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1Department of Physiology, Medical College of Wisconsin, 2Department of Physical Therapy, Marquette University, and 3Zablocki Veterans Affairs Medical Center, Milwaukee, Wisconsin
Submitted 8 August 2005 ; accepted in final form 20 February 2006
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ABSTRACT |
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I) in one group of studies and decreased I in a second group and the difference between the groups was significant (t = 9.16, P < 0.001). In one group of studies (n = 8), FA with 50 and 80% CO2 significantly increased (P < 0.05) I by 16 and 12%, respectively, and significantly increased (P < 0.05) heart rate by 13 and 9%, respectively. In contrast, in another group of studies (n = 6), FA with 25 and 50% CO2 significantly decreased (P < 0.05) I by 7 and 10%, respectively. In this group oxygen consumption was decreased during dialysis with 80% CO2. On the basis of histology, we estimate that the increased and decreased responses were associated with FA primarily in the rCFN and cCFN, respectively. We conclude that there are CO2/H+-sensitive neurons in the CFN that do not uniformly affect breathing. In addition, the significant changes in heart rate and oxygen consumption during FA indicate that the CFN can also influence non-respiratory-related control systems. microdialysis; focal acidosis
I) 24% in the awake state but did not affect I while asleep. In contrast, Nattie and Li (23, 24) found that FA in the medullary raphe increased I by 20% only during sleep, whereas FA in the NTS increased I during sleep by 16% and during wakefulness by 28%. The data of Nattie and colleagues suggest that the ventilatory response to FA at different sites is state dependent and that the contribution of each site to the hyperpnea during global brain acidosis is not uniform (20–24).
Nattie and colleagues (20–24) created a FA with mock cerebral spinal fluid (mCSF) equilibrated with different levels of CO2 delivered to the nucleus being studied using the technique of reverse microdialysis. Using the same technique as Nattie et al., Hodges et al. (12) found 8 and 12% increases in I with single-site FA in the medullary raphe in awake goats, but they found no effect during sleep. In addition, Hodges et al. found an average of 15–25% increase in I during the last 15 min of FA at multiple raphe sites, suggesting a graded effect of FA depending on the volume of tissue stimulated (13). The increase in I during multiple-site stimulation was similar to exposing the animal to 3% inspired CO2. Hodges et al. (13) suggested that the hyperpnea observed during global brain acidosis might be due to the cumulative effect of stimulation at widespread chemoreceptor sites. This view contrasts the specialized chemoreceptor theory, which states that only a small number of intracranial chemoreceptor neurons are highly sensitive and primarily mediate the hyperpnea during global brain acidosis (8, 9, 27, 29).
Several lines of evidence suggest that there are CO2/H+-chemosensitive neurons in the rostral CFN (rCFN). Bilateral lesions of the rCFN in anesthetized rats reduced the mean phrenic activity and frequency of breathing when inspired CO2 was increased to 7% (40). Moreover, in anesthetized, paralyzed, and spontaneously breathing cats, extracellular neuronal recordings in the rCFN showed enhanced neuronal firing that correlated with increases in phrenic nerve firing rate when inspired CO2 was raised to 7% (41). Finally, unilateral injections of 20 nl of 50 µM acetazolamide into the rCFN significantly elevated expired minute ventilation (E) and tidal volume (VT) by 46 and 31.7%, respectively (42). These data led to the present study, whose purpose was to test the hypothesis that in awake goats FA in the rCFN will increase breathing, but that the magnitude of the increase in breathing will be less than the 260–300% increase in ventilation above normocapnic values generated by systemic hypercapnia.
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METHODS |
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Surgical Procedures
Instrumentation surgery. An initial surgery was performed to elevate a 5-cm segment of the carotid arteries. In this and subsequent surgeries, the goats were initially anesthetized with a combination of ketamine and xylazine, intubated, and mechanically ventilated 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. After surgery, the goats received the antibiotic ceftifur sodium (2 mg/kg) daily as an antibiotic for 1 wk.
Microtubule implantation surgery.
