RESUMEN
Oxytocin is an important regulator of the social brain. In some animal models of autism, notably in Magel2tm1.1Mus-deficient mice, peripheral administration of oxytocin in infancy improves social behaviors until adulthood. However, neither the mechanisms responsible for social deficits nor the mechanisms by which such oxytocin administration has long-term effects are known. Here, we aimed to clarify these oxytocin-dependent mechanisms, focusing on social memory performance. Using in situ hybridization (RNAscope), we have established that Magel2 and oxytocin receptor are co-expressed in the dentate gyrus and CA2/CA3 hippocampal regions involved in the circuitry underlying social memory. Then, we have shown that Magel2tm1.1Mus-deficient mice, evaluated in a three-chamber test, present a deficit in social memory. Next, in hippocampus, we conducted neuroanatomical and functional studies using immunostaining, oxytocin-binding experiments, ex vivo electrophysiological recordings, calcium imaging and biochemical studies. We demonstrated: an increase of the GABAergic activity of CA3-pyramidal cells associated with an increase in the quantity of oxytocin receptors and of somatostatin interneurons in both DG and CA2/CA3 regions. We also revealed a delay in the GABAergic development sequence in Magel2tm1.1Mus-deficient pups, linked to phosphorylation modifications of KCC2. Above all, we demonstrated the positive effects of subcutaneous administration of oxytocin in the mutant neonates, restoring hippocampal alterations and social memory at adulthood. Although clinical trials are debated, this study highlights the mechanisms by which peripheral oxytocin administration in neonates impacts the brain and demonstrates the therapeutic value of oxytocin to treat infants with autism spectrum disorders.
Asunto(s)
Trastorno Autístico , Oxitocina , Animales , Antígenos de Neoplasias/uso terapéutico , Trastorno Autístico/tratamiento farmacológico , Hipocampo/metabolismo , Ratones , Oxitocina/uso terapéutico , Proteínas , Receptores de Oxitocina/metabolismo , Conducta SocialRESUMEN
Epidemiological studies have provided contradictory data on the deleterious sequels of cesarean section (C-section) delivery and their links with developmental brain disorders such as Autism Spectrum Disorders. To gain better insight on these issues, we have now compared physiological, morphological, and behavioral parameters in vaginal, term, and preterm C-section delivered mice. We report that C-section delivery does not lead to long-term behavioral alterations though preterm C-section delivery modifies communicative behaviors in pups. Moreover, C-section delivery neither alters the gamma-aminobutyric acid (GABA) developmental excitatory to inhibitory shift nor the frequency or amplitude of glutamatergic and GABAergic postsynaptic currents in hippocampal pyramidal neurons. However, these neurons present an underdeveloped dendritic arbor at birth in pups born by C-section delivery, but this difference disappears 1 day later suggesting an accelerated growth after birth. Therefore, C-section delivery, with prematurity as an aggravating factor, induces transient developmental delays but neither impacts the GABA developmental sequence nor leads to long-term consequences in mice. The deleterious sequels of C-section delivery described in epidemiological studies might be due to a perinatal insult that could be aggravated by C-section delivery.
Asunto(s)
Cesárea/efectos adversos , Discapacidades del Desarrollo/epidemiología , Nacimiento Prematuro , Animales , Conducta Animal/fisiología , Región CA3 Hipocampal/metabolismo , Región CA3 Hipocampal/patología , Región CA3 Hipocampal/fisiopatología , Femenino , Masculino , Ratones , Embarazo , Células Piramidales/metabolismo , Células Piramidales/patologíaRESUMEN
Epidemiological and experimental studies suggest that maternal immune activation (MIA) leads to developmental brain disorders, but whether the pathogenic mechanism impacts neurons already at birth is not known. We now report that MIA abolishes in mice the oxytocin-mediated delivery γ-aminobutyric acid (GABA) shift from depolarizing to hyperpolarizing in CA3 pyramidal neurons, and this is restored by the NKCC1 chloride importer antagonist bumetanide. Furthermore, MIA hippocampal pyramidal neurons at birth have a more exuberant apical arbor organization and increased apical dendritic length than age-matched controls. The frequency of spontaneous glutamatergic postsynaptic currents is also increased in MIA offspring, as well as the pairwise correlation of the synchronized firing of active cells in CA3. These alterations produced by MIA persist, since at P14-15 GABA action remains depolarizing, produces excitatory action, and network activity remains elevated with a higher frequency of spontaneous glutamatergic postsynaptic currents. Therefore, the pathogenic actions of MIA lead to important morphophysiological and network alterations in the hippocampus already at birth.
