1Laboratoire de Neurobiologie des
Réseaux Sensorimoteurs,
Prime, L.,
Y. Pichon, and
L. E. Moore.
N-Methyl-D-Aspartate-Induced
Oscillations in Whole Cell Clamped Neurons From the Isolated Spinal
Cord of Xenopus laevis Embryos.
J. Neurophysiol. 82: 1069-1073, 1999.
The patch-clamp
technique was used to measure the effect of
N-methyl-D-aspartate (NMDA) on
Xenopus embryonic neurons in an isolated, but intact
spinal cord. Whole cell recordings were done at external calcium
concentrations of 1 mM. NMDA alone (50-200 µM) or in association
with 10 µM serotonin or glycine induced oscillatory activity in most
presumed motoneurons, which were therefore considered part of rhythm
generating networks. In the presence of TTX, one-half of these neurons
maintained this activity. The oscillations fell into two main
categories: voltage-dependent, low-frequency (0.3-0.5 Hz) and
voltage-independent, high-frequency (3-8 Hz) oscillations. NMDA alone
induced TTX-insensitive oscillations in one-third of the neurons;
however, the percentage of neurons showing oscillations was greater in
the presence of exogenous 5-hydroxytryptamine (5-HT) or glycine.
Because these observations were made at embryonic stages where little
or no serotonergic innervation exists, it is likely that NMDA-induced
intrinsic oscillatory activity in Xenopus embryonic
neurons does not require 5-HT.
Rhythmic activity in neural systems is
associated with the activity of both conditional pacemaker neurons and
oscillatory networks. In many cases, part of this oscillatory behavior
is linked to the activation of one category of glutamate receptors: N-methyl-D-aspartate (NMDA) receptors. This is
true for lower vertebrates such as the lamprey in which TTX-resistant
membrane potential oscillations are induced in the spinal cord by NMDA (Wallen and Grillner 1987 Prehatching and hatching (32-37/38 stages of Nieuwkoop
and Faber 1956 The two ends of the preparations were fixed on the bottom of the
dish using two glass suction electrodes that were used for extracellular recording of spontaneous activity. Neurons located in
ventral quarters of the cord, presumed to be motoneurons, were selected
for the experiments. Whole cell patch-clamp measurements (Hamill
et al. 1981 The external Ringer solution contained (in mM) 110 NaCl, 3 KCl, 1 CaCl2, 1 MgCl2, 2.4 NaHCO3, and 10 HEPES buffered at pH 7.4. The
internal (pipette) solution contained (in mM) 90 K-gluconate, 10 KCl,
10 EGTA, 10 HEPES, 2 MgCl2, 3 ATP, and 0.5 GTP
buffered at pH 7.3. NMDA (50-200 µM) and 1 µM TTX were added to
the perfusing solution. In 50% of the experiments, 10 µM
5-hydroxytryptamine (5-HT) or 10 µM glycine was also added to the
perfusing solution to stabilize the NMDA oscillations (Hochman
et al. 1994b NMDA induced rhythmic activity in whole cell clamped neurons
The neurons had a mean resting potential of
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
), as well as amphibian embryos
and larvae in which the locomotor activity is associated with the
activation of NMDA receptors of the spinal motoneurons (Dale and
Roberts 1984
; Reith and Sillar 1998
;
Roberts et al. 1995
; Scrymgeour-Wedderburn et al.
1997
; Sillar and Simmers 1994a
,b
; Soffe
1993
, 1996
) and for the CNS of mammals
(Durand 1993
; Hochman et al. 1994a
,b
;
Kiehn et al. 1996
; MacLean et al. 1997
,
1998
; Tell and Jean 1993
; Vincent et al. 1996
).
METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
) Xenopus laevis embryos were obtained
from the local laboratory culture. The eggs, which were taken from
adult females injected with human chorionic gonadotrophin (HCG), were
fertilized with sperm and kept at room temperature (15-24°C). The
embryos were anesthetized using 0.5 mg/ml tricaïne methane
sulfate (MS222, Sigma). The CNS surrounded by myotomes was cut away
using sharp needles and transferred to a solution of 0.5 mg/ml dispase
(Sigma) in the external Ringer solution. The preparation was gently
agitated for ~20 min and then thoroughly rinsed with enzyme-free
saline. The spinal cord was dissected free and transected at the level of the otic capsule. This procedure removed the rostral part of the
hindbrain where the raphe nuclei containing the serotonergic neurons
are located (Reith and Sillar 1998
; Sillar et al.
1992
, 1995
; Van Mier et al.
1986
). The spinal cord preparation was then placed ventral side
up in a continuously perfused 200-µl chamber.
