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The Journal of Neuroscience, March 1, 1999, 19(5):1647-1656
Activity-Dependent Metaplasticity of Inhibitory and Excitatory
Synaptic Transmission in the Lamprey Spinal Cord Locomotor
Network
David
Parker and
Sten
Grillner
Nobel Institute for Neurophysiology, Department of Neuroscience,
Karolinska Institute, S-17177, Stockholm, Sweden
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ABSTRACT |
Paired intracellular recordings have been used to examine the
activity-dependent plasticity and neuromodulator-induced metaplasticity of synaptic inputs from identified inhibitory and excitatory
interneurons in the lamprey spinal cord. Trains of spikes at 5-20 Hz
were used to mimic the frequency of spiking that occurs in network
interneurons during NMDA or brainstem-evoked locomotor activity. Inputs
from inhibitory and excitatory interneurons exhibited similar
activity-dependent changes, with synaptic depression developing during
the spike train. The level of depression reached was greater with lower stimulation frequencies. Significant activity-dependent depression of
inputs from excitatory interneurons and inhibitory crossed caudal
interneurons, which are central elements in the patterning of network
activity, usually developed between the fifth and tenth spikes in the
train. Because these interneurons typically fire bursts of up to five
spikes during locomotor activity, this activity-dependent plasticity
will presumably not contribute to the patterning of network activity.
However, in the presence of the neuromodulators substance P and 5-HT,
significant activity-dependent metaplasticity of these inputs developed
over the first five spikes in the train. Substance P induced
significant activity-dependent depression of inhibitory but
potentiation of excitatory interneuron inputs, whereas 5-HT induced
significant activity-dependent potentiation of both inhibitory and
excitatory interneuron inputs. Because these metaplastic effects are
consistent with the substance P and 5-HT-induced modulation of the
network output, activity-dependent metaplasticity could be a potential
mechanism underlying the coordination and modulation of rhythmic
network activity.
Key words:
synaptic plasticity; metaplasticity; spinal cord; lamprey; neuropeptide; substance P; 5-HT
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INTRODUCTION |
Fast synaptic transmission plays a
major role in patterning the activity of neural networks underlying
motor and cognitive tasks (Singer, 1993 ; Calabrese, 1995). Although
short and long-term activity-dependent plasticity of synaptic
transmission has been analyzed extensively in relation to learning and
memory (Milner et al., 1998), little attention has focused on its role
in patterning the output of rhythmic neural networks.
Locomotion in vertebrates is generated by rhythmically active spinal
cord networks (see Kiehn et al., 1997). In the lamprey, the types and
properties of neurons in the locomotor network are known in some detail
(Buchanan and Grillner, 1987). As in other vertebrates (see Kiehn et
al., 1997), symmetrical networks on each side of the lamprey spinal
cord are coupled through glycinergic reciprocal inhibitory connections
(Buchanan, 1982). The strength of this reciprocal inhibitory input is
an important factor in determining the frequency of network activity
(Grillner and Wallén, 1980; Hellgren et al., 1992). An increase
in reciprocal inhibition slows the frequency of network activity by
delaying the activation of the contralateral network, whereas a
reduction increases the frequency of network activity by allowing the
contralateral network to become active sooner (Hellgren et al., 1992).
Excitatory drive at the segmental level is mediated by glutamatergic
interneurons (Buchanan and Grillner, 1987). A general increase in
excitatory drive to the network, either experimentally or in computer
simulations, results in an increase in the frequency of network
activity (Brodin et al., 1985; Hellgren et al., 1992).
Here, the activity-dependent synaptic plasticity of inputs from four
types of identified network interneurons in the lamprey have been
examined: glycinergic inhibitory and glutamatergic excitatory crossed
caudal interneurons (CCINs; Buchanan, 1982), glycinergic lateral
interneurons (LINs; Buchanan, 1982), and glutamatergic excitatory
interneurons (EINs; Buchanan and Grillner, 1987). Inhibitory CCINs and
EINs mediate reciprocal inhibition and excitatory drive, respectively
(Buchanan, 1982; Buchanan and Grillner, 1987), and are thus important
elements in the regulation of network activity. Because the roles of
these interneurons are known, the activity-dependent plasticity of
their inputs can be directly related to effects on the coordination of
network activity.
Activity-dependent plasticity is also plastic, an effect termed
"metaplasticity" (Abraham and Bear, 1996). Although this term was
introduced to describe the effects of previous activity on the
induction or expression of synaptic plasticity, metaplastic effects can
also occur in the presence of neuromodulators (Sombati and Hoyle, 1984;
Fisher et al., 1997). To determine whether network modulation can be
associated with the metaplasticity of interneuron inputs, the effects
of the neuromodulators substance P and 5-HT, which have been studied in
detail on the locomotor network (Harris-Warrick and Cohen, 1985;
Wallén et al., 1989; Parker and Grillner, 1998; Parker et al.,
1998), were examined. The results of this study suggest little
contribution of activity-dependent plasticity to the patterning of
network activity under control conditions, but that substance P and
5-HT induced activity-dependent metaplasticity could contribute to the
modulation of network activity.
