We investigated whether neurons in two chemosensitive areas of the medulla oblongata [nucleus of the solitary tract (NTS) and ventrolateral medulla (VLM)] respond to hypercapnia differently than neurons in two nonchemosensitive areas of the medulla oblongata [inferior olive (IO) and hypoglossal nucleus (Hyp)]. Medullary brain slices from preweanling Sprague-Dawley rats were loaded with 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein, and intracellular pH (pHi) was followed in individual neurons at 37 degrees C with the use of a fluorescence imaging system. Most neurons from the NTS and VLM did not exhibit pHi recovery when CO2 was increased from 5 to 10% at constant extracellular HCO3- concentration [extracellular pH (pHo) decreased approximately 0.3 pH unit] (hypercapnic acidosis). However, when CO2 was increased from 5 to 10% at constant pHo (isohydric hypercapnia), pHi recovery was seen. In contrast, all neurons from the IO and Hyp exhibited pHi recovery during hypercapnic acidosis. All pHi recovery in the four areas studied was inhibited by 1 mM amiloride and unaffected by 0.5 mM 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid. These data indicate that 1) pHi regulation differs between neurons in chemosensitive (NTS and VLM) and nonchemosensitive (IO and Hyp) areas of the medulla, 2) pHi recovery is due solely to Na+/H+ exchange in all four areas, and 3) Na+/H+ exchange is more sensitive to inhibition by extracellular acidosis in NTS and VLM neurons than in IO and Hyp neurons.

brain stem; central chemoreceptor; carbon dioxide; fluorescence imaging; respiration; Na ϩ /H ϩ exchange THE LEVEL OF CO 2 /H ϩ in the blood is carefully regulated by certain neurons in several areas of the medulla oblongata. These neurons are collectively known as central chemoreceptors. The areas in which these neurons are located are known as chemosensitive areas and include the ventrolateral medulla (VLM), the nucleus of the solitary tract (NTS), and the medullary raphe (16). It is hypothesized that an increased level of CO 2 /H ϩ stimulates the central chemoreceptors, which in turn, via the respiratory central pattern generator neurons (which they presumably innervate), increase ventilation (8). The stimulus to the central chemoreceptors has been the subject of much debate. It has been hypothesized that the stimulus may be an increase in molecular CO 2 , a decrease in extracellular pH (pH o ), a decrease in intracellular pH (pH i ), or a combination of any of the three (1,7,16,24,25,30). We have previously shown that both pH i and pH o may play a role in central chemosensitivity (22).It would seem logical that if a change in pH is the major signaling pathway by which central chemoreceptors monitor a change in blood CO 2 /H ϩ , the manner in which these cells respond to acid/base disturbances should be different from that of cells that are not chemoreceptors (nonchemoreceptors). In a previous study, we found that Na ϩ /H ϩ exchange is the only pH i -regulating mechanism involved during recovery from intracellular acidification in neurons from both chemosensitive (NTS and VLM) and nonchemosensitive [hypoglossal nucleus (Hyp) and inferior olive (IO)] areas of the medulla (22). We also found that neurons from chemosensitive areas (NTS and VLM) respond with a maintained intracellular acidification during hypercapnic acidosis, but exhibit pH i recovery during isohydric hypercapnia. This is in contrast to neurons from nonchemosensitive areas (Hyp and IO) that exhibit pH i recovery even during hypercapnic acidosis (22). These findings suggest that the Na ϩ /H ϩ exchanger is more easily inhibited by a decrease of pH o in neurons from chemosensitive areas versus nonchemosensitive areas.The major aim of the present study was to examine pH i regulation in greater detail in individual neurons from chemosensitive and nonchemosensitive areas of the medulla to investigate whether other differences in pH i regulation are present. It must be noted that these data are from neurons in known chemosensitive areas (16) but that the individual neurons themselves may or may not be chemoreceptors. Our data show the following: 1) intrinsic buffering power (␤ int ) is the same in all neurons tested; 2) removal of extracellular chloride at steady-state pH i results in intracellular alkalinization in all Hyp, IO, and VLM neurons but results in intracellular acidification in most NTS neurons, suggesting that Cl Ϫ /HCO 3 Ϫ exchange is present in all Hyp, IO, and VLM neurons but not in most NTS neurons; 3) steady-state pH i is more depende...

