Systematic comparison of adeno‐associated virus and biotinylated dextran amine reveals equivalent sensitivity between tracers and novel projection targets in the mouse brain

Abstract

As an anterograde neuronal tracer, recombinant adeno-associated virus (AAV) has distinct advantages over the widely used biotinylated dextran amine (BDA). However, the sensitivity and selectivity of AAV remain uncharacterized for many brain regions and species. To validate this tracing method further, AAV (serotype 1) was systematically compared with BDA as an anterograde tracer by injecting both tracers into three cortical and 15 subcortical regions in C57BL/6J mice. Identical parameters were used for our sequential iontophoretic injections, producing injections of AAV that were more robust in size and in density of neurons infected compared with those of BDA. However, these differences did not preclude further comparison between the tracers, because the pairs of injections were suitably colocalized and contained some percentage of double-labeled neurons. A qualitative analysis of projection patterns showed that the two tracers behave very similarly when injection sites are well matched. Additionally, a quantitative analysis of relative projection intensity for cases targeting primary motor cortex (MOp), primary somatosensory cortex (SSp), and caudoputamen (CP) showed strong agreement in the ranked order of projection intensities between the two tracers. A detailed analysis of the projections of two brain regions (SSp and MOp) revealed many targets that have not previously been described in the mouse or rat. Minor retrograde labeling of neurons was observed in all cases examined, for both AAV and BDA. Our results show that AAV has actions equivalent to those of BDA as an anterograde tracer and is suitable for analysis of neural circuitry throughout the mouse brain.

Distinct Representation and Distribution of Visual Information by Specific Cell Types in Mouse Superficial Superior Colliculus

“…Injection of adeno-associated virus (AAV) can retrogradely label cells whose axons target the region injected; the number of retrograde labeled cells depends on the particular brain region and other factors (Harris et al, 2012;Wang et al, 2014). After sSC injections of virus encoding nonconditional fluorescent protein expression, we observed retrogradely labeled neurons in several regions known to provide input to the sSC: retina, layer 5 of visual cortex, and PBg.…”

“…5 A, B). Previous studies of (unidentified) mouse sSC cells also described directionally selective neurons but differed in whether the preferred directions of these cells were uniformly distributed (Wang et al, 2010) or strongly biased for upward motion (Dräger and Hubel, 1975). We found that the preferred directions of directionally selective WF cells (those with DSI Ͼ 0.25), but not the other cell types, deviated significantly from equal distribution into the four cardinal directions ( p Ͻ 0.01; Fig.…”

“…One possibility is that horizontal cells internalize and/or transport beads and cholera toxin inefficiently. We took advantage of the fact that adenovirus injections retrogradely label neurons (Harris et al, 2012;Wang et al, 2014) and injected virus coding for Cre-dependent expression of GFP into the PBg or LGN of Gad2-Cre mice. All of the retrogradely labeled cells we recorded in the sSC of these mice were horizontal cells ( Fig.…”

Major progress towards elucidating brain wiring diagrams

“…Their companion paper, Wang et al, (2014) "Systematic comparison of adeno-associated virus and biotinylated dextran amine reveals equivalent sensitivity between tracers and novel projection targets in the mouse brain", provided the crucial technical foundation for Oh et al, (2014). They compared anterograde neuronal tracing using either EGFP-expressing AAV, or biotinylated dextran amine (BDA).…”

“…While both methods gave robust anterograde labeling, minor retrograde labeling of neurons was also observed for both AAV and BDA. Oh et al (2014) defined the axonal projections from 295 non-overlapping brain regions using an AAV vector expressing EGFP under the control of the human Synapsin I promoter. High-resolution projection data obtained from both cortical and subcortical mapping, were integrated with 863 grey-matter zones annotated within the Allen Reference Atlas.…”