Electrophysiological characterization of nicotinic acetylcholine
receptors in cat petrosal ganglion neurons in culture:
Effects of cytisine and its bromo derivatives
Rodrigo Varasc, Viviana Valdésc, Patricio Iturriaga-Vásquezb, Bruce K. Casselsb,
Rodrigo Iturriagac, Julio Alcayagaa,⁎
aLaboratorio de Fisiología Celular, Facultad de Ciencias, Universidad de Chile, Casilla 653, Santiago, Chile
bLaboratorio de Química Biodinámica, Facultad de Ciencias, Universidad de Chile, Santiago, Chile
cLaboratorio de Neurobiología, Facultad de Ciencias Biológicas, P. Universidad Católica de Chile, Santiago, Chile
A R T I C L E I N F O A B S T R A C T
Petrosal ganglion neurons are depolarized and fire action potentials in response to
acetylcholine and nicotine. However, little is known about the subtype(s) of nicotinic
acetylcholine receptors involved, although α4 and α7 subunits have been identified in
petrosal ganglion neurons. Cytisine, an alkaloid unrelated to nicotine, and its bromo
derivatives are agonists exhibiting different affinities, potencies and efficacies at nicotinic
acetylcholine receptors containing α4 or α7 subunits. To characterize the receptors
involved, we studied the effects of these agonists and the nicotinic acetylcholine receptor
antagonists hexamethonium and α-bungarotoxin in isolated petrosal ganglion neurons.
Petrosal ganglia were excised from anesthetized cats and cultured for up to 16 days.
Using patch-clamp technique, we recorded whole-cell currents evoked by 5–10 s
applications of acetylcholine, cytisine or its bromo derivatives. Agonists and
antagonists were applied by gravity from a pipette near the neuron surface. Neurons
responded to acetylcholine, cytisine, 3-bromocytisine and 5-bromocytisine with fast
inward currents that desensitized during application of the stimuli and were reversibly
blocked by 1 μM hexamethonium or 10 nM α-bungarotoxin. The order of potency of the
agonists was 3-bromocytisine ≫ acetylcholine≅cytisine ≫ 5-bromocytisine, suggesting
that homomeric α7 neuronal nicotinic receptors predominate in cat petrosal ganglion
neurons in culture.
nAChR, nicotinic acetylcholine
PG, petrosal ganglion
The petrosal ganglion (PG) is the main sensory ganglion
containing the perikarya of primary afferent neurons of the
glossopharyngeal nerve, whose peripheral axons project
either through the carotid sinus nerve or the glossophar-
yngeal branch (Alcayaga et al., 1996; Stensaas and Fidone,
1977). The carotid sinus nerve conveys chemo- and
mechanosensory fibers from the carotid body and carotid
sinus, respectively, while the glossopharyngeal branch
contains chemo- and mechanosensory fibers from the
tongue and the pharynx. The transduction of chemical
⁎ Corresponding author. Fax: +56 2 271 2983.
E-mail address: email@example.com (J. Alcayaga).
stimuli involves specialized receptor cells in both the
carotid body and the gustatory papillae.
The current model of chemotransduction in the carotid
body states that chemoreceptor (glomus) cells release one or
more transmitters, which in turn increase the frequency of
sensory discharges originated from PG neurons peripheral
endings (Iturriaga and Alcayaga, 2004). In response to hypoxia
and hypercapnia, the carotid body releases acetylcholine
(ACh) from chemoreceptor cells (Fitzgerald et al., 1999), and
several studies indicate that ACh modulates the release of
catecholamines from cat and rabbit carotid body chemore-
ceptor cells (Hirasawa et al., 2002; Ishizawa et al., 1996;
Iturriaga et al., 2000; Shirahata et al., 1998).
