324 Brain Research.

534 (1990) 324-328

Elsevier

BRES 24404

Electrophysiological evidence for the reconstitution of chemosensory

units in co-cultures of carotid body and nodose ganglion neurons

J. Alcayaga* and C. Eyzaguirre

Department of Physiology, University of Utah School of Medicine, Salt Lake City, UT 84108 (U.S.A.)

(Accepted 21 August 1990)

Key words,: Carotid body; Glomus cell; Nodose ganglion neuron

The electrophysiological characteristics of nodose ganglion sensory neurons, cultured alone or co-cultured with carotid body tissue, were
compared. Some properties of the neurons and their response to acid (a carotid body 'natural' stimulus) changed in the presence of this tissue.
(a) The evoked action potential after-hyperpolarization was smaller and longer whereas spike amplitude and duration, and the passive
membrane properties remained unaltered. (b) Spontaneously occurring action potentials happened more frequently (16% vs 3%). (c) Acid
solutions induced appreciable depolarization, an increased discharge, or both, only in a population of co-cultured neurons. These changes
probably arose because of synaptic and/or trophic interactions between neurons and glomus cells.

The carotid body is a complex organ containing two
types of parenchymal cells, type I (glomus) and type II
(sustentacular), the glomus cells being innervated by
afferent terminals of the carotid nerve with which they
form synaptic contacts 26'27'35'36. The sensory nature of
this system was established early, and there is a great deal
of information on the carotid nerve afferent impulses,
responsible for respiratory and cardiovascular reflexes 9'13
and on possible mechanisms of transduction at the
glomus cell level 5'7'9"13'21'24'25. But, we know little about
the other side of the junction, the nerve terminals. These
elements present spontaneous depolarizing potentials at
'rest' which, upon stimulation, increase in frequency and
fuse into a relatively large depolarization. These re-
sponses are local, non-propagated, resembling synaptic
or generator (receptor) potentials 2°. However, these
observations are few and descriptive because it is ex-
tremely difficult to record from the endings.
The working hypothesis, frequently espoused by this
and other groups, is that carotid body excitation by
'natural' stimuli (hypoxia, hypercapnia, acidity) elicits
the carotid nerve discharge via a synaptic process. It
entails release of transmitters from the glomus cells which
depolarize the nerve terminals prior to setting up the
all-or-none discharge of the nerve fibers (for refs. see ref.
9). Thus, the purpose of the present work was to study
in detail the behavior of th~ neural (postsynaptic) side of
the junction, formed by the nerve terminals, at rest and
during stimulation. To diminish the problems presented
by intracellular recordings from the endings, we used a
different approach. Sensory neurons from the nodose
ganglion were cultured close to carotid body cells because
both tissues survive well in culture 1-3"6'14'15'18'28'29'34 and
there is formation of synapses between the neurons and
glomus cells 18. Intracellular recordings from the neuron
somata, accomplished by many investigators, should
detect not only action potentials but also (by electrotonic
spread) non-propagated responses originating at the end
of the neurites contacting carotid body ceils. For stimu-
lation we applied acid solutions, one of the carotid body
'natural' stimuli. As a control, nodose ganglion cells were
cultured alone and subjected to the same stimulus.
Nodose ganglia and carotid bodies were removed from
anesthetized male Sprague-Dawley rats (100-150 g) and
dissociated in modified Hanks' solution (Ca 2+, Mg 2÷-
free) containing trypsin (0.05%), coUagenase (0.2%),
and DNAse (150 U/ml) at 37 °C for 35-50 min. Some
co-cultures were prepared by cutting the carotid bodies
into 10-20 pieces, instead of using enzymatic dissocia-
tion, and pure nodose ganglion cell cultures were
similarly prepared. The ceils were plated on 35 mm Petri
dishes coated with either poly-L-lysine (100 /~g/ml) or
fibronectin (10 /~g/ml), and cultured in H a m ' s F-12,
supplemented with 10% fetal bovine serum, 10% heat-
inactivated horse serum, 20 mM HEPES, and 15 ng/ml of
7S nerve growth factor, that was changed every other

* Postdoctoral Fellow of the Organization of American States. On leave of absence from the Departamento de Bioiogia, Faeultad de Ciencias,
Universidad de Chile.
Correspondence: J. Alcayaga, Department of Physiology, University of Utah School of Medicine, 410 Chipeta Way, Research Park, Salt Lake
City, UT 84108, U.S.A.

