RESUMEN
Although the impact of aging on the function of the central nervous system is known, only a limited amount of information is available about accompanying changes affecting the cellular composition of the brain and spinal cord. In the present work we used the isotropic fractionator method to reveal aging-associated changes in the numbers of neuronal and non-neuronal cells harbored by the brain and spinal cord. The experiments were performed on 15-week, 7-month, 13-month, and 25-month-old female mice. The major parts of the brain were studied separately, including the isocortex, hippocampus, cerebellum, olfactory bulb, and the remaining part (i.e., 'rest of brain'). The proliferative capacity of each structure was assessed by counting the number of Ki-67-positive cells. We found no aging-dependent change when the cellular composition of the isocortex was studied. In contrast, the neuronal and non-neuronal cell numbers of the hippocampus decreased in the 7-25-month period. The neuronal cell number of the olfactory bulb showed positive age-dependence between 15 weeks and 13 months of age and presented a significant decrease thereafter. The cerebellum was characterized by an age-dependent decrease of its neuronal cell number and density. In the rest of brain, the non-neuronal cell number increased with age. The neuronal and non-neuronal cell numbers of the spinal cord increased, whereas its neuronal and non-neuronal densities decreased with age. The number of proliferating cells showed a marked age-dependent decrease in the hippocampus, olfactory bulb, and rest of the brain. In contrast, the number of Ki-67-positive cells increased with age in both the cerebellum and spinal cord. In conclusion, aging-dependent changes affecting the cellular composition of the mouse central nervous system are present but they are diverse and region-specific.
Asunto(s)
Envejecimiento/patología , Encéfalo/citología , Médula Espinal/citología , Animales , Peso Corporal , Recuento de Células , Femenino , Técnicas para Inmunoenzimas , Antígeno Ki-67/metabolismo , Ratones , Ratones Endogámicos C57BLRESUMEN
AIM: The immunosuppressive drug tacrolimus has several effects on the central nervous system. Besides its protective effect in hearing deficiencies, it is also considered to be able to cause tinnitus. In the present work, we attempted to describe its effects on a characteristic synapse of the auditory system that may be involved in the pathogenesis of tinnitus. METHODS/MATERIALS: Slices of the dorsal cochlear nucleus (200 microm thick) were prepared from 9- to 14-day-old Wistar rats. In response to stimulation targeting the superficial layer of the nucleus, we recorded excitatory postsynaptic currents (EPSCs) developing in the cell bodies of the pyramidal neurons using whole-cell voltage clamps. Inhibitory synaptic activity was inhibited by the application of bicuculline and strychnine. Short-term plasticity was investigated using high-frequency stimulation (50 Hz). Unambiguous identification of the investigated neurons was ensured by employing biocytin in the pipette solution, which allowed the confocal reconstruction of the cells after the functional measurements. A concentration of 1 micromol/L tacrolimus was applied extracellularly. RESULTS: Tacrolimus effectively and reversibly inhibited glutamatergic neurotransmission in the investigated synapse from -145 +/- 26 pA to -55 +/- 15 pA (n = 7; P = .00928). In contrast, EPSC amplitudes without failures were not significantly reduced (from -153 +/- 26 pA to -131 +/- 23 pA) in the presence of tacrolimus, but there were increased failure numbers of synaptic transmission. These data suggested that application of tacrolimus produced a combined pre- and postsynaptic inhibition. CONCLUSION: Tacrolimus affected short-term synaptic plasticity in the rat dorsal cochlear nucleus. It was also capable of inhibiting the glutamatergic neurotransmission. These effects suggested that tacrolimus may have neuroprotective effects in this structure.
