Exp Physiol 90.

3 pp 259269 259

Experimental Physiology Exchange of Views

Homing in on the specific phenotype(s) of central

respiratory chemoreceptors
G. B. Richerson1,2,3 , W. Wang1 , M. R. Hodges1 , C. I. Dohle1 and A. Diez-Sampedro1
1
Departments of Neurology and Cellular & Molecular Physiology, Yale University School of Medicine, New Haven, CT, USA
2
Veterans Affairs Medical Center, West Haven, CT, USA
3
Neurology, LCI-712, 15 York St, PO 208018, New Haven, CT 06520-8018, USA

To some it may seem that we now know less about respiratory chemoreception than we did
20 years ago. Back then, it was widely accepted that the central respiratory chemoreceptors
(CRCs) were located exclusively on or near the surface of the ventrolateral medulla (VLMS).
Now, instead, it is generally believed that there are widespread sites of chemoreception, and
there is little agreement on when and how each of these sites is involved in respiratory control.
However, those in the field know that this actually is progress, primarily because we have gone
from simply identifying candidate regions, to identifying specific neuronal subtypes that may be
the sensors. In this invited review, we have been asked to discuss some of the current controversies
in the field. First, we define the minimal requirements for a cell to be a CRC, and what assumptions
can not be made without more data. Then we review the evidence that two neuronal subtypes,
serotonergic neurones of the midline raphe and glutamatergic neurones of the retrotrapezoid
nucleus, are chemoreceptors. There is evidence supporting a role in respiratory chemoreception
for both types of neurone, as well as the other candidates, but there is also information that is
missing. Future work will need to focus on which of the candidates are indeed chemoreceptors,
what percentage of the overall response each one contributes, and how this percentage varies
under different conditions.
(Received 6 January 2005; accepted after revision 22 February 2005; first published online 25 February 2005)
Corresponding author G. B. Richerson: Neurology, LCI-712, 15 York St, PO 208018, New Haven, CT 06520-8018, USA.
Email: george.richerson@yale.edu

In recent years, the field of respiratory neurobiology
has gone from only having a rough idea about where a
subset of the central respiratory chemoreceptors (CRCs)
are located (somewhere near the VLMS) to identifying
numerous candidates for the specific neurones involved
(most of which are not near the VLMS). These candidates
include serotonergic neurones of the midline raphe
(Richerson, 2004), noradrenergic neurones of the locus
coeruleus (Pineda & Aghajanian, 1997; Putnam et al.
2004) and glutamatergic neurones of the retrotrapezoid
nucleus (Mulkey et al. 2004). Other candidate neurones
without known neurotransmitter content are located
in the nucleus tractus solitarius, preBotzinger complex
(PBC), hypothalamus and cerebellum (Dean et al. 1990;
Richerson, 1998; Feldman et al. 2003; Putnam et al.
2004). There are also non-serotonergic neurones in the
midline raphe that are inhibited by acidosis that have
been proposed to disinhibit respiratory output during
hypercapnia (Wang et al. 2001; Richerson, 2004).
Properties that all central respiratory chemoreceptors
must possess
There are two essential criteria that all chemoreceptors
must meet (Richerson, 1998; Putnam et al. 2004).
Intrinsic chemosensitivity to physiologically relevant
changes in P CO2 . A CRC must respond to changes in
CO2 that occur under non-pathological conditions in vivo,
and this response must be due to mechanisms intrinsic
to that cell. To prove that the response is intrinsic, all
influences from other cells must be eliminated. While
theoretically straightforward, this is not so easy in practice.
The only method that guarantees this is to physically
separate the candidate from all other cells. Of course,
this could eliminate modulatory effects that enable a
CRC to express intrinsic chemosensitivity. An alternative
is to combine a less complete method for physical
isolation (e.g. brain slices) with pharmacological blockade

