Hypertension Induced by Angiotensin II and a High Salt Diet Involves
Reduced SK Current and Increased Excitability of RVLM Projecting
Qing-Hui Chen, Mary Ann Andrade, Alfredo S. Calderon, and Glenn M. Toney
Exercise Science, Health and Physical Education Department, Michigan Technological University, Houghton, Michigan
Submitted 19 November 2009; accepted in final form 13 August 2010
Chen QH, Andrade MA, Calderon AS, Toney GM. Hypertension
and increased excitability of RVLM projecting PVN neurons. J Neuro-
physiol 104: 2329–2337, 2010. First published August 18, 2010;
doi:10.1152/jn.01013.2009. Although evidence indicates that activa-
tion of presympathetic paraventricular nucleus (PVN) neurons con-
tributes to the pathogenesis of salt-sensitive hypertension, the under-
lying cellular mechanisms are not fully understood. Recent evidence
indicates that small conductance Ca2?-activated K?(SK) channels
play a significant role in regulating the excitability of a key group of
sympathetic regulatory PVN neurons, those with axonal projections to
the rostral ventrolateral medulla (RVLM; i.e., PVN-RVLM neurons).
In the present study, rats consuming a high salt (2% NaCl) diet were
made hypertensive by systemic infusion of angiotensin II (AngII), and
whole cell patch-clamp recordings were made in brain slice from
retrogradely labeled PVN-RVLM neurons. To determine if the am-
plitude of SK current was altered in neurons from hypertensive rats,
voltage-clamp recordings were performed to isolate SK current. Re-
sults indicate that SK current amplitude (P ? 0.05) and density
(P ? 0.01) were significantly smaller in the hypertensive group. To
investigate the impact of this on intrinsic excitability, current-clamp
recordings were performed in separate groups of PVN-RVLM neu-
rons. Results indicate that the frequency of spikes evoked by current
injection was significantly higher in the hypertensive group
(P ? 0.05–0.01). Whereas bath application of the SK channel blocker
apamin significantly increased discharge of neurons from normoten-
sive rats (P ? 0.05–0.01), no effect was observed in the hypertensive
group. In response to ramp current injections, subthreshold depolar-
izing input resistance was greater in the hypertensive group compared
with the normotensive group (P ? 0.05). Blockade of SK channels
increased depolarizing input resistance in normotensive controls (P ?
0.05) but had no effect in the hypertensive group. On termination of
current pulses, a medium afterhyperpolarization potential (mAHP)
was observed in most neurons of the normotensive group. In the
hypertensive group, the mAHP was either small or absent. In the latter
case, an afterdepolarization potential (ADP) was observed that was
unaffected by apamin. Apamin treatment in the normotensive group
blocked the mAHP and revealed an ADP resembling that seen in the
hypertensive group. We conclude that diminished SK current likely
underlies the absence of mAHPs in PVN-RVLM neurons from hy-
pertensive rats. Both the ADP and greater depolarizing input resis-
tance likely contribute to increased excitability of PVN-RVLM neu-
rons from rats with AngII-Salt hypertension.
I N T R O D U C T I O N
Low-dose systemic infusion of angiotensin II in combination
with a high salt (2% NaCl) diet (AngII-Salt) leads to develop-
ment of arterial hypertension in rats. Like many forms of
hypertension in humans, the AngII-Salt model is salt-sensitive
and dependent on sustained activation of the sympathetic
nervous system (King and Fink 2006; King et al. 2008; Kline
et al. 1990; Osborn et al. 2007; Toney et al. 2010). At present,
the CNS sites and cellular mechanisms that mediate the in-
crease of sympathetic nerve activity are unknown.
Studies have demonstrated that acute central administration
of NaCl and AngII activate sympathetic-regulatory neurons in
the hypothalamic PVN via inputs they receive from forebrain
circumventricular organs (CVOs). Because forebrain CVOs
lack a blood-brain barrier, their neurons are capable of sensing
and responding to elevated levels of plasma Na?/osmolality
and circulating AngII (Barth and Gerstberger 1999; Bourque et
al. 2007; Brooks et al. 2001; Ferguson and Bains 1997;
Gutman et al. 1998; Shi et al. 2007, 2008; Stocker and Toney
2005; Toney et al. 2003). Synaptic activation of presympa-
thetic PVN neurons thereby translates increases of plasma
AngII and Na?concentration into elevations of sympathetic
nerve activity (Chen and Toney 2001; Schad and Seller 1975;
Shi et al. 2007, 2008; Yasuda et al. 2000). Increases of
sympathetic activity and arterial pressure (Chen and Toney
2001; Martin and Haywood 1992; Porter and Brody 1985) that
result from PVN neuronal activation are mediated through a
variety of efferent pathways (Chen and Toney 2003, 2010;
Kannan and Yamashita 1983; Stocker et al. 2004, 2006; Yang
and Coote 1998; Yang et al. 2001), including monosynaptic
connections with sympathoexcitatory neurons in the rostral
ventrolateral medulla (RVLM) (Stocker et al. 2006; Yang et al.
2001), the main source of excitatory synaptic input to sympa-
thetic preganglionic neurons in the spinal cord (Guyenet et al.
