Brain Research 988 (2003) 154–163

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Research report

ACh and ATP mediate excitatory transmission in cat carotid identified

chemoreceptor units in vitro
Rodrigo Varas a , Julio Alcayaga b , Rodrigo Iturriaga a , *
a
Laboratorio de Neurobiologıa´ , Departamento de Ciencias Fisiologicas
´ ´
, Facultad de Ciencias Biologicas ´
, P. Universidad Catolica de Chile, Casilla
114 -D, Santiago 1, Chile
b
´ Celular, Departamento de Biologıa
Laboratorio de Fisiologıa ´ , Facultad de Ciencias, Universidad de Chile, Casilla 653, Santiago 1, Chile

Accepted 21 July 2003

Abstract

Several molecules have been proposed as excitatory transmitters between glomus (type 1) cells and nerve terminals of petrosal ganglion
(PG) neurons in the carotid body (CB). We tested whether ACh and ATP have a role to play as excitatory transmitters in the cat CB by
recording intracellularly from identified PG neurons functionally connected to the CB in vitro. PG neurons projecting to the CB were
classified according to their intracellular responses as: (a) neurons with humped action potentials (hAP neurons) responding phasically to
long-lasting depolarizing pulses (53 / 67), and (b) neurons with smooth action potentials (non-hAP neurons) that fire tonically during
long-lasting depolarizations (14 / 67). CB stimulation by stop flow and / or acidosis induced activity in 28 of 39 hAP-type neurons, being
classified as chemosensory, but in none of the non-hAP neurons. Hexamethonium (10 mM) and suramin (100 mM) reversibly abolished
the increased discharges evoked in chemosensory neurons (8 / 9) by stop flow or acidosis. Moreover, 24 of 27 chemosensory neurons
responded to ganglionar application of ACh and ATP, while two neurons responded only to ACh and one to ATP. Mechanical deformation
of the carotid sinus induced firing activity in 10 of 13 non-hAP neurons, but in none of the hAP neurons tested. Interestingly, 4 / 10
non-hAP neurons, which responded to carotid sinus mechanical stimulation also responded to ganglionar application of ATP, but were
insensitive to ACh. Present results favor the hypothesis that ACh and ATP are excitatory transmitters in the cat CB, acting—at least—on
the PG neuron terminals in the CB.
 2003 Elsevier B.V. All rights reserved.

Theme: Sensory systems

Topic: Somatic and visceral afferents

Keywords: ACh; ATP; Carotid body; Co-transmission; Petrosal ganglion

1. Introduction CB [27]. However, the identity of the excitatory transmit-

The carotid body (CB) is the main chemosensory organ
that senses the levels of pO 2 , pCO 2 , and pH in the arterial
blood. In response to hypoxia, hypercapnia and acidosis,
the glomus (type 1) cells of the CB are expected to release
one or more transmitters that increase the frequency of
action potentials in the apposed nerve endings of petrosal
ganglion (PG) neurons [18]. Several molecules present in
glomus cells, such as acetylcholine (ACh), adenosine 59-
triphosphate (ATP), dopamine (DA) and substance P have
been postulated to be the excitatory transmitter(s) in the
*Corresponding author. Tel.: 156-2-686-2852; fax: 156-2-222-5515.
E-mail address: riturria@bio.puc.cl (R. Iturriaga).
ter between glomus cells and sensory afferent neurons is
still controversial. Among the putative excitatory transmit-
ters present in the CB, DA has received much attention
[27]. The proposal that DA is the excitatory transmitter
was strongly supported by the observation that hypoxia
produces DA release from the CB. Indeed, Fidone et al.
[19] found that after incubation with [ 3 H]tyrosine for 2–3
h, the amount of [ 3 H]DA released from rabbit CB super-
fused in vitro was roughly proportional to the degree of
hypoxia. However, simultaneous recordings of CB
chemosensory discharges and endogenous DA secretion
measured by amperometry have shown a clear dissociation
between chemosensory excitation and DA efflux induced
by natural stimuli [10,13,30,31]. In addition, the inhibitory

