Characterization of oligophrenin-1, a RhoGAP lost in patients affected with mental retardation: lentiviral injection in organotypic brain slice cultures.
Mutations in regulators and effectors of the Rho GTPases underlie various forms of mental retardation (MR). Among them, oligophrenin-1 (OPHN1), which encodes a Rho-GTPase activating protein, was one of the first Rho-linked MR genes identified. Upon characterization of OPHN1 in hippocampal brain slices, we obtained evidence for the requirement of OPHN1 in dendritic spine morphogenesis and neuronal function of CA1 pyramidal neurons. Organotypic hippocampal brain slice cultures are commonly used as a model system to investigate the morphology and synaptic function of neurons, mainly because they allow for the long-term examination of neurons in a preparation where the gross cellular architecture of the hippocampus is retained. In addition, maintenance of the trisynaptic circuitry in hippocampal slices enables the study of synaptic connections. Today, a multitude of gene transfer methods for postmitotic neurons in brain slices are available to easily manipulate and scrutinize the involvement of signaling molecules, such as Rho GTPases, in specific cellular processes in this system. This chapter covers techniques detailing the preparation and culturing of organotypic hippocampal brain slices, as well as the production and injection of lentivirus into brain slices.
Kasri
Nael Nadif
NN
Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, USA.
Govek
Eve-Ellen
EE
Van Aelst
Linda
L
D000818:Animals; D001921:Brain / Q000378:metabolism*; D003598:Cytoskeletal Proteins / Q000302:isolation & purification / Q000502:physiology*; D020690:GTPase-Activating Proteins / Q000302:isolation & purification / Q000502:physiology*; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D006624:Hippocampus / Q000502:physiology / Q000821:virology; D006801:Humans; D016086:Lentivirus; D009687:Nuclear Proteins / Q000302:isolation & purification / Q000502:physiology*; D009924:Organ Culture Techniques / Q000379:methods; D051381:Rats
D003598:Cytoskeletal Proteins; D020690:GTPase-Activating Proteins; D009687:Nuclear Proteins; C529605:OPHN1 protein, rat; C085379:rho GTPase-activating protein