| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
DEVELOPMENT AND TISSUE PLASTICITY
1Department of Physiology, Dartmouth Medical School, Lebanon, New Hampshire 03756-0001; 2Biology Department, University of Texas-San Antonio, San Antonio, Texas 78249
Submitted 9 May 2003 ; accepted in final form 4 August 2003
 |
ABSTRACT |
|---|
 TOP ABSTRACT METHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
|---|
isolated brain stem preparation; central carbon dioxide chemosensitivity; kainic acid; metamorphosis; amphibian; hypercapnia
The distribution of the central CO2 chemosensitive sites has been investigated less extensively in nonmammalian species, and rarely has the ontogeny of central CO2 chemosensitivity been investigated in any species. Such an investigation is virtually impossible in mammals since most of ontogeny is experimentally inaccessible during early fetal life. The bullfrog is, therefore, particularly appropriate for investigations of the distribution and ontogeny of central CO2 chemosensitivity because isolated brain stem preparations from the bullfrog are reliably robust and chemosensitive, and because bullfrog developmental stages are all free living and easily accessible. Torgerson et al. (43) conducted a series of transection studies in which the medulla of the tadpole was divided along the rostrocaudal axis. As a result of these studies, they concluded that the CO2 chemosensory sites migrated from a caudal site in early-stage tadpoles to a more rostral site in late-stage tadpoles and frogs. However, the location of chemosensory sites within the transactions was not further defined. A subsequent study using hypercapnic microinjections to identify chemosensory sites placed the central chemosensors in the ventral two-thirds of the medulla but did not define the rostrocaudal location of the chemosensory sites with any greater accuracy (40).
We hypothesized that caudal and rostral sites of CO2 chemosensitivity would exist on the ventral surface of the medulla of the tadpole at sites indicated by the transection studies. We were intrigued by the possibility that the rostral and caudal sites corresponded to similar superficial rostral and caudal sites in the ventral medulla of mammals (33), a finding that would imply a phylogenetically ancient function for these ventral chemosensory sites. We used focal perfusion of CO2 at discrete areas of the ventral medulla in this investigation to locate sites of chemosensitivity in the brain stem of bullfrogs over the entire range of developmental stages. Whereas focal CO2 perfusion assessed discrete sites in terms of their sufficiency in evoking the hypercapnic response, we used focal disruption to assess the necessity of each site. We expected to find similarities to the mammalian system; stimulation of a discrete site would be sufficient to evoke a partial ventilatory response to CO2, but not sufficient to evoke the full ventilatory response, and that each site's functionality would be necessary for the expression of ventilatory responses from the system at large.
|
METHODS |
|---|
|
TOPABSTRACTMETHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
|---|
Surgical preparation. Each animal was anesthetized by immersion in a cold 0.2 mM solution of tricaine methanesulfonate (MS222; Sigma, St. Louis, MO) in dechlorinated water buffered with NaHCO3 to pH 7.4. Under a dissecting microscope, the dorsal cranium was removed, the forebrain rostral to the optic lobes was resected, and the fourth ventricle was exposed by removing the choroid plexus. The brain stem and spinal cord were removed en bloc from the cranium and spinal canal, the dura mater was stripped away, and the brain was transected rostral to the optic tectum and caudal to the brachial nerve. During dissection, exposed tissues were superfused with cold artificial cerebrospinal fluid (aCSF) composed of (in mM) 104 NaCl, 4 KCl, 1.4 MgCl2, 10 D-glucose, 25 NaHCO3, and 2.4 CaCl2 equilibrated with 100% O2. The aCSF HCO3- concentration is similar to that of late-stage tadpole and frog plasma, but higher than the HCO3- concentration in early-stage tadpole plasma. This concentration has been used in our previous tadpole studies, and to ensure comparability between experiments on animals of different stages, the aCSF composition was kept consistent in all experiments.
The isolated brain stem was transferred to a low volume (0.5 ml) flow-through recording chamber (41, 42). Each isolated brain stem was supported ventral side up between coarse nylon mesh such that all surfaces were bathed with aCSF flowing from rostral to caudal regions at a rate of 5 ml/min. A supply of aCSF, equilibrated with O2-CO2 mixtures to produce the desired pH, flowed through glass tubing to the chamber. The pH of the aCSF was maintained at either pH 7.8 (normocapnia) or pH 7.4 (hypercapnia) by adjusting the fractional concentrations of O2 and CO2 in the equilibration gas. CO2 was monitored with a CO2 analyzer (Capstar-100, CWE, Ardmore, PA). After dissection, the brain stem was allowed to stabilize while superfused with aCSF at 22°C, pH 7.8, and 
9 Torr PCO2 for 1 h.
Nerve recording. Roots of cranial nerve (CN) 7 and spinal nerve (SN) 2 were drawn into glass suction electrodes pulled from 1-mm-diameter capillary glass-to-tip diameters of 30-60 µm. Whole nerve discharge was amplified (x100 by DAM-50 amplifier, World Precision Instruments, Sarasota, FL; x1,000 by model 1700 A-M Systems, Carlsborg, WA), filtered (1 Hz low pass to 500 Hz high pass) and integrated (full-wave rectified and averaged over 50 ms; MA-821/RSP, CWE, Ardmore, PA). Both amplified raw and integrated signals were digitized at 200 Hz/channel, and these neurograms were archived as computer files (Datapac, RUN Technologies, Mission Viejo, CA) for subsequent analysis.
