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1 Department of Organismal Biology and Anatomy, The University of Chicago, Chicago, Illinois 60637; 2 Department of Neurology, University of Freiburg, D-79106 Freiburg; and 3 Department of Pediatric Neurology, University of Göttingen, D-37073 Gottingen, Germany
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ABSTRACT |
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 Abstract
 Introduction
Methods
Results
Discussion
References
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Ramirez, J. M., U.J.A. Quellmalz, and B. Wilken. Developmental changes in the hypoxic response of the hypoglossus respiratory motor output in vitro. J. Neurophysiol. 78: 383-392, 1997. The transverse brain stem slice of mice containing the pre-Bötzinger complex (PBC), a region essential for respiratory rhythm generation in vitro, was used to study developmental changes of the response of the in vitro respiratory network to severe hypoxia (anoxia). This preparation generates, at different postnatal stages [postnatal day (P)0-22], spontaneous rhythmic activity in hypoglossal (XII) rootlets that are known to occur in synchrony with periodic bursts of neurons in the PBC. It is assumed that this rhythmic activity reflects respiratory rhythmic activity. At all examined stages anoxia led to a biphasic response: the frequency of rhythmic XII activity initially increased ("primary augmentation") and then decreased ("secondary depression"). In neonates (P0-7), anoxia did not significantly affect the amplitude of integrated XII bursts. Secondary depression never led to a cessation of rhythmic activity. In mice older than P7, augmentation was accompanied by a significant increase in the amplitude of XII bursts. A significant decrease of the amplitude of XII bursts occurred during secondary depression. This depression led always to cessation of rhythmic activity in XII rootlets. The anoxia-induced response of the respiratory rhythmic XII motor output is biphasic and changes during development in a similar way to the in vivo respiratory network. Whether this biphasic response is due to a biphasic response of the respiratory rhythm generator and/or to a biphasic modulation of the XII motor nucleus remains unresolved and needs further cellular analysis. We propose that the transverse slice is a useful model system for examination of the mechanisms underlying the hypoxic response.
The response of the mammalian respiratory system to hypoxia is biphasic (Cherniack et al. 1971 Preparation
Experiments were conducted on male and female mice (MRI-1 and Bahabor: 0-22 days) that were deeply anesthetized with ether and decapitated at the C3/C4 spinal level. The brain stem was isolated in ice-cold artificial cerebrospinal fluid (a-CSF) and further processed as described previously (Ramirez et al. 1996
Tissue oxygen measurements
Tissue oxygen in transverse slice preparations was measured with the use of oxygen tension (PO2)-sensitive electrodes (tip diameter 40 µm) connected to a chemical microsensor meter (Diamond General). Before every series of measurements, the PO2-sensitive electrode was polarized for 1 h ( Recording and data analysis
Recordings and data analysis were also described elsewhere (Ramirez et al. 1996
Under control conditions neither neonates (P0-6, Fig. 2A) nor more mature mice (P20-22, Fig. 2C) exhibited an anoxic core when PO2 was measured at different depths from the surface of the slice. Measurements were obtained in regions that contained either the pre-Bötzinger complex (Fig. 2) or the XII nucleus (not shown). Because of the flow of the a-CSF within the bath, the oxygen profiles were not symmetrical. The rostral surface of the transverse slice was more hyperoxic than the caudal surface (Fig. 2, A and C). The oxygen profile differed between neonates and more mature slices. Neonates were more hyperoxic than more mature preparations. Thus slightly lowering the oxygen levels in the perfusate would create an unreproducable distribution of oxygen in the slice: anoxic in the core and perhaps normoxic at the surface, depending a great deal also on the age of the slice. To guarantee reproducable and equal conditions in neonates and more mature slices, we perfused slices with 95% N2-5% CO2, which resulted within 30-50 s in anoxic conditions at all tissue levels irrespective of the slices. Figure 2, B and D, illustrates how PO2 levels at a depth of 200 mm changed on superfusion of the slice with 95% N2-5% CO2. In the following we call these severe hypoxic conditions anoxic.