C The Physiological Society 2005 DOI: 10.1113/expphysiol.2005.029843
260 G. B. Richerson and others Exp Physiol 90.3 pp 259269

of synaptic transmission. However, interconnections can
remain intact in brain slices (Richerson, 1998), and
none of the commonly used pharmacological methods is
complete. For example, lowering calcium and increasing
magnesium is not effective in blocking non-vesicular
neurotransmitter release or electrical transmission via gap
junctions, and the typical approach of applying glutamate
and GABA receptor antagonists does not block other forms
of neurotransmission.
Appropriate effects on respiratory output. A CRC must
have the capability of increasing respiratory output in
response to an increase in CO2 . This could be accomplished
if the neurone is part of the respiratory network,
or if it projects to respiratory neurones and releases
neurotransmitters that influence respiratory output.
Assumptions that cannot be made about central
respiratory chemoreceptors
Although assumptions are often made about the following
properties of CRCs, they cannot be substantiated until the
CRCs have been unequivocally identified.
Primary stimulus. It is often assumed that the primary
stimulus for central chemoreception is either intracellular
pH or extracellular pH, and there is good evidence
for this in some CRC candidates (Wang et al. 2002;
Putnam et al. 2004), but it remains possible that
some CRCs respond instead to HCO3 or molecular
CO2 .
Type of response to acidosis. It is often assumed that the
CRCs are neurones stimulated by acidosis and in turn
stimulate breathing, but a CRC could also be inhibited by
acidosis and then in turn release respiratory output from
tonic inhibition (Wang et al. 2001). There is a precedent
for such inhibitory respiratory chemoreceptors in reptiles
and birds (Powell et al. 1988). It is also possible that some
CRCs are glia.
Degree of chemosensitivity. At the systems level,
respiratory output is extremely sensitive to changes in
P CO2 , so it is often assumed that each individual CRC
neurone is equally sensitive. However, this is not necessary
because there are many opportunities for the response to
be amplified within the respiratory network. For example,
many neurones contain co-localized neuropeptides that
are only released after firing rate increases to higher levels,
and neuropeptides can have larger effects on postsynaptic
neurones than the primary neurotransmitter. There are
also other mechanisms that could lead to amplification
downstream, including divergence, convergence and
distributed chemosensitivity (Fig. 1).
Thus, it is tempting to believe that CRCs are extremely
sensitive to small changes in pH, but a neurone cannot
be excluded as a CRC simply because it has a small
response to acidosis. It is probably true that the degree
of chemosensitivity is an important factor in determining
the relevance of chemosensitivity of a CRC candidate to
respiratory control (Richerson, 1998; Putnam et al. 2004),
but none of the candidates can be excluded as long as they
respond to physiologically relevant changes in pH.

A B C
CRC #1 PBC
PBC
+26% +26%

CRC CRC #2 CRC PreM

PreM Resp
+26% +26% +26% +26%

PMN CRC #3 PMN

+26%

+100% +100% +100%

Figure 1. Potential mechanisms of downstream amplification of the response to CO2 include divergence,
convergence and distributed chemosensitivity
A, divergence: a single set of CRCs may have widely divergent projections. For example, a single pool of CRCs
might project to the PBC, premotor neurones (PreM) and phrenic motor neurones (PMNs). If the CRCs respond with
a 26% increase in firing, and stimulate each of these three sites by that amount, then the output of the system
would be doubled = (1.26)3 . B, convergence: three sets of CRCs might all converge onto a single respiratory
neurone (Resp). If each one increases their firing by 26%, and stimulates the target by that amount, then this
would also lead to a doubling of output. C, distributed chemosensitivity: if CRCs are stimulated by 26%, and they
project to PBC neurones that are also stimulated by 26%, and these in turn project to premotor neurones that are
stimulated by 26%, then the output of the system as a whole would also be doubled. Divergence, convergence and
distributed chemosensitivity are not mutually exclusive. Thus, the system as a whole could be extremely responsive
if all three mechanisms are employed, even if each individual neurone has only a modest response.

