RESUMO
Physiological stimuli operative during, for example, dehydration or lactation, induce neurohypophysial astrocytes (pituicytes) to undergo reversible morphological changes, which in turn may modulate the release of vasopressin and oxytocin. To study the molecular mechanisms of this morphological plasticity, we used primary cultures of rat pituicytes. During stimulation with adenosine, pituicytes become stellate, which is characterized by a round, phase-bright soma and complex arborization, implying major cytoskeletal modifications. Following addition of vasopressin or oxytocin, stellate pituicytes revert to a flat shape. The effects of both hormones are mediated by V(1a) receptor activation, which also induces biphasic Ca(2+) (i) signals in pituicytes. Stellation reversal requires Ca(2+)-dependent activation of Cdc42, a small GTPase known to impact on the cytoskeleton. V(1a) receptor activation by vasopressin or oxytocin also stimulates [(3)H]taurine efflux from cultured pituicytes. As taurine inhibits vasopressin output from neurohypophysial terminals, we postulate a negative-feedback mechanism whereby secreted vasopressin limits its own availability. This stop signal might be reinforced by shape changes elicited by vasopressin in pituicytes. These results support the concept that, during specific physiological states, pituicyte V(1a) receptor activation modulates the release of neurohypophysial hormones.
Assuntos
Hipófise/metabolismo , Hormônios Hipofisários/metabolismo , Receptores de Vasopressinas/metabolismo , Animais , RatosRESUMO
To obtain definitive evidence for a physiological allosteric modulatory role for endogenous brain ALLO on GABA(A) receptor function, we studied GABA(A) receptor activity under conditions in which the concentration of endogenous brain ALLO was decreased by about 80% for longer than 5 h following the administration of SKF 105111- 17beta-17-[bis (1methylethyl) amino carbonyl] androstane-3,5-diene-3-carboxylic acid (SKF), a potent inhibitor of 5alpha-reductases Type I and II. We used the in situ patch-clamp technique to record GABA-evoked currents and spontaneous inhibitory postsynaptic currents (sIPSCs) from pyramidal neurons in neocortical slices of vehicle- or SKF-treated mice. The potency, but not the efficacy, of exogenously applied GABA was decreased in slices from mice treated with SKF. When neocortical slices were treated in vitro for 3 h with 10 microM SKF, ALLO was also reduced (25-30%) and in addition, the GABA dose-response curve was shifted to the right; however this shift was not as marked as the shift in the slices obtained from mice treated with SKF, in keeping with the smaller decrease of the ALLO content in these slices. Furthermore, direct application of ALLO to these slices shifted the dose-response curve of GABA back toward a non-SKF treated profile. We then analyzed GABAergic sIPSCs in neocortical slices obtained from vehicle or SKF-treated mice. Mean decay time and charge transfer were significantly reduced by SKF treatment. The decay of sIPSCs was best fitted by two exponentials, but only the fast component was decreased in the SKF group. Direct application of ALLO (100 nM) normalizes the sIPSC kinetics in slices from ALLO depleted mice. No changes were detected in the amplitude or frequency of sIPSCs. These data demonstrate that endogenous ALLO physiologically regulates spontaneously induced Cl(-) current by acting on a specific recognition site, which is probably located on GABA(A) receptors (a receptor on a receptor), thereby prolonging inhibitory currents by facilitating conformational transition of the GABA-gated Cl(-) channel to an open state.
Assuntos
Pregnanolona/fisiologia , Receptores de GABA-A/fisiologia , Inibidores de 5-alfa Redutase , Regulação Alostérica , Androstanos/farmacologia , Animais , Canais de Cloreto/efeitos dos fármacos , Canais de Cloreto/fisiologia , GABAérgicos/farmacologia , Técnicas In Vitro , Masculino , Camundongos , Neocórtex/efeitos dos fármacos , Neocórtex/metabolismo , Técnicas de Patch-Clamp , Pregnanolona/metabolismo , Pregnanolona/farmacologia , Receptores de GABA-A/efeitos dos fármacos , Ácido gama-Aminobutírico/farmacologiaRESUMO
It is becoming increasingly evident that activation of ionotropic glutamate receptors leads to significant changes in cytosolic [K+] ([K+]c), a major determinant of the plasma membrane (PM) potential, Em. Since Em affects fluxes of key cations, such as Ca2+, it is important to precisely quantify [K+]c and Em in neurons exposed to glutamate receptor agonists. Here we studied the relationships between [K+]c and Em in primary cultures of cerebellar granule cells, and found that kainate elicits a rapid drop in [K+]c below 10 mM. Using patch electrodes containing 10 or 150 mM K+, we determined that kainate depolarizes the PM to -2 or -28 mV, respectively. Therefore, the actual PM depolarization elicited by kainate is much larger than that routinely measured with K+-rich electrodes.
