Download Enhanced NMDA receptor-mediated intracellular calcium signaling

Am J Physiol Regul Integr Comp Physiol 305: R414–R422, 2013.
First published June 19, 2013; doi:10.1152/ajpregu.00160.2013.
Enhanced NMDA receptor-mediated intracellular calcium signaling in
magnocellular neurosecretory neurons in heart failure rats
Javier E. Stern and Evgeniy S. Potapenko
Department of Physiology, Georgia Regents University, Augusta, Georgia
Submitted 1 April 2013; accepted in final form 18 June 2013
NMDA; vasopressin; supraoptic; glutamate; Ca2⫹
involving augmented sympathetic tone and elevated hormonal plasma levels, including
vasopressin (VP) and angiotensin II, among others (37, 43, 73),
is a key central nervous system pathophysiological process in
congestive heart failure (HF). Chronically elevated plasma VP
levels have been reported both in animal models and human
patients with HF (15, 19, 52, 62) being an important factor
contributing to altered fluid/electrolyte balance, as well as
detrimental myocardial effects (17, 18, 40, 44, 53). The importance of VP in HF is also underscored by several clinical
trials demonstrating that VP receptor antagonism efficiently
improves water balance and hemodynamic parameters in HF
patients (2, 9, 41). Thus the VP system is currently emerging as
a novel therapeutic target for the treatment of HF (14). Despite
its major impact on morbidity and mortality in HF patients (7),
the precise mechanisms contributing to neurohumoral activation, including elevated VP release in HF, remain incompletely
understood.
SYMPATHOHUMORAL ACTIVATION
Address for reprint requests and other correspondence: J. E. Stern, Dept. of
Physiology, Georgia Regents Univ., 1120 15th St., Augusta, GA 30912
(e-mail: [email protected]).
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The hypothalamic supraoptic (SON) and paraventricular
(PVN) nuclei are crucial centers involved in autonomic and
neuroendocrine regulation of the circulation (23, 60). Within
these nuclei, magnocellular neurosecretory cells (MNCs) directly control VP (and oxytocin) release into the circulation,
according to their degree and pattern of electrical activity (6,
51). A growing body of evidence supports an enhanced
glutamate excitatory action within the SON and PVN in HF
rats (27, 29, 32, 48, 49, 71). This includes elevated endogenous glutamate levels (29, 32), increased expression of
glutamate N-methyl-D-aspartate (NMDA) receptors (32),
and synaptic remodeling involving an increased predominance of glutamate excitatory over GABA inhibitory inputs
in HF rats (24, 48). This exacerbated glutamate excitatory
strength was shown to contribute to enhanced presympathetic and neurosecretory neuronal activity (24, 48), as well
as the concomitant increase in sympathohumoral activation
in HF rats (29, 32, 71).
NMDA receptors are one of the key glutamate receptors
influencing neuronal activity and sympathohumoral outflow
from the hypothalamus (25, 31, 32, 42, 61, 72). NMDA
receptor activation results in an influx of Ca2⫹, which in
addition to evoking a direct membrane depolarization, leads to
an increase in intracellular free Ca2⫹ levels (⌬Ca2⫹) (38). This
in turn affects a variety of downstream signaling pathways,
including activation of Ca2⫹-sensitive channels (47, 54) and
changes in the intracellular kinase/phosphatase balance (8), all
of which can in turn further alter neuronal excitability following NMDA receptor activation. Moreover, other signaling
mechanisms known to contribute to altered neuronal function
in HF rats, including nitric oxide (NO) and reactive oxygen
species (ROS) production (4, 22, 26, 68), are strongly dependent on or influenced by NMDA-mediated increases in intracellular Ca2⫹. The functional consequences of ⌬Ca2⫹ are
largely dependent on its magnitude and time course. Still, the
precise spatiotemporal dynamics of NMDA-⌬Ca2⫹ in key
neurons involved in the regulation of sympathohumoral activation has not been investigated yet. Moreover, whether a
change in NMDA-⌬Ca2⫹ dynamics contributes to altered neuronal activity and thus neurohumoral activation in HF rats, is
presently unknown. In this study, we performed simultaneous
patch-clamp electrophysiological recordings and fast confocal
Ca2⫹ imaging to characterize NMDA-⌬Ca2⫹ dynamics in
MNCs in sham and HF rats. Our results show an enhanced
NMDA-⌬Ca2⫹ signal in MNCs of HF rats, which was independent of the magnitude of the NMDA-mediated current, or
of the degree of NMDA-evoked firing activity. Moreover, our
results suggest that the enhanced NMDA-⌬Ca2⫹ acts as a
positive feedback mechanism contributing to increased MNC
membrane excitability in HF rats.
