RESUMO
Hippocampal alpha-calcium/calmodulin-dependent protein kinase II (alphaCaMKII) has been implicated in neuronal plasticity and spatial learning. In the present experiment, an adeno-associated virus (AAV) vector was designed to express alphaCaMKII driven by the U6 promotor. Microinfusion of this vector into the rat hippocampus increased alphaCaMKII immunoreactivity by approximately 73% (Western analysis) and improved performance in a water maze task. Locomotor activity and exploratory behavior in an open field task were not altered by the overexpression of alphaCaMKII. These data support a role for alphaCaMKII in spatial or explicit memory storage. The advantages of viral vectors for manipulating target proteins expression compared with genetically modified mouse models are discussed.
Assuntos
Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Regulação Enzimológica da Expressão Gênica/fisiologia , Hipocampo/fisiologia , Memória/fisiologia , Percepção Espacial/fisiologia , Animais , Comportamento Animal , Western Blotting , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina , Dependovirus/fisiologia , Comportamento Exploratório/fisiologia , Imuno-Histoquímica , Masculino , Aprendizagem em Labirinto , Atividade Motora/fisiologia , Ratos , Tempo de ReaçãoRESUMO
Hippocampal alpha-Ca2+/calmodulin-dependent protein kinase II (alpha-CaMKII) has been implicated in spatial learning, neuronal plasticity, epilepsy, and cerebral ischemia. In the present study, an adeno-associated virus (AAV) vector was designed to express green fluorescent protein (GFP) from the CBA promoter and a small hairpin RNA targeting alpha-CaMKII (AAV-shCAM) driven from the U6 promoter. The AAV-shCAM or control vector was microinfused into the rat hippocampus and behavioral testing conducted 19-26 days following surgery. Expression of the marker gene and alpha-CaMKII was evaluated 31 days following AAV infusion. GFP expression was localized to the hippocampus and extended +/-2 mm rostral and caudal from the injection site. Hippocampal alpha-CaMKII was significantly reduced following AAV-shCAM treatment as demonstrated using immunohistochemical and Western analysis. This suppression of alpha-CaMKII was associated with changes in exploratory behavior (open field task) and impaired place learning (water maze task). These results demonstrate the efficacy of a viral-based delivered shRNA to produce gene suppression in a specific circuit of the brain.