Studies performed to date indicate that changes in both
synaptic inputs to and intrinsic membrane properties of PVN-
RVLM neurons contribute to sustained activation of sympa-
thetic activity in hypertension. For example, Li and Pan re-
ported that in vitro spontaneous discharge of PVN-RVLM
neurons from spontaneously hypertensive rats is greater than
that of neurons from normotensive controls. The latter appears
to involve both greater glutamatergic and reduced GABAergic
activity (Li and Pan 2006; Li et al. 2008). Similarly, Sonner et
al. reported that potassium A-current is diminished among
PVN-RVLM neurons from rats with renal-vascular hyperten-
sion. They further demonstrated that the diminution of A-cur-
rent contributes to enhanced in vitro spontaneous discharge
(Sonner et al. 2008). In further exploring ion channel mecha-
nisms controlling PVN-RVLM neuronal excitability, we re-
Address for reprint requests and other correspondence: Q.-H. Chen, Exercise
Science, Health and Physical Education Department, Michigan Technological
University, Houghton, MI 49931-1295 (E-mail: email@example.com).
J Neurophysiol 104: 2329–2337, 2010.
First published August 18, 2010; doi:10.1152/jn.01013.2009.
23290022-3077/10 Copyright © 2010 The American Physiological Societywww.jn.org
cently reported that PVN-RVLM neurons express a prominent
current mediated by small conductance Ca2?-activated K?
(SK) channels. Because this current was determined to potently
suppress the intrinsic excitability of PVN-RVLM neurons
(Chen and Toney 2009), the present study was performed to
determine if PVN-RVLM neurons from rats with AngII-Salt
hypertension exhibit diminished SK current and if this contrib-
utes to enhancement of their excitability.
M E T H O D S
Animal preparation and AngII-Salt hypertension
Male Sprague-Dawley rats (n ? 25, 225-250g, Charles River Labs,
Wilmington, MA) were individually housed in a temperature con-
trolled room (22–23°C) with a 14 h:10 h light-dark cycle. Rats in the
normotensive (NT) group were placed on a normal salt diet (0.4%
NaCl), and rats in the AngII-Salt hypertensive (HT) group were
placed on a high salt diet (2% NaCl). Diets were otherwise identical
in calories from fat and protein as well as total carbohydrate and
sucrose (Research Diets, New Brunswick, NJ). After 1 wk on each
diet, animals were anesthetized with isoflurane (3% in O2) and
instrumented with a telemetry transmitter (Data Science) for recording
arterial blood pressure and heart rate. Each animal received daily
injections of penicillin G (30,000 U/100 g body wt sc) and buprenor-
phine (0.05 mg/kg sc) for 3 days after surgery. After recovering for
5–7 days, baseline values of arterial pressure and heart rate were
recorded for 7 days. Then an osmotic mini-pump (2ML2, Alzet) was
implanted subcutaneously to deliver AngII (150 ng · kg?1· min?1) in
the HT group or vehicle (saline) in the NT group. AngII and saline
were infused for 14 days prior to performing electrophysiological
studies. All experimental and surgical procedures were approved by
the Institutional Animal Care and Use Committee of The University
of Texas Health Science Center at San Antonio.
Retrograde labeling of PVN-RVLM neurons
Two to 3 days before implantation of osmotic mini-pumps (see
preceding text), PVN neurons were retrogradely labeled from the
ipsilateral RVLM as previously described (Cato and Toney 2005;
Chen and Toney 2009). Briefly, rats were anesthetized with isoflurane
(3% in O2), placed in a stereotaxic frame, and a small burr hole was
made to expose the cerebellum. A glass micropipette was lowered into
the pressor region of RVLM (coordinates: ?12.7 mm caudal to
bregma, 1.8 mm lateral to midline and 8.9 mm below the skull) and
rhodamine-containing microspheres were microinjected in a volume
of 50 nl. Location of the tracer was verified postmortem in histological
sections through the RVLM (Cato and Toney 2005; Chen and Toney
2009; Stocker et al. 2006).
After 2 wk of continuous AngII or saline infusion, rats were
anesthetized with isoflurane (3% in O2) and decapitated. Brains were
removed and placed in ice-cold cutting solution containing (in mM)
206 sucrose, 2 KCl, 2 MgSO4, 1.25 NaH2PO4, 26 NaHCO3, 1 CaCl2,
1 MgCl2, 10 D-glucose, and 0.4 ascorbic acid. Osmolality and pH
were adjusted to 290–295 mosmol l?1and 7.4, respectively. Solution
pH and pO2were maintained by equilibration with a 95% O2-5% CO2
gas mixture. Coronal slices through the PVN were cut to a thickness
of 300 ?m on a vibrating microtome (Leica VT 1000S; Leica,
Nussloch, Germany). Slices were incubated at room temperature
(24–26°C) for ?2 h in continuously gassed artificial cerebrospinal
fluid (ACSF) containing (in mM) 125 NaCl, 2 KCl, 2 MgSO4, 1.25
NaH2PO4, 26 NaHCO3, 2 CaCl2, 10 D-glucose, and 0.4 ascorbic acid
(osmolality: 295–300 mosmol l?1; pH 7.4). Slices were transferred to
a glass-bottomed recording chamber and viewed through an upright
microscope (E600FN, Nikon) equipped with DIC optics, epi-fluores-
cence, an infrared (IR) filter and an IR-sensitive video camera (C2400,
Hamamatsu, Bridgewater, NJ). An appropriate filter was used to
visualize neurons retrogradely labeled with rhodamine beads.
