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Departments of 1Physiology and 2Pediatrics, Medical College of Wisconsin, 3Zablocki Veterans Affairs Medical Center, and 4Department of Physical Therapy, Marquette University, Milwaukee, Wisconsin 53226
Submitted 12 September 2003 ; accepted in final form 11 December 2003
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ABSTRACT |
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6.8 or 6.3), increased (P < 0.05) inspiratory flow by 8 and 12%, respectively, while the animals were awake during the day, but not at night while they were awake or in non-rapid eye movement sleep. While the animals were awake during the day, there were also increases in heart rate and blood pressure (P < 0.05) but no significant change in metabolic rate or arterial PCO2. Dialysis with mCSF equilibrated with 25 or 80% CO2 reduced ECF pH by the same amount (25%) or three times more (80%) than when inspired CO2 was increased to 7%. During CO2 inhalation, the reduction in ECF pH was only 50% of the reduction in arterial pH. Finally, dialysis in vivo only decreased ECF pH by 19.1% of the change during dialysis in an in vitro system. We conclude that 1) the physiological responses to focal acidosis in the RO and RP are consistent with the existence of chemoreceptors in these nuclei, and 2) local pH buffering mechanisms act to minimize changes in brain pH during systemic induced acidosis and microdialysis focal acidosis and that these mechanisms could be as or more important to pH regulation than the small changes in inspiratory flow during a focal acidosis. central chemoreceptors; pH regulation; control of breathing
As a result of the summarized experiments, an important question arose: Why are there chemoreceptors at widespread sites in the brain? Two hypotheses have been formulated (20): 1) the chemoreceptor sites "can vary in effectiveness depending on the state of arousal," and 2) the overall sensitivity of the respiratory control system "relies on an additive or greater effect" of multiple CCR sites. If indeed the different CCR sites function in a state-dependent fashion, then multiple sites would seem necessary. The second hypothesis is an alternative perspective to the relatively small changes in breathing observed during FA. If an increase in breathing is important in the correction of a FA at one CCR site, an increase in breathing would be only a fraction of the ventilatory response when all CCRs are activated, as occurs during systemic respiratory acidosis. However, one consequence of a response to FA may be hypocapnia at other CCR sites. Indeed, Li and Nattie (14) reported RTN acidification-increased breathing, leading to a systemic hypocapnia (4.9 Torr), which "may inhibit other chemoreceptor sites." This result seems counterintuitive, as one may not expect that the respiratory control system would alter the overall output due to increased excitatory drive from one CCR site, particularly if other CCR sites inhibit or limit output. Thus it seems logical that evolution has created mechanisms and systems other than a ventilatory response to correct for a FA in the brain. However, it is unclear of what importance and relative contributions breathing and other mechanisms have in compensation of the FA in the brain.
Therefore, the purpose of the present study was to determine the effects of FA in the raphe during wakefulness and sleep in adult goats and to gain insight into the relative role of the ventilatory response to regulation of brain extracellular pH in the awake state. In light of previous findings, we hypothesized that breathing would increase in response to FA in the raphe nuclei but that the effect would be dependent on the state of arousal. We further hypothesized that, if an increase in breathing is observed, the magnitude of the effect will be a small fraction of the response to an equivalent systemic generated respiratory acidosis. Finally, we hypothesized that FA will significantly affect heart rate (HR), blood pressure (BP), metabolic rate, and body temperature due to the known influences of the medullary raphe on cardiovascular and metabolic functions.
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METHODS |
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Surgical Procedures
An initial surgery was performed to elevate a 5-cm segment of the carotid arteries and for electrode implantation. The goats were anesthetized initially with a combination of ketamine and xylazine, intubated, and mechanically ventilated. Throughout surgery, anesthesia was maintained with 1-1.5% halothane in oxygen. Under sterile conditions, the carotid arteries were isolated from the vagi and elevated superficial to the muscle, and the skin was sutured. Upper airway electromyographic (EMG) electrodes were implanted through an anterior midline incision in the neck into laryngeal and pharyngeal muscles. An EMG electrode was also placed into the diaphragm, and electroencephalographic (EEG) and electrooculographic (EOG) electrodes were implanted in the cranium and dorsal to the orbital ridge, respectively. After surgery, the goats received ceftifur sodium (2 mg/kg) daily as an antibiotic for 1 wk.
