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.

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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 previously reported (J Appl Physiol 89: 807-822, 2000) that < or =10 min of hyperbaric oxygen (HBO(2); < or = 2,468 Torr) stimulates solitary complex neurons. To better define the hyperoxic stimulus, we measured PO(2) in the solitary complex of 300-microm-thick rat medullary slices, using polarographic carbon fiber microelectrodes, during perfusion with media having PO(2) values ranging from 156 to 2,468 Torr. Under control conditions, slices equilibrated with 95% O(2) at barometric pressure of 1 atmospheres absolute had minimum PO(2) values at their centers (291 +/- 20 Torr) that were approximately 10-fold greater than PO(2) values measured in the intact central nervous system (10-34 Torr). During HBO(2), PO(2) increased at the center of the slice from 616 +/- 16 to 1,517 +/- 15 Torr. Tissue oxygen consumption tended to decrease at medium PO(2) or = 1,675 Torr to levels not different from values measured at PO(2) found in all media in metabolically poisoned slices (2-deoxy-D-glucose and antimycin A). We conclude that control medium used in most brain slice studies is hyperoxic at normobaric pressure. During HBO(2), slice PO(2) increases to levels that appear to reduce metabolism.

We developed a hyperbaric chamber for intracellular recording in rat brain stem slices during continuous compression and decompression of the tissue bath with the inert gas helium. Air, rather than helium, was also used as the compression medium in some cases to increase tissue nitrogen levels. An important feature is the chamber door, which opens or closes rapidly at 1 atmosphere absolute (ATA) for increased accessibility of the microelectrode. The door also closes and seals smoothly without disrupting the intracellular recording. Hyperbaric oxygen was administered during helium compression using a separate pressure cylinder filled with perfusate equilibrated with 2. 3-3.3 ATA oxygen. Measurements of tissue/bath PO(2) and pH confirmed that the effects of compression using helium or air could be differentiated from those due to increased PO(2). One hundred and thirteen neurons were studied during 375 compression cycles ranging from 1 to 20 ATA (mode 3.0 ATA). We conclude that it is technically feasible to record intracellularly from the same mammalian neuron while changing ambient pressure over a physiologically important range. These techniques will be useful for studying how various hyperbaric environments affect neurophysiological mechanisms.