Patch electrodes were pulled (Flaming/Brown P-97, Sutter Instru-
ment, Novato, CA) from borosilicate glass capillaries and polished to
a tip resistance of 4–5 M?. Electrodes were filled with a solution
containing (in mM) 135 K-gluconate, 10 HEPES, 0.1 EGTA, 1.0
MgCl2, 1.0 NaCl, 2.0 Na2ATP, and 0.5 Na2GTP (osmolality: 280–
285 mosmol l?1; pH 7.3). Note that a relatively low concentration of
EGTA (0.1 mM) was used to allow intracellular free Ca2?to accu-
mulate and activate SK channels during membrane potential depolar-
ization (Brenner et al. 2005; Chen and Toney 2009). Records were not
corrected for a liquid junction potential of ?15 mV. After achieving
a G? seal and whole cell configuration, cell capacitance, access
resistance, and resting membrane potential (Vm) were monitored until
stable. In voltage-clamp recordings, uncompensated series resistance
averaged 16 ? 2 M?. Cells that met the following criteria were
included in the analysis: action potential amplitude ?50 mV from
threshold to peak, input resistance (Rinput) ?0.5 G? (determined by
injection of ?20 pA from a holding potential of ?80 mV), resting Vm
negative to ?50 mV, and ?20% change in series resistance during the
recording. Recordings were made using an Axopatch 200B amplifier
and pCLAMP software (v10.0, Axon Instruments, Union City, CA).
Signals were filtered at 1 kHz, digitized at 10 kHz (Digidata 1322A,
Axon Instruments), and saved on a computer for off-line analysis.
Recording SK current
To study SK current, voltage-clamp recordings were performed
with intracellular solution containing the cAMP analogue 8-(4-chlo-
rophenylthio) 3=,5=-cyclic adenosine monophosphate (8CPT-cAMP,
50 ?M) to block the slow afterhyperpolarization current (Stocker et al.
1999). Recordings were performed in the presence of extracellular
tetrodotoxin (TTX, 1.0 ?M) and tetraethylammonium (TEA, 1.0
mM). Membrane potential was clamped at ?60 mV and stepped to
?10 mV for 100 ms. On returning Vmto ?60 mV, an outward tail
current was recorded. After recording a control tail current, apamin
(100 nM) was bath applied to selectively block SK channels. The tail
current recorded during treatment was subtracted from the corre-
sponding control tail current to isolate the SK current. Decay of the
SK current was analyzed by fitting the subtracted current with a
Testing neuronal excitability
Excitability of neurons from NT and HT rats was studied in
current-clamp mode in the absence of TTX, TEA, or 8CPT-cAMP.
With Vmadjusted to-80 mV by continuous negative current injection,
a series of square-wave current injections was delivered in steps of
?25 pA, each for a duration of 800 ms. To study the medium
afterhyperpolarization potential (mAHP) and afterdepolarization po-
tential (ADP), ?150 pA current injections (500 ms) were made from
a potential of ?60 mV. Between current injections, Vmwas returned
to ?60 mV for 5 s. Amplitude of the slow AHP was quantified 1.5 s
after termination of each current pulse. To determine the action
potential voltage threshold (Vt) and depolarizing Rinputbelow Vt, ramp
current injections (0.2 pA ms?1, 1,000 ms) were made from a
potential of ?80 mV. Square-wave and ramp current injections were
made in the same neurons. Effects of SK channel blockade on
neuronal excitability were determined by comparing current evoked
Vmand spike frequency responses under control conditions with
responses recorded from separate groups of neurons exposed to bath
applied apamin (100 nM). Note that current- and voltage-clamp
recordings were made from different groups of PVN-RVLM neurons.
2330Q.-H. CHEN, M. A. ANDRADE, A. S. CALDERON, AND G. M. TONEY
J Neurophysiol • VOL 104 • NOVEMBER 2010 • www.jn.org
All chemicals were obtained from Sigma-Aldrich (St Louis, MO)
except for TTX (Tocris Bioscience) and TEA (Fluka BioChemika).
Amplitude of the apamin-sensitive (SK) current was compared
across groups of neurons from NT and HT rats using an unpaired
t-test. Passive membrane properties of neurons from NT and HT rats
under control conditions were compared with corresponding values
determined in separate groups of neurons exposed to apamin using a
one-way ANOVA. Excitability tested with graded square-wave cur-
rent injections was compared across groups using a two-way
ANOVA. A similar approach was used for group comparisons of ISI
data for assessment of spike-frequency adaptation. When significant
main effects were found, Dunn’s post hoc tests were used for multiple
pair-wise comparisons. All statistical tests were performed using
Prism software (v5.0, GraphPad). Differences between means were
considered significant at P ? 0.05. Summary data are reported as
means ? SE.
R E S U L T S
Recordings were made from 46 PVN-RVLM neurons; 22
from NT rats (n ? 12) and 24 from HT rats (n ? 13). Before
performing brain slice studies, telemetric recordings in con-
scious rats revealed that baseline mean arterial pressure (MAP)
and heart rate (HR) were not different (P ? 0.05) between NT
(MAP: 97 ? 2 mmHg, HR: 412 ? 7 bpm) and HT (MAP:
101 ? 2 mmHg, HR: 393 ? 5 bpm) rats. On day 14 of AngII
administration, MAP was significantly (P ? 0.001) higher in
HT (134 ? 5 mmHg) than NT (101 ? 1 mmHg) rats.
Throughout the infusion protocols, there was no difference in
HR between groups (NT: 416 ? 4 bpm; HT: 402 ? 5 bpm).
Comparison of passive membrane properties
Table 1 shows resting Vm, Rinput, and whole cell capacitance
(Cm) of PVN-RVLM neurons from NT and HT rats. No
significant differences were identified either in the absence or
presence of bath applied apamin (100 nM). Note that values
under control conditions and during bath application of apamin
were determined from separate groups of neurons.
