The local neural circuitry underlying the control of breathing was studied by injecting nanoliter volumes of excitatory amino acids into discrete regions of cat brain stem. Experiments were performed on chloralose-urethane anesthetized, vagotomized, paralyzed, and artificially ventilated cats. Phrenic, intercostal, and recurrent laryngeal nerve discharges were recorded. Multibarrel pipettes were used for recording and pressure ejection of drugs or a dye for marking recording and ejection sites. Ejected volumes were directly monitored for every injection. Injections, proximal to neurons discharging with a respiratory periodicity, of as little of 200 fmol of L-glutamate in 200 pl of saline elicited marked, site-specific increases or decreases in respiratory motoneuronal discharge. N-Methyl-D-aspartic acid and homocysteic acid elicited similar site-specific alterations in respiratory motor output, although some details of the response could differ qualitatively. Responses to all the excitatory agents used were attenuated by concurrent injection of kynurenic acid, DL-2-aminoA-phosphonobutyric acid, or glutamic acid diethyl ester. There was no change in spontaneous phrenic nerve discharge in response to injections of equivalent or larger volumes of saline or lidocaine. These results indicate a heterogeneity in the spatial organization of the brain-stem neural circuitry underlying respiratory control, which has not been described previously. This injection technique may provide a mechanism for probing the neural circuitry underlying other behaviors.Brain function underlying behavior in mammals involves information transfer between neurons in complex networks. The nuclear and layered structure of the mammalian brain suggests that small aggregates of neurons act as functional units. It is of fundamental interest to determine the nature of this local information transfer and its consequences in terms of behavior. In this paper, we present an approach to study this problem using site-specific delivery of (sub)picomolar amounts of excitatory amino acids in nanoliter volumes. Using this approach, we present evidence of a neuronal mosaic underlying the control of breathing.Neurons that discharge with a respiratory periodicity are concentrated in the nucleus ambiguus-retroambigualis (ventral respiratory group, VRG) and the nucleus tractus solitarius of the medulla (Cohen, 1979; Feldman, in press;von Euler, 1983). Received Oct. 24, 1985; revised Jan. 21, 1986; accepted Jan. 22, 1986. The technical assistnce of Paul G. Bums and the helpful comments of Professor Oscar Hechter are gratefully acknowledged. We would like to thank Drs. Howard H. Ellenberger, Debra E. Weese-Mayer, and Jeffery C. Smith for their help in experiments at the latter stages of this study. This work was supported by NIH Grant NS-21036.Correspondence should be addressed to Jack L. The VRG extends rostrally from the spinomedullary junction to the level of the retrofacial nucleus. Neurons within the VRG can be divided into 2 populations based on their impuls...