Asunto(s)
Región CA3 Hipocampal/crecimiento & desarrollo , Región CA3 Hipocampal/inmunología , Potenciales de la Membrana , Embarazo/inmunología , Células Piramidales/inmunología , Ácido gamma-Aminobutírico/inmunología , Animales , Región CA3 Hipocampal/efectos de los fármacos , Dendritas/efectos de los fármacos , Dendritas/inmunología , Femenino , Ácido Glutámico/fisiología , Potenciales de la Membrana/efectos de los fármacos , Ratones Endogámicos C57BL , Poli I-C/administración & dosificación , Células Piramidales/citología , Células Piramidales/efectos de los fármacos , Miembro 2 de la Familia de Transportadores de Soluto 12/inmunologíaRESUMEN
Autism spectrum disorders (ASD) are neurodevelopmental disorders induced by genetic and environmental factors. In our recent studies, we showed that the GABA developmental shifts during delivery and the second postnatal week are abolished in two rodent models of ASD. Maternal treatment around birth with bumetanide restored the GABA developmental sequence and attenuated the autism pathogenesis in offspring. Clinical trials conducted in parallel confirmed the usefulness of bumetanide treatment to attenuate the symptoms in children with ASD. Collectively, these observations suggest that an alteration of the GABA developmental sequence is a hallmark of ASD. Here, we investigated whether similar alterations occur in the Shank3 mouse model of ASD. We report that in CA3 pyramidal neurons, the driving force and inhibitory action of GABA are not different in naïve and Shank3-mutant age-matched animals at birth and during the second postnatal week. In contrast, the frequency of spontaneous excitatory postsynaptic currents is already enhanced at birth and persists through postnatal day 15. Therefore, in CA3 pyramidal neurons of Shank3-mutant mice, glutamatergic but not GABAergic activity is affected at early developmental stages, hence reflecting the heterogeneity of mechanisms underlying the pathogenesis of ASD.
Asunto(s)
Región CA3 Hipocampal/metabolismo , Potenciales Postsinápticos Excitadores/fisiología , Ácido Glutámico/metabolismo , Proteínas del Tejido Nervioso/genética , Células Piramidales/metabolismo , Animales , Animales Recién Nacidos , Trastorno del Espectro Autista/genética , Trastorno del Espectro Autista/metabolismo , Modelos Animales de Enfermedad , Ratones , Ratones Noqueados , Proteínas de Microfilamentos , Proteínas del Tejido Nervioso/metabolismo , Técnicas de Placa-Clamp , Ácido gamma-Aminobutírico/metabolismoRESUMEN
UNLABELLED: We report that daily administration of the diuretic NKCC1 chloride co-transporter, bumetanide, reduces the severity of autism in a 10-year-old Fragile X boy using CARS, ADOS, ABC, RDEG and RRB before and after treatment. In keeping with extensive clinical use of this diuretic, the only side effect was a small hypokalaemia. A double-blind clinical trial is warranted to test the efficacy of bumetanide in FRX. CONCLUSION: This single case report showed an improvement of the scores of each test used after 3 months of treatment. Double-blind clinical trials are warranted to test the efficacy of bumetanide in FRX.
Asunto(s)
Bumetanida/uso terapéutico , Trastornos Generalizados del Desarrollo Infantil/complicaciones , Diuréticos/uso terapéutico , Síndrome del Cromosoma X Frágil/complicaciones , Síndrome del Cromosoma X Frágil/tratamiento farmacológico , Bumetanida/efectos adversos , Niño , Diuréticos/efectos adversos , Humanos , Hipopotasemia/inducido químicamente , MasculinoRESUMEN
GABA depolarizes immature neurons because of a high [Cl(-)](i) and orchestrates giant depolarizing potential (GDP) generation. Zilberter and coworkers (Rheims et al., 2009; Holmgren et al., 2010) showed recently that the ketone body metabolite DL-3-hydroxybutyrate (DL-BHB) (4 mM), lactate (4 mM), or pyruvate (5 mM) shifted GABA actions to hyperpolarizing, suggesting that the depolarizing effects of GABA are attributable to inadequate energy supply when glucose is the sole energy source. We now report that, in rat pups (postnatal days 4-7), plasma D-BHB, lactate, and pyruvate levels are 0.9, 1.5, and 0.12 mM, respectively. Then, we show that DL-BHB (4 mM) and pyruvate (200 µM) do not affect (i) the driving force for GABA(A) receptor-mediated currents (DF(GABA)) in cell-attached single-channel recordings, (2) the resting membrane potential and reversal potential of synaptic GABA(A) receptor-mediated responses in perforated patch recordings, (3) the action potentials triggered by focal GABA applications, or (4) the GDPs determined with electrophysiological recordings and dynamic two-photon calcium imaging. Only very high nonphysiological concentrations of pyruvate (5 mM) reduced DF(GABA) and blocked GDPs. Therefore, DL-BHB does not alter GABA signals even at the high concentrations used by Zilberter and colleagues, whereas pyruvate requires exceedingly high nonphysiological concentrations to exert an effect. There is no need to alter conventional glucose enriched artificial CSF to investigate GABA signals in the developing brain.
