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J Neurophysiol (November 1, 2002). 10.1152/jn.00978.2001
Submitted on 29 November 2001
Accepted on 18 July 2002
1National Institute on Drug Abuse, Intramural Research Program, Baltimore, Maryland 21224; and 2Neurosciences, Ottawa Health Research Institute, University of Ottawa, Ottawa, Ontario K1Y 4E9 Canada
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Oz, Murat and
Leo P. Renaud.
Angiotensin AT1-Receptors Depolarize Neonatal Spinal
Motoneurons and Other Ventral Horn Neurons Via Two Different
Conductances.
J. Neurophysiol. 88: 2857-2863, 2002.
Angiotensin receptors are highly expressed
in neonatal spinal cord. To identify their influence on neuronal
excitability, we used patch-clamp recordings in spinal cord slices to
assess responses of neonatal rat (5-12 days) ventral horn neurons to bath-applied angiotensin II (ANG II; 1 µM). In 14/34 identified motoneurons tested under current clamp, ANG II induced a slowly rising
and prolonged membrane depolarization, blockable with Losartan (n = 5) and (Sar1,
Val5, Ala8)-ANG II
(Saralasin, n = 4) but not PD123319 (1 µM each;
n = 4). Under voltage clamp
(VH 
65 mV), 7/22 motoneurons
displayed an ANG-II-induced tetrodotoxin-resistant inward current
(128 ± 31 pA) with a similar time course, an associated
reduction in membrane conductance and net current reversal at
98.8 ± 3.9 mV. Losartan-sensitive ANG II responses were also
evoked in 27/78 tested ventral horn "interneurons." By contrast
with motoneurons, their ANG-II-induced inward current was smaller
(39.9 ± 5.2 pA) and analysis of their I-V plots
revealed three patterns. In eight cells, membrane conductance decreased
with net inward current reversing at 103.8 ± 4.1 mV. In seven
cells, membrane conductance increased with net current reversing at
37.9 ± 3.6 mV. In 12 cells, I-V lines remained
parallel with no reversal within the current range tested.
Intracellular dialysis with GTP-
-S significantly prolonged the ANG
II effect in seven responsive interneurons and GDP-
-S significantly
reduced the ANG II response in four other cells. Peak inward currents were significantly reduced in all 13 responding neurons recorded in
slices incubated in pertussis toxin (5 µg/ml) for 12-18 h or in 12 neurons perfused with N-ethylmaleimide. Of 29 interneurons sensitive to pertussis toxin or N-ethylmaleimide treatment,
9 cells displayed a decrease in membrane conductance that
reversed at 101.3 ± 3.8 mV. In eight cells, membrane
conductance increased and reversed at 38.7 ± 3.4 mV.
In 12 cells, the I-V lines remained parallel with no
reversal within the current range tested, suggesting that both
conductances are modulated by pertussis toxin-sensitive G proteins.
These observations reveal a direct, G-protein-mediated depolarizing
action of ANG II on neonatal rat ventral horn neurons. They also imply
involvement of two distinct conductances that are differentially
distributed among different cell types.
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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In the peripheral
circulation, angiotensin (ANG II) is not only a powerful modulator of
vasomotor activity but can act through neurons in the subfornical
organ, a circumventricular organ, to initiate a behavioral response,
i.e., drinking, a response dependent on a central renin-angiotensin
system whose roles also include modulation of cardiovascular reflexes
(Phillips 1987
; Sumners et al. 1994). In
CNS, angiotensin-like immunoreactive neurons and fibers as well as ANG
II receptors, mainly of the AT1 type, have a wide
but differential distribution (see Gehlert et al. 1986;
Lind et al. 1984; Mendelsohn et al.
1984). That the latter participate in regulating neuronal
excitability is inferred from electrophysiological observations of
neuronal responsivity to exogenous ANG II (e.g., Bai and Renaud
1998; Ferguson and Washburn 1998; Li and
Guyenet 1996; Ono et al. 2001; Yang et
al. 1992). These features indicate considerable regional and
cell-specific expression of ANG II receptors.
Binding studies and receptor-expression analyses also indicate
plasticity within the central renin-angiotensin system. A high level of
expression during the first and second postnatal week and change during
ontogeny (Tsutsumi and Saavedra 1991) suggest a
physiologically important role in CNS development. One of these sites
may be in spinal cord, where ANG II-immunoreactive fibers and receptors
have been observed (Gehlert et al. 1986; Lind et al. 1984; White et al. 1988) and where ANG II
has a depolarizing action on motoneurons (Suzue et al.
