We have been developing several new anatomical methods to enhance our studies of coexisting transmitters in the brain. These include intensification of immunohistochemistry, double and triple labeling of single brain sections, new retrograde dyes such as fluorogold and in situ hybridization. In addition, we have been using these techniques to examine the effects of adrenalectomy on neuropeptide (dynorphin and vasopressin) in the hypothalamus and innervation of the phrenic motor nucleus. Finally, studies using antibodies specific to a subpopulation of dorsal root ganglion cells and studies tracing cholinergic projections to both substantia nigra and prefrontal cortex are underway.