Asunto(s)
Potenciales de Acción/efectos de los fármacos , Cuerpos Cetónicos/metabolismo , Neuronas/efectos de los fármacos , Neuronas/fisiología , Ácido Pirúvico/metabolismo , Ácido gamma-Aminobutírico/farmacología , Ácido 3-Hidroxibutírico/sangre , Ácido 3-Hidroxibutírico/farmacología , Potenciales de Acción/fisiología , Animales , Animales Recién Nacidos/sangre , Bicuculina/farmacología , Encéfalo/citología , Encéfalo/crecimiento & desarrollo , Bumetanida/farmacología , Calcio/metabolismo , Relación Dosis-Respuesta a Droga , Antagonistas de Aminoácidos Excitadores/farmacología , Femenino , Antagonistas de Receptores de GABA-A/farmacología , Técnicas In Vitro , Ácido Láctico/sangre , Masculino , Técnicas de Placa-Clamp/métodos , Ácido Pirúvico/farmacología , Ratas , Ratas Wistar , Transducción de Señal/efectos de los fármacos , Inhibidores del Simportador de Cloruro Sódico y Cloruro Potásico/farmacologíaRESUMEN
Brain slices incubated with glucose have provided most of our knowledge on cellular, synaptic, and network driven mechanisms. It has been recently suggested that γ-aminobutyric acid (GABA) excites neonatal neurons in conventional glucose-perfused slices but not when ketone bodies metabolites, pyruvate, and/or lactate are added, suggesting that the excitatory actions of GABA are due to energy deprivation when glucose is the sole energy source. In this article, we review the vast number of studies that show that slices are not energy deprived in glucose-containing medium, and that addition of other energy substrates at physiologic concentrations does not alter the excitatory actions of GABA on neonatal neurons. In contrast, lactate, like other weak acids, can produce an intracellular acidification that will cause a reduction of intracellular chloride and a shift of GABA actions. The effects of high concentrations of lactate, and particularly of pyruvate (4-5 mm), as used are relevant primarily to pathologic conditions; these concentrations not being found in the brain in normal "control" conditions. Slices in glucose-containing medium may not be ideal, but additional energy substrates neither correspond to physiologic conditions nor alter GABA actions. In keeping with extensive observations in a wide range of animal species and brain structures, GABA depolarizes immature neurons and the reduction of the intracellular concentration of chloride ([Cl(-)](i)) is a basic property of brain maturation that has been preserved throughout evolution. In addition, this developmental sequence has important clinical implications, notably concerning the higher incidence of seizures early in life and their long-lasting deleterious sequels. Immature neurons have difficulties exporting chloride that accumulates during seizures, leading to permanent increase of [Cl(-)](i) that converts the inhibitory actions of GABA to excitatory and hampers the efficacy of GABA-acting antiepileptic drugs.
Asunto(s)
Encéfalo/citología , Metabolismo Energético/efectos de los fármacos , Cuerpos Cetónicos/metabolismo , Neuronas/efectos de los fármacos , Ácido gamma-Aminobutírico/farmacología , Animales , Animales Recién Nacidos , Encéfalo/crecimiento & desarrollo , Metabolismo Energético/fisiología , Técnicas In Vitro , Ácido Láctico/metabolismo , Ácido Pirúvico/metabolismo , Simportadores de Cloruro de Sodio-Potasio/metabolismo , Miembro 2 de la Familia de Transportadores de Soluto 12 , Simportadores/metabolismo , Ácido gamma-Aminobutírico/metabolismo , Cotransportadores de K ClRESUMEN
OBJECTIVE: The mechanisms of epileptogenesis in Sturge-Weber syndrome (SWS) are unknown. We explored the properties of neurons from human pediatric SWS cortex in vitro and tested in particular whether gamma-aminobutyric acid (GABA) excites neurons in SWS cortex, as has been suggested for various types of epilepsies. METHODS: Patch-clamp and field potential recordings and dynamic biphoton imaging were used to analyze cortical tissue samples obtained from four 6- to 14-month-old pediatric SWS patients during surgery. RESULTS: Neurons in SWS cortex were characterized by a relatively depolarized resting membrane potential, as was estimated from cell-attached recordings of N-methyl-D-aspartate channels. Many cells spontaneously fired action potentials at a rate proportional to the level of neuronal depolarization. The reversal potential for GABA-activated currents, assessed by cell-attached single channel recordings, was close to the resting membrane potential. All spontaneously firing neurons recorded in cell-attached mode or imaged with biphoton microscopy were inhibited by GABA. Spontaneous epileptiform activity in the form of recurrent population bursts was suppressed by glutamate receptor antagonists, the GABA(A) receptor agonist isoguvacine, and the positive allosteric GABA(A) modulator diazepam. Blockade of GABA(A) receptors aggravated spontaneous epileptiform activity. The NKCC1 antagonist bumetanide had little effect on epileptiform activity. INTERPRETATION: SWS cortical neurons have a relatively depolarized resting membrane potential and spontaneously fire action potentials that may contribute to increased network excitability. In contrast to previous data depicting excitatory and proconvulsive actions of GABA in certain pediatric and adult epilepsies, GABA plays mainly an inhibitory and anticonvulsive role in SWS pediatric cortex.