After 
3 wk, a second surgery was performed to chronically implant MTs into the MRN. An occipital craniotomy was created, and the dura mater was excised to expose the posterior cerebellum and dorsal aspect of the medulla for visualization of the obex. The dorsal surface of the medulla, the obex, and the midline were all used as reference points for stereotaxic coordinates in the dorsoventral, rostrocaudal, and mediolateral planes. The implantation sites were always at the midline into either the raphe obscurus (RO) or raphe pallidus (RP). The MTs were stainless steel (25 and 18 gauge) or polyethylene tubing (PE), depending on the intended purpose. For pH measurements, either a single 70-mm PE-160 or two 70-mm PE-90 tubes were implanted. For studies when pH was not measured, a single 70-mm 18.5-gauge stainless steel MT was implanted. After placement, the MTs were secured with screws in the bone and dental acrylic.
Laboratory personnel monitored the goats continuously for a minimum of 24 h after the MT implantation surgery. A few goats were unable to maintain normal sternal recumbent posture and/or stand for 3-6 h postsurgery, and one animal was unable to stand for 2 days after the implant. However, after full recovery, these animals were studied. Food and water intake was monitored closely in all goats daily after the implantation surgery. Brain edema was minimized with dexamethasone injections (0.4 mg·kg-1·day-1 iv for 2 days, then decreasing by 0.05 mg·kg-1·day-1) three times a day for 1 wk. Infection was minimized with chloramphenicol injections (20 mg/kg iv) for 3 days and daily injections thereafter of ceftifur sodium (2 mg/kg) and gentamyacin (3 mg/kg). Buprenorphine was administered 3-12 h after implantation to minimize pain.
Experimental Procedures and Protocols
Physiological measurements. For all studies, a fitted mask was taped firmly to the snout, and a two-way breathing valve was attached to the mask. The inspired port of the valve was connected to a pneumotachograph and computerized data acquisition system to measure inspiratory flow (
I). The expired port was connected to a spirometer (Tissot) for collection of expired air and analysis of O2 and CO2 concentrations (daytime studies only). A chronically placed catheter in the elevated carotid artery was used to monitor arterial BP and HR and for arterial blood sampling to obtain pH and arterial PO2 and PCO2 values (model 278, Ciba-Corning). EMG recordings from the upper airway muscles and diaphragm were also collected by using the Grass recorder and computer system. Rectal temperature (Tre) of the animal was measured at regular intervals.
Mock cerebrospinal fluid preparation and content. The dialyslate for both in vitro and in vivo FA studies was 124 mM NaCl, 2.0 mM MgCl2, 2.0 CaCl2, and 26 mM NaHCO3 in sterile distilled H2O. Experimental pH and PCO2 levels of the mock cerebrospinal fluid (mCSF) were generated by bubbling CO2 gases [6.4% CO2-21% O2-balance N2 or 10, 25, 50, 80 (balance O2), or 100% CO2] while mixing the mCSF in a heated (39.0°C) tonometer.
MD probes. The CMA (CMA Microdialysis, Solna, Sweden) 11 MD probes (6-kDa molecular mass cut-off) had a 80-mm shaft length, a membrane length of 1 mm, and a membrane diameter of 0.25 mm. The CMA 12 MD probes (20-kDa molecular mass cutoff) had a 70-mm shaft length, a 2-mm membrane length, and a 0.5-mm membrane diameter. CMA 11 probes were used in the initial sets of experiments as others used them in rats (14, 16, 23, 24). pH and/or physiological changes induced by the CMA 11 probes were small. In the later studies, we used the CMA 12 probes to generate greater FA.
