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The Journal of Neuroscience, September 15, 2002, 22(18):8230-8237
Vanilloid-Sensitive Afferents Activate Neurons with Prominent
A-Type Potassium Currents in Nucleus Tractus Solitarius
Timothy W.
Bailey,
Young-Ho
Jin,
Mark W.
Doyle, and
Michael
C.
Andresen
Department of Physiology and Pharmacology, Oregon Health and
Science University, Portland, Oregon 97201-3098
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ABSTRACT |
Cranial visceral afferents innervate second-order nucleus tractus
solitarius (NTS) neurons via myelinated (A-type) and unmyelinated (C-type) axons in the solitary tract (ST). A- and C-type afferents often evoke reflexes with distinct performance differences, especially with regard to their frequency-dependent properties. In horizontal brainstem slices, we used the vanilloid receptor 1 agonist capsaicin (CAP; 100 nM) to identify CAP-sensitive and CAP-resistant
ST afferent pathways to second-order NTS neurons and tested whether
these two groups of neurons had similar intrinsic potassium currents. ST stimulation evoked monosynaptic EPSCs identified by minimal synaptic jitter (<150 µsec) and divided into two groups:
CAP-sensitive (n = 37) and CAP-resistant
(n = 22). EPSCs in CAP-sensitive neurons had longer
latencies (5.1 ± 0.3 vs 3.6 ± 0.3 msec;
p = 0.001) but similar jitter
(p = 0.57) compared with CAP-resistant
neurons, respectively. Transient outward currents (TOCs) were
significantly greater in CAP-sensitive than in CAP-resistant neurons.
Steady-state currents were similar in both groups.
4-Aminopyridine or depolarized conditioning blocked the TOC, but
tetraethylammonium had no effect. Voltage-dependent activation and
inactivation of TOC were consistent with an A-type
K+ current, IKA. In
current clamp, the activation of IKA reduced neuronal excitability and action potential responses to ST
transmission. Our results suggest that the potassium-channel
differences of second-order NTS neurons contribute to the differential
processing of A- and C-type cranial visceral afferents beginning as
early as this first central neuron. IKA can
act as a frequency transmission filter and may represent a key target
for the modulation of temporal processing of reflex responsiveness such
as within the baroreflex arc.
Key words:
sensory; vanilloid; glutamate; presynaptic modulation; autonomic; visceral; baroreceptor; baroreflex
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INTRODUCTION |
Cranial afferent information is
conveyed by a mixture of myelinated (A-type) and unmyelinated (C-type)
axons that form the solitary tract (ST) to synapse in the nucleus
tractus solitarius (NTS). These two afferent classes appear to have
important functional differences in their contributions to homeostatic
regulatory reflexes (Loewy, 1990 ; Andresen and Kunze, 1994). In
particular for example, A-type arterial baroreceptors have lightly
myelinated axons, higher conduction velocities, and lower pressure
thresholds compared with C-type baroreceptors (Kunze and Andresen,
1991). The reflexes evoked by selective activation of A- and C-type
baroreceptor afferents can have quite different frequency-response
characteristics (Fan and Andresen, 1998; Fan et al., 1999). Little is
known about the mechanisms responsible for these differences in A- and
C-type reflexes. Here we take advantage of the presence of the
vanilloid receptor 1 (VR1) on C-type axons (Fan and Andresen, 1998;
Doyle et al., 2002) and use capsaicin (CAP) to identify the general class of ST afferent input to second-order NTS neurons and examine the
properties of the postsynaptic neurons contacted by these two classes
of cranial visceral afferent CAP-sensitive and CAP-resistant neurons.
The cellular processes within and beyond the NTS that contribute to
differential processing of A- and C-type cranial visceral afferents are
unknown. Some of these autonomic pathways may have as few as two
central neurons (e.g., the cardiac parasympathetic pathway) (Standish
et al., 1994). The NTS contains the initial synapse on the second-order
neuron of these paths, but the second-order neurons are intermixed
within a heterogeneous population with varying intrinsic properties.