0363-6119/13 Copyright © 2013 the American Physiological Society
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Stern JE, Potapenko ES. Enhanced NMDA receptor-mediated
intracellular calcium signaling in magnocellular neurosecretory
neurons in heart failure rats. Am J Physiol Regul Integr Comp
Physiol 305: R414 –R422, 2013. First published June 19, 2013;
doi:10.1152/ajpregu.00160.2013.—An enhanced glutamate excitatory function within the hypothalamic supraoptic and paraventricluar
nuclei is known to contribute to increased neurosecretory and presympathetic neuronal activity, and hence, neurohumoral activation,
during heart failure (HF). Still, the precise mechanisms underlying
enhanced glutamate-driven neuronal activity in HF remain to be
elucidated. Here, we performed simultaneous electrophysiology and
fast confocal Ca2⫹ imaging to determine whether altered N-methylD-aspartate (NMDA) receptor-mediated changes in intracellular Ca2⫹
levels (NMDA-⌬Ca2⫹) occurred in hypothalamic magnocellular neurosecretory cells (MNCs) in HF rats. We found that activation of
NMDA receptors resulted in a larger ⌬Ca2⫹ in MNCs from HF when
compared with sham rats. The enhanced NMDA-⌬Ca2⫹ was neither
dependent on the magnitude of the NMDA-mediated current (voltage
clamp) nor on the degree of membrane depolarization or firing activity
evoked by NMDA (current clamp). Differently from NMDA receptor
activation, firing activity evoked by direct membrane depolarization
resulted in similar changes in intracellular Ca2⫹ in sham and HF rats.
Taken together, our results support a relatively selective alteration of
intracellular Ca2⫹ homeostasis and signaling following activation of
NMDA receptors in MNCs during HF. The downstream functional
consequences of such altered ⌬Ca2⫹ signaling during HF are discussed.
ENHANCED NEURONAL NMDA-⌬Ca2⫹ DURING HEART FAILURE
MATERIALS AND METHODS
Table 1. Summary data of echocardiography measurements
of left ventricular parameters obtained from sham and heart
failure rats
Sham
HF
EF, %
FS, %
LVIDd, mm
LVIDs, mm
80.1 ⫾ 4.0
34.8 ⫾ 1.6*
52.2 ⫾ 4.7
17.6 ⫾ 0.9*
7.6 ⫾ 0.3
10.2 ⫾ 0.2*
3.7 ⫾ 0.5
8.5 ⫾ 0.2*
Values are means ⫾ SE; n ⫽ 19 rats for each group. EF, ejection fraction;
FS, fractional shortening; LVID,d and s: left ventricle internal dimension
during diastole and systole respectively; HF, heart failure. *P ⬍ 0.0001 vs.
sham.
ments of NMDA-mediated currents. Recordings were obtained with
an Axopatch 200B amplifier (Axon Instruments, Foster City, CA)
from SON neurons using infrared differential interference contrast
(IR-DIC) videomicroscopy. The voltage output was digitized at 16-bit
resolution and 10 kHz and were filtered at 2 kHz (Digidata 1320A,
Axon Instruments). Data were discarded if the series resistance was
not stable throughout the entire recording (⬎20% change) (48, 58).
The NMDA receptor-mediated current (INMDA) in SON neurons was
assessed by measuring the peak and the integrated area of the evoked
change in holding current (Iholding) following a focal puff of NMDA
into the recorded cell using a picospritzer device (Toohey) connected
to a patch pipette positioned around 5–10 ␮m from the recorded cell,
which in most cases was located in the second layer of cells within the
slice preparation. A minimal pressure of 5– 8 PSI was used. Cell input
resistance and cell capacitance were calculated in voltage clamp using
a 5-mV pulse while holding the cells at ⫺70 mV. Spike threshold was
calculated based on the third derivative of the action potential waveform implemented by MiniAnalysis software (5, 56). Repetitive firing
activity was evoked by injecting depolarizing current pulses (80 pA)
of progressively increasing durations (0.1– 0.7 s), and plots of the
number of evoked spikes as a function of the current pulse duration
were generated. All drugs were purchased from Sigma-Aldrich (St.
Louis, MO).