Asunto(s)
Vías Auditivas/efectos de los fármacos , Núcleo Coclear/fisiología , Fibras Nerviosas/fisiología , Células Piramidales/fisiología , Transmisión Sináptica/fisiología , Tacrolimus/farmacología , Animales , Vías Auditivas/fisiología , Núcleo Coclear/efectos de los fármacos , Estimulación Eléctrica , Potenciales Postsinápticos Excitadores/efectos de los fármacos , Potenciales Postsinápticos Excitadores/fisiología , Procesamiento de Imagen Asistido por Computador , Inmunosupresores/farmacología , Microscopía Confocal , Fibras Nerviosas/efectos de los fármacos , Neuronas/efectos de los fármacos , Neuronas/fisiología , Técnicas de Placa-Clamp , Células Piramidales/citología , Células Piramidales/efectos de los fármacos , Ratas , Ratas Wistar , Sinapsis/efectos de los fármacos , Sinapsis/fisiología , Transmisión Sináptica/efectos de los fármacosRESUMEN
Although cholinergic modulation of the cochlear nucleus (CN) is functionally important, neither its cellular consequences nor the types of receptors conveying it are precisely known. The aim of this work was to characterise the cholinergic effects on giant cells of the CN, using electrophysiology and quantitative polymerase chain reaction. Application of the cholinergic agonist carbachol increased the spontaneous activity of the giant cells; which was partly the consequence of the reduction in a K(+) conductance. This effect was mediated via M4 and M3 receptors. Cholinergic modulation also affected the synaptic transmission targeting the giant cells. Excitatory synaptic currents evoked by the stimulation of the superficial and deep regions of the CN were sensitive to cholinergic modulation: the amplitude of the first postsynaptic current was reduced, and the short-term depression was also altered. These changes were mediated via M3 receptors alone and via the combination of M4, M2 and M3 receptors, when the superficial and deep layers, respectively, were activated. Inhibitory synaptic currents evoked from the superficial layer showed short-term depression, but they were unaffected by carbachol. In contrast, inhibitory currents triggered by the activation of the deep parts exhibited no significant short-term depression, but they were highly sensitive to cholinergic activation, which was mediated via M3 receptors. Our results indicate that pre- and postsynaptic muscarinic receptors mediate cholinergic modulation on giant cells. The present findings shed light on the cellular mechanisms of a tonic cholinergic modulation in the CN, which may become particularly important in evoking contralateral excitatory responses under certain pathological conditions.
Asunto(s)
Núcleo Coclear/metabolismo , Neuronas/metabolismo , Receptores Muscarínicos/metabolismo , Animales , Carbacol/farmacología , Colinérgicos/farmacología , Núcleo Coclear/efectos de los fármacos , Núcleo Coclear/fisiología , Electrofisiología , Femenino , Potenciales Postsinápticos Inhibidores/efectos de los fármacos , Potenciales Postsinápticos Inhibidores/fisiología , Masculino , Neuronas/efectos de los fármacos , Neuronas/fisiología , Técnicas de Cultivo de Órganos , Ratas , Ratas Wistar , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Although the hyperpolarization-activated non-specific cationic current (I(h)) plays important roles in determining the membrane characteristics of the spiral ganglion cells (SGCs), neither the exact types of the hyperpolarization-activated, cyclic nucleotide-gated, cation non-selective channel (HCN) subunits contributing to the molecular assembly of the relevant channels, nor their distribution pattern presented by the SGCs is known. In the present work immunolabeling and Western blot analysis were performed to describe the presence and distribution of all four known HCN subunits in the guinea-pig spiral ganglion. Besides determining the expression of the HCN1-HCN4 subunits by both type I and type II SGCs, the presence of possible apico-basal gradients in the expression patterns was also sought. The results indicate that both type I and type II SGCs express all four HCN subunits. The intensity of the immunolabeling of the cell surface membrane was generally strong, but it showed pronounced cell-to-cell variability. The Western blot experiments in combination with densitometry revealed that the amount of the HCN1 and HCN3 proteins was more significant in the apical than in the basal third of the guinea-pig cochlea. These findings not only imply potential heteromeric HCN channel formation of the spiral ganglion neurons, but they also offer a possible explanation of the previously reported heterogeneity of I(h) recorded in functional studies.
Asunto(s)
Canales Catiónicos Regulados por Nucleótidos Cíclicos/metabolismo , Expresión Génica/fisiología , Neuronas/metabolismo , Subunidades de Proteína/metabolismo , Ganglio Espiral de la Cóclea/citología , Animales , Canales Catiónicos Regulados por Nucleótidos Cíclicos/clasificación , Canales Catiónicos Regulados por Nucleótidos Cíclicos/genética , Cobayas , MasculinoRESUMEN
Amplification of the kcnk9 gene and overexpression of the encoded channel protein (TASK-3) seems to be involved in carcinogenesis. In the present work, TASK-3 expression of melanoma cells has been studied. For the investigation of TASK-3-specific immunolabelling, a monoclonal antibody has been developed and applied along with two, commercially available polyclonal antibodies targeting different epitopes of the channel protein. Both primary and metastatic melanoma cells proved to be TASK-3 positive, showing prominent intracellular TASK-3-specific labelling; mostly concentrating around or in the proximity of the nuclei. The immunoreaction was associated with the nuclear envelope, and with the processes of the cells and it was also present in the cell surface membrane. Specificity of the immunolabelling was confirmed by Western blot and transfection experiments. As TASK-3 immunopositivity of benign melanocytes could also be demonstrated, the presence or absence of TASK-3 channels cannot differentiate between malignant and non-malignant melanocytic tumours.
