Brain stem lesion size determined by DEAD red or conjugation of neurotoxinto fluorescent beads

Neurotoxin microinjected into the retrotrapezoid nucleus of anesthetized ra ts decreases phrenic activity and eliminates the response to CO2. In unanes thetized rats, such treatment has no effect on awake, resting breathing and decreases CO2 sensitivity by 40% (M. Akilesh, M. Kamper, A. Li, and E. E. Nattie. J. Appl. Physiol. 82: 469-479, 1997). One important factor in expla ining these disparate results is the actual size of the anatomic lesion. In the present study, we injected ibotenic acid into the retrotrapezoid nucle us of anesthetized rats and evaluated lesion size by using two new approach es: 1) DEAD red, a fluorescent probe that enters impaired cells through lea ky membranes and binds to nucleic acids, and 2) conjugation of toxin to flu orescent beads. With the use of DEAD red, the region containing labeled dyi ng cells was 313 +/- 104 nl (n = 4), six times larger than the initial inje cted volume, and the physiological effects on phrenic amplitude, the CO2 re sponse, and blood pressure began within minutes and were substantial. With conjugated toxin, in theory, neuronal damage would be limited to the region of detectable fluorescence (49 +/- 10 nl; n = 4). Effects on phrenic ampli tude, CO2 sensitivity, and blood pressure were absent until similar to 2 h postinjection. Control experiments, with 2 h of in vitro incubation of the neurotoxin-microbead conjugate and injection of the supernatant after centr ifugation, showed similar results that suggest release of conjugated neurot oxin. We conclude that DEAD red provides a useful means to monitor neuronal impairment in acute studies in vivo. Conjugation of neurotoxin to microbea ds may be less reliable in this regard.