Confocal calcium imaging. SON neurons were loaded through the
patch pipette with Fluo-5F pentapotassium salt (100 ␮M; Molecular
Probes, Carlsbad, CA), as previously described (13, 57). Once in the
whole cell mode, the dye was allowed to dialyze into the cell for at
least 15 min before the initiation of the recordings to allow complete
dialysis of the dye. Imaging was conducted using the Yokogawa real
time live cell laser confocal system combined with a highly sensitive
EMCCD camera (iXON⫹885, Andor Technology, South Windsor,
CT). Fluorescence images were obtained using diode-pumped solidstate laser (Melles Griot, Carlsbad, CA) at 488 nm and emitted light
at ⬎495. Images were acquired at a rate of 2 Hz. The fractional
fluorescence (F/F0) was determined by dividing the fluorescence
intensity (F) within a region of interest (6 ⫻ 6 pixels ⬃ 4.8 ⫻ 4.8 ␮m)
by a baseline fluorescence value (F0) determined from 30 images
before activation of NMDA receptors. Data were analyzed using
Andor IQ software (Andor Technology).
Statistical analysis. All values are expressed as means ⫾ SE.
Between-group differences (sham vs. HF) were compared using
unpaired t-tests or analysis of variance, as indicated followed by
Bonferroni post hoc tests. Differences were considered statistically
significant at P ⬍ 0.05, and n refers to the number of cells. All
statistical analyses were conducted using GraphPad Prism (GraphPad
Software, San Diego, CA).
RESULTS
Cardiac function values in sham and HF rats. Mean cardiac
function values obtained from sham and HF rats in this study
are summarized in Table 1. When compared with sham rats,
ligated rats showed a significant increased left ventricle internal dimension throughout the cardiac cycle, a decreased percentage of ejection fraction, and a decreased percentage fractional shortening (P ⬍ 0.0001 in all cases).
NMDA receptor activation evokes a similar INMDA, but
larger ⌬Ca2⫹ in MNCs in HF rats. Patch-clamp electrophysiological and/or imaging recordings were obtained from a total
of 95 SON MNCs obtained from sham (n ⫽ 48 MNCs from 19
rats) and HF rats (n ⫽ 47 MNCs from 19 rats). To study
NMDA receptor-mediated changes intracellular Ca2⫹ dynamics (NMDA-⌬Ca2⫹) in MNCs from sham and HF rats, simultaneous patch-clamp recordings (voltage-clamp mode) and fast
confocal Ca2⫹ imaging measurements were obtained from
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Animals and induction of HF-Male Wistar rats (150 –180 g) were
purchased from Harlan Laboratories (Indianapolis, IN). Rats were
housed at room temperature (24 –26°C) in a 12-h light-dark cycle
room and given free access to food and water. In a subset of
experiments, we also used male heterozygous transgenic VP-eGFP
Wistar rats (5– 6 wk old), in which VP neurons are endogenously
fluorescent (67). All procedures were carried out in agreement with
the Georgia Regents University Institutional Animal Care and Use
Committee guidelines. All protocols used for these studies were
submitted, reviewed, and approved by an independent committee
(IACUC). HF was induced by coronary artery ligation as previously
described (4). Briefly, animals were anesthetized with isoflurane 4%
and intubated for mechanical ventilation. A left thoracotomy was
performed and the heart exteriorized. The ligation was placed on the
main diagonal branch of the left anterior descending coronary artery.
Buprenorphine (Bruprenex C3 0.3 mg/kg sc; Butler Schein/NLS,
Dublin, OH) was given immediately after surgery to minimize postsurgical pain. Sham animals underwent the same procedure but the
coronary artery was not occluded. All animals were used 6 to 7 wk
after surgery. Transthoracic echocardiography (Vevo 770 system;
Visual Sonics) was performed 4 wk after surgery under light anesthesia. The left ventricle internal diameter, as well as the left ventricle
posterior and anterior walls diameter, were obtained throughout the
cardiac cycle from the short-axis motion imaging mode. Automatic
calculation using the parameters measured was obtained for ejection
fraction and fractional shortening. Mean cardiac function values
obtained from sham and HF rats are summarized in Table 1.
Hypothalamic slice preparation. Hypothalamic brain slices were
prepared according to methods previously described (48, 58). Briefly,
rats were deeply anesthetized with pentobarbital sodium (80 mg/kg ip)
and perfused through the heart with an ice-cold sucrose solution
[containing in mM: 200 sucrose, 2.5 KCl, 3 MgSO4, 26 NaHCO3,
1.25 NaH2PO4, 20 D-glucose, 0.4 ascorbic acid, 1 CaCl2, and 2
pyruvic acid (290 –310 mosmol/l)]. Rats were then quickly decapitated, brains dissected out, and coronal slices cut (300 ␮m thick) using
a vibroslicer (D.S.K. Microslicer, Ted Pella, Redding, CA). An
oxygenated ice-cold artificial cerebrospinal fluid (aCSF) was used
during slicing (containing in mM: 119 NaCl, 2.5 KCl, 1 MgSO4, 26
NaHCO3, 1.25 NaH2PO4, 20 D-glucose, 0.4 ascorbic acid, 2 CaCl2,
and 2 pyruvic acid; pH 7.4; 290 –310 mosmol/l). Slices were placed in
a holding chamber containing aCSF and kept at room temperature
until used.
