RESUMO
1. The Mg2+ block of Na+ and Ca2+ currents through high-voltage activated (HVA; L- and N-type) Ca2+ channels was studied in chick dorsal root ganglion neurones. 2. In low extracellular [Ca2+] (< 10(-8) M) and with Na+o and Cs+i as the main charge carriers (120 mM), HVA Na+ currents started to activate at -40 mV, reached inward peak values near 0 mV and reversed at about +40 mV. 3. Addition of 30-500 microM Mg2+ to the bath caused a strong depression of inward Na+ currents that was voltage and dose dependent (KD = 39 microM in 120 mM Na+ at -10 mV). The block was maximal at negative potentials (< -70 mV) and decreased with increasing positive potentials, suggesting that Mg2+ cannot escape to the cell interior. 4. Block of Ca2+ currents by Mg2+ was also voltage dependent, but by three orders of magnitude less potent than with Na+ currents (KD = 24 mM in 2 mM Ca2+ at -30 mV). The high concentration of Mg2+ caused a prominent voltage shift of channel gating kinetics induced by surface charge screening effects. To compensate for this, Mg2+ block of inward Ca2+ currents was estimated from the instantaneous I-V relationships on return from very positive potentials (+100 mV). 5. Inward Na+ and Ca2+ tail currents following depolarization to +90 mV were markedly depressed, suggesting that channels cleared of Mg2+ ions during strong depolarization are quickly re-blocked on return to negative potentials. The kinetics of re-block by Mg2+ was too fast (< 100 microseconds) to be resolved by our recording apparatus. This implies a rate of entry for Mg2+ > 1.45 x 10(8) M-1 S-1 when Na+ is the permeating ion and a rate approximately 3 orders of magnitude smaller for Ca2+. 6. Mg2+ unblock of HVA Na+ currents at +100 mV was independent of the size of outward currents, whether Na+, Cs+ or NMG+ were the main internal cations. 7. Consistent with the idea of a high-affinity binding site for Ca2+ inside the channel, micromolar amounts of Ca2+ caused a strong depression of Na+ currents between -40 and 0 mV, which was effectively relieved with more positive as well as with negative potentials (KD = 0.7 microM in 120 mM Na+ at -20 mV). In this case, the kinetics of re-block could be resolved and gave rates of entry and exit for Ca2+ of 1.4 x 10(8) M-1 S-1 and 2.95 x 10(2) s-1, respectively. 8. The strong voltage dependence and weak current dependence of HVA channel block by divalent cations and the markedly different KD values of Na+ and Ca2+ current block by Mg2+ can be well described by a previously proposed model for Ca2+ channel permeation based on interactions between the permeating ion and the negative charges forming the high-affinity binding site for Ca2+ inside the pore (Lux, Carbone & Zucker, 1990).
Assuntos
Bloqueadores dos Canais de Cálcio/farmacologia , Canais de Cálcio/metabolismo , Cálcio/metabolismo , Magnésio/farmacologia , Neurônios Aferentes/metabolismo , Bloqueadores dos Canais de Sódio , Canais de Sódio/metabolismo , Animais , Embrião de Galinha , Estimulação Elétrica , Eletrofisiologia , Gânglios Espinais/citologia , Gânglios Espinais/efeitos dos fármacos , Gânglios Espinais/metabolismo , Ativação do Canal Iônico/efeitos dos fármacos , Cinética , Potenciais da Membrana/fisiologia , Neurônios Aferentes/efeitos dos fármacos , Técnicas de Patch-Clamp , Sódio/metabolismoRESUMO
A system for rapid, local superfusion of cultured neurones and their neurites with various different test drugs is elucidated. An area of down to 30 micron diameter was superfused with the aid of two micropipettes, one for delivering the test solution and the other for its removal. Active removal of solution within the deadspace of the delivery pipette guarantees, on the one hand, fast and flexible pressure control and, on the other, enables the quick exchange (<1 s) of multiple solutions. By increasing the pressure in the superfusion pipette, the laminar stream between the pipettes was forced down onto the cell layer. The change from bath to superfusion solutions, evaluated by liquid junction potential changes, occurs in the order of 1 ms.