After 
3 wk, a second surgery was performed to chronically implant two microtubules (MT) into the CFN (n = 8) or elsewhere in the cerebellum as a control (n = 2). Arterial blood pressure (BP) and rectal temperature were continuously monitored throughout the duration of surgery. An occipital craniotomy was created, and the dura mater was excised to expose the dorsal cerebellum and dorsal aspect of the medulla for visualization of obex. The dorsal surface of the medulla, obex, and the midline were all used as reference points for stereotaxic coordinates in the dorsoventral, rostrocaudal, and mediolateral planes. The MTs used were 18-gauge stainless steel tubes 70 mm in length. The tips of the MTs were placed at 45° from the horizontal plane at the obex and then raised 18–21 mm above the obex and 1–2 mm lateral to the midline. We then advanced the MTs with the use of a micromanipulator until the tip made contact with the dorsal surface of the cerebellum, which served as our zero point for MT advancement into the cerebellum at 45°. An electrical stimulating electrode was inserted through the MTs and extended 2 mm beyond the tip of the MT. All but the last 1 mm of the stimulating electrode was insulated. The electrical stimulation electrode was grounded directly to the MTs or at a site on the neck of the goat. We used a Grass SD9 square-pulse stimulator to deliver a 80-Hz, 5-V, 1-ms pulse width, 200-ms duration, and a 2-ms delay for 30 s (1, 41, 42). The MTs were advanced 10 mm from the dorsal surface of the cerebellum and then we began to electrically stimulate the tissue. We advanced the MT and stimulating electrode in 1- or 2-mm increments until electrical stimulation elicited an increase in BP and heart rate (HR). Other studies have also shown these increases in BP and HR when the rCFN is electrically stimulated (1, 41, 42). At this location the MTs were secured with screws and dental acrylic to the bone.
Laboratory personnel monitored the goats continuously for a minimum of 24 h after the MT implantation surgery. Brain edema was minimized with dexamethasone injections (0.4 mg·kg–1·day–1 iv for 3 days, then decreasing by 0.05 mg·kg–1·day–1, tid) for 1 wk. Infection was minimized with chloramphenicol injections (20 mg/kg iv, tid) for 3 days, and daily injections thereafter of ceftifur sodium (2 mg/kg im, sid), and gentamicin (3 mg/kg im, sid). Buprenorphine was administered 3–12 h after implantation to minimize pain. Goats were recovered for a minimum of 2 wk or until baseline breathing and arterial PCO2 (PaCO2) returned to control levels, before studies commenced.
Physiological Measurements
For all studies, inspiratory flow was measured with a pneumotach by attaching a breathing valve to a custom-fitted mask secured firmly to the goat’s snout. The expired gas was collected in the spirometer to determine volume and concentrations CO2 and O2 needed for metabolic rate calculations. A chronically placed catheter in an elevated carotid artery was used to monitor BP and HR, and for arterial blood sampling to obtain pH, PO2, and PCO2 (model 278, Ciba-Corning). Rectal temperature of the animal was measured at regular intervals.
Assessment of CO2 Sensitivity
Breathing, BP, and HR were measured for 30 min before exposure to three levels of elevated inspired CO2 (2.5, 5.0, and 7.5% CO2 in room air). Arterial blood samples were drawn during the control period and during the 4th and 5th minute of each CO2 exposure level. The change in E and PaCO2 from room air breathing to all levels of CO2 was used to determine the slope of the relationship between E and PaCO2 and used as an index of CO2 sensitivity.
FA Studies
Studies began at least 2 wk after implantation. Throughout the protocol all goats were in good health, with stable baseline breathing and PaCO2 values. The CMA 12 (CMA Microdialysis, Solna, Sweden) microdialysis (MD) probes (20-kDa molecular weight cutoff) had a 70-mm shaft length, a 0.7-mm membrane diameter, and a 3-mm membrane. The dialysis probe was placed down the inner lumen of the microtubule and extended 3 mm beyond the end of the microtubule; thus 3 mm of the probe provided the surface area for diffusional exchange between the tissue and the dialysis probe. Previous experiments in our laboratory established that MD in the raphe nucleus with 50% CO2 and 80% CO2 (50 µl/min) decreased extracellular fluid pH by 0.6 and 0.9, respectively (12). These decreases in pH are 1.5 or 3 times greater than that observed with 7.5% inspired CO2 in room air, respectively (12). This degree of FA increased breathing about 10% of the increase in breathing observed with an equivalent systemically induced global brain acidosis.