Patch-clamp electrophysiology. Slices were bathed with solutions
(⬃2.0 ml/min) that were continuously bubbled with 95% O2-5% CO2
and maintained at 32°C. Thin-walled (1.5 mm od, 1.17 mm id)
borosilicate glass (G150TF-3, Warner Instruments, Sarasota, FL) was
used to pull patch pipettes (3– 4 M⍀) on a horizontal Flaming/Brown
micropipette puller (P-97, Sutter Instruments, Novato, CA). The
internal solution contained (in mM) 140 potassium gluconate, 0.2
EGTA, 10 HEPES, 10 KCl, 0.9 MgCl2, 4 MgATP, 0.3 NaGTP, and
20 phosphocreatine (Na⫹); pH 7.2–7.3. For voltage-clamp recordings,
a low Mg2⫹ aCSF (20 ␮M MgSO4) was used to facilitate measure-
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ENHANCED NEURONAL NMDA-⌬Ca2⫹ DURING HEART FAILURE
neurons loaded with Fluo-5F (100 ␮M) through the patch
pipette. Focal application of NMDA (50 ␮M, 1 s, 5– 8 PSI)
onto the recording neurons evoked a transient inward current
(INMDA) and a concomitant ⌬Ca2⫹ (Fig. 1, A and B). As shown
in Fig. 1C, the overall magnitude of INMDA was not different
between MNCs in sham and HF rats (n ⫽ 9 and 13, respectively, P ⫽ 0.8). Conversely, the magnitude of the NMDA⌬Ca2⫹ was significantly larger in MNCs from HF rats (⬃85%,
P ⬍ 0.05). When Ca2⫹ responses were normalized to the
underlying INMDA within each cell, a larger ⌬Ca2⫹/unit of
INMDA was still observed in HF compared with sham rats
(⬃125%, P ⬍ 0.01, Fig. 1C). Given similar cell capacitance
values between MNCs in sham and HF rats (Table 2), similar
results were observed when INMDA and ⌬Ca2⫹ values were
normalized by cell capacitance (not shown). Thus, despite
similar INMDA-evoked currents, a larger increase in intracellular Ca2⫹ was observed in MNCs from HF rats.
A major factor influencing the shape and time course of
intracellular Ca2⫹ signal is buffering by the endoplasmic reticulum via the sarco/endoplasmic reticulum Ca2⫹-ATPase
(SERCA) pump (66). Thus to determine whether the prolonged NMDA-⌬Ca2⫹ in HF rats was due to a diminished
ER Ca2⫹ buffering capacity, we repeated a set of experiments in slices preincubated with thapsigargin (2 ␮M, 45
min), an endoplasmic reticulum SERCA pump blocker (35).
We found that thapsigargin significantly prolonged the duration of the NMDA-⌬Ca2⫹, in MNCs from both sham and
HF rats (⌬Ca2⫹ decay time: sham-thapsigargin: 43.4 ⫾ 4.9
s; HF-thapsigargin: 56.9 ⫾ 7.4 s; n ⫽ 11 and 6, P ⬍ 0.05 vs.
the respective sham and HF groups in control ACSF, see
Fig. 1C for comparison). No changes in other ⌬Ca2⫹ parameters were observed (not shown).
NMDA receptor activation evokes an enhanced firing activity along with larger ⌬Ca2⫹ per action potential in HF rats. To
study NMDA responses under more physiological conditions,
experiments were performed also in the current-clamp mode, a
condition in which NMDA receptor activation evokes membrane depolarization and firing discharge. As summarized in
Table 2, no differences in resting membrane potential, input
resistance, or action potential threshold were observed between
sham and HF groups. Focal application of NMDA to the
recording cell (10 ␮M, 1 s) evoked a transient membrane
depolarization, an increased firing rate, and a concomitant
increase in intracellular Ca2⫹ (Fig. 2). The degree of NMDA
Table 2. Summary data of basic intrinsic membrane properties of MNCs in sham and HF rats
Sham
HF
Resting Vm, mV
Input Resistance, G⍀
Cell Capacitance, pF
Spike Threshold, mV
⫺72.9 ⫾ 0.8 (n ⫽ 30)
⫺73.4 ⫾ 0.9 (n ⫽ 24)
0.51 ⫾ 0.05 (n ⫽ 30)
0.52 ⫾ 0.04 (n ⫽ 24)
25.7 ⫾ 0.9 (n ⫽ 48)
27.3 ⫾ 1.1 (n ⫽ 47)
⫺45.0 ⫾ 1.2 (n ⫽ 30)
⫺44.7 ⫾ 1.0 (n ⫽ 24)
Values are means ⫾ SE; n ⫽ number of rats. Vm, membrane potential.