0006-8993 / 03 / $ – see front matter  2003 Elsevier B.V. All rights reserved.
doi:10.1016 / S0006-8993(03)03366-3
 R. Varas et al. / Brain Research 988 (2003) 154–163
effect of DA and the excitatory effects of D 2 antagonists
on cat CB chemoreception suggest a tonic inhibitory role
for DA within the CB [8,12,32,47]. Moreover, the
modulatory action of DA on ACh-induced responses [6]
and its inhibitory action on ATP-induced responses [5] in
the isolated PG in vitro suggest a postsynaptic action of
DA in the CB.
ACh is another interesting putative transmitter that
meets most of the criteria to be considered the excitatory
transmitter in the CB [21]. ACh is released from the CB in
response to hypoxia [22,23] and the application of ACh
and nicotinic agonists to the cat CB [17] and PG [4]
produces a dose-dependent excitation in the carotid sinus
nerve (CSN). ACh evokes depolarization and action
potentials in cultured cat PG neurons [44], and depolariza-
tion and fast inward currents in rat petrosal-jugular neu-
rons, effects that are mimicked by nicotine and blocked by
hexamethonium [49]. Moreover, immunocytochemical
studies have shown the presence of neuronal nicotinic
receptor on PG neurons [42]. The above evidence supports
the hypothesis that ACh is an excitatory transmitter in the
CB, but other observations indicate that ACh is not the
exclusive excitatory molecule. Indeed, the same doses of
cholinergic blockers that abolished CB chemosensory
excitation induced by ACh reduced only partially the
chemosensory response to hypoxia [22]. Moreover, hexa-
methonium failed to abolish the hypoxia-induced depolar-
ization of petrosal-jugular neurons co-cultured with glomus
cells [48,50]. Thus, it is likely that other excitatory
molecule(s) could be released from glomus cells besides
ACh. A candidate to be the excitatory co-transmitter in the
CB is ATP [48]. Application of ATP to the CB [43] and
the PG [1] of the cat generates dose-dependent bursts of
action potentials in the CSN. In cultured rat petrosal–
jugular neurons, ATP induces a fast depolarizing inward
current that is mimicked by a,b-methylene ATP and
blocked by suramin, a P2X receptor antagonist [48]. Using
a reconstituted rat chemosensory system, obtained from
CB and petrosal–jugular cells in culture, Zhang et al. [48]
have recently suggested that ACh and ATP are co-released
from glomus cells. However, the establishment of synaptic
contacts between non-chemosensory neurons (i.e. gustative
chemosensory or mechanoreceptor neurons) present in the
petrosal–jugular complex and glomus cells cannot be ruled
out. Monti-Bloch et al. [36] reported that fibers that re-
innervate the CB transplanted in the tenuissimus muscle,
present spontaneous activity and respond to muscle stretch,
hypoxia, NaCN, ACh and nicotine, because of dual
innervation of glomus cells and muscle spindles. Thus,
glomus cells are capable of inducing chemosensory prop-
erties in axons that normally subserve a mechanosensory
function. Nevertheless, Zhang et al. [48] recorded the
effects of suramin and mecamylamine on chemosensory
discharges in the isolated rat CB preparation in vitro. They
found that suramin and mecamylamide applied separately
produced a graded and reversible inhibition of chemosen-
155
sory response to hypoxia, while the combined administra-
tion of both blockers eliminated the hypoxia-induced
response. Thus, they stated that those results raise the
possibility that co-release of ACh and ATP may mediate
the hypoxic transduction in the intact CB [48]. However, it
is not known if ACh and ATP may serve as co-transmitters
in the CB of other species, because species differences
may account for the irreconcilable effects of some putative
transmitters in the CB [27].
Accordingly, we studied the role played by ACh and
ATP on CB chemoreception using an in vitro preparation
consisting of the carotid bifurcation and the PG. In this
preparation, the PG remains functionally connected to the
carotid bifurcation and the CB through the CSN, allowing
the recording of chemosensory and barosensory neurons in
the PG that are excited by stimuli applied in the CB (stop
flow) and carotid sinus (mechanical stimulation), respec-
tively [7,24]. Thus, we tested if a combination of nicotinic
and purinergic blockers was able to abolish chemosensory
responses of identified carotid PG chemosensory neurons
to well known CB stimuli in vitro, such as stop flow
[3,7,24] and acidosis [16,17].
2. Materials and methods
2.1. CB-PG preparation
Adult cats of either sex (2.0–3.5 kg) were anaesthetized
with sodium pentobarbitone (40 mg / kg, i.p.) and supple-
mentary doses (8–12 mg, i.v.) were given when necessary
to maintain a surgical level of anesthesia. The cat was
placed in supine position and the trachea was cannulated.
The external and common carotid arteries were ligated, and
the carotid bifurcation containing the CB, the CSN, and the
petrosal ganglion were removed as a whole and placed in
ice-chilled, Ca 21 –Mg 21 -free modified Hanks’ solution.
The same procedure was repeated contralaterally. After
bilateral removal of the tissue, the cat was sacrificed with
an overdose of sodium pentobarbitone (200 mg / kg, i.v.).
The protocol was approved by the Ethical Committee of
´
the Facultad de Ciencias Biologicas of the P. Universidad
´
Catolica de Chile, and was performed according to the
Guiding Principles for the Care and Use of Animals of the
American Physiological Society, and the National Insti-
tutes of Health Guide for the Care and Use of Laboratory
Animals (NIH Publications No. 80-23, revised 1996).
The preparation was gently dissected from surrounding