Applications of ACh to the isolated rat and cat PG
neurons in culture evoke inward currents, depolarization
and action potentials, effects mimicked by nicotine and
blocked by hexamethonium (Koga and Bradley, 2000; Varas
et al., 2000; Zhong and Nurse, 1997). Moreover, applications
of ACh to the superfused cat PG generate bursts of action
potentials conducted along the carotid sinus nerve, while
only a few spikes are observed in the glossopharyngeal
branch in response to the highest doses of ACh (Alcayaga
et al., 1998). The neural response evoked by ACh is dose-
dependent and reversibly antagonized by hexamethonium
and mecamylamine. Similarly, recordings from cat PG
neurons functionally connected to the carotid body in
vitro show that application of ACh to their somata
depolarizes these neurons, having no effect on PG mechan-
osensory neurons (Varas et al., 2003). Thus, the electro-
physiological data indicate that a population of PG neurons
projecting through the carotid sinus nerve are selectively
activated by ACh acting on nicotinic ACh receptors located
in the somata of these neurons.
Nicotinic cholinergic receptors (nAChRs) are widely distrib-
uted in vertebrates, including different regions of the central
and peripheral nervous systems (Hogg et al., 2003). Neuronal
nAChRs are pentameric structures resulting from the ensem-
ble of 12 subunits (α2–α10 and β2–β4), of which at least
subtypes are known to be present in other sensory ganglia
(Genzen et al., 2001; Lips et al., 2002; Liu et al., 1998). The
subunit composition of nACh heteromeric receptors deter-
mines features such as conductance and kinetics of desensi-
tization (Fenster et al., 1997), Ca2+permeability and agonist
and/or antagonist affinities. Homomeric α7-nACh receptors
have the highest Ca2+permeability, the fastest desensitization
kinetics of nACh receptors and are blocked by α-bungarotoxin.
In contrast, heteromeric receptors have much lower Ca2+
permeability and are mostly insensitive to α-bungarotoxin
(Fenster et al., 1997; Gotti et al., 1997). Immunohistochemical
studies have shown the presence of α7 and α4 nACh subunits
in the nerve endings as well as in the perikarya of cat PG
neurons that innervate the carotid body, suggesting that α7
and α4β2 receptors predominate in these neurons (Ishizawa et
al., 1996; Shirahata et al., 1998). However, little is known about
the functional type of nAChRs that mediate the electrophys-
iological response of PG neurons to ACh. Accordingly, we
characterized the currents elicited by ACh in PG neurons using
the nAChR agonist cytisine and its bromo derivatives 3-
bromocytisine (3-Br-cy) and 5-bromocytisine (5-Br-cy) to
distinguish between the α7 and α4β2 nicotinic cholinergic
receptor subtypes (Houlihan et al., 2001; Slater et al., 2003).
2.1.ACh-evoked currents in cultured PG neurons
At an imposed membrane resting potential of −60 mV, ACh
(0.02–1 mM) evoked fast-desensitizing, dose-dependent tran-
sient inward currents in 17 out of 24 (71%) PG neurons in
culture (Figs. 1 and 3A). To avoid the effects of receptor
desensitization, at least 5 min were allowed between
successive ACh applications. The nicotinic blocker hexame-
thonium (1 μM) reversibly suppressed ACh-evoked inward
currents in all the cases studied (Figs. 1A, B). Fig. 1 shows the
inward currents induced by ACh in 3 different neurons.
Superfusion for 3 min with Hanks' solution containing 1 μM
hexamethonium completely abolished the inward current
(Figs. 1A, B), an effect that was reversible in all the 17
neurons studied. Similarly, the nicotinic blocker α-bungar-
otoxin (10 nM) reversibly blocked the ACh-evoked inward
current in 5 of 6 PG neurons studied (83%). Fig. 1C shows
that the inward current evoked by ACh (500 μM) in one
neuron was reversibly blocked by superfusion of α-bungar-
otoxin (10 nM) for 3 min. The blocking effect α-bungarotoxin
was reversible in two neurons in which the recording was
stable after a 10-min period of wash out.