0006-8993/90/$03.50 (~) 1990 Elsevier Science Publishers B.V. (Biomedical Division)

 325

day. Some pure ganglion cell cultures were treated for 3 hyperpolarization became smaller and longer. Differ-
days, beginning on the third day, with 3.7/zM cytosine ences with previous work should not be too surprising
arabinofuranoside. since the species (cat vs rat), the ganglia (petrosal vs
The cultures were placed on the stage of an inverted nodose) and environmental conditions (adult intact ani-
phase-contrast microscope and observed at 100-400x. mal vs culture) are all quite different.
Neurons, cultured for 3-78 days (mean = 18 days), were An important difference between our two types of
superfused with 1.1-1.3 ml/min of Earle's balanced cultures was the number of neurons that presented
saline, containing 26 mM NaHCO 3 (pH 7.37-7.47), spontaneous activity. Cells from pure nodose ganglion
equilibrated with 95% 0 2 and 5% CO 2 at room temper- cultures seldom presented spontaneous activity (~-3%),
ature. Intracellular penetrations of the neuron somata whereas about 16% of the neurons in co-cultures had
with glass microelectrodes (resistance 60-100 MI2) al- spontaneously occurring spikes. In both instances, the
lowed recordings of membrane and action potentials as action potentials were usually preceded by a slow (up to
well as the input resistance, via a bridge circuit. Acid 40 ms) depolarization of up to 10 mV (Fig. 3A),
applications were made by adding either 20-100/zl of suggesting that the spontaneous activity originated near
lactic (5.3%) or acetic acid (0.12 N) to the 2 ml bath, or the recording site. Low levels of spontaneous discharges
by pressure pulses which ejected Earle's solution supple- in nodose cell cultures have been associated with the
mented with 20 mM HEPES (pH 6.3-7), contained in a presence of non-neuronal cells or target tissues 6. In our
micropipette situated 10-100/zm from the recorded cell. experiments, the abundance of non-neuronal cells may
Recordings of membrane and action potentials, pH
and temperature were stored on FM tape (DC to 2500
Hz) for later analysis. The signals were digitized off-line
using an analog-to-digital converter, whose sampling rate
(up to 38 kHz) and gain were adjusted to obtain adequate
resolution of a particular event. The resulting data files
were stored on diskettes, analyzed and displayed using
custom-developed programs as well as commercial
spreadsheets and graphics software.
Nodose ganglion neurons generally appeared as round
cells with perikarya of 20-60/zm, standing out from the
cellular background because of their high birefringency
(Fig. 1). Their morphological characteristics were similar
to those described for newborn and adult rat nodose
ganglion neurons 2,3,6. Isolated and clustered carotid body
cells could be well recognized during the first days in
culture (Fig. 1), but as the non-neuronal background
became confluent and more dense their identification
became difficult. However, glomus cells survive well in
culture for long periods of time 15'28.
The passive and active electrical properties of nodose
ganglion neurons (Fig. 2) were similar to those previously
reported in vitro 3'16,23,33, and the time in culture did not
obviously change these parameters. Neurons cultured
alone and co-cultured with carotid body tissue had similar
electric characteristics, except that the action potential of
co-cultured neurons had smaller and longer after-hyper-
polarizations (Fig. 2). No obvious explanation is available
for these differences. Peripheral, but not central, axot-
omy increases the action potential duration and markedly
reduces the amplitude and duration of the spike after-
hyperpolarization of the cat petrosal ganglion neurons 4, Fig. 1. Phase-contrast micrograph of a 6-day co-culture of carotid
17 These changes are reverted after peripheral body fragment and nodose ganglion cells. A neuron appears as a
reinnervation 4. Our cultured neurons reacted differently highly birefringent cell (small arrow) near a cluster of carotid body