Comparison of SK current
PVN-RVLM neurons express a prominent SK current (Chen
and Toney 2009). Here an apamin-sensitive SK current was
recorded in six of six neurons tested from NT rats. Figure 1A
shows representative tail current traces before and after bath
application of apamin (100 nM). Digital subtraction of these
traces yielded the SK current (inset). Peak SK current ampli-
tude and density averaged 66 ? 12 pA (Fig. 1C, left) and
3.3 ? 0.8 pA/pF (C, right), respectively. Figure 1B shows
representative tail current traces before and after apamin and
the SK current (inset) of a neuron from an HT rat. In the HT
group (n ? 7), peak current amplitude and density were
significantly less than corresponding values in the NT group
and averaged 16 ? 8 pA (Fig. 1C, left, P ? 0.05) and 0.5 ? 0.3
pA/pF (C, right, P ? 0.01), respectively.
SK current regulates PVN-RVLM neuronal excitability
As reported earlier (Chen and Toney 2009), PVN-RVLM
neurons lacked spontaneous discharge at resting Vm, but depo-
larizing current injections consistently evoked repetitive action
potential firing. SK current regulation of excitability among
neurons from NT and HT rats was investigated by comparing
the relationship between the amplitude of injected current and
the frequency of evoked discharge in the absence (control) and
presence of the SK channel blocker apamin (100 nM).
Figure 2A shows repetitive action potential firing of neurons
from NT rats in response to ?200 pA current pulses in the
absence (top) and presence (bottom) of apamin. Under control
conditions, the frequency of spiking among neurons from NT
rats (Fig. 2A, top) was significantly lower than that of neurons
from HT rats (B, top, and C, left vs. right, P ? 0.05–0.01).
Blockade of SK channels with apamin significantly increase
discharge frequency in the NT group (Fig. 2, A, bottom, and C,
left, P ? 0.05–0.01), but had no effect in the HT group (Fig.
Passive membrane properties of PVN-RVLM neurons
?62 ? 2
?63 ? 2
?66 ? 2
?60 ? 2
1.2 ? 0.1
1.2 ? 0.1
1.3 ? 0.1
1.3 ? 0.1
25 ? 2
24 ? 2
28 ? 2
23 ? 2
PVN-RVLM, paraventricular nucleus - rostral ventrolateral medulla; Vm,
resting membrane potential; Cm, membrane capacitance; Rinput, input resis-
tance; NT, normotensive; HT, hypotensive.
K current amplitude (
current density (pA/
and HT rats. A: representative traces from a neuron of a NT rat showing
outward tail current following step (100 ms) depolarization of Vm(?60 to ?10
mV) in the absence (black trace) and presence (gray trace) of apamin (100
nM). B: outward tail current in a neuron from a HT rat in the absence (black
trace) and presence (gray trace) of apamin (100 nM). Note: Insets in A and B
show the net SK current obtained by subtraction (control - apamin).
C: summary data show the amplitude (left) and density (right) of SK current
for cells in each treatment group. All recordings were performed with TTX
(0.5 ?M) and TEA (1.0 mM) in the bath and with 8-(4-chlorophenylthio)
3=,5=-cyclic adenosine monophosphate (8CPT-cAMP, 50 ?M) in the intracel-
lular solution. *P ? 0.05, **P ? 0.01 vs. NT (unpaired Mann-Whitney test).
Comparison of SK current among PVN-RVLM neurons from NT
2331DIMINISHED NEURONAL SK CURRENT IN A RAT MODEL OF HYPERTENSION
J Neurophysiol • VOL 104 • NOVEMBER 2010 • www.jn.org
2, B, bottom, and C, right). As a result, peak discharge
frequency during SK channel blockade was similar across
groups (Fig. 2, C, left vs. right).
Excitability of PVN-RVLM neurons was also compared by
examining the slope of stimulus-response curves for current
pulses between 0 and ?125 pA, which represented the linear
portion of the relationship (Fig. 2D, left). The slope of the
stimulus-response curve under control conditions was signifi-
cantly greater for neurons from the HT than NT group (HT:
0.31 ? 0.04 Hz/pA; NT: 0.18 ? 0.03 Hz/pA; P ? 0.05).
Compared with control conditions, the slope was significantly
greater (P ? 0.05) in the NT group (0.35 ? 0.03 Hz/pA,)
exposed to apamin. Apamin treatment was without affect in the
HT group (0.39 ? 0.04 Hz/pA).
We next sought to determine if increased excitability was
associated with a reduced rate of interspike interval (ISI)
prolongation (i.e., spike frequency adaptation) in neurons from
HT rats. Spike-frequency adaptation was compared by exam-
ining the slope of the ISI-ISI number curve (Fig. 2D, right). In
the NT group, the slope of the liner fit of ISI-ISI number curve
was significantly greater than that of the HT group (NT: 1.4 ?
