Adenovirus-rnediated expression of a reporter gene in thalamocortical cocultures

Abstract

Organotypic cocultures of thalamic and cortical explants have recently been used to study the development of the thalamocortical axonal network in the mammalian neocortex. To explore the possibility of genetically manipulating organotypic explants, rat thalamocortical (TC) cocultures were infected with the recombinant adenovirus, Adv/RSVβgal. Infection of the cortical explants resulted in long-term expression (2 weeks) of the reporter gene (β-galactosidase) with no significant alterations to the structural integrity of the explants. By micro-injecting the adenoviruses into cortical explants a significant degree of spatial control over reporter gene expression was obtained. Dil-labeled axonal projections from thalamic explants into infected ( n = 116) and control cortical ( n = 120) explants were also analyzed. There was no significant difference in the extent or degree of TC ingrowth into infected or control cortical explants. Thalamic explants were also efficiently infected with the Adv/RSVβgal virus. While the pattern and extent of TC ingrowth from infected thalamic explants was similar to controls, the percentage of viable, infected thalamic explants was decreased. These experiments were necessary precursors for future studies using recombinant adenoviruses and organotypic cocultures. Genetic manipulation of these cocultures should enable the dissection of proteins involved in the development of axonal networks in the mammalian neocortex, using a system amenable to direct manipulation and observation.