Download Endogenous release of 5-HT modulates the plateau phase of NMDA

J Neurophysiol 112: 30 –38, 2014.
First published April 16, 2014; doi:10.1152/jn.00582.2013.
Endogenous release of 5-HT modulates the plateau phase of NMDA-induced
membrane potential oscillations in lamprey spinal neurons
Di Wang,1,2 Sten Grillner,2 and Peter Wallén2
1
Department of Physiology, Liaoning Medical University, Jinzhou, People’s Republic of China; and 2Nobel Institute for
Neurophysiology, Department of Neuroscience, Karolinska Institutet, Stockholm, Sweden
Submitted 15 August 2013; accepted in final form 15 April 2014
plateau properties; pacemaker-like oscillations; spinal serotonin system; calcium-dependent potassium channels; locomotor network
IN ALL VERTEBRATES INVESTIGATED, the networks underlying locomotion are located in the spinal cord, while they are activated by glutamatergic reticulospinal neurons in the brain
stem, acting through ␣-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid and N-methyl-D-aspartic acid (NMDA)
receptors (Grillner 2006; Ohta and Grillner 1989). Descending
pathways and intraspinal neurons using 5-HT as a transmitter
also play an important role in many vertebrates (Jordan et al.
2008; Schotland et al. 1995). 5-HT acts through voltagedependent calcium channels, which in turn, control the postspike afterhyperpolarization via calcium-dependent potassium
(KCa) channels [lamprey: Hill et al. (2003) and Wallén et al.
Address for reprint requests and other correspondence: P. Wallén, Nobel
Institute for Neurophysiology, Dept. of Neuroscience, Karolinska Institutet,
SE-171 77 Stockholm, Sweden (e-mail: [email protected]).
30
(1989); rat: Bayliss et al. (1995) and Blomeley and Bracci
(2005); turtle: Hounsgaard and Kiehn (1989); frog: Scroggs
and Anderson (1989) and Sun and Dale (1998)] and also acts
through presynaptic inhibition [lamprey: Buchanan and Grillner (1991) and Schwartz et al. (2005); mouse: Garcia-Ramirez
et al. (2014); frog: Sillar and Simmers (1994b)]. In the lamprey, administration of 5-HT agonists slows down the locomotor activity and makes the bursts more intense (Harris-Warrick
and Cohen 1985). Conversely, administration of 5-HT antagonists during locomotor activity leads to a pronounced increase
of the locomotor frequency (Zhang and Grillner 2000).
In the lamprey, the locomotor networks can be activated by
superfusion of NMDA to the isolated spinal cord preparation
(Grillner et al. 1981). The motor activity can be recorded from
the ventral roots or from motoneurons and interneurons intracellularly. The key elements of the segmental and intersegmental networks responsible for generating the locomotor activity
have been identified, and they consist of ipsilateral excitatory
interneurons and commissural inhibitory interneurons (Buchanan and Grillner 1987; Grillner 2003; Kozlov et al. 2009).
In the isolated spinal cord, after cellular interactions have
been blocked through administration of TTX, the activation of
NMDA receptors generates pacemaker-like oscillations in
many neurons, which rely on the voltage-dependent properties
of NMDA receptors in interaction with voltage-dependent
potassium and KCa channels (Wallén and Grillner 1987). NMDA
receptor-mediated, TTX-resistant membrane potential oscillations can contribute to the generation of locomotor activity and
have also been demonstrated in other species, including rodents (Hochman et al. 1994; MacLean et al. 1997; Masino et al.
2012; Schmidt et al. 1998) and amphibians (Li et al. 2010;
Reith and Sillar 1998; Scrymgeour-Wedderburn et al. 1997). In
the lamprey, these oscillations vary in a characteristic way
between different spinal cord preparations. In some preparations, there is a pronounced, depolarized plateau phase, which
can be prolonged further by a blockade of KCa channels via
administration of 5-HT or specific antagonists, such as apamin
(El Manira et al. 1994). In other preparations, there are instead
more short-lasting depolarizations without any plateau phase
[cf. Wang et al. (2013)], lasting approximately 200 –300 ms. In
the present study, we characterize these two types of oscillations and show that they depend on the level of endogenous
release of 5-HT in the spinal cord preparations. The plateaulike oscillations are converted to the second type of oscillations
lacking a plateau phase when 5-HT antagonists are administered, and conversely, plateau-like oscillations can be induced
or prolonged by 5-HT agonists. These properties most likely
0022-3077/14 Copyright © 2014 the American Physiological Society
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Wang D, Grillner S, Wallén P. Endogenous release of 5-HT
modulates the plateau phase of NMDA-induced membrane potential
oscillations in lamprey spinal neurons. J Neurophysiol 112: 30 –38,
2014. First published April 16, 2014; doi:10.1152/jn.00582.2013.—
The lamprey central nervous system has been used extensively as a
model system for investigating the networks underlying vertebrate
motor behavior. The locomotor networks can be activated by application of glutamate agonists, such as N-methyl-D-aspartic acid
(NMDA), to the isolated spinal cord preparation. Many spinal neurons
are capable of generating pacemaker-like membrane potential oscillations upon activation of NMDA receptors. These oscillations rely on
the voltage-dependent properties of NMDA receptors in interaction
with voltage-dependent potassium and calcium-dependent potassium
(KCa) channels, as well as low voltage-activated calcium channels.
