Retrograde neuronal tracing with a deletion-mutant rabies virus

Abstract

We have constructed a deletion-mutant rabies virus encoding EGFP and find it to be an excellent tool for studying detailed morphology and physiology of neurons projecting to injection sites within the mammalian brain. The virus cannot spread beyond initially infected cells yet, unlike other viral vectors, replicates its core within them. The cells therefore fluoresce intensely, revealing fine dendritic and axonal structure with no background from partially or faintly labeled cells.

Figure 1

The recombinant rabies virus SADΔG-EGFP. (a) In SADΔG-EGFP the glycoprotein (G) open reading frame is replaced with that of EGFP. EGFP mRNA is transcribed using the authentic G transcription start (black arrowhead) and stop-polyadenylation signals (black bar). Viral open reading frames, light gray boxes; noncoding sequences, gray; transcription signals, black. N, P, M and L denote the remaining viral genes. (b) An example of retrograde labeling with SADΔG-EGFP in mouse brain. These are widefield microscopic images of intrinsic EGFP fluorescence in unstained tissue, 6 d after injection. Top, deep-layer cortical pyramidal cells infected after injection in thalamus. Scale bar, 150 µm. Inset, injection site. Scale bar, 750 µm.

Figure 2

Comparison of SADΔG-EGFP and lentivirus. These confocal images of intrinsic fluorescence, taken back to back using identical instrument settings, show cortical pyramidal cells retrogradely infected either with a lentivirus pseudotyped with the rabies virus glycoprotein and expressing EGFP driven by the cytomegalovirus promoter, or with the recombinant rabies virus SADΔG-EGFP. (a–c) Lentivirus. (d) The rabies virus SADΔG-EGFP. Scale bars, 150 µm.