blocked. Our data show that in lamina II of the dorsal horn, NMDA receptors significantly affect neuronal excitability even in the absence of co-activation of AMPA receptors.
Assuntos
Animais Recém-Nascidos/fisiologia , Receptores de N-Metil-D-Aspartato/fisiologia , Medula Espinal/fisiologia , Potenciais de Ação/fisiologia , Animais , Bicuculina/farmacologia , Estimulação Elétrica/métodos , Potenciais Pós-Sinápticos Excitadores/efeitos dos fármacos , Potenciais Pós-Sinápticos Excitadores/fisiologia , Antagonistas GABAérgicos/farmacologia , Glicinérgicos/farmacologia , Técnicas In Vitro , Inibição Neural/efeitos dos fármacos , Inibição Neural/fisiologia , Técnicas de Patch-Clamp , Ratos , Raízes Nervosas Espinhais/fisiologia , Estricnina/farmacologia , Sinapses/efeitos dos fármacos , Sinapses/fisiologiaRESUMO
Inward currents to glutamate receptor agonists, quisqualate (QA), kainate (KA) and N-methyl-D-aspartate (NMDA) were examined in spinal dorsal horn neurons by whole-cell voltage-clamp techniques after acute dissociation. Neurons were dissociated from the superficial dorsal horn (laminae I/II) of the adult rat (8-16 weeks old) spinal cords by enzymatic and mechanical treatment. The KA-induced current was sustained during KA application, while the QA- and NMDA-induced currents were attenuated. The NMDA response was augmented dose-dependently by addition of glycine (10(-7)-5 X 10(-6) M) and became obscure in the absence of glycine. The NMDA current was depressed by D-2-amino-5-phosphonovaleric acid (APV). Analyses of dose-response curves of these inward currents indicate that both the QA and KA currents were competitively blocked by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), while the NMDA current was blocked non-competitively.
Assuntos
Canais Iônicos/fisiologia , Neurônios/metabolismo , Receptores de Neurotransmissores/fisiologia , Medula Espinal/citologia , 2-Amino-5-fosfonovalerato/farmacologia , 6-Ciano-7-nitroquinoxalina-2,3-diona , Animais , Animais Recém-Nascidos/fisiologia , Ácido Caínico/farmacologia , N-Metilaspartato/farmacologia , Fibras Nervosas/metabolismo , Fibras Nervosas/fisiologia , Quinoxalinas/farmacologia , Ácido Quisquálico/farmacologia , Ratos , Receptores de GlutamatoRESUMO
This study tested quantitatively the degree to which selective activation of NMDA receptors increases the intracellular concentration of free Ca2+ ions ([Ca2+]i) in neurons isolated from the rat spinal cord dorsal horn and grown in culture. The results show that activation of NMDA receptors, without additional depolarizing stimuli, produce [Ca2+]i increases in the neuronal cell bodies in the presence of physiological concentrations of Mg2+. Furthermore, this phenomenon occurs during synaptic activation of NMDA receptors. Thus, it cannot be assumed that in physiological extracellular [Mg2+], there is an absolute requirement for additional membrane depolarization before NMDA receptor activation stimulates significant increases in [Ca2+]i.
Assuntos
Cálcio/metabolismo , Neurônios/metabolismo , Receptores de AMPA/metabolismo , Receptores de N-Metil-D-Aspartato/fisiologia , Medula Espinal/metabolismo , Animais , Biotransformação/fisiologia , Células Cultivadas , Gânglios Espinais/citologia , Gânglios Espinais/metabolismo , Magnésio/metabolismo , Magnésio/fisiologia , Ratos , Medula Espinal/citologia , Sinapses/fisiologiaRESUMO
The effects of suramin and pyridoxal-phosphate-6-azophenyl-2',4'-disulfonic acid (PPADS) on glutamatergic synaptic transmission were studied on dorsal horn lamina II neurons of rat spinal cord slice preparation and cultured dorsal horn neurons. Suramin at 100 microM significantly suppressed the amplitude of the evoked excitatory postsynaptic currents (EPSCs) by 33%, miniature EPSC (mEPSC) amplitude was decreased by 46% and the mEPSC frequency also decreased by 41%. PPADS at 50 microM had little effect on either the evoked EPSCs or mEPSCs. The lack of effect of PPADS on glutamatergic synaptic transmission suggests that the effect of suramin is less likely to be mediated by P2x receptors. When whole-cell (+/-)-alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) currents evoked by glutamate were examined, both suramin and PPADS showed no inhibition of peak amplitude. However, the onset of glutamate-evoked whole-cell currents became significantly slowed by suramin but not by PPADS. The suppression of synaptic transmission by suramin may be due, in part, to the slowed onset of glutamate-evoked AMPA currents. These results suggest that the analgesic effects of suramin shown in cancer patients and animal pain models may not be solely due to its antagonism to purinoceptors. PPADS is probably a more suitable antagonist for the study of synaptic P2x receptor function at excitatory synapses mediated by AMPA receptors.
