Nobel Institute for Neurophysiology, Department of Neuroscience, Karolinska Institute, S-17177 Stockholm, Sweden
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ABSTRACT |
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Parker, David. Presynaptic and Interactive Peptidergic Modulation of Reticulospinal Synaptic Inputs in the Lamprey. J. Neurophysiol. 83: 2497-2507, 2000. The modulatory effects of neuropeptides on descending inputs to the spinal cord have been examined by making paired recordings from reticulospinal axons and spinal neurons in the lamprey. Four peptides were examined; peptide YY (PYY) and cholecystokinin (CCK), which are contained in brain stem reticulospinal neurons, and calcitonin-gene-related peptide (CGRP) and neuropeptide Y (NPY), which are contained in primary afferents and sensory interneurons, respectively. Each of the peptides reduced the amplitude of monosynaptic reticulospinal-evoked excitatory postsynaptic potentials (EPSPs). The modulation appeared to be presynaptic, because postsynaptic input resistance and membrane potential, the amplitude of the electrical component of the EPSP, postsynaptic responses to glutamate, and spontaneous miniature EPSP amplitudes were unaffected. In addition, none of the peptides affected the pattern of N-methyl-D-aspartate (NMDA)-evoked locomotor activity in the isolated spinal cord. Potential interactions between the peptides were also examined. The "brain stem peptides" CCK and PYY had additive inhibitory effects on reticulospinal inputs, as did the "sensory peptides" CGRP and NPY. Brain stem peptides also had additive inhibitory effects when applied with sensory peptides. However, sensory peptides increased or failed to affect the amplitude of reticulospinal inputs in the presence of the brain stem peptides. These interactive effects also appear to be mediated presynaptically. The functional consequence of the peptidergic modulation was investigated by examining spinal ventral root responses elicited by brain stem stimulation. CCK and CGRP both reduced ventral root responses, although in interaction both increased the response. These results thus suggest that neuropeptides presynaptically influence the descending activation of spinal locomotor networks, and that they can have additive or novel interactive effects depending on the peptides examined and the order of their application.
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INTRODUCTION |
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Neuropeptides are contained in supraspinal and
spinal systems (see Nyberg et al. 1995). Although the
effects of neuropeptides have been examined in some detail on sensory
inputs to the spinal cord (see Nyberg et al. 1995
), and
to some extent directly on spinal circuits (Barthe and Clarac
1997
; Barthe and Grillner 1995
; Fisher
and Nistri 1993
; Parker et al. 1998b
;
Suzue et al. 1981
), little is known of their effects on
descending inputs to the spinal cord. In this study, the analysis of
neuropeptide-mediated modulation in the lamprey has been extended to
examine their effects on descending reticulospinal synaptic transmission.
Reticulospinal inputs activate or modulate locomotor networks in lower
and higher vertebrates (Jordan 1986; McClellan
1986
; Wannier et al. 1998
). Modulation of these
inputs may thus be required to ensure that they are appropriate to the
ongoing behavior of the animal. The organization of reticulospinal
systems in the lamprey is known in some detail. Reticulospinal axons
make excitatory glutamatergic (Brodin et al. 1988b
) or
inhibitory glycinergic (Wannier et al. 1995
) connections
with motor neurons, and have neuron specific connections with network
interneurons (see Brodin et al. 1988b
). In addition to
glutamate and glycine, reticulospinal neurons also contain several
putative modulatory transmitters, including peptide YY (PYY),
cholecystokinin (CCK), and serotonin (5-HT) (see Brodin et al.
1988b
). In this study, the endogenous neuropeptides PYY, CCK,
neuropeptide Y (NPY), and calcitonin gene-related peptide (CGRP) have
been examined on reticulospinal synaptic transmission. PYY is contained
in the somata of reticulospinal neurons in the middle and anterior
rhombencephalic reticular nuclei, and in fibers in the dorsolateral and
ventromedial regions of the spinal cord (Brodin et al.
1989
). CCK is contained in the somata of reticulospinal neurons, and in fibers in the ventromedial region of the spinal cord
(Brodin et al. 1988a
,b
). NPY is contained in cell bodies and fibers in the spinal dorsal horn and dorsal column (Brodin et al. 1989
), whereas CGRP co-localizes with 5-HT and bombesin in primary afferents (Brodin et al. 1988a
).