Confirmation and quantification of dialysis-induced acidosis in vitro. A 100- or 400-µl Eppendorf tube (reservoir) was filled with mCSF equilibrated with 6.4% CO2 (pH 7.3). The custom-made, metal-metal oxide pH electrode and Ag-AgCl reference electrode (WPI) were passed through two small holes bored in the reservoir cap and then connected to a pH meter (Corning 315 pH/Ion). Two 19-gauge stainless steel tubes were passed through two additional holes in the cap to provide the inflow and outflow of mCSF. All ports in the reservoir cap were sealed, and a baseline millivolt value was obtained. Flow (60 µl/min) was then initiated and maintained for a period of 30-40 min, at which time the CO2/H+ concentration (or flow rate) was altered, and the cycle was repeated several times.
Additionally, acidosis was generated with the CMA 11 or CMA 12 probes in this same environment. The MD probe and pH electrode were inserted into the reservoir (<250 µm apart), and subsequent pH measurements were obtained at multiple levels of mCSF pH and flow rates.
In vivo pH measurements during elevated inspired CO2. To obtain extracellular fluid (ECF) pH measurements in awake, adult goats (n = 10; 27 trials), the pH electrode was inserted through the implanted MTs and 1-2 mm into the brain tissue. A skin reference electrode was placed on a shaven area on the neck, and both the pH and reference electrodes were connected to a pH meter. We monitored I, BP and HR, upper airway and diaphragm EMG activity, and expired gas O2 and CO2 concentrations. After a 30-min control period while breathing room air, the animals were then exposed to three levels of elevated inspired CO2 (inspired fraction of CO2 = 0.025, 0.05, and 0.075 in room air), followed by a 15-min recovery period. Millivolt readings were recorded every minute during this period. Arterial blood samples were drawn during the control period (room air) and over the last 2 min at each level of inspired CO2. On completion of the study, the probe was removed, and an in vitro calibration was performed (see below).
After insertion into the goat, the pH (mV) readings of the electrode drifted upward or downward at a constant rate (see Fig. 1); thus the absolute pH could not be measured. We decided that the drift was acceptable when the measurements were 
0.1 mV/min. With the use of a linear equation derived from a best fit line describing all data points during control and recovery periods, an interpolated baseline was calculated (Fig. 1). In most cases, the calculated baseline fit well with pre- and post-CO2 measurements, suggesting a representative baseline. The change in millivolts due to a change in brain pH was determined by subtracting the experimental values during the 4th and 5th min of each level of CO2 exposure from the expected values from the interpolated equation.
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In vivo MD and pH measurements. Studies began 2 wk after implantation, a guideline previously described (35). Throughout the protocol, all 13 goats were in good health, with stable baseline breathing and arterial PCO2 values. During the first experiments performed in some animals, shivering occurred. These data were excluded from the final analysis of all physiological data. ECF pH was measured at a site distal (1.4 mm away) to the MD probe in two additional animals. Also, in two other animals, ECF pH was measured during MD at night.
ECF pH measurements were made in the RO or RP of awake goats during FA studies (n = 10; 69 trials): 47 trials with the use of the CMA 11 MD probe, and 22 with the use of the CMA 12 MD probe. The MD probe was inserted into the MT, along with the pH electrode, such that the MD probe and pH electrode were 215 ± 45 µm apart. The pH readings were monitored for a period of 0.5-1 h to reach a stable baseline. Millivolt readings were recorded each minute throughout a 30-min control period, throughout dialysis (20-45 min), and for 15-30 min after termination dialysis. An in vitro calibration was performed after the completion of the studies (see below). The change in millivolts observed with in vivo MD was calculated the same as during systemic acidosis.
pH electrode calibration. The pH electrode was calibrated in a heated water bath containing five pH buffer solutions at a temperature of 39.0°C. From millivolt readings for each pH level (pH = 4.0, 6.86, 7.0, 7.38, and 10), a linear calibration curve was calculated to be 40.09 ± 1.39 mV/pH unit.
FA during sleep. To consolidate sleep, the animals were sleep restricted (not allowed to lie down) during the daytime hours before the sleep study (9 PM to 3 AM). At the time of study, the goats were instrumented for physiological measurements, and the MD probes were inserted as described above. The dialysis line was passed through a port in the environmental chamber wall and connected to the infusion pump so as to isolate the animal from both the investigator and equipment. I, BP and HR, Tre, EMG, EEG, and EOG activities were then monitored for a control period of 15-30 min; blood samples were drawn; then the animal was allowed to lie down; and the lights were switched off. MD studies were then performed throughout the 6-h study, where the dialysis periods were between 30 and 45 min in duration, followed by at least 30 min of recovery between dialysis periods. Blood samples were also drawn 5 min before and 15 min after termination of dialysis.
