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3 Department of Biology, St. Lawrence University, Canton, New York 13617; 1 Department of Biology, Allegheny College, Meadville, Pennsylvania 16335; and 2 Departments of Physiology and Medicine, Dartmouth Medical School, Lebanon, New Hampshire 03756
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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We examined intracellular pH (pHi) regulation in the retrotrapezoid nucleus (RTN), a CO2-sensitive site, and the hypoglossal nucleus, a nonchemosensitive site, during development (postnatal days 2-18) in rats. Respiratory acidosis [10% CO2, extracellular pH (pHo) 7.18] caused acidification without pHi recovery in the RTN at all ages. In the hypoglossal nucleus, pHi recovered in young animals, but as animal age increased, the slope of pHi recovery diminished. In animals older than postnatal day 11, the pHi responses to hypercapnia were identical in the hypoglossal nucleus and the RTN, but hypoglossal nucleus and RTN neurons could regulate pHi during intracellular acidification at constant pHo at all ages. Recovery of pHi from acidification in the RTN depended on extracellular Na+ and was inhibited by amiloride but was unaffected by DIDS, suggesting a role for Na+/H+ exchange. Hence, pHi regulation during acidosis is more effective in the hypoglossal nucleus in younger animals, possibly as a requirement of development, but in older juvenile animals (older than postnatal day 11), pHi regulation is relatively poor in chemosensitive (RTN) and nonchemosensitive nuclei (hypoglossal nucleus).
chemoreceptor; brain slice
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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PREVIOUS IN VIVO AND IN VITRO studies examining the effects of acidic stimuli on neuronal electrical activity or respiratory output identified brain stem neurons that are sensitive to changes in CO2/pH (10, 11, 20, 34, 43). These neurons are located at sites within the medulla oblongata implicated in central chemoreception, suggesting that they are the central chemoreceptors for the respiratory control system (29). It has been hypothesized that the neural output of putative medullary CO2 chemoreceptor cells increases as intracellular pH (pHi) decreases during hypercapnia (21, 32). Moreover, it has been proposed in vertebrates (45) and invertebrates (13) that optimal chemoreceptor design would require that the acid sensors demonstrate sustained acidification during hypercapnia.
As in many biological systems, the neural circuitry responsible for respiratory control is subject to developmental changes. For example, the greatest increases in dendritic length, dendritic branching, and synapse formation occur during the first 3 wk of life in the nucleus tractus solitarii (NTS) and the ventrolateral medulla (VLM), two sites implicated in CO2 chemoreception (41). There are also developmental changes in the ventilatory response to CO2 during the postnatal period in rats. A ventilatory response to CO2 is present at birth, but during the 1st wk of life, the ventilatory effect of hypercapnia wanes and reaches a nadir at ~8 days of age. After this period, the hypercapnic ventilatory response increases and stabilizes at ~2-3 wk of age (3, 48). Moreover, the pattern of breathing in whole animals during hypercapnia in the neonate differs from that in the adult rat (27). Neonatal animals predominantly increase tidal volume during hypercapnia, rather than breathing frequency, but adult rats increase breathing frequency and tidal volume (24). In addition to the changes in whole animal ventilatory control, there may be developmental changes in chemosensory function of individual neurons. Wang and Richerson (55) found marked developmental changes in spontaneous neuronal firing of cells located in one chemosensitive site, the medullary raphé. The firing rate of only 3% of the cells in the raphé increased in response to CO2 in rats at postnatal day 9, but in 18% of the raphé cells in rats at postnatal day 14, the firing rate increased during hypercapnia. These findings from in vivo and in vitro studies of respiratory control suggest that the neural circuits and the ionic currents that underlie CO2 chemosensory responses undergo significant postnatal development.
In most vertebrate cells, pHi regulation after
acidification is achieved by membrane-bound transport systems, often
including an amiloride-sensitive Na+/H+
exchanger (NHE) and one or more HCO
The dichotomous pattern of pHi regulation among brain stem nuclei in brain slices from neonatal rats may be at odds with the pattern of pHi regulation in vivo in the brain of adult animals, in which a more homogeneous pattern of poor pHi regulation occurs. 31P nuclear magnetic resonance (NMR) spectroscopy measurements of pHi in the brain of adult rats during sustained hypercapnia demonstrated surprisingly poor pHi regulation (33). The rats were anesthetized, and the spectroscopic measurements provided an average pHi value, derived from neurons and glia, for the entire brain beneath the NMR probe, which was over the midline of the posterior skull. Hence, regional variation of pHi regulation may be masked by these NMR measurements, but pHi regulation was, nonetheless, absent when animals inspired 5% CO2 and slow and incomplete when animals inspired 10% CO2 for a sustained period (3 h) even in nonchemosensitive regions of the brain.