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MATERIALS AND METHODS |
Adult male and female lampreys (Lampetra fluviatilis)
were used in all experiments. Animals were anesthetized with tricaine methanesulphonate (MS-222; Sandoz, Basel, Switzerland), and the spinal
cord and notochord were removed. The spinal cord was isolated and
placed ventral side up in a Sylgard-lined (Sikema, Stockholm, Sweden)
chamber and superfused with Ringer's solution containing (in
mM): 138 NaCl, 2.1 KCl, 1.8 CaCl2, 1.2 MgCl2, 4 glucose, 2 HEPES, and 0.5 L-glutamine, which was bubbled with O2. The
chamber was kept at a temperature of 8-12°C.
Intracellular recordings were made from the cell bodies of spinal cord
neurons using thin-walled micropipettes filled with 3 M
potassium acetate and 0.1 M potassium chloride. An Axoclamp 2A amplifier (Axon Instruments, Foster City, CA) was used for voltage
recording and current injection. Data were acquired and stored on a
computer using an analog-to-digital interface (Digidata 1200; Axon
Instruments) and Axon Instruments software (pClamp 6). Neurons were
identified according to previously established criteria (Buchanan,
1993). Except where stated, motor neurons were used as the postsynaptic
target cells. These were identified by recording orthodromic spikes in
the adjacent ventral root after suprathreshold current injection into
their somata. Inhibitory or excitatory CCINs were identified by their
ability to elicit monosynaptic IPSPs or EPSPs, respectively, in
contralateral motor neurons, and by recording orthodromic spikes on the
contralateral caudal region of the spinal cord, at least ten segments
from the presumed CCIN. EINs were identified by their ability to elicit monosynaptic EPSPs in ipsilateral motor neurons. Lateral interneurons were identified by their shape and position in the spinal cord and by
recording orthodromic spikes on the ipsilateral caudal region of the
spinal cord at least ten segments from the presumed LIN. CCINs, LINs,
and EINs were recorded in either the same segment or at most one
segment rostral to the postsynaptic neuron. Monosynaptic EPSPs and
IPSPs were identified by their ability to follow reliably and with
constant latency after presynaptic stimulation at 20 Hz.
During NMDA and brainstem stimulation-evoked locomotor activity,
network interneurons fire between two to five spikes at frequencies of
5-30 Hz, depending on the burst duration (Buchanan and Cohen, 1982;
Buchanan and Kasicki, 1995), although spiking for up to 1 sec can occur
during slower network activity (D. Parker, unpublished data). In
this study, intracellular stimulation of the presynaptic neuron with
trains of twenty spikes at frequencies of 5-20 Hz was used to
approximate physiological activity. The highest frequency used was 20 Hz, because at this frequency the PSPs had largely decayed
before the onset of the next PSP in the train, thus facilitating the
measurement of the amplitude of individual PSPs. Spike trains were
evoked at 1 min intervals. Recovery of the activity-dependent changes
was examined by triggering presynaptic spikes at 1 Hz for 1-4 sec
after the end of the spike train. When excitatory inputs were examined,
5 µM strychnine was added to the Ringer's solution to
block glycinergic IPSPs. When examining IPSPs, CNQX (10 µM) and APV (100 µM) were used to block
glutamatergic inputs. Only postsynaptic neurons with a low level of
spontaneous synaptic inputs were used, to minimize the occurrence of
spontaneous inputs during the spike train. Substance P can increase
synaptic inputs and induce membrane potential oscillations in motor
neurons and network interneurons (Parker and Grillner, 1998). Because
the synaptic input and oscillations are cyclical, with a period of several minutes, trials were only performed when synaptic inputs to the
cell were low. In all cases, the membrane potential in control and in
the presence of substance P or 5-HT was kept constant by injecting
depolarizing or hyperpolarizing current using single-electrode current
clamp. The output of the sample and hold amplifier was monitored
continuously to ensure complete voltage settling.
Drugs were applied to the bath using a peristaltic pump. A 1 µM concentration of substance P was used in all
experiments, because this concentration has been shown to have
significant long-lasting effects on the locomotor network (Parker and
Grillner, 1998; Parker et al., 1998). A 10 µM
concentration of 5-HT was used, because this concentration has been
shown to modulate sensory and reticulospinal synaptic transmission
(Buchanan and Grillner, 1991; El Manira et al., 1997). Substance P and
5-HT were applied for 10 min. Two or three trials were performed at
each frequency in control and in the presence of the modulator.
n numbers in the text refer to the number of pairs examined.
The different trials for each pair were averaged. No more than two
pairs were examined in a single piece of spinal cord. When modulators
were applied, only one pair was examined in each piece of cord. Values in the text refer to the EPSP or IPSP amplitude as percent of control.