Assuntos
Eletrofisiologia/métodos , Perfusão/métodos , Eletrofisiologia/instrumentação , Desenho de Equipamento , Neurônios/fisiologia , Perfusão/instrumentação , Fatores de TempoRESUMO
Reduction of voltage-activated Ca2+ currents by intracellular application of guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) through ultraviolet (UV) photolysis of the caged compound, is followed by a re-augmentation to control levels within 10 min, independently of the divalent cation used. The Ca2+ current inhibition by the gamma-aminobutyric acid type B (GABAB) receptor agonist baclofen, which is also thought to be mediated by a GTP-binding protein (G-protein), is potentiated when GTP gamma S is uncaged during agonist superfusion. The authors suggest that GTP gamma S activates G-protein-dependent pathways that are not activated by the baclofen receptor.
Assuntos
Baclofeno/farmacologia , Canais de Cálcio/efeitos dos fármacos , Guanosina 5'-O-(3-Tiotrifosfato)/farmacologia , Hipocampo/efeitos dos fármacos , Animais , Técnicas In Vitro , Cinética , RatosRESUMO
Voltage-dependent Ca2+ channels appear to constitute a central component of signal transduction cascades in neuronal growth cones. By means of spatially selective superfusion in the subcellular range growth cone, Ca2+ channels were investigated quantitatively. Both principal types of Ca2+ channels, low voltage activated and high voltage activated Ca2+ channels were present in growth cones of cells regenerating neurites in culture as well as growth cones of differentiating neuronal precursor cells. Studies concerning their spatial distribution revealed a remarkable clustering of Ca2+ channels at the growth cone. Their possible functional roles in neurite growth, axonal pathfinding, and synaptogenesis are discussed.
Assuntos
Canais de Cálcio/fisiologia , Neurônios/fisiologia , Animais , Canais de Cálcio/biossíntese , Moléculas de Adesão Celular/fisiologia , Comunicação Celular , Diferenciação Celular , Membrana Celular/fisiologia , Neuritos/fisiologia , Neuroglia/fisiologia , Neurônios/citologia , Transdução de Sinais , Sinapses/fisiologiaRESUMO
Excitatory postsynaptic long-term potentiation (LTP) was observed after restricted glutamate or metabotropic agonist application to somata and proximal neurites of identified presynaptic neurons. LTP-induction in this way entailed delays of minutes that correlated with lengths of afferent fibres. Potentiation failed to occur in cells pretreated with colchicine to degrade their microtubular transport matrix. The results suggest fast axonal transport to be a message carrier in the acquisition process of LTP.
Assuntos
Ácido Glutâmico/farmacologia , Hipocampo/efeitos dos fármacos , Hipocampo/fisiopatologia , Potenciação de Longa Duração , Neurônios/efeitos dos fármacos , Terminações Pré-Sinápticas/efeitos dos fármacos , Animais , Axônios/metabolismo , Transporte Biológico/efeitos dos fármacos , Células Cultivadas , Colchicina/farmacologia , Eletrofisiologia , Hipocampo/citologia , Ratos , Ratos WistarRESUMO
G protein-mediated downregulation of current through neuronal voltage-gated Ca2+ channels is well known. We now report that G protein activation by GTP gamma S increases the Ba2+ conductance of high-voltage-activated Ca2+ channels of chick dorsal root ganglion (DRG) cells. This occurs with a delay of minutes during which the channels are inhibited by the activated G proteins. The Ba2+ current (IBa) showed an absolute enhancement by a factor near 2, 15 min after GTP gamma S application. However, by utilizing prior observations of the voltage dependence of the inhibitory action we could demonstrate that the G protein-inhibited component of IBa, was still present. Moreover, the achieved amount of IBa disinhibition showed little variation throughout the experiments. This indicates that the increase in IBa is not due to a relief of the inhibitory action of activated G proteins but to the slow appearance of a distinct upregulating action, probably through a different pathway. Augmentation of IBa was eliminated by pertussis toxin (PTX) infusion or pretreatment, but was also prevented by intracellularly infusing protein kinase C (PKC) inhibitors together with GTP gamma S. The upregulation of neuronal Ca2+ channels thus appears to be exerted through a messenger pathway upstream of PKC activation that involves G proteins. Augmentation of Ca2+ currents (ICa) was observed only with strong intracellular [Ca2+] buffering, which suggests a control of the upregulating action by even moderate increase in intracellular [Ca2+].