We compared the response to hypercapnia (10%) in neurons and astrocytes among a distinct area of the retrotrapezoid nucleus (RTN), the mediocaudal RTN (mcRTN), and more intermediate and rostral RTN areas (irRTN) in medullary brain slices from neonatal rats. Hypercapnic acidosis (HA) caused pH(o) to decline from 7.45 to 7.15 and a maintained intracellular acidification of 0.15 +/- 0.02 pH unit in 90% of neurons from both areas (n = 16). HA excited 44% of mcRTN (7/16) and 38% of irRTN neurons (6/16), increasing firing rate by 167 +/- 75% (chemosensitivity index, CI, 256 +/- 72%) and 310 +/- 93% (CI 292 +/- 50%), respectively. These responses did not vary throughout neonatal development. We compared the responses of mcRTN neurons to HA (decreased pH(i) and pH(o)) and isohydric hypercapnia (IH; decreased pH(i) with constant pH(o)). Neurons excited by HA (firing rate increased 156 +/- 46%; n = 5) were similarly excited by IH (firing rate increased 167 +/- 38%; n = 5). In astrocytes from both RTN areas, HA caused a maintained intracellular acidification of 0.17 +/- 0.02 pH unit (n = 6) and a depolarization of 5 +/- 1 mV (n = 12). In summary, many neurons (42%) from the RTN are highly responsive (CI 248%) to HA; this may reflect both synaptically driven and intrinsic mechanisms of CO(2) sensitivity. Changes of pH(i) are more significant than changes of pH(o) in chemosensory signaling in RTN neurons. Finally, the lack of pH(i) regulation in response to HA suggests that astrocytes do not enhance extracellular acidification during hypercapnia in the RTN.

Putative chemoreceptors in the solitary complex (SC) are sensitive to hypercapnia and oxidative stress. We tested the hypothesis that oxidative stress stimulates SC neurons by a mechanism independent of intracellular pH (pH(i)). pH(i) was measured by using ratiometric fluorescence imaging microscopy, utilizing either the pH-sensitive fluorescent dye BCECF or, during whole cell recordings, pyranine in SC neurons in brain stem slices from rat pups. Oxidative stress decreased pH(i) in 270 of 436 (62%) SC neurons tested. Chloramine-T (CT), N-chlorosuccinimide (NCS), dihydroxyfumaric acid, and H(2)O(2) decreased pH(i) by 0.19 +/- 0.007, 0.20 +/- 0.015, 0.15 +/- 0.013, and 0.08 +/- 0.002 pH unit, respectively. Hypercapnia decreased pH(i) by 0.26 +/- 0.006 pH unit (n = 95). The combination of hypercapnia and CT or NCS had an additive effect on pH(i), causing a 0.42 +/- 0.03 (n = 21) pH unit acidification. CT slowed pH(i) recovery mediated by Na(+)/H(+) exchange (NHE) from NH(4)Cl-induced acidification by 53% (n = 20) in CO(2)/HCO(3)(-)-buffered medium and by 58% (n = 10) in HEPES-buffered medium. CT increased firing rate in 14 of 16 SC neurons, and there was no difference in the firing rate response to CT with or without a corresponding change in pH(i). These results indicate that oxidative stress 1). decreases pH(i) in some SC neurons, 2). together with hypercapnia has an additive effect on pH(i), 3). partially inhibits NHE, and 4) directly affects excitability of CO(2)/H(+)-chemosensitive SC neurons independently of pH(i) changes. These findings suggest that oxidative stress acidifies SC neurons in part by inhibiting NHE, and this acidification may contribute ultimately to respiratory control dysfunction.

We studied the development of chemosensitivity during the neonatal period in rat Nucleus tractus solitarii (NTS) neurons. We determined the percentage of neurons activated by hypercapnia (15% CO2) and assessed the magnitude of the response by calculating the chemosensitivity index (CI). There were no differences in the percentage of neurons that were inhibited (9%) or activated (44.8%) by hypercapnia or in the magnitude of the activated response (CI 164±4.9%) in NTS neurons from neonatal rats of all ages. To assess the degree of intrinsic chemosensitivity in these neurons we used chemical synaptic block medium and the gap junction blocker carbenoxolone. Chemical synaptic block medium slightly decreased basal firing rate but did not affect the percentage of NTS neurons that responded to hypercapnia at any neonatal age. However, in neonates aged