connective tissue and transferred to a chamber with two-
compartments (1.5 ml each) separately superfused, and
communicated through a small channel. The carotid
bifurcation, with the carotid sinus and CB, was pinned to
the bottom of one compartment and the PG to the other,
while the CSN was passed through the communicating
channel filled with mineral oil to assess electrical isolation.
Each compartment was independently superfused with
156 R. Varas et al. / Brain Research 988 (2003) 154–163
Hanks’ solution (in mM: NaCl 137, CaCl 2 1.3, MgSO 4
0.8, KCl 5.4, KH 2 PO 4 0.4, Na 2 HPO 4 0.3, D-glucose 5.6,
HEPES 5, and NaHCO 3 4.0), pH 7.43 at 30 8C and
equilibrated with air. The flow rate in both compartments
was maintained at 1.2 ml / min with a perfusion pump
controlling the input and output of the saline solution. The
capsule of the PG was opened and the CSN was placed on
a pair of platinum–iridium electrodes for electrical stimu-
lation. No further dissection was performed to avoid nerve
fibers damage.
2.2. Intracellular recordings
The PG neurons were impaled, under microscopic
guidance, with glass pulled microelectrodes filled with KCl
3 M (20–50 MV) and connected to an electrometer that
allows the recording of resting membrane and action
potentials and the injection of current. The preparation was
grounded with an Ag–AgCl reference electrode placed in
one of the outflows of the chamber. The recordings were
displayed in an oscilloscope and stored in an analog–
digital videotape system (Instrutech, USA). Data were
digitized off-line at 10 KHz with a Digitada 1200 analog–
digital board (Axon Instruments, USA) and analyzed using
Axoscope 8.2 data acquisition software (Axon Instru-
ments). The resting membrane potential was measured and
the input resistance was calculated from the steady-state
change in membrane potential attained during the applica-
tion of hyperpolarizing currents (0.1–0.5 nA). Somatic
action potentials in PG neurons were evoked with de-
polarizing currents (2–5 nA) injected during 5 ms through
the intracellular electrode. The duration of the action
potentials was measured from its threshold to the point
where its falling phase crossed the resting membrane
potential. The duration of the after-hyperpolarization was
measured from the later point until membrane potential
returned to the resting level. All measurements were made
while the neurons maintained stable resting membrane
potential, and were able to fire action potentials. Axonal
conduction time was estimated from the time elapsed
between the electrical stimulation of the CSN and the
recording of the action potential. At the end of the
experiment, the conduction distance was estimated from
the distance between the stimulation electrodes and the
center of the PG, ranging 5–19 mm. The conduction
velocity was calculated as the ratio between the conduction
distance and the axonal conduction time.
2.3. Identification of carotid chemosensory and
barosensory neurons
Petrosal ganglion neurons were classified as carotid
neurons when the electrical stimulation of the CSN evoked
depolarization and action potentials in the impaled cell.
Carotid neurons were classified as chemosensory neurons
if their discharges increased in response of stop-flow and /
or acidosis applied to the CB, or as barosensory neurons if
their discharges increased in response to the mechanical
stimulation of the carotid sinus with a puff of Hanks’
solution.
2.4. Drugs
Nicotine, ACh, ATP, and a,b-methylene ATP were
freshly prepared in Hanks’ solution, diluted to desired
concentration (0.01–1 mM) and delivered by pressure
ejection from a nearby pipette positioned at approximately
100 mm from the ganglion surface or, in some cases, over
the CB. Hexamethonium and suramin were applied in
combination to the CB superfusate to block the responses
to stop flow or acidosis. All drugs were obtained from
Sigma (USA), except for nicotine that was obtained from
ICN (USA).
2.5. Data analysis
The data was expressed as means6S.E.M. To assess
statistical differences for two samples we used one-tailed
Student’s t-test, and for multiple samples we used the
Friedman test followed by multiple comparisons using the
Conover test. To compare the responses elicited by ACh
and ATP on baro- and chemosensory neurons (categorical
variable) we used the chi-square test. The level of signifi-
cance between differences was established at P,0.05 in all
statistical tests.
3. Results
We recorded intracellularly a total of 360 neurons from
11 preparations, that presented stable membrane potentials
and action potentials in response to CSN or glos-
sopharyngeal nerve electrical stimulation. From this popu-
lation, 67 neurons (18.6%) responded only to CSN stimu-
lation, while the remaining 293 (81.4%) responded only to
stimulation of the glossopharyngeal nerve.
3.1. Electrical properties of petrosal neurons projecting
through the CSN
Out of 67 neurons that responded to CSN stimulation,
54 had action potentials with a hump on the falling phase
(hAP), a prolonged after-hyperpolarization and fired phasi-
cally during long lasting depolarizing current pulses in-
jected in their somata (Fig. 1). The remaining 13 neurons
showed fast action potential without a hump on the falling
phase (non-hAP) and fired tonically during long lasting
depolarizing pulses (Fig. 2). From the 54 hAP neurons, 44
were classified as A-type neurons because of their axonal
conduction velocity (CV) faster than 2 m / s (mean CV5
11.562.0 m / s) and the remaining 10 hAP neurons were
classified as C-type neurons because they show CV slower
 R. Varas et al. / Brain Research 988 (2003) 154–163 157