Neuromodulators, including neuropeptides, often co-localize with other
modulators and/or fast-acting transmitters. The potential for their
co-release provides the opportunity for modulator/transmitter interactions (Brezina et al. 1996; Harris-Warrick
and Marder 1991
; Kupfermann 1991
). In addition
to effects caused by their co-release, the "modulatory tone"
resulting from the relatively slow actions of neuromodulators allows
for interactions between effects, even when their release is temporally
or spatially independent. Several examples of modulator interactions
have been shown (Brezina et al. 1996
; Dickinson
et al. 1997
; Jorge-Rivera et al. 1998
;
Wood 1995
), suggesting that these effects may be common
occurrences in the nervous system. These interactions result in linear
or nonlinear effects on cellular properties and network outputs (see Kupfermann 1991
), the resulting complexity and subtlety
produced providing a possible explanation for the widespread occurrence and large number of different modulators in the nervous system (see
Kupfermann 1991
). In addition to their individual
effects, interactions between neuropeptides were thus also examined here.
The results of this study suggest that the peptides presynaptically inhibit monosynaptic reticulospinal synaptic inputs, and that they have additive or novel interactive effects depending on the combination of peptides used and the order of their application. This peptidergic modulation is associated functionally with a reduction in the strength of motor responses evoked by brain stem stimulation. Peptidergic modulatory systems can thus act individually or synergistically to control the strength of descending synaptic inputs to the spinal cord.
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METHODS |
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Adult male and female lampreys (Lampetra fluviatilis; Petromyzon marinus) were used in this study. No species or sex-related differences were seen. Lampreys were anesthetized with tricaine methane sulfonate (MS-222; Sandoz, Switzerland) and the spinal cord and notochord removed. The spinal cord was isolated and the connective tissue and meninx primitiva removed from the dorsal and ventral surfaces. The cord was then placed ventral side up in a silicone elastomer (Sylgard; Sikema, Stockholm)-lined chamber kept at 8-12°C, and superfused with Ringer 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 and the pH adjusted to 7.4 with 1 M NaOH. A plastic net was placed over the cord and pinned into the Sylgard to improve stability.
Paired intracellular recordings were made from reticulospinal axons and
the somata of motor neurons and unidentified neurons in the gray matter
region of the spinal cord using thin-walled glass micropipettes filled
with 4 M K acetate (resistances of ~40 M). Müller cell axons
(see Brodin et al. 1988b
) were impaled in the lateral
part of the ventromedial column, 1-2 cm rostral to the postsynaptic
neuron and at least 1 cm from the cut end of the spinal cord (see
Wickelgren et al. 1985
). Motor neurons were identified
by recording 1:1 orthodromic spikes in the adjacent ventral root after
current injection into their somata. Reticulospinal axons with
conduction velocities of at least 2 m/s were identified by recording
their antidromic and orthodromic extracellular spikes on the caudal and
rostral ends of the spinal cord. Strychnine (5 µM) was used to block
glycinergic inputs. Postsynaptic neurons with a high level of
spontaneous excitatory postsynaptic potentials (EPSPs) were not used,
because this activity could complicate measurement of the evoked EPSP.
High divalent cation Ringer was not used to reduce spontaneous
activity, because this Ringer can affect the
N-methyl-D-aspartate (NMDA) component of the
EPSP (data not shown) and thus could have potentially affected the
peptidergic modulation. Spikes were evoked in reticulospinal axons by
1-ms depolarizing current pulses (10-60 nA) or on rebound from
hyperpolarizing current pulses (5-40 ms, 10-60 nA). Spiking on
rebound was preferred because it prevented the reticulospinal spike
from being obscured by a stimulation artifact. EPSPs were evoked every
30-60 s to prevent activity-dependent changes. Monosynaptic potentials
were identified by their short, constant latency after presynaptic stimulation at frequencies of 10-20 Hz.
In some experiments (see RESULTS) the ventromedial region of the spinal cord rostral to the recorded neuron was stimulated extracellularly with a glass suction electrode to elicit compound depolarizations in postsynaptic neurons. The lateral tract and gray matter regions were lesioned on both sides of the cord between the stimulation and recording sites so that the origin of the postsynaptic input was limited to that from axons in the medial region of the spinal cord. Because the cord was placed ventrally, this input presumably originates from reticulospinal axons in the ventromedial column, although effects on dorsal column axons cannot be ruled out. No consistent differences were observed in the results of experiments using intracellular or extracellular stimulation.
An Axoclamp 2A amplifier (Axon Instruments) was used for amplification and in discontinuous current-clamp mode for current injection. The membrane potential of the postsynaptic cell was kept constant in control and in the presence of neuropeptides by current injection. Axon Instruments software (Axotape and pClamp6, Axon Instruments) was used for writing and triggering stimulation protocols and data acquisition and analysis using a 486 PC-computer equipped with an A/D interface (Digidata 1200, Axon Instruments).