Upon membrane depolarization, influx of calcium will activate KCa
channels, which in turn, will contribute to repolarization and termination of the depolarized phase. The appearance of the NMDAinduced oscillations varies markedly between spinal cord preparations; they may either have a pronounced, depolarized plateau phase
or be characterized by a short-lasting depolarization lasting approximately 200 –300 ms without a plateau. Both types of oscillations
increase in frequency with increased concentrations of NMDA. Here,
we characterize these two types of membrane potential oscillations
and show that they depend on the level of endogenous release of 5-HT
in the spinal cord preparations. In the lamprey, 5-HT acts to block
voltage-dependent calcium channels and will thereby modulate the
activity of KCa channels. When 5-HT antagonists were administered,
the plateau-like oscillations were converted to the second type of
oscillations lacking a plateau phase. Conversely, plateau-like oscillations can be induced or prolonged by 5-HT agonists. These properties
are most likely of significance for the modulatory action of 5-HT on
the spinal networks for locomotion.
ENDOGENOUS 5-HT RELEASE AND NMDA OSCILLATIONS IN LAMPREY
play a role in the generation of locomotor activity by the spinal
networks.
MATERIALS AND METHODS
ventral roots were drawn into the tips of glass suction electrodes for
motoneuron identification. The chamber was perfused continuously
with oxygenated lamprey physiological solution containing (in mM):
138 NaCl, 2.1 KCl, 1.8 CaCl2, 1.2 MgCl2, 4 glucose, 2 HEPES, and
0.5 L-glutamine, bubbled with O2 and adjusted to pH 7.4 with NaOH.
The experimental chamber was cooled to maintain the preparation at
6 –10°C.
Drugs used were NMDA (Tocris, Bristol, UK); TTX (final concentration 1.5 ␮M; Sigma); 5-HT (1 ␮M; Tocris), spiperone hydrochloride,
a 5-HT antagonist (10 ␮M; Tocris); NAD 299 hydrochloride, a specific
5-HT1A antagonist [(3R)-3-(dicyclobutylamino)-8-fluoro-3,4-dihydro-2H1-benzopyran-5-carboxamide hydrochloride, 10 –20 ␮M; Tocris]; and
nimodipine, an L-type calcium channel antagonist [1,4-dihydro-2,6dimethyl-4-(3-nitrophenyl)-3,5-pyridinedicarboxylic acid 2-methyloxyethyl
1-methylethyl ester, 10 ␮M; Tocris]. All drugs were applied to the
perfusion solution. NMDA was applied in incremental concentration
steps of 50 ␮M, from 50 to 200 ␮M or in one case, to 250 ␮M.
Electrophysiology. To induce NMDA receptor-dependent, TTXresistant oscillations, NMDA and TTX were added in the physiological solution (Wallén and Grillner 1987). For intracellular recordings,
sharp microelectrodes were filled with 3 M KAc and 0.1 M KCl
B
-48
2
1 100µM NMDA
oscillation frequency (Hz)
A
150µM NMDA
mV
-70
3 200µM NMDA
C
10s
4 250µM NMDA
8
½
100µM
150µM
200µM
Width
0.0
35
30
amplitude (mV)
cycle duration (s)
0.2
100
150
200
NMDA concentration (µM)
2
Amplitude
6
4
2
25
20
15
10
5
0
0
cell 2
cell 3
cell 4
cell 5
5
4
3
2
1
0
4
1
half-width/cycle duration
cell 1
half-width (s)
0.4
Cycle duration
1 10
3
0.6
0.5
cell 1
cell 2
cell 3
cell 4
cell 5
cell 1
cell 2
cell 3
cell 4
cell 5
0
cell 1
cell 2
cell 3
cell 4
cell 5
Fig. 1. Membrane potential oscillations depend on the N-methyl-D-aspartic acid (NMDA) concentration. A: NMDA-induced, TTX-resistant membrane potential
oscillations at different concentrations of NMDA in a lamprey spinal neuron, recorded in discontinuous current clamp mode. The average trough membrane
potential (⫺70 mV) was kept constant by direct current (DC)-current injection. The oscillation frequency was increased markedly when concentration was raised
from 100 to 150 ␮M (A1 and A2). At 200 and 250 ␮M (A3 and A4), no further change of oscillation frequency was apparent. Time and voltage calibrations in
A1 apply to all records. B: relation between NMDA concentration and oscillation frequency. Combined results from 9 neurons tested. The overall frequency range
was 0.08 – 0.54 Hz. The higher doses resulted in higher frequency, with the more marked increase commonly occurring from 100 to 150 ␮M. In several cells,
the dose-frequency relation tended to saturate at the highest dose (200 ␮M). Each point represents the average frequency from 10 cycles at each concentration
in an individual neuron. C: the influence of changing the NMDA concentration on the structure of the oscillatory cycle. Inset: the different parameters measured:
cycle duration, time from 1 depolarization peak to the next; amplitude, voltage deflection from trough to peak; half-width, duration of depolarized plateau at
half-maximal amplitude (½). These parameters were analyzed in 5 cells with plateau-like oscillations: cycle duration (C1), amplitude (C2), half-width (C3), and
half-width normalized to cycle duration (C4). Average values from 20 cycles recorded at each concentration. Student’s t-test, means ⫾ SD, comparisons of nearby
groups.