Assuntos
Glutamatos/fisiologia , Neurônios/efeitos dos fármacos , Fosfato de Piridoxal/análogos & derivados , Medula Espinal/efeitos dos fármacos , Suramina/farmacologia , Transmissão Sináptica/efeitos dos fármacos , Animais , Animais Recém-Nascidos , Potenciais Evocados/efeitos dos fármacos , Potenciais Pós-Sinápticos Excitadores/efeitos dos fármacos , Técnicas In Vitro , Neurônios/citologia , Antagonistas do Receptor Purinérgico P2 , Fosfato de Piridoxal/farmacologia , Ratos , Medula Espinal/citologia , Medula Espinal/fisiologia , Fatores de Tempo , Ácido alfa-Amino-3-hidroxi-5-metil-4-isoxazol Propiônico/farmacologiaAssuntos
Neurônios Aferentes/metabolismo , Neurotransmissores/metabolismo , Receptores de Ácido Caínico/metabolismo , 6-Ciano-7-nitroquinoxalina-2,3-diona/farmacologia , Potenciais de Ação/efeitos dos fármacos , Animais , Capsaicina/farmacologia , Células Cultivadas , Antagonistas de Aminoácidos Excitatórios/farmacologia , Gânglios Espinais/efeitos dos fármacos , Gânglios Espinais/embriologia , Ácido Caínico/farmacologia , Ratos , Receptores de Ácido Caínico/antagonistas & inibidores , Medula Espinal/efeitos dos fármacos , Medula Espinal/embriologiaAssuntos
Sistema Nervoso Central/fisiologia , Hipófise/fisiologia , Hormônio Liberador de Tireotropina/farmacologia , Ácido gama-Aminobutírico/farmacologia , Células Cultivadas , Cloretos/metabolismo , Células Clonais , Hipocampo/efeitos dos fármacos , Humanos , Canais Iônicos/fisiologia , Cinética , Ligantes , Potenciais da Membrana/efeitos dos fármacos , Hipófise/efeitos dos fármacos , Potássio/metabolismo , Relação Estrutura-Atividade , Ácido gama-Aminobutírico/metabolismoAssuntos
Aminoácidos/farmacologia , Cálcio/metabolismo , Neurônios/efeitos dos fármacos , Animais , Células Cultivadas , Ácido Caínico/farmacologia , N-Metilaspartato/farmacologia , Neurônios/metabolismo , Ratos , Receptores de Aminoácido , Receptores de Superfície Celular/efeitos dos fármacos , Receptores de Superfície Celular/metabolismo , Medula Espinal/efeitos dos fármacos , Medula Espinal/metabolismoRESUMO
Spinal cord neurons were dissociated from 13-day embryonic mice and grown in culture for 1-28 days. Sodium currents of neurons in culture for 1-2 days were compared with those in culture for 2-4 weeks, using the whole-cell voltage clamp method. Rapid neurite outgrowth created space clamp limitations so that unclamped neuritic sodium action potentials prevented accurate analysis of sodium current properties. Therefore neurons were bathed in sodium-free solution and brief puffs of sodium were delivered to the cell soma so that only somatic sodium currents were recorded. Sodium currents of neurons at 1-2 days in culture had voltage-dependent activation and inactivation characteristic of these channels, both in mature cultured spinal neurons and in other preparations. However, the estimated channel density on the soma of neurons 1-2 days in culture was less than two channels per micron2. Since the available sodium conductance (as measured by action potential rise rates) increases during development of spinal cord neurons in culture (Westbrook and Brenneman, 1984), we suggest that changes in channel density and/or distribution, rather than in channel kinetics, may underlie the increase in sodium conductance.
Assuntos
Canais Iônicos/fisiologia , Sódio/fisiologia , Medula Espinal/fisiologia , Potenciais de Ação , Animais , Células Cultivadas , Condutividade Elétrica , Potenciais da Membrana , Camundongos , Medula Espinal/citologia , Fatores de TempoRESUMO
1. Excitatory amino acid (EAA) receptors mediate synaptic transmission in many areas of the vertebrate CNS. To map the distribution of the EAA receptors, three agonists selective for EAA receptor subtypes [kainate, quisqualate, and N-methyl-D-aspartate (NMDA)] were applied by pressure to the cell bodies and neurites of voltage-clamped, embryonic rat spinal cord neurons in culture. 2. Current loss along the neurite between the site of activation and the recording electrode at the soma was estimated independently of variations in receptor density by focal application of high [K+] solution. This estimate was used to compensate agonist-evoked responses for current loss due to leak. K(+)-evoked current amplitudes progressively decreased as applications were made at more distal positions along the neurite. 3. Response amplitudes to EAAs showed a monotonic decay, similar to that with high [K+] solution, in only a few of the cells tested with each agonist. The majority of neurons had areas of high agonist sensitivity along the neurites, implying a nonuniform density of receptors. Such regions sometimes occurred at the most proximal segment of the neurite. Most neurites had distal regions of agonist sensitivity that ranged from 40 to 300% higher than at the soma. 4. The relative distributions of the three EAA receptors were compared by applying two agonists to the same sites along neurites and observing variations in normalized response amplitudes. When comparing NMDA versus non-NMDA receptor distributions on the same neurite, approximately 40% of the cells had similar patterns to two agonists, and the remaining 60% had different patterns. When sensitivity to the two non-NMDA agonists kainate and quisqualate were compared, about one-half of the cells tested had different patterns of agonist sensitivity. 5. Areas of high sensitivity persisted after block of calcium channels by addition of La3+ to the bath solution and after prevention of evoked transmitter release by a low [Ca2+]/high [Mg2+] solution. 6. These results show that spinal cord neurons can have regions of high agonist sensitivity to NMDA, kainate, and quisqualate along their neurites and that the sensitivity to any one of the EAA receptor subtypes can be elevated independently of the others.