We reevaluated pHi regulation during hypercapnia in brain stem slices prepared from rats over a larger range of ages (postnatal days 2-18) to try to reconcile the divergent patterns of pHi regulation in neonatal and adult animals and to test the hypothesis that the pattern of pHi regulation evolved over the course of development. We examined pHi regulation during hypercapnia in one chemosensitive nucleus and one nonchemosensitive nucleus within the brain stem. It was our hypothesis that pHi regulation would remain consistently poor in the chemosensitive brain stem nucleus but change in the nonchemosensitive nucleus from a pattern of effective pHi regulation early in development to a pattern of poor pHi regulation in older animals.
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METHODS |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Slice Preparation
Medullary tissue slices were prepared from 2- to 18-day-old Sprague-Dawley rat pups of either gender, as previously described (52). These procedures were performed in accordance with the guidelines stated in the Guide for the Care and Use of Laboratory Animals as put forth by the Public Health Service, National Institutes of Health. Briefly, the animal was killed by rapid decapitation, and the brain was quickly removed from the skull and submerged in chilled (6-8°C) control saline (see below) for 2-3 min. The caudal cerebellum was removed, and a tissue block was prepared from the medulla oblongata. Transverse medullary slices (120- to 150-µm thick) from a region extending rostrally from the obex to the pons were sectioned in chilled, oxygenated control saline using a Vibratome and transferred to a holding chamber containing control saline at room temperature (22°C).Solutions
For the medullary brain slice, the control, CO2/HCOThe nigericin calibration solution contained (in mM) 104 KCl, 2.4 CaCl2, 1.3 MgSO4, 1.24 KH2PO4, 10 glucose, 25 NMDG-HEPES, 25 K-HEPES,
and 4 × 10
3 nigericin. A calibration curve was
constructed by titrating the pH of the calibration solutions from 5.8 to 8.6 pH units with KOH or HCl. A one-point calibration was performed
at the end of each experiment using nigericin solutions that were
titrated to pH 7.3 at 37°C (6). After each calibration,
the entire perfusion apparatus was soaked and washed three times in
70% ethanol in water and subsequently rinsed repeatedly in distilled
water to remove any retained nigericin. All reagents were purchased
from Sigma (St. Louis, MO) or Fisher Scientific (Pittsburgh, PA).
Superfusion Chamber
Single brain slices loaded with 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF; see below) were removed from the holding chamber and transferred to a superfusion chamber described previously (46). Brain slices were immobilized in the chamber using a platinum wire and nylon grid (12). The total chamber volume was ~1 ml, and the chamber was continuously superfused with control or test solutions using syringe pumps at a rate of ~2 ml/min. The time required for complete exchange of the chamber solution was ~1 min. Solutions entering the chamber were heated to 37°C using a servo-controlled stage heater (WPI, Sarasota, FL), and a continuous stream of 95% O2-5% CO2 was directed over the air-fluid interface. The pH of superfused solutions was monitored using a pH electrode (Beetrode, WPI).Measurement of pHi
Slices were loaded with the pH-sensitive fluoroprobe BCECF (Molecular Probes, Eugene, OR) by incubation in a 20 µM solution of BCECF-AM in control saline bubbled with 95% O2-5% CO2 for 20-30 min at room temperature. BCECF-loaded slices were transferred to a holding chamber containing control saline (no dye) bubbled with 95% O2-5% CO2 at room temperature and maintained in this solution in the dark until the time of experimentation. The pH was measured in single, BCECF-loaded slices placed in a chamber mounted on the stage of an upright microscope (model E600FN, Nikon, Melville, NY) and continuously superfused with control saline. The pHi of individual neurons was measured within discrete areas (~0.042 mm2 at ×400 magnification) of the medulla by alternately exciting the area with light from a 75-W xenon lamp (Chiu Industries, Kings Park, NY) at wavelengths of 500 ± 10 and 440 ± 10 nm for 0.3-0.8 s. The excitation filters were switched by a computer-controlled filter wheel (Lambda 10-2, Sutter Instruments, Novato, CA). The emitted fluorescence from the intracellular BCECF was filtered using a 510-nm dichroic mirror (model C5700, Nikon), intensified, and measured with a cooled charge-coupled device camera (Photometrics, Tucson, AZ). The digital images were acquired and processed with Axon Imaging Workbench software (Axon Instruments, Foster City, CA). The rate of BCECF leakage is a measure of neuronal viability (4), and a rate of leakage <1%/min is consistent with viable neurons. The average rate for BCECF loss in our cells (measured as the rate of fall of the pH-insensitive fluorescence at 440-nm excitation) was ~0.3%/min, and results from neurons in which the rate of BCECF leakage exceeded 1%/min were not analyzed.Data Analysis