Mock Cerebrospinal Fluid Preparation and Content
The dialysate for 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 mCSF were generated by bubbling CO2 gases [6.4% CO2-21% O2-balance N2 or 25, 50, 80 (balance O2)], while mixing the mCSF in a heated (39.0°C) tonometer. I, HR, mean arterial BP, and oxygen consumption (O2) were measured continuously or at 5-min intervals during 15 min of control, 45 min of microdialysis, and for 15 min after terminating the dialysate flow. Arterial blood was drawn during the final 5-min period of the control, microdialysis, and recovery periods. Four different mCSF pH and PCO2 were dialyzed: 1) 6.4% CO2 (pH 
7.32, PCO2 43), 2) 25% CO2 (pH 6.85, PCO2 175), 3) 50% CO2 (pH 6.55, PCO2 > 250), and 4) 80% CO2 (pH 6.35, PCO2 > 250). Often, three to four of these dialysis studies were completed in order of increasing percentages of CO2 on a single day, but on other days only a single dialysis study was completed. Dialysis was performed in one or both microtubules with a flow rate (50 µl/min) identical to that previously described (12).
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 cerebellum was then removed, postfixed in 4% paraformaldehyde solution for 24 h, and cryoprotected in a 30% sucrose solution. The cerebellum was then frozen and serial sectioned (25 µm) in a horizontal or transverse plane, and the sections were 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, 0.9–1 mm along the rostral-caudal axis.
Data and Statistical Analyses
We calculated I, BP, HR, breathing frequency, VT, O2. Periods in which the goat was unstable, for example shivering, were excluded from the final analysis of all physiological data. The calculated breath-by-breath data were binned into 5-min averages for the control, MD, and recovery periods. All bins were divided by the 15-min control mean for normalization (% of control). The mean data were statistically analyzed by using one-way ANOVA for repeated measures to compare each of the 5-min binned data during MD and recovery to the 15-min averaged control value for all of the physiological variables. Two-way ANOVA was used to compare MD with 25, 50, and 80% CO2 against MD with 6.4% CO2 (control level for MD). If the two-way ANOVAs found significance between any of the three levels of FA vs. MD with 6.4% CO2, then a Bonferroni post hoc analysis was used in attempt to isolate the specific time points of significant changes. An unpaired t-test was performed in order compare the peak increases and decreases in I during MD with 50 and 80% CO2. The threshold for significance was set at P < 0.05.
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RESULTS |
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The baseline ventilatory status was unchanged by microtubule implantation into the cerebellum (n = 10). Resting PaCO2 was 40.8 ± 0.75 Torr before implantation and 42.2 ± 0.86 Torr after microtubule implantation. The slope of the systemic CO2 sensitivity (change in E/change in PaCO2) was 2.02 ± 0.24 and 1.93 ± 0.19 before and after implantation, respectively. In addition, the average percent increase in ventilation above baseline during systemic hypercapnic acidosis was not altered by microtubule implantation. For example, the percent increase in ventilation between room air and 7% inspired CO2 was 263.5 ± 21.3 and 301.4 ± 35.5% before and after implantation, respectively.
Location of Implanted MT
Figure 1 depicts the postmortem identification of the microtubule placement. The squares and triangles indicate placement of MD probe tips during FA. The black squares indicate the tip of the dialysis probes (dashed lines) where an increase in I was produced during FA with 50 and 80% CO2, and the black triangles indicate the tips where a decrease in I was produced during FA with 25 and 50% CO2. On the basis of our previous measurements of tissue pH during FA (12), we estimated the area affected by dialysis, which is represented for one goat (asterisk) in Fig. 1 (enclosed area that surrounds the exposed MD probe area < 19 mm2).
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There were no physiological effects of dialysis with 6.4% CO2. However, there were significant changes during the dialysis with higher levels of mCSF PCO2, but the changes differed significantly (t = 9.16, P < 0.001) between the two groups of studies. In one group, dialysis with 50% CO2 increased I and VT both significantly greater than control and greater than during FA with 6.4% CO2 (Fig. 2, Table 1, P < 0.05, n = 8). Dialysis with 80% CO2 also significantly increased I above control (Fig. 2, Table 1, P < 0.05, n = 8), but there was no dose-dependent effect. Finally, in this group, HR during MD with 50 and 80% CO2 was significantly greater than control and greater than the HR during MD with 6.4% CO2 (Table 1, P < 0.05, n = 8). In this group, we estimate that 40 ± 7.0 and 18.7 ± 5.6% of the rCFN and caudal CFN (cCFN), respectively, were acidified during dialysis. There was no correlation between the magnitude of the I response to the estimated area of tissue acidosis in the CFN during dialysis with 50 or 80% CO2 (Table 2).
