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We investigated mechanisms of CO2/H+ chemoreception in the respiratory centre of the medulla by measuring membrane potentials of pre-inspiratory neurons, which are putative respiratory rhythm generators, in the brainstem–spinal cord preparation of the neonatal rat. Neuronal response was tested by changing superfusate CO2 concentration from 2% to 8% at constant HCO3− concentration (26 mm) or by changing pH from 7.8 to 7.2 by reducing HCO3− concentration at constant CO2 (5%). Both respiratory and metabolic acidosis lead to depolarization of neurons with increased excitatory synaptic input and increased burst rate. Respiratory acidosis potentiated the amplitude of the neuronal drive potential. In the presence of tetrodotoxin (TTX), membrane depolarization persisted during respiratory and metabolic acidosis. However, the depolarization was smaller than that before application of TTX, which suggests that some neurons are intrinsically, and others synaptically, chemosensitive to CO2/H+. Application of Ba2+ blocked membrane depolarization by respiratory acidosis, whereas significant depolarization in response to metabolic acidosis still remained after application of Cd2+ and Ba2+. We concluded that the intrinsic responses to CO2/H+changes were mediated by potassium channels during respiratory acidosis, and that some other mechanisms operate during metabolic acidosis. In low-Ca2+, high-Mg2+ solution, an increased CO2 concentration induced a membrane depolarization with a simultaneous increase of the burst rate. Pre-inspiratory neurons could adapt their baseline membrane potential to external CO2/H+ changes by integration of these mechanisms to modulate their burst rates. Thus, pre-inspiratory neurons might play an important role in modulation of respiratory rhythm by central chemoreception in the brainstem–spinal cord preparation.
Neuronal mechanisms of central chemoreception of respiration are not fully understood. Chemosensitive regions have been found in the ventral medullary surface (Mitchell et al. 1963; Schlaefke et al. 1970; Loeschcke et al. 1970). Because no intrinsically chemosensitive neuron has been identified there, Loeschcke (1982) suggested that the synaptic mechanism located in the chemosensitive areas of the ventral surface of the medulla is responsible for the chemosensitivity of respiration. Chemosensitive sites have also been found in the nucleus tractus solitarii (NTS), the medullary raphe, the retrotrapezoid nucleus (RTN), the rostral aspect of the ventral respiratory group, the pre-Bötzinger complex, the fastigial nucleus of the cerebellum and the region of the locus coeruleus (Dean et al. 1989; Coates et al. 1993; Kawai et al. 1996; Oyamada et al. 1998; Nattie, 1999, 2000, 2001; Nattie & Li, 2002). Intrinsic chemosensitive neurons have been found in the NTS (Dean et al. 1989, 1990) as well as in the ventral respiratory area and ventral surface (Onimaru et al. 1989; Kawai et al. 1996; Okada et al. 2002). Recently, Mulkey et al. (2004) found intrinsic CO2-sensitive neurons in the RTN that were located close to the ventral surface of the rostral medulla. Serotonergic neurons in the medulla have also been shown to be chemosensitive to CO2/H+ (Richerson, 2004). These chemosensitive neurons might be the primary chemoreceptors of respiration. Although such neurons are intrinsically chemosensitive to CO2/H+, the neuronal mechanisms mediating an increase in respiratory rhythm in response to hypercapnia are unknown. In a recent review, Feldman et al. (2003) emphasized that many cell types and molecular mechanisms are involved in respiratory chemoreception.
The in vitro brainstem–spinal cord preparation from newborn rat is known to respond to hypercapnia mainly by an increase in respiratory frequency (Issa & Remmers, 1992; Okada et al. 1993b; Kawai et al. 1996); however, it has also been suggested that the amplitude of phrenic nerve activity could increase independently in response to hypercapnia (Harada et al. 1985). In the awake adult rat preparation, hypercapnia increases both respiratory frequency and tidal volume (Li & Nattie, 2002). The in vivo vagotomised cat preparation, tidal volume is known to increase only in response to hypercapnia. Focal stimulation of the RTN in the awake rat increases tidal volume alone (Li et al. 1999), whereas focal stimulation of the NTS increases both respiratory frequency and tidal volume (Nattie & Li, 2002). The NTS and the carotid body or another central chemoreceptor site of the brainstem may contribute to the response of respiratory frequency (Li & Nattie, 2002). The in vitro brainstem–spinal cord preparation from newborn rat does not have the carotid body and contains the NTS as well as many chemoreceptor sites of the medulla. Because the in vitro preparation spontaneously generates a respiratory rhythm for more than 8 h and extracellular conditions can easily be modified, the preparation is useful for pursuit of the neuronal mechanisms of CO2/H+ chemoreception and respiratory rhythm modulation (Suzue, 1984; Ballanyi et al. 1999).