Asunto(s)
Melanoma/química , Canales de Potasio de Dominio Poro en Tándem/análisis , Animales , Línea Celular Tumoral , Proteínas Fluorescentes Verdes/análisis , Humanos , Huésped Inmunocomprometido , Inmunohistoquímica , Melanocitos/citología , Melanocitos/metabolismo , Melanoma/metabolismo , Melanoma/patología , Ratones , Canales de Potasio de Dominio Poro en Tándem/inmunología , Canales de Potasio de Dominio Poro en Tándem/metabolismo , Ratas , Proteínas Recombinantes de Fusión/análisisRESUMEN
Depolarization-activated outward currents of bushy neurones of 6-14-day-old Wistar rats have been investigated in a brain slice preparation. Under current-clamp, the cells produced a single action potential at the beginning of suprathreshold depolarizing current steps. On voltage-clamp depolarizations, the cells produced a mixed outward K+ current that included a component with rapid activation and rapid inactivation, little TEA+ sensitivity, a half-inactivation voltage of -77 +/- 2 mV (T = 25 degrees C; n = 7; Mean +/- S.E.M.) and single-exponential recovery from inactivation (taurecovery= 12 +/- 1 ms at -100 mV; n=3). This transient component was identified as an A-type K+ current. Bushy cells developed a high-threshold TEA-sensitive K+ current that exhibited less prominent inactivation. These characteristics suggested that this current was associated with the activation of delayed rectifier K+ channels. Bushy neurones also possessed a low-threshold outward K+ current that showed partial inactivation and high 4-aminopyridine sensitivity. Part of this current component was blocked by 200 nmol/l dendrotoxin-I. Application of 100 micromol/l 4-aminopyridine changed the firing behaviour of the bushy neurones from the primary-like pattern to a much less rapidly adapting one, suggesting that the low-threshold current might have important roles in maintaining the physiological function of the cells.
Asunto(s)
Potenciales de Acción/fisiología , Núcleo Coclear/citología , Núcleo Coclear/fisiología , Neuronas Aferentes/fisiología , Canales de Potasio/fisiología , Animales , Electrofisiología , Neuronas Aferentes/citología , Técnicas de Cultivo de Órganos , Técnicas de Placa-Clamp , Ratas , Ratas WistarRESUMEN
Extracellular application of glutamate elicited cytoplasmic Ca2+ transients in freshly dissociated rat neurones of the dorsal cochlear nucleus (DCN) (identified as pyramidal cells) with half-maximal concentration of 513 micromol/l while saturating doses (5 mmol/l) of this neurotransmitter caused transients of 46.1 +/- 3.0 nmol/l on an average. The genesis of these glutamate-evoked Ca2+ transients required extracellular Ca2+. When [Mg2+]o was 1 mmol/l, the NMDA receptor antagonist AP5 (100 micromol/l) had no effects while 100 micromol/l CNQX and 10 micromol/l NBQX, inhibitors of the AMPA receptors, greatly decreased the glutamate-induced Ca2+ transients (a decrease of 92 and 57%, respectively). When facilitating the activation of the NMDA receptors (50 micromol/l glycine, 20 micromol/l [Mg2+]o) in the presence of 100 micromol/l CNQX, Ca2+ transients of 55.4 +/- 13.1 nmol/l could be produced. Block of the voltage-gated Ca2+ channels (200 micromol/l Cd2+) decreased the Ca2+ transients to approx. 50%. The data indicate that under our control experimental circumstances the glutamate-induced Ca2+ transients of the isolated DCN neurones are produced mainly by Ca2+ entry through voltage-gated Ca2+ channels and AMPA receptors. However, when the activation of the NMDA receptors may take place, these receptors also contribute significantly to the genesis of the glutamate-evoked cytoplasmic [Ca2+] elevations.
Asunto(s)
Señalización del Calcio/efectos de los fármacos , Señalización del Calcio/fisiología , Núcleo Coclear/efectos de los fármacos , Núcleo Coclear/fisiología , Ácido Glutámico/farmacología , Neuronas/efectos de los fármacos , Neuronas/fisiología , Animales , Animales Recién Nacidos , Células Cultivadas , Relación Dosis-Respuesta a Droga , Ratas , Receptores AMPA/efectos de los fármacos , Receptores AMPA/metabolismo , Receptores de Glutamato/efectos de los fármacos , Receptores de Glutamato/metabolismoRESUMEN
In this work, the distributions of some acid-sensitive two-pore-domain K+ channels (TASK-1, TASK-2 and TASK-3) were investigated in the rat and human cerebellum. Astrocytes situated in rat cerebellar tissue sections were positive for TASK-2 channels. Purkinje cells were strongly stained and granule cells and astrocytes were moderately positive for TASK-3. Astrocytes isolated from the hippocampus, cerebellum and cochlear nucleus expressed TASK channels in a primary tissue culture. Our results suggest that TASK channel expression may be significant in the endoplasmic reticulum of the astrocytes. The human cerebellum showed weak TASK-2 immunolabelling. The pia mater, astrocytes, Purkinje and granule cells demonstrated strong TASK-1 and TASK-3 positivities. The TASK-3 labelling was stronger in general, but it was particularly intense in the Purkinje cells and pia mater.