and after-hyperpolarization were similar in both types of

Fig. 1. Intracellular recording from the soma of a carotid PG neuron with
humped action potential (hAP). (A) Electrical stimulation (5 mV, 2 ms) of
the CSN evokes a hAP in the neuron. (B) Long-lasting depolarizing
current injection (2 nA, 100 ms) through the microelectrode evokes a
single action potential in this petrosal neuron.
neurons. However, hAP neurons had larger input resistance
(30.161.0 vs. 18.064.2 MV; P,0.05), longer action
potential duration (4.660.5 vs. 1. 160.1 ms; P,0.05), and
longer after-hyperpolarization duration (40.963.8 vs.
25.064.7 ms; P,0.05) than non-hAP neurons. On the
other hand, non-hAP neurons had significantly faster CV
than hAP neurons (31.861.9 vs. 11.562.0 m / s; P,0.05).
The electrical properties of hAP and non-hAP C-type
neurons are summarized in Table 1. We did not find
significant differences in resting membrane potential,
neither in input resistance, nor amplitude of the action
potential or the after-hyperpolarization, nor CV. However,
hAP C-type neurons showed longer action potential
(4.460.7 vs. 1.760.3 ms, P,0.05) and after-hyperpolari-
zation duration (41.765.6 vs. 12.861.4 mV, P,0.05) than
non-hAP C-type neurons.

3.2. Carotid chemosensory, barosensory and unidentified

neurons

Carotid PG neurons were classified as chemosensory

Fig. 2. Intracellular recording from the soma of a carotid PG neuron with
non-humped action potential (non-hAP). (A) Electrical stimulation (5 mV,
2 ms) of the CSN evokes a non-hAP in the neuron. (B) Long-lasting
depolarizing current injection (2 nA, 100 ms) through the microelectrode
evokes multiple action potentials in this petrosal neuron.
than 1.5 m / s (CV51.260.1 m / s). From the 13 non-hAP
neurons, 10 were classified as A-type neurons (CV5
31.861.8 m / s), while the remaining 3 were classified as
C-type neurons (CV51.160.8 m / s).
Table 1 summarizes the electrical properties of hAP and
non-hAP for A-type neurons. The resting membrane
potential and the amplitudes of the evoked action potential
neurons if they increased their frequency of discharge in
response to superfusate stop flow or application of an
acidic Hanks’ puff in the CB chamber. We tested the
effects of stop flow in 39 of 54 hAP neurons and in all
non-hAP neurons. We found that 28 of the 39 hAP neurons
increased their frequency of discharge in response to stop
flow, while the remaining 11 neurons failed to respond to
stimulation of the CB or to the carotid sinus, although one
of them responded to ACh and ATP. From these 28 hAP
neurons, 24 were A-type neurons and the remaining four
were C-type neurons. None of the non-hAP neurons
responded to stop flow. It is noteworthy that only hAP
neurons responded to CB stimulation increasing their
discharge, thus allowing to characterize them as chemosen-
sory neurons. Most of the carotid chemosensory neurons
showed spontaneous irregular discharges, but some of
them were silent in normoxia. Fig. 3 shows the response to
a 3-min stop flow of one carotid chemosensory PG neuron.
The frequency of chemosensory discharges increased after

