RESUMEN
Fast synaptic communication uses diffusible transmitters whose spread is limited by uptake mechanisms. However, on the submicron-scale, the distance between two synapses, the extent of glutamate spread has so far remained difficult to measure. Here, we show that quantal glutamate release from individual hippocampal synapses activates extracellular iGluSnFr molecules at a distance of >1.5 µm. 2P-glutamate uncaging near spines further showed that alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-Rs and N-methyl-D-aspartate (NMDA)-Rs respond to distant uncaging spots at approximately 800 and 2000 nm, respectively, when releasing the amount of glutamate contained in approximately five synaptic vesicles. The uncaging-induced remote activation of AMPA-Rs was facilitated by blocking glutamate transporters but only modestly decreased by elevating the recording temperature. When mimicking release from neighboring synapses by three simultaneous uncaging spots in the microenvironment of a spine, AMPA-R-mediated responses increased supra-additively. Interfering with extracellular glutamate diffusion through a glutamate scavenger system weakly reduced field synaptic responses but not the quantal amplitude. Together, our data suggest that the neuropil is more permissive to short-range spread of transmitter than suggested by theory, that multivesicular release could regularly coactivate nearest neighbor synapses and that on this scale glutamate buffering by transporters primarily limits the spread of transmitter and allows for cooperative glutamate signaling in extracellular microdomains.
Asunto(s)
Ácido Glutámico , Receptores AMPA , Ácido Glutámico/farmacología , Hipocampo/fisiología , Neurópilo/metabolismo , Receptores AMPA/fisiología , Receptores de N-Metil-D-Aspartato/metabolismo , Sinapsis/fisiología , Ácido alfa-Amino-3-hidroxi-5-metil-4-isoxazol Propiónico/farmacologíaRESUMEN
Reduced heart rate variability (HRV) may predispose to sudden unexpected death in epilepsy (SUDEP). We ascertained whether HRV predicts SUDEP in chronic epilepsy using a case-control design and investigated parameters of inter-ictal HRV in 14 patients (7 had died from SUDEP). No HRV parameter was associated with SUDEP. Thus, although altered HRV might be involved in SUDEP, HRV parameters are not clear-cut predictors for SUDEP.
Asunto(s)
Muerte Súbita Cardíaca/etiología , Epilepsias Parciales/complicaciones , Frecuencia Cardíaca/fisiología , Corazón/fisiopatología , Adulto , Sistema Nervioso Autónomo/fisiopatología , Enfermedades del Sistema Nervioso Autónomo/mortalidad , Enfermedades del Sistema Nervioso Autónomo/fisiopatología , Estudios de Casos y Controles , Electroencefalografía , Epilepsias Parciales/mortalidad , Epilepsias Parciales/fisiopatología , Femenino , Corazón/inervación , Humanos , Masculino , Selección de Paciente , Valor Predictivo de las PruebasRESUMEN
Cultured neurons from bdnf-/- mice display reduced densities of synaptic terminals, although in vivo these deficits are small or absent. Here we aimed at clarifying the local responses to postsynaptic brain-derived neurotrophic factor (BDNF). To this end, solitary enhanced green fluorescent protein (EGFP)-labeled hippocampal neurons from bdnf-/- mice were compared with bdnf-/- neurons after transfection with BDNF, bdnf-/- neurons after transient exposure to exogenous BDNF, and bdnf+/+ neurons in wild-type cultures. Synapse development was evaluated on the basis of presynaptic immunofluorescence and whole-cell patch-clamp recording of miniature postsynaptic currents. It was found that neurons expressing BDNF::EGFP for at least 16 h attracted a larger number of synaptic terminals than BDNF-deficient control neurons. Transfected BDNF formed clusters in the vicinity of glutamatergic terminals and produced a stronger upregulation of synaptic terminal numbers than high levels of ambient BDNF. Glutamatergic and GABAergic synapses reacted differently to postsynaptic BDNF: glutamatergic input increased, whereas GABAergic input decreased. BDNF::EGFP-expressing neurons also differed from BDNF-deficient neurons in their dendrite morphology: they exhibited weaker dendrite elongation and stronger dendrite initiation. The upregulation of glutamatergic synaptic input and the BDNF-induced downregulation of GABAergic synaptic terminal numbers by postsynaptic BDNF depended on tyrosine receptor kinase B activity, as deduced from the blocking effects of K252a. The suppression of dendrite elongation was also prevented by block of tyrosine receptor kinase B but required, in addition, glutamate receptor activity. Dendritic length decreased with the number of glutamatergic contacts. These results illuminate the role of BDNF as a retrograde synaptic regulator of synapse development and the dependence of dendrite elongation on glutamatergic input.