Reticulospinal EPSPs often have chemical and electrical components (see
Brodin et al. 1988b). The amplitude of the
chemical EPSP was measured as the peak amplitude above the baseline
excluding the electrical EPSP. EPSPs in which the electrical component
obscured the peak of the chemical EPSP were not used. Where the
electrical EPSP was clearly separate from the chemical component, its
peak amplitude was also measured. In experiments in which the
reticulospinal action potential was free of stimulation artifacts,
spikes were averaged (n = 10) in control and in the
presence of a peptide, and the spike amplitude (the peak potential
reached above the baseline before the spike), spike duration (at
half-height), and the afterhyperpolarization (AHP) amplitude (the peak
hyperpolarized membrane potential reached immediately after the spike)
were measured.
Postsynaptic responses to glutamate were examined by pressure applying
glutamate (1 mM) from a micropipette onto the surface of the spinal
cord above the neuron being recorded. Drugs applied in this way readily
gain access to the somata and dendrites of spinal cord neurons.
Pressure pulses of between 20 and 200 ms were used to give clear,
consistent responses. Tetrodotoxin (TTX; 1.5 µM) was used to block
indirect effects due to the action of glutamate on nearby neurons.
Responses were evoked at 1-min intervals. Miniature glutamatergic EPSPs
(mEPSPs) were examined in the presence of TTX (1.5 µM) and strychnine
(5 µM) (see Parker and Grillner 1998). mEPSPs were
digitized at 2 kHz, and their amplitude and frequency (measured as the
interval between successive events) measured off-line using the
MiniAnalysis program (Jaejin Software). mEPSPs were detected by their
ability to exceed a preset threshold. All detected events were examined
to ensure that they had characteristic rapid rise and slow decay times.
Drugs were bath applied using a peristaltic pump. The following
peptides were used: peptide YY (PYY), neuropeptide Y (NPY), cholecystokinin octapeptide (CCK; RBI, Natick, MA), and
calcitonin-gene-related peptide (CGRP; Sigma, Stockholm, Sweden).
Peptides were applied for 10 min; it took ~4-5 min for the solution
in the bath to be replaced. Peptides were applied at concentrations of
100 nM. This concentration is physiologically relevant (Duggan
1995) and has previously been used to examine peptidergic
modulation in the lamprey (Parker and Grillner 1996
;
Parker et al. 1998a
,b
; Ullström et al.
1999
). Higher peptide concentrations (500 nm to 1 µM) result either in effects that require long wash-off times, possibly due to
difficulties in washing peptides out of the tissue, or they evoke
physiologically relevant long-term changes (Parker et al. 1998b
; Ullström et al. 1999
). Interactive
effects of the peptides were examined by applying one peptide until a
plateau inhibitory effect was evoked (typically between 15 and 30 min),
and then applying the second peptide in the presence of the first
peptide. Locomotor activity was evoked by applying NMDA (50-200 µM)
to isolated spinal cord-notochord preparations (see Parker et
al. 1998b
). Peptide effects were examined on the burst
frequency and coefficient of variation (CV), the latter being defined
as the standard deviation of the cycle duration divided by the mean
cycle duration (see Parker et al. 1998b
).
The functional effect of reticulospinal modulation was examined on
ventral root responses evoked by extracellular stimulation of
reticulospinal somata in the brain stem. A fine-tipped glass pipette
filled with Ringer was used to stimulate regions in the "locomotor
strip" (see McClellan and Grillner 1984). The spinal cord was left attached to the notochord, and the lateral tracts and
gray matter regions of the spinal cord were lesioned bilaterally ~2
cm rostral to the stimulation site. Recordings were made from spinal
ventral roots at least 5 cm caudal to the stimulation site. A petroleum
jelly (Vaseline) barrier divided the preparation into brain stem and
spinal cord pools. Peptides were applied to the spinal cord pool to
modulate reticulospinal inputs. Ventral root responses were rectified
and integrated to give a measure of the evoked response (see
Ullström et al. 1999
).
Numbers in the text refer to the number of experiments performed. Only
one experiment was performed in each piece of cord, with at most two
pieces of cord being taken from the same animal. EPSP or action
potential modulation is expressed as percent of control ± SE.
Unless stated otherwise, statistical significance was examined using
two-tailed paired t-tests. All values, whether an effect was
seen or not, were included in the statistical analysis. For the
analysis of mEPSP amplitudes, a Kolmogorov-Smirnov test was performed
on cumulative probability plots of amplitude and frequency histograms.