Sleep state was determined by standard EEG and EOG and behavioral criteria (28). The awake state was defined as low-voltage, mixed-frequency EEG with concurrent behavior of head holding, alerting response to random ambient noises and eye blinks. Slow-wave [non-rapid eye movement (NREM)] sleep was defined as a synchronized low-frequency EEG (2 Hz) with an amplitude two to three times greater than that found during wakefulness and concurrent absence of rapid eye movements (REMs). REM sleep was defined as a desynchronization of the EEG with relatively low-voltage, mixed-frequency EEG with frequent REMs in the EOG channels and postural muscle atonia indicated by inability of head holding. REMs were distinguished from eye blinks as they were defined as sharp waves with an amplitude of >30 µV from baseline in the EOG tracing and regularly confirmed visually with twitching movements.
Histological studies. After completion of these protocols, the animals were euthanized (Beuthanasia), and the brain was perfused with PBS solution (pH = 7.35-7.4) and 4% paraformaldehyde fixative in PBS. The medulla was then removed and placed in a 4% paraformaldehyde solution for 24-48 h and then placed in a 30% sucrose solution. The medulla was then frozen and serial sectioned (20-25 µm) in a transverse plane, and the sections adhered to chrom alum-coated slides. The tissue was then stained with hematoxylin and eosin, coverslipped, and examined microscopically. The MT implantation site was identifiable by visualization of an area of absent or disrupted tissue, which extended over a finite rostrocaudal distance (0.9-1.1 mm). The implantation site was defined as being at the tip (ventralmost aspect) and middle of the MT-induced tissue disruption. The identification of which MRN the MT reached (and presumably the FA) was achieved by taking into consideration the calculated rostral distance (in mm) from the obex and distance from the ventral surface of the medulla, as well as the published data from the anatomic atlas of the goat medulla (7).
Data and statistical analyses. We calculated I, mean arterial BP, HR, frequency, I rate [tidal volume-to inspiratory time ratio (VT/TI)], and metabolic rate [O2 uptake (O2)].
Due to the differences observed in the capabilities of the CMA 11 and CMA 12 MD probes in creating acidosis in vitro and in vivo, the data obtained for all MD studies were segregated based on the type of probe and the level of CO2. For each goat, the results of 30-s binned data for the daytime MD studies for all parameters were divided by the control period mean to obtain 30-s bin means as a percentage of the control value. The control, MD, and recovery (%control) data for all goats were then further binned into 5-min averages, giving rise to three 5-min control means (15-min control period), six to nine 5-min MD means (depending on MD duration), and three 5-min recovery means. O2 data were binned to 5-min periods and averaged for each of the control, MD, and recovery periods. Arterial PCO2 and Tre values were averaged for each of the control, MD, and recovery periods. For each parameter, the individual mean values for each time period for each goat were statistically analyzed by using a one-way ANOVA and Bonferroni post hoc analysis.
For nighttime studies, the data were further segregated based on the state of the animal (wakefulness, NREM, or REM sleep). This was achieved by first averaging the breath-by-breath data into 30-s bins or epics for the control and MD periods. The epics were then scored as either awake, NREM, or REM sleep. Then data were sorted into one of four data groups and averaged: awake control, NREM control, awake MD, and NREM MD. Insufficient data were obtained during REM sleep to permit a meaningful statistical analysis. A one-way ANOVA was performed across all four groups at a given CO2 level and for each of the probe types used to test for significance (P < 0.05).
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RESULTS |
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Figure 2 shows the results of postmortem analyses of the MT location, where each symbol represents the ventral-most aspect of an individual MT. All of the implanted MT were in or within 1.5 mm dorsal to either the RO or RP. Insertion of the MD and/or pH electrode extended 1-2 mm beyond (more dorsal) the dorsal-most aspect of the MT, indicating that the pH measurements and FA were within the targeted nuclei. We also noted tissue disruption at or ventral to the implantation site, as indicated by scarring and/or dead neurons by the hematoxylin and eosin-stained tissue (data not shown), likely from the insertion of the tissue pH electrode and/or MD probe.