Focal perfusion. A stainless steel Babcock spinal needle with a 40-µm-diameter tip (Popper & Sons, New York) was used to construct the focal-perfusion pipette (13). A 20-ml glass syringe, held in a syringe pump (Orion Research, Boston, MA), was connected to the spinal needle by polyethylene tubing. The sleeve of the spinal needle was connected to the laboratory vacuum line. The syringe pump was set to dispense 0.25 ml/min. The dispensed volume plus some aCSF from the recording chamber was immediately removed by vacuum up the sleeve. Thus only a small area on the brain stem was exposed to the perfusion solution. To determine the area perfused by the pipette, aCSF containing trypan blue (1 mg/ml; Sigma) was applied with the pipette, and immediately after this perfusion, the brain stems were viewed with a dissecting microscope, and the stained areas were measured by using a graduated reticule. These measurements indicated that focal perfusion affects an area 500 ± 35 µm in diameter; this represents 25-35% of the width of the isolated brain stem, depending on the age and size of the animal.
Before focal perfusion in each isolated brain stem was studied, a baseline response to normocapnia was first established by superfusing with aCSF at pH 7.8 (equilibrated with 1.3% CO2-98.7% O2) for 20 min. We increased the CO2 of the aCSF superfusate to a hypercapnic level (equilibrated with 4.7% CO2-95.3% O2; pH 7.4) for 20 min to confirm that the preparation was able to generate a hypercapnic response. After the baseline fictive lung frequency was reestablished by superfusing with normocapnic aCSF for 20-40 min, focal perfusions were carried out. Brain stems from 46 tadpoles and frogs received focal perfusions of hypercapnic aCSF (the same as used for bath application of hypercapnia) applied for 20 min to the ventral medullary surface adjacent to CN 5, CN 8, CN 10, or SN 2. Using brain stems from four late-stage tadpoles, we focally perfused normocapnic aCSF (the same as used for bath application of normocapnia) for 20 min to the ventral medullary surface adjacent to CN 5 and CN 10. This focal normocapnic perfusion was carried out while the remainder of the brain stem was bathed by hypercapnic aCSF. In all unilateral focal perfusion studies, we varied the side of brain stem (left vs. right) that was perfused.
In addition to applying different levels of CO2, the focal perfusion pipette was used to create lesions within identified chemosensitive sites in the ventral medulla. Lesions were created by either a 13-min focal perfusion of aCSF containing 1 mg/ml protease (Sigma) or a 10-min focal perfusion of aCSF containing 1 mM kainic acid (Sigma). After a lesion was created, the baseline fictive lung frequency was reestablished by superfusing with normocapnic aCSF for 40-60 min, and the capacity for a hypercapnic response was tested. First, hypercapnic aCSF was focally perfused at the lesion site for 20 min to confirm that chemosensitivity at that site had been eliminated. Then we applied hypercapnic aCSF in the bath for 20 min to test the effect of the lesion on the brain stem's overall hypercapnic response. Isolated brain stems from 16 bullfrogs of all developmental groups received a protease lesion adjacent to CN 5; 18 received a protease lesion adjacent to CN 10; 10 received a kainic acid lesion adjacent to CN 5; and 17 received kainic acid lesion adjacent to CN 10.
Distribution of cells affected protease or kainic acid lesion. The fluorescent, nucleic acid stain DEAD red (Molecular Probes, Eugene, OR) was used to determine the location of cells whose function was impaired by the focal perfusion of either protease or kainic acid. DEAD red is a cell-impermeant dye that can only diffuse across leaky or otherwise compromised cell membranes (18). Brain stems were completely immersed in 300 µl of aCSF containing DEAD red (1:50 dilution) for 15 min, and the brain stems were frozen in OCT compound (Fischer, Pittsburgh, PA) and sliced in a cryostat into 20-µm-thick coronal sections. Tissue sections were mounted on glass slides and photographed by using an epifluorescence microscope (BH2 Olympus, Melville, NY) equipped with a 480- to 550-nm excitation and 590-nm emission filter set and a charge-coupled device camera (Optronics, Goleta, CA). Images were saved on a personal computer and analyzed by using ImagePro software (Media Cybernetics, Silver Springs, MD).
