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In mammals, an increase in arterial CO2 partial pressure (PCO2) is the most powerful stimulus for ventilation, acting through the peripheral and central chemoreceptors (for reviews, see Nattie, 1999; Richerson et al. 2001). The prevailing view is that changes in arterial PCO2 produce their effects by inducing changes in the intracellular pH (pHi) of chemoreceptor cells (Peers & Buckler, 1995; Wiemann et al. 1998; Filosa et al. 2002; Wang et al. 2002). The peripheral chemoreceptors are in the carotid and aortic bodies, which contain chemosensitive type I or glomus cells with neuronal properties. The identities of the central chemoreceptor neurones have not yet been unequivocally defined. Chemosensitive neurones are present in many brainstem nuclei that are linked to respiratory control, including the ventrolateral medulla (VLM), nucleus of the tractus solitarius (NTS), medullary raphe, locus coeruleus, and the hypothalamus (Richerson, 1998; Nattie, 1999). Indeed, in many of these regions, inducing local acidosis causes ventilation to increase (Nattie, 1999). It remains to be proven which of these chemosensitive neurones are responsible for the normal ventilatory response to small, physiological changes in pH/CO2, but accmulating evidence suggests that serotonergic neurones within the medullary raphe nuclei are likely to play an important role (Richerson et al. 2001; Wang et al. 2001; Richerson, 2004). Most types of cells, when subjected to a respiratory acid–base disturbance (a pH change produced by a change in PCO2) or a metabolic acid–base disturbance (a pH change produced by a change in [HCO3−] at a fixed PCO2), exhibit a pHi change that is 20–30% as large as the change in extracellular pH (pHo) (Ellis & Thomas, 1976; Vaughan-Jones, 1986; Tolkovsky & Richards, 1987; Glunde et al. 2002). By contrast, the chemosensitive type I cells of the carotid body respond to the above-mentioned acid–base disturbances with unusually large changes in pHi (Buckler et al. 1991), having a ΔpHi/ΔpHo ratio of 60–70% without recovery during sustained exposure. Similarly high ΔpHi/ΔpHo ratios have also been reported in subsets of neurones in the NTS and locus coeruleus (Richerson, 1998; Ritucci et al. 1998; Filosa et al. 2002), two regions that contain putative central chemoreceptor neurones, as well as in chemoreceptor neurones of the pulmonate terrestrial snail (Goldstein et al. 2000). Consult the review by Putnam (2001) for a discussion of pHi regulation in neurones in chemosensitive brain regions, and the review by Chesler (2003) for a more general discussion of pH regulation in the brain. The above observations have led to the concept that the absence of a pHi recovery ensures that the primary stimulus – intracellular acidosis – continues to drive ventilation as long as the respiratory acidosis persists (i.e. the steady-state ΔpHi/ΔpHo is large). The implicit assumption is that, when subjected to sustained respiratory acidosis, the normal response of non-chemosensitive neurones is to return pHi nearly to baseline levels in an attempt to stabilize the intracellular milieu (i.e. the steady-state ΔpHi/ΔpHo is small). In determining whether putative central chemoreceptor neurones have a unique pHi response to extracellular acid–base disturbances, it is important to compare this response to that of non-chemosensitive neurones. Such an analysis is the purpose of the present study, in which we have examined the pHi response to acid–base disturbances in neurones from the medullary raphe versus the hippocampus. We chose to work with cultured neurones because (1) the preparation is ideal for monitoring pHi while changing extracellular composition rapidly and effectively, and (2) cultured medullary raphe neurones retain the electrophysiological response to acidosis first identified in slices (Wang et al. 1998). Thus, working with cultured neurones would allow us to compare our pHi data directly with electrophysiological data previously obtained on cultured medullary raphe and hippocampal neurones under the same conditions (Wang & Richerson, 2000). In culture, a subset (∼20%) of medullary raphe neurones responds to respiratory acidosis (a shift from 5% to 9% CO2) by tripling their firing rate; these neurones are all serotonergic (Richerson, 1995; Wang et al. 2001). A second subset of medullary raphe neurones (∼20%) responds to respiratory acidosis in the