Unless stated otherwise, statistical significance of effects seen with
individual stimulation frequencies, either in control or in the
presence of neuromodulators, was examined using a two-tailed paired
t test. A one-way ANOVA was used to examine the significance
of effects between different stimulation frequencies, and a Tukey test
was used for post hoc comparisons of group means.
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RESULTS |
Activity-dependent plasticity of interneuron inputs
During NMDA and brainstem stimulation-evoked locomotor activity,
network interneurons fire between two to five spikes at frequencies of
5-30 Hz, depending on the burst duration (Buchanan and Cohen, 1982;
Buchanan and Kasicki, 1995). A train of twenty stimuli applied to
inhibitory CCINs at frequencies of 5-20 Hz resulted in
frequency-dependent effects on the amplitude of the monosynaptic IPSP
in contralateral motor neurons (Fig. 1).
At 5 Hz, depression developed gradually over successive IPSPs, reaching
a significant plateau level of depression (i.e., one at which no
further depression occurred through the remainder of the stimulation
train) by the tenth spike in the train (59 ± 8%;
p < 0.05; n = 10; Fig.
1A). At 10 Hz, the depression did not begin to
develop until after the first five spikes, but it again reached a
significant plateau level of depression by the tenth spike in the train
(67 ± 6%; p < 0.05; n = 10;
Fig. 1B). At 20 Hz, the IPSP was initially
nonsignificantly facilitated over the first five spikes in the train
(106 ± 11%; p > 0.05), a nonsignificant plateau
level of depression to ~80% of control developing between the fifth
and tenth spikes in the train (81 ± 20%; p > 0.1; n = 10; Fig. 1C,D).
Although the magnitude of the synaptic depression was reduced with an
increase in stimulus frequency, there was no significant difference
between the plateau level reached at 5 and 10 Hz
(p > 0.05; one-way ANOVA). The plateau level
reached at 20 Hz was significantly different, however, to that at 5 and
10 Hz (p < 0.001, one-way ANOVA). Recovery of
the depression had usually occurred within 1-2 sec of the end of the stimulus train (Fig. 1D). This pattern of
activity-dependent plasticity occurred consistently in all of the
CCIN-motor neuron (MN) pairs examined (n = 10). The
activity-dependent changes of the IPSP amplitude were not associated
with a change in the CCIN spike amplitude or duration over the course
of the spike train (Fig. 1D), because there was no
significant correlation between the amplitude of the IPSP and the spike
width (r2 = 0.087) or amplitude
(r2 = 0.056) measured at the soma, (data
not shown).
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Figure 1.
Activity-dependent plasticity of inhibitory CCIN
inputs. Effects of 5 (A), 10 (B), and 20 (C) Hz
stimulation of the presynaptic CCIN on the amplitude of the IPSP
recorded in a contralateral motor neuron. In this and subsequent
figures, the inset shows a schematic diagram of one
segment of the lamprey spinal cord. Filled
circles indicate glycinergic connections, bars
indicate glutamatergic connections. Inhibitory CCINs inhibit all
contralateral neurons. The neurons that are studied on each figure are
highlighted. In all figures, IPSP or EPSP values are normalized to the
amplitude of the initial PSP in the train. Dashed lines
on the graphs indicate the level of the initial IPSP, and, where
appropriate, the plateau level of depression reached. D,
Traces from a motor neuron and a CCIN showing the effects of 20 Hz
stimulation on the amplitude of the monosynaptic IPSP. The
dashed line indicates a delay of 1 sec before a test
spike was given to measure the extent of the recovery from depression.
At the end of each trace, the 1st and 20th spike in the train and the
1st, 5th, and 20th IPSP are overlaid on an expanded time scale. Note
that there is no activity-dependent effect on the CCIN spike. Summed
data from 10 inhibitory CCIN-MN pairs is shown in
A-C. Calibration: 0.5 mV (for the
synaptic input), 30 mV (spikes), 200 msec; 0.5 mV, 20 msec (for the
expanded traces).
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Synaptic inputs from glutamatergic EINs exhibited a similar pattern of
activity-dependent plasticity as CCINs. At 5 Hz, the depression again
developed gradually during the spike train to reach a final level of
56 ± 15% of control (p < 0.05), although no clear plateau level was reached. At 10 Hz, a plateau level of
depression was reached between the fifth and tenth spikes in the train
(57 ± 9%; p < 0.05). At 20 Hz, the monosynaptic
EPSP reached a plateau level of depression between the fifth and tenth spikes in the train (77 ± 20%; p > 0.05;
n = 11; Fig. 2). The depression that developed was thus again less at higher stimulus frequencies. As with CCIN inputs, there was no significant difference between the level of depression reached at 5 and 10 Hz
(p > 0.05; one-way ANOVA), but the plateau
reached at 20 Hz was significantly different to the depression at 5 and
10 Hz (p < 0.001; one-way ANOVA). Recovery of
the depression again usually occurred within 1-2 sec of the end of the
stimulus train (Fig. 2D). These effects also occurred
consistently in the different EIN-MN pairs examined (n = 10 of 11; Fig. 2A-C), although in one
pair a significant plateau level of depression of the EPSP occurred
after the first two spikes in the train at 20 Hz (data not shown). In
addition, in some pairs nonsignificant facilitation
(p > 0.05) could develop during the first five
spikes in the train, particularly at higher frequencies (5 Hz,
n = 3; 10 Hz, n = 2; 20 Hz,
n = 9; Fig. 2A-C). As
with inhibitory CCINs, no effect on the presynaptic spike was seen over
the course of the spike train (Fig. 2D), there again being no correlation between the EPSP amplitude and the somatic spike
width (r2 = 0.091) or amplitude
(r2 = 0.117; data not shown).