Data analyses and statistics. Burst activity patterns in the neurograms were designated as either buccal or lung on the basis of the amplitude of the integrated nerve activity and the presence or absence of coincident firing in both CN 7 and SN 2, as previously described (17, 42). Buccal bursts had lower amplitude in CN 7 than lung bursts and no coincident firing in SN 2, whereas lung bursts were of greater amplitude in CN 7 with coincident firing in SN 2. Our laboratory previously demonstrated (39) that changes in the frequency of fictive lung ventilation are the primary manifestation of the CO2 response. The amplitude of fictive lung ventilation and the pattern of gill ventilation are unresponsive to hypercapnia. Thus only the frequency of fictive lung ventilation was quantified by counting the number of lung bursts in 2 consecutive minutes of each treatment condition.
Frequencies of fictive lung ventilation were analyzed by using a repeated-measures ANOVA in which metamorphic group (4 levels) was a continuous between-subject factor and CO2 level (room air vs. hypercapnia) was a within-subject factor (GLM procedure, Systat 9.0, Chicago, IL). When ventilatory responses to focal perfusion of CO2 were analyzed within a developmental group, site of CO2 application was a between-subject factor and CO2 level (normocapnia vs. hypercapnia) was a within-subject factor. When ventilatory responses to lesions were compared, CO2 was analyzed within a chemosensitive site, lesion type (protease vs. kainic acid) and developmental group were between-subject factors, and CO2 level (normocapnia vs. hypercapnia) was a within-subject factor. When an ANOVA indicated that significant differences existed between the treatment groups, multiple preplanned comparisons were made by using t-values adjusted by the Bonferroni method. Values reported in the text represent means ± SD.
|
RESULTS |
|---|
|
TOPABSTRACTMETHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
|---|
|
Lung frequency responses to bath-applied CO2. Mean frequencies of fictive lung ventilation are presented in Fig. 2 as a function of developmental group and level of CO2 (normocapnia or hypercapnia). During normocapnia, there is a linear increase in lung burst frequency with increased development (P < 0.001, r2 = 0.50; higher order curve fits were not significant). Under normocapnic conditions, lung burst frequency increased an average of 4.22 bursts/min between successive developmental groups from 0.58 ± 0.13 burst/min in early tadpoles to 11.66 ± 1.86 bursts/min in frogs. Under hypercapnic conditions, lung burst frequency increased an average of 6.77 bursts/min between successive developmental groups from 3.27 ± 0.42 burst/min in early tadpoles to 20.47 ± 3.29 bursts/min in frogs, and again the increase in lung burst frequency was significant (P < 0.001, r2 = 0.43). Moreover, the changes in lung burst frequency between normocapnic and hypercapnic treatments within each metamorphic group were also significant (P < 0.001 for all groups), but the increase in lung burst frequency was significantly greater in the older metamorphic groups (the slope of the hypercapnic line was significantly greater than the slope of the normocapnic response; P < 0.001). Thus development and CO2 each caused an increase in lung burst frequency, and bullfrogs at each metamorphic stage exhibited a hypercapnic response. Although the absolute size of the hypercapnic response increased as tadpoles aged, the relative change in frequency was remarkably similar across stages; at each stage, hypercapnia caused a 2- to 2.5-fold increase in lung burst frequency.
|
Responses to focal perfusion of CO2 at CN 8 or SN 2. Mean frequencies of fictive lung ventilation are presented in Fig. 3 as a function of developmental group, CO2 level, and CO2 focal perfusion site. Values for bath normocapnia and hypercapnia are taken from Fig. 2 and repeated in this figure for the sake of comparison. For each developmental stage, lung burst frequency during focal perfusion of CO2 adjacent to either CN 8 or SN 2 was not different from the lung burst frequency measured during normocapnia (P = 0.10, 0.62, 0.09, and 0.71 for early-, middle-, and late-stage tadpoles, and frogs, respectively). Thus the ventral medullary sites adjacent to CN 8 and SN 2 are not chemosensitive in the bullfrog brain stem.