2.2.Currents evoked by cytisine and its bromo derivatives
Cytisine and 3-Br-cy evoked dose-dependent, transient
inward currents in 15 of the 17 (88%) neurons responding
to ACh (Figs. 3B–D). In contrast, 5-Br-cy only induced
currents in 10 out of the 15 (59%) neurons responding to
ACh, all of which had responded to cytisine. It is worth
mentioning that only those neurons that responded to ACh
showed currents evoked by cytisine or its brominated
derivatives. Fig. 2 shows a comparison of the responses
evoked by 100 μM applications of ACh, cytisine, 5-Br-cy and
3-Br-cy in a single PG neuron. All these agents produced
transient inward currents that receded within 4 s even
when the agonists were still being applied to the neuron.
For this single concentration of 100 μM, ACh and cytisine
evoked currents of similar amplitude, while 3-Br-cy pro-
duced a larger inward current (about 140%) and 5-Br-cy
evoked a smaller inward current (only 40%). Superfusion of
1 μM hexamethonium for 3 min completely and reversibly
blocked the currents evoked by ACh, cytisine and 5-Br-cy,
but failed to completely block the currents evoked by 3-Br-
cy at concentrations higher than 100 μM (data not shown).
Dose–response curves evoked by ACh, cytisine and its
Responses induced by increasing concentrations of ACh,
cytisine, 3-Br-cy and 5-Br-cy are shown in Fig. 3. ACh and
cytisine (20 μM–1 mM) produced dose-dependent inward
currents of similar amplitude, with a threshold concentra-
tion of 10–20 μM and reaching maxima at concentrations
near 0.5–1 mM (Figs. 3A, B). The maximal currents evoked by
the highest concentrations of ACh and cytisine were in the
order of 1 nA for neurons of about 45 μm of diameter.
However, the neurons were more sensitive to 3-Br-cy, which
produced currents of larger amplitude, reaching their
maximum near 50–100 μM (Fig. 3C). In contrast, 5-Br-cy
only induced very small inward currents in a neuron of
similar size (Fig. 3D).
Fig. 4 summarizes the dose-dependent curves for
inward currents evoked by ACh, cytisine, 3-Br-cy and 5-
Br-cy in 6 neurons. The dose–response curves induced by
ACh and cytisine had similar Imax(−1.02 vs. −1.12 nA) and
ED50(182 vs. 168 μM) (Fig. 4, Table 1), but that evoked by 3-
Br-cy was shifted to the left by one order of magnitude
(EC50 = 16.5 μM) and showed a larger Imax (−1.28 nA). In
contrast, the currents evoked by 5-Br-cy attained a very
small amplitude and showed a much higher EC50(1264 μM)
(Fig. 4, Table 1). Therefore, the rank order of potencies was:
3-Br-cy ≫ ACh ≅ cytisine ≫ 5-Br-cy.
The present results show that ACh evoked transient inward
currents in 71% of PG neurons studied. The proportion of ACh-
sensitive neurons found here agrees with previous studies in
rat (Zhong and Nurse, 1997) and cat cultured PG neurons
Fig. 1 – Inward currents evoked by ACh in three cultured cat PG neurons. Superfusion with Hanks' solution containing 200 or
500 μM ACh (pipette concentration) generates a rapidly inactivating inward current which is reversibly blocked by 1 μM
hexamethonium (A, B) and 10 nM α-bungarotoxin (C). Vh, holding membrane potential at −60 mV.
(Varas et al., 2000). With a holding membrane potential of −60
mV, ACh evoked a fast inactivated inward current, whose
amplitude showed a dose-dependent increase, with a
Fig. 2 – Inward currents elicited by 100 μM (in the pipette)
concentrations of ACh, cytisine, 3-bromocytisine (3-Br-Cy)
and 5-bromocytisine (5-Br-Cy) in a PG neuron in culture. The
bar indicates the duration of the superfusion of the drugs. Vh,
holding membrane potential at −60 mV.