Data were collected from ~160 brain slices from 80 animals. Steady-state pHi values were obtained by averaging at least five contiguous values at a point in the pHi vs. time record when pHi was stable. The initial fall in pHi after acidification was measured as the lowest value within 4 min of changing the perfusate from the control to the hypercapnic superfusate. We estimated the intrinsic buffer power (
int) of neurons by calculating the ratio of the change
in intracellular bicarbonate to the change in pHi after changing from the control superfusate (pHo 7.48) to the
hypercapnic superfusate (pHo 7.18). Intracellular
bicarbonate was estimated using the Henderson-Hasselbalch equation, a
pK'a for carbonic acid of 6.12, a
CO2 solubility of 0.03 mM/mmHg, and the measured pHi. Bicarbonate buffering was calculated by multiplying
the estimated intracellular bicarbonate concentration by 2.303. Bicarbonate buffering power at the midpoint of pHi recovery
during hypercapnic acidosis was calculated using the
Henderson-Hasselbalch relationship and the pHi at the
midpoint of pHi recovery: log[(10initial
pHi + 10final pHi)/2],
where initial and final refer to the end points of the linear portion
of pHi recovery. The total buffer power was the sum of int and the bicarbonate buffering power. The proton flux
at the midpoint of the hypercapnic exposure
(JH,mid) was calculated by multiplying the total
buffering power at the midpoint of recovery by the rate of
pHi recovery (38, 47). Recovery rates of
pHi were calculated by performing least-squares regression
on the linear portion of the pHi vs. time traces (Fig.
1). Values are means ± SE.
Statistical comparisons were made using a repeated-measures or
factorial ANOVA, as appropriate, in the General Linear Model procedure
of Systat (SPSS Science, Chicago, IL). P 
0.05 was considered statistically significant.
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RESULTS |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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pHi, int, and pHi Recovery
in the Hypoglossal Nucleus and RTN
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The initial effects of exposing cells in each nucleus to hypercapnia
(10% CO2-90% O2, pHi 7.18) are
summarized in Table 2. Exposing the
hypoglossal nucleus to solutions mimicking hypercapnic acidosis (10%
CO2-90% O2, pHo 7.18) decreased
pHi to 7.03 ± 0.01. The extent of acidification
during hypercapnia was not dependent on age (P = 0.18).
Exposing RTN neurons to solutions mimicking hypercapnic acidosis
decreased pHi to 6.98 ± 0.01, but the extent of
acidification during hypercapnia was dependent on age
(P < 0.03). The pHi fell more in younger
animals (younger than postnatal day 11; pHi
6.94 ± 0.01, n = 27 cells) than in older animals
(postnatal day 11 and older; pHi 7.00 ± 0.02, n = 61). Although the control pHi
values did not differ between the hypoglossal nucleus and the RTN, the
fall in pHi during acidosis was significantly greater in
the RTN than in the hypoglossal nucleus (P < 0.01).
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Estimates of int are summarized in Table
3. The estimates of int
were somewhat variable, as shown by the relatively large SE. However,
significant differences existed between nuclei. The int
in the hypoglossal nucleus was 40.6 ± 1.7 meq · l1 · pH unit1, and
this value increased significantly as developmental age increased
(P = 0.0001). The RTN had a lower int
(17.0 ± 1.1 meq · l1 · pH
unit1), but int also increased in older
animals (P = 0.0001). The rate at which
int increased as animals aged was similar in the hypoglossal nucleus and the RTN (there was no interaction between age
and nucleus, P = 0.53), even though the absolute value
of int at any age was lower in the RTN than in the
hypoglossal nucleus (P < 0.0001).
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There were major age-related differences in the rate of pHi
recovery and the rate of proton flux between nuclei and among animals
of different ages. The rates of pHi change during sustained hypercapnia are shown for the hypoglossal nucleus and RTN in Fig. 2. Recovery of pHi was
apparent in the hypoglossal nucleus in younger animals, but the rate of
recovery diminished as animal age increased. The loss of
pHi recovery as a function of age was highly significant
(P < 0.0001). In contrast, there was little or no
evidence of pHi recovery in the RTN at any age studied and no age-related change in the slope of pHi recovery
(P = 0.38).
