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

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 ϩ ], 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 ] generate rhythm with a high sensitivity to neuromodulators for extended time periods, whereas spontaneous "in vitro apnea" 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 ϩ ].

Cumulative lesioning of respiratory interneurons disrupts and precludes motor rhythms in vitro

“…Therefore, we assume that, on average, fewer than 13 neurons were detected at superficial levels because of slicing-related tissue damage, and that fewer than 13 neurons were detected at deeper levels because of limited dye penetration and scattering of laser pulses (33). Even if technical limitations prevented their detection, we assume an average uniform density of 13 inspiratory neurons per 10 μm per side in the preBötC, which occupies 250 μm in the rostrocaudal axis, bilaterally (9,25,34). Thus, we calculate 650 inspiratory neurons comprise the preBötC.…”

Tripartite Purinergic Modulation of Central Respiratory Networks during Perinatal Development: The Influence of ATP, Ectonucleotidases, and ATP Metabolites

“…Once rhythmic activity was lost (15-45 min in E17-E21; Ն60 min in P0 -P4), it was reactivated by raising extracellular K ϩ . In P0 -P4 slices, rhythm was reactivated with 9 mM K ϩ , as is typical for postnatal slice preparations (range is 8 -10 mM K ϩ ) (Smith et al, 1991;Funk et al, 1993;Tryba et al, 2003;Ruangkittisakul et al, 2006). However, as reported anecdotally, 9 mM K ϩ was associated with the generation of slow, largeamplitude, intermittent bursts of nonrespiratory activity in E17-E21 slices Ren et al, 2006) that prevents measurement of baseline inspiratory activity and drug effects.…”

“…Aside from the demonstration in Xenopus tadpoles in which ATP initiates and ADO accumulation helps terminate activity (Dale and Gilday, 1996), evidence of its significance for sensorimotor integration in functioning mammalian motor networks is lacking. The objective of this study is to explore the potential significance of ATP distance to VIIc) (Ruangkittisakul et al, 2006). The spatial relationships between reference nuclei and molecular markers of the preBötC (NK1 receptor labeling) is only available in the parasagittal plane for embryonic animals (Pagliardini et al, 2003), not in the transverse plane of rhythmic medullary slices.…”

Amyloid β (Aβ) Peptide Directly Activates Amylin-3 Receptor Subtype by Triggering Multiple Intracellular Signaling Pathways

“…Ca 2ϩ Imaging-Dynamic changes of the free cytosolic Ca 2ϩ concentration were monitored with confocal microscopy as described previously (21). For this, AMY3-HEK293 cells were plated on glass coverslips precoated with poly-L-ornithine and incubated at 37°C for 12-36 h with DMEM, 10% FBS, and…”