) were made on single neurons emerging from the main
body of the cord (Desarmenien et al. 1993
). The
electrode resistance was 10-12 M
before the seal was made. White
noise measurements (Moore et al. 1993
) were used to
estimate an electrode series resistance of 20-30 M
in the whole
cell current clamp. All experiments were done with an Axoclamp-2A
amplifier (Axon Instruments) in a constant current (bridge) mode. The
signals were filtered at 2 kHz using a 24-dB/octave Flat delay (Linear Phase) filter (Rockland Systems) and digitized with a 12-bit A/D converter (Labmaster, Scientific Solutions). Data acquisition and some
analysis was done with ACQUIS1 (Bio-Logic/CNRS License) software. In
addition, Sigmaplot (Jandel Scientific) was used for statistical
analysis and figure preparation.
). It should be noted that the whole cell patch
recording was done in Ringer solution containing a relatively normal
calcium concentration of 1 mM, whereas most previous microelectrode
measurements required high calcium levels of 4 mM.
RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
53 ± 13 mV
(mean ± SD, N = 38), and in most cases, clear
excitatory synaptic potentials up to 10 mV were observed. Bath
application of 50-200 µM NMDA, with or without exogenous glycine,
caused a 10 to 30 mV depolarization of the membrane potential generally
associated with an increase in synaptic noise and oscillatory spike
activity. A hyperpolarizing current enhanced the membrane potential
oscillatory response underlying the action potentials (Figs.
1A and
2A) in 30 of the 38 tested
neurons. The means ± SD of the frequencies (Hz) and amplitudes
(mV) of these NMDA-induced oscillations fell into two categories: slow
oscillations (Fig. 1A), 0.5 ± 0.13 Hz (20.9 ± 10.1 mV, N = 3), 0.6 ± 0.3 Hz (26.7 ± 18.6 mV, N = 5) with glycine and 0.6 Hz (33.1 mV,
N = 1) Hz with 5-HT; and fast oscillations (Fig.
2A), 3.9 ± 2.6 Hz (15.6 ± 6.0 mV,
N = 9), 12.1 Hz (36.6 mV, N = 1) with
glycine and 5.3 ± 3.9 Hz (20.8 ± 9.6 mV, N = 6) Hz with 5-HT, where N is the number of neurons in each category. As can be seen from this data, the rhythmic activity was
present with (13 neurons) or without serotonin (5-HT) or glycine (12 neurons).
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Fig. 1.
Slow oscillatory activity induced by
N-methyl-D-aspartate (NMDA) in presumed
motoneurons from an isolated spinal cord of a Xenopus
embryo. A: slow (0.55 ± 0.05 Hz,
n = 11) sinusoidal activity induced by 200 µM
NMDA alone. A 80-pA current (as indicated) was used to hold the
membrane potential to about
70 mV. Note the existence of a few
truncated spikes on top of the (48.7 ± 2.9 mV,
n = 11 peak-to-peak) depolarizations.
B1 and B2: oscillatory activity in the
presence of NMDA and tetrodotoxin (TTX); 1: beginning of
NMDA application; 2: relatively stable activity after
22 s in NMDA (0.72 ± 0.4 Hz, 33.4 ± 4.2 mV,
n = 15). A
5-pA current was continuously injected
in this neuron. C: effects of NMDA, TTX, and glycine on
another neuron. C1: the 1st part of the recording shows
the electrical activity induced by 50 µM NMDA. At the arrow, the
holding current was changed from
20 pA to
30 pA, and 1 µM TTX + 10 µM glycine were added to the bath. The activity was transformed
into large-amplitude (50 ± 7.5 mV, n = 5)
low-frequency (0.5 ± 0.05 Hz, n = 5)
irregular oscillations that are illustrated with an expanded scale in
C2. D1-D4: effects of current on the amplitude and
frequency of the slow oscillatory activity (same neuron as in
C1 and C2). D1: reduction
of the injected current from
30 pA (as indicated) to
20 pA,
reversibly inhibited the oscillations (interval between the 2 arrows)
resulting in a constant membrane potential at its plateau value of
20
mV. D2: increase of the current to
40 pA
hyperpolarized the cell by ~15 mV to
75 mV, increased the amplitude
of the oscillations to 55 mV, and decreased their frequency.
D3: a transient increase of the injected current from
40 pA (as indicated) to
55 pA reversibly inhibited the activity
(interval between the 2 arrows). D4: plot of the
relationship between the intensity of the injected current and the
frequencies and amplitudes of the oscillations (mean values) for the
same neuron. Means ± SD for the frequencies and amplitudes are
given for the number, n, of oscillations used.
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Fig. 2.
Fast oscillatory activity induced by NMDA in a (presumed) motoneuron of
an isolated spinal cord of a Xenopus embryo.