|
Responses to focal perfusion of CO2 at CN 5 or CN 10. Mean frequencies of fictive lung ventilation are presented in Fig. 4 as a function of developmental group, CO2 level, and CO2 focal perfusion site. Values for bath normocapnia and hypercapnia are the same as in Fig. 2 and are repeated in this figure for the sake of comparison. Applying CO2 to the ventral medullary surface adjacent to either CN 5 or CN 10 elicited a hypercapnic response in all but the late-stage tadpoles. In early- and middle-stage tadpoles and frogs, lung burst frequency increased above the normocapnia values when CO2 was perfused adjacent to CN 5 (frequency increased from 0.84 ± 0.33 to 5.30 ± 1.98 bursts/min for early-stage tadpoles, P < 0.001; from 0.76 ± 0.33 to 4.57 ± 2.47 bursts/min for middle-stage tadpoles, P < 0.001; from 8.58 ± 2.46 to 19.75 ± 6.83 bursts/min for frogs, P < 0.001). Similarly, when CO2 was perfused adjacent to CN 10 in early- and middle-stage tadpoles and frogs, lung burst frequency increased over normocapnic values (frequency increased from 0.42 ± 0.26 to 4.75 ± 0.73 bursts/min for early-stage tadpoles, P < 0.001; from 0.85 ± 0.17 to 3.50 ± 0.81 bursts/min for middle-stage tadpoles, P < 0.001; from 10.67 ± 2.40 to 17.83 ± 4.64 bursts/min for frogs, P < 0.001). During hypercapnic stimulation of the ventral medulla adjacent to CN 5 and CN 10, brain stems from late-stage tadpoles exhibited lung burst frequencies of 6.90 ± 3.23 and 8.40 ± 3.68, respectively. These frequencies were not different from those measured during normocapnia (P = 0.067). Thus the ventral medullary surface adjacent to CN 5 and CN 10 contains central chemosensitive sites in bullfrogs of most developmental stages, the exception being late-stage tadpoles, and each CO2-sensitive site is capable, after only unilateral stimulation, of recapitulating the entire hypercapnic response to equivalent bath-applied stimulation.
|
Responses to focal normocapnia at CN 5 and CN 10 during bath application of CO2. Mean frequencies of fictive lung ventilation are presented in Fig. 5 as a function of CO2 level and application of a normocapnic stimulus to the chemosensitive sites adjacent to CN 5 and CN 10. These experiments were performed only on late-stage tadpoles because they were meant to determine why brain stems from this developmental group exhibited a hypercapnic response to bath-applied CO2 but not to focal perfusion of CO2 at any one site on the brain stem. Lung burst frequency during bath application of CO2 (28.50 ± 4.60 bursts/min) was higher (P = 0.002) than that during normocapnia (14.33 ± 4.83 bursts/min) or during coincident bath application of CO2 and focal perfusion of normocapnic aCSF to either the CN 5 chemosensitive site (14.67 ± 5.81 bursts/min) or the CN 10 chemosensitive site (14.50 ± 5.07 bursts/min). Thus the sites adjacent to CN 5 and CN 10 are chemosensitive, but the hypercapnic response of isolated brain stems from late-stage tadpoles requires simultaneous bilateral stimulation of both of these chemosensitive sites.
|
Responses to CO2 after unilateral rostral lesions (CN 5). Mean frequencies of fictive lung ventilation are presented in Fig. 6 as a function of developmental group, CO2 level, and unilateral damage caused by either protease (Fig. 6A) or kainic acid (Fig. 6B) applied within the chemosensitive site adjacent to CN 5 in the ventral medulla. There was a developmental increase in lung burst frequency regardless of CO2 level or lesion treatment (P < 0.001), and this increase was consistent with the previously demonstrated developmental increase in lung frequency (see Fig. 2). The ability of the isolated brain stem to generate a hypercapnic response was influenced by the type of lesion created (there was a significant interaction between the effect of CO2 and lesion type; P = 0.003). Figure 6A shows the frequency of lung bursts under normocapnic control conditions and hypercapnic conditions after the protease lesion was created. Irrespective of the developmental group, bath application of hypercapnia elicited an increase in lung burst frequency despite the protease lesion in the rostral chemosensitive site. Although protease lesions in the rostral site did not affect the overall hypercapnic response of the brain stem, they did eliminate the chemosensitivity of that site. Focal application of hypercapnic aCSF to the rostral chemosensitive site after a protease lesion had been made elicited no increase in the frequency of fictive lung ventilation in any animal (data not shown). Figure 6B shows the frequency of lung bursts under normocapnic control conditions and hypercapnic conditions after a lesion was created with kainic acid applied to the ventral medullary surface adjacent to CN 5. Here, both focal and bath application of hypercapnia failed to elicit a change in lung burst frequency after lesion placement at the CN 5 site. Thus, although the ability of the isolated brain stem to respond to hypercapnia was not compromised by the protease lesion in the rostral chemosensitive site, the hypercapnic response could no longer be elicited when a lesion was made at the same site with kainic acid.