Previous studies suggested that the transient A-type voltage-gated
K+ current
IKA is one of the varied features of
these NTS neurons (Dekin and Getting, 1984, 1987; Dekin et al., 1987;
Moak and Kunze, 1993; Paton et al., 1993). The presence of
IKA affects the excitability and
firing characteristics of neurons (Rudy, 1988; Malin and Nerbonne, 2001), but the relationship of these differences to the overall function of the nucleus is unknown. Here we report the unexpected finding that CAP can be used to distinguish two different groups of
second-order neurons (Doyle et al., 2002) that differ in their expression of K+ currents. The
IKA currents are larger in
CAP-sensitive second-order NTS neurons; this difference in
IKA expression reduces overall excitability and the ability of ST activation to evoke action potentials. Thus, preferential expression of
IKA in NTS neurons with input from
VR1-containing afferents contributes to the differential processing of
C-type cranial visceral afferents within the NTS.
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MATERIALS AND METHODS |
NTS slices. Horizontal slices of the hindbrain of
male Sprague Dawley rats (150-350 gm; Charles River Laboratories,
Wilmington, MA) were prepared as described previously (Doyle et
al., 2002). All animal procedures were conducted with the approval of
the University Animal Care and Use Committee in accordance with U.S. Public Health Service Policy on Humane Care and Use of Laboratory Animals and the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Solutions, recording conditions, and the focus on medial portions of the NTS (mNTS) were as described previously (Doyle et al., 2002).
Electrophysiological recordings. Patch recording electrodes
(1.8-3.5 M ) were filled with a solution composed of (in
mM): 10 NaCl, 130 K gluconate, 11 EGTA, 1 CaCl2, and 2 MgCl2, 2 ATP, 0.2 GTP, 10 HEPES, pH 7.3, 295 mOsm. Selection criteria were based on
the recording stability and the monosynaptic jitter of ST EPSC latency
(Doyle et al., 2002). In addition to voltage-clamp recordings (Axopatch
200A; Axon Instruments, Foster City, CA) described previously (Doyle et
al., 2002), some experiments used single-electrode continuous voltage-clamp and current-clamp recordings in the same neurons (Axoclamp 2A; Axon Instruments).
For assessments of outward current responses, voltage-clamp protocols
included command voltage steps used to evoke transient outward currents
(TOCs) and steady-state currents (SSCs). The voltage dependence of the
activation of these currents was assessed by initially conditioning at
 90 mV (500 msec) followed by longer activation steps (1200 msec) to
voltages ranging from 100 to 0 mV. Voltage-dependent inactivation of
TOCs was assessed with conditioning steps (500 msec) ranging from 100
to 0 mV that were followed by a longer test step (1200 msec) to 10 mV
(see Fig. 2). SSCs were measured near the end of the second long step
in each protocol. The peak of the TOCs was measured after capacitive transients had subsided at 3-5 msec after the initiation of the long
step and SSC subtracted. In some experiments, current-clamp recordings
were used to assess the effect of activating TOCs on the firing
properties of second-order neurons by injecting depolarizing and
hyperpolarizing steps of current to determine the effect on current-evoked firing and ST-evoked responses (see Figs. 6 and 7).
Tetraethylammonium (TEA) and 4-aminopyridine (4-AP) were obtained from
Sigma (St. Louis, MO). Both TEA and 4-AP were dissolved in extracellular solution on the day of the experiment.
All data are presented as averages ± SEM. Statistical comparisons
were made using either unpaired Student's t test,
repeated-measures (RM) ANOVA, or one-way ANOVA followed by Fisher's
PLSD post hoc analysis when appropriate (see individual
results) (Statview 4.57; Abacus Concepts, Calabasas, CA).
p < 0.05 indicated a significant difference.