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Figure 2.
Activity-dependent plasticity of EIN inputs.
Effects of 5 (A), 10 (B),
and 20 (C) Hz stimulation of the presynaptic EIN
on the amplitude of the monosynaptic EPSP in an ipsilateral motor
neuron (inset). D, Traces from a motor
neuron and an EIN showing the effects of 20 Hz stimulation on the
amplitude of the monosynaptic EPSP. The dashed line
again indicates a delay of 1 sec before a test spike was given to
measure the extent of the recovery from depression. Expanded traces of
the 1st and 20th spike in the train and the 1st, 5th, and 20th EPSP are
also shown. Note that there is again no activity-dependent effect on
the EIN spike. Summed data from 11 EIN-MN pairs is shown in
A-C. Calibration: 0.5 mV (for the
synaptic input), 30 mV (spikes), 200 msec; 0.5 mV, 20 msec (for the
expanded traces).
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In addition to examining inhibitory CCIN and EIN inputs, the
activity-dependent plasticity of two other connections, excitatory CCIN
inputs to contralateral motor neurons (Buchanan, 1982) and glycinergic
LIN inputs to ipsilateral CCINs (Buchanan, 1982) have also been
examined. Excitatory CCIN inputs exhibited a similar pattern of
activity-dependent plasticity as inhibitory CCINs (Fig. 3A), although the depression
that developed was greater than that seen with inhibitory CCIN inputs
(p < 0.001; one-way ANOVA; Figs. 1A-C, 3A). At 5 Hz, depression
developed gradually to reach a plateau level between the fifth and
tenth spikes in the train (34 ± 5%; p < 0.01;
n = 4). As with inhibitory CCIN and EIN inputs, at 10 Hz the level of depression reached was less than that at 5 Hz (37 ± 4%; p < 0.01); the depression also took longer to
reach a plateau level. At 20 Hz there was again a nonsignificant
facilitation over the first three spikes (104 ± 9%;
p > 0.05), which was followed by depression to 52 ± 7% of control (p < 0.01). The depression at
20 Hz did not reach a plateau level, it instead increased continuously during the spike train. There was again no significant difference between the plateau level reached at 5 and 10 Hz
(p > 0.05; one-way ANOVA), whereas the final
level at 20 Hz was significantly different to that at the other two
frequencies (p < 0.001; one-way ANOVA). With
LIN inputs (n = 4) at each frequency, a significant
plateau level of depression to 50-55% of control developed between
the fifth and tenth spikes in the train (p < 0.01; Fig. 3B). The plateau level of depression reached was
not significantly different for the different stimulation frequencies
(p > 0.05; one-way ANOVA), although the
depression developed faster at lower stimulation frequencies (Fig.
3B).
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Figure 3.
Activity-dependent plasticity of excitatory CCIN
and LIN inputs. A, Effects of 5, 10, and 20 Hz
stimulation of presynaptic excitatory CCINs on the amplitude of the
monosynaptic EPSP in a contralateral motor neuron
(inset). B, Effects of 5, 10, and 20 Hz
stimulation of the presynaptic LIN on the amplitude of the monosynaptic
IPSP in an ipsilateral CCIN (inset). Summed data from
four pairs is shown on each graph.
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Significant activity-dependent synaptic plasticity of inputs from
identified network interneurons thus develops during physiological spike trains. However, with the exception of excitatory CCIN and LIN
inputs at 5 and 10 Hz, and inhibitory CCIN inputs at 5 Hz, significant
activity-dependent plasticity only occurred in the latter part of the
spike train, typically between the fifth and tenth spikes. Because
network interneurons have been reported to fire up to five spikes
during locomotor activity (Buchanan and Cohen, 1982; Buchanan and
Kasicki, 1995), the activity-dependent plasticity that develops after
this time will presumably only be relevant when network interneurons
fire relatively long bursts of action potentials, and thus will
presumably not contribute significantly to the patterning of network
activity during the normal range of locomotor frequencies.
