Inspiratory augmenting bulbospinal neurons express both glutamatergic and enkephalinergic phenotypes

Many of the inspiratory augmenting (I-AUG) neurons of the rostral ventral respiratory group (rVRG) are premotor neurons that excite phrenic motor neurons during inspiration, probably by releasing glutamate. In the present study, we demonstrate that these neurons are indeed glutamatergic, in that their cell bodies contain vesicular glutamate transporter-2 (VGLUT2) mRNA and spinal terminals from neurons in the region of the rVRG contain VGLUT2 protein. We also demonstrate by using parallel in situ hybridization and immunocytochemical evidence that most rVRG inspiratory premotor neurons are enkephalinergic. After iontophoretic deposits of biotinylated dextran amine (BDA) in the area of the rVRG, many BDA-labeled terminals in the ventral horn of cervical spinal cord (C4-C5) were immunoreactive for enkephalin and VGLUT2. Injections of Fluoro-Gold amidst phrenic motor neurons in C4-C5 labeled neurons in the area of the rVRG that contained both VGLUT2 mRNA and preproenkephalin (PPE) mRNA as revealed by double in situ hybridization. Thirty-eight bulbospinal I-AUG neurons were recorded in the rVRG and filled with biotinamide by using the juxtacellular labeling technique. Every biotinamide-filled cell tested was positively labeled for VGLUT2 mRNA (n = 14), and most of the cells tested in a separate population exhibited PPE mRNA (16/18). We conclude that most of the phrenic inspiratory premotor neurons of the rVRG are glutamatergic neurons that may also release enkephalins.