Assuntos
Cerebelo/efeitos dos fármacos , Agonistas de Aminoácidos Excitatórios/farmacologia , Ácido Caínico/farmacologia , Potássio/metabolismo , Animais , Animais Recém-Nascidos , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Células Cultivadas , Cerebelo/citologia , Cerebelo/metabolismo , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Ratos , Ratos Sprague-DawleyRESUMO
Allopregnanolone (ALLO), a potent positive-allosteric modulator of the action of GABA at GABA(A) receptors, is synthesized in the brain from progesterone by the sequential action of two enzymes: 5alpha-reductase and 3alpha-hydroxysteroidoxidoreductase. The concentration of ALLO in various parts of the mouse brain varies substantially, from 15 pmol/g in the olfactory bulb, to approximately 6 pmol/g in the frontoparietal cortex, and 2.7 pmol/g in the cerebellum. The systemic administration of 48 micromol/kg of the Type I and Type II 5alpha-reductase inhibitor, (17beta)-17-[bis (1-methylethyl) amino carbonyl)] androsta-3, 5-diene-3-carboxylic acid (SKF 105,111), reduced brain ALLO content by 80-90% in 30 min; the rate constant (k) of ALLO decrease in each brain area can be utilized to establish the rate of ALLO biosynthesis, which is higher in the olfactory bulb (62 pmol/g/h) than in the frontoparietal cortex (24 pmol/g/h) or cerebellum (11 pmol/g/h). The duration of the righting reflex loss elicited by the potent GABA(A) receptor agonist muscimol was reduced in SKF 105,111-treated ALLO-depleted mice. SKF 105,111 treatment had no effect on muscimol metabolism or on brain levels of pregnenolone and progesterone; however, the brain levels of 5alpha-DHP, the precursor of ALLO, were also decreased. Administration of ALLO at a dose of 15 micromol/kg i.p. by itself did not alter the muscimol-induced loss of the righting reflex; but it completely blocked the effect of SKF 105,111. To elucidate the possible molecular mechanism by which a decrease of brain ALLO content can shorten the duration of the righting reflex loss elicited by muscimol, we patch-clamped neocortical pyramidal neurons of mice pretreated with SKF 105,111 or vehicle, and studied the efficiency of muscimol in eliciting Cl- currents. The current amplitude was significantly smaller in neurons from SKF 105,111-treated mice, especially at lower doses (0.1-1 microM) of muscimol, and the muscimol dose-response (0.1-10 microM) relationship displayed cooperativity (nH=1.4). These data suggest that ALLO synthesized in brain plays an important physiological permissive role in the modulation of GABA-gated Cl- channel function.
Assuntos
Encéfalo/metabolismo , Pregnanodionas/metabolismo , Pregnanolona/biossíntese , Receptores de GABA-A/metabolismo , 5-alfa-Di-Hidroprogesterona , Animais , Encéfalo/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Agonistas GABAérgicos/farmacologia , Masculino , Camundongos , Muscimol/farmacologia , Células Piramidais/efeitos dos fármacos , Células Piramidais/fisiologia , Receptores de GABA-A/efeitos dos fármacos , Transmissão Sináptica/efeitos dos fármacos , Transmissão Sináptica/fisiologiaRESUMO
Cajal-Retzius (CR) cells were discovered at the end of the 19th century but, surprisingly, the exploration of their physiological properties is only now beginning, as we near the end of the 20th century. A few papers addressing these properties have appeared recently, but incomplete data generally give the arguably misleading impression that CR cells are similar to other neocortical neurons, and therefore may perform analogous functions. It is one of the motives of this review to dispel such conceptions. Although CR cells display features of 'regular' neurons, including excitability and responsiveness to neurotransmitters, their function is probably limited to the primary implementation of cortical circuits. A strong indication in support of this idea is the fact that CR cells appear at the onset of neocorticogenesis and disappear at the end of neuronal migration.
