Department of Pediatrics, Division of Respiratory Medicine, Yale University School of Medicine, New Haven, Connecticut 06520
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ABSTRACT |
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Donnelly, David F..
Developmental Changes in Membrane Properties of Chemoreceptor
Afferent Neurons of the Rat Petrosal Ganglia.
J. Neurophysiol. 82: 209-215, 1999.
Carotid body
chemoreceptors increase their responsiveness to hypoxia in the
postnatal period, but the mechanism for this increase is unresolved.
The purpose of the present study was to examine developmental changes
in cellular characteristics of chemoreceptor afferent neurons in the
petrosal ganglia with the underlying hypothesis that developmental
changes occur and may account for the developmental increase in
chemoreceptor responsiveness. Chemoreceptor complexes (carotid body,
sinus nerve, glossopharyngeal nerve, and petrosal ganglia) were
harvested from rats, aged 3-40 days, and intracellular recordings were
obtained from petrosal ganglion neurons using sharp electrode
impalement. All chemoreceptor neurons across ages were C fibers with
conduction velocities <1 m/s and generated repetitive action
potentials with depolarization. Resting membrane potential was
61.3 ± 0.9 (SE) mV (n = 78) and input
resistance was 108 ± 6 M
and did not significantly change with
age. Cell capacitance was 32.4 ± 1.7 pF and did not change with
age. Rheobase averaged 0.21 ± 0.02 nA and slightly increased with age.
Action potentials were followed by an afterhyperpolarization of
12.4 ± 0.6 mV and time constant 6.9 ± 0.5 ms; only the time
constant decreased with age. These results, obtained in rat,
demonstrate electrophysiologic characteristics which differ
substantially from that previously described in cat chemoreceptor
neurons. In general developmental changes in cell characteristics are
small and are unlikely to account for the developmental increase in chemoreceptor responsiveness with age.
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INTRODUCTION |
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Carotid body chemoreceptors transduce a decrease
in arterial PO2 into an increase in sinus nerve
afferent activity, which in turn stimulates respiration. This reflex is
weak at birth, resulting in the generation of few action potentials,
even during strong stimulation (Blanco et al. 1984), and
the magnitude of the response matures during the first few weeks after
birth (Blanco et al. 1984
; Kholwadwala and
Donnelly 1992
). Several investigators have suggested that the
maturation process primarily occurs at the level of the glomus cell
a
secretory cell that is presynaptic to the afferent nerve terminals and
that responds to hypoxia by increasing intracellular calcium and
enhancing catecholamine secretion (Urena et al. 1994
).
In support of this contention, the magnitude of the anoxia-induced
calcium transient as well as the level of catecholamine secretion has
been shown to increase in the postnatal period (Donnelly and
Doyle 1994
; Sterni et al. 1995
).
In contrast to the developmental data available on glomus cell function
in this period, no datum is currently available on maturational changes
in the characteristics of the primary afferent neuron. However, changes
in afferent neuron characteristics may potentially play a critical role
in determining the level of spiking activity for a given stimulus. For
instance, in one model of hypoxia transduction, the afferent spike is
initiated by the summation of excitatory events at the nerve terminals
(Hayashida et al. 1980). From this model, it is obvious
that a change in excitability (e.g., threshold, input resistance) could
greatly affect the translation of these excitatory synaptic events into
the output train.
The purpose of the present study was to examine developmental changes
in membrane properties of chemoreceptor afferent neurons with the
underlying assumption that the soma and nerve terminals share similar
membrane characteristics. Thus observations at the soma may be useful
in understanding processes that take place in the nerve terminal. Such
similarity is well established for baroreceptor afferent fibers that
possess stretch activated channels in the soma and peripheral processes
(Kraske et al. 1998). The hypothesis of the present
study was that developmental changes occur and may explain the
developmental increase in chemoreceptor responsiveness.