|
Responses to CO2 after caudal unilateral lesions (CN 10). Mean frequencies of fictive lung ventilation are presented in Fig. 7 as a function of developmental group, CO2 level, and unilateral damage caused by either protease (Fig. 7A) or kainic acid (Fig. 7B) applied within the chemosensitive site adjacent to CN 10 on the ventral medulla. There was a developmental increase in lung burst frequency regardless of CO2 level or lesion type (P < 0.001), and this increase was consistent with the previously demonstrated developmental increase in lung frequency (see Fig. 2). The ability of the isolated brain stem to generate a hypercapnic response was eliminated by the creation of either type of lesion within the caudal chemosensitive site. There was no main effect of CO2 level (P = 0.234) and no interaction between either CO2 level and lesion type (P = 0.722) or CO2 and developmental group (P = 0.111). Thus the response to focal and bath-applied hypercapnia was effectively abolished in all metamorphic groups by both lesion types at the CN 10 site. Figure 7A shows the frequency of lung bursts under normocapnic control conditions and hypercapnic conditions after protease was applied to the ventral medullary surface adjacent to CN 10. Hypercapnia failed to elicit an increase in lung burst frequency under these conditions. Figure 7B shows the frequency of lung bursts under normocapnic control conditions and hypercapnic conditions after a lesion was created with kainic acid applied to the ventral medullary surface adjacent to CN 10. Here again, hypercapnia failed to elicit an increase in lung burst frequency under these conditions in any of the developmental groups. Thus either type of lesion in the caudal chemosensitive site eliminated the hypercapnic response, suggesting that the caudal site is necessary for expression of the hypercapnic response.
|
Normocapnic lung frequency before and after unilateral lesion in a chemosensitive site. Mean frequencies of fictive lung ventilation during normocapnia are presented in Fig. 8 as a function of lesions that disrupted overall hypercapnic sensitivity or disrupted hypercapnic sensitivity only at the lesion site. Focal perfusion of kainic acid to either chemosensitive site or protease to the caudal site had similar effects. These lesions, which disrupted the brain stem's overall chemosensitivity, also caused a decrease in lung burst frequency during normocapnia (P = 0.009). In contrast, focal perfusion of protease to the rostral chemosensitive site disrupted the chemosensitivity of that site when stimulated by focal hypercapnic but did not disrupt the response to bath-applied hypercapnic stimulation and had no effect on the frequency of lung bursts during normocapnia (P = 0.618)
|
Cell destruction after protease and kainic acid treatment. The locations of cells destroyed by either protease or kainic acid lesions in the ventral medulla are illustrated in Fig. 9 by photomicrographs of DEAD red staining in representative slices from the brain stems of three different late-stage tadpoles. Figure 9, A-C, compares the penetration of protease and kainic acid into the ventral medulla. Untreated brain stems (Fig. 9A) exhibited sporadic staining throughout, but the densest staining was at the outermost edges of the medulla. Brain stems treated with focal perfusion of protease (Fig. 9B) showed an intense area of staining at the site of perfusion and extending an average of 50 ± 20 µm below the ventral surface of the medulla. Brain stems treated with focal perfusion of kainic acid (Fig. 9C) showed an even more intense staining at the site of perfusion, which extended an average of 100 ± 20 µm below the ventral surface of the medulla. Figure 9, E-L, illustrates the distribution throughout the medulla of cells affected by protease and kainic acid lesions within the chemosensitive sites. Kainic acid was focally perfused in the ventral medulla adjacent to CN 5 (Fig. 9D), and the resulting cell death is reflected by intense DEAD red staining in the slice containing that site. Intense staining is seen only at the site of perfusion (Fig. 9F) and not on the contralateral side adjacent to CN 5 (Fig. 9E). Lesions within one chemosensitive site did not destroy cells in the other chemosensitive site, as seen in the whole slice at the level of CN 10 (Fig. 9I) and the higher magnification of the contralateral side of that slice (Fig. 9J). Nor did the lesions destroy cells in any other area of the medulla, as seen in the whole slices at the level of CN 8 (Fig. 9G) and SN 2 (Fig. 9K) and the higher magnification of the contralateral (Fig. 9H) and ipsolateral (Fig. 9L) sides of the two slices, respectively. DEAD red staining, and therefore cell death, as a result of either protease or kainic acid lesion was restricted to the perfusion site and the ventral medulla 50-100 µm below the perfusion site.
|
|
DISCUSSION |
|---|
|
TOPABSTRACTMETHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
|---|
Hypercapnic responses over the course of development. The ontogeny of the neuroventilatory response to hypercapnia, evident as an increase in fictive ventilation, has been investigated previously in bullfrogs, but conflicting results were found as to whether a hypercapnic response was evident as an increase in fictive lung ventilation in the earliest stages of metamorphosis (39) or evident only in the latter half of metamorphosis (42). Once again, we found that a hypercapnic response was present from the earliest stages of bullfrog development. Furthermore, the strength of this response steadily increases over the course of development, and a constant level of hypercapnic stimulation elicits a greater increase in the frequency of fictive lung ventilation, although there is a relatively consistent 2- to 2.5-fold increase in lung frequency in each metamorphic group. There are two possible ways to interpret the interactions between chemosensors and the lung central pattern generator (CPG). As a first approximation, the interaction may be additive or multiplicative. If the hypercapnic stimulus adds to the ventilatory activity already present, then the increase in lung bursts during hypercapnia as metamorphosis progresses is evidence of a developmental increase in the neuroventilatory response to CO2. On the other hand, if CO2 multiplies the basic lung CPG output [and that is consistent with other aspects of the hypercapnic response in tadpoles (see below)], then there is no increase in responsiveness to CO2 during metamorphosis since the relative change in lung frequency during hypercapnia remained constant over the course of metamorphosis. However, there was a metamorphic change in the activity of the lung CPG since the normocapnic lung frequency increased during metamorphosis. Regardless of the nature of the interaction between chemosensors and the lung CPG or the neuronal mechanism of this ontogenetic change, it is clear that central CO2 chemoreceptors are present in the brain stem and influence the lung rhythm generator from the earliest stages of bullfrog metamorphosis.