Table 1
Electrical properties of carotid neurons
Vm Ri aAP dAP aAHP dAHP CV
(mV) (MV) (mV) (ms) (mV) (ms) (m / s)
A-type neurons
hAP (n544) 254.261.1 30.162.8* 75.264.1 4.660.6* 10.960.7 40.968.4* 11.562.0*
Non-hAP (n59) 256.763.0 14.360.8 73.664.6 1.260.2 11.661.8 25.065.0 31.861.9
C-type neurons
hAP (n510) 253.562.0 58.264.9 74.162.8 4.460.7* 11.761.3 41.765.6* 1.260.1
Non-hAP (n54) 253.462.9 47.164.9 69.366.9 1.760.3 11.060.7 11.662.0 1.160.1
Vm, membrane potential; Ri, input resistance; aAP, amplitude action potential; dAP, duration action potential; aAHP, amplitude of the after-
hyperpolarization; dAHP, duration after-hyperpolarization; CV, conduction velocity.
* P,0.05 Student’s test between hAP and non-hAP neurons.
158 R. Varas et al. / Brain Research 988 (2003) 154–163
Fig. 3. Stop flow-induced activity in a carotid chemosensory neuron. (A)
Spontaneous basal activity of a hAP neuron in control condition. (B)
After 1 min of stop flow. (C) Basal activity after 1 min of resumed flow.
about 1 min of stop flow, and returned to baseline after the
restitution of flow.
Ten non-hAP neurons responded to a puff of Hanks’
solution upon the carotid sinus with bursts of action
potential (Fig. 4A), thus allowing to classify them as
barosensory neurons. Eight of 10 mechanosensory neurons
were A-type, while the remaining two neurons were C-type
neurons. It is noteworthy that all of the barosensory
neurons have action potentials without a hump, and that
hAP neurons never responded to carotid sinus stimulation.
A total of 14 neurons that were activated by electrical
stimulation of the CSN failed to respond to chemical and
mechanical stimulation of the CB and the carotid sinus,
respectively. Based on the presence or absence of a hump
on the falling phase, we identify 11 unresponsive neurons
as hAP and three unresponsive as non-hAP neurons.
Fig. 4. Intracellular responses of a barosensory neuron. (A) Mechanical
stimulation of the carotid sinus (empty arrows). (B) Injection of current
into the soma (rectangular pulse) and application of ATP (filled arrow).
3.3. Effects of hexamethonium and suramin on
chemosensory responses elicited by stop flow and
acidosis
The superfusion of the CB with Hanks’ solution con-
taining either the nicotinic receptor blocker hexameth-
onium (10 mM) or the P2 purinergic receptor blocker
suramin (100 mM) only reduced the chemosensory excita-
tion induced by stop flow. However, when the CB was
superfused with Hanks’ solution containing both hexa-
methonium (10 mM) and suramin (100 mM), the
chemosensory excitation was completed blocked in five of
six neurons studied. In the remaining neuron, the applica-
tion of both drugs slightly decreased the chemosensory
excitation induced by stop flow. Fig. 5 shows the effects of
the application of hexamethonium, suramin and both drugs
combined on the excitatory chemosensory response to stop
flow in one hAP PG neuron. The inhibitory effect of
hexamethonium and suramin was completely reverted after
10 min of washing out the drugs. Fig. 6 summarizes the
effects of these drugs on the response of four hAP neurons
to stop flow. In addition, we tested the effects of stimula-
tion with an acidic stimulus in four CBs. A 200-ml bolus of
Hanks’ solution at pH 6.8–7.0 applied directly to the CB
increased the chemosensory discharges in three neurons
that also responded to stop flow. Fig. 7A shows that the
application of the acidic bolus to the CB elicited the firing
of three action potentials in the recorded neuron. After 5
min of superfusion with Hanks’ containing 10 mM hexa-
methonium and 100 mM suramin the same acid stimulation
of the CB did not evoke any response in the neuron (Fig.
7B). After 10 min of washing out the drugs, the acid bolus
Fig. 5. Effect of P2 and nicotinic ACh receptor blockade on stop
flow-induced excitation in one carotid chemosensory neuron. (A) Stop
flow-induced activity in control condition. (B) Response to stop flow after
5 min of superfusion with 10 mM hexamethonium. (C) Response to stop
flow after 5 min of superfusion with 100 mM suramin. (D) Response to
stop flow after 5 min of superfusion with a combination of 100 mM
suramin and 10 mM hexamethonium. (E) Response to stop flow after 10
min of washing out both blockers.
 R. Varas et al. / Brain Research 988 (2003) 154–163
Fig. 6. Summary of the effects of 10 mM hexamethonium (H), 100 mM
suramin (S) and 10 mM hexamethonium plus 100 mM suramin on
chemosensory responses to stop flow in four hAP neurons. bas, basal
discharge during control superfusion; C, discharge attained during stop
flow in control condition; R, discharge attained during stop flow after 10
min of washing out the drugs; fx, frequency of chemosensory discharge.
Friedman overall test, P58.15310 27 .
evoked again a chemosensory response (Fig. 7C). It is