opposite manner, decreasing firing rate to an average of 20–50% of control (Wang et al. 2001); none of these neurones is serotonergic. These two classes of medullary raphe neurones are excellent candidates for contributing to the physiological ventilatory response because of their extreme pH sensitivity. In addition, the chemosensitive serotonergic neurones are closely apposed to large arteries in the ventral medulla, where they could faithfully monitor arterial PCO2 (Bradley et al. 2002). A third subset (∼60%) of medullary raphe neurones does not change firing rate in response to acidosis. Here we compared the pHi responses of medullary raphe neurones with those of hippocampal neurones, none of which exhibit a large change in firing rate in response to acidosis (Wang & Richerson, 2000). We know of no systematic studies of the effects of respiratory and metabolic acidosis/alkalosis on steady-state pHi in either medullary raphe or hippocampal neurones. Working on organotypic cultures of rat medullary neurones, Wiemann and colleagues (Wiemann et al. 1998, 1999; Wiemann & Bingmann, 2001) found that respiratory acidosis generally causes a modest but sustained fall in pHi. In the present study, we used the fluorescent pH-sensitive dye BCECF and a video-imaging system to monitor pHi in primary neuronal/glial cultures subjected to extracellular respiratory acidosis and alkalosis as well as extracellular metabolic acidosis and alkalosis. In some experiments, we used immunocytochemistry to determine which neurones studied in the imaging experiments were serotonergic. We made the surprising observation that, in response to respiratory acid–base disturbances, as well as metabolic alkalosis, all medullary raphe and hippocampal neurones have similarly large ΔpHi/ΔpHo values, and neither group exhibits a significant pHi recovery during any of these three stresses. Moreover, among medullary raphe neurones, the responses of serotonergic and non-serotonergic neurones to respiratory disturbances were indistinguishable. The major exception to this pattern of uniformity was the response to metabolic acidosis: about 80% of medullary raphe neurones underwent a large, sustained, and reversible pHi decrease; the rest exhibited only a small pHi decrease. Conversely, less than 30% of hippocampal neurones underwent the large, sustained and reversible response; the majority exhibited only a small pHi decrease from which they rebounded after the removal of the insult. Based on our comparison of medullary raphe and hippocampal neurones, we conclude that a high ΔpHi/ΔpHo and a lack of pHi recovery are not unique to chemosensitive neurones.

Neurokinin-1 receptor (NK1R)-expressing neurones that are involved in chemoreception at the retrotrapezoid nucleus (Nattie & Li, 2002b) are also prominent at locations that contain medullary serotonergic neurones, which are chemosensitive in vitro. In medullary regions containing both types, we evaluated their role in central chemoreception by specific cell killing. We injected (2×100 nl) (a) substance P–saporin (SP-SAP; 1μm) to kill NK1R-expressing neurones, (b) a novel conjugate of a monoclonal antibody to the serotonin transporter (SERT) and saporin (anti-SERT-SAP; 1μm) to kill serotonergic neurones, or (c) SP-SAP and anti-SERT-SAP together to kill both types. Controls received IgG-SAP injections (1μm). There was no double-labelling of NK1R-immunoreactive (ir) and tryptophan-hydroxylase (TPOH)-ir neurones. Cell (somatic profile) counts showed that NK1R-ir neurones in the SP-SAP group were reduced by 31%; TPOH-ir neurones in the anti-SERT-SAP group by 28%; and NK1R-ir and TPOH-ir neurones, respectively, in the combined lesion group by 55% and 31% (P < 0.001; two-way ANOVA; P < 0.05, Tukey's post hoc test). The treatments had no significant effect on sleep/wake time, body temperature, or oxygen consumption but all three reduced the ventilatory response to 7% inspired CO2 in wakefulness and sleep by a similar amount. SP-SAP treatment decreased the averaged CO2 responses (3, 7 and 14 days after lesions) in wakefulness and sleep by 21% and 16%, anti-SERT-SAP decreased the responses by 15% and 18%, and the combined treatment decreased the responses by 12% and 12% (P < 0.001; two-way ANOVA; P < 0.05, Tukey's post hoc test). We conclude that separate populations of serotonergic and adjacent NK1R-expressing neurones in the medulla are both involved in central chemoreception in vivo.

