Trans-synaptic retrograde spread of rabies virus from the LH to the VON. (a) Schema of virus-mediated retrograde tracing. We first injected a mixture of adeno-associated viruses (AAVs) encoding CMV-GFPCre and CAG-Flex-TVA-mCherry either with or without CAG-Flex-rabies G into the lateral hypothalamus (LH). Two weeks later, SAD-dG-EGFP-EnvA was injected into the LH. We examined the distribution of EGFP-labelled presynaptic cells in the ventral olfactory nucleus (VON). (b) EGFP-labelled presynaptic neurons (green) were observed in the VON when rabies G-encoding AAV was concomitantly injected (right panels). Immunostaining for DARPP-32 (red) was used to discriminate the VON from the olfactory tubercle (OT) and nucleus accumbens (NAc). AON, anterior olfactory nucleus; VON, ventral olfactory nucleus; APC, anterior piriform cortex; OT, olfactory tubercle; NAc, nucleus accumbens. Scale bar: 100 μm. (c) The number of EGFP-labelled cells in the olfactory cortical areas. Data are shown as mean with individual plots. Statistical differences were tested between data of rabies G(+) in the VON vs. data of rabies G(+) in other areas by two-way ANOVA with post-hoc Tukey’s test. ND, not detected; *p < 0.05; **p < 0.01. (d) EGFP-labelled presynaptic neurons (green) in the mPFC (left), NAc (middle), and amygdala (right). Scale bar: 100 μm. (e) The number of EGFP-labelled cells in the VON, mPFC, NAc, and amygdala. Data for the VON are similar to the data in (c). Data are shown as mean with individual plots. Each colour represents the data from one mouse. ns, not significant; **p < 0.01; ***p < 0.001.