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Ethical approval. All experimental procedures were approved by
Stockholm Norra Försöksdjursetiska Nämnd, according to the Swedish regulations for the care and use of laboratory animals. Before
removal of the tissue, the animals were deeply anesthetized (MS-222,
tricaine methanesulfonate, 100 mg/l, dissolved in water; Sigma, St.
Louis, MO) and killed by decapitation.
In vitro lamprey spinal cord preparation. Experiments were performed using isolated spinal cord preparations of adult river lampreys
(Lampetra fluviatilis; n ⫽ 31) or in a few cases, American silver
lampreys (Ichthyomyzon unicuspis; n ⫽ 2). Results obtained from the
two species were indistinguishable. In previous studies, no differences
in cellular properties of spinal neurons in the two species have been
observed. A spinal cord piece comprising 10 –15 segments was
dissected from a region caudal to the gills. The spinal cord was
isolated from the notochord and placed with the ventral side up into a
Sylgard-lined (Dow Corning, Midland, MI) recording chamber. The
meninges were stripped from the ventral surface, and in most cases,
31
32
ENDOGENOUS 5-HT RELEASE AND NMDA OSCILLATIONS IN LAMPREY
(resistance 30 –50 M⍀). Cells were recorded in current clamp mode
using an AxoClamp 2A amplifier (Axon Instruments, Weatherford,
TX). The general membrane potential level was adjusted by direct
current injection when needed. In cases when recordings were started
before TTX application, motoneurons could be identified by recording
their axonal spike in the ventral root.
Student’s t-test was used for statistical comparisons of oscillation
cycle parameters (cf. Fig. 1C1, inset). Parameter values are given as
means ⫾ SD, with levels of significance: *P ⬍ 0.05, **P ⬍ 0.01, and
***P ⬍ 0.001.
RESULTS
A 1 100µM NMDA
3 200µM NMDA
2 150µM NMDA
-30
mV
-60
10s
B
1
2
25
100µM
150µM
200µM
20
35
30
amplitude(mV)
cycle duration(s)
25
15
10
20
15
10
5
5
0
0
cell 6
3
cell 7
cell 8
cell 9
cell 10
1
cell 6
cell 7
cell 8
cell 9
cell 10
cell 6
cell 7
cell 8
cell 9
cell 10
4 0.5
half-width/cycle duration
0.4
half-width(s)
Fig. 2. Lamprey spinal neurons may generate
spike-like oscillations that are also influenced
by the NMDA level. A: a lamprey spinal neuron generating spike-like NMDA oscillations.
The oscillation frequency was increased markedly when NMDA concentration was raised
from 100 (A1) to 150 ␮M (A2). At 200 ␮M
NMDA (A3), oscillation amplitude increased
further, whereas the frequency did not. Time
and voltage calibrations in A1 apply to all
records. B1–B4: the influence of changing the
NMDA concentration on the structure of the
oscillatory cycle in 5 neurons with spike-like
oscillations. Parameters analyzed were as in
Fig. 1C. The effects of varying the NMDA
concentration on the cycle structure were similar in the 2 types of oscillations. Average
values from 20 cycles recorded at each concentration. Student’s t-test, means ⫾ SD, comparisons of nearby groups.
0.5
0.3
0.2
0.1
0
0
cell 6
cell 7
cell 8
cell 9
cell 10
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Upon application of NMDA and in the presence of TTX, many
neurons in the lamprey spinal cord will generate endogenous,
pacemaker-like membrane potential oscillations (Wallén and
Grillner 1985, 1987). These oscillations are strongly voltage
dependent, and previous reports have also suggested a positive
correlation between the frequency of the oscillations and the
NMDA concentration (Huss et al. 2008; Sigvardt et al. 1985;
Wallén and Grillner 1987).
