- The study investigated the effects of in vivo hyperkalemia on neurons in the nucleus of the solitary tract (NTS) using brainstem slices from control rats, adrenalectomized rats, and rats pretreated with spironolactone. - Results showed that hyperkalemia increased the firing rate of NTS neurons and depolarized their membrane potential. Neurons from adrenalectomized rats did not fire in response to hyperkalemia. - Pretreatment with spironolactone partially prevented the effects of hyperkalemia on NTS neuronal excitability. This suggests that endogenous aldosterone increases the excitability of NTS neurons during hyperkalemia.

1. - Effect of hyperkalemia on the membrane potential of NTS
neurons from rats under KCl overload previous
submitted to ADX
25
mV
15 s
- 58 mV
TTX - 1 µM
(E)
- 53 mV
30 s
30
mV
Baseline KCl 8 mM Wash-out
- Effect of exogenous KCl on the membrane potential of
NTS neurons from hyperkalemic rats pre-treated with SP
Menegaz, D., Fazan F.S., Varanda, W.A., Menani, J.V., Zoccal, D.B., Colombari, D.S.A., Colombari, E.
Department of Physiology and Pathology, School of Dentistry, São Paulo State University - UNESP, Araraquara, São Paulo, Brazil.
 Male Holtzman rats (200 g) were decapitated and had their
brain cut coronally in the NTS area.
 Whole-cell patch-clamp recordings of NTS neurons were
performed in the current-clamp and voltage-clamp modes.
 The slices were continuously perfused with oxygenated aCSF
containing (in mM): 125 NaCl, 4 KCl, 1 MgCl2, 1.25 NaH2PO4,
25 NaHCO3, 25 glucose, and 2 CaCl2 with or without
aldosterone (1 µM) or TTX (1 µM) at room temperature
(osmolality 300 mOsm/kg.H2O, pH 7.3).
 For the experiments in vitro to mimic in vivo hyperkalemia,
aCSF solution contained (in mM): 121 NaCl, 8 KCl, 1 MgCl2,
1.25 NaH2PO4, 25 NaHCO3, 25 glucose, and 2 CaCl2
(osmolality 300 mOsm/kg.H2O, pH 7.3).
 The pipette internal solution contained (in mM): 125 K+
gluconate, 10 KCl, 2 MgCl2, 0.1 CaCl2, 5.5 EGTA, 2 Na-ATP
and 10 HEPES.
 3 experimental groups submitted to KCl overload: control;
adrenalectomized (ADX) or pre-treated with spirinolactone
(SP; 80 mg/kg, ip).
 Experimental protocols were approved by the Ethics
Committee for Animal Care and Use (CEUA 13/2018).
 A population of neurons in the nucleus of the solitary tract
(NTS) expresses both the mineralocorticoid receptor (MR)
and the enzyme 11β-hydroxysteroid dehydrogenase type 2
(HSD2), making these neurons responsive to aldosterone.
 The synthesis and release of aldosterone occur during
situations of hyponatremia and hyperkalemia.
 HSD2 neurons in the NTS are activated by sodium
deprivation, an important physiological stimulus for
aldosterone production, which stimulates sodium appetite.
 It is not known whether the excitability of NTS neurons
increases and/or sodium appetite is stimulated by the
increase in endogenous aldosterone in animals with
hyperkalemia caused by potassium overload
OBJECTIVE
RESULTS
- Brainstem slice containing the NTS and a single
neuron of the NTS with the recording micropipette
METHODS
Figure 1. (A) Coronal slice of the brainstem containing the NTS. Area postrema (AP).
(B) Neuron recorded from the NTS showing the position of the recording micro pipette (RP).
Figure 3. (A) Recording of silent NTS neurons of rats with adrenalectomy (ADX) submitted
to KCl overload (n = 8) and (B) perfused with exogenous aldosterone (1 µM). The latter
recording shows a depolarization followed by an increase in firing frequency of action
potentials after 10 minutes of aldosterone treatment (n = 15, 35% of responsive neurons).
Figure 5. Frequency of action potential (AP) in neurons of normokalemic (NormoK) rats (0.12
± 0.02 Hz, n = 35); neurons of NormoK rats perfused with exogenous aldosterone (0.38 ±
0.06 Hz, n = 15); firing neurons of hyperkalemic (HyperK) rats (0.73 ± 0.07 Hz, n = 20); silente
neurons of HyperK rats submitted to ADX (0.025 ± 0.02 Hz, n = 8); and neurons of
hyperkalemic rats pre-treated with SP (0.11 ± 0.03 Hz, n = 16). Results are expressed as
means ± SEM and statistical significance (**p < 0.01; ***p < 0.001) was determined by One-
way ANOVA followed by post-test Tukey.
- Resting membrane potential difference of NTS neurons
from control and KCl overload rats
CONCLUSIONS
SUPPORTED
ALDOSTERONE INCREASES THE EXCITABILITY OF NEURONS OF THE
NUCLEUS OF THE SOLITARY TRACT IN HYPERKALEMIC RATS
Figure 2. (A, C) Recording of silent NTS neurons from rats submitted to KCl overload (n =
20); (B) Recording of spontaneous inward currents in voltage-clamp mode (cell-attached
configuration) and (D) Spontaneous action potentials firing in the current-clamp mode
(whole-cell configuration) of NTS neurons from rats submitted to KCl overload (n = 20, 40
% of firing neurons). E) Addition of TTX (tetrodoxine) abolished the action potential firing
triggered by hyperkalemia (n = 6).