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RESULTS |
CAP-sensitive ST-evoked synaptic responses
The shock-to-shock variability of the EPSC response latencies to
electrical activation identifies mNTS neurons monosynaptically connected to the ST in horizontal brainstem slices (Doyle and Andresen,
2001). We selected neurons with an EPSC jitter of <150 µsec for
study as second-order neurons. Challenges with CAP (100 nM)
divided these neurons into two groups on the basis of the CAP
sensitivity of their EPSCs. ST-evoked EPSCs of CAP-sensitive mNTS
neurons were blocked by sustained exposure to CAP (Fig.
1A, left).
In the remainder of the mNTS second-order neurons, CAP did not alter
their ST-evoked EPSCs (CAP-resistant) (Fig. 1A, right). Note that ongoing spontaneous EPSCs were not blocked
in both classes of neurons. The ST-evoked EPSCs of CAP-sensitive neurons had a significantly longer latency on average (5.1 ± 0.3 msec; n = 37) (Fig. 1B,
left) compared with CAP-resistant mNTS neurons (3.6 ± 0.3 msec; n = 22; p = 0.0016;
Student's t test). However, latency alone poorly predicted
EPSC sensitivity to CAP blockade, because the distribution of latencies
for the two groups overlapped considerably (Fig. 1B,
right). The average jitter for CAP-sensitive (82.0 ± 4.8 µsec) and CAP-resistant (86.9 ± 7.6 µsec) neurons was not
different (p = 0.57; unpaired Student's t test).
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Figure 1.
CAP sensitivity of ST-evoked synaptic responses
in mNTS neurons. ST stimulation [five shocks (arrows)
at 20 msec intervals] consistently triggered EPSCs in these mNTS
neurons. Neurons with synaptic jitters (SD of latency) of <150 µsec
were selected for study and considered to be second-order mNTS neurons.
The holding potential was 70 mV. The sustained application of CAP
(100 nM) blocked ST-evoked EPSCs in CAP-sensitive neurons
but not in the remaining neurons (CAP-resistant). A,
Left, Nine successive ST-evoked EPSCs from one such
CAP-sensitive neuron before (top) and 5 min after
(bottom) CAP application. A,
Right, ST EPSCs from a representative CAP-resistant
neuron before (top) and 5 min after
(bottom) CAP application. B,
Left, Average latency of ST-evoked EPSCs in
CAP-sensitive neurons (hatched bar;
n = 37) was significantly longer than in
CAP-resistant neurons (solid bar; n = 22; *p = 0.0016; Student's
t test). B, Right,
Histogram (bin size, 0.5 msec) of the distribution for these
individual latencies in CAP-resistant (solid bars) and
CAP-sensitive (hatched bars) neurons. Although group
average latencies were different, the two overlapped
considerably.
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Prominent transient voltage-dependent outward currents in
CAP-sensitive mNTS neurons
The pattern of time- and voltage-dependent outward currents
activated by depolarization differed significantly between
CAP-sensitive and CAP-resistant mNTS neurons (Fig.
2). From a potential of 90 mV,
sustained depolarization elicited large outward currents in CAP-sensitive neurons that decayed over time (Fig.
2A, top). Conditioning such neurons at
progressively more depolarized potentials virtually eliminated these
early transient currents in CAP-sensitive neurons (Fig.
2B, top). These early transient,
voltage-dependent currents resemble A-type potassium currents (Pongs,
1999). In contrast, outward currents in CAP-resistant neurons were
dominated by sustained currents with little early transient component
(Fig. 2A,B, middle). Average peak SSCs
(Fig. 3) were significantly different in
CAP-sensitive compared with CAP-resistant neurons
(p < 0.0001; RM ANOVA). At activation voltages
from 60 to 0 mV, the average TOCs were significantly larger in
CAP-sensitive neurons compared with those observed in CAP-resistant
neurons (p < 0.0011; one-way ANOVA and
Fisher's PLSD). The SS component of these currents in the same neurons was indistinguishable between CAP-sensitive and CAP-resistant neurons
(p = 0.752; RM ANOVA) (Fig. 3B). The
outward current patterns were highly dependent on the membrane
potential preceding the depolarization step. From hyperpolarized
holding potentials (90 mV), depolarizing steps activated TOCs
beginning at more than 60 mV (Fig. 3A). These TOCs had
similar voltage-dependent activation in both CAP-sensitive and
CAP-resistant neurons (Fig. 3A). CAP-resistant neurons
generally had the smallest peak transient currents, whereas CAP-sensitive neurons had the largest (Fig. 3C). However,
the two groups overlap in the magnitude of peak TOCs (Fig.