The NMDA-induced, TTX-resistant membrane potential oscillations vary in shape between different spinal cord preparations; the oscillations may be “plateau like” with a pronounced,
depolarized plateau phase (cf. Fig. 1A) but may in other
preparations appear “spike like” with a short-lasting depolarization and no evident plateau phase (cf. Fig. 2A). Below, we
will characterize these two forms of oscillation by a quantitative analysis of the effects of varying the NMDA concentra-
tion. In addition, we describe the modulatory influence of
endogenous 5-HT release on the oscillations.
Dependence of the membrane potential oscillations on
NMDA concentration. In most cells of the lamprey spinal cord,
the threshold concentration of NMDA to induce membrane
potential oscillations is approximately 50 –100 ␮M (Wallén
and Grillner 1987). Figure 1A illustrates one neuron where 100
␮M NMDA induced rhythmic plateau oscillations at a rate of
⬃0.2 Hz (Fig. 1A1). Increasing the NMDA concentration to
150 ␮M (Fig. 1A2) caused an increase in frequency to ⬃0.25
Hz, whereas a higher concentration did not result in any
obvious, further increase in the rate of oscillations (Fig. 1, A3
and A4). In general, there was a tendency to reach “saturation”
⬃150 ␮M, with no further increase in the rate of oscillations
with higher levels of NMDA (Fig. 1B). During NMDA-induced fictive locomotor activity in the spinal network, a corresponding relation is seen (Brodin et al. 1985).
The influence of varying the NMDA concentration on different parameters characterizing the oscillatory behavior was
analyzed further in 10 spinal cord neurons (of which three were
identified as motoneurons), with five cells showing plateau-like
oscillations (Fig. 1C) and the other five cells showing spikelike oscillations (Fig. 2B). For plateau-like oscillations, the
cycle duration (see inset in Fig. 1C1 depicting measured
parameters) was reduced significantly, with higher NMDA
concentration in three of the five cells (Fig. 1C1), whereas one
cell (cell 4) showed a change in the opposite direction. The
ENDOGENOUS 5-HT RELEASE AND NMDA OSCILLATIONS IN LAMPREY
-40
B
2 150µM NMDA
1 100µM NMDA
0.50
oscillation frequency (Hz)
A
sponding to a range between 2% and 10% of the cycle (Fig.
2B4). Higher NMDA concentrations caused a significant increase of the normalized half-width in all five tested cells with
spike-like oscillations (Fig. 2B4). Even so, the half-width
values were typically ⬍10% of the cycle even with higher
levels of NMDA.
The NMDA-induced oscillations are strongly dependent on
the membrane potential level (Wallén and Grillner 1985,
1987). We therefore also analyzed the effects of varying the
NMDA concentration at two different holding potential levels
(n ⫽ 5; Fig. 3). Figure 3A illustrates spike-like oscillations
recorded at a holding potential of ⫺70 mV (Fig. 3, A1 and A2)
and at ⫺75 mV (Fig. 3, A3 and A4) and with two different
levels of NMDA. Increasing the concentration of NMDA
increased the oscillation frequency at both holding potential
levels, but the effect was more prominent at the ⫺70-mV level,
thus giving a steeper relation between NMDA concentration
and oscillation frequency (Fig. 3B). Corresponding results
were also found in four neurons displaying plateau-like oscillations (not illustrated).
The effects on the structure of the oscillation cycle of the cell
in Fig. 3, A and B, are illustrated in Fig. 3C. Corresponding to
the graph in Fig. 3B, the cycle duration (Fig. 3C1) was
significantly longer at the more hyperpolarized level with the
higher NMDA concentration (150 ␮M), whereas with 100 ␮M
NMDA, there was no difference. The oscillation amplitude
(Fig. 3C2) was significantly larger at ⫺75 mV and with both
NMDA concentrations. The half-width (Fig. 3C3) did not
mV
-80 Holding pot.-70mV
10s
Holding pot.-70mV
4 150µM NMDA
3 100µM NMDA
-70mV
-75mV
0.25
0.00
100
150
NMDA concentration (µM)
Holding pot.-75mV
100µM
150µM
1
2 40
35
+++
6
4
amplitude (mV)
cycle duration (s)
8
+++
++
0.3
30
25
20
15
10
2
0.2
0.1
5
0
0
-70mV
-75mV
0
-70mV
-75mV
+++
4
3
half-width (s)
C
half-width/cycle duration
Holding pot.-75mV
0.10
0.06
0.02
0
-70mV
-75mV
-70mV
-75mV
Fig. 3. NMDA oscillations depend on both NMDA concentration and membrane potential. A: at both holding potentials (Holding pot.), ⫺70 mV (A1 and A2)
and ⫺75 mV (A3 and A4), oscillation frequency increased with NMDA concentration. At the higher NMDA level (150 ␮M), depolarization of the holding
potential also caused a marked frequency increase (A2 and A4). Time and voltage calibrations in A1 apply to all records. B: relation between NMDA concentration
and oscillation frequency in the same neuron as in A. Each point represents the frequency from 10 cycles. At the more depolarized holding potential level, the
relation is steeper. C: cycle structure analysis of oscillations in the same neuron as in A (cf. Fig. 1C1, inset). Average values from 20 cycles of oscillation. Cycle
duration (C1) was increased significantly at the more hyperpolarized level and with the higher NMDA concentration, whereas the amplitude (C2) increased
significantly upon hyperpolarization with both high and low NMDA concentration. Half-width (C3 and C4) was decreased significantly at the more
hyperpolarized level with 150 ␮M NMDA but only when expressed as normalized values (C4). Student’s t-test, means ⫾ SD; comparison of 100 ␮M group vs.