noteworthy that one hAP neuron responded to acidic
stimuli, but not to stop flow. In this particular neuron, 10
mM hexamethonium was able to reversibly suppress the
chemosensory response induced by acid stimulation (data
not shown).
3.4. Somatic stimulation of PG neurons by ACh and
ATP
Since it has been proposed that some electrical prop-
Fig. 7. Effect of P2 and nicotinic ACh receptor blockade on acidic-
induced activity in a carotid chemosensory neuron. (A) Action potentials
evoked by a 200-ml puff of Hank’s solution at pH 6.8 applied to the
carotid body (filled arrow). (B) After 5 min of superfusion with 100 mM
suramin and 10 mM hexamethonium, the acidic-induced activity was
completely suppressed. (C) After 10 min of washing out the blockers, the
acidic-induced activity was recovered.
159
Fig. 8. Intracellular responses to somatic application of ACh and ATP in
two identified carotid chemosensory neurons. (A) Bolus application (200
ml) of ACh 1 mM (arrow) in one neuron. Responses to ACh (B) and ATP
(C) in another neuron.
erties and neurotransmitter receptors are shared by mem-
brane of somata and their respective nerve endings in
primary afferent neurons [4,26,40], we applied ACh and
ATP directly to the somata of identified carotid chemosen-
sory and barosensory neurons. Application of ACh and
ATP to the somata of chemosensory neurons produced a
sustained depolarization and generation of action potentials
in 24 of 27 chemoreceptor neurons studied, while the
remaining ones were stimulated only by ACh (2) or ATP
(1). Fig. 8 shows that applications of ACh (0.5 and 1 mM)
and ATP (0.2 mM) to the PG evoked sustained depolariza-
tions and action potentials in identified chemosensory
neurons. As shown in Table 2, four of 10 barosensory
neurons also responded to somatic application of ATP, but
were unresponsive to ACh. The pattern of the distribution
of the responses to ATP and ACh of barosensory and
chemosensory neurons was significantly different (P,
0.01; chi-square test).
4. Discussion
The present results show that the increase in discharges
of identified chemosensory PG neurons projecting through
the CSN induced by acid and stop-flow is reversibly
blocked by a combination of hexamethonium (10 mM) and
suramin (100 mM) applied to the CB. Either ACh or ATP
applied to the somata of these chemosensory neurons
induces firing of action potentials. Thus, our results
160 R. Varas et al. / Brain Research 988 (2003) 154–163
Table 2
Responses to ACh and ATP of identified carotid neurons
ACh ATP
(0.01–1 mM) (0.01–1 mM)
Chemosensory* (n527) 2 1
Barosensory (n510) 0 4
* P,0.001, chi-square test for contingency tables.
support the idea that ACh and ATP are both excitatory
transmitters in the cat CB and agree and extend previous
observations [48]. Recordings from rat petrosal–jugular
neurons co-cultured with glomus cells often show sponta-
neous and hypoxia-evoked excitatory postsynaptic re-
sponses [48]. Hexamethonium (100 mM) or suramin (50
mM) applied separately inhibited partially these responses,
while joint application inhibited completely the activity
induced by hypoxia in about 70% of the cases. This
suggests that both ACh and ATP are released and mediate
the excitatory hypoxic signaling between glomus cells and
sensory neurons [48]. Similarly, both suramin and hexa-
methonium reduced or abolished the hypoxia-induced
responses in the CB in vitro, while the responses were
virtually eliminated in the presence of both blockers [48].
However, the phenotype of gustatory or mechanoreceptor
neurons present in the petrosal–jugular complex could be
modified by the presence of CB cells in culture. It has been
reported that glomus cells are capable of inducing
chemosensory properties in axons that normally subserve a
mechanosensory function [36]. Moreover, some electrical
properties of rat nodose neurons were modified when
neurons were co-cultured with glomus cells, and acidic
stimulation induced depolarization and increased dis-
charges only in co-cultured neurons [2]. The present results
suggest that the modification of neuronal responses might
arise from synaptic and / or trophic interactions between
glomus cells and neurons. The present experiments, per-
formed in an in vitro preparation of the cat PG neurons
functionally connected to the CB, may prevent medium to
long-term changes induced by culture conditions.
For several decades, the identity of the excitatory
transmitter(s) in the CB has remained unsolved. Several
molecules present in the CB such as catecholamines, ACh,
peptides, and adenosine nucleotides have been considered
as potential excitatory transmitters in the junctions between
glomus cells and nerve terminals [27]. Among these
molecules, ACh has received great attention (for review,