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Physiological Effects of MD
MD with 6.4% CO2 (control dialysis, Fig. 3) or 50% CO2 (not shown) using the CMA 12 probe had no effect on measured ventilatory parameters during daytime awake studies. However, a sustained and significant increase in I, VT/TI, and HR was observed during the dialysis with 25% CO2 (P < 0.05, Fig. 4) and 80% CO2 (P < 0.05, Fig. 5). Curiously, MD with 6.4% CO2 using the CMA 11 probe had significant effects on I, breathing frequency, VT/TI, and HR (P < 0.05, data not shown). However, with few exceptions, there were no significant effects of MD on variables when dialyzing with 10, 25, 50, or 80% CO2 with the use of the CMA 11 MD probe (P > 0.05).
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During nighttime studies, I did not significantly change throughout periods of wakefulness and NREM sleep during control (pre-MD) or MD periods at all levels of CO2/H+ and for each of the probe types (P 0.665, Figs. 6 and 7).
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pH Measurements
The tissue pH electrode was shown to accurately reflect pH changes generated in an in vitro environment (Fig. 8). The ratio of the change in pH from baseline (7.290) detected by the custom-made pH electrode divided by the measured pH change (blood-gas analyzer) was roughly 1.0 (100%) when averaged over all levels of pH tested. In other words, the custom-made pH electrode measurements (with different infusate pH levels) were, overall, 98.7 ± 8.7% similar to the measured pH with the use of a blood-gas analyzer.
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Both MD probes were capable of creating an acidosis with dialysis in vitro. However, the CMA 12 MD probe generated a greater acidosis at each mCSF CO2 level than the CMA 11 probe (data not shown), likely due to the differences in the physical properties of each probe (described previously). The CMA 11 probes decreased pH by 14% of that in the dialysate in a volume of 100 µl. In contrast, the CMA 12 probes decreased pH by 55% of that in the dialysate.
After 4-5 min at each level of inspired CO2 (P 0.012) (Fig. 9), the decrease in brain pH is roughly 50% of that observed in the arterial blood, which likely reflects changes in cerebral blood flow that partially buffer the full acid load.
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During daytime studies, changes were observed in brain pH in most, but not all, MD experiments. Increasing mCSF CO2/H+ levels generated greater decreases in pH (Fig. 10). These changes were measured, on average, within 215 ± 45 µm of the MD site. In two goats, we were able to measure pH simultaneously within 215 and 1,400 µm of the MD site. We never observed a pH change at the distal site, which indicates that the MD-induced acidosis was indeed focal. In two other goats, we measured brain pH during night studies, and we found no pH change during any MD period while awake or during brief periods of NREM sleep. Despite the large differences in vitro, the in vivo pH data were similar for both probes at lower CO2 levels (6.4, 10, and 25% CO2). However, the CMA 12 MD probe generated greater decreases in ECF pH at higher levels of CO2 (50 and 80% CO2), and these pH changes were more consistently observed with the CMA 12 probe. Additionally, MD with the CMA 12 probe generated a much greater acidosis in vitro than in vivo at a given level of CO2, where the in vivo pH change was 19.1% of the change in vitro. These data highlight differences in the buffering capacities of the in vitro and in vivo (brain) environments.
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We attempted to gain further insight into the local buffering in the raphe by exposing two goats to elevated inspired CO2, FA, and a combination of the two stimuli while simultaneously measuring pH within 200 µm of the CMA 12 MD probe. In both experiments, pH was reduced when breathing 5% inspired CO2 (-0.049 ± 0.015 pH unit) and during MD with 80% CO2 (-0.061 ± 0.01 pH unit; Fig. 11). Brain pH decreased to a greater degree after 20 min of hypercapnia than after 5 min of exposure, suggesting that, over time, the brain is less buffered, or buffering mechanisms may become saturated. When the animal was exposed to 5% inspired CO2 in combination with MD with 80% CO2, the pH decreased by -0.173 ± 0.03, a pH change greater than would be expected (0.11 pH unit) if these effects were simply additive. These data may indicate that, when local buffering mechanisms are saturated with a systemic acid load, CO2 dialysis has a greater effect on lowering tissue pH at the site.