Sudden unexpected death in epilepsy (SUDEP) is the leading cause of death in patients with refractory epilepsy. Defects in central control of breathing are important contributors to the pathophysiology of SUDEP, and serotonin (5-HT) system dysfunction may be involved. Here we examined the effect of 5-HT neurone elimination or 5-HT reduction on seizure risk and seizure-induced mortality. Adult Lmx1b(f/f/p) mice, which lack >99% of 5-HT neurones in the CNS, and littermate controls (Lmx1b(f/f)) were subjected to acute seizure induction by maximal electroshock (MES) or pilocarpine, variably including electroencephalography, electrocardiography, plethysmography, mechanical ventilation or pharmacological therapy. Lmx1b(f/f/p) mice had a lower seizure threshold and increased seizure-induced mortality. Breathing ceased during most seizures without recovery, whereas cardiac activity persisted for up to 9 min before terminal arrest. The mortality rate of mice of both genotypes was reduced by mechanical ventilation during the seizure or 5-HT2A receptor agonist pretreatment. The selective serotonin reuptake inhibitor citalopram reduced mortality of Lmx1b(f/f) but not of Lmx1b(f/f/p) mice. In C57BL/6N mice, reduction of 5-HT synthesis with para-chlorophenylalanine increased MES-induced seizure severity but not mortality. We conclude that 5-HT neurones raise seizure threshold and decrease seizure-related mortality. Death ensued from respiratory failure, followed by terminal asystole. Given that SUDEP often occurs in association with generalised seizures, some mechanisms causing death in our model might be shared with those leading to SUDEP. This model may help determine the relationship between seizures, 5-HT system dysfunction, breathing and death, which may lead to novel ways to prevent SUDEP.

Sleep apnoea is a common disorder that leads to a variety of sequelae, including hypertension, heart failure, stroke and hypersomnolence. Unfortunately, the therapies are not ideal. Standard treatment is continuous positive airway pressure (CPAP) given to patients while they are sleeping. However, the mask is often not well tolerated, so many patients are not compliant with treatment. There is considerable interest among the scientific community in the neural mechanisms of respiratory control during sleep, in part because it is justifiably believed that defining these mechanisms may help to identify new treatments for sleep apnoea. The most obvious intervention would be to selectively increase respiratory output during sleep, and especially to increase upper airway dilatation. Long term facilitation (LTF) of breathing offers a window into a potential way to accomplish this goal. LTF is an increase in ventilation or of respiratory motor output (e.g. phrenic nerve activity) that is induced by a short episode of intermittent hypoxia, but that continues for a long period of time (>90 min) after return to normoxia. It is due in some cases to plasticity of the carotid body, and in other cases to plasticity of neurons involved in respiratory control. A lot of attention has been focused on LTF in part because it may be possible to selectively target the pathways involved in this respiratory plasticity for treatment. LTF has been widely studied, but remains poorly understood. Rather than being a single entity, this term encompasses phenomena that share some features, but differ in other respects. As referred to above, one form of LTF occurs within the carotid body, and is due to changes intrinsic to glomus cells that alter their response to hypoxia (Peng et al. 2003). A different form of LTF occurs as a result of strengthening of the synapse from respiratory pre-motor neurons onto phrenic motor neurons. It involves a serotonin-dependent increase in BDNF generation (Mahamed & Mitchell, 2007), and shares mechanisms with synaptic plasticity seen in hippocampal long-term potentiation (Richerson & Bekkers, 2004). This form of LTF is typically studied in anaesthetized rats (Mahamed & Mitchell, 2007), and is manifest as an increase in phrenic nerve output that does not begin until after a long delay (often >30 min). In contrast, the LTF that has been described in awake humans is manifest as an increase in ventilation that occurs immediately after termination of hypoxia, and may actually be a continuation of ‘progressive augmentation’, which begins during the hypoxia. These differences in time course suggest that the mechanisms of these forms of LTF may be very different. Thus, different forms of LTF require different patterns of intermittent hypoxia to elicit them, occur in different loci, and utilize different mechanisms. A number of important questions remain about LTF. What is the physiological significance of LTF? What are the optimal ways to elicit each of the forms of LTF? Which mechanisms are shared and which differ? And of particular significance to treatment of patients, is LTF adaptive or maladaptive? In a recent issue of The Journal of Physiology, Lee et al. (2009) present new data that intersect with many of these questions about LTF. They studied human patients with sleep apnoea, who