Characterization of Oligophrenin‐1, a RhoGAP Lost in Patients Affected with Mental Retardation: Lentiviral Injection in Organotypic Brain Slice Cultures

Abstract

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 (Rho-GAP), was one of the first Rho-linked MR gene identified. Upon characterization of OPHN1 in hippocampal brain slices, we obtained evidence for the requirement of OPHN1 in dendritic spine morphogenesis and synaptic 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, the maintenance of the tri-synaptic 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.

Novel intragenic deletion in OPHN1 in a family causing XLMR with cerebellar hypoplasia and distinctive facial appearance

“…At least six of the genes involved in XLMR encode regulators or effectors of Rho-GTPase proteins, suggesting an important role of the Rho signaling pathway in cognitive function (10). At least six of the genes involved in XLMR encode regulators or effectors of Rho-GTPase proteins, suggesting an important role of the Rho signaling pathway in cognitive function (10).…”

“…Affected individuals exhibit moderate to severe cognitive impairment with major language, behavioral problems, dysmetria and nystagmus. The pathophysiological role of OPHN1, however, remains poorly understood (10). The OPHN1 gene is highly expressed in fetal and adult brain, and it encodes a protein with a Rho-GTPase-activating domain (rhoGAP).…”

“…The OPHN1 gene is highly expressed in fetal and adult brain, and it encodes a protein with a Rho-GTPase-activating domain (rhoGAP). The pathophysiological role of OPHN1, however, remains poorly understood (10).…”

Myelination and Axonal Electrical Activity Modulate the Distribution and Motility of Mitochondria at CNS Nodes of Ranvier

“…The slices were cultured on Millicell organotypic culture inserts (Millipore, Billerica, MA, USA) in 6-well plates containing 50% minimal essential medium, 25% horse serum, 25% HBSS, 6mg/ml D-glucose, 1× Glutamax (Invitrogen) and penicillin/streptomycin. Two hours after plating, the lentiviral vectors were injected into the Purkinje cell layer using a glass microinjection needle (Kasri et al, 2008). …”

Acute Hippocampal Slice Preparation and Hippocampal Slice Cultures

“…Various labeling techniques have been successfully applied to image neuronal and glial cells in hippocampal slice cultures, including: (1) immunohistochemistry; (2) bath application, injection, or ballistic delivery (8) of fluorescent structural or physiological indicators; and (3) biolistic (9) or viral transfection (10) to express cDNAs or siRNAs in a subset of neuronal or glial cells. In this chapter, we describe a method for labeling the soma, dendritic arbors, and axon of a subset of neurons in slice cultures using the biolistic delivery of cDNA encoding a fluorescent protein.…”