Because respiratory rhythm of the neonatal rat is thought to be generated in the rostral ventrolateral medulla (RVL) (Onimaru et al. 1987; Errchidi et al. 1991; Ballanyi et al. 1999) or the pre-Bötzinger complex (Smith et al. 1991), or in both (Mellen et al. 2003; Feldman et al. 2003), chemosensitive neurons in these regions and chemosensitive neurons having connection with these regions are expected to be important modulators of the basic respiratory rhythm. Pre-inspiratory (Pre-I) neurons located in the RVL, including the recently identified para-facial respiratory group (pFRG; Onimaru & Homma, 2003), constitute a crucial part of the respiratory neuronal network and contribute to respiratory rhythmogenesis in the neonatal rat brainstem. We have proposed that Pre-I neurons are the primary respiratory rhythm generators and that they determine basic respiratory rhythm by triggering inspiratory activity in the brainstem–spinal cord preparation (reviewed by Ballanyi et al. (1999)). Therefore, clarification of the mechanisms of CO2/H+ chemoreception by Pre-I neurons is necessary for understanding modulation of respiratory rhythm by changes in CO2/H+ in this preparation. The neuronal mechanisms of chemoreception by Pre-I neurons have not been examined systematically and the channels involved in chemoreception have never been elucidated. Thus, we tested the response of Pre-I neurons to both respiratory and metabolic acidosis and determined whether the responses were mediated by intrinsic or synaptic mechanisms, or by both. Furthermore, we examined the channel mechanisms that are responsible for chemoreception. Some of our results have appeared in abstract form (Kawai et al. 1997).
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We found that the Pre-I neuron, the putative respiratory rhythm generator, is intrinsically and synaptically chemosensitive to both CO2 and H+. We also found that the intrinsic responses are mediated by potassium channels during respiratory acidosis and by another mechanism during metabolic acidosis. Pre-I neurons are potentially able to modulate the respiratory rhythm in response to CO2/H+ changes in the brainstem–spinal cord preparation.
Although it has been shown that the medullary respiratory network remains functionally intact in the isolated brainstem–spinal cord preparation of the neonatal rat (Suzue, 1984; Smith et al. 1990), the relation between the rhythm generated by the in vitro brainstem and that generated in vivo is not understood in detail. Moreover, we did not study the response of pacemaker neurons in the pre-Bötzinger complex (advocated by Smith et al. 1991) to changes in CO2/H+. Thus, we emphasize that considerable care should be exercised in extrapolating our experimental data to in vivo conditions.
Differences in responses of Pre-I neurons to respiratory acidosis and metabolic acidosis
The burst frequency of Pre-I neurons and the respiratory frequency of this preparation depend mainly on the pH of the superfusate. This conclusion is consistent with previous studies with this preparation (Onimaru et al. 1989; Kawai et al. 1996). Nevertheless, a change in CO2 concentration at the same pH level could affect these parameters in a situation where respiration was reduced at alkaline pH. Voipio & Ballanyi (1997) reported a similar effect on respiratory frequency and proposed a direct effect of CO2. When the stimulation of the respiratory network by H+ ion is reduced, the effect of CO2 on respiratory frequency may be apparent.
The effects on the amplitude of the drive potential of Pre-I neurons differed between respiratory acidosis and metabolic acidosis. A change in CO2 concentration resulted in a marked change in the amplitude of the drive potential of Pre-I neurons, whereas a pH change at the same 5% CO2 level induced a relatively smaller change in drive potential. Moreover, even at the same pH at the medullary surface, a change in CO2 level clearly affected the amplitude of the drive potential. As CO2 concentration increased at the same pH at the surface, the amplitude of the drive potential increased. We measured only the surface pH and did not measure the tissue pH where the neurons were located. Our preliminary experiments and a previous study (Okada et al. 1993a) showed that a change in pH induced by a change in CO2 concentration was much faster than that induced by a change in bicarbonate concentration even at a depth of 100–200 μm from the surface where most Pre-I neurons are located. However, 5 min after a change of bath CO2 concentration and 10 min after a change of bicarbonate concentration, the tissue pH stabilized. In the present experiments, we examined the effects of a change in CO2 concentration 10 min after the change of the bath solution. Moreover, the tissue pH change induced by a change in CO2 concentration was slightly smaller than that induced by a change in bicarbonate concentration (Okada et al. 1993a). Therefore, respiratory acidosis might induce smaller acidic shifts in the regions where the cell bodies are located than those induced by metabolic acidosis. Thus, it is possible that the CO2 molecule itself affects the drive potential of Pre-I neurons, although further study will be necessary to obtain a definite result and elucidate the mechanisms involved.
