To determine the neuronal connections in the periaqueductal gray (PAG) is important for studying modulation of neuronal activity of PAG to influence sympathetic responses. We had characterized projections from the left kidney to the midbrain PAG in adult male melanocortin-4 receptor (MC4R)-green fluorescent protein (GFP) transgenic mice by using retrograde tracing techniques of pseudorabies virus (PRV)-614 for direct visualization under two-photon immunofluorescence microscope. We found that injections of PRV-614 into the kidney resulted in retrograde infection of neurons in the ventrolateral sub-areas of PAG, and PRV-614/MC4R-GFP double-labeled neurons were detected in the ventrolateral sub-areas of PAG. These results indicated that a subpopulation of ventrolateral PAG neurons innervating renal tissues expressed MC4R, suggesting that deep brain stimulation of the ventrolateral PAG may influence renal function by melanocortinergic pathway.