cells (big arrow). Non-neuronal cells appear as background of flat
in the presence of the target tissue: the spike after- small cells. Bar = 100/zm.
 326
have reduced the incidence of spontaneous neuron
activity. But, co-cultures contained similar or greater
numbers of non-neuronal cells and spontaneous action
potentials occurred much more often suggesting that the
presence of glomus tissue was important in generating
such activity.
Acid applications to the superfusate or localized
pressure ejections produced variable effects on pure
nodose ganglion cells. Frequently, the neurons were
unaffected. In other cases, rapid depolarizations or
hyperpolarizations of up to 10 mV, although usually not
exceeding 5 mV, were recorded during the stimulation
period. Action potentials, evoked by suprathreshold
depolarizing pulses, were usually blocked (Fig. 3B) but
this effect was overridden by increasing the stimulus
strength, suggesting an acid-induced increase in thresh-
old.
Similar effects have been reported for cultured spinal
cord neurons in which iontophoretic H ÷ applications
induce a short-lasting, low amplitude depolarization and
an increase in spike threshold 19. It may entail an effect on
ion channels since acidity reduces sodium and potassium
conductances in the nodes of Ranvier of amphibian
sciatic nerve fibers 22. However, cultured mouse dorsal
root ganglion cells behave differently since their mem-
branes are not affected by acid 19.
Applications of acid to nodose ganglion neurons in
co-cultures produced small changes in resting potential
(depolarization or hyperpolarization) accompanied by an
80- lo6loz 83
-80
loo ~;~t~ 60
81
tj)
¢0
I--
Z 4£
s o 10,
I ~:;~, , o
-MP APa APd AHPa AHPcl Ro
Fig. 2. Neurons in pure nodose ganglion cell cultures (empty bars)
or in co-culture with carotid body cells (hatched bars). Abscissa:
mean values of -MP, resting (membrane) potential; APa, action
potential amplitude; APd, action potential duration; AHPa, after-
hyperpolarization amplitude; AHPd, after-hyperpolarization dura-
tion; Ro, input resistance. Ordinates: units are amplitudes of
different parameters measured in mV; durations in ms; and Ro in
MI2. Number of observations over each bar; asterisks indicate
statistically significant differences (Mann-Whitney U-test; P <
o.o5).
increase in spike threshold in about 80% of the cells (Fig.
3C). However, in the remaining 20%, a much larger
response was evoked. When acid was applied to the
superfusate and pH fell by 0.7 to 2.5 units, the neurons
depolarized by a maximum of 45 mV in about 30-60 s
before slowly returning to the baseline (Fig. 4A). The pH
changes had a similar time course (not shown), suggest-
ing that the membrane potential of these neurons was
readily affected by the acid in the solution. No action
potentials appeared during this slow depolarization.
Acid application by pressure ejection produced two
types of responses in co-cultured neurons. In some cases,
the acid elicited an increased discharge frequency during
the whole period of stimulation but only slight changes in
resting potential (Fig. 4B). This effect suggests that the
acid was acting at some distance from the neuron soma,
and that spikes initiated elsewhere were conducted along
the neurites before invading the soma. The small changes
in membrane potential may have resulted from a direct
effect of the acid on the neuron membrane, as observed
A
25
E 0
¢-i
25
-50
0 20 40 60 80
13
0 -- -
~E -40
-6(3,
0 1 0 20 30
c 35-
-700 10 ~0 30
T I M E (m~)
Fig. 3. Intracellular recordings from nodose ganglion neurons
co-cultured with carotid body tissue (A,C) or cultured alone (B). A:
spontaneous action potential preceded by a slow depolarization of
about 30 ms duration. B: responses evoked by depolarizing pulses
before and during pressure ejection of acid. C: similar to B, but acid
stimulation was accompanied by a slight hyperpolarization. During
acid applications depolarizing pulses did not evoke an action
potential, even when depolarization reached prior threshold level.