These differences in response to respiratory and metabolic acidosis have not previously been observed in respiratory modulated neurons. Takeda & Haji (1991) reported that respiratory acidosis increased the amplitude of phasic depolarization in inspiratory and post-inspiratory neurons in the ventral respiratory group of an in vivo cat preparation, although they did not examine the effect of metabolic acidosis. Wang et al. (2002) performed systematic experiments to determine the primary stimulus for chemosensitive neurons located in the medullary raphe nuclei. They suggested that a change in intracellular pH might be the primary stimulus for chemosensitivity in the neurons because chemosensitivity of raphe neurones can occur independently of changes in CO2 level, extracellular pH or bicarbonate level. This may be the case for the response to respiratory acidosis in Pre-I neurons because CO2 induces an intracellular acidification much faster than H+.
Synaptic mechanisms of CO2/H+ chemoreception of Pre-I neurons
The drive potential is a mixture of membrane potential fluctuations caused by synaptic potentials and activation of intrinsic conductance (Onimaru et al. 2003). The contribution of intrinsic conductance to pH effects was examined in the presence of TTX (see below). The contribution of synaptic potentials was analysed under voltage clamp. Although the space clamp was incomplete, especially during the burst phase, the results indicated that the frequency of EPSCs increased in response to an increase in CO2 concentration. The origin of neurons sending these EPSCs is unknown, but other Pre-I neurons and tonically active chemosensitive neurons in the RTN (Mulkey et al. 2004; Li & Nattie, 2002) and other brainstem regions (Richerson, 2004) should be considered (see below).
Intrinsic mechanisms of CO2/H+ chemoreception of Pre-I neurons
Because the depolarizing response and increase in the input resistance of Pre-I neurons to respiratory acidosis was retained after the suppression of synaptic transmission, our findings suggested that the response was mediated by an intrinsic mechanism. However, the results do not exclude the possible involvement of action potential-independent release of neuromodulators (e.g. Gourine et al. 2005) in the depolarizing response to respiratory acidosis. The response of the input resistance to a change in CO2 concentration was also retained in these neurons, further suggesting the contribution of an intrinsic channel mechanism in the membrane potential response. The response of the other neurons was a markedly decreased depolarizing shift in membrane potential after suppression of synaptic transmission, suggesting involvement of a synaptic mechanism.
Further study of the channel mechanisms found that a potassium channel blocker (Ba2+), in the presence of TTX, completely blocked the depolarizing shift in membrane potential of Pre-I neurons during respiratory acidosis and did not block it during metabolic acidosis, whereas an inorganic calcium channel blocker (Cd2+) had no significant effect. Thus, a decrease in the potassium conductance seems to be responsible for the depolarizing response of Pre-I neurons to respiratory acidosis.
Recently, a CO2/H+ chemosensitive potassium channel consisting of two members of the inward rectifier potassium channel family (Kir4.1 and Kir5.1) was reported by Xu et al. (2000). This potassium channel family enables neurons to detect intracellular acidification induced by hypercapnia and is expressed in brainstem neurons. If Pre-I neurons have Kir4.1 and Kir5.1 in their membranes, the intrinsic response to hypercapnia might be mediated by the CO2-sensitive inward rectifier potassium channel.
