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Exchange of Views |
1 Department of Pharmacology, University of Virginia, Charlottesville, VA 22908, USA
Dr Richerson and ourselves agree that central chemoreception (central respiratory chemoreception according to his terminology) is still poorly understood at any level, be it molecular, cellular or integrative (network level). The focus of the present debate is the phenotype of central chemoreceptor candidates. Although we subscribe to the principle that central chemoreception involves multiple clusters of pH-sensitive neurones (Feldman et al. 2003), we still have reservations concerning the theory that serotonergic neurones are chemoreceptors. The reason is that we place the highest premium on electrophysiological results obtained in vivo and we find the evidence that these cells respond appropriately to respiratory acidosis quite insufficient. These final comments must therefore be taken as a call for further experimentation. We express our thanks to Dr Richerson and to the Editor for the opportunity to engage in this constructive exchange of views.
RTN and chemoreception
There is substantial agreement that RTN neurones have properties consistent with chemoreceptors but the theory clearly still needs much elaboration (Mitchell, 2004). The evidence that pH acts directly on RTN neurones is stronger than Dr Richerson et al. acknowledge in the accompanying review. It includes, but is not limited to, the observation that the activation of the cells by pH is unaffected by antagonists of glutamate and P2X purinergic receptors. An essential piece of evidence is that bath alkalization increases the potassium conductance of these neurones in the presence of tetrodotoxin (TTX).
Still, it remains possible that acidification activates these neurones by causing the TTX-independent release of an unidentified factor from nearby non-neuronal cells. Dr Richerson et al. also downplay somewhat the significance of the observation that RTN neurones retain their response to hypercapnia after intrathecal administration of kynurenic acid in vivo. We did not simply ‘reason’ that this drug blocked the respiratory pattern generator. We demonstrated that this was the case by showing that kynurenic acid eliminates phrenic nerve activity and either silences respiratory neurones located in the ventrolateral respiratory group or renders their discharge pattern tonic and virtually insensitive to hypercapnia. The persistence of the response of RTN neurones to hypercapnia under these conditions is therefore highly unusual and suggests strongly that these cells are either directly sensitive to pH or at least are in very close physiological proximity to the pH detectors. Dr Richerson et al. also overestimate the gap that exists between the pH sensitivity of RTN neurones in vivo and in vitro. As shown in our review (Fig. 2), this gap narrows dramatically when the recording temperature is taken into consideration. A similar congruence is notably absent in the case of serotonergic cells. We agree that the ionic mechanisms responsible for the pH sensitivity of RTN neurones need to be more thoroughly investigated. We only showed that alkalization induced a potassium current that was relatively voltage-independent between –120 and –40 mV; the suggestion that TASK channels underlie this current is only one of many possibilities (Mulkey et al. 2004).
Finally, the notion that RTN neurones drive respiration derives mostly from prior and more global observations that impairment of the RTN via microinjection of neuronal depressant drugs or by cooling produces massive decreases in breathing (Feldman et al. 2003). It is logical to explain these global effects by a selective reduction in the activity of the hypercapnia-sensitive neurones of RTN since the rest of the active RTN neurones are presympathetic neurones probably very peripherally involved in breathing, but logic does not constitute proof. In the future it will be crucial to identify the exact neuronal targets of RTN neurones. These targets may include premotor neurones that control pump and airway muscles and may also include neurones that control the parasympathetic outflow to the bronchi (Perez Fontan & Velloff, 1997). Also one should not ignore the possibility raised by others before (Okada et al. 2002) that RTN might harbour several functional classes of chemosensitive neurones just like there are several functional classes of presympathetic blood-pressure regulating neurones in the region of the ventrolateral medulla that overlies RTN (Dampney et al. 2002).
Are serotonergic neurones CO2 detectors and central chemoreceptors?