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Exp Physiol 90.3 pp 259269 Phenotype(s) of respiratory chemoreceptors
Compartment sensed. Ventilation increases in response
to acidic solutions in the cerebrospinal fluid (CSF) space,
but that does not mean that the CRCs were necessarily
designed specifically to sample this compartment. An
alternative is that CRCs sample tissue pH near large
arteries. As Virchow-Robin spaces that surround large
penetrating arteries are filled with CSF, experimental
changes in CSF pH would alter pH along them.
This possibility is consistent with the conclusion of
Pappenheimer et al. (1965) that the CRCs are located two-
thirds to three-fourths of the distance along the functional
[gradient of pH] between CSF and blood.
Transduction mechanism. Many ion channels are sensitive
to changes in pH (Putnam et al. 2004). Although some have
been linked to the response of individual CRC candidates,
none have been shown to be present exclusively in CRCs,
or to be critical for central respiratory chemoreception at
the systems level.
Projections to specific respiratory nuclei. In principle,
CRCs could target any or all respiratory nuclei. It would be
possible to conclude that the respiratory pattern generator
was a target of CRCs if hypercapnia consistently changed
respiratory rate after the pulmonary stretch receptors were
eliminated by vagotomy. However, this is usually not the
case. Instead, the existing data are equally consistent with
CRCs influencing respiratory output by stimulating either
the respiratory pattern generator, premotor neurones,
respiratory motor neurones, or all three of these cell
groups.
Uniqueness of chemoreceptors to respiratory control.
The term central chemoreceptor is frequently used
to describe neurones that drive respiratory output in
response to hypercapnia. However, the term central
respiratory chemoreceptor has been used in this review
because this is a more accurate description of these
neurones. The term central chemoreceptor should be
more correctly used in a broader sense to describe any
neurone that responds to changes in CO2 or pH and
modulates any brain function, not just breathing. There are
many non-respiratory brain functions that are sensitive to
hypercapnia. As many of these have a higher threshold
for activation (e.g. anxiety), it is likely that central
chemoreceptors that induce these non-respiratory effects
are activated at higher levels of CO2 .
Evidence for central respiratory chemoreceptors
in the raphe and retrotrapezoid nucleus
Serotonergic neurones of the raphe. The evidence in
support of serotonergic neurones being CRCs has recently