150 ␮M group: *P ⬍ 0.05, ***P ⬍ 0.001; comparison of ⫺70 mV vs. ⫺75 mV group: ⫹⫹P ⬍ 0.01, ⫹⫹⫹P ⬍ 0.001.
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trough-to-peak amplitude, ranging between 13 and 28 mV,
showed a variable dependence on NMDA concentration among
the cells (Fig. 1C2). The width of the depolarized plateau at
half-maximal amplitude (half-width; see inset in Fig. 1C1) was
typically ⬃1 s or longer and showed an increase with higher
NMDA levels in four cells and a decrease in one neuron (Fig.
1C3). Normalized to the cycle duration, the half-width ranged
between ⬃13% and 57% of the cycle duration (Fig. 1C4).
NMDA-induced oscillations characterized by shorter depolarization peaks, without a prolonged plateau, are illustrated in
Fig. 2. The spike-like oscillations also increased in frequency,
with higher concentrations of NMDA (Fig. 2A). As with
plateau-like oscillations, a saturation level was generally seen
for spike-like oscillations in the relationship between NMDA
concentration and rhythm frequency. In general, the cycle
durations as well as the amplitudes were within the same range
for cells with plateau- and spike-like oscillations. The effects
of varying the NMDA concentration on different oscillatory
parameters were also similar in cells displaying spike-like
oscillations (Fig. 2B) compared with those described above for
plateau-like oscillations. In all of the five cells analyzed, cycle
duration became significantly shorter with higher concentrations of NMDA (Fig. 2B1). Four of the five cells showed a
significant increase in oscillation amplitude with higher
NMDA levels (Fig. 2B2). The brief depolarization peaks of
spike-like oscillations consequently occupied a small proportion of the cycle, with the half-width varying between 0.2 and
0.4 s at the lowest NMDA concentration (Fig. 2B3), corre-
33
ENDOGENOUS 5-HT RELEASE AND NMDA OSCILLATIONS IN LAMPREY
A
1
2
Control (150µM NMDA)
B
1
10µM spiperone 24min
2
2.5
-35
half-width/cycle duration
34
-70
3
10µM spiperone 40min
5s
4
10µM spiperone + 1µM 5-HT 12min
half-width (s)
2
mV
1.5
1
0.5
0
C
2
Control (150µM NMDA)
half-width/cycle duration
5s
4
10µM NAD + 1µM 5-HT 41min
half-width (s)
10µM NAD + 1µM 5-HT 27min
Control spip
spip
+ 5-HT
0.3
mV
3
0.1
2
1.2
-65
0.2
00
spip
+ 5-HT
D
1
10µM NAD 22min
0.3
0.8
0.4
0
Control NAD NAD
+ 5-HT
0.2
0.1
0
Control NAD NAD
+ 5-HT
Fig. 4. Plateau-like oscillations are converted to the spike-like form after application of 5-HT antagonists. A and B: effects of spiperone. A1: control; plateau-like
membrane potential oscillations induced by bath application of 150 ␮M NMDA in the presence of TTX. A2 and A3: after the application of spiperone (10 ␮M),
the depolarized plateaus were gradually shortened to finally become spike like (A3). A4: the spike-like oscillations were converted back to the plateau-like form
after application of 5-HT (1 ␮M). B: effects on the half-width of the depolarized phase of the cell in A. Spiperone (spip) significantly reduced the half-width
duration (B1: from 1.86 s to 0.07 s; B2: normalized values, from 0.40 to 0.02), whereas the addition of 5-HT partly counteracted the effect. Average values from
10 to 22 cycles. Student’s t-test, means ⫾ SD, comparisons of nearby groups. C and D: effects of the 5-HT1A receptor antagonist NAD 299. C1: control;
plateau-like membrane potential oscillations induced by bath application of 150 ␮M NMDA in the presence of TTX. C2: after application of NAD 299 (10 ␮M),
the plateau-like oscillations were modified to become spike like, and upon addition of 5-HT (1 ␮M; C3 and C4), they were converted back to the plateau-like
form. D: effects on the half-width of the depolarized phase of the cell in C. NAD 299 significantly reduced the half-width duration (D1: from 0.95 s to 0.48 s;
D2: normalized values, from 0.22 to 0.14), whereas the addition of 5-HT counteracted the effect. Average values from 17 to 24 cycles. Student’s t-test, means ⫾ SD,
comparisons of nearby groups. Traces in A and C, respectively, are from continuous recordings in discontinuous current clamp mode and with the trough
membrane potential held at ⫺70 mV (A) and ⫺66 mV (C) by DC-current injection. Time and voltage calibrations in A1 apply to all traces in A, and calibrations
in C1 apply to all traces in C.
change with the holding potential, except when expressed as
normalized half-width and with the higher NMDA concentration (Fig. 3C4).
