New lentiviral vector to selectively target glycinergic neurons in the brainstem

Abstract

Inhibitory interactions between populations of inspiratory and expiratory neurons within the brainstem respiratory network are paramount for the operation of the respiratory CPG. However, the exact role that populations of inhibitory glycinergic neurones play in rhythm and pattern generation is unknown. The lack of information results from technical difficulties in identifying and targeting these neurones in vivo. To overcome this problem, we explored the sequences 5′ upstream of the glycine transporter‐2 gene (GlyT2), a reliable marker for glycinergic neurons, and generated lentiviral vectors (LV) to express far‐red fluorescent protein (Katushka1.3), which were then, tested in vivo using microinjections into the rat brainstem. All fragments longer than 1.5 kb conferred specific expression in glycinergic neurons with the 1.5 kb one providing the strongest transcriptional activity. We then injected the 1.5 kb LV into different sites of the brainstem of transgenic mice expressing enhanced green fluorescent protein (eGFP) in glycinergic neurons to confirm the specificity of this vector and then compared sites with abundant glycinergic innervation, e.g. gigantocellular reticular nucleus, with sites where this innervation was absent, e.g. inferior olive. We concluded that this novel vector allows selective targeting of glycinergic neurons within the brainstem respiratory network. Support: NIH R01 NS057815, BHF RJ/07/006.