0.2 ms/ISI; HT: 0.4 ? 0.2 ms/ISI; P ? 0.05), suggesting that
spike-frequency adaptation under control conditions was re-
duced in HT compared with NT neurons. The slope of ISI-ISI
number curve in the NT group exposed to apamin was signif-
icantly less than that of the NT group under control conditions
(0.7 ? 0.2 ms/ISI, P ? 0.05 vs. NT-control). Compared with
the control HT group, slope was unchanged in the HT group
exposed to apamin (0.3 ? 0.1 ms/ISI, P ? 0.05 vs. HT-
SK current regulates Vtand depolarizing Rinputin
To determine whether action potential voltage threshold (Vt)
and subthreshold depolarizing Rinputare altered in neurons
from HT rats, ramp current injections (0.2 pA ms?1, 1 s
duration) were performed. To evaluate regulation by SK chan-
nels, responses were compared across groups of control neu-
rons and neurons exposed to apamin (100 nM). Prior to
initiating each current ramp, Vmwas set to ?80 mV by
negative current injection. Currents needed to hyperpolarize
NT-Control (n = 8)
NT-Apamin (n = 8)
HT-Control (n = 10)
?HT-Apamin (n = 7)p
(200 pA, 0.8 s)
e frequency (Hz)
0 50 100150200
0 50 100 150200
NT-Control (n = 8)
HT-Control (n = 10)
HT-Apamin (n = 7)
NT-Control (n = 8)
NT-Apamin (n = 8)
?HT-Apamin (n = 7)
NT-Apamin (n = 8)
HT-Control (n = 10)
† † †
11 13 11 1399
of PVN-RVLM neurons from NT and HT rats. A: voltage traces
showing representative responses of PVN-RVLM neurons from
NT rats to a 200 pA depolarizing current injection in the
absence (top, control) and presence (bottom) of the SK channel
blocker apamin (100 nM). Note that traces were recorded from
2 different neurons. B: representative responses of 2 different
PVN-RVLM neurons from HT rats to depolarizing current
injections in the absence (top, control) and presence (bottom) of
apamin. C: graphs showing the relationship between the ampli-
tude of injected current and the frequency of evoked discharge
for cells from NT (left) and HT (right) rats in the absence
(control) and presence of apamin. Note that the maximum
discharge frequency achieved was lower in the NT (left) than
HT (right) group under control conditions and that apamin
increased discharge in the NT group (left) but not the HT group
(right). D: the slope of the linear portion of current-discharge
response (0–125 pA) curves is shown for neurons from NT and
HT rats in the absence (control) and presence of apamin. Note
that under control conditions the slope was significantly greater
for neurons of the HT group than the NT group. Apamin
significantly increased the slope of the response of the NT
group but not the HT group (left). Interspike intervals (ISIs)
plotted for trains of current-evoked action potentials revealed
that average ISI and spike-frequency adaptation (ISI prolonga-
tion) were greater (right) among neurons from NT than HT rats.
*P ? 0.01, **P ? 0.001 apamin vs. control groups from NT
rats (2-way ANOVA). †P ? 0.05, ††P ? 0.01 HT vs. NT
groups in the absence of apamin (2-way ANOVA). #P ? 0.05
vs. NT groups in the absence of apamin (1-way Kruskall-Wallis
Effect of SK channel blockade on excitability
2332Q.-H. CHEN, M. A. ANDRADE, A. S. CALDERON, AND G. M. TONEY
J Neurophysiol • VOL 104 • NOVEMBER 2010 • www.jn.org
Vmwere not different across NT (?17 ? 6 pA) and HT
(?12 ? 3 pA) groups (P ? 0.38) and were not different in
groups exposed to apamin (NT-apamin: ?17 ? 7 pA; P ?
0.98 vs. NT-control; HT-apamin: ?16 ? 5 pA; P ? 0.38 vs.
Figure 3A shows representative discharge responses of neu-
rons from NT rats. Note that Vmdepolarized gradually and
action potentials were seen at Vt(Fig. 3A, top and bottom).
Blockade of SK channels with apamin increased the rate of
depolarization such that Vtwas reached earlier during the
current ramp (Fig. 3A, bottom). Thus apamin increased depo-
larizing Rinputmeasured below Vt. Figure 3B shows represen-
tative responses of a neuron from the control HT group (top)
and the apamin exposed HT group (bottom). Note that the
subthreshold depolarization rate of the control HT neuron (Fig.
3B, top) was greater than that of the control NT neuron (A,
top). Figure 3C shows summary data of depolarizing Rinput
(top), action potential firing rate (middle), and Vt(bottom) for
separate groups of neurons from control and apamin treated NT
and HT animals. Depolarizing Rinputwas significantly greater
among neurons of the control HT group compared with the
control NT group (P ? 0.05). Blockade of SK channels
significantly increased depolarizing Rinputof neurons in the NT
group but was without affect on neurons of the HT group (Fig.
3C, top, P ? 0.05). Similar to discharge responses to depolar-
izing current pulses (Fig. 2C), action potential firing rate during
current ramps was significantly greater for neurons in the HT
group compared with NT controls. Apamin increased the firing
rate of neurons in the NT group but was without affect in the
HT group (Fig. 3C, middle, P ? 0.05). Note that there was no
difference in Vtbetween control NT and HT groups and
separate groups exposed to apamin (Fig. 3C, bottom).
SK current contributes to the mAHP and masks an ADP in
To explore a potential role for reduced SK current in the
increased excitability observed among neurons from HT rats,
we compared effects of SK channel blockade on evoked
mAHPs and ADPs. Among neurons of the NT group under
control conditions (Fig. 4A, top), a train of action potentials
was induced by delivering a depolarizing current pulse (150
pA, 500 ms) from a potential of ?60 mV. Note that a
prominent mAHP was observed following termination of the
action potential train in eight of eight neurons tested. In the
presence of apamin (Fig. 4A, bottom), a shoulder-shaped ADP
(0.2 pA ms-1, 1.0 s)
on Vt and depolarizing Rinput of PVN-
RVLM neurons from NT and HT rats.
