, 2004) injected into the cortex transduces almost exclusively

neurons locally near the injection site. The GFP is soluble and diffuses along the dendrites and axons of the transduced neurons, including long-range axonal projections. Lenti-GFP can therefore be used as an unequivocal anterograde anatomical tracer (Ferezou et al., 2007; Broser et al., 2008a). Whereas VSV-G pseudotyped lentivirus only transduces neurons with somata Panobinostat purchase within a few hundred microns of the cortical injection site, other viral vectors behave quite differently. Adeno-associated viruses (AAVs) are physically much smaller, so they can diffuse further, transducing neurons across larger brain regions. Different serotypes of AAV have different properties and, like adenovirus and rabies virus, some AAVs can be retrogradely transported after axonal uptake of vector (Taymans et al., 2007; Hollis et al., 2008). AAV serotype 6 (AAV6; Grimm et al., 2003) binds to heparin (like AAV serotype 2, but different from other serotypes) and probably because of this binding it diffuses less in the brain than many other AAV serotypes. Nonetheless, neurons transduced with AAV6 are found

far from the injection site, presumably because of retrograde transport (Kaspar et al., 2003; Towne et al., 2008, 2010). Injection of AAV6 encoding a ‘humanized’ cre-recombinase (AAV6-Cre; BMN 673 nmr Shimshek et al., 2002; Fig. 3F) into Rosa floxed-LacZ cre-reporter mice (Soriano, 1999), allows staining of transduced neurons with the blue XGal chromogenic substrate. If the AAV6-Cre vector is injected into the neocortex, it is taken up

DOK2 by axon boutons near the injection site (while also transducing neurons with somata near the injection site). The AAV6-Cre is then retrogradely transported to the nucleus of neurons with axonal projections to the injection site, and the subsequent expression of cre-recombinase can be monitored in cre-reporter mice. AAV6-Cre can therefore be used as a retrograde vector for anatomical labelling of neurons projecting to the injection site. Both the classical anatomical tracers and the viral vectors can be injected simultaneously to allow labelling of both anterograde and retrograde connectivity from a single well-defined injection site. Voltage-sensitive dye imaging reveals that activity within the C2 barrel column rapidly propagates to neighboring cortical columns (Fig. 2). This spread is likely to be mediated, at least in part, by the extensive local axonal projections of the pyramidal neurons located in the C2 barrel column. Injections into the C2 barrel column of the anterograde tracers Lenti-GFP (Fig. 4A and B; Dittgen et al., 2004) or BDA (Fig. 4C) indicate that C2 barrel cortex neurons extend axonal arborizations into layers 2/3 and layers 5/6, almost across the entire extent of S1 barrel cortex. The density of axons is highest close to the C2 barrel column and decreases across the neighboring cortical columns (Brecht et al.

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