Figure 5A illustrates the bullfrog in vitro brainstem-spinal cord preparation. In bullfrogs in vivo, lung ventilation cycles involve activation of both glottal dilator (value to the lung) and buccal floor pump muscles. The general motor behavior associated with breathing persists in the isolated brainstem-spinal cord in vitro and can be recorded with extracellular suction electrodes through cut spinal nerve II (hypoglossal nerve; top) that innervates the buccal floor, and the caudal portion of cranial nerve X (vagus; bottom) that innervates the glottis (Sakakibara, 1984a). Since activation of both groups of motoneurons are required for lung ventilation, coactivation of buccal floor and glottal-related motor outflow confirms respiratory-related central pattern generator activity associated with lung ventilation (i.e. a fictive lung breath; Figure 5A) (Sanders and Milsom, 2001). Here, we analyzed respiratory bursts recorded from the CN X nerve root as it contains the glottal dilator motoneurons (Kogo et al., 1994) that we showed to exhibit up-scaling of AMPA receptors. Figure 5B shows example CN X root recordings from control (green; top) and winter inactivity (blue; bottom) in vitro brainstem-spinal cord preparations before (left) and after application of 4 μM DNQX (right). Dashed boxes across the compressed recordings represent minimum and maximum burst amplitudes with each preparation. Both control and winter inactivity preparations underwent relatively similar decreases in respiratory burst frequency during bath application of DNQX; however, only preparations from winter inactivity bullfrogs experienced a decreased in burst amplitude. This can be further observed in the expanded example integrated motoneuron population burst in the inset. Figure 5C1–E1 shows mean changes in fictive lung burst amplitude, area, and frequency in response to 4 μM DNQX expressed as a percent of baseline (n = 4 brainstems per group; two-tailed Mann Whitney test). In winter inactivity bullfrogs, DNQX led to a significant decrease in burst amplitude and area compared to control bullfrogs. Figure 5C2–E2 show raw values used to generate each of the means in Figure 5C1–E1. DNQX caused a decrease in fictive lung burst frequency in both groups of frogs despite winter inactivity frogs starting from a higher baseline (Figure 5E2). Absolute comparisons for burst amplitude and area characteristics were not performed as these are only relative measures. Means for each analysis are shown as a solid green or blue line. Scattered circles in each graph are individual points that generate the mean. Error bars are standard error of the mean (SEM). *p<0.05.