A: representative voltage traces from two
different PVN-RVLM neurons from NT rats
showing the response to ramp current injec-
tion in the absence (top, control) and pres-
ence (bottom) of the SK channel blocker
apamin (100 nM). B: representative voltage
traces from 2 different PVN-RVLM neurons
from HT rats showing the response to ramp
current injection in the absence (top, control)
and presence (bottom) of apamin. In A and
B, membrane potential depolarized gradu-
ally and action potential discharge began at a
discrete voltage threshold (Vt). C: summary
data showing that subthreshold depolarizing
input resistance (Rinput, top) and spike fre-
quency (middle) were greater under control
conditions for neurons in the HT than NT
group. Apamin increased both Rinputand
spike frequency only in the NT group. Vt
(bottom) was similar across groups under
control conditions and was unchanged by
apamin. *P ? 0.05 vs. NT groups under
control condition; NS, P ? 0.05 (1-way
Effect of SK channel blockade
2333 DIMINISHED NEURONAL SK CURRENT IN A RAT MODEL OF HYPERTENSION
J Neurophysiol • VOL 104 • NOVEMBER 2010 • www.jn.org
was observed following the spike train in six of eight neurons.
The peak amplitude and decay time constant of ADPs averaged
?8 ? 1 mV (Fig. 4C, left) and 479 ? 79 ms (C, right),
respectively. The two neurons that did not show an obvious
ADP during apamin treatment had smaller amplitude mAHPs
(?4 ? 1 mV) compared with those recorded under control
condition (n ? 8; ?7 ? 1 mV).
Traces in Fig. 4B show representative ADPs recorded from
neurons in the HT group. In the absence of apamin (top), an
ADP was recorded in four of seven neurons. The peak ampli-
tude and decay time constant of ADPs averaged ?7 ? 1 mV
(Fig. 4C, left) and 312 ? 80 ms (C, right), respectively. The
remaining three cells in the HT group did not have an obvious
ADP and showed a much smaller mAHP (?4 ? 2 mV)
compared with that in the NT group under control conditions
(P ? 0.05). In the presence of apamin (Fig. 4B, bottom), an
ADP was observed in five of seven neurons. The average peak
amplitude of these ADPs was ?8 ? 1 mV (Fig. 4C, left) and
the decay time constant averaged 514 ? 139 ms (C, right).
Similarly, cells in the HT group that did not have an obvious
ADP in the presence of apamin had smaller amplitude mAHPs
(?2.3 ? 1.4 mV) compared with cells from NT rats under
control conditions. There was no difference in the peak ampli-
tude or decay time constant of ADPs between control NT and
HT groups and corresponding groups exposed to apamin.
It should be noted that an apamin-insensitive slow AHP was
observed in eight of eight neurons from the NT group and
seven of seven neurons from the HT group. Peak amplitude of
slow AHPs did not differ across groups (NT: ?2.8 ? 0.9 mV;
HT: ?2.4 ? 0.8 mV).
D I S C U S S I O N
This study investigated the role of SK channels in regulating
excitability of PVN-RVLM neurons from NT control and
AngII-Salt HT rats. We found that the amplitude of whole cell
SK current was reduced and that neuronal excitability was
increased in the HT group compared with NT controls. Excit-
ability of neurons from NT but not HT rats was significantly
increased by SK channel blockade. The rate of subthreshold
depolarization during ramp current injections was greater
among neurons from HT than NT rats, indicating that depo-
larizing Rinputwas greater in the HT group. During SK channel
blockade, however, a greater increase in subthreshold depolar-
izing Rinputwas observed in the NT group than the HT group,
suggesting that SK current slows sub-threshold depolarization
considerably more in the NT group. The latter is consistent
with loss of SK current among neurons from the HT group. To
explore potential mechanisms underlying greater excitability of
neurons from HT rats, the SK channel-mediated mAHP was
analyzed and its amplitude was found to be significantly
reduced in the HT compared with the NT group. Moreover,
most neurons from HT rats had a prominent ADP. Blockade of
SK channels not only inhibited the mAHP in neurons from NT
rats but also uncovered an ADP resembling that observed in the
HT group. A striking observation was that SK channel block-
ade in the HT group had no obvious effect on either the mAHP
0 8 0.8
P amplitude (mV)
P decay Tau (s)
0 2 0.2
the ADP of PVN-RVLM neurons from NT and HT rats.
A: representative voltage traces showing the effect of SK channel
blockade with apamin (100 nM) on the mAHP recorded in neurons
from NT rats. In the absence of apamin (top), a train of action
potentials was induced by a depolarizing current pulse (150 pA,
500 ms). Following termination of action potential firing, a mAHP
was observed. In the presence of apamin (bottom), the mAHP
disappeared and ADP was revealed. B: representative voltage
traces showing the effect of apamin (100 nM) on the mAHP and
ADP of cells from HT rats. In the absence (top) and presence
(bottom) of apamin, an ADP was observed. C: summary data
showing the maximum ADP amplitude (left) and decay time
constant (right) of cells from NT and HT rats. NS, P ? 0.05 (1-way
Effect of SK channel blockade on the mAHP and
2334 Q.-H. CHEN, M. A. ANDRADE, A. S. CALDERON, AND G. M. TONEY
J Neurophysiol • VOL 104 • NOVEMBER 2010 • www.jn.org
or the ADP. Taken together, these findings indicate that dimin-
ished SK current likely contributes to increased in vitro excit-
ability of PVN-RVLM neurons from rats with AngII-Salt
hypertension. This appears to be accomplished, at least in part,
by a blunting of the mAHP that opposes an ADP in these
neurons and by enhancing subthreshold depolarizing Rinput.