3C). Inactivation of the normalized TOC in CAP-sensitive
neurons (Fig. 4) was steeply dependent on
the voltage in the range nearest the resting potential from 70 to
50 mV. Together, these results suggest that second-order neurons
connected to VR1-containing afferents express larger TOCs than neurons
connected to afferents without VR1. The TOCs in these mNTS neurons
resemble A-type K+ currents
IKA (Dekin and Getting, 1987; Moak and
Kunze, 1993).
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Figure 2.
Voltage-dependent activation and inactivation of
outward currents in two representative second-order mNTS neurons, one
CAP-sensitive and one CAP-resistant. A, Prolonged
depolarization evoked prominent TOCs in CAP-sensitive neurons but not
in CAP-resistant neurons. All neurons were held at 70 mV before each
test sequence. Neurons were preconditioned at 90 mV for 500 msec
followed by step commands to test potentials (100 to 0 mV in 10 mV
increments). For clarity, only six representative current responses
(A, bottom) are displayed. The large
outward current to sustained depolarization of the CAP-sensitive neuron
(top) rapidly decayed. Current traces from a
representative CAP-resistant neuron (middle) show little
such TOC. B, Conditioning neurons at depolarized
potentials suppressed the TOC component in the CAP-sensitive neuron
(top) but had little effect on the outward currents of
CAP-resistant (middle) neurons (representative neurons).
Whole-cell currents were measured in response to step voltage commands.
Between sweeps, neurons were held at 70 mV. Conditioning steps varied
in 10 mV increments from 100 to 0 mV. Conditioning was followed by a
prolonged (1200 msec) step to 10 mV. For clarity, only 5 of the 11 sweeps are shown. Voltage step protocols are outlined in the
bottom panels.
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Figure 3.
Summary of average outward currents in
CAP-sensitive (n = 37) and CAP-resistant
(n = 22) mNTS neurons. Voltage-dependent TOCs (Peak SS) and SSCs. Voltage step protocols were identical to those in
Figure 2. Points are averages ± SEM. A,
CAP-sensitive neurons (solid line) had a significantly
larger TOC compared with CAP-resistant neurons (dashed
line; p < 0.0001; RM ANOVA). Comparison of
the TOC observed at specific voltages revealed that TOCs were larger in
CAP-sensitive neurons beginning at 60 mV and extending to 0 mV
(one-way ANOVA; *p < 0.001). B,
Average SSCs were similar (p = 0.752; RM
ANOVA) in CAP-sensitive (solid line) and CAP-resistant
(dashed line) neurons. C, Distribution of
peak TOCs in CAP-sensitive and CAP-resistant mNTS second-order neurons.
CAP-sensitive neurons (hatched bars) generally expressed
larger TOCs than did CAP-resistant neurons (solid bars),
although there is some overlap.
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Figure 4.
Summary of mean activation and inactivation
relationships for TOCs of CAP-sensitive neurons. Voltage protocols were
identical to those in Figure 2. Average normalized peak TOCs ± SEM (n = 37) are plotted against activation voltage
( ) or conditioning voltage ( ). These voltage-dependent activation
and inactivation characteristics of TOCs resemble those reported for
A-type, 4-AP-sensitive potassium currents.