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METHODS |
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Experimental preparation
Both carotid body/sinus nerve/petrosal ganglia (chemoreceptor
complex) were isolated from 28 rats, aged 3-40 days, using a methodology adapted from the original work of Belmonte and Gallego who
isolated the chemoreceptor complex from adult cats (Belmonte and
Gallego 1983). For the isolation, rats were anesthetized by placement in a closed chamber in which the atmosphere was saturated with methoxyflurane vapor. After deep anesthesia as evidenced by an
absence of motor movements, outside of respiration, the rats were
removed and decapitated. The carotid body was exposed by dissecting
medial to the carotid artery and removing the superior cervical
ganglia. The petrous bone was split and the central portion of the
vagal and glossopharyngeal nerves were dissected free and reflected
over the carotid bifurcation. The bifurcation and associated nerves
were cut free and placed in chilled, oxygenated (95%
O2-5% CO2) Ringer saline
[containing (in mM) 125 NaCl, 3 KCl, 1 NaH2PO4, 2 CaCl2, 1.5 MgSO4, 26 NaHCO3, and 10 glucose].
Using sharp dissection, the area was cleaned with the removal of vagal fibers, carotid arteries, and connective tissue, and the remaining tissue was placed in a dilute solution of collagenase/trypsin (0.1%/0.01%) in oxygenated Ringer saline for 30 min to aid in further cleaning. After exposure to the enzymes, the connective tissue was gently pulled/cut away using a fine pipette and scalpel blade until the carotid body/sinus nerve/glossopharyngeal nerve and petrosal ganglia were easily visible and free of connective tissue. The chemoreceptor complex was transferred to a perfusion chamber mounted on the stage of an inverted microscope, and the preparation was superfused with Ringer saline equilibrated with 21% O2-5% CO2 and heated to 32-33°C.
Stimulus electrode
An insulated metal microelectrode (Frederick Haer) with a 5-µ exposed tip was advanced into the carotid body under visual control. While searching for a chemoreceptor unit or during unit characterization, a cathodal electrical stimulus (0.1-ms duration) was delivered through an optically isolated, constant current source (BAK instruments, BSI-1) to evoke an orthodromic action potential. Initial current level was set at 800 µA, which is ~10-20 times higher than the minimal current necessary to evoke an orthodromic action potential.
Intracellular recording
Intracellular recordings from the soma of petrosal neurons were
obtained used micropipettes filled with 3 M KCl and connected to an
electrometer (IE-210, Warner Instruments) the output of which was
filtered from 0 to 2 kHz. Tip resistance was 80-120 M. A
chemoreceptor petrosal neuron was identified based on the orthodromic
action potential initiated from the stimulus electrode placed in the
carotid body. Criteria for an acceptable recording included a stable
membrane potential for
5 min after cell puncture.
Passive properties
Characterization of passive membrane properties included measurement of resting membrane potential and input resistance. Input resistance was based on the steady-state magnitude of hyperpolarization during application of a 0.1-nA hyperpolarizing current. For measurement of membrane time constant, the charging transient at the start of application of a 0.1-nA hyperpolarizing current was fit to a single order exponential function (pClamp, Axon Instruments). Estimation of membrane capacitance was calculated by dividing the membrane time constant by the input resistance.
Spike properties
All spike properties were based on measurements obtained from orthodromic spikes elicited by current pulses applied to the carotid body. Control of the stimulus pulse and data acquisition were controlled by pClamp software (Axon Instruments) at 10-kHz digitization rate. Spike rise time was the time period for the spike to rise from 10 to 90% of its peak value. Action potential height was the difference between the peak of the action potential waveform and the resting membrane potential. Afterhyperpolarization potential (AHP) was the difference between the resting membrane potential and the maximum hyperpolarization after a spike. AHP time constant was estimated by a best-fit approximation of the post-AHP potential to a single exponential function.
Repetitive spiking properties
Rheobase was measured as the minimal current necessary to evoke at least one action potential. The stimulus current was generally applied for 100 ms and was incremented by 0.1 nA over the range of 0.1-2 nA. In some cases, the stimulus was applied between 0.05 and 1 nA in increments of 0.05 nA. Peak discharge frequency was calculated as the instantaneous frequency between the first and second spikes during application of a 0.8-nA depolarizing stimulus. If no repetitive spike discharge was observed, then it was deemed that the cell was unable to support repetitive spike generation.