Effects of neuromodulators on the induction of
activity-dependent plasticity
Neuromodulators can profoundly alter the output of the lamprey
locomotor network (see Harris-Warrick and Cohen, 1985; Parker et al.,
1998). This network modulation is associated with changes in the
cellular and synaptic properties of network neurons (Wallén et
al., 1989; Parker and Grillner, 1998). Because synaptic plasticity is
itself plastic (metaplasticity; Abraham and Bear, 1996), the effects of two neuromodulators, 5-HT and substance P, which have been
studied in detail on the locomotor network (Harris-Warrick and Cohen,
1985; Wallén et al., 1989; Parker and Grillner, 1998; Parker et
al., 1998), were examined to determine whether modulation of the
network output could be associated with the metaplasticity of
interneuron inputs. Neuromodulators were studied on EIN and inhibitory
CCIN inputs because these interneurons are thought to be of primary
importance in the patterning of network activity (Hellgren et al.,
1992). The tachykinin substance P (1 µM; Parker and
Grillner, 1998; Parker et al., 1998) did not affect the amplitude of
the first CCIN-evoked IPSP in the train compared with control (Fig.
4Di), consistent with
its failure to modulate the amplitude of single, low frequency-evoked
inhibitory CCIN inputs (Parker and Grillner, 1998). Substance P also
did not significantly affect the depression of CCIN inputs that
developed during 5 Hz stimulation (p > 0.1;
n = 5; Fig. 4A). However, it
significantly enhanced the depression of the monosynaptic IPSP at 10 and 20 Hz (p < 0.05; n = 5;
Fig. 4B,C,E).
Substance P reduced the IPSP amplitude to 16 ± 9% of control
after the first three spikes in the train at 20 Hz (Fig.
4C,Di,E) and to 45 ± 8% at 10 Hz (Fig. 4B,E).
Although the depression was enhanced by substance P, the amplitude of
the IPSP still usually recovered within 1-2 sec of the end of the stimulation train (data not shown). As with substance P, 5-HT (10 µM) had no consistent or significant effect on the
amplitude of the initial CCIN-evoked IPSP in the train compared with
control (Fig. 4Dii). However, in contrast to
substance P, 5-HT facilitated the amplitude of CCIN inputs at 5, 10, and 20 Hz to ~150% of control over the first five spikes in the
train (p < 0.05; n = 4; Fig. 4A-C,Dii). Although the
magnitude of the facilitation was not significantly different at the
different frequencies (p > 0.1; one-way ANOVA),
its duration differed, in that it was maintained throughout the
stimulation train at 5 Hz, recovered to control after ten spikes at 10 Hz, and developed into a depression of ~60% of control after the
first five spikes at 20 Hz (Fig.
4A-C,E).
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Figure 4.
Metaplasticity of inhibitory CCIN-evoked inputs.
Graphs showing the effects of 5 (A), 10 (B), and 20 (C) Hz
stimulation of CCINs on monosynaptic inputs to contralateral motor
neurons. Because the pattern of activity-dependent plasticity seen in
this study occurred consistently, control responses before application
of the neuromodulators were similar. Thus, control responses before
substance P and 5-HT application are combined on the graphs to simplify
comparison of their effects (substance P, 1 µM;  ;
n = 5; 5-HT, 10 µM;  ;
n = 4). Di, Traces showing the
effects of 20 Hz stimulation on the CCIN input to a contralateral motor
neuron in control (black line) and in the same cell
after the application of 1 µM substance P
(gray line). At the end of trace, the 1st and 5th
IPSPs in a train from a different experiment are shown on an expanded
time scale in control (black line) and in the presence
of substance P (gray line). Dii,
Traces showing the effects of 5 Hz stimulation on the CCIN input
(black line) and in the same cell after application of
10 µM 5-HT (gray line). The 1st and
5th IPSPs from a separate experiment are again shown in control
(black line) and in the presence of 5-HT
(gray line). Horizontal lines in
Di and Dii indicate the amplitude of the
initial IPSP in control (black line) and in substance P
or 5-HT (gray line), the overlap of the lines
indicating that there was no effect of 5-HT on the amplitude of the
initial IPSP in the train. E, Graph showing the effects
of stimulus trains at 5-20 Hz on CCIN IPSPs. Averages of the first
IPSP in the train (IPSPinitial), IPSPs during
the early part of the train (IPSP2-5), and the
final five IPSPs in the train (IPSP15-20) are
shown at each frequency in control and in the presence of substance P
(1 µM) or 5-HT (10 µM). Calibration: 1 mV,
200 msec (for the train), 20 msec (for the expanded trace).
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Low frequency-evoked EIN inputs are potentiated by substance P (1 µM; Parker and Grillner, 1998), and consequently
substance P increased the amplitude of the initial EPSP in the train
compared with control (Fig.
5Di). In addition to
increasing the amplitude of the initial EPSP, substance P
nonsignificantly reduced the depression of the EPSP at 5 and 10 Hz
(p > 0.05; n = 5; Fig.
5A,B). However, at 20 Hz, substance
P caused significant facilitation of the EPSP amplitude
(p < 0.05; n = 5; Fig.