A: effects of TTX (1 µM) and 5-hydroxytryptamine
(5-HT; 10 µM) on the electrical activity of the neuron previously
perfused with 50 µM NMDA with a holding current of 100 pA. The
action potentials were abolished, and the fast oscillatory activity
decreased from 13.1 ± 2.6 (n = 66) to
7.8 ± 1.7 Hz (n = 38). B:
changes of the injected current (as indicated on the bottom
trace) failed to modify the frequency of the oscillations,
whereas their amplitude and the time course were altered. The amplitude
increased with membrane depolarization, which was associated with the
appearance of spikelike events (see C and
D). E: the oscillations progressively
disappeared, and the membrane repolarized following return to a normal
drug-free bathing solution. F: plot of the relationship
between the intensity of the injected current and the frequencies and
amplitudes of the oscillations (mean values) for the same neuron.
NMDA-induced oscillations in the presence of TTX
In the presence of TTX, NMDA-induced oscillations were observed in
5 of 14 motoneurons, three with slow (Fig. 1, B1 and
B2) 0.6 ± 0.2 Hz (26.9 ± 5.8 mV,
N = 3) and two with fast 2.3 ± 0.7 Hz (14.8 ± 1.9 mV, N = 2) oscillations. The mean holding
potential in these experiments with or without TTX was 60 to
50 mV.
The observation of oscillations in TTX required a bias current of
5
to
50 pA. Serotonin in combination with NMDA led to four of seven
neurons showing TTX-resistant oscillations (Fig. 2), namely, 0.6 Hz
(31.5 mV, N = 1) and 4.6 ± 3.1 Hz (20.6 ± 5.4 mV, N = 3). Similarly, in glycine with NMDA (Fig.
1, C1, C2, and D1-D3), four of six cells had
oscillatory activity in TTX: 0.4 ± 0.1 Hz (23.2 ± 10.1 mV,
N = 3) and 7.5 Hz (10.2 mV, N = 1). The
finding that higher ratios of neurons showed NMDA-induced oscillations
with the addition of serotonin (4/7) or glycine (4/6) compared with NMDA alone (5/14) is consistent with the hypothesis that these two
amino acids enhance NMDA-induced intrinsic oscillatory behavior. The
maintenance of the fast oscillations in Fig. 2, A and
B, after TTX was added, suggests that although the synaptic
input contributes to the oscillatory activity, the rapid sinusoidal
form is due to the activation of intrinsic TTX-insensitive receptors.
Voltage dependency of the oscillations in TTX
The effects of membrane potential on the properties of the two
categories of oscillations were studied by injecting current through
the patch electrode. The voltage dependency of the two categories of
oscillations was clearly different. The frequency of the slower
patterned activity was sensitive to changes in the membrane voltage and
was similar to that observed in other preparations (Chub et al.
1997; Wallen and Grillner 1987
). The
oscillations, illustrated in Fig. 1, D1-D3, were
characterized by plateau potentials around
20 mV that had variable
durations and abrupt terminations. The frequency and plateau duration
of these oscillations decreased with membrane hyperpolarization,
whereas their amplitude increased (Fig. 1D4).
By contrast, the frequency of the fast oscillations was not modified by changes in membrane potential, but their amplitude was found to decrease with membrane hyperpolarization (Fig. 2F). This change was associated with a modification of the time course of the oscillations as shown on Fig. 2, C and D. The more hyperpolarized responses (Fig. 2C) were nearly sinusoidal; however, at depolarized potentials, a 20 to 30 mV abortive spike appeared on the rising phase of the oscillation (Fig. 2D). These findings suggest that increased amplitude is related to the turning on of voltage-dependent conductances that are activated in concert with the direct NMDA effects.
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These experiments clearly show that in the presence of TTX, NMDA
(50-200 µM) together with a negative current bias can induce oscillatory behavior in whole cell clamped (presumed) motoneurons of
the spinal cords of Xenopus embryos at stages 32-37/38.
This is in contrast to the slow (sinusoidal) pattern of activity
observed by Scrymgeour-Wedderburn et al. (1997) in
postembryonic and larval neurons of the same preparation, which
required exogenous 5-HT and NMDA. At least two differences in the
experimental procedures could be used to explain why 5-HT was needed in
their experiments and not in the present ones: 1) the
internal milieu of the cell was not the same since, in patch-clamped
neurons, the interior of the cell was perfused with the pipette
solution, and 2) the external calcium concentration was 1 mM
in the patch-clamp experiments compared with 4 mM in the intact
preparation. Because oscillations depend on a complex balance between
multiple conductances, it is not surprising that changes in both
external and interior environments would alter this behavior.
We cannot completely exclude the presence of endogenous serotonin
playing some role in NMDA-induced oscillations, nevertheless the levels
must be relatively low because our isolated spinal cord was
continuously perfused, and the serotonergic neurons of the brain stem
were not present. It has been suggested that 5-HT may have a direct
agonist effect on the NMDA receptor (Scrymgeour-Wedderburn et
al. 1997), however, its influence on fictive swimming at stage 37/38 (Sillar et al. 1992
) could be caused by other
mechanisms, such as an alteration in a potassium conductance. Although
5-HT may not be obligatory for the activation of the NMDA receptor, both the microelectrode and whole cell clamp measurements are consistent with a modulatory effect of 5HT on NMDA-induced oscillations.