see Ref. [21]). ACh is present in the CB, its synthesizing
enzyme—choline acetyltransferase—is localized in the
glomus cells of cats and rabbits [46], and a high affinity
sodium-dependent choline uptake mechanism has been
found in glomus cells of the cat CB [20,46]. Moreover,
ACh is released from the CB in response to electrical
stimulation [18], hypoxia, and hypercapnia [23]. On the
other hand, ACh increases chemosensory discharges in a
dose dependent-manner in most species, with the exception
ACh and ATP Unresponsive
(0.01–1 mM)
24 0
0 6
of the rabbit, where ACh depresses the activity [18]. The
excitatory effect of ACh is mediated by nicotinic receptors,
and the depression by muscarinic receptors that dominated
in rabbits. The excitatory effect of ACh is blocked by
hexamethonium and mecamylamine. Immunocytochemical
studies have showed the presence of both a4 and a7
subunits of the nicotinic ACh receptor in sensory afferents
apposed to glomus cells, on fibers of CSN and on somata
in PG neurons [42]. Recently, it has been reported the
presence of a3, a4, b2 subunits of the nicotinic receptor,
as well as m1 and m2 muscarinic receptors in cat glomus
cells and petrosal neurons [29]. The localization of nico-
tinic receptors on postsynaptic PG neurons agrees with the
results of the electrophysiological studies performed on the
whole PG and isolated PG neurons. The application of
ACh to the cat PG in vitro selectively increases the
antidromic discharges in the CSN, but ACh had little or no
effect on the neurons projecting through the glos-
sopharyngeal branch [4]. In cultured cat PG neurons, ACh
induces depolarization and spike generation [44], and
patch-clamp recordings show that ACh induces depolariza-
tion and inward currents in about 70% of rat [49] and 60%
of cat PG neurons [45], effect mimicked by nicotine and
blocked by hexamethonium [49]. Thus, these data taken
together strongly support the hypothesis that ACh is
released in response to hypoxia from the glomus cells and
increases the frequency of discharge in terminals of PG
neurons. However, a major problem to demonstrate that
ACh is the only excitatory transmitter in the CB is that
cholinergic antagonists that block the excitatory effect of
exogenous ACh are unable to abolish the excitatory
response to hypoxia. Indeed, a cocktail of nicotinic and
muscarinic antagonists only partially block the chemosen-
sory response to hypoxia, although very high doses of
atropine blocked the response to hypoxia [22]. Thus, it is
likely that glomus cells may co-release more than one type
of molecule in response to natural stimuli. ATP has been
proposed to be an excitatory transmitter in afferent
nociceptive sensory neurons in dorsal root, and in trigemi-
nal and nodose neurons [15]. Thus, the idea that ATP may
be co-released with ACh in the CB in response to hypoxia
is especially attractive [48]. Large amounts of adenine
nucleotides have been found in glomus cells stored in
granules, in addition to catecholamines and proteins [9].
Adenosine and ATP evoked a dose-dependent increase in
chemosensory discharge in the cat CB [35,43] and the
application of ATP to the cat PG induces a brief, dose-
 R. Varas et al. / Brain Research 988 (2003) 154–163
dependent, increase in discharges in both the CSN and the
glossopharyngeal branch [1]. However, the responses in
the CSN present lower thresholds and larger amplitudes
than those evoked in the glossopharyngeal branch [1].
Similarly, in rat petrosal–jugular neurons in culture, ATP
induces a dose-dependent depolarization, blocked by
suramin, and voltage-clamp recordings show that ATP
induces a fast, partially inactivating current [45,48]. The
pharmacological properties of ATP-induced responses in
rat and cat PG neurons suggest that these neurons express
P2X receptors. Immunostaining of PG with antibody
against the P2X 2 subunit indicates that this subunit is
present in a vast population of neurons in the PG and in its
peripheral processes within the CB [48]. Similarly, single
cell RT-PCR technique demonstrated the presence of
mRNA for P2X 2 and P2X 3 subunits in petrosal–jugular
neurons that respond to hypoxia in co-cultures with glomus
cells [39].
The proposition that ACh and ATP are the excitatory
transmitters in the rat and cat CBs may be not exclusive of
PG neurons, and other primary afferent neurons may use
these molecules as excitatory transmitters. Indeed, in the
rat, about 50% of neurons of the petrosal–jugular complex
projecting to the tongue respond to ACh with depolariza-
tion [33], indicating that—at least in the rat—this popula-
tion is also endowed with ACh receptors. Thus, ACh-
induced responses are not only confined to carotid PG
neurons projecting through the CSN, but species differ-
ences can account for the differences in the evoked