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Pharmacological characterization of outward currents
4-AP reliably blocks A-type K+
currents (Pongs, 1999) and identified
IKA in dissociated NTS neurons (Moak
and Kunze, 1993). In subsets of our CAP-sensitive mNTS neurons, we
tested whether the TOCs or SSC outward currents were sensitive to 4-AP
or TEA. 4-AP selectively depressed the TOC in a concentration-dependent manner (p = 0.0001) without altering the
sustained, steady-state level of the current (Fig.
5A, top right). In
the voltage range for activation (40 to 0 mV), both 1 and 5 mM 4-AP consistently reduced the group average
peak TOC (Fig. 5A, top right) compared with
controls (p < 0.026). 4-AP did not
significantly affect SSCs (p = 0.262) (Fig.
5A, bottom right). TEA (10 mM), in contrast, depressed only the SSC
(p = 0.0014) without altering the TOCs (p = 0.663) (Fig. 5B). Application of
TEA reduced SSCs at 10 and 0 mV (p < 0.0007) (Fig. 5B, right). Together, these results identify the TOC as the 4-AP-sensitive
IKA; this current appears to be
expressed much more in second-order mNTS neurons with CAP-sensitive afferent inputs.
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Figure 5.
Pharmacological sensitivity of outward currents in
CAP-sensitive mNTS neurons. TOC was calculated as the peak SSC and
SSC measured at the end of the test step. Command voltage step
protocols are indicated in the bottom traces of
A and B. Average values are means ± SEM in each summary relationship. A,
Left, Three current traces from a representative
experiment generated by the step command in the bottom
trace show the effects of 1 and 5 mM 4-AP on TOCs
and SSCs. 4-AP reduced the TOC in this neuron in a dose-dependent
manner but did not affect the SSCs. The middle trace is
a difference current obtained by subtracting the control and 5 mM 4-AP traces to yield the net 4-AP sensitive current.
Right, Summary data for 1 and 5 mM 4-AP on
TOCs and SSCs in five CAP-sensitive neurons. Points are
averages ± SEM in controls ( ), with 1 mM 4-AP
(), and 5 mM 4-AP ( ). Both 1 and 5 mM
4-AP caused a significant reduction in voltage-activated TOCs compared
with controls (p = 0.0001; RM ANOVA). Both 1 and 5 mM 4-AP reduced the TOCs observed at 40 through 0 mV (#p < 0.025; one-way ANOVA and
Fisher's PLSD). SSCs were not affected by 4-AP (bottom
right; p = 0.262; RM ANOVA). These results
suggest that the TOC observed in CAP-sensitive mNTS neurons is an
A-type potassium current. B, Left, Two
representative current traces evoked by steps to zero potential from
90 mV conditioning levels. The middle trace is a
difference current obtained by subtracting the control and 10 mM TEA traces to yield the net TEA-sensitive current.
Right, Summary data for TEA (10 mM) in seven
CAP-sensitive neurons. TEA had no effect on the TOC
(p = 0.6626; RM ANOVA) but reduced the SSCs
(p = 0.0014; RM ANOVA) at 10 and 0 mV
(*p < 0.0007; one-way ANOVA with Fisher's
PLSD).
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K+ current differentially modulates synaptic
excitation in CAP-sensitive and CAP-resistant mNTS neurons
Such substantial differences in K+
currents between CAP-sensitive and CAP-resistant mNTS neurons should
have an important impact on the ability of afferent synaptic inputs to
excite the postsynaptic neurons and, therefore, to determine the
characteristics of the sensory throughput of these pathways. To assess
this, we measured responses to current injection (Fig.
6) and to ST activation (Fig. 7) under current clamp in representative
CAP-sensitive and CAP-resistant second-order neurons. Equivalent
hyperpolarizing current injection evoked a delayed excitation in
CAP-sensitive neurons to subsequent depolarizing current injection
(Fig. 6B, top, arrow), and this was not present in CAP-resistant neurons (Fig. 6B,
middle). The length of this delay was modulated by both the
magnitude and the duration of membrane hyperpolarization in a manner
consistent with the activation of IKA
(data not shown). In CAP-sensitive second-order neurons, the generation
of action potentials during synaptic activation should be quite
dependent on membrane potential history. ST stimulation reliably evoked
action potentials in these neurons at steady resting potentials (Fig.