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Fig. 1. N-methyl-D-aspartate (NMDA) receptor activation in magnocellular neurosecretion cells (MNCs) of heart failure (HF) rats evokes a similar NMDA
receptor-mediated current (INMDA) but a larger NMDA-mediated ⌬Ca2⫹. A: representative examples showing INMDA (top traces), and the concomitant increase
in ⌬Ca2⫹ (F/F0, bottom traces), following a focal application of NMDA (50 ␮M, 1 s, arrows) to a MNC in a sham and HF rats. B: example of the Fluo5-loaded
neuron in the sham rat before (left) and after (right) NMDA application. Note the increases Fluo-5 fluorescence following NMDA receptor activation both in
the soma and a proximal dendrite (arrow). C: summary data showing the mean INMDA area (top left), ⌬Ca2⫹ area (lower left), ⌬Ca2⫹ decay time (top right) and
mean ⌬Ca2⫹ area-to-INMDA area ratio (bottom right) in MNCs from sham and HF rats (n ⫽ 9 and 13, respectively). *P ⬍ 0.05 and **P ⬍ 0.01 vs. respective
sham, unpaired t-tests.
ENHANCED NEURONAL NMDA-⌬Ca2⫹ DURING HEART FAILURE
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receptor-mediated membrane depolarization and firing discharge was significantly larger in MNCs from HF rats (P ⬍
0.05 in both cases, see Fig. 2B, n ⫽ 30 and 24 in sham and HF
rats, respectively). In a proportion of these cells, we were able
to reliably quantify ⌬Ca2⫹ responses. In these MNCs (n ⫽ 16
and 18 in sham and HF rats, respectively), the magnitude and
time course of the NMDA receptor-evoked ⌬Ca2⫹ was significantly larger in HF rats. Thus both the ⌬Ca2⫹ peak amplitude
and area were significantly larger in HF compared with sham
rats (⬃145 and 210%, respectively, P ⬍ 0.001) (Fig. 2C).
While a strong tendency for a slower monoexponential decay
time course was observed in MNCs from HF rats, differences
did not reach statistical significance (P ⫽ 0.08). Conversely, no
differences in the ⌬Ca2⫹ rise time were observed between
experimental groups (not shown).
The larger NMDA-⌬Ca2⫹ observed in HF rats could be the
result of the larger depolarization and/or number of action
potentials triggered by NMDA receptor activation in these rats.
However, when we normalized within each MNC, the overall
magnitude of the evoked ⌬Ca2⫹ by either the area of the
membrane depolarization or the total number of spikes evoked,
a significantly larger ⌬Ca2⫹ response was still observed in
MNCs of HF rats (P ⬍ 0.01 and P ⫽ 0.05, respectively, n ⫽
16 and 18 in sham and HF rats, respectively, Fig. 3, A and B).
In addition, no significant correlation was observed between
the magnitude of the NMDA-⌬Ca2⫹ and the NMDA-⌬mV
(R2 ⫽ 0.19 and 0.08 for sham and HF rats, respectively).
Moreover, the enhanced NMDA-⌬Ca2⫹ in HF rats persisted
when action potentials were blocked in slices pretreated with
tetrodotoxin (TTX, 1 ␮M) (P ⬍ 0.05, n ⫽ 7 and 4 in sham and
HF rats, respectively, Fig. 3D). Finally, to verify that these
changes occur in vasopressin neurons, we obtained a few
recordings from endogenously fluorescent eGFP-VP MNCs.
Similar to nonidentified cells, we found a significantly larger
NMDA-⌬Ca2⫹ in identified eGFP-VP neurons in HF rats
[⌬Ca2⫹ area (F/F0 ⫻ s): sham ⫽ 20.1 ⫾ 8.9; HF: 63.3 ⫾ 11.2,
P ⬍ 0.05, n ⫽ 4 and 11, respectively], as well as larger
⌬Ca2⫹/unit of membrane depolarization (sham ⫽ 0.12 ⫾ 0.04;
HF: 0.65 ⫾ 0.16, P ⬍ 0.05, n ⫽ 4 and 11, respectively).
Taken together, these results indicate that the enhanced
NMDA-⌬Ca2⫹ in MNCs in HF rats was not due to the larger
NMDA-mediated depolarization/firing discharge. Conversely,
they support an overall increased ⌬Ca2⫹ entry per unit of
NMDA-mediated membrane excitation.