Assuntos
Potenciais de Ação/fisiologia , Neocórtex/citologia , Neurônios/fisiologia , Potenciais de Ação/efeitos dos fármacos , Animais , N-Metilaspartato/farmacologia , Neocórtex/efeitos dos fármacos , Neurônios/citologia , Neurônios/efeitos dos fármacos , Ratos , Receptores de GABA/efeitos dos fármacos , Receptores de GABA/fisiologia , Receptores de Glutamato/efeitos dos fármacos , Receptores de Glutamato/fisiologia , Ácido gama-Aminobutírico/farmacologiaRESUMO
Although an important secretory function of Cajal-Retzius (CR) cells has been discovered recently, the precise electrical status of these cells among other layer I neurons in particular and in cortical function in general is still unclear. In this paper, early postnatal CR cells from rat neocortex were found to express an inactivating K current whose molecular substrate is likely to be the Kv1.4 channel. Both electrophysiological and immunocytochemical experiments revealed that expression of this A-type current is down-regulated in vivo and virtually disappears by the end of the second postnatal week. At this time, CR cells have become capable of evoked repetitive firing, and their action potentials are larger and faster, yet these electrical properties still appear incompatible with a role in cortical network function, as inferred from comparisons with other cortical neurons. Also at this time, a large proportion of CR cells display spontaneous spiking activity, which suggests the possibility of additional roles for these cells. We conclude that the loss of A channels along with an increase in Na channel density shape the changes in excitability of postnatal CR cells, in terms of both the patterns of evoked firing and the emergence of spontaneous spiking.
Assuntos
Animais Recém-Nascidos/fisiologia , Neocórtex/fisiologia , Neurônios/fisiologia , Potássio/fisiologia , Potenciais de Ação/fisiologia , Envelhecimento/fisiologia , Animais , Animais Recém-Nascidos/crescimento & desenvolvimento , Regulação para Baixo/fisiologia , Condutividade Elétrica , Imuno-Histoquímica , Neocórtex/citologia , Ratos , Ratos Endogâmicos F344 , Tempo de Reação/fisiologiaRESUMO
Allopregnanolone [3alpha-hydroxy-5alpha-pregnan-20-one] (ALLO), a potent neurosteroid that positively modulates gamma-aminobutyric acid (GABA) action at various GABA(A) receptor subtypes is synthesized in nanomolar concentrations and stored non uniformly in various brain structures of mammals. We have measured brain ALLO content and its precursors by negative ion chemical ionization-mass-spectrometry after purification and separation of the different steroids with HPLC and gas chromatography. Our procedure measures steroids in the femtomolar range with structural information and unsurpassed selectivity. We were able to establish an association between the decrease in content of ALLO in mouse brain cortex elicited by either long-lasting social isolation or by the administration of 17beta-17 [bis (1-methylethyl) amino carbonyl] androstane-3,5-dilene-3-carboxylic acid (SKF 105111). an inhibitor of Types I and II 5alpha reductases, and the shortening of the righting reflex loss elicited by pentobarbital (PBT). SKF 105111 added to cortical brain slices in concentrations up to 10(-5) M failed per se to alter GABAergic currents or their potentiation by PTB recorded from pyramidal neurons. Fluoxetine (1.45 or 2.9 micromol/kg i.p.) doses that fail to change the PTB-induced loss of righting reflex and the level of brain ALLO in group-housed mice normalized both parameters in socially-isolated mice. In addition, we could detect both fluoxetine actions in socially isolated mice pretreated with doses of p-chlorophenylalanine (1.2 mmol/kg i.p. at 72, 48, and 24 h) that substantially inhibit brain serotonin 5HT synthesis as shown by an 80% drop of brain 5HT content. These studies for the first time have provided evidence suggesting that the endogenous cortical stores of ALLO physiologically upregulate GABAergic tone and by such a mechanism play a permissive or facilitatory role on the PTB-induced loss of the righting reflex. In the absence of such a permissive physiological influence by endogenous ALLO, the righting reflex inhibition by PTB is down regulated.