A general conclusion from these findings is thus that for both
types of membrane potential oscillations, higher levels of
NMDA receptor activation will result in an increased oscillation frequency until a saturation level is reached. Also, in most
cases, there is an increase in oscillation amplitude and in
half-width duration with higher NMDA concentrations.
Serotonergic modulation of membrane potential oscillations. It
is thus clear that the NMDA-induced membrane potential oscillations may vary between different spinal cord preparations. In
some, the oscillations are plateau like, whereas in other preparations, there are instead spike-like oscillations, lasting approximately 200 –300 ms without any plateau phase. One may
then ask whether this difference reflects different states of the
preparations and if the two forms of oscillations may occur in
the same cell.
Differences in the functional state may be caused by influence from various modulatory systems. The duration of the
depolarized plateau is prolonged by a partial blockage of KCa
channels by apamin, and these channels are also depressed by
5-HT (El Manira et al. 1994; Wallén et al. 1989; Wallén and
Grillner 1987). The possibility that the oscillations may be
subject to an endogenous modulation by the spinal 5-HT
system was first investigated by bath application of spiperone,
a 5-HT antagonist (Hoyer et al. 1994; Metwally et al. 1998;
Zhang and Grillner 2000). Spiperone was previously shown to
increase the frequency of fictive locomotion induced by
NMDA, as well as the variability of the rhythmic activity
(Zhang and Grillner 2000), indicating an endogenous release of
5-HT during fictive locomotion.
When spiperone was applied during plateau-like oscillations
(Fig. 4, A and B), the plateau phase became progressively
shorter (Fig. 4A2), and eventually, the oscillations were converted to the spike-like form, with no plateau phase (Fig. 4A3;
n ⫽ 6). When 5-HT was then applied, the oscillations partly
reverted back to the plateau-like form (Fig. 4A4). Spiperone
thus caused a marked and significant reduction in the halfwidth of the depolarized phase (Fig. 4B), seen in all six cells
tested. Due to the possibility that spiperone may, in addition,
act as a dopamine receptor antagonist (Metwally et al. 1998;
Seeman and Van Tol 1994), we also tested the effect of the
specific 5-HT1A antagonist NAD 299 (Johansson et al. 1997)
(Fig. 4, C and D; n ⫽ 6), which like spiperone, altered the
plateau-like oscillations to become more spike like (Fig. 4C2),
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1
-45
Control spip
0.4
ENDOGENOUS 5-HT RELEASE AND NMDA OSCILLATIONS IN LAMPREY
C
A
1
-50
Control (150µM NMDA)
-55
mV
1
Control (150µM NMDA)
mV
-70
5s
-75
5s
2
1µM 5-HT 5min
2
1µM 5-HT 9min
3
1µM 5-HT + 10µM spiperone 5min
3
1µM 5-HT + 20µM NAD 19min
D
1
0.8
0.4
0
0.2
0.1
1.2
0.8
0.4
0
Control 5-HT 5-HT
+ spip
2
2
1.6
half-width (s)
half-width/cycle duration
2 0.3
half-width/cycle duration
B
1 1.2
half-width (s)
plateau-like oscillations, an effect that was markedly counteracted by subsequent application of either of the two antagonists
(Fig. 5, B and D). Thus 5-HT will act to promote a transition
from spike-like to plateau-like oscillations, whereas blocking
the influence of 5-HT will convert the oscillations back to the
spike-like form. An altered level of 5-HT tone in the spinal
cord may thus modulate the oscillatory activity and cause a
switch from one form of oscillation to the other.
5-HT indirectly blocks KCa channels through an action on
voltage-dependent calcium channels (Hill et al. 2003). Moreover, 5-HT acts on low-threshold L-type calcium channels
(CaV 1.3) to modulate the postinhibitory rebound (PIR) response in lamprey spinal neurons (Wang et al. 2011). We have
reported recently that during NMDA-induced membrane potential oscillations, low voltage-activated (LVA) calcium channels of the CaV 1.3 subtype will contribute (Wang et al. 2013).
