RESUMEN
Although functional glycinergic synapses have not been identified in the hippocampus, neurons in this area express Cl(-) permeable extrasynaptic glycine receptors (GlyRs). In experiments on CA3 pyramidal neurons on postnatal day 0-6 rat hippocampal slices, we detected robust GlyR activity as a tonic current and as single-channel events. Glycine release was independent of neuronal activity or extracellular Ca(2+). The endogenous GlyR activity was strongly enhanced by inhibition of the glycine-transporter-1 (GlyT1). Blockade of GlyT1 also caused a profound increase in the baseline current induced by exogenous glycine. Inhibition of GlyT1 reduced the frequency of spontaneous network events known as field giant depolarizing potentials (fGDPs) and of the unit activity in the absence of synaptic transmission. This inhibitory action on fGDPs was mimicked by applying 2 µm glycine or 0.1 µm isoguvacine, a GABAA-receptor agonist. Furthermore, 2 µm glycine suppressed unit spiking in the absence of synaptic transmission. Hence, despite the well known depolarizing Cl(-) equilibrium potential of neonatal hippocampal neurons, physiologically relevant extracellular glycine concentrations can exert an inhibitory action. The present data show that, akin to GABA uptake, GlyT1 exerts a powerful modulatory action on network events in the newborn hippocampus.
Asunto(s)
Proteínas de Transporte de Glicina en la Membrana Plasmática/fisiología , Hipocampo/fisiología , Inhibición Neural/fisiología , Receptores de Glicina/fisiología , Animales , Animales Recién Nacidos , Femenino , Glicina/fisiología , Proteínas de Transporte de Glicina en la Membrana Plasmática/agonistas , Proteínas de Transporte de Glicina en la Membrana Plasmática/antagonistas & inhibidores , Masculino , Técnicas de Cultivo de Órganos , Ratas , Ratas Wistar , Receptores de Glicina/agonistasRESUMEN
Depolarizing and excitatory GABA actions are thought to be important in cortical development. We show here that GABA has no excitatory action on CA3 pyramidal neurons in hippocampal slices from neonatal NKCC1(-/-) mice that lack the Na-K-2Cl cotransporter isoform 1. Strikingly, NKCC1(-/-) slices generated endogenous network events similar to giant depolarizing potentials (GDPs), but, unlike in wild-type slices, the GDPs were not facilitated by the GABA(A) agonist isoguvacine or blocked by the NKCC1 inhibitor bumetanide. The developmental upregulation of the K-Cl cotransporter 2 (KCC2) was unperturbed, whereas the pharmacologically isolated glutamatergic network activity and the intrinsic excitability of CA3 pyramidal neurons were enhanced in the NKCC1(-/-) hippocampus. Hence, developmental expression of KCC2, unsilencing of AMPA-type synapses, and early network events can take place in the absence of excitatory GABAergic signaling in the neonatal hippocampus. Furthermore, we show that genetic as well as pharmacologically induced loss of NKCC1-dependent excitatory actions of GABA results in a dramatic compensatory increase in the intrinsic excitability of glutamatergic neurons, pointing to powerful homeostatic regulation of neuronal activity in the developing hippocampal circuitry.