4C), which was maintained throughout the stimulation train
(Fig. 5C,Di,E). In contrast to substance P, 5-HT (10 µM) markedly depressed the
amplitude of the initial EPSP in the train (30 ± 12%;
p < 0.01; n = 5; Fig. 5Dii). As with substance P, 5-HT (10 µM)
failed to significantly affect the depression of the EPSP amplitude at
5 Hz (p > 0.05; n = 5; Fig.
5A) but caused significant facilitation of the EPSP amplitude at 10 and 20 Hz (p < 0.05;
n = 5; Fig. 5B,C).
Although the magnitude of the effect was greatest at 10 Hz, the
facilitation had decayed to control by the end of the spike train,
whereas at 20 Hz it was maintained throughout the spike train (Fig.
5E). The facilitation of EIN inputs evoked by substance P
and 5-HT again usually recovered within 1-2 sec of the end of the
stimulation train (data not shown). The metaplasticity of EIN and
inhibitory CCIN synaptic inputs was not associated with an effect on
the spike amplitude or duration (CCIN: substance P, spike
amplitude r2 = 0.14, spike duration
r2 = 0.11; 5-HT: spike amplitude
r2 = 0.12, spike duration
r2 = 0.16; EIN: substance P, spike
amplitude r2 = 0.17, spike duration
r2 = 0.09; and 5-HT: spike amplitude
r2 = 0.14, spike duration
r2 = 0.21). It was not possible to
maintain recordings from EINs long enough to examine recovery of the
metaplastic effects of 5-HT of substance P, but in CCINs, the effects
of 5-HT (n = 3 of 5) and substance P (n = 3 of 5) had partially recovered after washing for between 1-2 hr
(data not shown).
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Figure 5.
Activity-dependent plasticity and metaplasticity
of excitatory synaptic inputs. Graphs showing the effects of 5 (A), 10 (B), and 20 (C) Hz stimulation of EINs on monosynaptic inputs
in contralateral motor neurons. As in Figure 4, because the pattern of
activity-dependent plasticity in control was similar, summed control
data from EIN to motor neuron pairs before substance P (1 µM; ; n = 5) and 5-HT (10 µM; ; n = 5) application are shown
on the graphs. Di, Traces showing EIN inputs to a
postsynaptic ipsilateral motor neuron at 20 Hz in control (black
line) and the modulation of the effects by 1 µM
substance P (gray line). The 1st and 5th EPSPs in
a train from a different experiment to that in Di are
also shown on an expanded time scale in control (black
line) and in the presence of substance P (gray
line). Dii, Traces showing plasticity of EIN
inputs evoked at 20 Hz in control (black line) and its
modulation by 10 µM 5-HT (gray
line). The 1st and 5th EPSPs in a train from a different
experiment to that in Di are also shown on an expanded
time scale in control (black line) and in the presence
of 5-HT (gray line). The horizontal
lines on the traces in Di and Dii
indicate the amplitude of the initial EPSP in control
(black) and in substance P or 5-HT
(gray). E, Graph showing the
effects of stimulus trains at 5-20 Hz on EIN-evoked EPSPs. The first
EPSP in the train (EPSPinitial), EPSPs during
the early part of the train (EPSP2-5), and
during the final five EPSPs in the train
(EPSP15-20) are shown. These effects are shown
for control cords, and in the presence of substance P (1 µM) or 5-HT (10 µM). Calibration: 0.5 mV,
200 msec (for the train), 20 msec (for the expanded trace).
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The substance P-evoked metaplasticity of EIN inputs is
not NMDA-dependent
The substance P-mediated potentiation of single low
frequency-evoked EIN inputs is caused by an increase in transmitter
release and the postsynaptic potentiation of NMDA, but not AMPA,
receptors (Parker and Grillner, 1998; Parker et al., 1998). To
determine whether the potentiation of NMDA responses could contribute
to the facilitation of EIN inputs during the spike train, the effects of substance P on EIN inputs were examined in the presence of the NMDA
receptor antagonist AP-5 (100 µM). Although AP-5 reduced the amplitude of the initial EPSP to 80% of control (data not shown),
it did not affect the pattern of activity-dependent plasticity evoked
in control at 5-20 Hz (Fig.
6Ai) or the substance
P-mediated facilitation of EIN inputs during 20 Hz stimulation
(n = 4; Fig. 6Ai,Aii). The metaplasticity of EIN
inputs by substance P thus does not appear to involve the modulation of
NMDA receptors. Although the actual mechanism remains to be examined,
5-HT and substance P appear to share a common mechanism at some point,
because previous application of substance P occluded the facilitating
effect of 5-HT on EIN inputs (n = 3; Fig.
6B).
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Figure 6.