7A, left). However, when preceded by significant
membrane hyperpolarization, the same ST stimuli often failed to produce
action potentials (Fig. 7A, right). In contrast,
in a CAP-resistant neuron without a notable IKA, membrane hyperpolarization did
not reduce ST-evoked action potentials (Fig. 7B). Together,
these results demonstrate that the differential expression of
IKA in CAP-sensitive mNTS second-order neurons will result in very different processing of visceral afferent inputs.
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Figure 6.
The differential expression of A-type potassium
currents produces different firing properties in second-order
CAP-sensitive and CAP-resistant mNTS neurons. A,
Representative voltage traces measured in current-clamp mode from a
CAP-sensitive (toptrace) and a CAP-resistant
(middle trace) neuron show evoked firing produced by
current injection shown in the current command below (+40 pA). Both
neurons fire repeated action potentials with little or no delay
after the onset of current injection. B, Representative
voltage traces measured in current-clamp mode from the same
CAP-sensitive (top trace) and CAP-resistant
(middle trace) neurons in A showing
evoked firing after negative current-evoked membrane hyperpolarization
and subsequent depolarization (100 and +40 pA, step current command
shown in bottom trace). The hyperpolarization caused a
delay in the production of current-evoked action potentials
(arrow) in the CAP-sensitive neuron. In contrast, there
was no delay in firing produced by hyperpolarization in the
CAP-resistant neurons. The CAP-sensitive neuron from which these
voltage traces were recorded displayed a large
IKA-like current. The CAP-resistant neuron
did not contain such a current. The delay in evoked firing was likely
caused by hyperpolarization-induced removal of inactivation of A-type
potassium currents.
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Figure 7.
A-type potassium currents have an impact on the
processing of ST-evoked responses in second-order mNTS neurons.
A, Representative sequential voltage traces measured in
current-clamp mode from a representative CAP-sensitive neuron (same
neuron as Fig. 6) showing ST-evoked membrane potential responses.
Top traces show a complete, 2.5 sec test with ST-evoked
EPSPs and action potentials. Bottom traces show expanded
sweeps of five consecutive tests to better display responses to bursts
of five ST shocks (triangles). Note that the voltage
scaling of the expanded sweeps truncates action potentials. ST
stimulation in controls produced EPSPs that reliably generated action
potentials (A, left). Only 2 of 20 ST
shocks failed to trigger an action potential in this example.
Introduction of a hyperpolarizing current step (top,
long trace) suppressed action potentials but reliably
produced EPSPs to ST shocks in this CAP-sensitive neuron
(A, right). B, Tests
similar to those shown in A were conducted on a
representative CAP-resistant neuron (same neuron as Fig. 6). ST
stimulation (triangles) reliably produced EPSPs that
generated action potentials with and without conditioning
hyperpolarization. These observations suggest that the expression of
A-type currents has a differential impact on the processing of afferent
inputs in CAP-sensitive and CAP-resistant second-order mNTS
neurons.
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DISCUSSION |
Second-order mNTS neurons with VR1-containing afferents express
larger A-type K+ current
Our studies have identified two groups of second-order neurons
within the mNTS that have very different patterns of postsynaptic expression of K+ current subtypes. The
sensitivity of ST-activated EPSCs to block by sustained CAP exposure
divided the neurons into two groups, CAP-sensitive and CAP-resistant.
CAP blocks ST-evoked EPSCs by the presynaptic activation of VR1
receptors (Doyle et al., 2002). CAP-sensitive neurons had significantly
larger absolute TOCs than CAP-resistant NTS neurons. The SSCs
were similar in all mNTS second-order neurons. Both the voltage- and
time-dependent characteristics of the TOC and the pharmacological
sensitivity were consistent with A-type potassium currents,
IKA. Our results demonstrate that in
CAP-sensitive neurons, the activation of
IKA reduced neuronal excitability and depressed the generation of action
potentials by ST-evoked synaptic transmission. This preferential
expression of IKA in VR1-containing
pathways suggests that differences in presynaptic afferents are coupled
with key postsynaptic differences at the second-order neuron in the mNTS.