1981) and lateral horn neurons (Lewis and Coote
1993). There are few details on mechanisms of ANG II action on
spinal neurons.
During patch-clamp recordings to evaluate functional evidence for peptide receptors on thoracolumbar neurons in neonatal spinal cord, we observed that a subpopulation of motoneurons and unidentified ventral horn neurons were responsive to exogenous ANG II. We now report that these neurons display ANG-II-induced membrane depolarization and inward currents that engage two separate conductances: a potassium conductance in motoneurons and/or a presumed nonselective cationic conductance in interneurons.
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METHODS |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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The spinal cords of methoxyflurane-anesthetized Sprague-Dawley rats of either sex (5-12 days old) were removed after a dorsal thoracolumbar laminectomy and placed in ice-cold (4°C) artificial cerebrospinal fluid (ACSF). ACSF was composed of (in mM) 127 NaCl, 26 NaHCO3, 3.1 KCl, 1.2 MgCl2, 2.4 CaCl2, and 10 D-glucose (pH 7.35; osmolarity 290-305 mosmol) and was gassed with 95% O2-5% CO2. Transverse 350-450 µm sections from the Th7 to L5 segments were cut on a vibratome, equilibrated in ACSF at room temperature, and continuously superfused at 4-6 ml/min in a recording chamber.
Using the blind whole cell patch-clamp technique, neurons were recorded
using Axopatch 1A or an Axopatch 1D amplifier (Axon Instruments, Foster
City, CA). Micropipettes were filled with (in mM) 130 K-Gluconate, 10 KCl, 10 NaCl, 1 MgCl2, 10 HEPES, 1 EGTA, 1 GTP,
and 2 Mg-ATP, adjusted to a pH of 7.3 with Tris buffer. Lucifer yellow
(1 mg/ml) was included for later visualization and morphological
identification using methods described earlier (Kolaj and Renaud
1998). Corrections of the liquid-junction potentials (approximately 10 mV) were performed off-line. Data was filtered on-line at 2 kHz. Digidata 1200 interface and version 7 of pCLAMP software were used on-line to generate clamp commands. Motoneurons were
identified by their all-or-nonantidromic responses to ventral stimulation applied with a concentric bipolar electrode (1-10 V,
duration 0.02 s) and/or by their morphology and evidence of an
axon projecting toward the ventral root. In this study, we applied the
term interneuron to any other neuron.
ANG II, a peptide receptor antagonist (Sar1,
Val5, Ala8)-ANG II
(Saralasin), a nonpeptide AT2 receptor antagonist
PD123319, GTP, GTP--S, GDP--S, pertussis toxin, and tetrodotoxin
were from Sigma-RBI (St. Louis, MO). Agents were dissolved in ACSF
at their final concentrations, and delivered by bath application at a
perfusion rate of 4-6 ml/min. Losartan, a nonpeptide
AT1 receptor antagonist, was obtained from Merck
(Rathway, NJ). For statistical evaluation, we used paired or unpaired
Student's t-test as indicated in the text. Results are
presented as means ± SE.
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RESULTS |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Motoneurons
Data obtained from 56 motoneurons located in (within Rexed laminae
VIII and IX) displayed a mean resting membrane potential of 71.4 ± 1.9 mV and input resistance of 60.9 ± 4.3 M
.
ANG-II-INDUCED POSTSYNAPTIC MEMBRANE DEPOLARIZATION AND INWARD
CURRENT.
In 14/34 neurons tested while recording in current-clamp mode, bath
application of ANG II (1 µM; 30 s) initiated a slowly rising
(60-90 s to peak) membrane depolarization that reached a plateau of
11.6 ± 3.2 mV and was sufficient to trigger a burst of action
potentials in five cells (Fig.
1A). Some desensitization seems likely since washout intervals of 30-45 min were required to
regain full recovery. Application of an AT1
receptor antagonist Losartan, (1 µM; n = 5 cells) was
without effect on resting membrane properties but completely blocked
the ANG-II-induced membrane depolarizations. Similar results were
achieved with Saralasin (1 µM; n = 4 cells). However,
an AT2 receptor antagonist PD123319 (at 1 µM)
(cf. Kang et al. 1992) was without effects on
ANG-II-induced depolarizations (Fig. 1B; n = 4 cells).