Fig. 3 – Whole cell inward currents evoked by increasing concentrations of ACh (A: 20, 50,100, 200, 500 and 1000 μM), cytisine
(B: 10, 20, 100, 200, 500 and 1000 μM), 3-bromocytisine (C: 2, 5, 10, 20, 50 and 100 μM) and 5-bromocytisine (D: 100, 200, 500
and 1000 μM) in four cultured PG neurons of similar size (diameter 45 μm). Bars indicate the applications of the agonists.
Successive doses were applied at 5 min intervals, at a holding membrane potential (Vh) of −60 mV.
Fig. 4 – Dose–response curves for ACh (filled circles), cytisine
(filled triangles), 3-bromocytisine (3-Br-cy, filled diamonds)
and 5-bromocytisine (5-Br-cy, filled squares) in 6 PG neurons,
at a holding membrane potential of −60 mV. The abscissa
corresponds to the pipette concentration of the agonists.
thresholdof 10–20 μM, reaching a maximumbetween0.5 and 1
mM. Thus, our data on PG neurons obtained from adult cats
agree with those obtained from PG neurons of perinatal rats
(Koga and Bradley, 2000; Zhong and Nurse, 1997). The ACh-
induced current was reversibly blocked by 1 μM hexametho-
nium, a muchlower concentration than the 200 μM requiredto
block the ACh-induced inward current in rat PG neurons
(Zhong and Nurse, 1997). Even more, 10 nM α-bungarotoxin
blocked the current induced by ACh in 83% of the PG neurons
The proportion of PG neurons projecting through the
carotid sinus nerve represents approximately 15–20% of the
total number of neurons in the ganglion, and only a half of the
above proportion probably projects to the carotid body
(Alcayaga et al., 1996; Berger, 1980; Claps and Torrealba,
1988; McDonald, 1983). However, more than two thirds of our
neurons in culture showed sensitivity to ACh. A possibility is
that PG neurons that project through the glossopharyngeal
nerve also present ACh sensitivity. This agrees with the idea
that ACh is the excitatory transmitter between the gustatory
cells of the tongue and glossopharyngeal nerve endings
(Landgren et al., 1954), and the observation that, when
previously marked with Fluorogold applied to the tongue,
50% of the rat PG neurons in culture respond to ACh with
inward currents (Koga and Bradley, 2000), similarly to the
results reported here. Nevertheless, it is also possible that
culture conditions favor the survival of PG neurons that
project to the carotid body or the presence of trophic factors,
as NGF, may induce the upregulation of nAChRs. In addition,
modification of the trophic interaction between PG neurons
and their target tissues due to culture conditions may induce
an over-expression of nAChRs in the membranes of the PG
neurons in culture. This idea is supported by the observation
that scarce antidromic discharges are recorded from the
glossopharyngeal branch in response to ACh applied to the
PG superfused in vitro (Alcayaga et al., 1998).
A large percentage (88%) of the PG neurons that responded
to ACh also responded to cytisine with inward currents of
similar intensity and profile than those elicited by ACh. It
must be noted that a long series of reports by Russian
researchers led Anichkov and Belen'kii (1963) to conclude
that cytisine is “one of the most powerful carotid chemore-
ceptor stimulants” and to use cytisine as a respiratory
analeptic. Furthermore, cytisine applied to the superfused
PG in vitro elicits antidromic discharges in the carotid sinus
nerve (Alcayaga et al., unpublished observations).
Cytisine has a low micromolar affinity for α7 homomeric
nAChRs (Flores et al., 1992; Pabreza et al., 1991), but it is a full
agonist, with higher efficacy than ACh at these receptors.