Asunto(s)
Corteza Cerebral/fisiopatología , Inhibición Neural/fisiología , Neuronas/fisiología , Síndrome de Sturge-Weber/fisiopatología , Ácido gamma-Aminobutírico/metabolismo , Potenciales de Acción/efectos de los fármacos , Potenciales de Acción/fisiología , Bumetanida/farmacología , Corteza Cerebral/efectos de los fármacos , Diazepam/farmacología , Epilepsia/tratamiento farmacológico , Epilepsia/fisiopatología , Antagonistas de Aminoácidos Excitadores/farmacología , Agonistas del GABA/farmacología , Moduladores del GABA/farmacología , Agonistas de Receptores de GABA-A , Humanos , Técnicas In Vitro , Lactante , Ácidos Isonicotínicos/farmacología , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Inhibición Neural/efectos de los fármacos , Neuronas/efectos de los fármacos , Receptores de GABA-A/metabolismo , Receptores de Glutamato/metabolismo , Inhibidores del Simportador de Cloruro Sódico y Cloruro Potásico/farmacología , Miembro 2 de la Familia de Transportadores de Soluto 12RESUMEN
The NMDA subtype of glutamate receptors (NMDAR) at excitatory neuronal synapses plays a key role in synaptic plasticity. The extracellular signal-regulated kinase (ERK1,2 or ERK) pathway is an essential component of NMDAR signal transduction controlling the neuroplasticity underlying memory processes, neuronal development, and refinement of synaptic connections. Here we show that NR2B, but not NR2A or NR1 subunits of the NMDAR, interacts in vivo and in vitro with RasGRF1, a Ca(2+)/calmodulin-dependent Ras-guanine-nucleotide-releasing factor. Specific disruption of this interaction in living neurons abrogates NMDAR-dependent ERK activation. Thus, RasGRF1 serves as NMDAR-dependent regulator of the ERK kinase pathway. The specific association of RasGRF1 with the NR2B subunit and study of ERK activation in neurons with varied content of NR2B suggests that NR2B-containing channels are the dominant activators of the NMDA-dependent ERK pathway.
Asunto(s)
Proteínas Quinasas Activadas por Mitógenos/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Transducción de Señal/genética , Sinapsis/enzimología , ras-GRF1/metabolismo , Animales , Animales Recién Nacidos , Sitios de Unión/genética , Señalización del Calcio/efectos de los fármacos , Señalización del Calcio/fisiología , Células Cultivadas , Feto , Hipocampo/metabolismo , Humanos , Mutación/genética , N-Metilaspartato/farmacología , Plasticidad Neuronal/genética , Técnicas de Cultivo de Órganos , Péptidos/farmacología , Estructura Terciaria de Proteína/genética , Ratas , Receptores de N-Metil-D-Aspartato/efectos de los fármacos , Receptores de N-Metil-D-Aspartato/genética , Transmisión Sináptica/genética , ras-GRF1/antagonistas & inhibidores , ras-GRF1/genéticaRESUMEN
The relative contribution of kainate receptors to ongoing glutamatergic activity is at present unknown. We report the presence of spontaneous, miniature, and minimal stimulation-evoked excitatory postsynaptic currents (EPSCs) that are mediated solely by kainate receptors (EPSC(kainate)) or by both AMPA and kainate receptors (EPSC(AMPA/kainate)). EPSC(kainate) and EPSC(AMPA/kainate) are selectively enriched in CA1 interneurons and mossy fibers synapses of CA3 pyramidal neurons, respectively. In CA1 interneurons, the decay time constant of EPSC(kainate) (circa 10 ms) is comparable to values obtained in heterologous expression systems. In both hippocampal neurons, the quantal release of glutamate generates kainate receptor-mediated EPSCs that provide as much as half of the total glutamatergic current. Kainate receptors are, therefore, key players of the ongoing glutamatergic transmission in the hippocampus.