Asunto(s)
Potenciales de Acción/genética , Hipocampo/citología , Células Piramidales/fisiología , Simportadores de Cloruro de Sodio-Potasio/deficiencia , Potenciales de Acción/efectos de los fármacos , Potenciales de Acción/fisiología , Factores de Edad , Animales , Animales Recién Nacidos , Benzodiazepinas/farmacología , Biofisica , Calcio/metabolismo , Estimulación Eléctrica/métodos , Antagonistas de Aminoácidos Excitadores/farmacología , Potenciales Postsinápticos Excitadores/efectos de los fármacos , Técnicas In Vitro , Ratones , Ratones Noqueados , Red Nerviosa/efectos de los fármacos , Red Nerviosa/fisiología , Técnicas de Placa-Clamp , Células Piramidales/efectos de los fármacos , Quinoxalinas/farmacología , Miembro 2 de la Familia de Transportadores de Soluto 12 , Simportadores/genética , Simportadores/metabolismo , Regulación hacia Arriba/fisiología , Ácido gamma-Aminobutírico/farmacología , Cotransportadores de K ClRESUMEN
Members of the SLC4 bicarbonate transporter family are involved in solute transport and pH homeostasis. Here we report that disrupting the Slc4a10 gene, which encodes the Na(+)-coupled Cl(-)-HCO(3)(-) exchanger Slc4a10 (NCBE), drastically reduces brain ventricle volume and protects against fatal epileptic seizures in mice. In choroid plexus epithelial cells, Slc4a10 localizes to the basolateral membrane. These cells displayed a diminished recovery from an acid load in KO mice. Slc4a10 also was expressed in neurons. Within the hippocampus, the Slc4a10 protein was abundant in CA3 pyramidal cells. In the CA3 area, propionate-induced intracellular acidification and attenuation of 4-aminopyridine-induced network activity were prolonged in KO mice. Our data indicate that Slc4a10 is involved in the control of neuronal pH and excitability and may contribute to the secretion of cerebrospinal fluid. Hence, Slc4a10 is a promising pharmacological target for the therapy of epilepsy or elevated intracranial pressure.
Asunto(s)
Encéfalo/metabolismo , Encéfalo/patología , Líquido Cefalorraquídeo/metabolismo , Antiportadores de Cloruro-Bicarbonato/líquido cefalorraquídeo , Antiportadores de Cloruro-Bicarbonato/fisiología , Regulación de la Expresión Génica , Mutación , Neuronas/metabolismo , Simportadores de Sodio-Bicarbonato/líquido cefalorraquídeo , Simportadores de Sodio-Bicarbonato/fisiología , Animales , Conducta Animal , Transporte Biológico , Antiportadores de Cloruro-Bicarbonato/genética , Eliminación de Gen , Concentración de Iones de Hidrógeno , Iones , Aprendizaje , Ratones , Ratones Noqueados , Modelos Genéticos , Simportadores de Sodio-Bicarbonato/genéticaRESUMEN
Endogenous activity is a characteristic feature of developing neuronal networks. In the neonatal rat hippocampus, spontaneously occurring network events known as "Giant Depolarizing Potentials" (GDPs) are seen in vitro at a stage when GABAergic transmission is depolarizing. GDPs are triggered by the CA3 region and they are seen as brief recurrent events in field-potential recordings, paralleled by depolarization and spiking of pyramidal neurons. In the light of current data, GDPs are triggered by the glutamatergic pyramidal neurons which act as conditional pacemakers, while the depolarizing action of GABA plays a permissive role for the generation of these events in in vitro preparations. From an in vivo perspective, GDPs appear to be an immature form of hippocampal sharp waves.
Asunto(s)
Potenciales Postsinápticos Excitadores/fisiología , Hipocampo/fisiología , Red Nerviosa/fisiología , Neuronas/fisiología , Ácido gamma-Aminobutírico/fisiología , Animales , Animales Recién Nacidos , Hipocampo/crecimiento & desarrollo , Células Piramidales/crecimiento & desarrollo , Células Piramidales/fisiología , Ratas , Receptores de GABA-A/fisiologíaRESUMEN
Tonic activation of GABA(A) receptors takes place before the development of functional synapses in cortical structures. We studied whether inefficient GABA uptake might explain the presence of a tonic GABA(A)-mediated current (I(GABA-A)) in early postnatal hippocampal pyramidal neurons. The data show, however, that the tonic I(GABA-A) is enhanced by the specific blocker of GABA transporter-1 (GAT-1), NO-711 (1-[2-[[(Diphenylmethyleneimino]oxy]ethyl]-1,2,5,6-tetrahydro-3-pyridinecarboxylic acid hydrochloride), at birth in rat CA3 pyramidal neurons. NO-711 also prolonged the duration of GABA transients during endogenous hippocampal network events (known as giant depolarizing potentials) at postnatal day 0. The endogenous tonic I(GABA-A) was seen and it was enhanced by NO-711 in the presence of tetrodotoxin, which itself had only a minor effect on the holding current under control conditions. This indicates that the source of interstitial GABA is largely independent of action-potential activity. The tonic I(GABA-A) in neonatal CA3 pyramidal neurons was increased by zolpidem, indicating that at least a proportion of the underlying GABA(A) receptors contain gamma2 and alpha1-alpha3 subunits. The present data point to a significant role for GAT-1 in the control of the excitability of immature hippocampal neurons and networks.