Spontaneous action potential and response to cyanide
In seven cells that initially were identified as chemoreceptor afferent fibers based on orthodromic electrical stimulation, the response to brief application was tested. Cyanide (0.5 mM, 2 s) was applied locally to the carotid body through micropipette using positive ejection pressure (Picospritzer, General Value). Cells, which responded by an increase in spontaneous discharge rate, were considered as confirmed chemoreceptor fibers and used as a comparison population for the larger sample.
Data analysis
Each variable was plotted against the age of the rat from which the chemoreceptor complex was harvested. A linear regression against age was calculated as well as the 95% confidence interval for the regression line (Systat). A significant effect of age on the measured variable was established if the regression slope was significantly different from 0 at the 0.05 level. Data also were grouped by age into three age groups: 1-10 days, 11-20 days, and >20 days and analyzed for an effect of age using ANOVA. Post hoc testing was accomplished Student's t-test.
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RESULTS |
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Conduction velocity
Application of a cathodal electrical stimulus to the carotid body evoked an orthodromic action potential in both the older (Fig. 1) and in the newborn rat (Fig. 2). The latency between the stimulus and evoked spike was 3.53 ± 0.13 (SE) ms for the entire population (n = 78) but significantly increased ~2.5-fold with age (P < 0.001; Fig. 3). This was primarily due to an increase in conduction distance because the estimated conduction velocity for the population was 0.52 ± 0.02 m/s and increased only slightly with age (P < 0.05; Table 1).
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Passive properties
RESTING MEMBRANE POTENTIAL AND INPUT RESISTANCE.
Resting membrane potential for the entire population was 61.3 ± 0.9 mV (n = 78); however, there was considerable
variability among individual cells. This seemed in part due to a
variable response to electrode impalement that usually caused a brief
depolarization followed by a hyperpolarization beyond
80 mV, followed
by a slow depolarization and stabilization. ANOVA analysis of resting
membrane potential showed no difference among the three age groups
(Table 1). Input resistance, as measured with a hyperpolarization step of 0.1 nA averaged 108 ± 6 M
, and, like the resting membrane potential, did not change significantly with age (Table 1).
MEMBRANE TIME CONSTANT AND CAPACITANCE. Membrane time constant was estimated by fitting the charging transient for a 0.1-nA hyperpolarization pulse to a single-order exponential. Average membrane time constant was 3.1 ± 0.2 ms for the entire population. Time constant significantly decreased with age (P < 0.001, Fig. 4, Table 1) with approximately a 50% decrease over the period of 3-40 days. Dividing each time constant by the input resistance yielded an estimate of membrane capacitance that averaged 32.3 ± 1.7 pF for the entire population. Age had no significant effect on capacitance among the three age groups (Table 1).
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Active properties
SPIKE PROPERTIES.
Spike height for the population averaged 58.4 ± 1.3 mV and was
not significantly different among the three age groups (Table 1).
However, the spike rise time, which averaged 0.44 ± 0.2 ms for
the entire population, did significantly decrease with age (P < 0.05; Table 1). The spike was followed by an AHP
that averaged 12.4 ± 0.6 mV and had an average time constant of
~6.9 ± 0.5 ms (Figs. 1 and 2). The AHP amplitude was not
significantly different among age groups (Fig. 4, Table 1), but the
time constant shortened significantly (P < 0.05) by
~40% during the developmental time period (Fig. 4, Table 1).
Differentiation of the spike-wave failed, in all instances, to
demonstrate a "hump" on the falling phase (Figs. 1 and 2). A hump
on the falling phase is characteristic of many chemoreceptor neurons
from the adult cat and suggests the presence of a significant calcium
current in cat neurons (Belmonte and Gallego 1983).
RHEOBASE AND REPETITIVE DISCHARGE ACTIVITY. The minimum injected current needed to elicit an action potential averaged 0.21 ± 0.02 nA for the entire population and slightly, but significantly (P < 0.05), increased with age (Fig. 4, Table 1). Injections of greater amounts of current evoked multiple action potentials in every cell tested (Figs. 1 and 2), and, during injection of +0.8 nA the average spiking rate was 139 ± 5 Hz. Age had no significant effect on the spiking rates observed during this current injection (Table 1).