The substance P-mediated metaplasticity of EIN
inputs is not NMDA-dependent. Ai, Graph showing the
effects of 20 Hz stimulation of EINs on the amplitude of the
monosynaptic EPSP in ipsilateral motor neurons. The pattern of the
activity-dependent plasticity and the substance P-evoked metaplasticity
of EIN inputs was not affected when examined in the presence of the
NMDA receptor antagonist AP-5 (100 µM). Data from four
pairs is shown on this graph. Aii, Traces showing EIN
inputs after 20 Hz stimulation in the presence of AP-5 (100 µM; black line) and in the presence of
AP-5 and substance P (1 µM; gray line). At
the end of trace, the 1st and 5th EPSPs in a train from a different
experiment are shown on an expanded time scale in control (black
line) and in the presence of substance P and AP-5
(gray line). B, Graph showing the
lack of effect of 5-HT (10 µM) on EIN inputs already
potentiated by 1 µM substance P. Data from three pairs is
shown in B.
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DISCUSSION |
The results of this study show that synaptic inputs from
identified interneurons in the lamprey spinal cord exhibit
activity-dependent plasticity during spike trains at physiological
frequencies and that the induction and expression of this plasticity
can be altered by neuromodulators, thus providing examples of
metaplasticity (Abraham and Bear, 1996; Fischer et al., 1997).
Although interneuron inputs exhibited activity-dependent depression
during spike trains at physiological frequencies, significant depression of EIN and CCIN inputs, which are thought to be of central
importance to the patterning of network activity (Hellgren et al.,
1992), usually occurred between the fifth and tenth spikes in the
train, the only exception being inhibitory CCIN inputs evoked at 5 Hz,
which were significantly depressed by the third spike in the train.
Because these network neurons typically fire bursts of up to 5 spikes
at frequencies of 5-30 Hz during the normal frequency range of network
activity (0.5-10 Hz; Buchanan and Cohen, 1982; Buchanan and Kasicki,
1995), activity-dependent changes of synaptic inputs from these
interneurons will presumably only contribute to the regulation of
network activity when interneurons fire longer spike trains, as may
occur during slower locomotor activity or postural changes. In
contrast, inputs from excitatory CCINs and LINs showed significant
depression over the first five spikes in the train at 5 and 10 Hz.
Although the role of these interneurons in network activity have either
not been studied or are uncertain, activity-dependent plasticity of
their inputs may also contribute to the patterning of network activity.
The mechanisms underlying the activity-dependent or metaplastic changes
have not yet been examined. The depression that was evoked in each type
of interneuron was greater at lower stimulus frequencies. This result
suggests against transmitter depletion as a mechanism underlying the
synaptic depression, because the depression would be expected to
increase with increasing stimulation frequencies (Parker, 1995a). It is
possible, however, that partial replenishment of releasable transmitter
stores during higher stimulation frequencies contributes to the effects
seen (Wang and Kaczmarek, 1998).
The pattern of activity-dependent plasticity of interneuron inputs in
this study was consistent across all of the pairs examined. This is in
contrast to the situation in other systems, in which variability in the
strength of synaptic inputs has been shown (see Markram et al., 1998
and references therein). With the exception of the LINs, all of the
experiments reported here used motor neurons as postsynaptic targets,
and in all cases, the presynaptic interneurons were recorded in either
the same segment, or at most one segment rostral to the postsynaptic
neuron. Because populations of interneurons will presumably act
together to pattern activity in any one segment, it may be expected
that the pattern of activity-dependent synaptic inputs examined here
will not be markedly different. However, the input from these
interneurons to other postsynaptic targets, for example to other
network interneurons, or to postsynaptic neurons located more rostrally
or caudally, may exhibit differences in the pattern and expression of
activity-dependent changes that may be relevant to the coordination of
network activity at the segmental or intersegmental level.
Neuromodulator-mediated metaplasticity
Although significant activity-dependent plasticity of EIN and
inhibitory CCIN inputs did not occur during the first five spikes in
control conditions, the situation was very different when 5-HT or
substance P was applied. 5-HT evoked activity-dependent
facilitation of EIN and inhibitory CCIN inputs, whereas substance P
evoked activity-dependent facilitation of EIN inputs but enhanced the activity-dependent depression of inhibitory CCIN-evoked IPSPs. Identified network interneuron inputs thus exhibit neuron and modulator-specific metaplasticity (Abraham and Bear, 1996; Fischer et al., 1997). In contrast to the activity-dependent changes in control, these metaplastic effects were pronounced over the initial part of the spike train and can thus presumably contribute to the
modulation of the network output during the normal frequency range of
locomotor activity.
The substance P and 5-HT-mediated metaplasticity of inhibitory CCIN
inputs is consistent with the effects of these modulators on the
network output, namely an increase and a decrease, respectively, in the
frequency of network activity (Harris-Warrick and Cohen, 1985; Parker
et al., 1998). The substance P-mediated depression of CCIN inputs
during the spike train will reduce the strength of reciprocal
inhibitory drive during network activity. This will allow the
contralateral network to escape from inhibition earlier, thus resulting
in an increase in the frequency of network activity (Hellgren et al.,
1992). In contrast, 5-HT facilitated inhibitory CCIN inputs. This will
increase the strength of reciprocal inhibitory drive during the CCIN
burst, delaying the onset of activity in the contralateral network, and
thus reducing the frequency of network activity (Hellgren et al.,
1992). The substance P and 5-HT-mediated metaplasticity of inhibitory
CCIN inputs is thus consistent with the effects of these modulators on
the network output.