Asunto(s)
Factor Neurotrófico Derivado del Encéfalo/fisiología , Dendritas/fisiología , Ácido Glutámico/fisiología , Hipocampo/fisiología , Sinapsis/fisiología , Ácido gamma-Aminobutírico/fisiología , Animales , Factor Neurotrófico Derivado del Encéfalo/genética , Células Cultivadas , Factor de Crecimiento Epidérmico/genética , Femenino , Expresión Génica/fisiología , Hipocampo/citología , Hipocampo/crecimiento & desarrollo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Plasticidad Neuronal/fisiología , Neuronas/fisiología , Neuronas/ultraestructura , Técnicas de Placa-Clamp , Embarazo , Receptor de Factor de Crecimiento Nervioso/fisiología , Receptor trkB/fisiología , TransfecciónRESUMEN
Brain-derived neurotrophic factor is known to modulate the function of GABAergic synapses, but the site of brain-derived neurotrophic factor action is still a matter of controversy. This study was aimed at further dissecting the functional alterations produced by brain-derived neurotrophic factor treatment of GABAergic synaptic connections in cultures of the murine superior colliculus. The functional consequences of long-term brain-derived neurotrophic factor treatment were assessed by analysis of unitary evoked and delayed inhibitory postsynaptic currents in response to high frequency stimulation of single axons. It was found that brain-derived neurotrophic factor facilitated the asynchronous release, but had no effect on the probability of evoked release, the size of the readily releasable pool, and the paired-pulse behavior of evoked inhibitory postsynaptic currents. However, the amplitudes of evoked inhibitory postsynaptic currents, delayed inhibitory postsynaptic currents and miniature inhibitory postsynaptic currents were significantly reduced. Non-stationary fluctuation analysis revealed a decrease in the open channel number at the miniature/evoked inhibitory postsynaptic current peak, but no effect on the mean GABA(A) receptor single channel conductance. Quantitative immunocytochemistry uncovered a significant elevation of presynaptic levels of glutamic acid decarboxylase 65. Together, these findings indicate that brain-derived neurotrophic factor treatment induces pre- as well as postsynaptic changes. What effect predominates will depend on the presynaptic activity pattern: at low activation rates brain-derived neurotrophic factor-treated synapses display a pronounced postsynaptic depression, but at high frequencies this depression is fully compensated by an enhancement of asynchronous release.
Asunto(s)
Factor Neurotrófico Derivado del Encéfalo/fisiología , Glutamato Descarboxilasa/metabolismo , Isoenzimas/metabolismo , Receptores de GABA/fisiología , Sinapsis/metabolismo , Transmisión Sináptica/fisiología , Ácido gamma-Aminobutírico/fisiología , Animales , Células Cultivadas , Estimulación Eléctrica , Electrofisiología , Potenciales Postsinápticos Excitadores/efectos de los fármacos , Técnica del Anticuerpo Fluorescente , Inmunohistoquímica , Ratones , Ratones Endogámicos C57BL , Técnicas de Placa-Clamp , Receptores de GABA-A/fisiología , Colículos Superiores/citología , Colículos Superiores/fisiologíaRESUMEN
During postnatal development, the retinocollicular pathway undergoes activity-dependent refinement, resulting in the precise retinotopic map seen in adults. Previous studies established that retinal efferents reach the mouse superior colliculus (SC) by embryonic day 16. Morphologically, synapses were found in the rat SC before birth. As part of an extended project aimed at understanding the development of synaptic transmission in the visual layers of the SC, we report here the presence of functionally active synapses immediately after birth. Circuit activity in mouse SC neurons was detected in horizontal slices of the visual layers using cell-attached voltage clamp. The spontaneous discharge of action potentials was abolished by glutamatergic blockers and facilitated by bicuculline, showing that circuit activity is based on synaptic transmission and that the action of gamma-aminobutyric acid is inhibitory. Using whole-cell voltage clamp, spontaneous glutamatergic postsynaptic currents as well as miniature GABAergic postsynaptic currents were recorded on postnatal day 1. Excitatory and inhibitory postsynaptic currents could also be evoked by electrical stimulation. Glutamatergic postsynaptic currents comprised both (S)-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid and N-methyl-D-aspartate receptor-mediated components. The early function of glutamatergic and GABAergic synaptic transmission in the visual layers of SC suggests that SC neurons are able to process information originating from retinal axons immediately after birth.
Asunto(s)
Bicuculina/análogos & derivados , Ácido Glutámico/metabolismo , Colículos Superiores/crecimiento & desarrollo , Colículos Superiores/fisiología , Transmisión Sináptica/fisiología , Ácido gamma-Aminobutírico/metabolismo , 2-Amino-5-fosfonovalerato/farmacología , Animales , Animales Recién Nacidos , Bicuculina/farmacología , Antagonistas de Aminoácidos Excitadores/farmacología , Potenciales Postsinápticos Excitadores/efectos de los fármacos , Potenciales Postsinápticos Excitadores/fisiología , Antagonistas del GABA/farmacología , Ratones , Ratones Endogámicos C57BL , Antagonistas Nicotínicos/farmacología , Técnicas de Cultivo de Órganos , Quinoxalinas/farmacología , Ratas , Ratas Wistar , Especificidad de la Especie , Transmisión Sináptica/efectos de los fármacos , Tubocurarina/farmacologíaRESUMEN
One important aspect of utilizing transgenic mice is the need to genotype them in order to distinguish mice that carry a disrupted gene or a transgene from mice that do not. Current methods for genotyping include isolation of genomic DNA from tail biopsies followed by PCR amplification. Particularly, both digestion of tail tissue using proteinase K as well as resuspension of purified DNA are time-consuming and were usually carried out overnight. Here, we describe a rapid and robust method for the genotyping of bdnf targeted mice which allows us to determine the genotype of newborn mice at the day of birth within 6 h. After a freezing-thawing step tail tissue is digested in less than 2 h, and the DNA is precipitated, resuspended and ready for PCR in about 60 min. The method could be easily adapted to a variety of different mutant mice and especially should benefit neuroscientists interested in using animals with known genotype very early in postnatal development.