Asunto(s)
Cerebelo/metabolismo , Proteínas del Tejido Nervioso/biosíntesis , Canales de Potasio de Dominio Poro en Tándem , Canales de Potasio/biosíntesis , Animales , Astrocitos/metabolismo , Western Blotting , Células Cultivadas , Electroforesis en Gel de Poliacrilamida , Retículo Endoplásmico/metabolismo , Hipocampo/metabolismo , Humanos , Inmunohistoquímica , Microscopía Fluorescente , Piamadre/metabolismo , Células de Purkinje/metabolismo , Ratas , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , TransfecciónRESUMEN
A hyperpolarization-activated current recorded from the pyramidal cells of the dorsal cochlear nucleus was investigated in the present study by using 150- to 200-microm-thick brain slices prepared from 6- to 14-day-old Wistar rats. The pyramidal neurones exhibited a slowly activating inward current on hyperpolarization. The reversal potential of this component was -32 +/- 3 mV (mean +/- SE, n = 6), while its half-activation voltage was -99 +/- 1 mV with a slope factor of 10.9 +/- 0.4 mV (n = 27). This current was highly sensitive to the extracellular application of both 1 mM Cs+ and 10 microM ZD7288. The electrophysiological properties and the pharmacological sensitivity of this current indicated that it corresponded to a hyperpolarization-activated non-specific cationic current (Ih). Our experiments showed that there was a correlation between the availability of the h-current and the spontaneous activity of the pyramidal cells, suggesting that this conductance acts as a pacemaker current in these neurones. Immunocytochemical experiments were also conducted on freshly isolated pyramidal cells to demonstrate the possible subunit composition of the channels responsible for the genesis of the pyramidal h-current. These investigations indicated the presence of HCN1, HCN2 and HCN4 subunits in the pyramidal cells.
Asunto(s)
Cóclea/metabolismo , Canales Iónicos/metabolismo , Proteínas Musculares/metabolismo , Animales , Canales Catiónicos Regulados por Nucleótidos Cíclicos , Canales Regulados por Nucleótidos Cíclicos Activados por Hiperpolarización , Potenciales de la Membrana/fisiología , Canales de Potasio , RatasRESUMEN
The cochlear nucleus is the site in the auditory pathway where the primary sensory information carried by the fibres of the acoustic nerve is transmitted to the second-order neurones. According to the generally accepted view this transmission is not a simple relay process but is considered as the first stage where the decoding of the auditory information begins. This notion is based on the diverse neurone composition and highly ordered structure of the nucleus, on the complex electrophysiological properties and activity patterns of the neurones, on the activity of local and descending modulatory mechanisms and on the presence of a highly sophisticated intracellular Ca2+ homeostasis. This review puts emphasis on introducing the experimental findings supporting the above statements and on the questions which should be answered in order to gain a better understanding of the function of the cochlear nucleus.
Asunto(s)
Núcleo Coclear/fisiología , Neuronas/fisiología , Animales , Vías Auditivas/fisiología , Calcio/metabolismo , Núcleo Coclear/citología , Citoplasma/metabolismo , Electrofisiología , HomeostasisRESUMEN
Enzymatically isolated type I spiral ganglion neurons of the guinea pig have been investigated in the present study. The identity of the cells was confirmed by using anti-neuron-specific enolase immunostaining. The presence and shredding of the myelin sheath was also documented by employing anti-S100 immunoreaction. The membrane characteristics of the cells were studied by using the whole-cell patch-clamp technique. The whole-cell capacitance of the cells was 9 +/- 2 pF (n = 51), while the resting membrane potential of the cells was -62 +/- 9 mV (n = 19). When suprathreshold depolarizing stimuli were applied, the neurons fired a single action potential at the beginning of the stimulation. It was confirmed in this study that type I spiral ganglion cells possess a hyperpolarization-activated nonspecific cationic current (Ih). The major characteristics of this current component were unaffected by the enzyme treatment. Type I spiral ganglion cells also expressed various depolarization-activated K+ current components. A high-threshold outward current was sensitive to 1-10 mm TEA+ application. The ganglion cells also expressed a relatively small, but nevertheless present, transient outward current component which was less sensitive to TEA+ but could be inhibited by 100 micro m 4-aminopyridine. A DTX-I-sensitive current was responsible for some 30% of the total outward current (at 0 mV), showed rapid activation at membrane potentials positive to -50 mV and demonstrated very little inactivation. However, inhibition of the highly 4-AP- or DTX-I-sensitive component did not alter the rapidly inactivating nature of the firing pattern of the cells.