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Because int differed between nuclei and changed as a
function of animal age, the absolute values of the slope of
pHi change during hypercapnic acidosis might not accurately
depict proton flux rates (JH,mid), which depend
on the total buffering power (int + bicarbonate
buffering) and the rate of change of pHi. Therefore, we
estimated JH,mid during hypercapnic acidosis in the hypoglossal nucleus and RTN at different postnatal ages.
Despite differences in buffering power, the changes in
JH,mid mirrored the changes in the slope of
pHi during hypercapnia. Proton flux rates fell in the
hypoglossal nucleus as animal age increased and became negative at
postnatal day 16. The decline in
JH,mid as age increased was highly significant
(P < 0.001). There was no effect of age on
JH,mid in the RTN (P = 0.87).
Once we found that pHi recovery was less in older animals
in the nonchemosensitive hypoglossal nucleus, we examined
pHi recovery in additional nonchemosensitive nuclei to
determine whether the loss of pHi recovery in older animals
was a general phenomenon within the brain stem or unique to the
hypoglossal nucleus. We examined pHi recovery in the facial
nucleus and medial vestibular nucleus in animals on postnatal day
14, and in both nuclei, the rate of pHi recovery was
negligible (the mean slope in both nuclei was 
0.003 pH U/min, which
was not significantly different from zero). The int was
~37 meq · l1 · pH unit1
in each of these nuclei, which was similar to the int
observed in the hypoglossal nucleus. This value is larger than the
int in the RTN, and as a result, the total buffering
power was also greater in the facial and medial vestibular nuclei. As a
result, JH,mid was actually more negative in the
facial and medial vestibular nuclei than in the RTN. Hence, these other
nonchemosensitive brain stem nuclei also lack pHi
regulation in older animals.
Effect of pHo on pHi Regulation
In previous studies of pHi regulation during acidosis in CO2-chemosensitive and nonchemosensitive regions in the brain stem in younger animals (postnatal day 12 and younger), pHi regulation was inhibited when pHo was also reduced, but pHi regulation was apparent when only pHi (and not pHo) was reduced by the acidic stress (45). We examined the effect of pHo on pHi regulation during hypercapnic acidosis (pHi and pHo drop) and after an ammonia prepulse (pHi falls, but pHo remains constant) in the hypoglossal nucleus and RTN of animals at postnatal days 16 and 17, respectively. The pHi profile over the course of the experiment and the sequence of test conditions are shown in representative neurons from both nuclei in Fig. 3. We usedint calculated
from the initial change in pHi and calculated the change in
bicarbonate during the hypercapnic acidosis to derive an estimate of
int, and we measured the rate of pHi change
per minute during the linear portion of pHi "recovery" during hypercapnia and after the ammonia prepulse. From these measurements, we calculated JH,mid during each
period of acidosis in each of the nuclei. The average proton flux rates
were 0.001 ± 0.038 meq · l1 · min1 during
hypercapnia in the hypoglossal nucleus (n = 12) and
0.033 ± 0.032 meq · l1 · min1 in the RTN
(n = 16). These values are not significantly different from each other (P > 0.5). Compared with the
hypercapnic JH,mid, the average
JH,mid after the prepulse increased to
1.316 ± 0.072 meq · l1 · min1 in the
hypoglossal nucleus and 0.800 ± 0.063 meq · l1 · min1 in the RTN.
Both values significantly exceed the
JH,mid during hypercapnia (P < 0.001 in both cases). Furthermore, the JH,mid was significantly greater in the hypoglossal nucleus than in the RTN
after the ammonia prepulse (P < 0.001), even though
the rates were similar during hypercapnia. The pHi was
significantly lower at the midpoint of the linear portion of the
pHi recovery phase in the RTN than in the hypoglossal
nucleus (P < 0.001), which is consistent with the
lower buffering power in the RTN shown in Tables 1-3. However,
within each nucleus, pHi was similar during hypercapnia and
after the NH4Cl prepulse protocol. Therefore, it appears
that the inhibitory effect of pHo on pHi
regulation was greater in the hypoglossal nucleus, since
JH,mid increased more than in the RTN when
pHo was held constant after the ammonia prepulse and the
inhibitory effect of pHo was absent.