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65 mV) and
in the presence of 1 µM TTX, 1 µM ANG II applications to 22 motoneurons yielded seven cells that responded with a slowly developing
postsynaptic response characterized by inward current (peak of
128 ± 31 pA) that slowly recovered over 8-10 min (Fig.
2A). Comparison of
instantaneous I-V plots before and at the peak of the ANG II
responses revealed net ANG II currents (difference between control and
peak ANG II effect) whose slope indicated a 19.1% reduction in
membrane conductance (from 11.3 ± 1.8 to 8.9 ± 1.3 nS). The
mean net ANG II current reversed at 98.8 ± 3.9 mV,
approximating the estimated equilibrium potential for potassium ions
under these conditions (Fig. 2B). Increasing extracellular
concentration of K+ to 10 mM shifted the reversal
potential to 68 ± 2.3 mV (Fig. 2C; n = 3 cells) implying mediation via potassium channels.
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Interneurons
A population of 78 cells labeled as interneurons based on failure
of antidromic activation displayed a significantly less negative
resting membrane potential (59.8 ± 1.4 mV; P < 0.05, Student's t-test) and higher input resistance
(179.8 ± 14.3 M; P < 0.05, Student's
unpaired t-test) than motoneurons.
ANG-II-INDUCED POSTSYNAPTIC RESPONSES.
Recordings from 78 neurons in voltage-clamp mode in the presence of TTX
yielded 27 neurons that responded to ANG II (1 µM) with a mean inward
current of 37.9 ± 5.2 pA (Fig.
3A). Although different in
magnitude from the response in motoneurons, the time course of these
ANG-II-induced current was virtually identical (compare Figs.
2A and 3A). Similarly, responses to ANG II were blocked by prior application of Saralasin (1 µM; 3/3 cells) and Losartan (1 µM; 4/4 cells), but not by PD123319 (1 µM, 4/4), all without effect on resting membrane properties (Fig. 3A).
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100 mV (102.6 ± 4.3 mV). Thus the data from this group resembled that from the
motoneuron population, suggesting mediated of the ANG II response via
reduction in conductance for potassium ions. By contrast, in another
seven cells, the slope of the net ANG II current reflected an
increase in conductance (from 4.8 ± 0.6 to 5.3 ± 1.2 nS, 11,3% increase) with current reversal close to 40 mV
(38.4 ± 4.1 mV). These values could reflect mediation of the
ANG II response via increase in a nonselective cationic conductance. In
the remaining 12 cells, the mean net ANG-II-induced current displayed a
slight reduction (only 3.6% decrease) in membrane conductance (from
5.1 ± 1.2 to 4.9 ± 1.1 nS), with no reversal within the
voltage range tested.
Because ANG II receptors belong to family of G-protein-coupled
receptors, we first compared the control data (containing 1 mM GTP)
with data obtained 5 min after establishment of seals using pipettes
that contained GTP--S (0.4 mM), a nonhydrolyzable derivative of GTP
that activates G protein in an irreversible manner (Gilman
1987). Of 18 cells recorded and tested with ANG II, 7 cells
responded. The maximum amplitudes of their response (38.7 ± 6.1 pA) were not significantly different from control, but the recovery
phase was greatly prolonged by the presence of GTP--S in the pipette
(Fig. 4A).
Of 7 cells treated with GTP--S, membrane conductance
increased in two cells (reversed close to EK+),
decreased in two cells (reversed close to 40 mV), and was unchanged in the remaining three cells (with no reversal potential detected in the range of 10 to 120 mV). We also dialyzed four cells
with GDP--S (1 mM for 10 min), a stable analogue of GDP that
competitively inhibits G protein binding by GTP (Gilman
1987). The data from four cells indicated that mean
ANG-II-induced inward current was significantly reduced (17.4 ± 3.7 vs. 37.9 ± 5.2 pA in 27 control cells; P < 0.05, Student's unpaired t-test, Fig. 4B).