responses. Unexpectedly, we found that ATP excites four
of 10 baroreceptor neurons. However, it has been shown
recently that ATP stimulates primary sensory mechanical
neurons [41]. Substantial evidence suggest that ATP
participates in the initiation of impulses in some sensory
fibers [38]. P2X receptors, which are a family of ligand-
gated ion channels responsive to ATP, are present in
nociceptive sensory neurons in dorsal root, and in trigemi-
nal and nodose neurons [15].
Our results confirm and extend previous findings show-
ing that cat PG A-type neurons projecting to the CB can be
distinguished by the presence or absence of an inflexion
(‘hump’) in the repolarizing phase of the action potential
[7]. In addition, our results indicate that a similar distinc-
tion can be made within the PG C-type neurons that have
not been previously described. In our preparation, neurons
presenting hAPs comprised about 80% of the recorded A-
and C-type neurons projecting through the CSN. This
proportion is similar to that previously reported for A-type
neurons in the cat PG projecting through the CSN [7] or
the glossopharyngeal branch [37]. The electrical properties
of hAP and non-hAP neurons were largely similar, with
hAP neurons presenting action potentials and after-hy-
perpolarizations of significantly longer duration than non-
hAP neurons. Similar differences had been described for
cat PG A-type neurons [7,37]. C-type neurons presented
161
larger input resistance than A-type neurons, which resist-
ance was related to CV. Thus, A-type hAP neurons present
slower CV and larger input resistance than non-hAP
neurons, suggesting that hAP neurons comprise a popula-
tion of cells smaller than non-hAP neurons [25,28,34].
Stimulation of the CB increased the basal frequency of
discharge, or initiated the discharge, in a subset of neurons
responding to CSN stimulation and characterized by the
presence of hAPs, and that were insensitive to the me-
chanical stimulation of the carotid sinus. It has been
reported that cat PG A-type neurons that respond to CB
stimulation have humped action potentials, a prolonged
after-hyperpolarization, and fire phasically during long-
lasting intracellular depolarizing pulses [7]. These PG
chemosensory A-type neurons, termed H-type, were the
most abundant (88.25%) responsive cells projecting
through the CSN. Our results agree with this previous
report, but in addition of A-type neurons (24 of 28), we
found a population of responsive chemosensory C-type
neurons (4 of 28) characterized by their hAP, long after-
hyperpolarization, and phasic response to long-lasting
intracellular depolarizations. Chemosensory C-type neu-
rons with hAP and non-hAP have been recorded in the cat,
but a thorough characterization of these neurons is not
available [7]. In the rat, all chemosensory units recorded
correspond to non-hAP C-type neurons [11,14]. Thus, in
our preparation, all neurons that respond to CB stimulation
present similar action potential shape and spiking charac-
teristics, but comprise both A- and C-type neurons.
Mechanical stimulation of the carotid sinus induced
responses in both A- and C-type neurons characterized by
non-hAPs, short after-hyperpolarizations and tonic dis-
charge during long-lasting depolarization. The characteris-
tics of C-type and A-type baroreceptor neurons were
almost indistinguishable, diverging only in their CVs.
These neurons were not activated by CB stimulation. It has
been reported that the most common PG baroreceptor
neurons (7.5%), termed F-type neurons, have non-humped
action potentials with short after-hyperpolarizations, and
fast conduction velocities [7]. A less abundant barorecep-
tor A-type neurons (4.25%), termed S-type, present slower
conduction velocities, a hump similar to that present in
H-type neurons, and a shorter after-hyperpolarization than
chemosensory neurons [7]. All our baroreceptor A-type
units can be classified as F-type neurons, but we did not
find any S-type baroreceptor neuron. However, S-type
barosensory neurons represent only a small population (4
of 94) of the neurons previously reported [7].
In summary, our data indicate that most identified
carotid chemosensory neurons in the PG respond to
somatic application of either ACh or ATP. Moreover, the
responses evoked in carotid PG neurons by stop-flow and
acidosis applied in the CB can be blocked by nicotinic and
purinergic blockers. Therefore, ACh and ATP play an
excitatory role in the cat CB.
162 R. Varas et al. / Brain Research 988 (2003) 154–163
Acknowledgements
Work supported by grant 2010133 of the National Fund
for Scientific and Technological Development, Chile. We
´ for her help in the
would like to thank Carolina Larraın
preparation of this manuscript.
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