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261
been reviewed in detail (Richerson, 2004), and is
highlighted here.
When neurones of the rat medullary raphe are isolated
and grown in tissue culture, the majority of those that
are serotonergic (73%) are highly chemosensitive to
physiologically relevant changes in pH. Their response is
maintained after blocking glutamate and GABA receptors
(Wang et al. 2001). Although tissue culture potentially
alters the properties of neurones, the same response
also occurs in serotonergic neurones of the midline in
rat medullary slices, and is maintained in both high-
magnesium/low-calcium solution and in the presence of
glutamate and GABA receptor antagonists (Richerson,
1995; Wang & Richerson, 1999; Bradley et al. 2002). The
intrinsic response of these neurones is extremely high,
with a mean increase in firing rate to 300% of control
in response to a decrease in extracellular pH from 7.4 to
7.2 (Wang et al. 1998, 2001, 2002). The magnitude of this
response appears to be large compared to that of the other
CRC candidates (Putnam et al. 2004, 2005). In addition,
this intrinsic chemosensitivity does not appear in rat raphe
neurones in vitro until they are at least 12 days old (Wang &
Richerson, 1999), which is the age at which the ventilatory
response to hypercapnia begins to mature in the rat in vivo
(Serra et al. 2001; Stunden et al. 2001), consistent with
the pH response of raphe neurones contributing to the
response in the intact animal.
A subset of serotonergic medullary raphe neurones (six
of 27; 22%) increase their firing rate in awake, behaving
cats to 160% of control in response to inhalation of 8%
CO2 (Veasey et al. 1995). The response of these neurones in
vivo is blunted during sleep, as is the ventilatory response of
the animal. When the response of these neurones in vivo is
compared to predicted changes in tissue pH (Fig. 2), there
are three notable observations: (1) levels of hypercapnia
sufficient to strongly stimulate ventilation in vivo (fraction
of inspired CO2 (F iCO2 ) = 0.050.07) induce changes in
tissue pH that are small compared to those used in even the
most conservative in vitro experiments (Wang et al. 2002);
(2) some serotonergic neurones are sensitive to very small
changes in pH in vivo; and (3) some serotonergic neurones
that are sensitive to hypercapnia in cats in vivo would not
have been detected if F iCO2 was limited to 0.07.
Serotonergic neurones in the midline have dendrites
that are closely associated with the basilar artery and its
penetrating branches, coming within 0.5 m of the lumen
of these large arteries (Bradley et al. 2002). As tissue
pH in the raphe nuclei more closely reflects changes in
arterial pH in response to hypercapnia than many other
medullary nuclei (Lamanna et al. 2003), the location of
these neurones would help them to monitor better the
effectiveness of ventilation.
Serotonergic neurones of the raphe project widely to
medullary respiratory neurones, as well as phrenic and
hypoglossal motor neurones (Richerson, 2004). These
262 G. B. Richerson and others
neurones release serotonin, substance P and thyrotropin-
releasing hormone, all three of which stimulate breathing
in vivo, promote respiratory output of the PBC in vitro,
and enhance excitability of respiratory motor neurones in
vitro (Richerson, 2004). Blockade of serotonin receptors
also blocks respiratory bursts generated by the PBC in vitro
(Richerson, 2004).
Ventilation increases when acidosis is induced
selectively within the medullary raphe of rats and goats
in vivo by focal application of either acetazolamide or CO2
(Feldman et al. 2003; Hodges et al. 2004b). Selective lesions
of serotonergic neurones with intraventricular or focal
injections of 5,7-dihydroxytryptamine, or focal injections
of saporin conjugated to an antibody to the serotonin
transporter, lead to a reduction of the ventilatory response
to hypercapnia in vivo (Nattie et al. 2004; Richerson, 2004).
Figure 2. Serotonergic neurone firing rate in vivo (from Veasey
et al. 1995) compared to brain acidbase status compiled from
the work of three different investigators
A, measured changes in arterial P CO2 (P a,CO2 ) and brain pH in response
to various levels of inspired CO2 . Shown are measurements of P a,CO2
(
) and CSF pH () in rats (Siesjo et al. 1972), tissue pH in goats ( e)
(Hodges et al. 2004a) and tissue pH in rats (
) (Li & Nattie, 2002).
Tissue pH was measured as a change from baseline, so those points
are normalized to the same pH as CSF at an inspired CO2 of 0%.
B, changes in firing rate of six serotonergic neurones of the medullary
raphe in response to changes in CO2 . From Veasey et al. (1995).
Exp Physiol 90.3 pp 259269
Thus, there is now compelling evidence, accumulated
from a variety of both in vivo and in vitro approaches,
in support of serotonergic neurones within the midline
raphe being CRCs (Feldman et al. 2003; Richerson, 2004).
However, there are many questions that remain, including
how and when they play a role in the intact animal.
Serotonergic neurones of the ventolateral medulla
(VLM). There is heterogeneity of serotonergic neurones,
including differences in location, cell shape, projections
and neuropeptide content. There is also heterogeneity
of their intrinsic chemosensitivity, as only 73% of
medullary midline (i.e. raphe) serotonergic neurones
meet established criteria for intrinsic chemosensitivity
(Wang et al. 2001), and among those, there is substantial
variability in the degree of chemosensitivity (Wang et al.
1998, 2002). Although there is chemosensitivity in a subset
of serotonergic neurones from the two regions examined
to date (medullary raphe and dorsal raphe), there is no
evidence that every serotonergic neurone in the CNS is a
CO2 /pH sensor. However, this remains a viable hypothesis.
The intrinsic response of serotonergic neurones in the
parapyramidal region of the VLM has not yet been studied.
However, as these neurones are closely associated with
large arteries, and a subset of them project to respiratory
neurones within the medulla, as well as phrenic motor
neurones in the spinal cord (Richerson, 2004), it is possible
that they are also CRCs, and that they contributed to
localization of chemoreceptors to the surface of the VLM
during early experiments (Loeschcke, 1982).
Recently, Guyenet and coworkers concluded that VLM
serotonergic neurones are not CRCs. In this review, we have
been asked to critique the evidence for that conclusion.
Their assessment was based on two observations. The
first was that only a small percentage of tryptophan
hydroxylase-immunoreactive neurones in the marginal
layer (ML) of the VLMS project to the PBC and rostral
ventral respiratory group (rVRG) (Weston et al. 2004).
The authors state that the serotonergic neurones of the
ML probably do not contribute to central [respiratory]
chemoreception, and instead mediate sympathetic
autonomic adjustments to [acidification of the ventral
medullary surface]. This possibility is consistent with our
previous hypothesis that many CNS serotonergic neurones
are central chemoreceptors that mediate non-respiratory
changes in response to hypercapnia (Richerson, 2004).
However, it is premature to conclude that none of
the serotonergic neurones on the VLMS are CRCs for
the follow reasons: (1) the subset of VLM serotonergic
neurones that do project to the PBC and rVRG could
be CRCs; (2) the serotonergic neurones in the ML that
project to the spinal cord (Weston et al. 2004) may
be CRCs that stimulate phrenic motor neurones; and
(3) some ML serotonergic neurones may project to other
medullary respiratory nuclei that were not assessed, such as


