Substance P reconfigures the neural respiratory network in ventrolateral medulla in the neonatal rat brainstem in vitro

The effects of SP on the respiratory activity in the rat (0–4 days old) neonatal brainstem-spinal cord preparation were investigated. Respiratory activity was recorded from C4 ventral roots and intra-cellularly, from three types of respiration-related neurons: pre-inspiratory (Pre-I or biphasic E, n = 19), three subtypes of inspiratory (InspI-III, n = 18), expiratory (Exp, n = 11), and tonic neurons (n = 11) in the ventrolateral medulla (VLM). There were two types of respiratory rhythm in respiration-related neurons and in integrated C4 nerve activity under normal experimental conditions, with marked similarities to normal eupneic respiration and sighs. These two types of bursts were similar to patterns described recently in rhythmic brainstem slices in neonatal mice [1]. In control, the frequency of eupneic-like bursts varied in a range of 7–11 bursts per minute, whereas the frequency of the sigh-like activity was 10–30 times slower at 0.2–0.6 bursts per minute. Bath application of SP (10 nM-1 μM) caused a biphasic effect on eupneic-like respiratory frequency. There was a pronounced dose-dependent decline of burst rate (by 48% from control level of 9.35 ± 1.8 (mean ± SEM) bursts per minute, n = 28) after the onset the SP application (phase P1), followed by a weaker dose-dependent increase (by 17.5%) in burst rate (phase P2) (P < 0.001, two-way ANOVA). The biphasic effect of SP on inspiratory burst rate was associated with sustained membrane depolarization (in a range of 0.5–13 mV) of respiration-related and tonic neurons. C4 bursting before and after SP application was synchronized with neuronal activity of respiration-related neurons. Unlike the biphasic effect shown in the eupneic-like activity, the sigh-like burst frequency was considerably and gradually increased. This elevated sigh-like activity could reset normal respiration rhythm, so that in some cases the respiration activity was represented exclusively by the sigh-like pattern. As with the first pattern, this frequency increase depended on SP concentration (P < 0.001, one-way ANOVA), though the range of the changes was an order bigger as compared to normal respiratory pattern, and reached 650%. There was significant correlation (R = 0.537, P < 0.0001) between the eupneic-like frequency decrease during P1 and the sigh-like rhythm increase, and P1 phase corresponded in time to maximum slope of increase in sigh-like frequency. In turn, the P2 phase in the eupneic-like bursting coincided in time with maximum sigh-like rhythm developed during SP application. Thus, the biphasic effect of SP on eupneic-like respiratory activity was superimposed on a monotonic increase of the sigh-like pattern frequency. Our results suggest that a) a single respiratory network in the VLM might reproduce or generate two different respiratory patterns; b) within this respiratory network SP exerts a general excitatory effect on respiration-related and tonic neurons; c) the transient changes in neuronal activity in the VLM produced by SP may underlie the biphasic effect in the eupneic-like bursting, as well as the changes in the sigh-like pattern frequency, and d)SP can transform the particular type of the interaction of eupneic-like and sigh-like patterns, which compose the normal respiratory activity in the neonatal brainstem, and thus, reconfigure the neural network controlling respiratory rhythm generation.