Assuntos
Pentobarbital/farmacologia , Pregnanolona/metabolismo , Reflexo Anormal/efeitos dos fármacos , Inibidores de 5-alfa Redutase , Animais , Fluoxetina/farmacologia , Fluoxetina/uso terapêutico , Masculino , Camundongos , Córtex Pré-Frontal/efeitos dos fármacos , Córtex Pré-Frontal/metabolismo , Reflexo Anormal/fisiologia , Inibidores Seletivos de Recaptação de Serotonina/farmacologia , Inibidores Seletivos de Recaptação de Serotonina/uso terapêuticoRESUMO
Using in situ patch-clamp techniques in rat telencephalic slices, we have followed resting potential (RP) properties and the functional expression of NMDA receptors in neocortical Cajal-Retzius (CR) cells from embryonic day 18 to postnatal day 13, the time around which these cells normally disappear. We find that throughout their lives CR cells have a relatively depolarized RP (approximately -50 mV), which can be made more hyperpolarized (approximately -70 mV) by stimulation of the Na/K pump with intracellular ATP. The NMDA receptors of CR cells are subjected to intense postnatal upregulation, but their similar properties (EC50, Hill number, sensitivity to antagonists, conductance, and kinetics) throughout development suggest that their subunit composition remains relatively homogeneous. The low RP of CR cells is within a range that allows for the relief of NMDA channels from Mg2+ blockade. Our findings are consistent with the hypothesis that CR cells may degenerate and die subsequent to uncontrolled overload of intracellular Ca2+ via NMDA receptor activation by ambient glutamate. In support of this hypothesis we have obtained evidence showing the protection of CR cells via in vivo blockade of NMDA receptors with dizocilpine.
Assuntos
Potenciais da Membrana/fisiologia , Neurônios/metabolismo , Neurônios/fisiologia , Receptores de N-Metil-D-Aspartato/metabolismo , 2-Amino-5-fosfonovalerato/farmacologia , Potenciais de Ação/fisiologia , Trifosfato de Adenosina/farmacologia , Envelhecimento , Animais , Morte Celular/efeitos dos fármacos , Córtex Cerebral/efeitos dos fármacos , Córtex Cerebral/fisiologia , Maleato de Dizocilpina/farmacologia , Embrião de Mamíferos , Antagonistas de Aminoácidos Excitatórios/farmacologia , Imuno-Histoquímica , Técnicas In Vitro , Meglumina/farmacologia , Potenciais da Membrana/efeitos dos fármacos , N-Metilaspartato/agonistas , N-Metilaspartato/metabolismo , Neurônios/efeitos dos fármacos , Fármacos Neuroprotetores/farmacologia , Técnicas de Patch-Clamp , Piperazinas/farmacologia , Ratos , Ratos Endogâmicos F344 , Receptores de N-Metil-D-Aspartato/genética , Receptores de N-Metil-D-Aspartato/fisiologia , Tetrodotoxina/farmacologia , Regulação para Cima/fisiologiaRESUMO
Using appropriate internal standards, quantitative reverse transcripase-polymerase chain reaction (RT-PCR), and cerebellar granule cell (CG) in primary cultures we have quantified the expression of mRNAs encoding for GluR1-4 DL-alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptor subunits during neuronal maturation in vitro. GluR1 is the mRNA that increases during CG maturation; the expression changes of the other GluR mRNAs are minimal and the translation products of these mRNAs change with a similar pattern. During CG maturation, there is an 8- to 10-fold increase in the GluR1 FLOP mRNA and a twofold increase in the expression of FLOP mRNA for GluR4 and GluR4C. The GluR1 FLIP mRNA increases, but by a smaller extent. We found that the GluR2 mRNA is completely edited at its Q/R site during CG maturation. The increase on the expression of GluR1 FLIP and FLOP and of GluR4 FLOP mRNA variants during development is associated with a 10-fold increase in AMPA-mediated Na+ currents and in the increased amplification of this current by 7-chloro-3-methyl-3,4 dihydro-2H-1,2,4 benzothiadiazine S-S-dioxide (IDRA21) or by 6-chloro-3,4 dihydro-3-(2-norbornen-5-yl)-7-sulfamoyl-1,2,4-benzothiadiazine 1,1 dioxide (cyclothiazide [CT]).