Asunto(s)
Potenciales Postsinápticos Excitadores/fisiología , Ácido Glutámico/metabolismo , Hipocampo/metabolismo , Terminales Presinápticos/metabolismo , Receptores AMPA/metabolismo , Receptores de Ácido Kaínico/metabolismo , Vesículas Sinápticas/metabolismo , Animales , Dinorfinas/metabolismo , Estimulación Eléctrica , Antagonistas de Aminoácidos Excitadores/farmacología , Potenciales Postsinápticos Excitadores/efectos de los fármacos , Antagonistas del GABA/farmacología , Hipocampo/citología , Hipocampo/efectos de los fármacos , Interneuronas/citología , Interneuronas/efectos de los fármacos , Interneuronas/metabolismo , Cinética , Masculino , Fibras Musgosas del Hipocampo/efectos de los fármacos , Fibras Musgosas del Hipocampo/metabolismo , Fibras Musgosas del Hipocampo/ultraestructura , Técnicas de Cultivo de Órganos , Terminales Presinápticos/efectos de los fármacos , Células Piramidales/citología , Células Piramidales/efectos de los fármacos , Células Piramidales/metabolismo , Ratas , Ratas Wistar , Receptores AMPA/antagonistas & inhibidores , Receptores de Ácido Kaínico/antagonistas & inhibidores , Bloqueadores de los Canales de Sodio/farmacología , Transmisión Sináptica/efectos de los fármacos , Transmisión Sináptica/fisiología , Vesículas Sinápticas/efectos de los fármacosRESUMEN
During postnatal development of the rat hippocampus, gamma-aminobutyric acid (GABA) switches its action on CA3 pyramidal cells from excitatory to inhibitory. To characterize the underlying changes in the GABA reversal potential, we used somatic cell-attached recordings of GABA(A) and N-methyl-D-aspartate channels to monitor the GABA driving force and resting membrane potential, respectively. We found that the GABA driving force is strongly depolarizing during the first postnatal week. The strength of this depolarization rapidly declines with age, although GABA remains slightly depolarizing, by a few millivolts, even in adult neurons. Reduction in the depolarizing GABA driving force was due to a progressive negative shift of the reversal potential of GABA currents. Similar postnatal changes in GABA signalling were also observed using the superfused hippocampus preparation in vivo, and in the hippocampal interneurons in vitro. We also found that in adult pyramidal cells, somatic GABA reversal potential is maintained at a slightly depolarizing level by bicarbonate conductance, chloride-extrusion and chloride-loading systems. Thus, the postnatal excitatory-to-inhibitory switch in somatic GABA signalling is associated with a negative shift of the GABA reversal potential but without a hyperpolarizing switch in the polarity of GABA responses. These results also suggest that in adult CA3 pyramidal cells, somatic GABAergic inhibition takes place essentially through shunting rather than hyperpolarization. Apparent hyperpolarizing GABA responses previously reported in the soma of CA3 pyramidal cells are probably due to cell depolarization during intracellular or whole-cell recordings.
Asunto(s)
Diferenciación Celular/fisiología , Hipocampo/crecimiento & desarrollo , Hipocampo/metabolismo , Inhibición Neural/fisiología , Neuronas/metabolismo , Ácido gamma-Aminobutírico/metabolismo , Envejecimiento/metabolismo , Animales , Animales Recién Nacidos , Membrana Celular/efectos de los fármacos , Membrana Celular/fisiología , Canales de Cloruro/efectos de los fármacos , Canales de Cloruro/metabolismo , Hipocampo/citología , Interneuronas/citología , Interneuronas/efectos de los fármacos , Interneuronas/metabolismo , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Inhibición Neural/efectos de los fármacos , Neuronas/citología , Neuronas/efectos de los fármacos , Células Piramidales/citología , Células Piramidales/efectos de los fármacos , Células Piramidales/metabolismo , Ratas , Receptores de GABA-A/efectos de los fármacos , Receptores de GABA-A/metabolismo , Receptores de N-Metil-D-Aspartato/efectos de los fármacos , Receptores de N-Metil-D-Aspartato/metabolismo , Transmisión Sináptica/efectos de los fármacos , Transmisión Sináptica/fisiologíaRESUMEN
We report that the oxytocin-mediated neuroprotective γ-aminobutyric acid (GABA) excitatory-inhibitory shift during delivery is abolished in the valproate and fragile X rodent models of autism. During delivery and subsequently, hippocampal neurons in these models have elevated intracellular chloride levels, increased excitatory GABA, enhanced glutamatergic activity, and elevated gamma oscillations. Maternal pretreatment with bumetanide restored in offspring control electrophysiological and behavioral phenotypes. Conversely, blocking oxytocin signaling in naïve mothers produced offspring having electrophysiological and behavioral autistic-like features. Our results suggest a chronic deficient chloride regulation in these rodent models of autism and stress the importance of oxytocin-mediated GABAergic inhibition during the delivery process. Our data validate the amelioration observed with bumetanide and oxytocin and point to common pathways in a drug-induced and a genetic rodent model of autism.