Anoxia-induced qualitative alterations in rhythmic XII nerve activity
In neonates and more mature slices, anoxia induced a transient tonic activation of the XII nerve, which is seen as an increased background activity in Fig. 3A, bottom. The rhythmic XII bursts were affected in a development-dependent manner. In neonates, the frequency of rhythmic XII bursts was augmented (Fig. 3A, top), but the intensity of rhythmic XII burst activity was unchanged (Fig. 3B, top). Subsequently, anoxia led to a slight reduction of the frequency, also without altering the intensity of the XII bursts. This depression in the frequency of XII burst activity was maintained for anoxic periods lasting in some slices for>1 h, never leading to cessation of respiratory activity (Fig. 3A, top) in all examined neonatal slices (n = 55, P0-7).
Evaluation of the anoxic effects on rhythmic XII activity
The XII activity was integrated to better analyze the anoxic effect under in vitro conditions (Fig. 4). In the integrated recording, the tonic activation of the XII nerve was characterized by an upward deflection (Fig. 4B, ALTERATIONS IN THE AMPLITUDE OF XII BURSTS.
The majority of neonatal slices exhibited no significant amplitude effects in response to anoxia (Fig. 4A), as shown in the sequential histogram in Fig. 5A. Sequential histograms were used to quantify the percentage change in XII amplitude at different developmental stages. Each filled circle in Fig. 5C represents the average change evaluated for one slice preparation from 15 consecutive cycles before anoxia and 15 consecutive slices during the peak of the augmentation phase (Fig. 5C) and 15 consecutive cycles during the peak of depression (Fig. 5D; on average 10 min after the onset of anoxia). Although individual neonatal slices (obtained during the 1st postnatal wk) exhibited anoxia-induced amplitude effects (Fig. 5C), the average percentage change in amplitude, as evaluated from sequential histograms of 10 neonatal slices, was not significantly different from control values. This was the case during both augmentation (Fig. 5C, white histogram bar, n = 10) and depression (Fig. 5D, white histogram bar, n = 10).
BIPHASIC ALTERATION IN THE CYCLE LENGTH OF RHYTHMIC INTEGRATED XII ACTIVITY.
The frequency of rhythmic XII activity increased usually 1-2 min after the onset of anoxia (Fig. 4, A and B). In the sequential histograms in Fig. 6, augmentation is shown as a decrease in cycle length of XII burst activity for a P0 (Fig. 6A) and P8 (Fig. 6B) slice preparation. Sequential histograms were also used to quantify the percentage change in cycle length at different developmental stages. Each filled circle in Fig. 6C represents the average percentage change evaluated for one slice preparation from 15 consecutive cycles before anoxia and 15 consecutive cycles during the peak of the augmentation phase. The scatter plot indicates that the percentage decrease in cycle length was not dependent on the age of the transverse slices. The average percentage value obtained from 10 slices during the first postnatal week (P0-7, Fig. 6C, white bar) was not significantly different from that obtained from 15 slices during the second postnatal week (P8-15, Fig. 6C, shaded bar).
The transverse slice of mice containing the pre-Bötzinger complex exhibited a biphasic response to anoxia. As discussed below, age-specific changes in the response of rhythmic XII activity to anoxia as observed in this in vitro preparation resembled hypoxic responses that were previously reported in other more complex preparations.
Central nervous structures contained within the transverse slice are sufficient to generate a biphasic response to anoxia
Numerous studies have indicated that the hypoxic response is biphasic, consisting of an initial increase in ventilation that is followed within a few minutes by a decline in ventilation reaching, in nenoates, values below baseline levels (e.g., Neubauer et al. 1990 Anoxic response in more mature mammals
In contrast to neonates, mature mammals respond under in vivo conditions with a sustained augmentation to anoxia that involves an increase in the amplitude of phrenic nerve activity (Richter et al. 1991 Developmental changes in the anoxia response
As mentioned in the previous two paragraphs, differences exist in the response of the neonatal and more mature respiratory system to anoxia. The comparison between the neonatal spinal cord-brain stem preparation and the isolated adult perfused brain stem suggests that the developmental changes are due to changes in the central respiratory network (Ballanyi et al. 1992 Absence of gasping in response of the transverse rhythmic slice to anoxia
It has been suggested that the rhythmic activity generated by the transverse slice may reflect gasping rather than respiratory activity (Fung et al. 1994 Conclusions
Our study indicates that the anoxia-induced changes in the transverse slice are very similar to those reported for the intact respiratory system. This is an important finding, because it clearly indicates that the neuronal structures captured by the 600- to 700-µm-thick transverse slice are sufficient to generate many characteristic features that have previously been described for the intact respiratory system. In addition, the developmental changes in the hypoxic response are consistent with findings obtained in numerous other preparations in which the hypoxic response matures within the first 3 wk of postnatal development. The transverse slice of mice from animals ranging in age from P0 to 22 therefore provides a functionally relavant model system for examining, at systems and cellular levels, the neuronal mechanisms underlying the complex response of the respiratory system to hypoxia.