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Exp Physiol 90.3 pp 259269 Phenotype(s) of respiratory chemoreceptors
the nucleus tractus solitarius, hypoglossal motor nucleus,
and caudal VRG.
The second observation was that out of 37 recordings
from serotonergic neurones in the parapyramidal region of
anaesthetized rats in vivo, none increased their firing rate
by more than 30% in response to an increase in end-tidal
P CO2 from 5% to 10% (Mulkey et al. 2004). The conclusion,
however, that none of the serotonergic neurones in the
VLM are CRCs cannot be justified, in our opinion, by
these data alone, for the following reasons.
(1) Anaesthesia may have suppressed the response of
serotonergic neurones, as it does the ventilatory response
to hypercapnia (Pavlin & Hornbein, 1986). (2) There is
no evidence that all CRCs have an extremely large response
to hypercapnia in vivo. Of the 37 serotonergic neurones,
19 actually did increase their firing rate above 0%, and
it cannot be assumed that their response has no effect
on respiratory output (see above). (3) In unanaesthetized
cats, some CO2 -responsive serotonergic neurones require
an F iCO2 of 0.080.09 for activation to be detected (Fig. 2B),
which roughly corresponds to an end-tidal CO2 of 10%
(Fig. 2A). As rats have a much smaller hypercapnic
ventilatory response than cats (Putnam et al. 2004), some
chemosensitive serotonergic neurones in rats may require
a larger stimulus, especially during anaesthesia. Although
a high level of CO2 may be required for activation to be
detected, lower levels of CO2 may increase firing in these
neurones enough to have a sizeable effect on breathing
if there are downstream mechanisms of amplification
employed (Fig. 1). (4) It is difficult to interpret data
from neurones within an intact neural network. There
are extensive inhibitory connections between serotonergic
neurones, and when one subset is activated by hypercapnia
they may inhibit others that are less sensitive. (5) Lastly,
the data were obtained from only a limited subset of VLM
serotonergic neurones. All but four were from neurones
deep to the surface, but there may be differences between
those on the surface and those in the parenchyma, as well
as those at different rostro-caudal levels of the medulla.
Mulkey et al. (2004) also made the conclusion that
the collective evidence does not support the view that
serotonergic neurones in general, or the serotonergic cells
located at the VLMS in particular, are CO2 detectors
in vivo. This conclusion, however, is not addressed
specifically by the authors data, because observations
on a small subset of VLM serotonergic neurones, which
are a small minority of medullary serotonergic neurones,
are not necessarily relevant to all serotonergic neurones,
including those in the raphe nuclei. Recordings are needed
from each of the subsets of serotonergic neurones over
a larger range of CO2 levels. This protocol must also be
performed in unanaesthetized animals, and in different
behavioural states, particularly because sleep may shift the
dominant CRC (Nattie, 1999; Mitchell, 2004). To date, this
type of approach has been limited to midline serotonergic


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263
neurones, and only those in the medulla that are deep to
the ventral surface (Veasey et al. 1995). Thus, there are no
published data yet that directly address the possibility that
the majority of serotonergic neurones are CO2 sensors in
vivo.
Glutamate neurones of the retrotrapezoid nucleus. The
retrotrapezoid nucleus (RTN) was first recognized as
potentially being involved in control of breathing because
it projects extensively to respiratory nuclei (Smith et al.
1989). Later, Nattie et al. (1993) demonstrated that a
large percentage of neurones in the RTN are stimulated
by hypercapnia in decerebrate cats. When Li & Nattie
(1997) then demonstrated that ventilation was increased
in response to CO2 dialysis in the RTN of rats in vivo it
became clear that the RTN might contain CRCs.
In this review, we have been asked to examine recent
evidence, provided by Mulkey et al. (2004), for the
possibility that the RTN contains glutamatergic CRCs.
They recorded from the RTN in anaesthetized rats in
vivo, and confirmed the work of Nattie et al. (1993)
that there is a subset of neurones that increases their
firing rate in response to hypercapnia. They then showed
that these neurones continue to have a robust response
to hypercapnia after application of kynurenic acid (a
glutamate receptor antagonist) to the ventricular and
subarachnoid spaces, or after intravenous injection of
morphine. It was reasoned that kynurenic acid or
morphine blocked the respiratory pattern generator,
and it was implied that this very large response in vivo
was due to intrinsic chemosensitivity of RTN neurones.
Although this is an exciting possibility, the data do not yet
support this conclusion, because the methods used by the
investigators would not be expected to block all synaptic
input into RTN neurones (Mitchell, 2004). Kynurenic
acid would only block glutamatergic neurotransmission,
and even this may have been incomplete due to lack of
penetration into the brain parenchyma. Morphine would
have a general inhibitory effect on neuronal activity,
but would not specifically affect synaptic transmission.
Thus, the majority of synaptic inputs into RTN neurones
would be unaffected, including serotonin, noradrenaline
(norepinephrine), glycine, GABA, neuropeptides, purines,
NO and gap junctions. Thus, there are insufficient data
to determine whether the response to hypercapnia was
intrinsic or due to synaptic input from CRCs elsewhere
(Fig. 3).
Mulkey et al. (2004) found that RTN neurones in
brain slices respond to changes in pH, albeit significantly
less vigorously than in vivo, and concluded that the pH
sensitivity of these neurones is intrinsic. However, this
conclusion is also premature. Glutamate and purinergic
P2X receptors were blocked, yet other types of synaptic
transmission (see above) may have contributed to the
firing rate response in vitro. Evidence was obtained
264 G. B. Richerson and others Exp Physiol 90.3 pp 259269