Evoked repetitive action potential firing activity results in
similar ⌬Ca2⫹ in MNCs from sham and HF rats. The results
above suggest that NMDA receptor activation per se, independent of action potential firing, is sufficient to evoke differences
in NMDA-⌬Ca2⫹ dynamics between MNCs in sham and HF
rats. To study in more details the relationship between repetitive action potential firing and Ca2⫹ dynamics in MNCs,
repetitive firing was evoked by injecting depolarizing current
pulses of incremental durations (80 pA, 0.1, 0.4, and 0.7 s),
while monitoring the magnitude of the evoked ⌬Ca2⫹ (n ⫽ 9
and 10 in sham and HF, respectively). As shown in Fig. 4, the
firing discharge in MNCs increased progressively in both
groups as a function of the injected current (P ⬍ 0.0001,
two-way ANOVA). However, no differences in the number of
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Fig. 2. NMDA receptor activation in current-clamp mode evokes an enhanced membrane depolarization, firing discharge, and ⌬Ca2⫹ in MNCs from HF rats.
A: representative examples showing NMDA-mediated depolarization and firing activity (top traces) and the concomitant increase in ⌬Ca2⫹ (bottom traces),
following a focal application of NMDA (10 ␮M, 1 s, arrows) to a MNC in a sham and HF rats. The bottom insets show the Fluo5-loaded neuron in the sham
rat before and after NMDA application. B: summary data showing the mean depolarizing peak amplitude, depolarizing area, and number of evoked action
potentials, respectively, following a focal NMDA application in MNCs from sham and HF rats (n ⫽ 30 and 24, respectively). C: summary data showing the mean
peak ⌬Ca2⫹ amplitude, decay time, and area following a focal NMDA application in MNCs from sham and HF rats (n ⫽ 16 and 18, respectively). *P ⬍ 0.05
and **P ⬍ 0.01 vs. respective sham, unpaired t-tests. Scale bar in inset ⫽ 20 ␮m.
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ENHANCED NEURONAL NMDA-⌬Ca2⫹ DURING HEART FAILURE
evoked actions potentials were observed between sham and HF
rats (P ⫽ 0.5, two-way ANOVA). Similarly, the mean ⌬Ca2⫹
area increased progressively in both groups as a function of the
injected current (P ⬍ 0.0001, two-way ANOVA), but no
differences between groups were observed (P ⫽ 0.8, two-way
ANOVA). Finally, when the magnitude of the evoked ⌬Ca2⫹
was normalized to the number of evoked action potentials for
each current step, no differences were observed between experimental groups (P ⫽ 0.2, two-way ANOVA).
DISCUSSION
Using a combination of patch-clamp electrophysiology with
simultaneous fast confocal Ca2⫹ imaging, we characterized
NMDA-⌬Ca2⫹ in MNCs from sham and HF rats. We show
that: 1) while the magnitude of INMDA was similar between
MNCs in the two experimental groups, a larger ⌬Ca2⫹ was
evoked in HF rats; 2) in current-clamp mode, NMDA receptor
activation evoked an enhanced membrane depolarization and
Fig. 4. Repetitive action potential firing evoked by direct
current injection resulted in similar ⌬Ca2⫹ in MNCs from
sham and HF rats. A: representative examples of 3 bursts of
action potentials evoked by progressively increasing current
pulse duration (0.1, 0.4, and 0.7 s, respectively, top traces),
and the respective ⌬Ca2⫹ (bottom traces) obtained from a
MNCs in a HF rat. B: summary data showing mean number
of evoked action potentials (top), mean changes in ⌬Ca2⫹
area (middle), and mean ⌬Ca2⫹ area/number of action
potential (APs, bottom) in MNCs from sham and HF (n ⫽
9 and 10 in sham and HF, respectively). Note the lack of
differences between sham and HF rats in any of the parameters measured.
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Fig. 3. Enhanced NMDA-mediated ⌬Ca2⫹ per
unit of membrane depolarization or evoked action poetential in MNCs of HF rats. Summary
data showing a significant increase in NMDAmediated ⌬Ca2⫹ in MNCs from HF rats, when
data were normalized either by the total area of
the NMDA-mediated depolarization (A) or by
the total number of evoked action potentials (B).
In C, a plot of the NMDA-evoked ⌬Ca2⫹ area
as a function of the NMDA-evoked depolarizing area is shown (n ⫽ 16 and 18 in sham and
HF rats, respectively). D: summary data showing that the larger increase in NMDA-mediated
⌬Ca2⫹ per unit of membrane depolarization in
HF rats persisted in the presence of tetrodotoxin
(1 ␮M, n ⫽ 7 and 4 in sham and HF rats,
respectively). *P ⫽ 0.05 and **P ⬍ 0.01 vs.
respective sham.