Assuntos
Processamento Alternativo/genética , Benzotiadiazinas/farmacologia , Cerebelo/citologia , Regulação da Expressão Gênica no Desenvolvimento , Neurônios/citologia , Receptores de AMPA/metabolismo , Animais , Cálcio/metabolismo , Diferenciação Celular , Células Cultivadas , Cerebelo/efeitos dos fármacos , Eletrofisiologia , Canais Iônicos/antagonistas & inibidores , Canais Iônicos/metabolismo , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Nitrendipino/farmacologia , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Edição de RNA/genética , Ratos , Ratos Sprague-Dawley , Receptores de AMPA/agonistas , Receptores de AMPA/genética , Receptores de AMPA/imunologia , Sódio/metabolismo , Ácido alfa-Amino-3-hidroxi-5-metil-4-isoxazol Propiônico/farmacologiaRESUMO
1. To characterize membrane properties that might be relevant to the function and fate of Cajal-Retzius (CR) cells, the pharmacological and physiological effects of GABA acting at GABAA receptors were studied in CR cells from embryonic (E18) and postnatal (P11-13) slices of rat neocortex. 2. From the embryonic to the postnatal stage, GABA-induced maximum current almost tripled, the EC50 increased from 38 to 74 microM, and the Hill number increased from 1.4 to 1.9. Muscimol-elicited currents were qualitatively and quantitatively similar to those produced by GABA. 3. GABA-induced changes in the amplitude of large-conductance Ca2+-activated K+ channel current recorded on-cell from E18 CR cells were consistent with depolarization. 4. GABA-mediated depolarization of embryonic and postnatal CR cells was studied directly with perforated-patch recording techniques. Ten micromolar and 1 mM GABA, respectively, depolarized E18 CR cells to -27 +/- 1 and -25 +/- 3 mV. These same concentrations of GABA depolarized P11 CR cells to -36 +/- 3 and -23 +/- 3 mV. 5. In postnatal cortex, GABA (100 microM) increased the firing rate of CR cells 7.3-fold. By contrast, the firing of hippocampal pyramidal cells from slices of the same age (P12) was totally and reversibly blocked by GABA. 6. These experiments suggest that contrary to its postnatal inhibitory shift observed in other cells, the depolarizing effect of GABA remains in CR cells from E18 until their virtual disappearance.
Assuntos
Hipocampo/citologia , Receptores de GABA-A/fisiologia , Ácido gama-Aminobutírico/farmacologia , Animais , Animais Recém-Nascidos , Cálcio/fisiologia , Eletrofisiologia , Feminino , Agonistas GABAérgicos/farmacologia , Hipocampo/efeitos dos fármacos , Hipocampo/embriologia , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Muscimol/farmacologia , Técnicas de Patch-Clamp , Canais de Potássio/efeitos dos fármacos , Canais de Potássio/fisiologia , Gravidez , Ratos , Receptores da Bradicinina/metabolismo , Receptores de GABA-A/efeitos dos fármacosRESUMO
Using in situ patch-clamp techniques, we have studied K current expression in rat telencephalon from embryonic day 12 to 21. For cells recorded in the ventricular zone, the K current consisted of a delayed rectifier and a large-conductance calcium-activated component, and displayed little variation from embryonic day 12 to 21. Cells recorded in pial regions could be separated into two classes: radially oriented, putatively migrating cells, and cells tangentially oriented in layer I, which were assumed to be Cajal-Retzius cells. When using a voltage-clamp protocol that included a prepulse to -120 mV, Cajal-Retzius cells displayed a larger density of total K current than radial cells, and both types revealed an inactivating component (IA). The proportion of this component increased from embryonic day 18 to 21 in both cell types, although the amplitude of total K current, in the respective cell type, did not vary. This suggested a concomitant decrease in delayed rectifier current, which was verified directly with an appropriate protocol. The activation rate of the delayed rectifier current was slower for ventricular zone cells than for radial or Cajal-Retzius cells. IA was studied in Cajal-Retzius cells and displayed a strikingly negative (approximately -100 mV) voltage of half-maximal steady-state inactivation. Tetraethylammonium ions only blocked the non-inactivating component(s) of K current whereas 4-aminopyridine appeared to decrease both inactivating and non-inactivating components. The quantitative changes in K current expression are likely to underlie the overall increase in excitability of differentiating cells. On the other hand, the observation of qualitative differences among channel properties opens an interesting area of investigation into their physiological significance.