Asunto(s)
Trastorno Autístico/inducido químicamente , Trastorno Autístico/genética , Citoprotección , Oxitocina/metabolismo , Ácido gamma-Aminobutírico/metabolismo , Animales , Trastorno Autístico/metabolismo , Conducta Animal , Bumetanida/administración & dosificación , Cloruros/metabolismo , Modelos Animales de Enfermedad , Femenino , Proteína de la Discapacidad Intelectual del Síndrome del Cromosoma X Frágil/genética , Intercambio Materno-Fetal , Ratones , Parto , Embarazo , Ratas , Ácido Valproico/farmacologíaRESUMEN
The mechanisms controlling pain in newborns during delivery are poorly understood. We explored the hypothesis that oxytocin, an essential hormone for labor and a powerful neuromodulator, exerts analgesic actions on newborns during delivery. Using a thermal tail-flick assay, we report that pain sensitivity is two-fold lower in rat pups immediately after birth than 2 days later. Oxytocin receptor antagonists strongly enhanced pain sensitivity in newborn, but not in 2-day-old rats, whereas oxytocin reduced pain at both ages suggesting an endogenous analgesia by oxytocin during delivery. Similar analgesic effects of oxytocin, measured as attenuation of pain-vocalization induced by electrical whisker pad stimulation, were also observed in decerebrated newborns. Oxytocin reduced GABA-evoked calcium responses and depolarizing GABA driving force in isolated neonatal trigeminal neurons suggesting that oxytocin effects are mediated by alterations of intracellular chloride. Unlike GABA signaling, oxytocin did not affect responses mediated by P2X3 and TRPV1 receptors. In keeping with a GABAergic mechanism, reduction of intracellular chloride by the diuretic NKCC1 chloride co-transporter antagonist bumetanide mimicked the analgesic actions of oxytocin and its effects on GABA responses in nociceptive neurons. Therefore, endogenous oxytocin exerts an analgesic action in newborn pups that involves a reduction of the depolarizing action of GABA on nociceptive neurons. Therefore, the same hormone that triggers delivery also acts as a natural pain killer revealing a novel facet of the protective actions of oxytocin in the fetus at birth.
RESUMEN
The neuronal potassium-chloride co-transporter 2 [indicated thereafter as KCC2 (for protein) and Kcc2 (for gene)] is thought to play an important role in the post natal excitatory to inhibitory switch of GABA actions in the rodent hippocampus. Here, by studying hippocampi of wild-type (Kcc2(+/+)) and Kcc2 deficient (Kcc2(-/-)) mouse embryos, we unexpectedly found increased spontaneous neuronal network activity at E18.5, a developmental stage when KCC2 is thought not to be functional in the hippocampus. Embryonic Kcc2(-/-) hippocampi have also an augmented synapse density and a higher frequency of spontaneous glutamatergic and GABA-ergic postsynaptic currents than naïve age matched neurons. However, intracellular chloride concentration ([Cl(-)](i)) and the reversal potential of GABA-mediated currents (E(GABA)) were similar in embryonic Kcc2(+/+) and Kcc2(-/-) CA3 neurons. In addition, KCC2 immunolabeling was cytoplasmic in the majority of neurons suggesting that the molecule is not functional as a plasma membrane chloride co-transporter. Collectively, our results show that already at an embryonic stage, KCC2 controls the formation of synapses and, when deleted, the hippocampus has a higher density of GABA-ergic and glutamatergic synapses and generates spontaneous and evoked epileptiform activities. These results may be explained either by a small population of orchestrating neurons in which KCC2 operates early as a chloride exporter or by transporter independent actions of KCC2 that are instrumental in synapse formation and networks construction.
RESUMEN
In the developing hippocampus, GABA exerts depolarizing and excitatory actions and contributes to the generation of neuronal network driven giant depolarizing potentials (GDPs). Here, we studied spike time coding at immature GABAergic synapses and its impact on synchronization of the neuronal network during GDPs in the neonatal (postnatal days P2-6) rat hippocampal slices. Using extracellular recordings, we found that the delays of action potentials (APs) evoked by synaptic activation of GABA(A) receptors are long (mean, 65 ms) and variable (within a time window of 10-200 ms). During patch-clamp recordings, depolarizing GABAergic responses were mainly subthreshold and their amplification by persistent sodium conductance was required to trigger APs. AP delays at GABAergic synapses shortened and their variability reduced with an increase in intracellular chloride concentration during whole-cell recordings. Negative shift of the GABA reversal potential (E(GABA)) with low concentrations of bumetanide, or potentiation of GABA(A) receptors with diazepam reduced GDPs amplitude, desynchronized neuronal firing during GDPs and slowed down GDPs propagation. Partial blockade of GABA(A) receptors with bicuculline increased neuronal synchronization and accelerated GDPs propagation. We propose that spike timing at depolarizing GABA synapses is determined by intracellular chloride concentration. At physiological levels of intracellular chloride GABAergic depolarization does not reach the action potential threshold and amplification of GABAergic responses by non-inactivating sodium conductance is required for postsynaptic AP initiation. Slow and variable excitation at GABAergic synapse determines the level of neuronal synchrony and the rate of GDPs propagation in the developing hippocampus.
