RESUMO
Brain-derived neurotrophic factor has been implicated in higher cognitive functions, and several neurological and psychiatric disorders. Recently, a variant brain-derived neurotrophic factor (BDNFMet), having a substitution referred to as Val66Met, was reported as a product of a bdnf allele with a common single nucleotide polymorphism. It has been reported that BDNFMet is impaired in its potential for activity-dependent release. We sparsely transfected cultured hippocampal neurons with BDNFMet or wild-type BDNFVal cDNAs and examined the amount of GABA-synthetic enzyme glutamic acid decarboxylase 65 (GAD65) in the adjacent region, probably in the GABAergic synapses. BDNFMet transfection increased the GAD65 level to the same extent as transfection with BDNFVal. Our findings suggest that the activity-independent secretion of brain-derived neurotrophic factor may be sufficient to induce inhibitory regulation.
Assuntos
Fator Neurotrófico Derivado do Encéfalo/fisiologia , Metionina/genética , Polimorfismo Genético , Sinapses/metabolismo , Valina/genética , Ácido gama-Aminobutírico/metabolismo , Animais , Fator Neurotrófico Derivado do Encéfalo/genética , Células Cultivadas , Embrião de Mamíferos , Glutamato Descarboxilase/metabolismo , Hipocampo/citologia , Imuno-Histoquímica/métodos , Isoenzimas/metabolismo , Neurônios/citologia , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Probabilidade , Ratos , Ratos Wistar , Sinapses/efeitos dos fármacos , Transfecção/métodosRESUMO
Mossy fibers, the dentate granule cell axons, are generated throughout an animal's lifetime. Mossy fiber paths and synapses are primarily restricted to the stratum lucidum within the CA3 region. Brain-derived neurotrophic factor (BDNF), a neurotrophin family protein that activates Trk neurotrophin receptors, is highly expressed in the stratum lucidum in an activity-dependent manner. The addition of a Trk neurotrophin receptor inhibitor, K252a, to cultured hippocampal slices induced aberrant extension of mossy fibers into ectopic regions. BDNF overexpression in granule cells ameliorated the mossy fiber pathway abnormalities caused by a submaximal dose of K252a. A similar rescue was observed when BDNF was expressed in CA3 pyramidal cells, most notably in mossy fibers distal to the expression site. These findings are the first to clarify the role of BDNF in mossy fiber pathfinding, not as an attractant cue but as a regulator, possibly acting in a paracrine manner. This effect of BDNF may be as a signal for new fibers to fasciculate and extend further to form synapses with neurons that are far from active BDNF-expressing synapses. This mechanism would ensure the emergence of new independent dentate gyrus-CA3 circuits by the axons of new-born granule cells.
Assuntos
Axônios/metabolismo , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Fibras Musgosas Hipocampais/metabolismo , Animais , Axônios/efeitos dos fármacos , Bioensaio , Carbazóis/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Células HEK293 , Humanos , Alcaloides Indólicos/farmacologia , Fibras Musgosas Hipocampais/efeitos dos fármacos , Peptídeos/metabolismo , Células Piramidais/metabolismo , RatosRESUMO
Low-cost, simple procedures for organotypic tissue cultures are desirable for high-throughput biological experiments such as large-scale medical/drug screening. We present a practical and economical method to cultivate brain slices using hydrophilic filtration membranes. With a cost reduction of more than 90%, this technique allows us to prepare hippocampal slice cultures that are morphologically and functionally indistinguishable from those obtained by the widely used Millicell-CM method.