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Physiological Response to FA
FA generated by MD in CCR sites (RTN, MRN) increases ventilation in a state-dependent manner (16, 23). MD with 25% CO2 into the raphe increased minute ventilation by 15-20% during sleep but not wakefulness in rats (23). We report in goats that FA in the RO or RP does not significantly alter ventilation during wakefulness or NREM sleep at night, but it increased ventilation during daytime wakefulness. The magnitude of the effect with MD with 25 and 80% CO2 (CMA 12) on ventilation was relatively small (8.2-12.0%) and less than that observed in rats. The lack of effects during the nighttime studies might be a reflection of a similarly small response that falls within physiological variation. The difference in results may also be a species variation, but there are other alternative explanations. The FA generated in our studies affected primarily caudal medullary raphe regions. In contrast, FA generated in the rat studies by Nattie and Li (23) was primarily in more rostral medullary raphe regions. It is possible that the difference in the state-dependent effects may be related to the different regions tested in each study. Additionally, the effective radius or "sphere of influence" of FA in our studies may not have reached a similar percentage of chemoreceptive neurons. In unanesthetized rats, pH changes with FA in the RTN were limited to a radius of <600 µm (14). We have not systematically determined the effective radius of acidification. However, in two goats, we measured a pH change 215 µm from the MD site, but there was no change in pH at a distance of 1,400 µm from the site. Thus it is unclear whether there was a difference in the sphere of influence between the goat and rat studies.
There also may be differences in local pH buffering mechanisms between the goat and rat, although there are no data to speak to this potential difference. The finding that we found no physiological changes at night may also reflect a difference in local CO2/H+ regulation in the RO or RP during the night. In two goats, we measured ECF pH changes while dialyzing during nighttime wakefulness and NREM sleep and found little or no change in pH at the site. These data may imply that, during the nighttime hours, pH regulation in the raphe is potentially altered from the daytime wakefulness state. In any case, the magnitude of the effects on breathing of FA in the raphe is small but significant in both species, which tends to validate the hypotheses that there are CCRs in the raphe nuclei.
It has been postulated that 5-HT neurons (a subpopulation of MRN) modulate state-dependent changes in cardiovascular function, but they are relatively unaffected by afferent information (2, 13). MRN (more specifically 5-HT) neurons innervate central nervous system sites involved in sympathetic activities and BP regulation, and pharmacological manipulations at these sites implicate 5-HT involvement. Additionally, electrical stimulation of the raphe magnus and pallidus neurons in anesthetized rats selectively reduced arterial blood flow to the tail cutaneous vascular bed, but it did not affect blood flow to the mesenteric arterial vasculature (5). In addition to the observed increases in breathing with FA of the RO and RP, we noted significant increases in HR (11.2-13%) and BP (5.2%). Our results indicate that FA in the MRN is capable of significant alterations in BP and HR, which, to our knowledge, have not been specifically addressed in previous investigations.
Although unclear, the medullary raphe is thought to play a role in thermogenesis (4, 13). By altering sympathetic drive to brown fat tissue (heat production) and/or increasing resistance in peripheral vascular beds important in heat loss, stimulation of raphe neurons can induce increases in body temperature (4). Dialysis of the GABAA agonist muscimol into the rapheparapyramidal region induced 2°C decrease in body temperature in rats (21) and sleeping piglets (18). FA in the raphe of awake rats, however, did not significantly alter O2 or body temperature (23). Our data are consistent with the latter observation, indicating that O2 and Tre were not significantly altered with FA. Additionally, in some goats, shivering and subsequently increases in Tre were observed, but it is unlikely that these data reflect an effect of acidosis per se, as it most often occurred with dialysis with mCSF with a pH of 7.3. Conceivably, dialysate may have cooled in route to the MD probe, initiating a temperature-sensitive thermoregulatory response. However, it seems unlikely that this is the case, as shivering during MD experiments seems to be the exception rather than the rule.
