Vertical Bias in Dendritic Trees of Non-pyramidal Neocortical Neurons Expressing GAD67-GFP In Vitro

X. Jin, P. H. Mathers, G. Szabo, Z. Katarova, A. Agmon
2001 Cerebral Cortex  
The neocortical neuropil has a strong vertical (orthogonal to pia) orientation, constraining the intracortical flow of information and forming the basis for the functional parcellation of the cortex into semi-independent vertical columns or 'modules'. Apical dendrites of excitatory pyramidal neurons are a major component of this vertical neuropil, but the extent to which inhibitory, GABAergic neurons conform to this structural and functional design is less well documented. We used a gene gun to
more » ... transfect organotypic slice cultures of mouse and rat neocortex with the enhanced green fluorescent protein (eGFP) gene driven by the promoter for glutamic acid decarboxylase 67 (GAD67), an enzyme expressed exclusively in GABAergic cells. Many GAD67-GFP expressing cells were highly fluorescent, and their dendritic morphologies and axonal patterns, revealed in minute detail, were characteristic of GABAergic neurons. We traced 150 GFP-expressing neurons from confocal image stacks, and estimated the degree of vertical bias in their dendritic trees using a novel computational metric. Over 70% of the neurons in our sample had dendritic trees with a highly significant vertical bias. We conclude that GABAergic neurons make an important contribution to the vertical neocortical neuropil, and are likely to integrate synaptic inputs from axons terminating within their own module. Materials and Methods GAD67-GFP DNA Preparation A 9 kb segment of the GAD67 gene's 5′ regulatory region, including the first intron, drives expression of a lacZ reporter transgene in hippocampus and neocortex of transgenic mice in a partially cell-type specific pattern (Katarova et al., 1998) . To improve specificity, we used a slightly longer segment, and to allow visualization of the reporter gene product in living tissue, we fused it in-frame to eGFP. We cloned a 10.3 kb segment of the murine GAD67 genomic DNA, including the 5′ upstream regulatory region, the first (untranslated) exon and intron and a portion of exon 2, from the HindIII site (at the 5′ end) to the BamHI site (at the 3′ end) (Katarova et al., 1998) , and inserted it in-frame between the HindIII and the BamHI insertion sites of the pEGFP-1 vector (Clontech, Palo Alto, CA). The resulting construct encodes a fusion protein consisting of the first 17 amino acids of GAD67, four amino acids of the vector and the full-length eGFP. Organotypic Culture Preparation Organotypic cortical slice cultures were prepared using a previously described method (Stoppini et al., 1991) . Postnatal day 2-7 mouse or rat
doi:10.1093/cercor/11.7.666 pmid:11415968 fatcat:vavwefgairhftlpodzp3l6hcde