Assuntos
Canais de Cálcio/fisiologia , Proteínas de Ligação ao GTP/fisiologia , Guanosina 5'-O-(3-Tiotrifosfato)/farmacologia , Neurônios/fisiologia , Animais , Bário/fisiologia , Embrião de Galinha , Eletrofisiologia , Gânglios Espinais/efeitos dos fármacos , Gânglios Espinais/fisiologia , Toxina Pertussis , Proteína Quinase C/antagonistas & inibidores , Proteína Quinase C/fisiologia , Regulação para Cima , Fatores de Virulência de Bordetella/farmacologiaRESUMO
The formation of synapses between cultured rat thalamic neurons was studied with electrophysiological and immunocytochemical methods. Thalamic neurons in culture form predominantly glutamatergic synapses. Already after 3 days in vitro glutamatergic miniature EPSCs occurred spontaneously and their frequency was strongly increased after K+ depolarization, while GABAergic mIPSCs were found after K+ depolarization at lower frequency. This demonstrates that both, excitatory glutamatergic and inhibitory GABAergic synapses were functional in close succession to initial neurite outgrowth. Synapses formed independent of spontaneous electrical activity, which was absent during the first week in culture. Spontaneous action potentials appeared during the second week and chronic action potential blockade by addition of tetrodotoxin reduced neuronal survival and the number of glutamatergic synapses per neuron. During in vitro differentiation the number of synapsin I immunoreactive presynaptic terminals and the frequency of spontaneous glutamatergic miniature EPSCs increased closely correlated, while the frequency of GABAergic mIPSCs after K+ depolarization did not increase. Thus, the continous formation of presynaptic terminals, including possible maturation of transmitter release, appeared to underlie the increase in mEPSC frequency. Analysis of miniature EPSC amplitudes at different stages in vitro revealed an increase in amplitudes, suggesting synaptic differentiation after initial establishment of functional transmission in glutamatergic synapses. This process was synapse specific as amplitudes of GABAergic mIPSCs were invariant.
Assuntos
Ácido Glutâmico/fisiologia , Neurônios/fisiologia , Sinapses/fisiologia , Potenciais de Ação/efeitos dos fármacos , Potenciais de Ação/fisiologia , Animais , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Eletrofisiologia , Processamento de Imagem Assistida por Computador , Imuno-Histoquímica , Potenciais da Membrana/fisiologia , Técnicas de Patch-Clamp , Potássio/farmacologia , Terminações Pré-Sinápticas/metabolismo , Ratos , Ratos Wistar , Tetrodotoxina/farmacologia , Tálamo/citologiaRESUMO
1. Northern blot analysis and cell transfection were used in conjunction with whole-cell current recordings to examine the involvement of the immediate early genes, c-fos and c-jun, in the expression of calcium channel currents. 2. Phaeochromocytoma cells (PC12 clone) were exposed to nerve growth factor (NGF) and to depolarizing concentrations of KCl for 60 min every day. Cells challenged with NGF developed extensive networks of neurites within 3 days. Cells depolarized periodically retained their undifferentiated morphology even after 5 days of treatment. 3. The maximal amplitude of high-voltage-activated calcium currents (ICa) increased from the control level of 117.8 +/- 48.3 (mean +/- S.D.) to 387.2 +/- 90.1 pA within 3 days of NGF treatment. omega-Conotoxin (5-10 microM) inhibited 24.6 +/- 8.5% of ICa in undifferentiated cells and 57.8 +/- 6.9% in NGF-treated cells. 4. The levels of c-fos and c-jun mRNAs increased transiently during each daily exposure to NGF. The level of c-fos mRNA also increased transiently during repeated KCl-induced depolarizations but c-jun mRNA remained low or absent. 5. Naive PC12 cells were transiently co-transfected with expression plasmids that contained the full length of c-fos and c-jun cDNA. After 2 days following transfection, the PC12 cells could be grouped according to the size of ICa. In 56% of cells, ICa was similar to control currents (106.1 +/- 37.4 pA). In the remaining 44% of cells, ICa showed a 2.2-fold enhancement with respect to control cells. Transfection of only c-fos had no effect on ICa but, in 24% of cells transfected with c-jun, ICa was 176.6 +/- 124.6 pA. Since periodic membrane depolarization induced c-fos but not c-jun mRNA, c-jun transfection was combined with a high-K+ treatment over 3 days. In 18% of treated cells, ICa was 3.7 times larger than control currents. Morphological differentiation was not observed in transfected cells. 