Brain H+ Concentration Regulation
Decades ago, it was established that the brain pH is regulated by independent mechanisms from those regulating arterial blood pH (29). During chronic systemic metabolic acidosis or alkalosis, cerebrospinal fluid (CSF) pH is changed from control by only 10% of the change in the arterial blood pH, where the blood-brain barrier is one of the protective mechanisms acting to limit the pH alterations in the brain (10). During chronic respiratory pH disturbances, CSF pH is not as well protected. For example, during chronic high-altitude exposure, CSF and blood pH are nearly equally changed from control levels and remain significantly alkaline (8, 11). However, in the acute phase of altitude exposure, CSF pH does not increase (0.05) nearly as much as blood pH (0.10) (9, 11). In this case, it is thought that increased brain production of lactic acid is the primary local mechanism for compensation for the acute respiratory alkalosis.
Along with the ventilatory data, a major aspect of the present study is several examples of local brain pH regulation. For a given change in arterial blood pH during acute systemic respiratory acidosis, the pH change in the RO or RP was 49.4% of that observed in the arterial blood, and preliminary results from similar studies at other medullary nuclei indicate that the change in brain ECF pH is 54.8% of the pH change in the arterial blood. Thus these data indicate that the brain is capable of buffering about one-half of the acid load during an acute CO2-induced systemic acidosis.
The pH change created with MD in vivo is 20% of the pH change generated in vitro, which also lends credence to the idea of local buffering in the brain. In addition, the FA created during in vivo MD experiments often exhibited a return toward baseline levels after reaching the peak decrease in pH, which also suggests that there are local mechanisms that actively guard against changes in pH. The combined effect of dialysis with 80% CO2 and 5% inspired CO2 (-0.173) was greater than the sum of each effect individually (-0.061 and -0.049, respectively). It is possible that a greater acidosis occurs at the site of dialysis as a result of partial or full saturation of protective buffering mechanisms by first inducing a systemic respiratory acidosis.
In summary, these observations highlight local H+ concentration regulatory mechanisms in the brain. It seems quite likely that a major contributor to this local pH buffering to a FA could be an increase in local medullary blood flow. Increases in CO2 and/or decreased pH are well known and potent stimuli in increasing cerebral blood flow. We have no data to directly speak to the local pH buffering mechanisms per se.
Conclusions
We conclude that the increase in ventilation elicited by FA at single sites in the medullary raphe clearly indicates that these nuclei are capable of acting as central respiratory CO2/H+ chemoreceptors. Furthermore, we conclude that FA can induce significant changes in cardiovascular variables, which speak to the vast influence that the raphe nuclei have on nonrespiratory physiological control systems. Finally, pH measurements in the raphe of conscious, freely moving animals during focal or systemic acidosis lead us to conclude that powerful local pH buffering mechanisms in the brain guard against acidosis.
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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.
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M. R. Hodges, C. Opansky, B. Qian, S. Davis, J. Bonis, J. Bastasic, T. Leekley, L. G. Pan, and H. V. Forster Transient attenuation of CO2 sensitivity after neurotoxic lesions in the medullary raphe area of awake goats J Appl Physiol, December 1, 2004; 97(6): 2236 - 2247. [Abstract] [Full Text] [PDF]
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M. R. Hodges, P. Martino, S. Davis, C. Opansky, L. G. Pan, and H. V. Forster Effects on breathing of focal acidosis at multiple medullary raphe sites in awake goats J Appl Physiol, December 1, 2004; 97(6): 2303 - 2309. [Abstract] [Full Text] [PDF]
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N. C. Taylor, A. Li, A. Green, H. C. Kinney, and E. E. Nattie Chronic fluoxetine microdialysis into the medullary raphe nuclei of the rat, but not systemic administration, increases the ventilatory response to CO2 J Appl Physiol, November 1, 2004; 97(5): 1763 - 1773. [Abstract] |
) or CMA 12 MD probes (X) were used for focal acidosis studies represent the ventralmost aspect of the microtubules. An arrow (top) and scale (bottom, in mm) are also included for rostrocaudal reference from the obex. Note that all sites of focal acidosis were 1-2 mm ventral to the symbol positions, indicating that all probes were within either the RO or RP.