Ventral medullary sites of CO2 chemosensitivity. In mammals, the specific sites that contribute to central chemosensitivity have been investigated extensively. Among the possible "proofs" of chemosensitivity, the most unequivocal evidence that an area of the brain stem is involved in respiratory chemosensitivity is that discrete application of CO2 and/or H+ in the particular area leads to an increase in ventilation or neuroventilatory activity (3, 9, 31, 37). Reported here is similar evidence that there are two sites of CO2 chemosensitivity on the ventral surface of the bullfrog medulla, a rostral one adjacent to the roots of CN 5 and a caudal one adjacent to the roots of CN 10. Chemosensitivity is not a general property of the ventral medullary surface in bullfrogs, as shown by the failure of similar hypercapnic stimulation at other ventral medullary sites adjacent to CN 8 and SN 2 to stimulate ventilatory activity (Fig. 3), but is a property specific to these two locations. Thus, like mammals, the bullfrog ventral medulla has identifiable sites of chemosensitivity, but the locations of these sites do not correspond to the rostral (28), intermediate (35), and caudal (23) areas of the ventral medulla identified in mammals.
Focal stimulation within either site of central chemosensitivity in the bullfrog elicits a hypercapnic response that is statistically indistinguishable from the response to equivalent CO2 stimulation of the entire isolated brain stem. In mammals, discrete stimulation at any one of the central chemosensitive sites elicits a significant increase in ventilation that is 15-40% of the increase seen when the body's entire complement of chemosensitive areas (central and peripheral) are stimulated simultaneously by an increase in arterial PCO2 in intact animals. Thus it appears that the various sites of mammalian central chemosensitivity are each capable of contributing partially to the hypercapnic response. The capacity to initiate only a portion of the total ventilatory response in mammals likely reflects three issues. There is a complicated algebraic relationship among the central CO2 chemoreceptors in intact unanesthetized animals. The peripheral chemoreceptors contribute to the ventilatory response to CO2 in intact animals, and other respiratory and nonrespiratory inputs provide tonic excitation to the respiratory centers and enhance the ventilatory response for any level of chemical drive. The details of interactions among central CO2 chemoreceptors depend on the animal's arousal state and the presence or absence of anesthesia, but in no case is the relationship mathematically straightforward (simple addition among sites or multiplication). In respect to peripheral chemoreceptors in mammals, the peripheral chemoreceptors contribute up to 50% of the ventilatory response to transitory changes in PCO2 and/or pH (25). Thus peripheral chemoreceptor input may add to the stimulatory influences of central chemoreceptors and/or augment the sensitivity of these receptors. Because the present study used isolated brain stem preparations in the tadpole, the response of ventral medullary chemosensitive sites to focal stimulation was never compared with a whole body hypercapnic response, i.e., one measured when both peripheral and central chemoreceptors were stimulated. The response of the isolated brain stem to bath application of hypercapnic aCSF is likely to represent only part of the response of the intact whole body hypercapnic response (27). Nonetheless, the central chemosensory sites in most bullfrog developmental stages have a clearer pattern of interdependence of function than in mammals since each ventral medullary site of chemosensitivity is capable of eliciting an equal hypercapnic response when it alone is stimulated, but no response can be elicited if the neurons in either region are destroyed.
Location of chemosensory sites within the ventral medulla. In addition to confirming that neuronal output from the two ventral medullary chemosensitive sites is necessary for both the hypercapnic ventilatory response and baseline ventilation, the kainic acid lesion studies provide information about the location of the chemosensory neurons associated with these sites. The kainic acid lesions indicate that the chemosensory neurons associated with each site all lay within 100 µm of the surface where the kainic acid was applied. Staining of neurons destroyed by kainic acid was limited to this area, which precludes the possibility that chemosensitivity of the sites was conferred by projections to these areas from neurons located elsewhere in the brain stem. The protease lesions provide further information about the location of the chemosensory neurons associated with these sites, as well as the CO2 sensors associated with these neurons. Focal perfusion of protease destroyed cells within 50 µm of the surface where it was applied. Because protease lesions at the caudal chemosensitive site eliminated the overall hypercapnic ventilatory response, the chemosensory neurons associated with this site are likely located within 50 µm of the ventral medullary surface adjacent to the roots of CN 10. At the rostral site, focal perfusion of protease destroyed the chemosensitivity of that site alone without affecting the overall hypercapnic ventilatory response or baseline ventilation. Protease treatment seemed to destroy the chemosensitivity of neurons at this site without destroying the neurons themselves. A possible explanation is that the neurons lie 50 to 100 µm below the ventral surface, but they possess CO2-sensing elements that project toward the surface and lie within 50 µm of it. Chemosensitive neurons with such projections have been identified in the neonatal rat brain stem (22). According to our studies, these CO2-sensing components consist of protein moieties for their CO2 sensitivity because protease destroyed that sensitivity. However, destruction of these protein moieties does not breach the integrity of the cell membrane because protease treatment did not allow the DEAD red stain to penetrate the cell. Thus, at both sites, CO2 sensors lie within 50 µm of the ventral surface. In the caudal site, these sensors are located close to the cell body, whereas in the case of the rostral site these protein-bearing sensors appear to be located on projections up to 100 µm away from the cell body. A similar superficial ventral location has been implicated in mammals (33).