RESUMEN
Oxytocin (OXT) exerts multiple effects in the adult central nervous system. However, little is known about the effects of OXT on foetal neurons during delivery, at the time when a surge of OXT occurs. In a recent study, the effects of OXT on gamma-aminobutyric acid (GABA) signalling have been reported in foetal and newborn rats. In the immature rat hippocampal and neocortical neurons at birth, endogenous OXT induced a switch in the action of GABA from excitatory to inhibitory. This excitatory-to-inhibitory switch was caused by a switch in the polarity of the GABAergic responses from depolarizing to hyperpolarizing, reflecting a decrease in the intracellular chloride concentration. The effects of OXT were mimicked and occluded by bumetanide, a selective blocker of the chloride co-transporter NKCC1, suggesting that the effects of OXT involve inhibition of NKCC1. Neuronal death caused by anoxic-aglycaemic episodes was substantially delayed in the foetal hippocampus by endogenous OXT. These findings suggest that OXT plays important role in the preparation of the foetal brain to delivery.
Asunto(s)
Encéfalo/embriología , Oxitocina/farmacología , Ácido gamma-Aminobutírico/fisiología , Potenciales de Acción/fisiología , Animales , Parto Obstétrico , Femenino , Feto/fisiología , Humanos , Trabajo de Parto/fisiología , Lactancia/fisiología , Neuroglía/fisiología , Neuronas/fisiología , Embarazo , Simportadores de Cloruro de Sodio-Potasio/fisiología , Miembro 2 de la Familia de Transportadores de Soluto 12RESUMEN
The timing of the developmental switch in the GABA(A) mediated responses from excitatory to inhibitory was studied in Wistar rat CA3 hippocampal pyramidal cells using gramicidin perforated patch-clamp and extracellular recordings. Gramicidin perforated patch recordings revealed a gradual developmental shift in the reversal potential of synaptic and isoguvacine-induced GABA(A) mediated responses from -55 +/- 4 mV at postnatal days P0-2 to -74 +/- 3 mV at P13-15 with a midpoint of disappearance of the excitatory effects of GABA at around P8. Extracellular recordings in CA3 pyramidal cell layer revealed that the effect of isoguvacine on multiple unit activity (MUA) switched from an increase to a decrease at around P10. The effect of synaptic GABA(A) mediated responses on MUA switched from an increase to a decrease at around P8. It is concluded that the developmental switch in the action of GABA via GABA(A) receptors from excitatory to inhibitory occurs in Wistar rat CA3 pyramidal cells at around P8-10, an age that coincides with the transition from immature to mature hippocampal rhythms. We propose that excitatory GABA contributes to enhanced excitability and ictogenesis in the neonatal rat hippocampus.
Asunto(s)
Hipocampo/crecimiento & desarrollo , Ácido gamma-Aminobutírico/fisiología , Potenciales de Acción/efectos de los fármacos , Potenciales de Acción/fisiología , Factores de Edad , Animales , Animales Recién Nacidos , Bicuculina/farmacología , Canales de Calcio/efectos de los fármacos , Electrofisiología , Potenciales Postsinápticos Excitadores/efectos de los fármacos , Potenciales Postsinápticos Excitadores/fisiología , Femenino , Antagonistas de Receptores de GABA-A , Gramicidina , Hipocampo/efectos de los fármacos , Hipocampo/fisiología , Potenciales Postsinápticos Inhibidores/efectos de los fármacos , Potenciales Postsinápticos Inhibidores/fisiología , Ácidos Isonicotínicos/farmacología , Masculino , Técnicas de Placa-Clamp , Células Piramidales/efectos de los fármacos , Células Piramidales/fisiología , Ratas , Ratas Wistar , Receptores de GABA-A/efectos de los fármacos , Receptores de GABA-A/fisiología , Ácido gamma-Aminobutírico/farmacologíaRESUMEN
Developing networks follow common rules to shift from silent cells to coactive networks that operate via thousands of synapses. This review deals with some of these rules and in particular those concerning the crucial role of the neurotransmitter gamma-aminobuytric acid (GABA), which operates primarily via chloride-permeable GABA(A) receptor channels. In all developing animal species and brain structures investigated, neurons have a higher intracellular chloride concentration at an early stage leading to an efflux of chloride and excitatory actions of GABA in immature neurons. This triggers sodium spikes, activates voltage-gated calcium channels, and acts in synergy with NMDA channels by removing the voltage-dependent magnesium block. GABA signaling is also established before glutamatergic transmission, suggesting that GABA is the principal excitatory transmitter during early development. In fact, even before synapse formation, GABA signaling can modulate the cell cycle and migration. The consequence of these rules is that developing networks generate primitive patterns of network activity, notably the giant depolarizing potentials (GDPs), largely through the excitatory actions of GABA and its synergistic interactions with glutamate signaling. These early types of network activity are likely required for neurons to fire together and thus to "wire together" so that functional units within cortical networks are formed. In addition, depolarizing GABA has a strong impact on synaptic plasticity and pathological insults, notably seizures of the immature brain. In conclusion, it is suggested that an evolutionary preserved role for excitatory GABA in immature cells provides an important mechanism in the formation of synapses and activity in neuronal networks.
