High Sensitivity to Neuromodulator-Activated Signaling Pathways at Physiological [K+] of Confocally Imaged Respiratory Center Neurons in On-Line-Calibrated Newborn Rat Brainstem Slices

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Abstract
The pre-Bötzinger complex (PBC) inspiratory center remains active in a transverse brainstem slice. Such slices are studied at high (8–10 mm) superfusate [K+], which could attenuate the sensitivity of the PBC to neuromodulators such as opiates. Findings may also be confounded because slice boundaries, drug injection sites, or location of rhythmogenic interneurons are rarely verified histologically. Thus, we first generated PBC slices with defined boundaries using novel “on-line histology” based on our finding that rostrocaudal extensions of brainstem respiratory marker nuclei are constant in newborn rats between postnatal days 0–4. At physiological superfusate [K+] (3 mm), 500- and 600-μm-thick slices with the PBC in the center and the caudal boundary 0.70 and 0.76 mm caudal to the facial motonucleus generated rhythm for >2 and ∼4 h, respectively. Rhythm was abolished by low nanomolar concentrations of the μ-opiate receptor agonist DAMGO ([d-Ala2,N-Me-Phe4, Gly5-ol]enkephalin). After spontaneous arrest of bursting, rhythm was reactivated at clinically relevant or physiological concentrations by 3,5-dihydroxyphenylglycine, thyrotropin-releasing hormone, or rolipram, each affecting distinct second-messenger pathways. Two-photon/confocal Ca2+imaging revealed that these agents reactivated the same PBC neurons initially active in 3 mm[K+]. The data show that “calibrated” PBC slices at physiological [K+] generate rhythm with a high sensitivity to neuromodulators for extended time periods, whereas spontaneous “in vitroapnea” is an important tool to study the interaction of signaling pathways that modulate rhythm. Our approaches and findings provide the basis for a pharmacological and structure–function analysis of the isolated respiratory center in a histologically well defined substrate at physiological [K+].