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Regulation of pHi in the RTN
The mechanisms of pHi regulation have been studied in the hypoglossal nucleus and inferior olive (nonchemosensitive regions) and in two medullary regions containing putative CO2 chemosensory neurons: the rostral VLM and the NTS (44, 45). In all four regions, an NHE mechanism dominated pHi recovery during acidosis. The NHE regulation in these CO2 chemosensory sites demonstrated unusual sensitivity to pHo, which inhibited the activity of the NHE mechanism. The pHi regulatory mechanisms in the RTN, also a putative site of CO2 chemosensory neurons, have not been described. Therefore, we tested the hypothesis that NHE also regulates pHi in the RTN under control conditions and during intracellular acidosis to determine whether the association of NHE inhibition and CO2 chemosensitivity is a more general phenomenon. We defined the RTN as a thin layer of cells in the VLM extending from the rostral tip of the facial nucleus caudally to the rostral retrofacial nucleus and lying within 400-600 µm of the ventral medullary surface in its most rostral extent and within 100-300 µm of the ventral medullary surface at its most caudal extent. The RTN is bounded laterally by the spinotrigeminal tract and medially by the parapyramidal region (30).Effects of amiloride on pHi recovery in RTN neurons.
We examined the effect of amiloride on pHi regulation
during normocapnic control conditions (5% CO2,
pHo 7.48) and hypercapnic conditions (10% CO2,
pHo 7.18). The responses of pHi to these conditions in representative neurons are shown in Fig.
4 (normocapnia) and Fig.
5 (hypercapnia). In both cases, amiloride
significantly reduced the rate of pHi recovery. We
estimated the rate of pHi change during the linear portion
of each test condition: normocapnia during and after amiloride exposure
and hypercapnia before and after amiloride exposure. We do not have an
estimate of int for the neurons tested under normocapnic
conditions, but for this analysis, we assumed that int
was similar in the normocapnic and hypercapnic neurons. We compared
JH,mid during normocapnia after amiloride was
removed from the perfusate with JH,mid during hypercapnia before amiloride was added to the perfusate. The proton flux rate was significantly greater during normocapnia (1.09 ± 0.09 meq · l1 · min1,
n = 11) than during hypercapnia (0.29 ± 0.09 meq · l1 · min1,
n = 12, P < 0.001). The
pHi was actually lower during normocapnic pHi
recovery after treatment with amiloride (Fig. 4) than during the
initial phase of hypercapnia (Fig. 5). This results in a lower intracellular bicarbonate concentration, a lower total buffering power,
and a lower estimate of JH,mid during
normocapnic recovery. However, JH,mid in the
absence of amiloride was still greater during normocapnia than during
hypercapnia, a finding that emphasizes the potent inhibitory effect of
pHo on pHi regulation. The proton flux rate
fell under both conditions when amiloride was added to the perfusate:
JH,mid was 1.16 ± 0.09 meq · l1 · min1 during
normocapnia and 1.06 ± 0.08 meq · l1 · min1 during
hypercapnia. These flux rates were significantly different from the
flux rates without amiloride (P < 0.001) and
significantly different from each other (P < 0.001).
It is worth noting that an amiloride-sensitive mechanism was active
during normocapnia, since amiloride caused pHi to fall.
This is not the case in the rostral VLM or NTS, where amiloride caused
no change in pHi under control normocapnic conditions
(44).
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int equal to the
average value of neurons in the RTN in animals older than postnatal
day 11 to calculate JH,mid. The
initial JH,mid was 0.026 ± 0.002 meq · l1 · min1, and
JH,mid fell significantly to 0.001 ± 0.001 meq · l1 · min1 when
amiloride was present in the perfusate (P < 0.001, n = 6). The rate rose to 0.007 ± 0.003 meq · l1 · min1 once
amiloride was no longer present; this rate was significantly greater
than the proton flux rate in the presence of amiloride (P < 0.001) but also less than the rate before
application of amiloride when pHi was lower as well
(P < 0.001).
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Effect of Na+-free solutions on
pHi recovery in RTN neurons.
We examined the effect of removing extracellular Na+ on
pHi regulation after an ammonia prepulse. The
pHi profiles from representative neurons in the RTN from an
animal at postnatal day 15 are shown in Fig.