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; Shapiro et al. 1994). To
evaluate the role of PTX-sensitive G proteins in ANG-II-induced
currents, slices were incubated for 12-18 h either in ACSF or in ACSF
containing PTX, and the magnitudes maximal ANG-II-induced currents were
compared. In the control cells, we also tested bath-applied NEM (50 µM), a sulfhydryl-alkylating agent shown to block G-protein-effector
interactions by alkylating the 
-subunits of PTX-sensitive
GTP-binding protein (Shapiro et al. 1994; Viana
and Hille 1996). The advantage of using NEM was that it allowed
us to examine ANG-II-induced currents before and after inhibition of
PTX-sensitive G proteins within the same cell.
In control slices, mean ANG-II-induced inward currents in 13 of 29 cells tested were 38.4 ± 4.1 pA. The mean ANG-II-induced inward
current was significantly reduced to 16.8 ± 2.1 in PTX treated
neurons (Fig. 4C, P < 0.05, Student's
unpaired t-test). Further analysis of the net ANG-II-induced
currents for individual cells revealed three differing patterns (Fig.
5). One group of four neurons
demonstrated a net ANG-II-induced conductance that decreased
from a control of 4.4 ± 0.5 to 3.7 ± 0.6 nS and reversed close to 100 mV (101.2 ± 4.1 mV). In another four cells, the slope of the net ANG-II-induced current reflected an
increase in conductance (from 4.6 ± 0.6 to 5.1 ± 1.1 nS) with current reversal close to 40 mV (39.4 ± 3.7 mV).
In the remaining five cells, the mean net ANG-II-induced current
displayed a slight reduction in membrane conductance (from 4.9 ± 1.2 to 4.7 ± 0.9 nS), with no reversal within the voltage range
tested.
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100 mV
(102.9 ± 3.4 mV). In another four cells, the slope of the net
ANG II current showed an increase in conductance (from
2.7 ± 0.5 to 3.1 ± 0.8 nS) with current reversal close to
40 mV (37.7 ± 2.8 mV). In the remaining seven cells, the mean
net ANG-II-induced current displayed a slight reduction in membrane
conductance (from 2.9 ± 0.7 to 2.6 ± 0.8 nS), with no
reversal within the voltage range tested.
In 12 of 13 cells in the control group described earlier, a 5-min
exposure treatment with 50 µM NEM was seen to reduce the ANG-II-induced inward current to 31.4% of control values (12.2 ± 3.6 vs. 38.1 ± 4 pA in controls, P < 0.05, Student's paired t-test, Fig. 4C). Further
analysis revealed that all patterns of conductance were sensitive to
NEM pretreatment (Fig. 5A-C). In four cells with current
reversal close to 100 mV, ANG-II-induced inward currents were
significantly reduced from 39.2 ± 4.3 (controls) to 14.6 ± 3.5 pA (P < 0.05, Student's paired
t-test). In another four cells with current reversal close
to 40 mV, ANG-II-induced inward currents were significantly reduced
from 36.9 ± 4.1 (controls) to 11.6 ± 3.2 pA
(P < 0.05, Student's paired t-test). In
the remaining four cells with no current reversal within the voltage
range tested, ANG-II-induced inward currents were significantly reduced
from 40.2 ± 4.3 (controls) to 12.9 ± 3.4 pA
(P < 0.05, Student's paired t-test).
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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The observation that exogenous ANG II induces membrane depolarization and inward currents in a subpopulation of neonatal spinal ventral horn neurons, including identified motoneurons, implies the presence of functional angiotensin receptors. These receptors are of the AT1 subtype because responses were sensitive to pretreatment with Losartan, a selective AT1 receptor antagonist, but not with PD123319, which selectively blocks angiotensin AT2 type receptors.
Our analyses of I-V relationships suggest that more than one
membrane conductance underlies the depolarizing action of ANG II. In
both motoneurons and a subpopulation of unidentified neurons, the
ANG-II-induced inward currents were consistently associated with
reduction in a membrane conductance that reversed close to the potassium equilibrium potential and shifted appropriately with the
transmembrane potassium gradient. In these neurons, the linearity and
reversal potential for the net ANG-II-induced currents indicate
involvement of a voltage-independent potassium conductance that
contributes to resting membrane potential, often referred to as a leak
conductance. Similar potassium-mediated responses to ANG II can be seen
in rat bulbospinal (Li and Guyenet 1996) and median
preoptic neurons (Bai and Renaud 1998) and hamster submandibular ganglion neurons (Ikegami et al. 2000). In
rat adrenal glomerulosa cells, ANG II suppresses a potassium
conductance that in brain stem motoneurons is attributed to a family of
two-pore domain pH-sensitive channels, named TASK-1 (Czirjak
and Enyedi 2002; Czirjak et al. 2000;
Talley and Bayliss 2002; Talley et al. 2000,
2002). Whether similar channels are responsible for ANG-II-induced inward currents in neonatal spinal motoneurons remains
to be established.