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I by 7 and 10%, respectively. In this group breathing frequency was significantly decreased 6% (Table 3, P < 0.05, n = 6) during dialysis with 50% CO2, VT was decreased (Table 3, P < 0.05, n = 6) 4–6% during MD at 25, 50, 80% CO2, and O2 was significantly decreased 6% (Table 3, P < 0.05, n = 6) during dialysis with 80% CO2. In this group, we estimate that 47.5 ± 11.0 and 23.2 ± 4.0% of the cCFN and rCFN, respectively, were acidified during dialysis. Again, there was no relationship between the I response to the area of dialysis during FA with 50 or 80% CO2 (Table 4).
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In six of the goats, one microtubule was implanted primarily in the cCFN and one microtubule was implanted primarily in the rCFN. There were no significant changes in most measured variables during simultaneous dialysis in both microtubules (Fig. 4, P > 0.05, n = 6). An exception was dialysis with 25 and 50% CO2, which significantly increased VT (Table 5, P < 0.05, n = 6) and 80% CO2, which significantly decreased breathing frequency (Table 5, P < 0.05, n = 6) relative to MD with 6.4% CO2. In two goats in which both microtubules were placed primarily in the rCFN, one goat exhibited a dose-dependent increase in I when bilaterally dialyzing with 50% CO2, and the other goat did not exhibit a dose-dependent effect.
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In two goats we implanted two microtubules in the cerebellum outside the CFN and performed FA. During these FA studies there were no changes in any measured respiratory or cardiovascular parameters compared with control (Fig. 5).
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DISCUSSION |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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I was increased from control, whereas when the FA was predominantly in the cCFN, I was decreased from control. Additionally, FA in the CFN altered HR and metabolic rate, indicating that the CFN can influence non-respiratory-related control systems. Physiological Responses to FA
Li et al. (15) found in rats that FA in the RTN increased I 24% in the awake state but did not affect I while asleep. In contrast, Nattie and Li (23, 24) found that FA in the medullary raphe increased I by 20% only during sleep, whereas FA in the NTS increased I during sleep by 16% and during wakefulness by 28%. The data of Nattie and colleagues suggest that the ventilatory response to FA at different sites is state dependent and that the contribution of each site to the hyperpnea during global brain acidosis is not uniform (20–24).
In awake goats, FA at a single site in the medullary raphe increased I in a dose-dependent manner by a maximum of 12%, but FA during non-rapid eye movement sleep did not significantly affect I. The dose-dependent effect of FA in the raphe of awake goats was further shown by findings that FA at multiple raphe sites increased I by a maximum of 25%. This effect is relatively small compared with the 260–300% increase during global brain acidosis in awake goats; thus the authors speculated that the response to global brain acidosis reflects the cumulative effect of chemoreceptor activity at widespread brain stem sites.
The unique finding in the present study is that FA in different regions of the CFN had different effects on breathing of awake goats. These findings are presumably due to differences in effects of FA on neuronal discharge rates. For example, data from in vitro preparations show that some raphe neurons increase, whereas other neurons decrease discharge rates with hypercapnic acidosis (29). Similarly, Erlichman et al. (49) used the brain stem slice preparation and observed groups of neurons that depolarize with acidosis that are located within 200–600 µM of groups of neurons that hyperpolarize with acidosis.
In contrast to the effects of FA in the raphe of awake goats, FA in the CFN does not elicit a dose-dependent response. Specifically, the increases rCFN and decreases cCFN in ventilation did not correlate with the level of mCSF PCO2 and pH, nor with the estimated volume of the CFN that was acidified. These data may indicate that CFN chemoreceptor neurons are in some manner fundamentally different from raphe chemoreceptor neurons. On the other hand, these data may simply reflect the apparent heterogeneity of CFN chemoreceptor neurons. Conceivably, neurons stimulated and inhibited by acidosis do not exclusively and uniformly exist in the rCFN and cCFN, but rather they are intermingled to a varying degree and/or each type is located in only a portion of the rCFN and cCFN. Moreover, the acidosis with the dialysis was not restricted to the CFN; thus chemoreceptor neurons outside the rCFN and the cCFN may have contributed to the response. However, this possibility seems unlikely because in two control animals (Fig. 5) in which the FA was clearly outside the CFN there appears to have been no effect on physiological variables. In any event, it is possible that dialysis in the rCFN with 80% CO2 did not have more of an effect on breathing than dialysis with 50% CO2, because in a population of heterogeneous neurons within and outside of the CFN the neurons inhibited by 80% CO2 were affected more than the neurons stimulated by 80% CO2. Similarly, increasing the volume of acidosis (Table 2) in the rCFN may not increase the ventilatory response, because the additional volume may contain primarily neurons inhibited by acidosis. Clearly, it is apparent that chemoreceptor neurons in the CFN are heterogeneous in their response characteristics and/or in their distribution within the nucleus.
