A herpes simplex viral vector expressing green fluorescent protein can be used to visualize morphological changes in gh-density neuronal culture

High-density cultures of mammalian neurons offer a model system for studies of brain development, but the morphological features of individual neurons is difficult to ascertain. We show that a herpes virus vector expressing a bioluminescent protein allows detailed morphometric analyses of living neurons in complex culture environments. Expression of enhanced green fluorescent protein (eGFP) was constitutively driven in neurons using the herpes simplex virus amplicon system. This system allowed us to make novel observations regarding development in high-density cultures from rat hippocampus and cerebellum. After the phase of initial neurite outgrowth, maturing neurons continue to show rapid remodeling of the neurite branches (0.79 ± 0.11 µ µm/h per neurite; mean ± SEM, n=8), and displacement of the soma within the neurite arbor (1.35 ± 0.74 µ µm/h). These results demonstrate that a substantial capacity for morphological plasticity persists in maturing mammalian CNS neurons after cessation of net neurite outgrowth in early development. Dispersed cultures of fetal neurons are commonly used as a model system to study brain development. When plated at higher densities that more closely approximate the connectivity found in vivo, the morphological features of individual neurons become indistinguishable among the dense tangle of the neuropil formation. Under these conditions, routine light microscopic analyses are not useful in studying morphological changes. We have employed a viral vector to induce expression of green fluorescent protein in dense heterogeneous cultures of neurons and glia. Using fluorescent microscopy, we demonstrate that detailed morphological analysis of single neurons in dense cultures is feasible. We have employed time-lapse photography to image the remodeling of neurites and somata of living neurons transfected with a virus engineered to express green fluorescent protein as an abundant cytoplasmic marker. Green fluorescent protein (GFP) is a bioluminescent molecule that has several advantages over other reporter genes: (1) its fluorescence does not depend on cofactors or substrates; (2) the protein is stable and not subject to rapid bleaching; (3) and visualization of living cells can be performed without obvious detriment to viability (Chalfie et al. 1994). Enhanced green fluorescent protein (eGFP) is a genetically modified, codon-optimized variant of the native protein found in the jellyfish Aequoria victoria (Zhang et al. 1996). Its fluorescence is 5 times brighter than that of the native protein when visualized with systems designed for fluorescein optics. GFP is finding great utility in the field of developmental neurobiology as a reporter gene and bioluminescent marker. The …