6. In PC12 cells co-transfected with c-fos and c-jun or treated with high K+ after transfection of c-jun, omega-conotoxin (5-10 microM) inhibited 68.7 +/- 11.9% of ICa when the current amplitude was in the range of 200-600 pA. since similar concentrations of omega-conotoxin blocked 19.2 +/- 5.4% of ICa in control cells, the current increase induced by c-fos and c-jun was supported by up to 11-fold enhancement of the omega-conotoxin-sensitive component of ICa.(ABSTRACT TRUNCATED AT 400 WORDS)
Assuntos
Neoplasias das Glândulas Suprarrenais/patologia , Neoplasias das Glândulas Suprarrenais/fisiopatologia , Canais de Cálcio/fisiologia , Feocromocitoma/patologia , Feocromocitoma/fisiopatologia , Proteínas Proto-Oncogênicas c-fos/fisiologia , Proteínas Proto-Oncogênicas c-jun/fisiologia , Regulação para Cima/fisiologia , Neoplasias das Glândulas Suprarrenais/química , Animais , Northern Blotting , DNA de Neoplasias/análise , DNA de Neoplasias/genética , Relação Dose-Resposta a Droga , Regulação Neoplásica da Expressão Gênica , Fatores de Crescimento Neural/farmacologia , Células PC12 , Feocromocitoma/química , Cloreto de Potássio/farmacologia , Proteínas Proto-Oncogênicas c-fos/análise , Proteínas Proto-Oncogênicas c-fos/genética , Proteínas Proto-Oncogênicas c-jun/análise , Proteínas Proto-Oncogênicas c-jun/genética , RNA Mensageiro/análise , RNA Mensageiro/genética , Ratos , TransfecçãoRESUMO
The regulation of calcium channel currents (ICa) induced by daily stimulation (1 hr) with 10 microM glutamate was studied in full differentiated hippocampal cells in culture. We report a specific enhancement of the high-voltage-activated current type (HVA ICa) ongoing over days. The density of HVA ICa increased about twofold after the second glutamate session, and this enhancement was still observed after the fifth day of treatment, while low-voltage-activated calcium currents (LVA ICa) remained unchanged. During glutamate application, a transient increase of intracellular calcium (Cai) was observed, followed by a slow decay within 2-3 min, and substantial recovery in about 10 min. Similarly, Cai transients induced by periodic membrane depolarization mimicked the long-term effect of glutamate on ICa. These results demonstrate for the first time an increase of ICa in a time frame of days. Since the effect of glutamate on ICa was prevented by cycloheximide, neosynthesis of channel proteins presumably supports this enhancement.
Assuntos
Canais de Cálcio/fisiologia , Glutamatos/farmacologia , Hipocampo/fisiologia , Neurônios/fisiologia , 2-Amino-5-fosfonovalerato/farmacologia , Animais , Cálcio/metabolismo , Canais de Cálcio/efeitos dos fármacos , Diferenciação Celular , Células Cultivadas , Cicloeximida/farmacologia , Condutividade Elétrica/efeitos dos fármacos , Estimulação Elétrica , Eletrofisiologia/métodos , Embrião de Mamíferos , Ácido Glutâmico , Cinética , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Neurônios/efeitos dos fármacos , Cloreto de Potássio/farmacologia , Células Piramidais/efeitos dos fármacos , Células Piramidais/fisiologia , Ratos , Fatores de TempoRESUMO
The time courses of the gamma-aminobutyric acid type B (GABAB) receptor-mediated inhibition of excitatory synaptic transmission and of action potential-evoked calcium currents were studied in hippocampal neurons in vitro with step-like changes of a saturating baclofen concentration. Inhibition mediated by postsynaptic GABAB receptors was excluded pharmacologically. Both presynaptic inhibition and reduction of calcium currents developed and declined exponentially with similar time constants of about 0.2 and 3 s, respectively. The close correlation of the time courses indicates that fast, G protein-mediated depression of voltage-gated calcium channels and thus direct reduction of the presynaptic calcium influx may contribute to the GABAB receptor-induced inhibition of excitatory synaptic transmission in hippocampal neurons in vitro.