Asunto(s)
Neuronas/fisiología , Transmisión Sináptica/fisiología , Ácido gamma-Aminobutírico/fisiología , Animales , Plasticidad Neuronal/fisiología , Convulsiones/fisiopatología , Transducción de Señal/fisiología , Sinapsis/fisiologíaRESUMEN
We report a signaling mechanism in rats between mother and fetus aimed at preparing fetal neurons for delivery. In immature neurons, gamma-aminobutyric acid (GABA) is the primary excitatory neurotransmitter. We found that, shortly before delivery, there is a transient reduction in the intracellular chloride concentration and an excitatory-to-inhibitory switch of GABA actions. These events were triggered by oxytocin, an essential maternal hormone for labor. In vivo administration of an oxytocin receptor antagonist before delivery prevented the switch of GABA actions in fetal neurons and aggravated the severity of anoxic episodes. Thus, maternal oxytocin inhibits fetal neurons and increases their resistance to insults during delivery.
Asunto(s)
Feto/fisiología , Hipocampo/embriología , Inhibición Neural , Neuronas/fisiología , Oxitocina/fisiología , Parto , Ácido gamma-Aminobutírico/fisiología , Potenciales de Acción , Animales , Animales Recién Nacidos , Benzamidas/farmacología , Cloruros/metabolismo , Femenino , Hipoxia Fetal/fisiopatología , Agonistas de Receptores de GABA-A , Antagonistas de Receptores de GABA-A , Hipocampo/citología , Hipocampo/fisiología , Técnicas In Vitro , Indoles/farmacología , Intercambio Materno-Fetal , Oxitocina/farmacología , Técnicas de Placa-Clamp , Embarazo , Ratas , Ratas Wistar , Receptores de GABA-A/fisiología , Receptores de Oxitocina/antagonistas & inhibidores , Transducción de Señal , Inhibidores del Simportador de Cloruro Sódico y Cloruro Potásico , Simportadores de Cloruro de Sodio-Potasio/metabolismo , Miembro 2 de la Familia de Transportadores de Soluto 12 , Vasotocina/análogos & derivados , Vasotocina/farmacologíaRESUMEN
The immature brain is prone to seizures but the underlying mechanisms are poorly understood. We explored the hypothesis that increased seizure susceptibility during early development is due to the excitatory action of GABA. Using noninvasive extracellular field potential and cell-attached recordings in CA3 of Sprague-Dawley rat hippocampal slices, we compared the developmental alterations in three parameters: excitatory actions of GABA, presence of the immature pattern of giant depolarizing potentials (GDPs) and severity of epileptiform activity generated by high potassium. The GABA(A) receptor agonist isoguvacine increased firing of CA3 pyramidal cells in neonatal slices while inhibiting activity in adults. A switch in the GABA(A) signalling from excitation to inhibition occurred at postnatal day (P) 13.5 +/- 0.4. Field GDPs were present in the form of spontaneous population bursts until P12.7 +/- 0.3. High potassium (8.5 mm) induced seizure-like events (SLEs) in 35% of slices at P7-16 (peak at P11.3 +/- 0.4), but only interictal activity before and after that age. The GABA(A) receptor antagonist bicuculline reduced the frequency or completely blocked SLEs and induced interictal clonic-like activity accompanied by a reduction in the frequency but an increase in the amplitude of the population spikes. In slices with interictal activity, bicuculline typically caused a large amplitude interictal clonic-like activity at all ages; in slices from P5-16 rats it was often preceded by one SLE at the beginning of bicuculline application. These results suggest that, in the immature hippocampus, GABA exerts dual (both excitatory and inhibitory) actions and that the excitatory component in the action of GABA may contribute to increased excitability during early development.