7. We calculated
JH,mid during the recovery from acidification after Na+ was removed and after Na+ was
restored using int derived from Table 3. During the
initial pHi recovery phase before Na+ was
removed, some of the neurons had not established a stable recovery
rate, and data were not analyzed from this period of the study. The
rate of proton flux when Na+ was removed was 0.419 ± 0.028 meq · l1 · min1
(n = 14). After Na+ was restored, the rate
of change was 0.418 ± 0.029 meq · l1 · min1. These
rates of change are significantly different (P < 0.001). Thus pHi recovery during acidosis requires
extracellular Na+. NHE, which requires extracellular
Na+, is the primary exchange mechanism active during
acidosis in many brain stem nuclei (44), and when NHE is
present, but extracellular Na+ is removed, the exchanger
may run in "reverse," effectively acid loading the cell.
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Effects of DIDS on pHi regulation in RTN neurons.
In addition to NHE, many cells rely on a variety of bicarbonate
transporters to regulate pHi. We examined the role of
Cl/HCO/HCOint from Table 3. The estimated proton flux rate during
recovery from the prepulse-induced acidosis was 0.462 ± 0.065 meq · l1 · min1 when DIDS
was present and 0.520 ± 0.084 meq · l1 · min1 after DIDS
was removed from the perfusate. These values are not significantly
different (n = 25, P = 0.521);
therefore, there is no evidence of a DIDS-sensitive mechanism of
pHi regulation active during recovery from acidosis in
these neurons.
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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There are three main findings in this study. First, pHi regulation was more effective in younger animals (no older than postnatal day 11) in the hypoglossal nucleus, as Ritucci et al. (44, 45) previously described. Second, as animals aged, pHi regulation deteriorated in the hypoglossal nucleus, and after postnatal day 11, pHi regulation was similar during hypercapnia in all medullary nuclei tested regardless of whether a nucleus was a putative chemosensory region. The reduced capacity for pHi regulation in the hypoglossal nucleus was associated with the emergence of inhibition of pHi regulation by pHo. Finally, pHi regulation in the RTN seemed to depend on an NHE mechanism that is similar to the mechanisms described for other chemosensory areas of the brain stem (44, 45).
Critique of Methods
The interpretation of our results relies on estimates of proton flux. These estimates, in turn, depend on the directly measured rate of pHi change per unit time and the calculated total buffer power (int + bicarbonate buffering). Calculations
of int vary significantly in different tissues and
organisms (47). The variability arises, in part, from the
particular method of acid loading used to determine int,
and using CO2 as the acid stress, instead of an ammonia
prepulse or propionate, leads to the lowest estimates of
int among these possible acid-loading protocols
(22). We did not modify the perfusate constituents or add
inhibitors of pHi regulation (i.e., amiloride or stilbene
derivatives) when we made estimates of int, and we did
not project the linear portion of the pHi profile during
hypercapnia back to time 0 to try to estimate the change in
pHi associated with hypercapnia more accurately as some
investigators have done (47). Nonetheless, our estimates of int are similar to other estimates of neuronal
int when hypercapnia was used to estimate
int (22, 47). This probably occurs because we measured the hypercapnic fall in pHi within 4 min after
hypercapnia was applied, and this is close to time 0, since
some of the delay in the fall in pHi after the stimulus was
applied reflects the washin time of our perfusate chamber and the
tissue slice. Furthermore, the fall in pHo associated with
hypercapnia can inhibit pHi regulatory mechanisms and limit
the effect of proton or bicarbonate transport mechanisms on
pHi in the short time between application of the stimulus
and the measurement of the initial pHi value used to calculate int. Thus our calculated values of
JH,mid are derived from estimates of buffering
power, but they are likely to provide an accurate representation of
proton fluxes in the conditions we studied.
Within the brain slice, there are glia and neurons. We are confident that cells in which we measured pHi are neurons. On the basis of electrophysiological and immunohistochemical criteria, we showed previously that the BCECF-loaded cells in which we determine pHi are neurons rather than glia (46). Furthermore, cells loaded with BCECF after the procedure we used are rapidly killed by the neurotoxin kainic acid (data not shown). Although glia do have glutamate receptors, exposure to kainic acid is not lethal to glia (17). Finally, we should note that the RTN is a heterogeneous nucleus with multiple neuronal types. Our conclusions refer only to the average responses of all neurons within the RTN; we have no information about differences that may exist between neurons within the RTN or the hypoglossal nucleus.