Assuntos
Baclofeno/farmacologia , Canais de Cálcio/fisiologia , Hipocampo/fisiologia , Neurônios/fisiologia , Quinoxalinas/farmacologia , Receptores de GABA-B/fisiologia , Transmissão Sináptica/fisiologia , Potenciais de Ação/efeitos dos fármacos , Potenciais de Ação/fisiologia , Animais , Canais de Cálcio/efeitos dos fármacos , Células Cultivadas , Eletrofisiologia/métodos , Embrião de Mamíferos , Potenciais Evocados/efeitos dos fármacos , Potenciais Evocados/fisiologia , Proteínas de Ligação ao GTP/metabolismo , Cinética , Ratos , Receptores de GABA-B/efeitos dos fármacos , Transmissão Sináptica/efeitos dos fármacosRESUMO
In neurones cultured from chick dorsal root ganglia, application of NH4Cl (3-45 mM) produced a transient inward current followed by a sustained current at negative holding potentials. Methylamines, hydrazine and guanidine were not able to mimic the effects of NH4Cl. The transient, but not the steady current, was inactivated during successive applications of NH4Cl. Challenge with acidic solutions (pH 6.0-6.4) or Ca(2+)-free solutions induced similar currents and abolished NH4Cl-induced transients. Exposure to 10 mM NH4Cl transiently increased cytoplasmic free Ca2+, which then fell to a sustained plateau. NH4Cl-induced membrane depolarization and the concomitant elevation in intracellular Ca2+ can play an important role in modulation of neuronal activity.
Assuntos
Cloreto de Amônio/farmacologia , Cálcio/fisiologia , Citoplasma/fisiologia , Neurônios Aferentes/fisiologia , Amilorida/farmacologia , Animais , Cádmio/farmacologia , Galinhas , Eletrofisiologia , Gânglios Espinais/citologia , Gânglios Espinais/fisiologia , Neurônios Aferentes/efeitos dos fármacosRESUMO
Digital imaging of fura-2 fluorescence and the voltage-clamp technique were combined to study cytoplasmic free Ca2+ concentration, [Ca]i, in neurons cultured from chick dorsal root ganglia. Depolarizing pulses raised [Ca]i to a new steady-state level which was achieved earlier in neurites than in the soma. The rise in [Ca]i during stimulated bursting or rhythmic activity was also faster in neurites. After stimulation [Ca]i recovered monoexponentially in the soma and biexponentially in neurites. Application of 50 mM KCl produced membrane depolarization and a concomitant increase of [Ca]i. During wash-out [Ca]i often declined to an intermediate steady-state level at which it stayed for several minutes. Thereafter the resting level of [Ca]i was quickly restored. [Ca]i recovery was delayed after treating the cell with 2 microM thapsigargin, an inhibitor of the Ca2+ pump of internal Ca2+ stores. Caffeine (10 mM) transiently increased [Ca]i. A second caffeine application produced smaller [Ca]i changes due to the prior depletion of Ca2+ stores, which could be replenished by brief exposure to KCl. Thapsigargin (2 microM) transiently increased [Ca]i both in the standard and Ca(2+)-free solution. [Ca]i transients due to caffeine and thapsigargin started in the cell interior, in contrast to [Ca]i changes evoked by membrane depolarization, which were noticed first at the cell edge. Caffeine and thapsigargin induced a transient inward current which persisted in the presence of 1 mM La3+ and in Ca(2+)-free solutions, but which was greatly diminished in Na(+)-free solutions. The effects of caffeine and thapsigargin were mutually exclusive both in the generation of [Ca]i transients and in the inward current induction.