By contrast, the ANG-II-induced inward currents in a population of
interneurons were associated with an increase in membrane conductance that reversed around 40 mV, suggestive of a nonselective cationic conductance. This is similar to the ANG-II-induced responses in rat supraoptic neurons where net currents also reverse close to 40
mV (Yang et al. 1992). In a subpopulation of rat adrenal glomerulosa cells, net ANG II currents reverse around 10 mV
(Lotshaw and Li 1996), and in ANG-II-responsive
subfornical organ neurons, a net current that reverses closer to 30
mV has been reported (Ono et al. 2001). Whether these
differences reflect variations in underlying cationic conductances
remains to be clarified, but they do suggest cell specificity in
coupling of ANG II receptors to ion channels. Moreover, we speculate
that in some neurons, ANG II receptors can couple to both potassium and
cationic conductances, in which situation the net ANG -II-induced
conductance could be reflected in a nearly parallel shift, as observed
here in a subpopulation of ventral horn interneurons.
The binding of angiotensin to AT1 receptors can
activate a number of intracellular signaling pathways, mediated through
heterotrimeric G proteins (see Sumners et al. 1994 for
review). These may including Gq
to stimulate
phosphoinositide hydrolysis, subsequent activation of protein kinase C
and a rise in intracellular calcium, Gi to inhibit adenylate cyclase, and a
G
/Ras/Raf-1 pathway that increases
mitogen-activated protein (MAP) kinase activities. While it remains
undefined as to the identity of G proteins linking AT1 receptors to the conductances described
above, the linkage must involve PTX-sensitive G proteins given the
marked reduction in both types of conductances after treatment with PTX
or NEM (Fig. 5).
The present observations bear a striking resemblance to the response of
neonatal spinal neurons to other peptides including thyrotropin-releasing hormone (Kolaj et al. 1997),
vasopressin (Kolaj and Renaud 1998; Oz et al.,
2001), and substance P (Yasuda et al., 2001).
Two issues that derive from these observations include the sources for
ligands for these receptors, and the physiological roles that peptide
receptors may have in neuronal function in neonatal spinal cord. One
source may derive from the cerebrospinal fluid, which is known to
contain a variety of neuropeptides, including some that display a
circadian rhythmicity in their concentrations (Reppert et al.
1987). Neuronal pathways may provide a source since
angiotensin-like immunoreactivity has been observed in adult spinal
cord (Lind et al. 1984); however, information is lacking for neonatal tissue. Whatever the origin, angiotensin receptors may
have specific functional links to neuronal development and survival.
This is suggested by their ability to undergo developmental changes
(Milas et al. 1991), to participate in recovery
following axotomy (Palkovits 1995), and to modulate
neurite outgrowth from cultured embryonic neurons (Iwasaki et
al. 1991; Sood et al. 1990) and by their
neurotrophic action in cultured explants of spinal cord (Ikeda
et al. 1989; Yang et al. 2001). It is possible
that their influence on excitability of neonatal spinal cord neurons is
important for their subsequent development.
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FOOTNOTES |
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Address for reprint requests: M. Oz, National Institute on Drug Abuse, Intramural Research Program, 5500 Nathan Shock Dr., Baltimore, MD, 21224 (E-mail: moz{at}intra.nida.nih.gov).
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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)
determined by subtraction of these I-V values displays
reversal approximately 
), note a decrease in membrane conductance with a
reversal potential approximately 
), note the shift in reversal potential
toward the Nernstian predicted equilibrium potential of 
(n = 5). B:
ANG-II-induced currents are mediated by pertussis toxin (PTX)-sensitive
G proteins. Histograms (left to right):
normalized control ANG -II-induced current recorded after overnight
incubation in ACSF (control, n = 13), in ACSF
containing PTX (5 µg/ml, n = 16), after 5 min
incubation with 50 µM N-ethylmaleimide
(n = 12), and in cells recorded with electrodes
containing GDP-
in
A-C) analyses indicated 3 patterns in
I-V relationships: current that reversed at 
in A-C;
n = 13). In 5 of 16 cells, currents were reversed
at