Steady-State pHi Measurements
There were no systematic differences in neuronal pHi between the putative CO2-sensitive nucleus (RTN) and the CO2-insensitive nucleus (hypoglossal nucleus). Ritucci et al. (45) obtained a similar result when studying the inferior olive and hypoglossal nucleus (CO2 insensitive) and the NTS and rostral ventral medulla (CO2 sensitive). We did find differences inint between the
RTN and the hypoglossal nucleus and between early and late
developmental ages of both nuclei. Buckler et al. (8)
found relatively low int in isolated type I cells of the
carotid body, which are also CO2 sensitive. A low buffering
power permits changes in pHo to be reflected by similar
changes in pHi, although the ultimate steady-state
pHi will depend on the effectiveness of
pHi-regulating mechanisms. Buckler et al. (8)
speculated that the steep pHi-pHo relationship
in the carotid body was an important aspect of acid sensing by this
organ. In contrast, Ritucci et al. (44) found no
differences in int among the chemosensory and
nonchemosensory nuclei they studied, and buffer power was not different
between CO2 chemosensory and nonchemosensory regions in the
pulmonate snail Helix aspersa (18). Thus it
seems that buffer power may be lower in some CO2-sensitive
neurons (e.g., the RTN), and although this may enhance the sensitivity
to pHo changes in the absence of effective pHi
regulation, it is not an essential element in the chemosensory process.
Finally, int increased within the RTN and hypoglossal
nucleus as the animals aged. Haworth et al. (19) observed
a similar trend in ventricular myocytes over the course of neonatal development.
Steady-State pHi Regulation in RTN Neurons
Depending on the type of vertebrate neurons studied, some authors have attributed pHi regulation during acidification to a single proton transport system, NHE (16, 26, 28, 36, 51), while other authors implicated an additional role for HCORecovery of pHi During Hypercapnic Acidosis
We have shown that pHi recovery in CO2/HCOOver the course of development, pHi regulation in the hypoglossal nucleus became less effective during hypercapnia. We believe that this reflects the emergence in older animals of a pHi regulatory mechanism that is increasingly sensitive to inhibition by pHo. Low pHo inhibits NHE activity (2), and susceptibility to inhibition by pHo may differ among NHE isoforms. Developmentally mediated changes in the expression of NHE isoforms 1 and 3 have been described in the brain, although not specifically in the medulla (25). Thus it is possible that the progressive decrease in pHi regulation during hypercapnic acidosis in hypoglossal neurons as animals aged reflected a change in NHE isoform expression. However, NHE mechanisms are also susceptible to modulation by a variety of second messengers (35), and a developmental change in second-messenger expression affecting NHE function might also increase the sensitivity of pHi regulation by NHE to pHo.
A possible alternate explanation for the decline in pHi regulatory capacity as animals age is a generalized loss of pHi regulatory capacity. For example, the number of NHE proteins per unit cell volume might decline as animals mature. We believe that this is unlikely, because older animals still regulate pHi well during intracellular acidosis when pHo is kept at the control value (Fig. 3), and the loss of pH regulatory activity, therefore, seems specifically related to inhibition of pHi regulation by pHo. We do not have measurements of maximum pHi regulatory activity during acidosis as a function of age to address this issue. Studies to examine this hypothesis in which we measure JH,mid during isohydric hypercapnia or NH4Cl prepulse studies over the course of development are underway but beyond the scope of this report.
pHi as a Unique Characteristic of Chemosensory Function
In theory, a neuron with a single pHi regulatory mechanism inhibited by small decreases in pHo would make an ideal chemosensor of hypercapnic acidosis. Such a neuron would not "regulate" the stimulus, and decreases in pHo associated with elevated CO2 would be rapidly transduced into a sustained decrease in pHi that would show no accommodation during acidification (13, 45). Previous studies using brain slices from neonatal rats showed that neurons located in putative chemoreceptor sites within the medulla of young animals (postnatal days 0-11) failed to regulate pHi in response to hypercapnic acidosis, whereas neurons in nonchemoreceptor sites showed rapid regulation toward normocapnic values (45). Hence, the pHi responses to hypercapnia in chemosensitive regions of the brain stem are in keeping with the ideal theoretical design of a chemosensor, and nonchemosensory regions seemed to regulate pHi well in animals younger than postnatal day 11. However, the most important finding in this study is our observation that pHi regulation in chemosensitive and nonchemosensitive sites is not different among nuclei in juvenile animals (older than postnatal day 11). The hypoglossal nucleus, RTN, medial vestibular nucleus, and facial nucleus failed to show pHi recovery during physiological levels of hypercapnic acidosis in older animals. Neurons studied in animals older than postnatal day 11 had similar pHi responses to hypercapnic challenge regardless of the brain stem site tested. Thus the profile of pHi regulation during hypercapnic acidosis in older animals is not a unique characteristic reflecting chemosensory function, and putative chemoreceptors and