Asunto(s)
Potenciales de Acción , Canales de Potasio/fisiología , Ganglio Espiral de la Cóclea/fisiología , 4-Aminopiridina/farmacología , Animales , Femenino , Cobayas , Inmunohistoquímica , Masculino , Potenciales de la Membrana/fisiología , Técnicas de Placa-Clamp , Bloqueadores de los Canales de Potasio/farmacología , Canales de Potasio/efectos de los fármacos , Ganglio Espiral de la Cóclea/efectos de los fármacos , Tetraetilamonio/farmacologíaRESUMEN
1. A hyperpolarization-activated non-specific cation current, I(h), was examined in bushy cell bodies and their giant presynaptic terminals (calyx of Held). Whole-cell patch clamp recordings were made using an in vitro brain slice preparation of the cochlear nucleus and the superior olivary complex. The aim was to characterise I(h) in identified cell bodies and synaptic terminals, to examine modulation by presynaptic cAMP and to test for modulatory effects of I(h) activation on synaptic transmission. 2. Presynaptic I(h) was activated by hyperpolarizing voltage-steps, with half-activation (V(1/2)) at -94 mV. Activation time constants were voltage dependent, showing an e-fold acceleration for hyperpolarizations of -32 mV (time constant of 78 ms at -130 mV). The reversal potential of I(h) was -29 mV. It was blocked by external perfusion of 1 mM CsCl but was unaffected by BaCl(2). 3. Application of internal cAMP shifted the activation curve to more positive potentials, giving a V(1/2) of -74 mV; hence around half of the current was activated at resting membrane potentials. This shift in half-activation was mimicked by external perfusion of a membrane-permeant analogue, 8-bromo-cAMP. 4. The bushy cell body I(h) showed similar properties to those of the synaptic terminal; V(1/2) was -94 mV and the reversal potential was -33 mV. Somatic I(h) was blocked by CsCl (1 mM) and was partially sensitive to BaCl(2). Somatic I(h) current density increased with postnatal age from 5 to 16 days old, suggesting that I(h) is functionally relevant during maturation of the auditory pathway. 5. The function of I(h) in regulating presynaptic excitability is subtle. I(h) had little influence on EPSC amplitude at the calyx of Held, but may be associated with propagation of the action potential at branch points. Presynaptic I(h) shares properties with both HCN1 and HCN2 recombinant channel subunits, in that it gates relatively rapidly and is modulated by internal cAMP.
Asunto(s)
Vías Auditivas/fisiología , Tronco Encefálico/fisiología , Cationes/metabolismo , Canales Iónicos/fisiología , Terminaciones Nerviosas/fisiología , Terminales Presinápticos/fisiología , Sinapsis/fisiología , Animales , Electrofisiología , Técnicas In Vitro , Ratas , Ratas Endogámicas , Transmisión Sináptica/fisiologíaRESUMEN
Neurones isolated from various parts of the brain are used extensively for electrophysiological and immuncytochemical studies, as well as to investigate their Ca(2+) homeostasis. In this work we report on an isolation technique that yielded neurones suitable for functional studies targeting the investigation of their Ca(2+) handling mechanisms. The cell isolation involved enzymatic dissociation with combined collagenase/pronase treatment and gentle mechanical trituration. At the end of the isolation the cells were incubated in a cell culture incubator (CO2 concentration = 5.1%) at 37 degrees C in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% heat-inactivated horse serum. The vitality of the isolated cells was indicated by their low intracellular Ca(2+) concentrations (17.2 +/- 0.5 nM; n = 38) and by their ability to produce large Ca(2+) transients on depolarization. These Ca(2+) transients were rapidly terminated and the resting intracellular Ca(2+) concentration was quickly restored proving that isolation did not compromise the Ca(2+) homeostatic mechanisms of the nerve cells. The technique allowed reliable, long (45-60 min) and reproducible measurements of Ca(2+) currents on these neurones as well as the recording of their intracellular Ca(2+) concentration. Our results indicate that incubation in DMEM with horse serum markedly increases the number of surviving neurones after the enzyme treatment, and their Ca(2+) homeostasis can be studied for significantly longer periods of time.
