Retrograde tracing with a fluorescent dye (Fast Blue) combined with immunohistochemistry was used to identify putative neurotransmitter(s) at the phrenic motor nucleus in the cat. Fast Blue was injected bilaterally into the diaphragm of five cats, where each phrenic nerve enters the muscle. Seven days later the animals were perfusion fixed and tissue sections from the fourth, fifth, and sixth cervical spinal cord segments were analyzed using a fluorescence microscope. Retrogradely labeled fluorescent phrenic motor neuron cell bodies appeared in all of the segments but primarily in sections from the fifth segment. The same or adjacent transverse sections were then used for the demonstration of the distribution of the neurotransmitters 5-hydroxytryptamine (5HT), substance P, and thyrotropin-releasing hormone (TRH) in the area of the phrenic motor nucleus using the indirect immunofluorescence technique. The most conspicuous neurotransmitters found at the phrenic motor nucleus were 5-HT and substance P. We observed dense and diffuse fiber networks throughout the ventral horn which contains the phrenic motor nucleus. These fibers contained varicosities in close proximity to phrenic motor neurons. In addition to 5-HT-and substance Pcontaining nerve endings, some fibers containing TRH were also found in the area of the phrenic motor nucleus. These results are consistent with earlier physiological data suggesting that 5-HT, substance P, and TRH are important neurotransmitters and/or neuromodulators involved in central control of respiration.Our knowledge of central respiratory neurons is extensive with regard to their anatomical localization and their electrophysiological properties (Cohen, 1979; Mitchell and Berger, 1981). However, very little is known about the neurotransmitters of these neurons. One approach commonly used to obtain information about this point has been to inject a putative CNS neurotransmitter, an agonist or antagonist of the neurotransmitter or a pre-

Significant progress has been made in identifying the nuclei and pathways involved in central control of respiration in mammals. This knowledge provides a framework from which to focus on the question of which putative neurotransmitters may be involved in central respiratory control. Application of chemical neuroanatomical methods, particularly immunohistochemistry, is a powerful approach to this important question. This commentary discusses how immunohistochemical approaches have been used so far to extend our knowledge of central respiratory neurotransmitters and also how they can be further applied to gain new information. The results of immunohistochemical studies provide a rationale for subsequent physiological and pharmacological studies of respiratory neurons where specific actions of putative neurotransmitters can be evaluated.