nonchemoreceptors are equally sensitive to the inhibitory effects of extracellular acidification in older juvenile animals. Our findings are consistent with previous studies measuring brain pHi using spectroscopic techniques in which goats exposed to sustained hypercapnia (5 and 10% inspired CO2) for several hours demonstrated a prompt fall in pHi within the brain but little or no recovery of pHi (33). In all CO2 chemosensory cells studied thus far (8, 18, 44), pHi regulation is inhibited when pHo falls. However, the results from the hypoglossal nucleus, facial nucleus, and medial vestibular nucleus in older animals indicate that the converse is not true; nonchemosensory neurons do not necessarily regulate pHi well when pHo falls during hypercapnia. Thus poor pHi regulation is not uniquely associated with CO2 chemosensory neurons. Goldstein et al. (18) reached a similar conclusion in studies of CO2 chemosensory and nonchemosensory regions of the pulmonate snail H. aspersa.Perspectives
The emergence of poor pHi regulation in nonchemosensitive regions of the brain stem as the animal ages is paradoxical given the important role of pHi in a plethora of cellular processes (39). The pHi, however, may modulate events associated with growth and mitosis. For example, intracellular alkalinization and effective pHi regulation may be required for growth and development (23) or at least "permit" cellular processes important for growth and development (38). Hence, we suspect that neurons in younger animals, unless they must contribute to the CO2 chemosensory response of the whole animal, will require effective pHi regulation as a necessary function to maintain and promote development. As the animal ages and development is completed, the strategy of pHi regulation may switch from a pattern essential for growth and development to a pattern better suited to enhance survival of terminally differentiated neurons without growth potential.We do not believe that sustained acidification during hypercapnia in older animals evolved to serve the needs of chemosensitivity. We believe that sustained intracellular acidification of neurons during hypercapnic acidosis has a neuroprotective effect, and the evolution of this pattern of pHi regulation in chemosensory neurons was secondary to its neuroprotective effects. Extracellular and intracellular acidification are natural consequences of ischemia and brain injury, and neurons exposed to extracellular acidification during ischemic stress in vivo or hypoxic, anoxic, or traumatic stress in vitro swell and may lyse (15, 31). Cell death has been attributed to elevated levels of intracellular Ca2+ and intracellular Na+. Intracellular Ca2+ and, to a lesser extent, Na+ rise after excitotoxic glutamate receptor activation. Na+ also rises by virtue of the action of NHE activity in response to the intracellular acidosis. As a result of the increase in intracellular Na+, the Na+/Ca2+ exchanger may also operate in reverse and further load the cell with Ca2+. The extent of cell death can be minimized during conditions of extracellular acidification by blocking Na+ entry with Na+ substitutes (15, 50) or specific blockers of Na+ channels (1, 7) or inhibitors of NHE such as amiloride (1, 14). In vivo studies of ischemia have demonstrated the efficacy of administration of such drugs in minimizing neuronal damage (37). Moreover, reducing pHo inhibits pHi regulation and is neuroprotective in tissue culture models of ischemia (53). Thus we believe that the inhibition of NHE during hypercapnic acidosis is a mechanism whereby all neurons minimize the likelihood of cellular lysis and death during ischemia and anoxia. Our data suggest that this neuroprotective effect is differentially expressed over the course of development in different brain stem nuclei. Nuclei containing putative chemoreceptors appear to exhibit the proton inhibition of NHE early in life, a response that is also sensible in terms of the animal's need to have a ventilatory response to CO2 after parturition. Nonchemosensory neurons may be resistant to the inhibitory effects of pHo because of the need for effective pHi regulation and alkalinization during processes of growth and development. However, as the animal ages and growth is less important in neurons, the neuroprotective effect of inhibiting NHE emerges and predominates, and pHo inhibits pHi regulation during extracellular acidosis in older animals. Thus, once the brain stem has matured and established appropriate synaptic targets, the pattern of pHi regulation changes to serve a neuroprotective function during periods of ischemia.
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ACKNOWLEDGEMENTS |
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We thank Dr. Robert W. Putnam for helpful review of the manuscript.
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FOOTNOTES |
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This work was supported in part by National Science Foundation Grant IBN-98-10809 (J. S. Erlichman) and grants from Merck-American Association for the Advancement of Science (P. Wages) and the Phelps Fund (P. Wages).
Address for reprint requests and other correspondence: J. S. Erlichman, Dept. of Biology, St. Lawrence University, Canton, NY 13617 (E-mail: jerlichman{at}stlawu.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 7 June 2001; accepted in final form 14 August 2001.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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