RESPONSE TO CYANIDE. In the above sample, identification of purported chemoreceptor afferent neurons was based on the ability to orthodromically activate the afferent from within the carotid body. To test the chemoresponsiveness of these cells, cyanide (0.5 mM) was "spritzed" on the carotid body in seven cases. In five of these cells, an increase in spontaneous spiking activity was observed readily after cyanide application (Fig. 5). Analysis of the spontaneous and evoked action potentials for these five cells were consistent with the averages obtained on the initial sample and the spontaneous action potentials were identical to those obtained using orthodromic electrical stimulation (Fig. 5).
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DISCUSSION |
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This is the first study to characterize the electrophysiological properties of petrosal chemoreceptor neurons of the rat and to examine developmental changes in the neuronal characteristics. Previous studies examined chemoreceptor neurons of the adult cat, and it is now apparent that there is a marked species difference between rat and cat in a number of variables. These issues will be considered in the following text, but it is first worth considering the issue of developmental changes in chemoreceptor responsiveness.
Previous results from our laboratory and others have demonstrated a
large developmental change in chemoreceptor responsiveness in the
newborn period. Fetal and newborn chemoreceptors generate significantly
fewer spikes in response to a strong hypoxic stimulus than do
chemoreceptors from older animals (Blanco et al. 1984; Kholwadwala and Donnelly 1992
). The enhanced spiking
activity may be due to a greater level of stimulation of the nerve
terminals or, potentially, due to changes in the sensitivity of the
nerve terminals to stimulation. Regarding the first possibility,
considerable data have been developed showing developmental changes in
the characteristics of the presynaptic element, the glomus cell. First, the magnitude of the hypoxia-induced increase in intracellular calcium
increases with development (Sterni et al. 1995
), and
this is correlated with an increase in the magnitude of hypoxia-induced catecholamine release from these cells (Donnelly and Doyle
1994
). On the other hand, it is currently unclear whether
glomus cell secretion of catecholamines causes the increase in afferent
nerve activity. Under several experimental conditions, there is a
dissociation between the magnitude of catecholamine secretion and
magnitude of nerve activity (Buerk et al. 1997
;
Donnelly 1996b
; Iturriaga et al. 1996
),
suggesting that hypoxia transduction may, at least in part, be due to
release of other transmitters such as substance P (Prabhakar et
al. 1993
) or acetylcholine (Fitzgerald and Shirahata 1994
). Alternatively, the nerve terminal itself may have an
endogenous sensitivity to hypoxia (Mitchell et al.
1972
).
Regardless of whether the nerve terminals are a site of hypoxia
transduction or not, the nerve terminals represent a potential site for
maturational changes in organ function. Although direct electrophysiologic study of the nerve terminals would be the preferred experiment, such recordings are extremely difficult due to their small
size (McDonald 1981), and, instead, recordings from the soma were used as a proxy for the membrane characteristics. From this
standpoint, the developmental changes are relatively minor and are
unlikely to account for the developmental increase in chemoreceptor
responsiveness. The lack of significant changes in resting potential or
input resistance suggests that no major changes occur in the passive
characteristics of these neurons. Rheobase, which may have been
expected to increase with development, actually decreased slightly with
development. Furthermore peak repetitive firing rates for chemoreceptor
afferents are considerably faster than that observed using
physiological stimulation and do not change with age. Thus the low
spiking rates in the newborn period are not due to a limitation of the
afferent neuron in supporting repetitive action potential generation.
Given that the membrane characteristics are not different with
development, a better explanation for the maturational increase in
activity is the enhancement in glomus cell stimulus/secretion coupling
with development (Donnelly and Doyle 1994
) or the
maturational increase in the number of axonal terminals per parent axon
with a corresponding increase in the number of spike generating sites
(Kondo 1976
). In addition, it is well established that
survival of the chemoreceptor afferent fibers as well as transmitter
phenotype is dependent on oxygen and depolarizing stimuli in the fetal
and newborn period (Erickson et al. 1998
;
Hertzberg et al. 1995
). How these phenotypic changes are
related to electrophysiologic characteristics currently is unexplored
and may form a useful focus for future studies.