INTRODUCTION
Abstract
Introduction
Methods
Results
Discussion
References
; England et al. 1995; Fregosi et al. 1987; Haddad and Mellins 1984; Maruyama et al. 1989; Richter et al. 1991; Scott et al. 1990; St. John and Bianchi 1985). An initial increase in the frequency of ventilation (augmentation) is followed by a decrease (depression) that can terminate in a cessation of ventilation (apnea). The extent of this biphasic response to hypoxia changes during postnatal development. Neonatal mammals, including pre- and full-term human infants, typically exhibit a brief augmentation phase that is followed by a sustained depression often persisting for some time after normoxic conditions are reintroduced (Bureau et al. 1984; Grunstein et al. 1981; Lawson and Long 1983; Rigatto 1979). In adult mammals, including humans, hypoxia causes a sustained hyperventilation accompanied by an increased amplitude in phrenic and hypoglossal (XII) nerve activity, which during severe hypoxia can also lead to a depression and apnea (Hwang et al. 1983b; Richter and Acker 1989; Richter et al. 1991; Weiskopf and Gabel 1975). Thus the major developmental difference is 1) the inability of the newborn to sustain enhanced ventilation at the initial period of hypoxia and 2) a depression in neonates that can be maintained at a low frequency for a long period (Haddad and Mellins 1984). During depression in chemodenervated adult animals, the frequency of respiration falls below baseline levels, leading within a short time to apnea (Neubauer et al. 1990; Richter et al. 1991). Neonates seem in general much more resistant to anoxia than adults (Ballanyi et al. 1992; Bomont et al. 1992; Richter and Ballanyi 1996).
; Ramirez et al. 1996). This preparation contains the pre-Bötzinger complex, a region essential for respiratory rhythm generation (Smith et al. 1991). Because this medullary slice spontaneously generates respiratory rhythmic activity at all postnatal stages from postnatal day (P)0 to 22, it has previously been used as a model to study postnatal changes in neuromodulation (Funk et al. 1994; Quellmalz et al. 1995) and mechanisms of respiratory rhythm and pattern generation (Ramirez and Richter 1996; Ramirez et al. 1996; Richter et al. 1996). Here we examined whether this transverse slice preparation also retains typical properties of the intact respiratory system in response to hypoxia. More specifically, we wanted to know whether the isolated respiratory network exhibits a biphasic hypoxic response and whether this response undergoes developmental changes similar to those described for the intact network under in vivo conditions. Respiratory activity was monitored with the use of the extracellularly recorded XII motor output. It is well established that the XII motor output is rhythmically active in phase with inspiration (Smith et al. 1990; Withington-Wray et al. 1988). Our study indicates that postnatal changes do occur in the response to severe hypoxia over a time period of 14 days. The in vitro preparation of the transverse slice preparation reveals a biphasic response similar to that of the in vivo network. We propose that the hypoxic reponse of the central respiratory network can now be studied in more detail under in vitro conditions.
METHODS
Abstract
Introduction
Methods
Results
Discussion
References
). The most important steps to obtain slices containing the pre-Bötzinger complex are therefore only briefly summarized here. The brain stem was secured in a vibratome with the rostral end upward, tilted at an angle of 20° to the plane of the razor blade. Thin (100-200 µm thick) slices were sectioned serially from rostral to caudal until reaching the rostral boundary of the pre-Bötzinger complex, which was recognized by cytoarchitectonic landmarks such as inferior olive, nucleus of the solitary tract, XII nucleus, and nucleus ambiguus, but the the facial nucleus was no longer present. The rhythmic slice that was contained within 650-700 µm caudal of this rostral boundary (Fig. 1) was immediately transferred into a recording chamber and submerged under a stream of a-CSF (29°C; flow rate 10 ml/min). The preparation was stabilized for 30 min in a-CSF containing (in mM) 128 NaCl, 3 KCl, 1.5 CaCl2, 1 MgSO4, 24 NaHCO3, 0.5 NaH2PO4, and 30 D-glucose equilibrated with carbogen (95% O2-5% CO2) at 27°C, pH 7.4. The potassium concentration was raised to 8 mM over a period of another 30 min to obtain regular rhythmic activity lasting for up to 13 h. Anoxia was induced by bubbling the a-CSF with 95% N2-5% CO2, pH 7.4. To avoid O2 diffusion, all tubings except of those within the roller pump were made of stainless steel.
