Evaluating the control: minipump implantation and breathing behavior in the neonatal rat

Abstract

We evaluated genioglossus (GG) gross motoneuron morphology, electromyographic (EMG) activities, and respiratory patterning in rat pups allowed to develop without interference (unexposed) and pups born to dams subjected to osmotic minipump implantation in utero (saline-exposed). In experiment 1, 48 Sprague-Dawley rat pups (Charles-River Laboratories), ages postnatal day 7 (P7) through postnatal day 10 (P10), were drawn from two experimental groups, saline-exposed (n = 24) and unexposed (n = 24), and studied on P7, P8, P9, or P10. Pups in both groups were sedated (Inactin hydrate, 70 mg/kg), and fine-wire electrodes were inserted into the GG muscle of the tongue and intercostal muscles to record EMG activities during breathing in air and at three levels of normoxic hypercapnia [inspired CO2 fraction (FiCO2 ): 0.03, 0.06, and 0.09]. Using this approach, we assessed breathing frequency, heart rate, apnea type, respiratory event types, and respiratory stability. In experiment 2, 16 rat pups were drawn from the same experimental groups, saline-exposed (n = 9) and unexposed (n = 7), and used in motoneuron-labeling studies. In these pups a retrograde dye was injected into the GG muscle, and the brain stems were subsequently harvested and sliced. Labeled GG motoneurons were identified with microscopy, impaled, and filled with Lucifer yellow. Double-labeled motoneurons were reconstructed, and the number of primary projections and soma volumes were calculated. Whereas pups in each group exhibited the same number (P = 0.226) and duration (P = 0.093) of respiratory event types and comparable motoneuron morphologies, pups in the implant group exhibited more central apneas and respiratory instability relative to pups allowed to develop without interference.

Keywords: apnea; control; motoneuron; upper airway.

Fig. 1.

Experimental schematic of whole body flow through chamber setup, showing location of the force transducer used to detect chest wall motions and genioglossus and intercostal electromyographic electrode locations.

Fig. 2.

Average number (± SE) of central apneas recorded in 2-min recording windows exhibited by saline-exposed (n = 24) (gray bars) and unexposed (n = 24) (black bars) pups spanning postnatal days 7–10 (P7–P10). There were no significant differences detected between saline-exposed and unexposed pups in regard to either apneic frequency (P = 0.226) or apnea duration (P = 0.725) across the age span.

Fig. 3.

An illustrative recording of a mixed apnea characterized by absence of EMG activity in pump and upper airway muscles followed by increasing EMG activation in both muscles followed by airway reopening and resumption of breathing. Black bar, central apnea; white bar, hypopnea.

Fig. 4.

Original chest wall recordings obtained from an unexposed (A) and a saline-exposed (B) pup. Solid lines show the respiratory trace, with a lagged or time-delayed copy of the trace indicated by a dashed trace. In each case, the offset between the traces is a result of the lag, the magnitude of which is represented by the horizontal arrows. Here, b and d in each panel show traces at lags equal to integer multiples of the period, and c shows the shift of an incomplete number of cycles in each case (unexposed and saline exposed) and highlights how well previous respiratory behavior predicts future respiratory behavior. Shown in e are the ACFs of the entire recording for unexposed and saline-exposed pups. Vertical dashed lines highlight peaks in each ACF the magnitude of which indicate how well actual and predicted chest wall traces align at each of the time points displayed in b–d.

Fig. 5.

A: individual ACF values for saline-exposed and unexposed pups displayed side by side with group average (± SE) ACF values. Relative to unexposed pups, saline-exposed pups exhibit overall lower average ACF values indicative of lower respiratory stability both within and across breaths (*P = 0.017). B: distribution of ACFs as a function of breath number and treatment group shows comparable rates of decline in stability as a function of the time (P < 0.001). Thus, once instability is triggered, the trajectory of that instability and the subsequent return to eupneic breathing is comparable for both groups.