Identification of purported chemoreceptor neurons was based on the
ability to orthodromically generate an afferent spike and the response
to chemostimulating agents was not routinely tested. Thus some
nonchemoreceptive neurons may have contributed to the sampled
population, perhaps due to electrical stimulation of C-fiber axons that
pass over the carotid body. However, it seems likely that the level of
contamination is small. C-fiber axons of passage are difficult to
stimulate electrically and require very high voltages or stimulus
currents. For instance, the threshold for C-fiber activation using
direct sciatic nerve stimulation is >100 V (Traub and Mendell
1988), which is beyond the voltage compliance of the stimulus
isolator used in the present study. In addition, the majority (5/7) of
cells that were tested for chemosensitivity showed spontaneous spiking
activity and an increase in activity after cyanide application. Spike
characteristics for these cells were consistent with the larger
population identified by orthodromic electrical stimulation.
Species dependence
In contrast to previous studies on cat carotid body
(Belmonte and Gallego 1983), virtually all rat
chemoreceptor afferents were C fibers
-a result generally consistent
with observation that >86% of rat sinus nerve fibers are unmyelinated
(McDonald 1983
). Estimated somal capacitance was also
small at 32 pF. This characterization is consistent with the
histological assessment of cell characteristics after retrograde tracer
application to the rat carotid body, which primarily labeled the
smallest cells of the petrosal ganglia (Finley et al.
1992
). In contrast to the rat, the vast majority of cat
chemoreceptor petrosal neurons were reported to have conduction
velocities >10 m/s (Belmonte and Gallego 1983
), and
somal capacitance approximately three times larger than the rat
(Belmonte and Gallego 1983
).
A second major difference between cat and rat appears to be
characteristics of action potential and ability to generate multiple spikes. Rheobase for the rat cells averaged ~0.2 nA with many cells
initiating spikes at lower levels of current injection. For cat cells,
the comparable value is ~1 nA (Belmonte et al. 1988;
Gallego et al. 1987
). Cat cells also fail to generate
multiple spikes at higher levels of current injection, and, instead,
generate only one to two spikes (Belmonte and Gallego
1983
). In contrast, all of the rat chemoreceptor cells
generated multiple spikes during strong depolarizations. The ionic
basis for this species difference is unclear since action potentials of
both species are followed by AHPs of similar magnitude (Belmonte
and Gallego 1983
). However, the duration of the AHP is almost
10 times longer in the cat cells, perhaps impairing the ability to
initiate new spikes before depolarization-induced inactivation of the
Na+ current. The long AHP in the cat cells
appears to be caused by a pronounced calcium current during the action
potential and activation of calcium-dependent K+
currents (Belmonte and Gallego 1983
). This calcium
current may not be shared with rat cells because no hump on the falling
phase of the action potential was observed in any recordings from the rat.
Given these differences, it is perhaps surprising that the afferent
spike trains from rat and cat chemoreceptors are similar. In both
cases, the afferent spike train approximates a Poisson random process
with some deviation due to the refractory period and a tendency of some
fibers to discharge in doublets (Donnelly 1996a;
Eyzaguirre and Koyano 1965
). Although the postspike
refractory period in the rat (~8 ms) is similar to the observed
duration of somal AHP, this is not true of the cat, which has a much
longer AHP (Belmonte and Gallego 1983
). It is currently
unclear why the long AHP is not manifest in the interspike interval
histogram of cat chemoreceptors, but the result suggests that the cat
nerve terminals may have different membrane characteristics than the soma.
In summary, the present results demonstrate that electrophysiologic characteristics of chemoreceptor rat petrosal neurons are relatively stable in the postnatal period and developmental changes in the cellular characteristics are unlikely to account for the postnatal increase in chemoreceptor responsiveness. However, the results also demonstrate that rat chemoreceptor neurons differ substantially from those previously reported from adult cat, particularly in conduction velocity, rheobase and ability to support multiple action potential generation. It is hoped that knowledge of the excitable properties of the afferent fibers will ultimately allow us to model the spike generation process of the arterial chemoreceptors.
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ACKNOWLEDGMENTS |
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This work supported by National Heart, Lung, and Blood Institute Grant HL-46149.
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FOOTNOTES |
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Address for reprint requests: D. F. Donnelly, Division of Respiratory Medicine, Dept. of Pediatrics, Yale University School of Medicine, 333 Cedar St., New Haven, CT 06520.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 30 July 1998; accepted in final form 5 March 1999.
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REFERENCES |
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