Assuntos
Neurônios/metabolismo , Receptores de Superfície Celular/metabolismo , Medula Espinal/metabolismo , Animais , Axônios/efeitos dos fármacos , Axônios/fisiologia , Canais de Cálcio/fisiologia , Células Cultivadas , Eletrofisiologia , Embrião de Mamíferos/citologia , Embrião de Mamíferos/metabolismo , Ativação do Canal Iônico , Ácido Caínico/farmacologia , N-Metilaspartato/farmacologia , Neurônios/ultraestrutura , Neurotransmissores/metabolismo , Ácido Quisquálico/farmacologia , Ratos , Receptores de Aminoácido , Medula Espinal/citologia , Medula Espinal/embriologia , Distribuição TecidualRESUMO
1. The effects of lanthanum ions (La3+) on voltage-gated calcium currents (VGCCs) and excitatory amino acid (EAA)-evoked currents were characterized using cultured or acutely dissociated neurons from the dorsal horn of the rat spinal cord. 2. VGCCs evoked by depolarizing voltage steps were reversibly blocked by La3+ with an apparent log dissociation constant Kd of 163 nM. 3. La3+ antagonism of currents evoked by NMDA was less potent, with an EC50 (half-maximal effective concentration) of 2 microM. The block of NMDA-evoked currents was voltage independent and non-competitive with respect to activation of the NMDA receptor. 4. La3+ had both enhancing and blocking actions on currents evoked by kainate or by quisqualate; concentrations of La3+ between 1 and 100 microM enhanced kainate- and quisqualate-evoked currents, while the currents were blocked by concentrations of La3+ greater than 100 microM. Both the blocking and the enhancing actions of La3+ were independent of membrane potential. 5. An enhancing dose of La3+ shifted the dose-response curve for kainate to lower concentrations of agonist without changing the maximum evoked current, and a similar leftward shift of the quisqualate dose-response curve occurred at non-saturating concentrations of quisqualate. This enhancement might occur either due to increased affinity of the receptor for ligand, or by increased concentration of ligand at the membrane surface; the latter effect could result from a reduction in the membrane surface charge. 6. The divalent cation Zn(2+)-mimicked the effects of La3+ on excitatory amino acid-evoked currents in dorsal horn neurons, but was less potent both as a blocker and as an enhancer. This suggests that La3+ and Zn2+ could act with different potencies at the same site or sites, and that La3+ may be a useful probe for the mechanisms of Zn2+ effects. 7. Since La3+ enhances kainate- and quisqualate-evoked responses at the same concentrations at which it suppresses VGCCs (and NMDA-gated currents), it can be a useful probe for separating VGCC activation from kainate- and quisqualate-induced depolarizations in experiments where voltage clamp is impractical.
Assuntos
Canais de Cálcio/efeitos dos fármacos , Ativação do Canal Iônico/efeitos dos fármacos , Lantânio/farmacologia , Medula Espinal/fisiologia , Animais , Células Cultivadas , Relação Dose-Resposta a Droga , Ácido Caínico/farmacologia , Potenciais da Membrana/efeitos dos fármacos , N-Metilaspartato/farmacologia , Ácido Quisquálico/farmacologia , Ratos , Zinco/farmacologia , Ácido gama-Aminobutírico/farmacologiaRESUMO
Painful stimuli to the skin initiate action potentials in the peripheral terminals of dorsal root ganglion (DRG) neurons. These action potentials propagate to DRG central terminals in the dorsal horn of the spinal cord, evoking release of excitatory transmitters such as glutamate onto postsynaptic dorsal horn neurons. P2X receptors, a family of ligand-gated ion channels activated by the endogenous ligand ATP, are highly expressed by DRG neurons. Immunoreactivity to P2X receptors has been identified in the dorsal horn superficial laminae associated with nociceptive DRG central terminals, suggesting the presence of presynaptic P2X receptors. Here we have used a DRG-dorsal horn co-culture system to show that P2X receptors are localized at presynaptic sites on DRG neurons; that activation of these receptors results in increased frequency of spontaneous glutamate release; and that activation of P2X receptors at or near presynaptic DRG nerve terminals elicits action potentials that cause evoked glutamate release. Thus activation of P2X receptors at DRG central terminals can modify sensory signal throughput, and might even initiate sensory signals at central synapses without direct peripheral input. This putative central modulation and generation of sensory signals may be associated with physiological and pathological pain sensation, making presynaptic P2X receptors a possible target for pain therapy.