Calcium Imaging Analysis of Cellular Responses to Hypercapnia and Hypoxia in the NTS of Newborn Rat Brainstem Preparation.

Hiroshi Onimaru,Itaru Yazawa,Isato Fukushi,Yasumasa Okada,Kotaro Takeda

doi:10.3389/fphys.2021.645904

Abstract

It is supposed that the nucleus of the solitary tract (NTS) in the dorsal medulla includes gas sensor cells responsive to hypercapnia or hypoxia in the central nervous system. In the present study, we analyzed cellular responses to hypercapnia and hypoxia in the NTS region of newborn rat in vitro preparation. The brainstem and spinal cord were isolated from newborn rat (P0-P4) and were transversely cut at the level of the rostral area postrema. To detect cellular responses, calcium indicator Oregon Green was pressure-injected into the NTS just beneath the cut surface of either the caudal or rostral block of the medulla, and the preparation was superfused with artificial cerebrospinal fluid (25–26°C). We examined cellular responses initially to hypercapnic stimulation (to 8% CO2 from 2% CO2) and then to hypoxic stimulation (to 0% O2 from 95% O2 at 5% CO2). We tested these responses in standard solution and in two different synapse blockade solutions: (1) cocktail blockers solution including bicuculline, strychnine, NBQX and MK-801 or (2) TTX solution. At the end of the experiments, the superfusate potassium concentration was lowered to 0.2 from 3 mM to classify recorded cells into neurons and astrocytes. Excitation of cells was detected as changes of fluorescence intensity with a confocal calcium imaging system. In the synaptic blockade solutions (cocktail or TTX solution), 7.6 and 8% of the NTS cells responded to hypercapnic and hypoxic stimulation, respectively, and approximately 2% of them responded to both stimulations. Some of these cells responded to low K+, and they were classified into astrocytes comprising 43% hypercapnia-sensitive cells, 56% hypoxia-sensitive cells and 54% of both stimulation-sensitive cells. Of note, 49% of the putative astrocytes identified by low K+ stimulation were sensitive to hypercapnia, hypoxia or both. In the presence of a glia preferential blocker, 5 mM fluoroacetate (plus 0.5 μM TTX), the percentage of hypoxia-sensitive cells was significantly reduced compared to those of all other conditions. This is the first study to reveal that the NTS includes hypercapnia and hypoxia dual-sensitive cells. These results suggest that astrocytes in the NTS region could act as a central gas sensor.

Highlights

The nucleus of the solitary tract (NTS) located in the dorsal medulla is the first relay station of sensory inputs involving autonomic functions and works as an integrative system that processes these inputs
A calcium indicator, Oregon Green 488 BAPTA-1 AM (200 μM; Invitrogen, Carlsbad, CA), was pressure-injected into the NTS just beneath the cut surface of either the rostral or caudal block preparation (Figure 1B), and the preparation was superfused with artificial cerebrospinal fluid (ACSF) composed of the following: 118 NaCl, 3 KCl, 1 CaCl2, 1 MgCl2, 26 NaHCO3, 1.2 NaH2PO4 and 30 glucose equilibrated with 95% O2 and 5% CO2, pH 7.4, at 25–26◦C (Okada et al, 2012)
We found that the NTS included cells that were sensitive to hypercapnia or hypoxia and some that were sensitive to both stimulations

Summary

Introduction

The nucleus of the solitary tract (NTS) located in the dorsal medulla is the first relay station of sensory inputs involving autonomic functions and works as an integrative system that processes these inputs. It is supposed that the NTS includes gas sensor cells responsive to hypercapnia and/or hypoxia in the central nervous system. It is known that the NTS region contributes to the regulation of the hypercapnic ventilatory response (Nattie and Li, 2002, 2008, 2009). CO2/H+ chemosensitive neurons were found in various subnuclei of the NTS (Dean et al, 2001; Nichols et al, 2008; Dean and Putnam, 2010; Huda et al, 2012). Recent studies showed that a subgroup of Phox2b-expressing neurons in the NTS exhibited intrinsic chemosensitivity to hypercapnic stimulation (Fu et al, 2017) and was presumed to participate in the hypercapnic ventilatory response (Fu et al, 2019)

Methods

Results

Conclusion