We recently demonstrated that PVN-RVLM neurons express
a prominent SK current (Chen and Toney 2009). The present
study extended this observation by determining that neurons
from rats with AngII-Salt hypertension have a significantly
diminished SK current. It should be noted that Sonner et al.
(2008) recently reported that PVN-RVLM neurons from rats
with renal-vascular hypertension have smaller amplitude tran-
sient outward current (IA) compared with neurons from nor-
motensive controls. Interestingly, they reported that the density
of IAwas not different between neurons from HT and NT rats,
suggesting that the reduction of IAin renal-vascular hyperten-
sion could reflect a smaller membrane surface area rather than
a reduction in channel expression or gating. Reduced mem-
brane surface area of PVN-RVLM neurons does not appear to
be a common feature of hypertension as the present study
found that both the amplitude and density of SK current were
significantly reduced in the AngII-Salt HT group compared
with NT controls (Fig. 1C). Mechanisms that contribute to
differential PVN-RVLM neuronal adaptive responses to renal-
vascular and AngII-Salt hypertension are not presently known,
but in the renal-wrap model of hypertension used by Sonner et
al., Haywood et al. reported reduced GABAAreceptor medi-
ated inhibition of the PVN. Mover, gabaergic synapses have
been reported to redistribute on dendrites and soma of PVN-
RVLM neurons in renal-vascular hypertensive rats (Biancardi
et al. 2010). Thus it appears that the function of both ligand-
and voltage-gated inhibitory channels is reduced in the PVN in
the renal-wrap model of renal-vascular hypertension. Interest-
ingly, the reduction of GABAergic inhibition does not appear
to reflect a reduction of GABAAreceptor expression in pro-
portion to reduced membrane surface area as GABAAreceptor
density in the PVN was reported to be unaltered in hyperten-
sive rats (Haywood et al. 2001).
Although increased in vitro spontaneous activity of PVN-
RVLM neurons from spontaneously hypertensive (Li and Pan
2006) and renal-vascular hypertensive (Sonner et al. 2008) rats
involves both synaptic and intrinsic mechanisms, effects of
AngII and a high salt diet on the discharge behavior of these
neurons has not been previously investigated. We found that in
vitro excitability is significantly greater among PVN-RVLM
neurons from HT than NT rats. Blocking SK channels caused
a significantly blunted increase in excitability of neurons from
HT rats compared with controls with maximum firing rates
during exposure to apamin being nearly the same in both
groups. These finding indicate that diminished SK channel
function (or expression) among PVN-RVLM neurons of HT
animals could contribute to their greater in vitro excitability
and perhaps to greater in vivo discharge as well.
During a train of action potentials, AHPs can develop and
increase the rate of ISI prolongation, i.e., spike-frequency
adaptation (Bond et al. 2005; Stocker 2004). SK channels can
mediate a mAHP that leads to spike-frequency adaptation
(Greffrath et al. 2004; Liu and Herbison 2008; Teshima et al.
2003), thereby regulating excitability. Studies have shown that
PVN-RVLM neurons not only display a prominent mAHP but
also undergo significant spike-frequency adaptation in re-
sponse to depolarizing current injections (Chen and Toney
2009; Stern 2001) and the neuropeptide AngII (Cato and Toney
2005). To determine whether a reduction of spike-frequency
adaptation underlies increased excitability of PVN-RVLM
neurons from our AngII-Salt HT rats, we analyzed the ISI
distributions of current-evoked action potential trains. We
determined the slope of the linear portion of the ISI-ISI number
curve (Fig. 2D, right) and found that it was significantly greater
in the NT than HT group under control conditions. Further-
more, we observed that blockade of SK channels with apamin
significantly increased the slope only in the NT group. These
data suggest that SK current normally contributes to spike-
frequency adaptation and that reduced SK current among
PVN-RVLM neurons from AngII-Salt HT likely contributes to
reduced spike-frequency adaptation and greater excitability of
It is important to emphasize that the present conclusion that
SK current normally regulates spike-frequency adaptation in
PVN-RVLM neurons is contrary to the conclusion we reached
in a recent report (Chen and Toney 2009). We previously
concluded that SK channels do not participate in spike-fre-
quency adaptation in PVN-RVLM neurons, but this was based
on our observation that the ratio of the 13th and 1st ISI under
control conditions was not significantly changed during SK
channel blockade with apamin. In the present study, linear
slope of ISI-ISI number curve was used to quantify spike-
frequency adaptation. Because of its apparently greater sensi-
tivity relative to comparing the ratio of th 13th and 1st ISI, a
significant role for SK channels in spike-frequency adaptation
To further explore mechanisms contributing to reduced
spike-frequency adaptation among neurons from HT rats, we
analyzed mAHPs and found their amplitudes to be significantly
smaller in neurons from HT than NT rats. Moreover, most
PVN-RVLM neurons from HT rats had a prominent ADP
under control conditions that was not present in the NT group.