Assuntos
Córtex Cerebral/embriologia , Canais de Potássio de Abertura Dependente da Tensão da Membrana , Canais de Potássio/fisiologia , Potássio/metabolismo , 4-Aminopiridina/farmacologia , Fatores Etários , Animais , Cálcio/farmacologia , Diferenciação Celular/fisiologia , Córtex Cerebral/química , Córtex Cerebral/citologia , Canais de Potássio de Retificação Tardia , Feminino , Feto/química , Feto/citologia , Feto/fisiologia , Corantes Fluorescentes , Isoquinolinas , Potenciais da Membrana/efeitos dos fármacos , Neurônios/química , Neurônios/citologia , Neurônios/fisiologia , Técnicas de Cultura de Órgãos , Técnicas de Patch-Clamp , Bloqueadores dos Canais de Potássio , Gravidez , Ratos , Ratos Sprague-Dawley , Tetraetilamônio/farmacologiaRESUMO
By using single-channel recording techniques, we measured the conductance (gK) of the Ca(2+)-activated Maxi-K+ channel from the embryonic rat brain, and examined its dependence on K+ ions present in equimolar concentrations on both sides of the membrane patch. With ionic strength maintained constant by substitution of N-methyl-D-glucamine for K+, gK has a sigmoidal dependence upon [K+]. This result has been obscured in previous work by variations in ionic strength, which has a marked effect on single-channel conductance, especially in the limit for which this variable approaches zero. The gK versus [K+] relationship is described, theoretically, by a three-barrier, two-binding-site model in which the barrier that an ion must cross to leave the channel is decreased as [K+] is increased.
Assuntos
Encéfalo/fisiologia , Canais de Potássio Cálcio-Ativados , Canais de Potássio/fisiologia , Potássio/farmacologia , Animais , Condutividade Elétrica , Embrião de Mamíferos , Cinética , Canais de Potássio Ativados por Cálcio de Condutância Alta , Meglumina/farmacologia , Potenciais da Membrana/efeitos dos fármacos , Concentração Osmolar , Técnicas de Patch-Clamp , Canais de Potássio/efeitos dos fármacos , RatosRESUMO
The pharmacological and biophysical properties of large-conductance Ca-activated K (BK) channels from embryonic rat telencephalic neuroepithelium were investigated with in situ patch-clamp techniques. A fraction of these channels exhibited properties characteristic of BK channels recorded in well differentiated cells, including normal gating mode (BKN channels). The vast majority of BK channels expressed distinctive properties, the most conspicuous being their buzz gating mode (BKB channels). BKB channels were insensitive to a concentration of charybdotoxin that completely and reversibly blocked BKN channels. In contrast with the strict dependence of BKN channel activation on cytoplasmic Ca, BKB channels displayed substantially high open probability (Po) after inside-out patch excision in a Ca-free medium. Intracellular trypsin down-regulated the Po of BKB channels, which then exhibited a greater sensitivity to cytoplasmic Ca, mainly in the positive direction (increased Po with increased Ca). This suggested a modulatory role for Ca as opposed to its gating role in BKN channels. Ca ions also reduced current amplitude of both types of channels. BKB channels were less voltage sensitive than BKN channels, but this was not correlated with their lower Ca sensitivity. We speculate that BKB channels may represent immature forms in the developmental expression of BK channels.
Assuntos
Cálcio/farmacologia , Canais de Potássio/efeitos dos fármacos , Canais de Potássio/fisiologia , Telencéfalo/citologia , Animais , Epitélio/química , Ativação do Canal Iônico/efeitos dos fármacos , Ativação do Canal Iônico/fisiologia , Neurônios/química , Técnicas de Patch-Clamp , Ratos , Telencéfalo/química , Compostos de Tetraetilamônio/farmacologia , Tripsina/farmacologiaRESUMO
We have investigated the effects of intracellular K+ and Rb+ on single-channel currents recorded from the large-conductance Ca(2+)-activated K+ (BK) channel of the embryonic rat telencephalon using the inside-out patch-clamp technique. Our novel observation concerns the effects of these ions on rapid flickering of channel openings. Specifically, flicker gating was voltage dependent, i.e., it was reduced by depolarization in the -60 to -10 mV range with equimolar concentrations of K+ ions (150 Ko+/150 Ki+). Removal of Ki+ resulted in significant flickering at all potentials in this voltage range. In other words, the voltage dependence of flicker gating was effectively eliminated by the removal of Ki+. This suggests that a K+ ion entering the channel from the intracellular medium binds, in a voltage-dependent manner, at a site that locks the flicker gate in its open position. No effects of changes in Ki+ were observed on the primary, voltage-dependent gate of the channel. The change in flickering did not cause a change in the mean burst duration, which indicates that the primary gate is stochastically independent of the flicker gate. Intracellular Rb+ can substitute for--and is even more effective than--Ki+ with regard to suppression of flickering. Substitution of Rbi+ for Ki+ also increased the mean burst duration for V > or = -30 mV. Both effects of Rbi+ were removed by membrane hyperpolarization.