CAP discrimination of A- and C-fiber afferent inputs to
the mNTS
The NTS receives a wide range of both fast-conducting, myelinated
afferents and slow-conducting, unmyelinated afferents from the viscera
(Loewy, 1990; Spyer, 1990; Andresen and Kunze, 1994). Many of these
afferents are well known to be activated by brief exposure to the VR1
agonist CAP (Marsh et al., 1987; Bevan et al., 1992; Li and Owyang,
1996; Malinowska et al., 2001). Neonatal CAP treatment permanently and
selectively eliminates C-type afferent neurons (Bevan and
Szolcsányi, 1990; Holzer, 1991; Szallasi and Blumberg, 1999).
Prolonged short-term application of CAP to the aortic depressor nerve,
a nerve trunk that contains only aortic baroreceptor afferent axons
(Sapru and Krieger, 1977; Kobayashi et al., 1999), selectively blocked
the conduction of C-type axons and their reflex effects (Fan and
Andresen, 1998). In another study (Doyle et al., 2002) and this study,
application of CAP to brainstem slices blocked ST-evoked EPSC
transmission to second-order mNTS neurons; we conclude that this
pharmacological sensitivity is consistent with the presence of VR1 on
C-type but not A-type cranial visceral afferent axons. These results in
ST afferent axons are consistent with the known properties of the
peripheral portions of cranial visceral afferents. Thus, CAP-sensitive
ST responses have longer average latencies than CAP-resistant ST pathways. Together, our results suggest that CAP pharmacologically discriminates between A- and C-fiber inputs to second-order neurons in
the mNTS.
Potassium channels in sensory integration
Potassium-selective ion channels display a wide range of gating
and kinetic properties that may shape cellular integration (Coetzee et
al., 1999; Pongs, 1999; Trussell, 1999; Choe, 2002), yet their
contribution within the fuller context of a physiological system is
poorly understood (Coetzee et al., 1999). Our present studies have
identified a differentiation of afferent pathways for two
pharmacological classes of visceral sensory afferents based on the
presence of VR1 and their consequent CAP sensitivity. These two
pathways contain second-order neurons with substantially different
K+ channel expression and correspond to A-
and C-fiber visceral afferent pathways. Previous studies of the
intrinsic properties of NTS neurons have proposed subgroups based on
morphological and electrophysiological differences, including
IKA (Dekin and Getting, 1987; Dekin et
al., 1987; Moak and Kunze, 1993; Schild et al., 1993; Tell and Bradley,
1994), but their relationship to functional processing within the NTS
was unknown. IKA finely modulates the
intrinsic firing rate, action potential threshold, and interspike
interval (Dekin and Getting, 1987; Dekin et al., 1987; Schild et al.,
1993; Pongs, 1999). Comprehensive models of the ionic currents of mNTS
neurons suggest an intimate interaction of voltage and time dependence,
with a key role for IKA in the modulation of these multiple aspects of neuronal excitability (Schild
et al., 1993). The removal of IKA from
model neurons yields a dichotomy of predicted performance results that
are quite similar to our present results from current-clamp recordings
in response to current injection. Ultimately, these findings indicate
that the transduction process of afferent action potential trains into spikes in the second-order mNTS neurons will be distinctly different through these two classes (CAP-sensitive and CAP-resistant) of afferent
pathways and should give rise to functionally distinct reflex responses.