Control 5-HT
5-HT
+ spip
0
Control 5-HT 5-HT
+ NAD
0.4
0.3
0.2
0.1
0
Control 5-HT 5-HT
+ NAD
Fig. 5. Spike-like oscillations are converted to the plateau-like form after application of 5-HT. A: 5-HT application, followed by addition of spiperone. A1: control;
spike-like membrane potential oscillations induced by bath application of 150 ␮M NMDA in the presence of TTX. A2: after the application of 5-HT (1 ␮M),
the oscillation peaks were prolonged to become plateau like. A3: the plateau-like oscillations were converted back to the spike-like form after addition of
spiperone (10 ␮M). B: effects on the half-width of the depolarized phase of the cell in A. 5-HT significantly increased the half-width duration (B1: from 0.21 s
to 0.94 s; B2: normalized values, from 0.09 to 0.24), whereas the addition of spiperone abolished the effect. Average values from 7 to 10 cycles. Student’s t-test,
means ⫾ SD, comparisons of nearby groups. C: 5-HT application, followed by addition of NAD 299. C1: control; spike-like membrane potential oscillations
induced by bath application of 150 ␮M NMDA in the presence of TTX. C2: after the application of 5-HT (1 ␮M), oscillations were converted to become plateau
like. C3: the plateau-like oscillations were converted back to the spike-like form after addition of NAD 299 (20 ␮M). D: effects on the half-width of the
depolarized phase of the cell in C. 5-HT significantly increased the half-width duration (D1: from 0.25 s to 1.36 s; D2: normalized values, from 0.07 to 0.32),
whereas the addition of NAD 299 counteracted the effect. Average values from 6 to 19 cycles. Student’s t-test, means ⫾ SD, comparisons of nearby groups.
Traces in A and C, respectively, are from continuous recordings in discontinuous current clamp mode and with the trough membrane potential held at ⫺70 mV
(A) and ⫺74 mV (C) by DC-current injection. Time and voltage calibrations in A1 apply to all traces in A, and calibrations in C1 apply to all traces in C.
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and after subsequent application of 5-HT, the plateau phase
was re-established (Fig. 4, C3 and C4). Like spiperone, NAD
299 caused a significant reduction in the half-width (Fig. 4D).
Thus by blocking 5-HT receptors, the plateau-like oscillations
are converted to the spike-like form, suggesting that 5-HT is
endogenously released and causes a prolongation of the plateau
phase. This modulatory action of 5-HT may primarily be
mediated via 5-HT1A receptors.
The converse experiment is illustrated in Fig. 5, where the
control oscillations are of the spike-like form (Fig. 5, A1 and
C1). After application of 5-HT, the oscillations were converted
to the plateau-like form (Fig. 5, A2 and C2; n ⫽ 6). When
spiperone was also applied, the oscillations again became spike
like (Fig. 5A3), as was the case upon application of NAD 299
(Fig. 5C3). Application of 5-HT thus caused a significant
prolongation of the half-width of the depolarized phase during
35
36
ENDOGENOUS 5-HT RELEASE AND NMDA OSCILLATIONS IN LAMPREY
We therefore tested whether the modulatory influence of 5-HT
on the oscillations is exerted solely via an action on L-type
calcium channels or if other routes of action may also contribute. Application of the L-type calcium channel antagonist
nimodipine prolongs the plateau phase of the oscillations, as
does 5-HT (Fig. 6, A and B) (Wang et al. 2013). In the presence
of nimodipine, 5-HT was, however, still able to prolong the
plateaus further (Fig. 6C; n ⫽ 3), suggesting that the action of
5-HT on NMDA-induced membrane potential oscillations is
not only exerted via a blockade of L-type calcium channels
(CaV 1.3).
DISCUSSION
A
Control (150µM NMDA)
-40
mV
-60
5s
B
10µM Nimodipine 17 min
C
10µM Nimodipine + 1µM 5-HT 12 min
Fig. 6. Blockade of L-type calcium channels does not occlude the effect of
5-HT application. A: control; spike-like membrane potential oscillations induced by bath application of 150 ␮M NMDA in the presence of TTX.
B: following application of the L-type calcium channel blocker nimodipine,
oscillations became more plateau like (cf. Wang et al. 2013). C: in the presence
of nimodipine, 5-HT application further prolonged the oscillation plateaus.
Time and voltage calibrations in A apply to all traces.
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NMDA can induce pacemaker-like oscillations in different
neurons in the lamprey spinal cord and in other species (Hochman et al. 1994; Li et al. 2010; Masino et al. 2012; Sigvardt et
al. 1985; Wallén and Grillner 1987) after a blockade of interaction between the cells with tetrodotoxin. The generation of
these plateaus depends on an interaction between a number of
ion channels. The voltage dependence of the NMDA receptors
is central to the oscillations, but the depolarization is counteracted by voltage-dependent potassium channels, and LVA
calcium channels also contribute to the depolarization (Grillner
and Wallén 1985; Wang et al. 2013). The termination of the
plateau phase is due to activation of KCa channels and a closure
of the voltage-dependent NMDA receptors (El Manira et al.
1994; Wallén and Grillner 1987).
