INTEGRATIVE PHYSIOLOGY OF THE MOTOR OUTPUT

The present study focuses on the integrative properties of Central Nervous System (CNS), which coordinates the activity of multiple neural circuits in order to express the rhythmic motor output. The contribution of supraspinal structure to the integration of inputs arising from respiratory and locomotor neural networks, especially at birth, is still underestimated. To study the neurogenic drive responsible for tailoring rhythmic motor responses, we introduced the ex vivo central nervous system (CNS) from neonatal rodents with legs attached. The preparation allowed to extracellularly record a stable and spontaneous respiratory rhythm for more than 4h from spinal ventral roots (VRs). Serial surgical ablations unveiled the contribution of supraspinal structures to the respiratory motor output. Precollicular decerebration reduced respiratory burst duration and frequency, while pontobulbar transection increased them. To explore whether sensory afferents from limbs modulate respiration during physical activity, we designed a novel experimental ex vivo platform. Hindlimbs of isolated CNS preparation from neonatal rats were kept attached to a robotic device (Bipedal Induced Kinetic Exercise, BIKE) driving passive pedaling at calibrated speeds. Brief sessions (5 min) of BIKE at maximum pedaling speed (3.5 Hz) augmented the respiratory rate in preparations with slow respiration in control, while a longer BIKE session (25 min) was required to slow down respiratory pace in preparations with intrinsically fast respiratory rhythm. However, regardless of the baseline respiratory frequency, BIKE always decreased duration of single bursts. Notably, Surgical ablation of suprapontine structures completely prevented modulation of breathing after intense training. Like exercise, hypoxia is a multifaced stimulus. We wanted to assess if the respiratory modulation provided by Intermittent Hypoxia (IH) might rely on a neurogenic component, already effective at birth and involving the hypothalamus among suprapontine structures. To mimic IH ex vivo, CNS isolated from 0-3-day old rats was perfused with four to eight brief (5-min) bouts of mild-hypoxic/normocapnic modified Krebs solution, spaced by normoxic episodes, during continuous electrophysiological recordings from upper cervical ventral roots. IH protocol did not modify bath pH, but medullary and hypothalamic areas encountered lowered oxygen tension, more severe after the second postnatal day, with a partial recovery after each hypoxic bout. Single exposures to mild hypoxia were well tolerated and frequently elicited a spontaneous episode of irregular baseline activity in both, whole CNS preparations and spinal cords. IH transiently increased amplitude of respiratory bursts and stably sped up rhythm in neonates (P0-1) for up to 45 min after the end of the protocol. Contrarywise, IH ceased breathing activity after the second postnatal day. Respiratory facilitation mediated by IH faded in the absence of suprapontine structures. Identical modulatory effects were observed with IH supplied through a HEPES buffer solution. Interestingly, IH increased c-Fos expression in hypothalamic areas, suggesting its selective activation during IH, which was confirmed by the absence of consistent c-Fos labeling in the hippocampus. Field recordings from hypothalamus revealed the appearance of a slow rhythmic pattern of discharges after IH. The current work contributes to clarify modulatory suprapontine influences on the respiratory motor output at birth and how neural outputs elicited by physiological challenge, like IH and exercise, are integrated in the central nervous system to control respiratory tone during development.