ENHANCED NEURONAL NMDA-⌬Ca2⫹ DURING HEART FAILURE
enhancement, rather than a reduction, in the NMDA-⌬Ca2⫹
signal indicate that Ca2⫹ release from the ER does not contribute to the ⌬Ca2⫹ signals following NMDA receptor activation in MNCs. Clearly, future studies will be needed to
elucidate alternative mechanisms contributing to the altered
NMDA receptor-mediated Ca2⫹ waveform in HF rats.
We observed a similar enhanced NMDA-⌬Ca2⫹ in the
current clamp mode, in which NMDA receptor activation
evoked also a larger membrane depolarization along with a
more pronounced firing discharge in MNCs of HF rats. Still,
the larger NMDA-⌬Ca2⫹ response persisted when action potentials were blocked by TTX, indicating that activation of
NMDA receptors per se (and not the Ca2⫹ influx associated
with action potential firing) was associated to the prolonged
Ca2⫹ signal in HF rats. The implications of the more robust
NMDA-mediated membrane depolarization and firing discharge in MNCs of HF rats is discussed further below. Notably, no differences in ⌬Ca2⫹ dynamics between sham and HF
rats were observed following trains of action potentials evoked
by direct membrane depolarization. In this case, most of the
Ca2⫹ contributing to the ⌬Ca2⫹ originates from influx via
voltage-gated Ca2⫹ channels. Thus these results suggest that
Ca2⫹ homeostasis is not globally affected in MNCs from HF
rats, but rather, that the source of Ca2⫹ (i.e., NMDA receptors),
and their spatial proximity to specific buffering mechanism are
key determining factors (46).
What are the possible downstream consequences of the
enhanced ⌬Ca2⫹ following NMDA receptor activation in HF?
Critical inhibitory signals within the SON and PVN, such as
NO and GABA, are both Ca2⫹ dependent and can be influenced directly or indirectly by NMDA receptor activation (3,
50). Moreover, the efficacy of both NO and GABA actions
have been shown to be diminished in HF rats, contributing in
turn to neurohumoral activation in this disease. For example, a
blunted NO production within the PVN is recognized as a key
mechanism contributing to sympathohumoral activation in HF
rats (4, 68, 73). While a diminished neuronal NO synthase and
endothelial NO synthase expression has been reported in the
SON/PVN of HF rats (4, 70), the possibility that an blunted
NMDA-⌬Ca2⫹ response contributes to diminished NOS activity and consequently, NO production, has not been explored.
While not directly tested in this study, our results showing an
enhanced, rather than a blunted, Ca2⫹ response would argue
against this possibility. Similarly, a blunted GABAergic inhibitory function has been reported in the hypothalamus of HF rats
(24, 48, 69). We recently demonstrated, both in SON MNCs
and presympathetic PVN neurons, an NMDA-mediated, Ca2⫹dependent potentiation of GABAA receptor function, which
serves as a counterbalancing inhibitory feedback mechanism to
restrain overexcitation following NMDA receptor activation
(50). Importantly, this NMDA-Ca2⫹-GABAA coupling was
blunted in MNCs from HF rats, likely contributing to NMDAdriven neurohumoral activation in HF (50). While the precise
mechanisms underlying the blunted NMDA-GABAA receptor
coupling are still unknown, the present results argue against a
diminished ability of NMDA receptors to evoke a sufficiently
large change in intracellular Ca2⫹ as a contributing factor.
Another important finding in this study is that NMDA
receptor activation in HF, despite evoking a similar underlying
INMDA current than in sham rats, resulted in a more pronounced
membrane depolarization and firing discharge in the former.
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firing discharge, along with a larger ⌬Ca2⫹ in HF, compared
with sham rats; 3), the NMDA-⌬Ca2⫹ per unit of membrane
depolarization or per individual action potential was larger in
HF rats; and 4) the number of action potentials and concomitant ⌬Ca2⫹ evoked by direct delivery of depolarizing steps of
increasing durations was similar between sham and HF rats.
While an enhanced glutamate function within the SON/PVN
of HF rats was previously reported (27, 29, 32, 48, 71), the
precise underlying mechanisms and cellular consequences of
the exacerbated glutamate function in HF rats remain to be
fully elucidated. In this study, we focused on the functional
efficacy of NMDA receptors and their actions on membrane
excitability and intracellular Ca2⫹ dynamics. We report here
that focal and transient activation of NMDA receptors evoked a
similar NMDA-mediated inward current in MNCs from sham and
HF rats. Despite a similar INMDA magnitude between the two
experimental groups, a larger NMDA-⌬Ca2⫹ was observed in HF
rats. Multiple factors shape the magnitude and waveform of a
Ca2⫹ transient such as those following NMDA receptor activation. These include the total number of NMDA receptors, the
single-channel Ca2⫹ permeability of NMDA receptors, release of
Ca2⫹ from intracellular stores, as well as intracellular Ca2⫹
buffering and cytosolic clearance mechanisms. The fact that a
similar INMDA current was evoked in sham and HF rats would
argue against an increased in NMDA receptor numbers or increased in Ca2⫹ permeability in this conditions, since Ca2⫹ influx
is a major component mediating INMDA (38). This is in agreement
with a previous study showing lack of changes in NMDA receptor
NR1 subunit mRNA expression in the SON in HF rats (32).