It has long been noted that these oscillations in the lamprey
can either have a pronounced, depolarized plateau phase or
show a continuous decrease of the amplitude after the peak
without a significant plateau. Different spinal cord preparations
have been found predominantly to express either of these two
types of pacemaker-like oscillations. We have here first characterized the properties of these two versions. Both types of
oscillations show a similar dependence on the level of NMDA
receptor activation, indicating that the same ion channel mechanisms underlie both types [cf. Wallén and Grillner (1987);
Wang et al. (2013)]. On the basis of these findings, we then set
out to investigate whether differences in modulatory, serotonergic influence may explain the presence of the two types of
oscillations in different preparations. We now show that this
appears to be the case and that the spinal 5-HT system indeed
may contribute to the shape of the oscillations. A related
finding is that Zhang and Grillner (2000) have shown that
during fictive locomotion, a blockade of 5-HT receptors led to
marked acceleration of the frequency of locomotor activity,
suggesting that there was a tonic release of 5-HT affecting the
locomotor frequency. This is most likely caused by the intraspinal 5-HT neurons located below the central canal (Schotland et al. 1995; van Dongen et al. 1985; Zhang et al. 1996).
The plateau-like oscillations induced by NMDA in the spinal
cord preparation were transferred to the spike-like form of
oscillations after a blockade of 5-HT receptors with spiperone
[cf. Zhang and Grillner (2000)] and also with NAD 299, a
specific 5-HT1A antagonist. This suggests that there is an
endogenous release of 5-HT also during the oscillations. Furthermore, since this release is maintained in the presence of
TTX, it may occur spontaneously in the absence of spikes. The
release of 5-HT causes a depression of KCa channel activation
(El Manira et al. 1994; Wallén et al. 1989) and thereby a
prolongation of the plateau, and consequently, a blockade of
5-HT receptors will enhance the level of KCa channel activity,
which will terminate the plateau-like oscillations earlier and
make them spike like. Conversely, an enhanced level of 5-HT,
as during external application in the present experiments, will
depress KCa channel activation and thereby transform the
spike-like oscillations to the more plateau-like version. Application of 5-HT is also known to slow down the locomotor
frequency (Harris-Warrick and Cohen 1985). A modulation of
TTX-resistant NMDA oscillations by 5-HT occurs in several
species, including rodents (Hsiao et al. 2002; MacLean et al.
1998; MacLean and Schmidt 2001; Masino et al. 2012;
Schmidt et al. 1998), amphibians (Reith and Sillar 1998;
Scrymgeour-Wedderburn et al. 1997; Sillar and Simmers
1994a; Sillar et al. 1992), and lamprey [present study and El
Manira et al. (1994); Wallén et al. (1989)].
5-HT will reduce KCa channel activation through an action on
voltage-dependent calcium channels (Hill et al. 2003). During
NMDA-induced membrane potential oscillations, which range
between approximately ⫺70 and ⫺40 mV, LVA calcium channels of the CaV 1.3 subtype contribute to KCa channel activation, in addition to calcium entry via other routes, notably
NMDA receptor channels (Alpert and Alford 2013; Nanou et
al. 2013; Wang et al. 2013). We have furthermore shown that
this subtype of LVA calcium channel also contributes to the
PIR response in lamprey spinal neurons and that 5-HT acts on
the CaV 1.3 subtype to modulate the PIR (Wang et al. 2011). In
the case of the 5-HT modulation of the NMDA-induced membrane potential oscillations studied here, the action appears not
to be mediated only via CaV 1.3 subtype calcium channels,
ENDOGENOUS 5-HT RELEASE AND NMDA OSCILLATIONS IN LAMPREY
ACKNOWLEDGMENTS
We are grateful to Dr. Brita Robertson for valuable comments on the
manuscript.
GRANTS
Support for this study was provided by the Swedish Research Council
(Medicine, project no. 3026; Natural and Engineering Sciences, project no.
1496), the European Union (project nos. QLG3-CT-2001-01241 and HealthF2-2007-201144, FP7), and the Karolinska Institute Foundations. Support for
the study was also provided by a grant from the National Natural Science
Foundation of China (grant no. 31371198) and the Natural Science Foundation
of Liaoning Province, People’s Republic of China (grant no. 201202144), and
by grants from the Scientific Research Foundation for the Returned Overseas
Chinese Scholars by the State Education Ministry and the Ministry of Human
Resources and Social Security of the People’s Republic of China (grant no.
2011LX007), received by D. Wang.
DISCLOSURES
No conflicts of interest, financial or otherwise, are declared by the authors.
AUTHOR CONTRIBUTIONS
Author contributions: S.G. conception and design of research; D.W. performed experiments; D.W. and P.W. analyzed data; D.W., S.G., and P.W.
interpreted results of experiments; D.W. and P.W. prepared figures; D.W.,
S.G., and P.W. drafted manuscript; S.G. and P.W. edited and revised manuscript; D.W., S.G., and P.W. approved final version of manuscript.
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