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FIG. 1.
Schematic representation of transverse brain stem slice obtained from mice together with a recording from the hypoglossal (XII) nerve. Shaded area in transverse slice indicates schematically the location of the pre-Bötzinger complex. XII rootlet activity was recorded with the use of a suction electrode (bottom trace). XII was integrated with the use of an electronic filter (top trace). Also indicated are the evaluated parameters, cycle length and amplitude. IO, inferior olive; NA, nucleus ambiguus; NTS, nucleus of the tractus solitarius; Sp5, spinal trigeminal nucleus; XII, XII motor nucleus; XII nerve, XII rootlet.
750 nA) and calibrated with the use of two a-CSF solutions that were saturated with either carbogen or a gas mixture containing 95% N2-5% CO2. We assume that the oxygen response properties of the electrode were linear. Given that we compared in this study only two states, i.e., control and anoxic conditions and not other degrees of hypoxia, the calibration with 95% N2-5% CO2 and 95% O2-5% CO2 should be sufficiently accurate to determine the absence of anoxia under control conditions and the presence of anoxic conditions during the perfusion with 95% N2-5% CO2. The oxygen-sensitive electrode was driven into the tissue in 50-mm steps with the use of a piezomicromanipulator.
). In short, activity from the peripheral end of cut XII rootlets was recorded extracellularly with suction electrodes (amplified 2,000 times and filtered: low pass 1.5 kHz, high pass 250 Hz; Fig. 1, bottom trace) and integrated electronically (Paynter filter, set at a time constant of 20-30 ms; Fig. 1, top trace). To quantify the average cycle length and amplitude of integrated XII nerve activity, data were analyzed on- and off-line with the use of two software programs that were written for personal computers. Data were digitized with a DT 2821 and Labmaster interface (Scientific Solutions).
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FIG. 4.
During anoxia-induced augmentation (gray area: anoxic stimulus), there is a dramatic increase in amplitude of integrated rhythmic XII activity ( 
) in a more mature mouse (B, P18), but not in a neonatal mouse (A, P2). Note relatively slow upward deflection in both neonatal and more mature mouse, indicating a tonic activation of the XII nerve (B, 
). Note also that the amplitude of XII activity in B remains increased even after the tonic activity of the XII nerve has returned to control values.
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FIG. 5.
Development-dependent effect of anoxia on amplitude of integrated rhythmic XII activity. Sequential histograms characterizing the effect of anoxia on the amplitude of rhythmic XII activity in a P0 (A) and P8 (B) mouse. Each dot represents the moving average of 3 consecutive bursts. Sequential histograms were used to evaluate average percentage changes in the amplitude of rhythmic XII bursts during augmentation (C) and depression (D). Average values were evaluated by averaging the percentage change values obtained from 15 consecutive cycles per slice before and during maximal augmentation or maximal depression. C and D: graphs show average amplitude values (ordinates) for individual slices obtained at different ages (abscissas) during maximal augmentation (C, 
) and maximal depression (D, 
). Histograms give average amplitude values for group data (white bars: 1st postnatal wk; shaded bars: 2nd postnatal wk).
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FIG. 6.
Development-dependent effect of anoxia on cycle length of rhythmic integrated XII activity. Sequential histograms characterize effect of anoxia on the cycle length in a P0 (A) and P8 (B) mouse. Each dot represents the moving average of 3 consecutive bursts. As also described in Fig. 5, sequential histograms were used to evaluate average percentage changes in cycle length of rhythmic XII bursts during augmentation (C) and depression (D). C and D: graphs represent average cycle length values (ordinates) for individual slices obtained at different ages (abscissas) during augmentation (C, ) and depression (D, ). Histograms give average cycle length values for group data (white bars: 1st postnatal wk; shaded bars: 2nd postnatal wk).
RESULTS
Abstract
Introduction
Methods
Results
Discussion
References
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FIG. 2.