Otherwise, cytisine nanomolar affinity for α4β2 receptors is
associated with a very low efficacy (0.04%) relative to that of
ACh (Slater et al., 2003). The bromo isosteres of cytisine have
shown widely different affinities and agonist potencies in
recombinant human α7, α4β2 and α4β4 nAChRs expressed in
cultured cells and in Xenopus oocytes (Houlihan et al., 2001;
Slater et al., 2003). According to Houlihan et al. (2001) and
Slater et al. (2003), cytisine and 3-Br-cy are full agonists at α7
nAChRs but partial agonists at α4β2 nAChRs. They also show
high potency and efficacy at α4β4 nAChRs. 5-Bromocytisine
has lower potency and is a partial agonist at α7 and α4β4
nAChRs, but it did not elicit any response at α4β2 nAChRs.
Thus, cytisine and its derivatives are useful tools to distin-
guish between the α7 and α4β2 nicotinic cholinergic receptor
Cytisine and its bromo derivatives evoked inward currents
only in neurons activated by ACh, and these currents were
blocked by hexamethonium. Cytisine shows high agonist
potency (EC50≈ nM) at α4 and β2 subunit-containing nicotinic
α7 nAChRs (Holladay et al., 1997; Houlihan et al., 2001; Slater et
al., 2003). Interestingly, we observed that 3-Br-cy was the most
potent agonist, with the lowest EC50(16.2 μM) of all agonists
used. The rank order of agonist potencies to evoke inward
(see Table 1). Taken together, with the very low efficacy of
cytisine and the relatively weak partial agonism of 3-bromo-
cytisine at α4 and β2 subunit-containing receptors, this
suggests that the current observed here corresponds mainly
to α7 nicotinic ACh receptors. The same rank order of potency
and relative efficacy of cytisine and its bromo derivatives has
been found for human α7 recombinant nAChRs expressed in
Xenopus oocytes or neuroblastoma cell lines (Houlihan et al.,
by ACh on PG neurons shows the same time-course of fast
inactivation that characterized the response of recombinant
human or rat dorsal root ganglion neurons expressing α7
nAChRs (Genzen et al., 2001; Houlihan et al., 2001).
Our electrophysiological recordings suggesting a promi-
nent contribution of α7 subunits agree with the immunohis-
tochemical studies showing the presence of the α7 subunitnot
only in nerve endings in the carotid body, but also in the
somata of PG neurons (Shirahata et al., 1998). Nevertheless, we
observed that 59% of the ACh-sensitive neurons (10/17) also
respondto 5-Br-cy, suggesting that the current evoked by 5-Br-
cy was due to the activation of nAChRs different from the α4β2
subtype, e.g. α4β4. An alternative explanation is that α4β2
nAChRs may differ between cat and human. However,
different specieshaveshownnAChRsof identical composition
(Itier and Bertrand, 2001; McGehee and Role, 1995), suggesting
that other combinations of nAChR subunits (such as α3 and
β2) found in the cat PG neurons (Hirasawa et al., 2002;
Shirahata et al., 1998) may be functional. It is important to
notice that 5 out of 15 neurons which responded to ACh,
cytisine and 3-Br-cy did not respond to 5-Br-cy, suggesting
Table 1 – Functional potency of ACh, cytisine and its
isosters 3-bromocytisine (3-Br-cy) and 5-bromocytisine
(5-Br-cy) on cultured cat PG neurons
Estimated values obtained by fitting the dose–response curves to
a non-linear regression according to the equation I = Imax /
[1 + (EC50 / X)n]. Imax = maximal current evoked by any given
agonist. EC50 = concentration required to evoke half-maximal
current amplitude. X = dose of a given agonist. n = calculated Hill
coefficient (N = 6 neurons).
that other nAChRs subtypes may exist in cat cultured PG
In summary, the present results show that PG neurons
responded to ACh, cytisine, 3-Br- and 5-Br-cytisine with fast
inward currents that desensitized rapidly (during stimuli
application) and were reversibly blocked by 1 μM hexametho-
nium or 10 nM α-bungarotoxin. The order of potency of the
Br-cytisine. Therefore, our results suggest that homomeric α7
nAChRs predominate in cat PG neurons in culture.