Asunto(s)
Proteínas Transportadoras de GABA en la Membrana Plasmática/fisiología , Células Piramidales/fisiología , Receptores de GABA-A/fisiología , Potenciales de Acción/efectos de los fármacos , Potenciales de Acción/fisiología , Anestésicos Locales/farmacología , Animales , Animales Recién Nacidos , Antagonistas del GABA/farmacología , Hipocampo/citología , Hipocampo/fisiología , Ácidos Nipecóticos/farmacología , Técnicas de Cultivo de Órganos , Oximas/farmacología , Técnicas de Placa-Clamp , Ratas , Ratas Wistar , Sinapsis/fisiología , Tetrodotoxina/farmacologíaRESUMEN
Febrile seizures are frequent during early childhood, and prolonged (complex) febrile seizures are associated with an increased susceptibility to temporal lobe epilepsy. The pathophysiological consequences of febrile seizures have been extensively studied in rat pups exposed to hyperthermia. The mechanisms that trigger these seizures are unknown, however. A rise in brain pH is known to enhance neuronal excitability. Here we show that hyperthermia causes respiratory alkalosis in the immature brain, with a threshold of 0.2-0.3 pH units for seizure induction. Suppressing alkalosis with 5% ambient CO2 abolished seizures within 20 s. CO2 also prevented two long-term effects of hyperthermic seizures in the hippocampus: the upregulation of the I(h) current and the upregulation of CB1 receptor expression. The effects of hyperthermia were closely mimicked by intraperitoneal injection of bicarbonate. Our work indicates a mechanism for triggering hyperthermic seizures and suggests new strategies in the research and therapy of fever-related epileptic syndromes.
Asunto(s)
Alcalosis Respiratoria/fisiopatología , Fiebre/fisiopatología , Convulsiones Febriles/fisiopatología , Alcalosis Respiratoria/inducido químicamente , Animales , Bicarbonatos , Temperatura Corporal , Encéfalo/crecimiento & desarrollo , Encéfalo/fisiopatología , Dióxido de Carbono/uso terapéutico , Modelos Animales de Enfermedad , Femenino , Fiebre/prevención & control , Embarazo , Ratas , Ratas WistarRESUMEN
The CA3 area of the mature hippocampus is known for its ability to generate intermittent network activity both in physiological and in pathological conditions. We have recently shown that in the early postnatal period, the intrinsic bursting of interconnected CA3 pyramidal neurons generates network events, which were originally called giant depolarizing potentials (GDPs). The voltage-dependent burst activity of individual pyramidal neurons is promoted by the well-known depolarizing action of endogenous GABA on immature neurons. In the present work, we show that a persistent Na+ current, I-Nap, accounts for the slow regenerative depolarization that triggers the intrinsic bursts in the neonatal rat CA3 pyramidal neurons (postnatal day 3-6), while a slow Ca2+ -activated K+ current, sI-K(Ca), is primarily responsible for the postburst slow afterhyperpolarization and consequent burst termination. In addition, we exploited pharmacological data obtained from intracellular recordings to study the mechanisms involved in network events recorded with field potential recordings. The data as a whole indicate that I-Nap and sI-K(Ca) are involved in the initiation and termination, respectively, of the pyramidal bursts and consequent network events underlying GDPs.