Oxygen tension (PO2) levels averaged from 12 neonatal slices [A, postnatal day (P)0-6] and 5 slices obtained at the age of P21 (C). Depth values correspond to depth from surface of slice in a region that also included the pre-Bötzinger complex. PO2 levels were measured in 50-µm steps starting at rostral surface of slice preparation (0 µm). Bath PO2 levels are indicated by an arrow in A and C. B and D: changes in PO2 levels during perfusion of slices with 95% N2-5% CO2. Note that these changes result in anoxia in both a P6 and P21 slice.
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FIG. 3.
Anoxic response of the respiratory system undergoes postnatal changes as indicated in A and B by nerve recordings from the XII nerve of a neonatal mouse (P1, top trace) and an 18-day-old mouse (bottom trace). A: recordings obtained during control, anoxia, and recovery. Time after onset of anoxia is also indicated. Note that the time scales for the neonate and more mature mouse were different, to best demonstrate the frequency effect. B: single bursts shown in a faster time scale to better demonstrate the development-dependent effect of anoxia on a single burst. Neonatal recording was performed at a lower aquisition rate than recording from the more mature mouse, which explains the difference in recording quality.
), which usually lasted 1-2 min in both neonates (Fig. 4A) and more mature animals (Fig. 4B). This tonic effect recorded from the XII nerve was not further evaluated, because it may be an effect specific for XII neurons. As mentioned in METHODS, modifications of rhythmic burst amplitude during the transient tonic activitation of the XII were difficult to interpret. Thus we further evaluated the effect of anoxia on the integrated rhythmic drive to the XII nerve before or after the tonic activation of the XII nerve (Fig. 4B, ). In this situation the intensity of the XII burst was reflected as the amplitude of integrated XII activity (Figs. 1 and 4B, ). The anoxic effects on the amplitude and cycle length of integrated rhythmic XII activity (see Fig. 1 for definition) were quantitatively analyzed for 25 slices between the ages of P0 and 15. Maximal depression in the frequency of rhythmic burst discharges was reached within 5-15 min. This was the time when amplitude and cycle length values were obtained for further analysis. Thus we often returned to carbogen perfusion in this time range without exposing the slices for much longer anoxic conditions (e.g., Fig. 6A). This reduced the time for the slice to fully recover to control values. Older ages were only qualitatively evaluated, because the number of older slices with a sufficiently good signal-to-noise ratio for a computer analysis was not enough to also justify a group of 3-wk-old slices. However, we did not see any obvious qualitative differences between 2- and 3-wk-old slices, as suggested also by the recordings shown in Figs. 3 and 4B.
) and depression (Fig. 5D, ) were also evident in animals older than P8. However, in contrast to the situation in neonates (Fig. 5A), the majority of slices of more mature animals exhibited a considerable increase in the amplitude of integrated XII bursts during augmentation (Fig. 5B). This was reflected in a significant increase in the average amplitude during augmentation (Fig. 5C, shaded bar) and significant decrease in amplitude during depression (Fig. 5D, shaded bar, evaluated 10 min after the onset of anoxia,n = 15). These alterations were significantly different from anoxia-induced amplitude effects as seen in animals younger than P7.
DISCUSSION
Abstract
Introduction
Methods
Results
Discussion
References
; Schwieler 1968). Although peripheral components, such as muscle mechanics or peripheral chemoreceptor drive, certainly contribute to the biphasic response of the respiratory system to anoxia, several studies have already indicated that the biphasic response of the respiratory system has a strong CNS component (Richter et al. 1991). Under in vivo conditions depression can be seen despite sustained stimulation of the carotid chemoreceptors during hypoxia, suggesting that central nervous mechanims are responsible for the decline of ventilation (Neubauer et al. 1990). In the transverse slices of neonatal and more mature mice, frequency of XII rhythmic activity declined to values that were below baseline levels, confirming that depression is primarily caused by CNS mechanisms. The depression typical for neonates, including human infants, is characterized by a decrease in respiratory frequency (Haddad and Mellins 1984; Rigatto 1979; Rigatto et al. 1975), which was also seen in the transverse slice of neonatal mice. Only cycle length, but not burst duration, was affected by anoxia (e.g., Fig. 3). Another similarity of the transverse slice to the in vivo hypoxic response was the decreased expression of augmentation in neonates (Haddad and Mellins 1984). We observed that augmentation was restricted to an increase in frequency without affecting the amplitude of the integrated XII burst. Previous in vivo studies attributed the relatively weak augmentation in neonates to a weakness of the response of peripheral chemoreceptors (Bureau et al. 1984). The absence of a strong centrally generated augmentation as seen in the neonatal transverse slice together with a weak peripheral chemoreceptor drive during anoxia could certainly explain the weak augmentation of neonatal mice in vivo.