Taken together, the kainic acid and protease studies indicate that neurons whose individual output is essential to both the hypercapnic ventilatory response and baseline ventilation are located on both sides of the ventral medulla in the outermost 100 µm near the roots of CN 5 and 10. The fact that unilateral lesions had such a large effect on chemosensitivity was somewhat surprising. We surmise that output from chemosensory neurons on both sides of the site is necessary for the hypercapnic response, although stimulation of one side is sufficient to elicit the response if the contralateral side is intact but not stimulated. To account for the unilateral effectiveness of lesions, the chemosensory neurons must have decussating projections between the two sides of the brain stem within a given site. Furthermore, the loss of chemosensory function when only one of two bilaterally symmetrical sites was inhibited suggests that there is a strong interdependence between the sites. Similar difficulties exist in mammalian studies, but the circumstances in which unilateral ablation has a large effect in mammals may help understand our results in tadpoles. In anesthetized rats and cats, unilateral lesions of small CO2-sensitive areas often wipe out all ventilatory activity for an extended time (30-90 min) and dramatically reduce the CO2 responsiveness when respiratory activity does resume (32). Some of the animals remain apneic once the lesion is made and regular ventilatory activity is never restored. In contrast, lesions in unanesthetized animals in exactly the same location have remarkably modest effects on ventilation and CO2 responsiveness (1). We did not study intact tadpoles, and the deafferented state of the isolated tadpole brain stem may resemble the anesthetic state in which tonic nonchemosensory excitatory inputs to the lung CPG were considerably reduced, and therefore, the importance of central chemosensory drive to the lung CPG was enhanced. The dramatic reduction in responsiveness when only one of a bilaterally paired structure was inhibited or ablated suggests that the CO2 sensors or neurons within the affected area have the effect of amplifying (multiplying) the respiratory output. Thus, if the amplifier multiplies by zero after ablation, the output of the system is zero. The system does not seem to be additive in respect to CO2 chemosensory responses, in which case ablation of a single site would simply reduce ventilation by a fixed amount. The system is plastic as well. If one waits long enough in mammals, the ventilatory output recovers (although chemosensory responses remain low; Ref. 32). Small, unilateral electrolytic lesions in one of the chemosensory sites often completely suppress lung bursts in tadpoles. About half the time, the lung rhythm is restored after 30-60 min (B. E. Taylor, unpublished observations). Thus the isolated tadpole brain stem demonstrates similar plasticity. Anesthesia and isolation of the brain stem seem to reduce the number of excitatory inputs to the respiratory system and reveal the multiplicative interaction between the lung CPG and chemosensory sites. In more intact systems, the chemosensors represent a much smaller fraction of the total drive to breath acting on the lung CPG, and consequently ablation of any single site has a much smaller effect in intact, unanesthetized animals. However, no studies have yet investigated the electrophysiological interactions or anatomic connections between adjacent sides within a chemosensory region in tadpoles to explore the cellular mechanisms whereby this apparent multiplicative interaction might originate.