Interestingly, blockade of SK channels in the NT group not
only reduced the mAHP but revealed an ADP resembling that
recorded in the HT group. Thus diminished SK current appears
to contribute to increased excitability of PVN-RVLM neurons
from HT rats by reducing the mAHP amplitude and uncovering
The present study confirmed our earlier report that blocking
SK channels increases sub-threshold depolarizing Rinputof
PVN-RVLM neurons (Chen and Toney 2009). Here we found
that neurons from AngII-Salt HT rats had significantly greater
subthreshold depolarizing Rinputcompared with those from
control rats. Blocking SK channels with apamin caused depo-
larizing Rinputto increase significantly more in neurons from
NT than HT rats. Collectively, these findings indicate that
apamin-sensitive depolarizing Rinputmight also contribute to
increased excitability of PVN-RVLM neurons from rats with
AngII-Salt hypertension, at least during the subthreshold phase
It should be mentioned that SK conductance does not appear
to be active at rest and therefore does not influence resting Vm
(approximately ?63 mV) or spontaneous discharge of PVN-
RVLM neurons from NT (Chen and Toney 2009) or HT rats
(present study). Based on our ramp current tests, however, it
appears that SK channels become active when depolarization
2335DIMINISHED NEURONAL SK CURRENT IN A RAT MODEL OF HYPERTENSION
J Neurophysiol • VOL 104 • NOVEMBER 2010 • www.jn.org
starts from a relatively hyperpolarized potential of ?80 mV.
This observation may be explained by hyperpolarization-in-
duced de-inactivation of T-type Ca2?channels (Lee et al.
2003; Shin et al. 2008). Accordingly, Ca2?influx could then
activate SK channels as neurons depolarize from a hyperpo-
larized potential. This possibility is worthy of further study
given a recent report that PVN-RVLM neurons do indeed
express a low voltage activated T-type Ca2?current (Lee et al.
2008). Another, perhaps less likely, possibility is that hyper-
polarization of Vmin our ramp current injection tests could
have activated hyperpolarization-activated cation channels
(HCN). Although parvocellular PVN neurons have been re-
ported to express HCN (Qiu et al. 2005), it is not known if
these HCN are Ca2?permeable like those expressed in dorsal
root ganglion neurons (Yu et al. 2004) and ventricular myo-
cytes (Yu et al. 2007). An argument against a role for HCN in
the present study is that currents needed to adjust Vmto ?80
mV prior to initiation of current ramps was not different
between NT and HT groups and were unaltered by bath
application of apamin. Thus at hyperpolarized potentials, neu-
rons from NT and HT rats have similar input resistance and
may therefore have similar profiles of active ionic currents,
including those mediated by HCN.
It is well established that SK channels are gated by even
small increases of intracellular Ca2?(Bond et al. 2005; Fakler
and Adelman 2008; Sah and Faber 2002; Stocker 2004). Given
that Ca2?influx through voltage-dependent Ca2?channels can
activate SK channels, it is possible that a reduction of voltage-
dependent Ca2?influx could explain diminished SK current
observed among PVN-RVLM neurons from HT rats. This
appears plausible since, as mentioned in the preceding text,
PVN-RVLM neurons express a low voltage activated T-type
Ca2?current (Lee et al. 2008). Although activation of SK
channels by Ca2?through T-type Ca2?channels is known to
occur in dopaminergic midbrain neurons (Wolfart and Roeper
2002) and thalamic dendrites (Cueni et al. 2008), similar
information is not presently available for PVN-RVLM neu-
rons. It is also possible that diminished SK current among
neurons from HT rats could involve reduced expression of SK
channel proteins and/or reduced trafficking of SK channels to
the plasma membrane. In this regard, studies have reported that
expression of SK channel mRNA and protein is reduced in rat
mesenteric arteries during development of hypertension in-
duced by chronic infusion of AngII (Hilgers and Webb 2007).
No study of hypertension has yet compared SK channel
mRNA/protein expression levels in the CNS, and additional
studies are clearly needed to address this question.
It should be mentioned that SK channels can function at
presynaptic sites (Obermair et al. 2003; Roncarati et al. 2001)
to influence neurotransmission and excitability. In specific
regard to PVN-RVLM neurons, it is noteworthy that glutama-
tergic activity is dominant under conventional slice recording
conditions, i.e., Vmheld at ?80 mV (Chen and Toney 2009; Li
and Pan 2005). Whether enhanced excitability of PVN-RVLM
neurons from rats with AngII-Salt hypertension is due to
augmentation of glutamatergic excitation resulting from loss of
presynaptic SK current has not been investigated. This seems
unlikely, however, because available evidence indicates that
SK channel blockade with apamin does not affect glutamater-
gic miniature excitatory postsynaptic current activity among
PVN-RVLM neurons from NT rats (Chen and Toney 2009).
Therefore elevated PVN-RVLM neuronal excitability in AngII-Salt
hypertension appears more likely to be caused by diminished
postsynaptic SK current (Fig. 1).
In summary, the present study revealed that SK current is
diminished among PVN-RVLM neurons from rats with AngII-
Salt hypertension compared with NT controls. Diminished SK
current appears to underlie the reduced mAHP in these neurons
and likely allows detection of a prominent ADP following a
train of action potentials. Reduced SK current in neurons from
HT rats may also contribute to greater depolarizing Rinputat
potentials below spike threshold. Collectively, these functional
alterations appear to contribute to the observed increase in
excitability of PVN-RVLM neurons from rats with AngII-Salt
hypertension. We speculate that increased excitability could
play an important role in the development and/or maintenance
of sympathetic activation in AngII-Salt hypertension. Future
studies will be needed to fully determine the contribution of
neuronal SK channel dysfunction and/or down-regulation in
the pathogenesis of hypertension.
A C K N O W L E D G M E N T S
We thank L. P. LaGrange for critically reviewing the manuscript.
G R A N T S
This study was funded by National Heart, Lung, and Blood Institute Grants
HL-088052 and HL-076312 to G. M. Toney and American Heart Association
Grants 0865107F and SDG2640130 (Q. H. Chen).
D I S C L O S U R E S
No conflicts of interest, financial or otherwise, are declared by the author(s).
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