, 1993) and XII nerve activity (Hwang et al. 1983a). In transverse slices of mice >1 wk old, we observed a significant augmentation that was manifested not only in an increased frequency of rhythmic XII activity, but in an increase of the amplitude of XII bursts (Fig. 3). Under control conditions, the pattern of XII activity in the transverse slice of mature mice (Ramirez et al. 1996) resembled that of adult cats and humans, being bell shaped and reaching a maximal value early in inspiration (Hwang et al. 1983a; Önal et al. 1981). At the onset of anoxia the burst amplitude of integrated XII rhythmic activity increased in the transverse slice. Previous in vivo experiments have similarly indicated that anoxia increases peak amplitude of phrenic (Richter et al. 1991) as well as XII bursts (Hwang et al. 1983a). This increase in XII nerve activity was essentially proportional to phrenic nerve activities (Hwang et al. 1983a,b).
). However, augmentation also occurs in chemodenervated animals (Miller and Tenney 1975; Moyer and Beecher 1942; Neubauer et al. 1990), leading to the hypothesis that a centrally derived excitation resides in the interaction between the cortex and diencephalon. An inhibitory hypoxic influence on the cortex would cause a disinhibitory influence on rate-facilitating structures in the diencephalon (Neubauer et al. 1990). However, because cortical and diencephalic structures are not present in the medullary slice preparation, other medullary mechanisms may also contribute to the hypoxia-induced augmentation.
; Richter et al. 1978; Trippenbach et al. 1990). Changes in ionic concentration will affect, under both in vivo and in vitro conditions, all levels of the respiratory network, modulating rhythm-generating neurons located within the pre-Bötzinger complex (e.g., Ballanyi et al. 1996), neurons within the Raphe nuclei that mediate CO2 effects (Richerson 1995), and XII neurons themselves (Haddad and Donnelly 1990; Jiang et al. 1992). The tonic activation of the XII rootlets was most likely due to the direct effect of anoxia on XII neurons. Also, the hypoxic effect on the amplitude and frequency of XII activity may be due to an effect on the excitability of XII neurons. Under in vitro conditions we previously reported that the XII motor output of more mature slices had a higher degree of cycle-to-cycle variability compared with the rhythmic activity in the ventrolateral reticular formation (Ramirez et al. 1996). Indeed, intracellular recordings obtained from individual pre-Bötzinger complex neurons of more mature mice showed that hypoxia changed in some cases the coupling ratio between the pre-Bötzinger complex and the XII nucleus from 3:1 to 1:1 (J. M. Ramirez, U.J.A. Quellmalz, B. Wilken, and D. W. Richter, unpublished data). Thus further cellular investigations will be necessary to examine at what levels of the respiratory network anoxia causes an augmentation in more mature slice preparations. Insights into these effects should also help in understanding of the in vivo hypoxic response.
; Richter and Ballanyi 1996). However, this comparison was based on two entirely different preparations, which allowed no characterization of the transition from a typical neonatal to a mature anoxic response. This became possible for the first time in the transverse slice. We demonstrated that adultlike properties in the anoxic response, such as an increase in the amplitude of XII burst during augmentation, were consistently observed as early as P8. In animals older than P8, prolonged exposure to anoxia led also to a cessation of rhythmic XII activity, which we interpret as absence of rhythmic activation of XII neurons due to central "apnea." Although the mechanisms that lead to the cessation of rhythmic XII activity cannot be determined from this study, cessation of XII rhythmic activity was clearly development dependent and not seen in animals younger than P7. In these animals depression was restricted to a decrease in the frequency of rhythmic discharge without significantly affecting the amplitude of XII bursts. The absence of a cessation of XII rhythmic activity in neonatal preparation has previously been demonstrated for the brain stem-spinal cord preparation (Ballanyi et al. 1995; Richter and Ballanyi 1996; Völker et al. 1995), indicating that the results obtained in this study for the transverse slice preparation are comparable with findings obtained in preparations that contain a larger portion of the respiratory network.