Anomalous chemosensory function in late-stage tadpoles. In late-stage tadpoles, focal stimulation of a single ventral medullary chemosensitive site is not sufficient to elicit a hypercapnic response. Rather, it is necessary that the two identified sites be simultaneously stimulated to produce a response. This necessity is illustrated by the disruption of an ongoing hypercapnic response by focal, unilateral application of normocapnic aCSF at either chemosensitive site. We believe that this ontogenetic change reflects a transient reduction in the robustness of chemosensitivity at both ventral medullary sites even though the overall chemosensory response to hypercapnia is not reduced. It appears that neither site loses its chemosensitivity (Fig. 4), but seemingly the individual signals of each site fall below the threshold of responsiveness (Fig. 5). If chemosensitivity is in fact less robust, one might look for a reduced cellular responsiveness (fewer cells and/or less-sensitive cells) or a reduced transmission from chemoreceptors to the lung rhythm generator. Because overall chemosensory responses consistently increase throughout bullfrog development, including the late tadpole stages, reduced cellular responsiveness seems unlikely. We think it is more likely that a neural reorganization, which is known to occur in late-stage tadpoles (4, 16), creates a temporary situation in which paired stimulation at both ventral medullary chemosensitive sites is necessary for a hypercapnic response. In a previous study (39), our laboratory found that acetazolamide produced a paradoxical reduction in the hypercapnic ventilatory response in late-stage tadpoles, a finding we also attributed to a loss of robustness in chemosensory responses. Thus, in two sets of experiments, we found that chemosensory function in late-stage tadpoles was more susceptible to disruption. The transition from aquatic to terrestrial life occurs in the late stages of metamorphosis, and a variety of sensory systems undergo a transformation associated with reduced sensory function at this stage in metamorphosis. The auditory system and visual systems seem to be rewired for aerial acoustic and visual function. In the case of the acoustic system, this may actually be associated with transient deafness (4). The dominant method of gas exchange changes at this stage of metamorphosis, and the PCO2 of blood rises dramatically in late metamorphosis. Thus we feel that it is likely that central CO2 chemosensory mechanisms are also undergoing a transformation that amplifies the interdependence of the rostral and caudal chemosensory sites as the system is prepared for aerial respiration.
Role of central chemoreceptors in respiratory rhythm generation. The lesion studies suggest that output from both the rostral and caudal chemosensitive sites is required for a hypercapnic response at any stage of development. In all but late-stage tadpoles, focal stimulation of a single ventral medullary chemosensitive site was sufficient to elicit a hypercapnic response, but only when the nonstimulated site was intact. Destroying cells with a kainic acid lesion at either site or a protease lesion at the caudal site disrupted the overall hypercapnic response of the brain stem. This suggests that, even when not stimulated, chemosensitive neurons at these sites have a tonic baseline output that contributes to the hypercapnic response. Nattie (30) and Cohen (10) have proposed that tonic baseline output from all chemoreceptor sites is required for full expression of the hypercapnic response in mammals. That the amphibian and mammalian brain stem share this characteristic suggests that integration of central chemosensory outputs from multiple sites is phylogenetically older than previously thought.
Central chemosensitivity sustains baseline, normocapnic ventilation as well as ventilatory responses to changes in CO2/H+ in mammals (29). Baseline ventilation in bullfrogs also seems to require input from central chemoreceptors. Lesions within the ventral medullary chemosensitive sites that eliminated the hypercapnic response also decreased the frequency of fictive lung ventilation during normocapnia. It is significant that, although the lesions decreased the frequency of fictive lung ventilation, they did not abolish it. Nor did they abolish fictive buccal ventilation. Because the destruction of central chemoreceptors did not prevent the generation of the lung or buccal rhythm, the central chemoreceptors are not obligatory components of the central respiratory rhythm generators, and the neurons responsible for central rhythm generation are probably not intrinsically CO2 chemosensitive.
Perspectives
During the late tadpole phase, the peak of metamorphosis, there is a transient change in the integration of chemosensation from the two ventral medullary sites such that stimulation of a single site is insufficient to elicit a hypercapnic response. The excitatory effects of acidosis are attenuated or less effectively communicated to the lung rhythm pattern generator, and the late-stage tadpole presents, therefore, an interesting conundrum regarding the ontogeny of central CO2 chemosensitivity. At the peak of metamorphosis, the bullfrog tadpole exhibits significant changes in the perception and/or transmission of chemosensory information, but at the close of metamorphosis, the bullfrog emerges with a central CO2 chemosensitivity that is remarkably similar to that which it possessed as a hatchling tadpole. The developmental stages apparent in tadpoles are recapitulated to some extent in mammals, but the period of mammalian development comparable to late-stage metamorphosis occurs early in fetal life and is inaccessible to study. Our study has suggested a homology of amphibian and mammalian central chemoreception because it identified similarities in the location and interplay between central chemosensitive sites. As we assess the reasons for the lack of robustness of chemosensitivity in late-stage tadpoles, we may extrapolate a similar respiratory transformation in fetal mammals, one that is not currently accessible to study, but a transformation that may represent a period of developmental vulnerability with implications for condition such as the sudden infant death syndrome in which maternal risk factors (alcohol and smoking) seem to modify the subsequent development of the respiratory system in the infant (20).
|
DISCLOSURES |
|---|
|
TOPABSTRACTMETHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
|---|
|
FOOTNOTES |
|---|
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.
Present address for B. E. Taylor: Dept. of Biology and Wildlife, University of Alaska-Fairbanks, Fairbanks, AK 99775.
|
REFERENCES |
|---|
|
TOPABSTRACTMETHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
|---|
| ||||||||||||||||||||||||||||||||||||||||||
) or hypercapnic aCSF (
). See text for details of statistical comparisons.