Dr Richerson et al. consider that the responsiveness of serotonergic cells to hypercapnia in vivo is a solidly established fact (Veasey et al. 1995). On this point, we disagree. With due respect to the authors of this very important and elegant study, they identified only 6 neurones that were activated by CO2 and the serotonergic nature of the responsive cells was not demonstrated. The only identification criteria were a slow regular discharge rate and the fact that the cells were inhibited by a serotonin-1 A receptor agonist given systemically. These criteria were selected on the reasonable assumption that medullary raphe serotonergic neurones should behave like those located in the dorsal raphe but the selected attributes have little specificity. Furthermore, the few cells that were activated by hypercapnia in the Veasey study only responded to a very high level of PCO2. These conditions would undoubtedly have strongly activated peripheral chemoreceptors, which are known to activate subsets of medullary serotonergic neurones (Erickson & Millhorn, 1991). In any event, our own study based on 37 cells, 24 of which were anatomically identified as serotonergic, revealed no activation of these cells by hypercapnia under conditions when RTN neurones were robustly stimulated and peripheral chemoreceptors not involved (Mulkey et al. 2004). As Dr Richerson et al. note, the serotonergic neurones of the medullary raphe display extraordinary phenotypic diversity (Guyenet et al. 2004) and are most likely heterogeneous functionally. The hypothesis that a small group of these cells might be highly sensitive to respiratory acidosis and that this cluster innervates the respiratory network is fascinating but requires proof. Currently available evidence that the midline medullary raphe contains CO2-sensitive cells involved in respiratory control is based on local tissue acidification by CO2 or carbonic anhydrase inhibition. It is a logical but unproven assumption that these procedures mimic the effect of respiratory acidosis on local neurones but there is also evidence to the contrary, at least in the case of carbonic anhydrase inhibitors. For instance, injections of this type of agent into the pre-Bötzinger complex increases respiratory frequency (Solomon et al. 2000), an effect that is arguably unphysiological since, as pointed out by Dr Richerson et al. themselves in this review, stimulation of central chemoreceptors by respiratory acidosis does not increase the breathing rate. In any event, the respiratory effects caused by acidification of the midline raphe could be equally well explained by assuming that the CO2 detectors are nonserotonergic given that the region contains a large variety of cell types. Considering the many effects of serotonin on respiration, the fact that chemical lesions of these cells attenuate central chemoreception does not provide compelling evidence that the serotonergic cells are CO2 detectors. The hypothesis that only a small proportion of serotonergic cells are chemoreceptors could definitely account for our negative results with the parapyramidal raphe but it introduces yet another logical contradiction. Why would so many serotonergic neurones, irrespective of anatomical location (dorsal or medullary raphe) (Richerson et al. 2001; Washburn et al. 2002), respond to acidification in vitro in a manner described by Dr Richerson et al. as exquisitely sensitive whereas so few of these cells display an even marginally detectable response in vivo, even in the absence of anaesthesia (Veasey et al. 1995; Mulkey et al. 2004)? Perhaps this discrepancy illustrates a more general problem that bedevils the field of central chemoreception, namely the difficulty in extrapolating in vitro data to the in vivo situation.
In vivo versus in vitro: is temperature the missing link?
In the preceding review, we have evoked some of the problems created by thick in vitro preparations (acidosis caused by reduced CO2 clearance) or neonate tissue (immature chemoreflexes) in the study of central chemoreception. Another issue is temperature. Cooling the ventral medullary surface is very effective at reducing central chemoreception and breathing (Millhorn et al. 1982). Is it possible that this approach has been so effective because of the unusually high temperature sensitivity of ventral surface chemoreceptors? As shown by the original data that we have incorporated into our invited review, the responsiveness of RTN neurones to pH is very temperature-sensitive (estimated Q10 of at least 3). At 35°C in vitro, their pH sensitivity (2 Hz per 0.1 pH unit; Fig 2 of accompanying invited review) is quite close to their estimated value in vivo (4 Hz per 0.1 unit change in arterial pH). The discrepancy may even be smaller given that in vivo recordings were done at a still higher temperature (37.5 °C) and the literature suggests that hypercapnia may produce slightly larger changes in brain pH that in arterial pH. Remarkably, the pH at which RTN cells become silent in vitro (approximately pH 7.5) is unchanged by temperature and therefore only the dynamic range of their response to pH is affected. Perhaps closer attention should be paid to the effect of temperature on the pH sensitivity of brainstem neurones recorded in vitro. Conceivably, the difference between chemoreceptors and neurones exhibiting insignificant responses to pH might become much more obvious at physiological temperature.
References
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