Assuntos
Cálcio/metabolismo , Canais de Potássio/metabolismo , Potássio/metabolismo , Rubídio/metabolismo , Animais , Fenômenos Biofísicos , Biofísica , Embrião de Mamíferos/metabolismo , Técnicas In Vitro , Líquido Intracelular/metabolismo , Ativação do Canal Iônico , Cinética , Potenciais da Membrana , Modelos Biológicos , Potássio/farmacologia , Canais de Potássio/química , Canais de Potássio/efeitos dos fármacos , Ratos , Rubídio/farmacologia , Telencéfalo/metabolismoRESUMO
1. In situ patch-clamp recordings of early embryonic large-conductance K+ channels consistently revealed (86% of patches) a complex behaviour characterized by noisy fluctuations between open and closed states of relatively short duration. 2. This behaviour is similar to the buzz mode, a type of gating observed only very rarely in some channels. Its prevalence in proliferative neuroepithelium may thus constitute a criterion of immaturity. 3. In 89% of patches, the buzz mode was converted to a 'normal' mode by intracellular exposure to trypsin. 4. These observations suggest that the immature channel protein includes (or is affected by) a cytoplasmic component, the presence or absence of which determines certain sets of conformational transitions.
Assuntos
Ativação do Canal Iônico/fisiologia , Canais de Potássio/fisiologia , Telencéfalo/metabolismo , Tripsina/farmacologia , Animais , Cálcio/fisiologia , Feminino , Hidrólise , Técnicas In Vitro , Cinética , Potenciais da Membrana/efeitos dos fármacos , Técnicas de Patch-Clamp , Canais de Potássio/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Telencéfalo/citologia , Telencéfalo/embriologiaRESUMO
A period of hypersensitivity to N-methyl-D-aspartate (NMDA) has been described during the early development of different types of neuron. Since activation of NMDA receptors can also induce rapid neuron death, the hypersensitivity to NMDA may be tightly controlled. In the present study we show that mouse cerebellar granule neurons become transiently hypersensitive to NMDA between days 10 and 14 after plating in a culture medium containing 30 mM K+. The NMDA sensitivity is higher when cells are cultured in the presence of an NMDA receptor antagonist [30 mM K+ plus 100 microM 3-((+/-)-2-carboxypiperazine-4-yl)-propyl-1-phosphonic acid (CPP)], and no hypersensitivity is observed when cells are cultured in the continuous presence of NMDA (12.5 mM K+ plus 100 microM NMDA). The high NMDA sensitivity in control cells is associated with a higher density of NMDA receptors than that measured in NMDA-treated cells, suggesting that the sensitivity to NMDA may be partly controlled by activity-dependent NMDA receptor down-regulation. We also examined the level of NMDA-zeta 1 mRNA and found no correlation between this parameter and the transient pattern of NMDA sensitivity. Such NMDA receptor plasticity may be of importance in the central nervous system, protecting developing cells from excitotoxicity at critical developmental stages.
Assuntos
Cerebelo/efeitos dos fármacos , Ácido Glutâmico/farmacologia , N-Metilaspartato/farmacologia , Plasticidade Neuronal/fisiologia , Receptores de N-Metil-D-Aspartato/análise , Animais , Células Cultivadas , Cerebelo/citologia , Cerebelo/crescimento & desenvolvimento , CamundongosRESUMO
Using the cell-attached configuration of the patch-clamp technique, we recorded large-conductance K channels (LCKs) from intact telencephala and cortical slices of embryonic (E) 12-14 and E21 rats, respectively. Conductance (about 200 pS with K(+)-rich pipettes), kinetics and sensitivity to fatty acids (FAs) were reminiscent of some features of the Ca-activated 'maxi-K' channel (BK), yet less than half the LCKs were Ca-sensitive. At E12-14, an even smaller proportion of the channels were strictly voltage-sensitive, that proportion rising to about 50% at E21. Ca and voltage sensitivities were observed independent of one another. The open probability of voltage-sensitive LCKs increased exponentially with depolarization rates similar to those reported for classical BKs. It is postulated that embryonic LCKs may constitute immature forms of BKs whose gating is regulated by endogenous FAs.