Asunto(s)
Calcio/metabolismo , Separación Celular/métodos , Separación Celular/normas , Núcleo Coclear/metabolismo , Homeostasis/fisiología , Membranas Intracelulares/metabolismo , Neuronas/metabolismo , Animales , Núcleo Coclear/citología , Núcleo Coclear/fisiología , Electrofisiología , Femenino , Inmunohistoquímica , Masculino , Neuronas/fisiología , Concentración Osmolar , Técnicas de Placa-Clamp , Ratas , Ratas WistarRESUMEN
The effects of divalent cations on voltage-activated Ca2+ channels and depolarization-evoked cytoplasmic [Ca2+] elevations were studied in pyramidal neurones isolated from the dorsal cochlear nucleus of the rat. Ca2+ currents were recorded using the whole-cell configuration of the patch-clamp technique. 10 micromol x l(-1) Cd2+ exerted a greater blocking effect on the high-voltage activated (HVA) currents than on the low-voltage activated (LVA) ones (decrease to 26.6+/-2.5% and to 87.8+/-2.1%, respectively). The blocking effect of 200 micromol x l(-1) Cd2+ was more pronounced and the difference between the effect on the HVA and LVA currents became smaller (decrease to 11.7+/-2.1% and to 32.4+/-2.7%, respectively). 200 micromol x l(-1) Ni2+ reduced the LVA component more effectively (to 77.6+/-5.4%) than the HVA one (to 86.9+/-2.6%). Cytoplasmic [Ca2+] changes were measured applying a fluorimetric technique (Fura-2). 10 micromol x l(-1) Cd2+ decreased the peak values of 50 mmol x l(-1) K+ depolarization-induced [Ca2]+i transients to 30.4+/-1.4% while 200 micromol x l(-1) Cd2+ caused a drop to 2.5+/-0.2%. 200 micromol x l(-1) Ni2+ decreased the peak of the transients to 69.6+/-2.9%. Comparison of the blocking effects of divalent cations on Ca2+ currents and [Ca2+]i transients supports further the conclusion that the depolarization-induced [Ca2+]i changes are produced mainly by the activation of the HVA Ca2+ channels.
Asunto(s)
Canales de Calcio/química , Calcio/metabolismo , Cationes , Núcleo Coclear/metabolismo , Células Piramidales/metabolismo , Animales , Citoplasma/metabolismo , Electrofisiología , Potasio/metabolismo , Ratas , Factores de TiempoRESUMEN
The significance of voltage-activated Ca2+ currents in eliciting cytoplasmic Ca2+ transients was studied in pyramidal neurones isolated from the rat dorsal cochlear nucleus using combined enzyme treatment/mechanical trituration. Increases in cytoplasmic Ca2+ concentration ([Ca2+]i) were evoked by K+-induced depolarizations (10-50 mM) and monitored by the Fura-2 fluorimetric technique. The acutely dissociated neurones had a resting [Ca2+]i of 17.2+/-0.5 nM. They possessed caffeine-sensitive Ca2+ stores which were empty at rest; these stores could be filled with Ca2+ entering from the extracellular space and were re-emptied quickly. The effects of various specific high-voltage-activated (HVA) Ca2+ channel antagonists (nifedipine, omega-agatoxin IVA and omega-conotoxin GVIA) on [Ca2+]i transients were tested. Analysis of the blocking effects of these agents on the [Ca2+]i, transients indicates that, in the pyramidal neurones of the dorsal cochlear nucleus, N-type Ca2+ channels are primarily responsible for producing the depolarization-induced increases in [Ca2+]i.