using voltage-clamp experiments that pH-sensitive TASK
channels are present in these neurones, but it remains
to be seen whether the changes in firing rate induced
by pH were due to modulation of TASK channels,
or if other mechanisms including synaptic input were
involved. Caution is also warranted in interpretation of
these data, as it is difficult to compare the degree of
chemosensitivity of RTN neurones with that of other
CRC candidates (Mitchell, 2004) because: (1) the pH
range used (6.97.5) is not physiological (Fig. 2A); and
(2) the methods of quantification were different from those
used by other investigators (Wang et al. 2002; Putnam
et al. 2004). Of note, a preliminary report using brain
slices (without blocking synaptic transmission) indicates
that the response of RTN neurones was smaller than
the intrinsic response of serotonergic neurones (Putnam
et al. 2004, 2005). The large response of RTN neurones
in vivo in the face of modest intrinsic chemosensitivity in
vitro makes it likely that there was a contribution from
incompletely blocked synaptic input in vivo (Mitchell,
2004). Alternatively, intrinsic chemosensitivity may have
been enhanced by the use of halothane, which activates
TASK channels. In addition, anaesthesia may have
exaggerated the response of these neurones, consistent
with the finding that anaesthesia can enhance the role of
the VLMS in chemoreception (Forster et al. 1997).
The new finding of this study (Mulkey et al.
2004) came from juxtacellular labelling combined with
immunohistochemistry. Using this approach, the authors
demonstrated that CO2 -activated neurones of the RTN
are glutamatergic. This is an important result, because it
now defines a specific phenotype for a putative CRC in
the RTN. Mulkey et al. (2004) found that RTN neurones
have processes in the ML of the VLM. They concluded
that glutamatergic neurones of the RTN are the long
sought-after VLMS chemoreceptors. This conclusion is
based primarily on the belief that CRCs are located
in the ML, where they could sense CSF pH. However,
as discussed above there is no definitive evidence that
there are CRCs in the ML, or that CRCs are designed
specifically to sense CSF pH. Therefore, the significance of
this anatomical observation remains unclear. In addition,
neurones of the VRG (Pilowsky et al. 1993; Kawai et al.
1996), and serotonergic neurones of the parapyramidal
region (Bradley et al. 2002) also have processes in the
ML, so this finding is not specific to glutamatergic RTN
neurones.
In summary, the evidence for chemoreception by
glutamatergic neurones of the RTN is provocative and
important, but requires further analysis. A key piece of
missing information is whether these neurones meet one
of the basic requirements of a chemoreceptor intrinsic
chemosensitivity or whether they simply transmit the
response of other neurones that are the actual sensors. If
they do possess intrinsic chemosensitivity, the magnitude
of their response is not unusually large. As discussed above,
this does not rule out a major contribution to respiratory
chemoreception, but the existing data do not support a
conclusion that these neurones are uniquely important. In
fact, the finding that the ventilatory response induced by
focal acidosis in the RTN is roughly equal to that of other
chemoreceptor regions (Li & Nattie, 1997; Feldman et al.
2003) suggests otherwise. There is also not yet evidence
that specific lesions of just the glutamate neurones in the
RTN leads to a decreased CO2 response at the systems
level. However, it is exciting that we have identified the