Assuntos
Desenvolvimento Embrionário e Fetal , Canais de Potássio/fisiologia , Telencéfalo/fisiologia , 8-Bromo Monofosfato de Adenosina Cíclica/farmacologia , Animais , Ácido Araquidônico/farmacologia , Cafeína/farmacologia , Calcimicina/farmacologia , GMP Cíclico/análogos & derivados , GMP Cíclico/farmacologia , Condutividade Elétrica , Embrião de Mamíferos , Feminino , Idade Gestacional , Técnicas In Vitro , Ativação do Canal Iônico , Cinética , Ácido Linoleico , Ácidos Linoleicos/farmacologia , Potenciais da Membrana/efeitos dos fármacos , Ácido Mirístico , Ácidos Mirísticos/farmacologia , Neurônios/fisiologia , Técnicas de Patch-Clamp , Gravidez , Ratos , Ratos Sprague-Dawley , Telencéfalo/embriologia , Fatores de TempoRESUMO
Using whole-cell patch-clamp techniques in situ (whole-tissue and tissue slices), we have studied two aspects of rat telencephalic cell development during the period of embryogenesis starting at E12. The first aspect was related to junctional coupling as revealed by low input resistance, intercellular dye spread and pharmacologic blockade. Coupling appeared to decrease with time, both in extent and occurrence. The second aspect dealt with cell excitability as revealed by voltage-dependent Na current (INa) expression. Immature action potentials and their underlying INaS were present in a small proportion of E12 cells. These currents were blocked 36% and 78% by 10(-7) M and 10(-6) M tetrodotoxin (TTX), respectively. From then onward, INaS got larger and more prevalent while no obvious changes in kinetics were observed. At E21, INaS were abolished by 10(-7) M TTX and channel density apparently was sufficient to support overshooting yet still immature action potentials.
Assuntos
Comunicação Celular , Embrião de Mamíferos/citologia , Embrião de Mamíferos/fisiologia , Desenvolvimento Embrionário e Fetal , Canais de Sódio/fisiologia , Telencéfalo/embriologia , Animais , Eletrofisiologia , Ratos , Canais de Sódio/efeitos dos fármacos , Tetrodotoxina/farmacologiaRESUMO
1. Calcium currents (ICa) were studied in voltage-clamped NCB-20 cells. In undifferentiated cells, voltage steps from hyperpolarized potentials (-80/-100 mV) essentially revealed transient ICa showing characteristics classically described for "T-type" channels. In about 50% of the cells, there was a residual current at the end of the step; no ICa was elicited from a holding potential of -50 mV. 2. In contrast, 100% of the cells differentiated with dibutyryl cyclic AMP (cAMP) displayed a residual current in addition to the transient one, and depolarizing steps from a holding potential of -50 mV induced a sustained current. In these cells, Bay K 8644 elicited both a negative shift in voltage dependence and a moderate increase of the sustained component. 3. Although these changes in Ca2+ channel physiology result from chemically induced differentiation, they might not be directly related to the concomitant morphologic differentiation. 4. In undifferentiated NCB-20 cells, T-type Ca2+ currents can be elicited in relative isolation.
Assuntos
Canais de Cálcio/fisiologia , Cálcio/metabolismo , Neurônios/fisiologia , Éster Metílico do Ácido 3-Piridinacarboxílico, 1,4-Di-Hidro-2,6-Dimetil-5-Nitro-4-(2-(Trifluormetil)fenil)/farmacologia , Animais , Bucladesina/farmacologia , Canais de Cálcio/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Cricetinae , Cricetulus , Células Híbridas , Ativação do Canal Iônico/efeitos dos fármacos , Potenciais da Membrana/efeitos dos fármacos , Camundongos , Neuroblastoma , Neurônios/citologia , Neurônios/efeitos dos fármacos , Células Tumorais CultivadasRESUMO
The activation of 5-HT3 receptors by 5-HT and 2-methyl-5-HT was studied with 'concentration-jump' techniques in voltage-clamped N1E-115 cells grown in culture. When applied to single cells with a fast perfusion technique, both agonists induced currents the on-rate kinetics of which were concentration-dependent. Based on the time constants of current kinetics and subsequent estimates of agonist association and dissociation rates, an apparent Kd of 1.3 microM was determined for 5-HT, a value in agreement with binding and functional studies. Receptor activation by 2-methyl-5-HT was slower, consistent with its lower potency as compared to the parent compound. In addition, the rise time of 2-methyl-5-HT-mediated currents was affected by hyperpolarizing membrane potential. The results show evidence of different molecular behaviors for the two agonists.