MNCs possess numerous Ca2⫹ buffering/clearance mechanisms, including plasmalemmal (PMCa) and endoplasmic reticulum (ER-SERCa) Ca2⫹ transport ATPases, and the mitochondrial Ca2⫹ selective uniporter (10), all of which have been
shown to efficiently shape somatic Ca2⫹ transients in these
neurons (28, 30). These Ca2⫹ buffering mechanisms slowly
decrease the levels of cytosolic free Ca2⫹, resulting in a slow
decaying Ca2⫹ time course following the initial transient rise.
Thus changes in the efficacy of Ca2⫹ buffering mechanisms
typically affect not only the peak, but mostly the decay phase,
of the Ca2⫹ transient. In the present study, we found that both
the peak and duration of the NMDA-⌬Ca2⫹ in MNCs from HF
rats were significantly enhanced compared with sham rats,
suggesting a compromised Ca2⫹ buffering.
To determine whether a blunted ER-SERCa buffering mechanism contributed to the prolonged NMDA-⌬Ca2⫹ in HF rats,
we compared the effects of thapsigargin (TG), a SERCa
blocker, between sham and HF rats. Our results showing that
TG prolonged the NMDA-⌬Ca2⫹ signal both in sham and HF
rats, indicate that the ER indeed acts as an important intracellular Ca2⫹ buffering mechanism shaping the NMDA-⌬Ca2⫹
waveform. This is in agreement with previous studies showing
that blockade of the ER-SERCa prolonged the decay of a
K⫹-induced increase in Ca2⫹ in MNCs (30). However, the fact
that TG prolonged the NMDA-⌬Ca2⫹ waveform in both experimental groups to a similar extent would argue against a
blunted ER-SERCa function during HF. Finally, in addition to
acting as a Ca2⫹ buffering organelle, the ER can also release
stored Ca2⫹, contributing to the overall NMDA-⌬Ca2⫹ waveform (12). Prolonged blockade of the SERCa with TG, as
performed in our study, also leads to the depletion of ER Ca2⫹
store (45). Thus our results showing that TG resulted in an
R419
R420
ENHANCED NEURONAL NMDA-⌬Ca2⫹ DURING HEART FAILURE
VP can also act in a diffusible manner to stimulate the activity
of neighboring presympathetic neurons, leading to an increased
renal sympathetic nerve activity (55). Activation of NMDA
receptor, and the subsequent increase in Ca2⫹, is a powerful
mechanism that stimulates dendritic peptide release from
MNCs (11). Thus it is reasonable to speculate that the enhanced NMDA-⌬Ca2⫹ reported here during HF may lead to an
exacerbated dendritic VP release, resulting in turn not only in
further increased VP neuronal activity, but also in a more
prominent recruitment of presympathetic neurons, both ultimately contributing to neurohumoral activation in HF. Future
studies assessing on one hand the precise mechanisms underlying exacerbated NMDA-⌬Ca2⫹ (e.g., intracellular Ca2⫹
buffering capacity), and on the other hand the overall impact of
such changes to sympathohumoral activation at the systems
level, are warranted.
GRANTS
This work was supported by National Heart, Lung, and Blood Institute R01
HL-090948 (to J. E. Stern).
DISCLOSURES
No conflicts of interest, financial or otherwise, are declared by the author(s).
AUTHOR CONTRIBUTIONS
Author contributions: J.E.S. conception and design of research; J.E.S. and
E.S.P. analyzed data; J.E.S. interpreted results of experiments; J.E.S. drafted
manuscript; J.E.S. edited and revised manuscript; J.E.S. approved final version
of manuscript; E.S.P. performed experiments; E.S.P. prepared figures.
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Perspectives and Significance
Neurohumoral activation is a critical pathological process
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clinical relevance. While an enhanced glutamate excitatory
action has been recognized as a crucial factor in HF, the precise
mechanisms contributing to elevated glutamate excitatory
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peptide release serves as an efficient autocrine mechanism by
which MNCs optimize their own firing activity in response to
specific physiological challenges (20, 33). Moreover, recent
studies from our laboratory indicate that dendritic release of
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