Petrosal ganglia were excised bilaterally from 10 adult cats
anesthetized with sodium pentobarbitone (40 mg/kg, i.p.). The
ganglia were placed in ice-chilled modified Hanks' solution (Ca2+–
Mg2+-free), minced into 15–20 pieces, and enzymatically dissoci-
ated under agitation at 38 °C in modified Hanks' solution
supplemented with 1 g/L collagenase, 0.5 g/L trypsin and 150,000
U/L DNAse for 30–60 min. The cell suspension was centrifuged for
10 min at 2000 × g, and the pellet suspended in F-12 nutrient
mixture supplemented with 100 mL/L horse serum, 100 mL/L fetal
bovine serum, NaHCO3(14 mM) and nerve growth factor (15 × 10−6
g/L). The cells were plated onto 35 mm Petri dishes previously
coated with poly-L-lysine (0.1 g/L) and maintained at 38 °C in
water-saturated, 5% CO2in air atmosphere. The culture medium
was changed every other day after a 3-day initial lag during which
the cultures were left undisturbed. The protocol was approved by
the Ethical Committees of the Facultad de Ciencias of the
Universidad de Chile and the Facultad de Ciencias Biológicas of
the P. Universidad Católica de Chile and meets the guidelines of
the National Fund for Scientific and Technological Research
The cultures were placed on the stage of an inverted
microscope with phase contrast and superfused (flow 1.2 mL/
min) at room temperature with Hanks' solution (in mM: NaCl 137,
CaCl21.3, MgSO40.8, KCl 5.4, KH2PO40.4, Na2HPO40.3, D-glucose
5.6, HEPES 5 and NaHCO34) at pH 7.43 equilibrated with room air.
Recordings were made with a patch-clamp amplifier (PC 501-A;
Warner Instrument Corp., USA) and 1.5-mm O.D. borosilicate glass
pulled electrodes (1–2 MΩ) filled with an intracellular solution (in
mM: KCl 135, NaCl 5, CaCl21, EGTA 10, HEPES 10, pH 7.2). Seal
formation and membrane breakthrough, from 25 to 65 μm
diameter neurons, was carried out in the current-clamp mode
and monitored by observing the response to one 2–10 pA
depolarizing current step lasting 50 ms.
The currents evoked by ACh, cytisine, 3-Br-Cy and 5-Br-Cy
were recorded in the whole cell voltage-clamp mode at a holding
potential (Vh) of −60 mV. Liquid junction potential between
microelectrodes and bath solution was corrected, and series
resistance and capacitive transients were electronically compen-
sated. All agonists were applied for 5–10 s by superfusion under
gravity flow from a pipette whose tip was located at about 100–200
μm from the neuron surface. Hexamethonium (1 μM) and α-
bungarotoxin (10 nM), in Hanks' solution, were applied for 3 min
through a superfusion pipette.
4.1.Purification of cytisine and its analogs
Cytisine was purified from Sophora secundiflora seeds using
standard methodology. 3-Br-cy and 5-Br-cy were obtained by
treating cytisine with slightly more than 1 M equivalent of
bromine in acetic acid. The brominated isomers were separated
by column chromatography on silica gel, crystallized to homoge-
neity and characterized by1H and13C NMR.
4.2. Statistical analyses
To compare the effects of ACh, cytisine and its bromo derivatives
onthe dose–responsecurves for the inward current (I),pooled data
of individual experiments were fitted to the following logistic
expression: I = Imax/ 1 + (EC50/ X)n, where Imax= maximal current
evoked by a given agonist, EC50 = agonist concentration that
evoked the half-maximal current, X = agonist concentration
delivered by the stimulus pipette and n = Hill coefficient.
Correlation coefficients for adjusted curves were higher than
0.90 (P b 0.01) for all conditions studied.
This work was supported by FONDECYT (National Fund for
Scientific and Technological Research, Chile) grants 1040638,
1040776 and 1010951.
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