Soffe (1996) reported that rapid rhythmic behavior,
similar to that induced by NMDA in whole cell clamped neurons, was
induced by non-NMDA agonists
(
-amino-3-hydroxy-5methyl-4-isoxazolepropionic acid and
kainate), but not NMDA. Some of the discrepancies between our
observations and those of Soffe (1993
,
1996
), as well as Scrymgeour-Wedderburn et al.
(1997)
, could also be reasonably well accounted for by differences in the techniques used to record the electrical activity of
the neurons. Thus the significantly (~5 times) higher impedance of
the patch-clamped neurons (601 ± 98.7 M
, n = 27) as compared with the microelectrode-impaled neurons (118 ± 17 M
) (Soffe 1990
) is indicative of a smaller leak that
could theoretically facilitate oscillatory activity.
Our slow voltage-dependent oscillations resemble the NMDA-induced
inherent oscillatory activity associated with fictive locomotion in the
lamprey (Wallen and Grillner 1985, 1987
).
NMDA-induced plateau potentials similar to those recorded in the
present experiments have been seen in a variety of preparations such as
the interneurons of the spinal cord of neonatal rats (Kiehn et
al. 1996
), cat neocortical neurons (Flatman et al.
1983
, 1986
), or isolated turtle spinal cords
(Guertin and Hounsgaard 1998
).
Besides the fact that 5-HT was needed in their experiments to obtain
the activity, the frequency of the oscillations recorded in larval
neurons by Scrymgeour-Wedderburn et al. (1997) and
Reith and Sillar (1998)
was voltage independent, whereas
it is strongly voltage dependent in one of our two types of
oscillations. Electrical coupling between the motoneurons in intact
Xenopus embryos (Perrins and Roberts 1995
)
has been proposed by Scrymgeour-Wedderburn et al. (1997)
as an explanation for the voltage independence of the NMDA-induced
oscillations in TTX of intact Xenopus larvae. It is
plausible that the experimental procedure used to isolate the spinal
cord in our experiments, which requires enzymatic treatment, partially
disrupts this coupling between the neurons, as well as synaptic
connections. This hypothesis is also consistent with the observation of
slow rhythmic oscillations with action potentials in Fig.
1A1 that resemble those observed by Reith and Sillar
(1998)
on Xenopus larvae when the inhibitory
synapses had been blocked by strychnine and bicuculline.
The fast oscillatory activity seen with or without TTX resembles in
some respects the "struggling" activity induced by 1 mM kynurenic
acid (Soffe 1993) in stage 37-38 Xenopus
embryos. Although some of the rapid behavior is a consequence of
network connections, our experiments clearly indicate that the fast
oscillations are also part of the intrinsic oscillatory properties of
some neurons. TTX-resistant voltage-independent fast oscillatory
activity, very similar to that observed in our experiments, has been
recorded with the patch-clamp technique in neonatal rat lumbar
motoneurons in vitro by MacLean et al. (1997
,
1998
). The mean frequency of these oscillations (~ 4.5 Hz) falls precisely in the range of frequencies observed in our
experiments (3-8 Hz).
The ionic basis of the slow oscillatory activity is most probably the
same as in the other preparations exhibiting a similar activity, i.e.,
the alternation of an inward (depolarizing) current corresponding to
the opening of nonselective NMDA-activated channels followed by an
outward potassium current corresponding to the opening of
calcium-activated potassium channels as described by Wallen and
Grillner (1987) (see also Moore and Buchanan
1993
; Moore et al. 1993
). The ionic basis of the
fast oscillations may simply be a continuum that includes the
voltage-dependent ionic channels, especially calcium (Gu and
Spitzer 1993
) and those of the delayed rectifier potassium
currents (Dale 1995
). The increase in amplitude of these
oscillations with membrane depolarization might correspond to the
activation of high-threshold channels, which have been observed in
isolated Xenopus neurons (Dale 1995
).
The physiological relevance of these intrinsic oscillatory properties
remains speculative. It is reasonable, however, to suggest that the
low-frequency oscillations might control the frequency of the swimming
episodes observed in Xenopus larvae as proposed by
Reith and Sillar (1998) and to assume that the
high-frequency oscillations are related to the struggling behavior of
the embryo.
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FOOTNOTES |
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Address for reprint requests: L. E. Moore, UPRESA-7060, Centre National de la Recherche Scientifique, Université Paris 5-7, 45 rue des Saint Pères, F-75270 Paris Cedex 06, France.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 28 December 1998; accepted in final form 5 April 1999.
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