A B C
CO2 CO2

CO2

CPG RTN RTN

CRCs

RTN CPG CPG

Figure 3. Three different interpretations of the effect of hypercapnia in vivo after application of
glutamate receptor antagonists to the surface of the medulla
A, CO2 acts (directly or indirectly) on the central pattern generator for breathing (CPG), which then drives RTN
neurones. This possibility can be excluded, but only if all synapses within the CPG are blocked. B, CO2 directly
stimulates RTN neurones acting as chemoreceptors, which stimulate the CPG. C, CO2 acts on a separate set
of CRCs that then stimulate RTN neurones via non-glutamatergic synapses. In our opinion, it is not possible to
differentiate between possibilities B and C using this in vivo approach.

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Exp Physiol 90.3 pp 259269 Phenotype(s) of respiratory chemoreceptors
phenotype of a neurone in the RTN that may be a CRC. It
will now be important to determine whether these specific
neurones meet the criteria for a CRC, and whether they
play a role under physiological or pathological conditions.
What information is still needed?
Now that so many CRC candidates have been identified,
the field can focus on the relative importance and
role(s) of each one. It is possible that they are all
redundant, that some are only important under certain
conditions (Nattie, 1999), or that some are not CRCs
at all. Nevertheless, none of the current candidates can
be excluded as actual chemoreceptors because there is
still incomplete information. It is unlikely that there
is a single experimental approach or preparation that
will unequivocally answer this question, but instead a
combination of in vivo and in vitro approaches will be
needed. In interpreting new data, it will be important to
apply rigorous criteria that do not rely on assumptions
that have not been validated, including the belief that
originated more than 20 years ago that the VLMS is the
sole site of CRCs. Given the modern tools in neuroscience,
the next 20 years should be exciting in establishing which
neurones mediate the respiratory and non-respiratory
responses to hypercapnia, and how dysfunction of these
chemoreceptors contributes to human disease.
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Experimental Physiology Exchange of Views
Re: Homing in on the specific phenotype(s) of central
respiratory chemoreceptors
Commentary by Patrice G. Guyenet, Ruth L. Stornetta, Douglas A. Bayliss and Daniel K. Mulkey
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
Exp Physiol 90.3 pp 259269
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Acknowledgements
We wish to thank Robert Putnam, Hannah Kinney and Eugene
Nattie for critical review of the manuscript. This work was
supported by NHLBI HL52539, NICHD HD36379 and the
VAMC.
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


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Exp Physiol 90.3 pp 259269 Commentary on Hot Topic Review
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


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267
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 P CO2 . 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-Botzinger
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.
268 P. G. Guyenet and others
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,
Exp Physiol 90.3 pp 259269
the difference between chemoreceptors and neurones
exhibiting insignificant responses to pH might become
much more obvious at physiological temperature.
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Exp Physiol 90.3 pp 259269 Commentary on Hot Topic Review 269

Experimental Physiology Exchange of Views

Authors response to P. G. Guyenets commentary

G. B. Richerson
Departments of Neurology and Cellular & Molecular Physiology, Yale University School of Medicine, New Haven, CT, USA

Most of the points raised by Guyenet et al. have already
been addressed in our commentary on their accompanying
review. We will not elaborate here, except to say that our
analysis of the existing data is quite different than theirs.
However, where we do agree is that more data are needed
to fully understand central respiratory chemoreception.
Thus, now is not the time to make firm conclusions,
but to gather more data. We disagree that data from
an in vivo approach has greater validity, but instead
strongly believe that a complete understanding of CO2 /pH
chemoreception will only come from the use of a variety
of approaches at all levels from molecules to the
network. When the data obtained at different levels is not
concordant, we need to reassess the assumptions made in
interpreting the data, rather than discarding results from a
particular approach. Until we in the field of respiratory
control can offer the scientific community a detailed
understanding of central respiratory chemoreception, it is
prudent to carefully and objectively evaluate the evidence
for and against each of the current hypotheses. We
appreciate the participation of Dr Guyenet and colleagues
in this fruitful debate, and share their enthusiasm for
further investigations to enhance our understanding of
central respiratory chemoreception.

C The Physiological Society 2005