The ventrolateral periaqueductal gray matter (vlPAG) controls the descending pain inhibitory system, however, it has been still unknown how neural activities of the vlPAG are regulated by higher brain regions. We anatomically explored brain regions that project to the vlPAG especially focusing on the higher brain regions, and the firing properties of neurons in vlPAG were investigated using VGAT-Venus transgenic and/or VGAT-Venus-ChAT-TdTomato transgenic rats. The retrograde tracer, cholera toxin subunit B that is fluorescently labeled with Alexa488 or Alexa647 was injected into the vlPAG. The CTB-positive neurons were principally distributed in the ventral hypothalamus, central amygdala, and cerebral cortex including the insular, prefrontal, and primary somatosensory cortices. The firing properties of vlPAG neurons were divided into 3 types in reference to their firing patterns and Venus/TdtTomato fluorescence. These results suggest that the vlPAG receives projections not only from brain regions that control emotion and sleep/awake cycles but also from the cerebral cortex and that we need to examine properties of these descending projections in each cell type.