presumably subject themselves to chronic intermittent hypoxia (CIH) at night, and compared them to healthy subjects. Both study groups had LTF in response to acute intermittent hypoxia while they were awake, but it was more robust in the sleep apnoea patients. Interestingly, pretreatment with antioxidants reduced LTF in the sleep apnoea patients to the level seen in the controls, suggesting that CIH enhanced LTF due to generation of reactive oxygen species (ROS) by chronic hypoxia that occurred at night. Generation of ROS is usually considered bad for you, so a natural conclusion is that treatment of sleep apnoea patients with antioxidants should be good for you. But shouldn't the increase in respiratory output seen with LTF be helpful in preventing hypoxia during sleep? As the authors point out, it may not be that simple. In some ways LTF may be helpful (Mahamed & Mitchell, 2007), but in other ways it may be maladaptive (Mateika & Narwani, 2009). These data point to some exciting possibilities for new treatments, but first require a better understanding of the mechanisms involved. There are several issues about LTF in awake humans that still need to be addressed. For example, the authors note that mild hypoxia may have had different effects on ROS than severe hypoxia. Those with severe hypoxia may induce a different form of LTF from those with mild hypoxia. Is the role of ROS different in the two cases? In this study the authors used supplemental CO2 under baseline conditions, because LTF is not elicited when patients are normocapnic. Why is there a dependence on CO2? Is this related to the fact that serotonin has a permissive effect on LTF, and hypercapnia is needed in order to increase serotonin release (Richerson, 2004)? How do studies in human patients given supplemental CO2 relate to the situation when patients are sleeping normally while breathing room air? What will be needed is data that examine whether it is better to treat patients with drugs that prevent LTF or enhance it, and whether it is better to increase or decrease ROS. Answering these questions may allow us to bypass hypoxia and directly target the pathways involved – ideally downstream of ROS generation – to specifically prevent apnoeas and augment motor output to the upper airway muscles. Accomplishing this goal could free many patients from the hassle of wearing a mask every night, and prevent sequelae in the many other patients who are non-compliant with CPAP.

The medullary raphe nuclei contain putative central respiratory chemoreceptor neurones that are highly sensitive to acidosis. To define the primary stimulus for chemosensitivity in these neurones, the response to hypercapnic acidosis was quantified and compared with the response to independent changes in P(CO2) and extracellular pH (pH(o)). Neurones from the ventromedial medulla of neonatal rats (P0-P2) were dissociated and maintained in tissue culture for long enough to develop a mature response (up to 70 days). Perforated patch clamp recordings were used to record membrane potential and firing rate while changes were made in pH(o), P(CO2) and/or [NaHCO(3)](o) from baseline values of 7.4, 5 % and 26 mM, respectively. Hypercapnic acidosis (P(CO2) 9 %; pH(o) 7.17) induced an increase in firing rate to 285 % of control in one subset of neurones ('stimulated neurones') and induced a decrease in firing rate to 21 % of control in a different subset of neurones ('inhibited neurones'). Isocapnic acidosis (pH(o) 7.16; [NaHCO(3)](o) 15 mM) induced an increase in firing rate of stimulated neurones to 309 % of control, and a decrease in firing rate of inhibited neurones to 38 % of control. In a different group of neurones, isohydric hypercapnia (9 % P(CO2); [NaHCO(3)](o) 40 mM) induced an increase in firing rate of stimulated neurones by the same amount (to 384 % of control) as in response to hypercapnic acidosis (to 327 % of control). Inhibited neurones also responded to isohydric hypercapnia in the same way as they did to hypercapnic acidosis. In Hepes-buffered solution, both types of neurone responded to changes in pH(o) in the same way as they responded to changes in pH(o) in bicarbonate-buffered Ringer solution. It has previously been shown that all acidosis-stimulated neurones in the medullary raphe are immunoreactive for tryptophan hydroxylase (TpOH-ir). Here it was found that TpOH-ir neurones in the medullary raphe were immunoreactive for carbonic anhydrase type II and type IV (CA II and CA IV). However, CA immunoreactivity was also common in neurones of the hypoglossal motor nucleus, inferior olive, hippocampus and cerebellum, indicating that its presence is not uniquely associated with chemosensitive neurones. In addition, under the conditions used here, acetazolamide (100 microM) did not have a significant effect on the response to hypercapnic acidosis. We conclude that chemosensitivity of raphe neurones can occur independently of changes in pH(o), P(CO2) or bicarbonate. The results suggest that a change in intracellular pH (pH(i)) may be the primary stimulus for chemosensitivity in these putative central respiratory chemoreceptor neurones.