Asunto(s)
Hipocampo/citología , Potenciales de la Membrana/fisiología , Neuronas/fisiología , Canales de Potasio Calcio-Activados/fisiología , Canales de Sodio/fisiología , Animales , Animales Recién Nacidos , Relación Dosis-Respuesta a Droga , Relación Dosis-Respuesta en la Radiación , Estimulación Eléctrica/métodos , Antagonistas de Aminoácidos Excitadores/farmacología , Hipocampo/crecimiento & desarrollo , Técnicas In Vitro , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/efectos de la radiación , Metales Pesados/farmacología , Técnicas de Placa-Clamp/métodos , Fenitoína/farmacología , Potasio/farmacología , Ratas , Ratas Wistar , Bloqueadores de los Canales de Sodio/farmacología , Tetrodotoxina/farmacologíaRESUMEN
Earlier studies indicate a crucial role for the interconnected network of intrinsically bursting CA3 pyramidal neurons in the generation of in vivo hippocampal sharp waves (SPWs) and their proposed neonatal in vitro counterparts, the giant depolarizing potentials (GDPs). While mechanisms involving ligand- and voltage-gated channels have received lots of attention in the generation of CA3 network events in the immature hippocampus, the contribution of ion-transport mechanisms has not been extensively studied. Here, we show that bumetanide, a selective inhibitor of neuronal Cl- uptake mediated by the Na+-K+-2Cl- cotransporter isoform 1 (NKCC1), completely and reversibly blocks SPWs in the neonate (postnatal days 7-9) rat hippocampus in vivo, an action also seen on GDPs in slices (postnatal days 1-8). These findings strengthen the view that GDPs and early SPWs are homologous events. Gramicidin-perforated patch recordings indicated that NKCC1 accounts for a large ( approximately 10 mV) depolarizing driving force for the GABAA current in the immature CA3 pyramids. Consistent with a reduction in the depolarization mediated by endogenous GABAA-receptor activation, bumetanide inhibited the spontaneous bursts of individual neonatal CA3 pyramids, but it slightly increased the interneuronal activity as seen in the frequency of spontaneous GABAergic currents. An inhibitory effect of bumetanide was seen on the in vitro population events in the absence of synaptic GABAA receptor-mediated transmission, provided that a tonic GABAA receptor-mediated current was present. Our work indicates that NKCC1 expressed in CA3 pyramidal neurons promotes network activity in the developing hippocampus.
Asunto(s)
Hipocampo/metabolismo , Red Nerviosa/metabolismo , Células Piramidales/metabolismo , Simportadores de Cloruro de Sodio-Potasio/metabolismo , Potenciales de Acción , Animales , Animales Recién Nacidos , Bumetanida/farmacología , Agonistas de Receptores de GABA-A , Hipocampo/efectos de los fármacos , Hipocampo/crecimiento & desarrollo , Interneuronas/efectos de los fármacos , Interneuronas/metabolismo , Red Nerviosa/efectos de los fármacos , Red Nerviosa/crecimiento & desarrollo , Células Piramidales/efectos de los fármacos , Ratas , Ratas Wistar , Receptores de GABA-A/metabolismo , Inhibidores del Simportador de Cloruro Sódico y Cloruro Potásico/farmacología , Miembro 2 de la Familia de Transportadores de Soluto 12 , Transmisión Sináptica , Ácido gamma-Aminobutírico/farmacologíaRESUMEN
Spontaneous periodic network events are a characteristic feature of developing neuronal networks, and they are thought to play a crucial role in the maturation of neuronal circuits. In the immature hippocampus, these types of events are seen in intracellular recordings as giant depolarizing potentials (GDPs) during the stage of neuronal development when GABA(A)-mediated transmission is depolarizing. However, the precise mechanism how GABAergic transmission promotes GDP occurrence is not known. Using whole-cell, cell-attached, perforated-patch, and field-potential recordings in hippocampal slices, we demonstrate here that CA3 pyramidal neurons in the newborn rat generate intrinsic bursts when depolarized. Furthermore, the characteristic rhythmicity of GDP generation is not based on a temporally patterned output of the GABAergic interneuronal network. However, GABAergic depolarization plays a key role in promoting voltage-dependent, intrinsic pyramidal bursting activity. The present data indicate that glutamatergic CA3 neurons have an instructive, pacemaker role in the generation of GDPs, whereas both synaptic and tonic depolarizing GABAergic mechanisms exert a temporally nonpatterned, facilitatory action in the generation of these network events.