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Improved Golgi-like Visualization in Retrogradely Projecting Neurons after EGFP-Adenovirus Infection in Adult Rat and Monkey

Ryohei Tomioka and Kathleen S. Rockland

Laboratory for Cortical Organization and Systematics, RIKEN Brain Science Institute, Saitama, Japan

Correspondence to: Ryohei Tomioka, PhD, Laboratory for Cortical Organization and Systematics, RIKEN Brain Science Institute, 2-1 Hirosawa, Wako-shi, Saitama 351-0198, Japan. E-mail: rtomioka{at}brain.riken.jp

An adenovirus vector was generated using a neuron-specific promoter synapsin I and enhanced green fluorescent protein (EGFP) reporter (AdSynEGFP). In addition, two modifications were identified that resulted in robust and reliable retrograde transport and EGFP expression after injection of the virus into three different brain regions in adult rats (medial prefrontal cortex, posterior thalamic nuclear group, and CA1). These are postinjection survival times of 14 days and addition of high concentrations of NaCl (600 mM) to the injection buffer. These modifications resulted in obvious improvement in the intensity of the EGFP signal and in the number of labeled cells. Use of anti-EGFP in immunofluorescence or immunoperoxidase processing further enhanced the signal so that Golgi-like filling of dendritic spines and axon collaterals was routinely achieved. Effectiveness of the AdSynEGFP for Golgi-like filling was confirmed in one rhesus monkey with injections in visual area V4. Because of the long-term viability of the infected neurons (at least up to 28 days in rats and 22 days in monkey), this AdSynEGFP is suitable for use in microcircuitry studies in combination with other fluorescently tagged elements, including anterogradely labeled extrinsic projections. The native EGFP signal (without antibody enhancement) may be sufficient for studies involving cultured cells or slices. (J Histochem Cytochem 54:539–548, 2006)

Key Words: cerebral cortex • dendritic spines • microcircuitry • NaCl • neuronal tracer • pyramidal neuron

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