Substance P and 5-HT both facilitated EIN inputs during the spike
train. Their effects differed, however, in that substance P facilitates
the amplitude of single low frequency-evoked EIN inputs (Parker and
Grillner, 1998) and thus increased the amplitude of the initial EPSP in
the train compared with control. 5-HT, however, markedly depressed the
amplitude of the initial EPSP in the train compared with control. Thus,
the facilitation of EIN inputs evoked by substance P develops from an
initial potentiation of the EPSP amplitude, whereas the 5-HT-mediated
facilitation develops from a marked reduction of the EIN input. The net
effect is that substance P potentiates EIN inputs during the train
compared with control (Fig. 5Di), whereas in the presence of
5-HT, EIN inputs during the train are not significantly affected
compared with control (Fig. 5Dii). At the network level,
increasing the general level of excitation results in an increase in
the frequency of network activity both experimentally (Brodin et al.,
1985) and in computer simulations (Hellgren et al., 1992), although specifically potentiating EIN inputs to CCINs slows simulated network
activity (Hellgren et al., 1992). Thus, although the increase in
excitatory drive is consistent with the effects of substance P on the
network output, the metaplasticity of EIN inputs to other network
components will need to be examined.
Contribution of cellular and synaptic mechanisms to the modulation
of the network output
Both cellular and synaptic mechanisms can contribute to the
patterning and modulation of network activity (Marder and
Calabrese, 1996; Marder et al., 1996), and thus individual cellular
or synaptic effects should not be taken in isolation in an attempt to
explain the mechanisms underlying network modulation. In addition to
examining the modulatory effects of substance P on synaptic
transmission, its effects have previously been studied in detail on
identified network interneurons at the cellular level (Parker and
Grillner, 1998). Substance P increases the excitability of EINs, and
this effect could act to increase EIN spiking during network activity from lower frequencies, where there is a nonsignificant potentiation of
EIN inputs in the presence of substance P, to higher frequencies, where
there is a significant potentiation of EIN inputs. Thus, cellular and
synaptic effects of substance P may act in parallel to facilitate EIN
inputs, and thus excitatory drive to the network. In contrast to its
effects on EINs, substance P reduces the excitability of CCINs (Parker
and Grillner, 1998), an effect that will again act in parallel with the
enhanced depression of IPSPs during the train to reduce the strength of
reciprocal inhibitory drive. Because the greatest activity-dependent
depression of CCIN inputs occurred at higher frequencies, there will be
a window of CCIN spiking frequencies between 10-20 Hz at which
significant activity-dependent depression will occur in parallel with a
substance P-mediated reduction in the number and frequency of CCIN
spikes (Parker and Grillner, 1998). Thus, substance P has complementary
modulatory effects on the cellular and synaptic properties of EINs and
CCINs. Depending on the circumstances, these effects could act
synergistically or independently to modulate the frequency of network activity.
Summary
In summary, the results of this study suggest that
activity-dependent plasticity of synaptic transmission from inhibitory CCINs and EINs plays little role in patterning network activity under
control conditions during locomotion. However, because interneuron inputs to motor neurons have only been examined here, further work is
needed before concluding that activity-dependent plasticity of these
interneuron inputs plays no role in patterning network activity,
because it is possible that significant activity-dependent plasticity
of these inputs occurs onto other network interneurons, as occurs for
LIN inputs to ipsilateral CCINs. Interneuron connections will need to
be examined directly, because it is not possible to extrapolate the
synaptic input from a single neuron to different postsynaptic targets
(see Markram et al., 1998 and references therein; Parker, 1995b).
Inputs from CCINs and EINs, however, do exhibit activity-dependent
metaplasticity, an alteration in the induction of the plasticity by
neuromodulators. These effects can be related directly to the
modulation of the network output during locomotor activity. Thus,
activity-dependent plasticity and metaplasticity need to be considered
as additional potential mechanisms underlying the coordination and
modulation of rhythmic network activity.
| |
FOOTNOTES |
Received Oct. 20, 1998; revised Dec. 14, 1998; accepted Dec. 16, 1998.
This work was supported by grants from the Swedish Brain Foundation and
the Swedish Medical Research Council (12589, 3026), and by funds from
the Karolinska Institute. Jeanette Hellgren, Erik Svensson, and Jesper
Tegnér provided constructive criticism of this manuscript.
Correspondence should be addressed to David Parker, Nobel Institute for
Neurophysiology, Department of Neuroscience, Karolinska Institute,
S-17177, Stockholm, Sweden.
| |
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Top
Abstract
Introduction
