Rabies virus-mediated trans-synaptic retrograde tracing from the VON. (a) Schema of virus-mediated neuronal pathway-specific retrograde tracing. We first injected a retrograde lentiviral vector encoding Cre (NeuRet-Cre) into the lateral hypothalamus (LH), and Cre-dependent adeno-associated virus (AAV) vector TVA-mCherry and rabies G into the VON. Two weeks after the first injection, EnvA-pseudotyped glycoprotein-deleted rabies virus encoding EGFP was injected into the VON. We then examined the distribution of EGFP-labelled presynaptic cells throughout the whole brain. (b) Coronal sections of the VON. Left, TVA-mCherry expressing neurons; middle, rabies-derived EGFP expressing neurons; right, colour merged with DAPI staining. Yellow cells indicate TVA-mCherry(+) EGFP(+) starter cells. AON, anterior olfactory nucleus; VON, ventral olfactory nucleus; OT, olfactory tubercle. Scale bar: 100 μm. (c) EGFP-labelled presynaptic cells in the OB. (d) The number of EGFP-labelled M/TCs in the OB. Data are shown as mean ± SD with individual plots. (e) Distribution of EGFP-labelled presynaptic cells projecting to the VON. AON, anterior olfactory nucleus; APC, anterior piriform cortex; OFC, orbitofrontal cortex; mPFC, medial prefrontal cortex; HDB, horizontal limb of the diagonal band; OT, olfactory tubercle; PPC, posterior piriform cortex. Scale bar: 100 μm. (f) The percentage of the number of EGFP-labelled cells in each region over total number of EGFP-labelled cells across the whole brain. Data are shown as mean ± SD with individual plots.