Asunto(s)
Canales de Calcio Tipo N/metabolismo , Calcio/metabolismo , Núcleo Coclear/citología , Potasio/farmacología , Células Piramidales/metabolismo , Animales , Bloqueadores de los Canales de Calcio/farmacología , Citoplasma/metabolismo , Colorantes Fluorescentes , Fura-2 , Técnicas In Vitro , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Nifedipino/farmacología , Células Piramidales/química , Células Piramidales/efectos de los fármacos , Ratas , Estimulación Química , Tetrodotoxina/farmacología , omega-Agatoxina IVA/farmacología , omega-Conotoxina GVIA/farmacologíaRESUMEN
Voltage-activated Ca(2+) currents have been studied in pyramidal cells isolated enzymatically from the dorsal cochlear nuclei of 6-11-day-old Wistar rats, using whole-cell voltage-clamp. From hyperpolarized membrane potentials, the neurones exhibited a T-type Ca(2+) current on depolarizations positive to -90 mV (the maximum occurred at about -40 mV). The magnitude of the T-current varied considerably from cell to cell (-56 to -852 pA) while its steady-state inactivation was consistent (E(50)=-88.2+/-1.7 mV, s=-6. 0+/-0.4 mV). The maximum of high-voltage activated (HVA) Ca(2+) currents was observed at about -15 mV. At a membrane potential of -10 mV the L-type Ca(2+) channel blocker nifedipine (10 microM) inhibited approximately 60% of the HVA current, the N-type channel inhibitor omega-Conotoxin GVIA (2 microM) reduced the current by 25% while the P/Q-type channel blocker omega-Agatoxin IVA (200 nM) blocked a further 10%. The presence of the N- and P/Q-type Ca(2+) channels was confirmed by immunochemical methods. The metabotropic glutamate receptor agonist (+/-)-1-aminocyclopentane-trans-1, 3-dicarboxylic acid (200 microM) depressed the HVA current in every cell studied (a block of approximately 7% on an average). The GABA(B) receptor agonist baclofen (100 microM) reversibly inhibited 25% of the HVA current. Simultaneous application of omega-Conotoxin GVIA and baclofen suggested that this inhibition could be attributed to the nearly complete blockade of the N-type channels. Possible physiological functions of the voltage-activated Ca(2+) currents reported in this work are discussed.
Asunto(s)
Canales de Calcio/fisiología , Núcleo Coclear/fisiología , Células Piramidales/fisiología , Animales , Especificidad de Anticuerpos , Baclofeno/farmacología , Bloqueadores de los Canales de Calcio/farmacología , Canales de Calcio/efectos de los fármacos , Núcleo Coclear/citología , Cicloleucina/análogos & derivados , Cicloleucina/farmacología , Agonistas del GABA/farmacología , Inmunoquímica , Técnicas In Vitro , Potenciales de la Membrana/fisiología , Nifedipino/farmacología , Técnicas de Placa-Clamp , Ratas , Ratas Wistar , Receptores de Glutamato Metabotrópico/agonistasRESUMEN
1. Whole-cell patch clamp recordings were made from bushy cells of the anterioventral cochlear nucleus (aVCN) and their synaptic terminals (calyx of Held) in the medial nucleus of the trapezoid body (MNTB). 2. Both high voltage-activated (HVA) and low voltage-activated (LVA) calcium currents were present in acutely dissociated aVCN neurones and in identified bushy neurones from a cochlear nucleus slice. 3. The transient LVA calcium current activated rapidly on depolarization (half-activation, -59 mV) and inactivated during maintained depolarization (half-inactivation, -89 mV). This T-type current was observed in somatic recordings but was absent from presynaptic terminals. 4. On the basis of their pharmacological sensitivity, P/Q-type Ca2+ channels accounted for only 6 % of the somatic HVA, while L-, N- and R-type Ca2+ channels each accounted for around one-third of the somatic calcium current. 5. The divalent permeabilities of these native calcium channels were compared. The Ba2+/Ca2+ conductance ratios of the somatic HVA and LVA channels were 1.4 and 0.7, respectively. The conductance ratio of the presynaptic HVA current was 0.9, significantly lower that that of the somatic HVA current. 6. We conclude that LVA currents are expressed in the bushy cell body, but are not localized to the excitatory synaptic terminal. All of the HVA current subtypes are expressed in bushy cells, but there is a strong polarity to their localization; P-type contribute little to somatic currents but predominate at the synaptic terminal; L-, N- and R-types dominate at the soma, but contribute negligibly to calcium currents in the terminal.