Assuntos
Cálcio/metabolismo , Neurônios Aferentes/metabolismo , Animais , Cafeína/farmacologia , Canais de Cálcio/efeitos dos fármacos , Canais de Cálcio/metabolismo , ATPases Transportadoras de Cálcio/antagonistas & inibidores , Células Cultivadas , Embrião de Galinha , Eletrofisiologia , Gânglios Espinais/citologia , Gânglios Espinais/efeitos dos fármacos , Gânglios Espinais/metabolismo , Neuritos/efeitos dos fármacos , Neuritos/metabolismo , Neurônios Aferentes/efeitos dos fármacos , Cloreto de Potássio/farmacologia , Espectrometria de Fluorescência , Terpenos/farmacologia , TapsigarginaRESUMO
1. The kinetics of glycine-sensitive, N-methyl-D-aspartate (NMDA) receptor desensitization were investigated in cultured neurones with the patch clamp technique. 2. The degree of fast NMDA-receptor desensitization was inversely related to glycine concentration. Thus, increasing concentrations of glycine from 30 nM to 2.5 microM potentiated desensitized NMDA responses (873% +/- 101%) to a greater degree than peak responses (260% +/- 27%). 3. The desensitization was due to a decrease in the affinity of glycine for the strychnine-insensitive, glycine modulatory site (glycineB site) following activation of the NMDA-receptor complex. Thus, the A50 for glycine in potentiating peak responses (77 nM, 95% confidence limited 58-104 nM) was five fold lower than that for plateau responses (399 nM, 340-468 nM). 4. The rate of desensitization was related to glycine concentration such that a reciprocal plot of desensitization rate (1/tau S-1) against glycine concentration had a slope of 9.5* 10(6) M-1 S-1. 5. Recovery from desensitization following step increases in glycine or L-alanine concentration in the continuous presence of NMDA (200 microM) reflected the association kinetics of the glycineB agonist used. 6. The rate and degree of NMDA receptor desensitization was independent of holding potential. 7. NMDA receptor desensitization was also evident at the single channel level. 8. The glycineB antagonist 7-chlorokynurenic acid (7-Chl-Kyn 3 and 10 microM) concentration-dependently induced an identical form of desensitization in the presence of 1 microM glycine. 9. In contrast, the competitive NMDA antagonist (+/-)-amino-phosphonovaleric acid (APV 30 to 300 microM) concentration-dependently antagonized and slowed the onset kinetics of NMDA responses.
Assuntos
Glicina/farmacologia , Neurônios/metabolismo , Receptores de N-Metil-D-Aspartato/efeitos dos fármacos , 2-Amino-5-fosfonovalerato/farmacologia , Aminoácidos/farmacologia , Animais , Células Cultivadas , Feminino , Cinética , Ácido Cinurênico/análogos & derivados , Ácido Cinurênico/farmacologia , N-Metilaspartato/farmacologia , Neurônios/efeitos dos fármacos , Gravidez , Ratos , Receptores de N-Metil-D-Aspartato/antagonistas & inibidores , Colículos Superiores/efeitos dos fármacos , Colículos Superiores/metabolismo , Sinapses/efeitos dos fármacos , Sinapses/metabolismoRESUMO
An electrogenic serotonin (5-HT) uptake process was characterized in the serotonergic Retzius-P cell synapse of the leech, and the simultaneous activation of this presynaptic reuptake and the postsynaptic response was monitored during evoked transmitter release. A presynaptic, Na(+)-dependent inward current upon application of 5-HT was isolated at membrane potentials between -80 and +60 mV. Its identification as a transmitter uptake current was confirmed by monitoring accumulation of the autofluorescent 5-HT analog 5,7-dihydroxytryptamine during activation of this current. To study the kinetics of 5-HT reuptake in functional synapses, transmitter release was stimulated by flash photolysis of the Ca(2+)-caging DM-nitrophen. The results demonstrate that reuptake activates with a minimal delay of less than a millisecond during synaptic transmission. It acts as a rapid transmitter removal system to determine the time course of the postsynaptic response and monitors the kinetics of transmitter clearance at the synaptic site.