in pure nodose cell cultures. In other cases, acid ejection
produced a rapid and large cell depolarization, accom-
panied by several spikes tiding on it, which was followed
by slow repolarization (Fig. 4C). Depolarization of the
neuron soma probably evoked a train of action potentials
once threshold was reached. This effect was not a
mechanical artifact, by fluid ejection, since deliberate
displacement of the cell with the recording electrode did
not produce any significant changes in membrane poten-
tial.
The presence of non-neuronal cells or other target
tissues reduces the spontaneous activity of cultured rat
nodose ganglion neurons 6. However, when the same
neurons are co-cultured with carotid body tissue there is
a higher incidence of spontaneous spike activity, which
can increase in frequency with acid stimulation. Thus,
carotid body parenchymal cells seem to restore or add
new qualities to the neurons TM as occurs in situ. Chemo-
reception, disrupted by axotomy, reappears after axonal
regenerationS'3~'36; also, mechanosensory fibers acquire
A
eO S responses, recorded from nodose neurons co-cultured
-55
-60
-65
ll[llillIll Jil il l .U.lrl.ll/
c acid-induced synaptic activity in co-cultures may be
T'°m'"
1OS
Fig. 4. Intracellular recordings of acid-induced responses from 3
different nodose ganglion neurons in co-culture. A: two consecutive
applications (arrows) of 50 pl of acetic acid (0.12 N) induced slow
depolarizations lasting more than 1 rain, with slower return to
baseline. Note that action potentials were not elicited. B: acid (pH
7.0) pressure ejections (between arrows), delivered as 200 ms pulses
at 1 Hz, produced small depolarization accompanied by increased
discharge frequency during stimulation. C: 4 pulses (as in B) of
pressure-ejected acid (pH 6.3) produced rapid neuron depolariza-
tion eliciting action potentials after second acid pulse (shown as a
step in the membrane potential) wkich continued during repolari-
zation.
327
chemosensory properties after a carotid body is trans-
planted to a muscle 27.
Functional restoration in vivo occurs concomitantly
with reappearance or formation of synapses between
neurons and glomus cells. These are more numerous
when neurons reinnervate their original targets rather
than non-specific ones 8'35'36. Something similar may have
happened in our nodose ganglion cell-carotid body
cultures (see also refs. 2, 18) although the original nature
of the sensory neurons (mechanical, chemical, etc.) is
unknown. Thus, one may expect fewer synapses if the
carotid body target is innervated by non-chemosensitive
neurons even when function is restored. In spite of the
existence of these synapses, trophic effects of glomus
cells on the sensory neurons 27 may also participate in the
addition and/or restitution of the chemosensory proper-
ties in co-cultures.
Acidity increases the chemosensory discharges both in
situ 3° and in freshly isolated tissue in vitro 1°, and this
response is reduced by synaptic blockers 12. Also, the
glomus cells are sensitive to acidity 11, which releases
neurotransmitters from them 32. Thus, the acid-induced
with carotid body tissue, may result from synaptic
interactions between glomus cells and neurons. This
assumption is supported by the recording of large
neuronal depolarizations accompanied by an increased
discharge during acid application. The distance between
the recording electrode and synapses may explain why no
spontaneous depolarizing potentials 2° were recorded at
rest and the occasional absence of neuron depolarization
during excitation. As in situ, these potentials are prob-
ably local, spreading electrotonically to the soma via the
thin conduits provided by the neurites. Their space
constant should be very short, allowing only the largest
evoked depolarizations to be detected in the soma. The
produced by transmitters released from the glomus cells
impinging on the neurite terminals. In similar co-cultures,
pressure ejections of two carotid body transmitters,
acetylcholine and dopamine, induced neuron depolariza-
tion and action potential trains which were respectively
blocked by hexamethonium and haloperidol TM. Although
similar tests have not been conducted for the acid-
induced responses there is evidence for interactions,
probably synaptic, between neurons and glomus cells. In
co-cultures, there is synapse formation between these
structures TM and only here this stimulus markedly depo-
larized the neurons and increased the frequency of
spontaneously occurring action potentials. Alternatively,
these acid-induced responses could result from an en-
dogenous neuronal chemosensory capability induced by
the glomus cells in co-culture. Although similar experi-
328
m e n t a l results c o u l d be o b t a i n e d in this situation, this
possibility m a y be s e c o n d a r y b e c a u s e of the p r e s e n t l y
k n o w n g l o m u s cell r e s p o n s e s to c h e m o s e n s o r y stimuli 5'
7.11,21,24,25
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The authors wish to thank Dr. K. English for her advice and help
in facilitating the use of her tissue culture facility. Also, the technical
assistance of Messrs. J. Fisher en B. Evans is gratefully acknowl-
edged. Work supported by Grants NS 05666 and NS 07938 from the
National Institutes of Health and Grant AHA 860662 from the
American Heart Association.
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