; Bureau et al. 1984; Moss et al. 1995), cats (Bonora et al. 1984), dogs (Haddad et al. 1982), and monkeys (Woodrum et al. 1981). Full-term human infants show a time course similar to those of the species mentioned above (Haddad and Mellins 1984; Rigatto 1979). However, maturation of the hypoxic response seems to be retarded in preterm infants, because typical neonatal responses to anoxia are still seen after >25 days of postnatal age (Rigatto 1979). These comparative studies of the hypoxic response indicate that maturation of the hypoxic response typically occurs within the first 3 wk of postnatal development in most mammals. This time course seems to be independent of the fact that different species are born at strikingly different neurological maturational states (Dobbing and Sands 1979).
). In support of this possibility is the persistence of rhythmic activity in neonatal slices during anoxia, which is indeed typical for gasping. In the experiments presented in this study we demonstrated that the persistence of rhythmic activity is only a transient feature present only during the early postnatal development of this network. The more mature slice preparation, however, responded to anoxia with an augmentation and subsequent cessation of XII rhythmic activity typical for the respiratory system. This finding can be regarded as a strong indication that rhythmicity generated by this preparation is respiration rather than gasping. Several in vivo lesion experiments indicated that there are two rhythm generators, which are located at different sites in the nervous system (Fung et al. 1994; St. John 1990; St. John et al. 1984). The rhythm generator for gasping is located in the lateral tegmental field of the medulla (Fung et al. 1994), whereas the respiratory rhythm generator for respiration is located more ventrolaterally in the pre-Bötzinger complex (Koshiya and Guyenet 1996; Ramirez et al. 1994; Schwarzacher et al. 1995; Smith et al. 1991). The existence of two separate rhythm generators for gasping and respiration is further supported by differences in physiological properties of these two activities (St. John 1990; St. John and Knuth 1981; St. John et al. 1989). On exposure to anoxia, there is an increase in the frequency of gasping, but, unlike respiration, this augmentation in frequency is not accompanied by an increase in the amplitude of gasping (St. John and Knuth 1981). In the transverse slice of more mature mice we found that augmentation in frequency is always accompanied by an increase in the amplitude of XII motor output (Figs. 3-5). Another indication for the XII motor output representing respiration rather than gasping is the fact that the slices were not hypoxic under control conditions (Fig. 2).
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ACKNOWLEDGEMENTS |
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We thank Dr. Diethelm W. Richter (University of Göttingen) for many interesting discussions during the course of this study and Drs. Jörg Schmidt (University of Göttingen) and Keir G. Pearson (University of Alberta) for writing the software that was essential for the analysis of the data presented in this study. We also appreciate the excellent technical assistance of A. Wei
e-Blanke.
This study is part of a doctoral thesis of U.J.A. Quellmalz. The study was supported by operating grants to J. M. Ramirez from Deutsche Forschungsgemeinschaft Ra 573/4-1 and Sonderforschungsbereich 406 A4.
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FOOTNOTES |
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Address for reprint requests: J. M. Ramirez, Dept. of Organismal Biology and Anatomy, The University of Chicago, 1027 East 57th St., Chicago, IL 60637.
Received 16 December 1996; accepted in final form 13 March 1997.
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REFERENCES |
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Abstract
Introduction
Methods
Results
Discussion
References
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 D. M. Robinson, H. Kwok, B. M. Adams, K. C. Peebles, and G. D. Funk Development of the ventilatory response to hypoxia in Swiss CD-1 mice J Appl Physiol, May 1, 2000; 88(5): 1907 - 1914. [Abstract] [Full Text] [PDF]
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I. C. Gibson and A. J. Berger Effect of Ethanol Upon Respiratory-Related Hypoglossal Nerve Output of Neonatal Rat Brain Stem Slices J Neurophysiol, January 1, 2000; 83(1): 333 - 342. [Abstract] [Full Text] [PDF]
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P. Telgkamp and J.-M. Ramirez Differential Responses of Respiratory Nuclei to Anoxia in Rhythmic Brain Stem Slices of Mice J Neurophysiol, November 1, 1999; 82(5): 2163 - 2170. [Abstract] [Full Text] [PDF]
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S L Mironov and D W Richter L-type Ca2+ channels in inspiratory neurones of mice and their modulation by hypoxia J. Physiol., October 1, 1998; 512(1): 75 - 87. [Abstract] [Full Text] [PDF]
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J M Ramirez, U J A Quellmalz, B Wilken, and D W Richter The hypoxic response of neurones within the in vitro mammalian respiratory network J. Physiol., March 1, 1998; 507(2): 571 - 582. [Abstract] [Full Text] [PDF]
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