Assuntos
Neurônios/fisiologia , Serotonina/fisiologia , Sinapses/fisiologia , Transmissão Sináptica/fisiologia , 5,7-Di-Hidroxitriptamina/farmacologia , Animais , Cálcio/farmacologia , Relação Dose-Resposta a Droga , Condutividade Elétrica , Fluorescência , Cinética , Sanguessugas , Estimulação Luminosa , Serotonina/farmacologia , Sódio/antagonistas & inibidores , Sódio/fisiologia , Zimeldina/farmacologiaRESUMO
1. Numerical methods were used to evaluate voltage space-clamp performance in the investigation of a voltage-dependent inward current similar to the noninactivating Ca current. In addition, the cell is equipped with a repolarizing system, represented by leak and outwardly rectifying outward conductances. The electrotonically compact model cell is represented by a cable with an electrotonic length of 1 space constant under control conditions, but that becomes effectively only 0.33 space constants during a 90% reduction of the leak and outward conductance. The cable is perfectly voltage clamped at one end. 2. The apparent voltage dependence, activation, and inactivation of the clamp current depend on the distribution of the membrane slope conductance along the cable; this depends on 1) the distribution of the inward current along the cable and 2) the amplitude of the inward current relative to the amplitudes of the leak and voltage-dependent outward currents. 3. Under control conditions, the membrane voltage decays steeply with distance from the command voltage at the clamp site to almost resting potential for most of the rest of the cable. This is because the leak and outward current are dominant over the inward current. The inward current is activated primarily at the clamped part of the cable. Clamp currents are activated instantaneously. The clamp-current current-voltage (I-V) relation is less steep with depolarization because the membrane potential for locations away from the clamp site lags behind the clamp potential. 4. When the conductances for leak and outward current are reduced by 90%, these conductances lose their dominance. The membrane slope conductance now has a range with negative values.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Simulação por Computador , Potenciais da Membrana/fisiologia , Modelos Neurológicos , Neurônios/fisiologia , Animais , Cálcio/fisiologia , Canais de Cálcio/fisiologia , Dendritos/fisiologia , Camundongos , Minicomputadores , Canais de Potássio/fisiologia , Células de Purkinje/fisiologia , Canais de Sódio/fisiologia , SoftwareRESUMO
We recorded from pairs of cultured, synaptically connected thalamic neurons. Evoked excitatory postsynaptic currents (EPSCs) reversed at +17 mV and were blocked reversibly by 1 mM kynurenic acid, a glutamate receptor antagonist. NMDA and non-NMDA receptors mediated excitatory post-synaptic responses, as shown by selective block of EPSC components with 50 microM (+/-)-2-amino-5-phosphonopentanoic acid and 10 microM 6,7-dinitroquinoxaline-2,3-dione, respectively. Inhibitory postsynaptic responses were evoked less frequently and were blocked by the GABAA receptor antagonist (-)-bicuculline methochloride. The pharmacological profiles of whole-cell calcium currents and evoked EPSCs were compared. With 50 microM cadmium chloride (Cd), whole-cell low voltage-activated (LVA) calcium currents were reduced in amplitude and high voltage-activated (HVA) calcium currents and excitatory synaptic transmission were completely blocked. This suggests that the residual calcium influx through LVA channels into the presynaptic terminal does not suffice to trigger transmitter release. A saturating concentration of omega-conotoxin GVIA (omega-CgTx) (2.5 microM) blocked one-third of whole-cell HVA calcium currents and evoked EPSCs. The dihydropyridine nifedipine (50 microM) reversibly reduced whole-cell HVA calcium currents in a voltage-dependent manner but not excitatory synaptic transmission. Cd and omega-CgTx did not alter amplitude distributions of miniature EPSCs, demonstrating that the inhibition of synaptic transmission was due to block of presynaptic calcium channels. We conclude that excitatory glutamatergic transmission in thalamic neurons in vitro was mediated mainly by HVA calcium currents, which were insensitive to omega-CgTx and nifedipine.
Assuntos
Cálcio/fisiologia , Neurônios/fisiologia , Sinapses/fisiologia , Transmissão Sináptica , Tálamo/fisiologia , Animais , Bloqueadores dos Canais de Cálcio/farmacologia , Células Cultivadas , Eletrofisiologia , Neurotransmissores/metabolismo , Ratos , Transmissão Sináptica/efeitos dos fármacos , Tálamo/citologiaRESUMO
Fast application of L-glutamate, AMPA (alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid) or kainate to cultured rat thalamic neurons revealed properties of non-NMDA (N-methyl-D-aspartate) receptors similar to those described in hippocampal neurons. The kinetics of non-NMDA receptor-mediated currents were altered by the addition of the dye Evans Blue (EB). Macroscopic desensitization was reduced and activation and deactivation kinetics were slowed. Delayed addition of EB, after desensitization of non-NMDA receptors, resulted in reactivation of desensitized receptors. Thus, both ion channel gating and entry into the desensitized state were affected. Evans blue also slowed the activation and the decay of glutamatergic miniature EPSCs (excitatory postsynaptic currents), demonstrating that receptor kinetics determine the time course of the synaptic response.
