Cell Responses of the Ventrolateral Medulla to PAR1 Activation and Changes in Respiratory Rhythm in Newborn Rat En Bloc Brainstem-Spinal Cord Preparations

Proteinase-activated receptor-1 (PAR1) is expressed in astrocytes of various brain regions, and its activation is involved in the modulation of neuronal activity. Here, we report effects of PAR1 selective agonist TFLLR on respiratory rhythm generation in brainstem-spinal cord preparations. Preparations were isolated from newborn rats (P0-P4) under deep isoflurane anesthesia and were transversely cut at the rostral medulla. Preparations were superfused with artificial cerebrospinal fluid (25–26 °C), and inspiratory C4 ventral root activity was monitored. The responses to TFLLR of cells close to the cut surface were detected by calcium imaging or membrane potential recordings. Application of 10 μM TFLLR (4 min) induced a rapid and transient increase of calcium signal in cells of the ventrolateral respiratory regions of the medulla. More than 88% of responding cells (223/254 cells from 13 preparations) were also activated by low (0.2 mM) K⁺ solution, suggesting that they were astrocytes. Immunohistochemical examination demonstrated that PAR1 was expressed on many astrocytes. Respiratory-related neurons in the medulla were transiently hyperpolarized (-1.8 mV) during 10 μM TFLLR application, followed by weak membrane depolarization after washout. C4 burst rate decreased transiently in response to application of TFLLR, followed by a slight increase. The inhibitory effect was partially blocked by 50 μM theophylline. In conclusion, activation of astrocytes via PAR1 resulted in a decrease of inspiratory C4 burst rate in association with transient hyperpolarization of respiratory-related neurons. After washout, slow and weak excitatory responses appeared. Adenosine may be partially involved in the inhibitory effect of PAR1 activation.