 Endogenous aldosterone increases the excitability of NTS
neurons of hyperkalemic rats.
 In vivo hyperkalemia is a new model for the study of
endogenous aldosterone without the involvement of the
renin-angiotensin system.
 In vitro hyperkalemia demonstrates a higher sensibility to
extracelular KCl in neurons of rats pre-treated with KCl
overload.
N
o
r
m
o
K
A
l
d
o
N
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m
o
K
C
o
n
t
r
o
l
H
y
p
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A
D
X
+
H
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p
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S
p
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+
H
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K
0.0
0.5
1.0
1.5
2.0
**
***
***
***
***
Frequency
(Hz)
- NTS neuronal firing rate in the different experimental models
C
ontrol
Silent
Firing
-100
-80
-60
-40
-20
Hyperkalemia
*
***
Resting
M
embrane
Potential
(mV)
Figure 6. Resting membrane potential difference of neurons from normokalemic rats
(-67 ±1.8 mV, n = 35) and hyperkalemic rats (silent neurons - 64.32 ±2.1 mV, n = 20,
firing neurons - 56 ±1.8 mV, n = 20) . Results are expressed as means ± SEM and
statistical significance (*p < 0.05; ***p < 0.001) was determined by One-way ANOVA
followed by post-test Tukey.
//
- Effect of exogenous KCl on the membrane potential
of NTS neurons from normokalemic rats
Figure 7. Recording of exogenous KCl (8 mM) on the membrane potential of neurons
of the NTS of normokalemic rats (n = 10).
- Effect of exogenous KCl on the membrane potential of
NTS neurons from hyperkalemic rats
Figure 8. Recording of exogenous KCl (8 mM) on the membrane potential of neurons
of the NTS of hyperkalemic rats (n = 6).
Intragastric KCl overload
(4.6 M, 10 mL/Kg)
Blood levels of KCl
(mEq/L)
Blood levels of
aldosterone
(ng/dL)
Control 4.0 ± 0.1 10.6 ± 2.8
3 hours 8.2 ± 0.7* 173 ± 15*
6 hours 7.7 ± 0.7* 203 ± 18*
9 hours 8.3 ± 0.1* 208 ± 27*
12 hours 5.3 ± 0.2 252 ± 44*
- Effect of hyperkalemia on the membrane potential of
NTS neurons from rats treated with KCl overload
HYPERKALEMIA PROTOCOL - KCl overload
Figure 9. Recording of exogenous KCl (8 mM) on the membrane potential of neurons
of the NTS of hyperkalemic rats pre-treated with spironolactone (SP) (n = 7).
To investigate the effects of in vivo hyperkalemia on the
membrane potential of NTS neurons in brainstem slices of
control rats, rats with adrenalectomy (ADX) or rats pre-treated
with spironololactone (SP).
25
mV
1 min
Aldosterone 1 µM
- 58 mV
- 60 mV
(A)
(B)
INTRODUCTION
- Effect of hyperkalemia on the membrane potential of NTS
neurons from rats under KCl overload pre-treated with SP
- 63 mV
- 65 mV
25
mV
5 s
(A)
(B)
Figure 4. (A, B) Recording of silent and partially active NTS neurons of rats pre-treated with
spirinolactone (SP) submitted to KCl overload (n = 16).
Baseline
Baseline
- 55 mV
10
mV
30 s
KCl 8 mM Wash-out
- 60 mV
30 s
30
mV
Baseline KCl 8 mM Wash-out
KCl
overload
Protocol 1
recordings
3 h
SP
3 h
Protocol 3
3 h
KCl
overload
recordings
48 h
ADX
Protocol 2
3 h
KCl
overload
recordings
0 mV
0 mV
N
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m
o
K
H
y
p
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r
K
S
p
i
r
o
+
H
y
p
e
r
K
-100
-80
-60
-40
-20 ns
Resting
Mem
brane
Potential
(m
V)
(A)
N
o
r
m
o
K
H
y
p
e
r
K
S
p
i
r
o
+
H
y
p
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r
K
0.0
0.5
1.0
1.5
***
*
***
Frequency
(Hz)
(B)
Figure 10. (A) Resting membrane potential difference of neurons from normokalemic
(NormoK) rats (- 58 ±1.9 mV, n = 10); neurons from hyperkalemic (HyperK) rats (-
61.03 ± 1.9 mV, n = 6) and neurons from hyperK rats pre-treated with SP (- 60.96 ±
2.4 mV, n = 7), perfused with exogenous KCl (8mM). (B) Frequency of action potential
(AP) of neurons from normoK rats (0.006 ± 0.002 Hz, n = 10); neurons from hyperK
rats (0.9 ± 0.09 Hz, n = 6), and neurons from hyperK rats pre-treated with SP (0.55
± 0.10 Hz, n = 7), perfused with exogenous KCl (8mM). Results are expressed as
means ± SEM and statistical significance (*p < 0.05; ***p < 0.001) was determined by
One-way ANOVA followed by post-test Tukey.
ELECTROPHYSIOLOGICAL RECORDINGS