Assuntos
Azul Evans/farmacologia , Neurônios/metabolismo , Receptores de Aminoácido/fisiologia , Sinapses/metabolismo , Tálamo/citologia , Animais , Feminino , Glutamatos/metabolismo , Ácido Ibotênico/análogos & derivados , Ácido Ibotênico/farmacologia , Ativação do Canal Iônico/efeitos dos fármacos , Gravidez , Ácido Quisquálico/farmacologia , Ratos , Receptores de Aminoácido/efeitos dos fármacos , Sinapses/efeitos dos fármacos , Ácido alfa-Amino-3-hidroxi-5-metil-4-isoxazol PropiônicoRESUMO
1. The whole-cell patch-clamp technique has been used to examine Ca channel currents carried by Ba (IBa) in rat hippocampal neurones. 2. Quinacrine selectivity decreased the high-threshold current activated by membrane depolarization from a holding potential of -70 mV. Neither the low-threshold Ca channel current nor the fast tetrodotoxin (TTX)-sensitive sodium current were affected by quinacrine. 3. Bath application of quinacrine caused a dose-dependent reduction of the peak amplitude of IBa. This effect was fast, voltage-independent, reversible and had a Kd of 30 +/- 5 microM. 4. The quinacrine-induced block did not change the time-course and the voltage dependence of IBa activation and deactivation. The inhibition revealed no use-dependence, ruling out an open channel block by quinacrine. 5. p-Bromophenacyl bromide had no effect on IBa suggesting the lack of involvement of phospholipase A2 in the action of quinacrine. In addition, the quinacrine-induced block was not related to the calmodulin pathway and internal quinacrine did not affect the peak amplitude of IBa. 6. The effect of quinacrine on the amplitude of IBa was dependent of the external pH, and suggested that only the single-protonated form of the drug can bind to the channel receptor with a Kd of 3 microM. Quinacrine and other substituted acridines can thus be useful for pharmacological and structure-activity studies of Ca channels.
Assuntos
Canais de Cálcio/efeitos dos fármacos , Hipocampo/metabolismo , Quinacrina/farmacologia , Animais , Relação Dose-Resposta a Droga , Eletrofisiologia , Hipocampo/citologia , Concentração de Íons de Hidrogênio , Técnicas In Vitro , RatosRESUMO
Previous studies resulted in conflicting conclusions that glutamate application either decreases or increases the activity of Ca2+ channels in hippocampal neurons. We studied whole-cell Ca2+ currents (ICa) in chick dorsal root ganglion neurons and rat hippocampal cells. For both cell types glutamate (1-30 microM) increased high-threshold Ca2+ current. It was independent of the charge carriers, Ca2+ or Ba2+. Low-threshold Ca2+ channel current and the fast sodium current were not changed with glutamate application. The effect developed within 1-2 min and then further facilitated after washout of the agonist. A second application of glutamate produced no additional increase in ICa. No changes in the time-course of whole-cell currents were observed, suggesting that glutamate recruits 'sleepy' Ca2+ channels. Whatever its mechanism, overlasting increase of ICa by glutamate may be important in neuronal plasticity.
Assuntos
Canais de Cálcio/efeitos dos fármacos , Glutamatos/farmacologia , Hipocampo/efeitos dos fármacos , Neurônios Aferentes/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Animais , Embrião de Galinha , Ácido Glutâmico , Hipocampo/citologia , RatosRESUMO
p21ras protein resembles the alpha subunit of trimeric G-proteins, which regulate ion channel function. We now report a modulation of Ca2+ channels in vertebrate sensory neurons by p21ras in addition to its role in cell growth and differentiation. Quantitative microinjection of oncogenic p21-H-ras into embryonic chick dorsal root ganglion neurons was performed. After 4 h the current density of the low-voltage-activated (LVA; T-type) Ca2+ channels was increased. However, in contrast to trimeric G-proteins, which inhibit high-voltage-activated (HVA) Ca2+ channels in chick dorsal root ganglion neurons, p21ras did not significantly affect HVA Ca2+ currents. To study the time course of p21ras action, guanosine triphosphate-preloaded p21ras was added to the patch pipette. Full-length ras was effective only after a delay of 20 - 30 min. C-terminal modification by cellular enzymes is required to activate full-length ras, and can account for the observed delay. Unexpectedly, C-terminal-truncated p21ras, which was found to be inactive in biological assays, enhanced LVA Ca2+ currents within minutes. This suggests a G-protein-like modulation of the LVA Ca2+ channel by p21ras. In an early phase of neuronal differentiation, dorsal root ganglion neurons express only LVA Ca2+ currents. The regulatory role of p21ras on LVA channels may therefore be particularly important during differentiation.