Divergent processing along A- and C-type afferent pathways in
the mNTS
The voltage and time dependence of
IKA predicts a substantial impact on
the processing of afferent inputs in the reflex pathways through the
NTS. The respective activation and inactivation relationships for
IKA in NTS neurons were steeply
dependent on membrane potential and suggest that the depolarizations
and hyperpolarizations that accompany excitatory and inhibitory
synaptic transmission will strongly affect
IKA activity and therefore neuronal
excitability in a physiologically critical range. GABAergic inhibitory
synaptic inputs are common on second-order mNTS neurons and frequently accompany afferent volleys through what appear to be local inhibitory circuits (Andresen and Yang, 1990; Mifflin and Felder, 1990; Andresen and Mendelowitz, 1996).
Our recordings were limited to the mNTS. This portion of the NTS
contains the greatest density of aortic baroreceptor terminal fields
(Mendelowitz et al., 1992). Selective activation of C-type afferents
from aortic baroreceptors evokes reflex responses that are markedly
different in their frequency-response relationships than for A-type
aortic baroreceptors (Fan et al., 1999). These reflex responses are
particularly dependent on the pattern of the afferent volley and,
therefore, the temporal distribution of afferent spikes entering
the CNS (Seagard et al., 1993; Fan and Andresen, 1998; Fan et al.,
1999). Burst patterns of stimulation elicited greater reflex changes in
blood pressure with A-type but not C-type afferents compared with
constant stimulus frequencies (Fan and Andresen, 1998). Part of this
frequency-dependent matching between the A-type, naturally
high-frequency baroreceptor action potential trains, with burst
responsiveness of A-type baroreflex responses, may well reflect a
relative paucity of IKA and the braking effects of its activation at the second-order neuron.
Baroreflex modulation via A-type
K+ current
An intriguing prospect of IKA is
its association with modulation by neurotransmitters (Gage, 1992;
Jänig and McLachlan, 1992; Deadwyler et al., 1995; Wang et al.,
1997). A wide range of neurotransmitters, including neuropeptides, are
found in the NTS; many of these appear to be differentially distributed
across A- and C-type afferents (Andresen and Kunze, 1994). Thus,
divergent processing of A- and C-fiber afferent inputs may be directed
toward the modulation of IKA.
Voltage-dependent K+ currents in the CNS
are modulated by neurotransmitters such as substance P (Haeusler and
Osterwalder, 1980), vasopressin (Oz et al., 2001), angiotensin II (Wang
et al., 1997), and calcitonin gene-related peptide (Zona et al., 1991).
Interestingly, our results indicate that
IKA is preferentially expressed in
second-order neurons with C-fiber afferents. This coupling of afferent
fibers containing peptidergic machinery with postsynaptic neurons
containing cellular targets provides a potential mechanism for the
modulation of afferent signal processing through cellular targets on
second-order neurons.
Overall, our results indicate that mNTS neurons that are contacted by
C-fiber cranial visceral afferents preferentially express greater
IKA compared with those connected to A
fibers. This offers an intriguing bias toward the lower frequencies of
transmission, with reduced fidelity to high frequencies of excitatory
visceral afferent inputs in the C-type pathway. Thus, even at the first central neuron, this frequency transmission in NTS resembles the frequency performance of the full C-type pathway for certain reflexes, such as the baroreflex. This nonuniform expression of potassium channels in second-order neurons likely contributes to the divergent processing of A- and C-fiber afferents in mNTS. Because of the important position of these neurons in the baroreflex loop,
IKA may represent a cellular target
for modulation of the baroreflex by neurotransmitters in various
physiological conditions that transform the reflex.
| |
FOOTNOTES |
Received April 5, 2002; revised June 10, 2002; accepted June 13, 2002.
This work was supported by National Institutes of Health Grant HL-41119
and by grants from the National Center of the American Heart Association.
Correspondence should be addressed to Dr. Michael C. Andresen,
Department of Physiology and Pharmacology, Oregon Health and Science
University, Portland, OR 97201-3098. E-mail: andresen{at}ohsu.edu.
M. W. Doyle's present address: Department of Biology, George Fox
University, Newberg, OR 97132-2697
| |
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ABSTRACT
INTRODUCTION