Asunto(s)
Canales de Calcio/metabolismo , Cóclea/citología , Cóclea/metabolismo , Neuronas/metabolismo , Terminales Presinápticos/metabolismo , Algoritmos , Animales , Separación Celular , Cicloleucina/análogos & derivados , Cicloleucina/farmacología , Estimulación Eléctrica , Electrofisiología , Técnicas In Vitro , Potenciales de la Membrana/fisiología , Fármacos Neuroprotectores/farmacología , Técnicas de Placa-Clamp , RatasRESUMEN
Under physiological conditions, calcium-dependent ([Ca2+]i-dependent) Cl- currents (ICl(Ca)) have been suggested to participate in the repolarizing processes. In this paper, the possible contribution of ICl(Ca) to transient inward currents and, hence to arrhythmias, has been studied in myocytes from the working myocardium and from the conductive system. Single atrial, ventricular and Purkinje cells, isolated enzymatically from rabbit heart, have been studied under whole-cell voltage-clamp and were internally perfused with the fluorescent Ca2+ indicator, fura-2 (100 microM). Ca2+ release from the sarcoplasmic reticulum was either induced by external application of caffeine or occurred spontaneously in Ca(2+)-overloaded cells. Membrane currents accompanying Ca2+ transients showed linear current-voltage characteristics between -60 and +80 mV as evidenced from fast voltage ramps. When intra- and extracellular Cl- concentrations were kept symmetrical in the absence of the Na(+)-Ca2+ exchange mechanism, transient currents had a reversal potential close to 0 mV. Reduction of external Cl- concentration shifted this reversal potential towards the new Cl- equilibrium potential. Neither the time course of the transient currents nor the shift in their reversal potentials was significantly affected by the presence of Na+. Approximately 90% of this current was blocked by the application of the Cl- channel blocker, anthracene-9-carboxylic acid (0.5 mM) at +80 mV. It is concluded, that [Ca2+]i-activated transient membrane currents in atrial, ventricular and Purkinje cells of rabbit heart are mainly due to the activation of a [Ca2+]i-dependent Cl- current.
Asunto(s)
Función Atrial , Calcio/fisiología , Canales de Cloruro/fisiología , Células de Purkinje/fisiología , Función Ventricular , Animales , Antracenos/farmacología , Cafeína/farmacología , Calcio/metabolismo , Canales de Calcio/fisiología , Canales de Cloruro/antagonistas & inhibidores , Cloruros/metabolismo , Femenino , Atrios Cardíacos/citología , Atrios Cardíacos/efectos de los fármacos , Ventrículos Cardíacos/citología , Ventrículos Cardíacos/efectos de los fármacos , Técnicas In Vitro , Masculino , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Miocardio/citología , Miocardio/metabolismo , Técnicas de Placa-Clamp , Células de Purkinje/efectos de los fármacos , Conejos , Retículo Sarcoplasmático/metabolismo , Intercambiador de Sodio-Calcio/fisiologíaRESUMEN
Granule cells are the most numerous neurons in the cochlear nucleus, but, because of their small size, little information on their membrane properties and ionic currents is available. We used an in vitro slice preparation of the rat ventral cochlear nucleus to make whole-cell recordings from these cells. Under current clamp, some granule neurons fired spontaneous action potentials and all generated a train of action potentials on depolarization (threshold current, 10-35 pA). Hyperpolarization increased the latency to the first action potential evoked during a subsequent depolarization. We examined which voltage-gated currents might underlie this latency shift. In addition to a fast inward Na+ current, depolarization activated two outward potassium currents. A transient current was rapidly inactivated by membrane potentials positive to -60 mV, while a second, more slowly inactivating current was observed following the decay of the transient current. No hyperpolarization-activated conductances were observed in these cells. Modelling of the currents suggests that removal of inactivation on hyperpolarization accounts for the increased action potential latency in granule cells. Such a mechanism could account for the 'pauser'-type firing patterns of the fusiform cells which receive a prominent projection from the granule cells in the dorsal cochlear nucleus.
Asunto(s)
Núcleo Coclear/fisiología , Potenciales Evocados Auditivos/fisiología , Neuronas/fisiología , Animales , Membrana Celular/fisiología , Núcleo Coclear/citología , Simulación por Computador , Gránulos Citoplasmáticos/fisiología , Gránulos Citoplasmáticos/ultraestructura , Electrofisiología , Técnicas In Vitro , Canales Iónicos/fisiología , Potenciales de la Membrana/fisiología , Modelos Neurológicos , Neuronas/ultraestructura , Técnicas de Placa-Clamp , RatasRESUMEN
The whole-cell configuration of the patch-clamp technique was employed to measure the transient outward potassium current in enzymatically isolated ventricular cells of spontaneously diabetic rats (BB/Wor) and mice (ob/ob). Healthy littermates (non-diabetic BB rats and lean mice) were used as controls. There was no significant difference between the non-diabetic and diabetic BB rats (Type I diabetes, IDDM) in the amplitude of either the current measured in the absence or the one found in the presence of 4-aminopyridine. The voltage dependence of the activation and steady-state inactivation was also similar in both populations, as no significant difference was observed in the rate of recovery from inactivation of Ito. The amplitudes of the total and 4-aminopyridine sensitive currents of lean and obese mice (Type II diabetes, NIDDM) were also similar. The voltage dependences of the activation and of the steady-state inactivation did not differ significantly, either. Our results might indicate certain limitations of the applicability of experiments carried out on genetically diabetic rats if the results are compared to those derived from the healthy littermates as controls.