Our present findings support the concept that the ventilatory response to systemic or global brain acidosis reflects the cumulative or additive effect of widespread brain chemoreceptor activity. Specifically, in six goats that had one microtubule in the cCFN and one in the rCFN, FA primarily in the rCFN increased I, but FA in the cCFN decreased I, but when we simultaneously created FA primarily in both the rCFN and the cCFN, there was no change in I. In two other goats, both microtubules were placed primarily in the rCFN, and in one of these the increases in I was greater during dialysis at both sites than with FA at a single rCFN site. In other words, the effect of FA at a stimulatory site can be offset by FA at an inhibitory site, and FA at two stimulatory sites can have additive effects.
There is no readily apparent explanation for why chemoreceptor neurons in close proximity to one another would or should have opposite effects on physiological functions. One possibility is that the cCFN and rCFN work together as a CO2 alert system. For example, one site might be important in generating a protective arousal stimulus that alerts an animal to change position to remove itself from the hypercapnic environment. Although our studies were not designed to investigate behavior, informal observations indicated that, during FA studies in the rCFN, the goats often began to turn their heads as if they were looking for something. They also often stomped their hooves and moved forward and backward as if they were trying to move away from the source of the FA. Similar behavioral changes were observed by Dormer and colleagues (1), who reported that when the voltage during electrical stimulation of the rCFN in awake dogs was increased, the dogs went from looking around, to moving around, and then at higher voltages becoming aggressive. In contrast, the goats often appeared relaxed during FA in the cCFN. To temper the more excitatory behavioral changes observed during FA in the rCFN, it is necessary for the cCFN to relax an animal. This tempering effect would allow an animal to be stimulated to change its location and move away from the source of the hypercapnia if possible, without becoming too excited and disoriented.
The findings that the CFN can influence HR, BP, and O2 may provide additional insight as to why there are widespread sites of CO2/H+ sensitivity. Hodges et al. (13) previously found that FA in the medullary raphe significantly increased BP, HR, and O2. Studies by Erlichman et al. (4a), in which they lesioned the LC, suggest that, in addition to being chemosensitive, this region also plays a role in arousal and descending pain control. Richerson et al. (30) suggested that pH sensitive serotonergic neurons in the midbrain raphe are also are involved in arousal and anxiety. Indeed, Hendricks et al. (10) demonstrated that mice lacking the Pet-1 ETS gene showed defective development of the 5-HT system, and this defect was associated with heightened anxiety and aggressive behavior in adults.
Others have shown that the CFN can modulate HR and BP (1, 2, 41, 42). In all studies at sites that electrical stimulation elicited an increase in ventilation there was a corresponding increase in HR and BP. The respiratory and cardiovascular changes occur together when the CFN is electrically stimulated. Only 
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-adrenergic receptor blockers injected intravenously before electrical stimulation have been shown to eliminate the changes in BP and HR and not affect the increases observed with ventilation (40). It is consistent with previous findings that we observed significant increases in HR with FA in the rCFN when microdialyzing with 50 and 80% CO2 that do correlate to a significant increase in I. FA primarily in the cCFN caused significant decreases ventilatory variables without significantly affecting cardiovascular variables. This might suggest that there are several cell types within the CFN with different receptor systems. It seems that perturbations in the CFN can influence either respiratory or cardiovascular variables or both.
We conclude that the changes in I elicited by unilateral FA of the CFN indicate that there are CO2/H+-sensitive neurons in the CFN of awake goats, but the effects on I of FA in the CFN are not uniform. Increases in I were observed when the FA was primarily in the rCFN and decreases in I were observed where the FA was primarily in the cCFN. The relatively small changes in I during FA in the rCFN and cCFN, relative to systemic hypercapnia, suggest that the CFN is not a dominant determinant of systemic CO2 sensitivity. Finally, these data provide further support for the concept that CO2/H+ chemoreceptors located at widespread sites in the brain do not all serve the same function.
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FOOTNOTES |
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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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, Tip of the dialysis probes (dashed lines) where an increase in 
and *, tips where a decrease in 
and 
each represent a different animal. The dashed line indicates the period of dialysis with 6.4, 25, 50, and 80% CO2. Values are means for unilateral MD with 6.4 (n = 2), 25 (n = 2), 50 (n = 2), and 80% (n = 1) CO2 in 2 areas outside the CFN in the cerebellum for each goat expressed as a percentage of control.