Because H+ is much less membrane-permeable than CO2, metabolic acidosis may not easily induce intracellular acidification of Pre-I neurons. This could explain why the responses of Pre-I neurons to respiratory acidosis and metabolic acidosis are different. Moreover, another family of putative leak potassium channels (KCNK) has been identified by molecular cloning (reviewed by Goldstein et al. 2001; Patel & Honore, 2001). Among these, subgroups of so-called TASK channel currents are inhibited by extracellular protons in a physiological pH range. TASK channel currents were found in neurons of the locus coeruleus and medullary raphe (Talley et al. 2000; Bayliss et al. 2001). If Pre-I neurons possess TASK channels, extracellular acidification would be able to modulate their resting membrane potentials. Some cases of metabolic acidosis might induce the depolarization of Pre-I neurons by this mechanism.
However, in response to metabolic acidosis, Pre-I neurons retained their depolarizing response following the blockade of both calcium and potassium channels. Such a response might be mediated by other intrinsic mechanisms not involving membrane conductance changes; however, these could not be identified in the present study. We suggest that the intrinsic mechanisms that account for the depolarizing responses of Pre-I neurons are different for respiratory and metabolic acidosis.
The mechanisms modulating respiratory activity in response to CO2/H+: functional considerations
CO2/H+ changes in a superfusate clearly affect the respiratory frequency of this preparation (Okada et al. 1993b; Kawai et al. 1996). We and other groups have provided evidence that the RVL plays a pivotal role in rhythmogenesis of respiration in the brainstem–spinal cord preparation (Onimaru et al. 1987; Errchidi et al. 1991). The neuronal network in the RVL that includes Pre-I neurons, recently renamed pFRG (Onimaru & Homma, 2003), is involved primarily in generation of the respiratory rhythm (Ballanyi et al. 1999). After suppression of synaptic transmission, some Pre-I neurons in the RVL showed rhythmic changes in membrane potentials (phasic bursts; see also Onimaru et al. 1995); the frequencies of the phasic bursts depended on their baseline membrane potentials, which were affected by CO2/H+ changes (Fig. 8; Onimaru et al. 1989). Such Pre-I neurons in the RVL possess an intrinsic burst-generating property. It has been reported that the respiratory frequency of this preparation closely correlated with the burst frequency of Pre-I neurons (Onimaru et al. 1998; Takeda et al. 2001; Mellen et al. 2003). Thus, the depolarizing response to increased CO2 concentration or decreased pH might directly result in an increase in respiratory frequency in the brainstem–spinal cord preparation. However, because we did not examine pacemaker neurons in the pre-Bötzinger complex (Smith et al. 1991), there remains a possibility that they also modulate respiratory frequency in response to CO2/H+ changes.
Of the many neurons, besides Pre-I neurons, that respond intrinsically to CO2/H+, at least three groups may be the primary chemosensors of respiration. The first group consists of relatively small neurons discovered by Okada et al. (2002). These neurons, which might be cholinergic (Chen et al. 1997), are located just beneath the surface of the ventral medulla and are connected to the deeper region. The second group consists of glutamatergic neurons, which are located in the retrotrapezoid nucleus in the rostral ventrolateral medulla; their pH sensitivity is intrinsic and involves a background K+ current (Mulkey et al. 2004). The third group consists of the serotonergic neurons in the ventral medulla that stimulate the neuronal network that controls breathing (Richerson, 2004). Pre-I neurons and the respiratory activity of this preparation are affected by serotonin (Onimaru et al. 1998). Recently, Gourine et al. (2005) suggested that release of ATP from the classical brainstem chemosensitive area participates to mediate the effect of CO2 on breathing. They stated that the immediate release of ATP from the chemosensitive structure plays a key role in central chemoreception. However, they did not elucidate the cellular sources and mechanisms underlying release of ATP in response to an increase in CO2 level. ATP also stimulates both Pre-I neurons and the respiratory activity of this preparation (A. Kawai, unpublished data). As Li & Nattie (2002) reported, multiple chemoreceptor sites and structures may interact to provide high CO2 chemosensitivity. Excitatory synaptic inputs onto Pre-I neurons were increased in response to increased CO2 concentration and decreased pH, and some Pre-I neurons were synaptically chemosensitive. Such Pre-I neurons might be stimulated by the superficial cholinergic, glutamatergic or serotonergic neurons and by the ATP-releasing structure in the medullary surface.
In conclusion, the frequency of the rhythmic respiratory bursts of Pre-I neurons is dependent on the baseline membrane potential that is modulated by the level of CO2/H+ as a result of the integration of both intrinsic membrane mechanisms and synaptic mechanisms. Thus, Pre-I neurons could contribute to control of respiratory frequency during central CO2/H+ chemoreception.