A significant effect was accepted when the Kolmogorov-Smirnov quotient
[QKS()] was <0.01. The CV and
mean of EPSP amplitudes were analyzed by dividing EPSPs into bins of 10. The mean, SD, and CV (SD/mean) were calculated for each block. Graphs of 1/CV2 against mean were plotted, the
values of the 1/CV2 and mean being normalized to
that of the initial bin.
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RESULTS |
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Neuropeptide-mediated modulation of reticulospinal synaptic transmission
The initial aim of this study was to determine the individual
effects of endogenous neuropeptides on monosynaptic glutamatergic transmission from reticulospinal axons. The brain stem peptides PYY and
CCK (100 nM) (Brodin et al. 1988a,b
,
1989
) and the sensory peptides CGRP and NPY (100 nM)
(Brodin et al. 1988a
, 1989
), all significantly (P < 0.05) reduced the amplitude of the
chemical component of the monosynaptic reticulospinal EPSP (Fig.
1, A and B). The
amplitude of the initial electrical component was not significantly
affected by any peptide (P > 0.1; see Fig. 1,
A and B).
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The pre- or postsynaptic locus of the synaptic modulation was also
examined. As shown in Fig. 1C, the reduction of the chemical component of the EPSP by the peptides was associated with a reduction in the inverse of the coefficient of variation
(1/CV2) of the EPSP (PYY,
R2 = 0.5; CCK,
R2 = 0.29; NPY,
R2 = 0.17; CGRP,
R2 = 0.76). Parallel changes in
1/CV2 and EPSP amplitudes are classically
attributed to presynaptic effects due to changes in quantal content
(np), either as a result of the reduction in the probability of
transmitter release (P), or the number of release sites
(n) (see Selig et al. 1995). However, this
method is ambiguous, because it requires that assumptions are made
about release sites and stimulation paradigms (Faber and Korn
1991
). Thus the locus of the synaptic modulation was examined
further. Because the results of this analysis were remarkably similar
for each of the peptides (see Table 1),
the effects of only one peptide, CGRP, are illustrated and described in
detail.
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Effect of CGRP on reticulospinal synaptic inputs
Bath application of CGRP (100 nM) significantly (P < 0.05) reduced the amplitude of reticulospinal synaptic inputs (71 ± 12.7%; mean ± SE, n = 10 of 12; Fig. 2, A1 and A2) and ventral column stimulation-evoked depolarizations (78.5 ± 32%; n = 4 of 6; data not shown) in motor neurons. In the remaining experiments, CGRP either increased (n = 2; mean increase 117 ± 7%) or had no effect on the synaptic input (n = 2). The latency from application to the maximal effect of CGRP was 14 ± 6 min. The effect of CGRP on synaptic inputs partially recovered on wash off (n = 5, mean recovery time 74 ± 14 min; Fig. 2A1).
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The synaptic modulation evoked by CGRP was not associated with an
effect on the postsynaptic input resistance, measured by injecting 1-nA
hyperpolarizing pulses into the somata of the postsynaptic neuron
(P > 0.1; Fig. 2B2), although it
occasionally caused a small depolarization of the postsynaptic membrane
potential (1.8 ± 0.7 mV; n = 8). As shown above,
CGRP also failed to significantly affect the amplitude of the
electrical component of the EPSP (Fig. 1A; 91 ± 8%,
P > 0.1). These results provide some support for a
presynaptic locus for the modulation of reticulospinal synaptic transmission. Further support was provided by the lack of a significant (P > 0.1) effect of CGRP on postsynaptic
depolarizations evoked by pressure-applied glutamate (1 mM;
n = 7; Fig. 2B1). However, because
exogenously applied glutamate may also activate extrasynaptic receptors, this result does not provide unequivocal support for the
lack of an effect of CGRP on postsynaptic responses. Thus the effect of
CGRP was also examined on spontaneous mEPSPs (see METHODS)
(Parker and Grillner 1998). CGRP (100 nM) failed to
significantly affect the amplitude of mEPSPs
[QKS(
) > 0.05;
n = 4; Fig. 2, C1 and C2], again
supporting a presynaptic locus for the synaptic modulation. Because
presynaptic inhibition is classically associated with a reduction in
the frequency of mEPSPs (Katz 1966
), the effect of CGRP
on the mEPSP frequency was also examined. Although the frequency was
reduced in three of four experiments, the reduction was not significant
[QKS(
) > 0.01;
n = 4; data not shown]. However, mEPSPs will also
originate from sources other than reticulospinal axons, including
excitatory network and sensory neurons. If these are not also
presynaptically modulated (see Peptidergic modulation of network
activity), an effect on mEPSP frequency may be obscured (see DISCUSSION).
CGRP thus appears to act presynaptically to inhibit reticulospinal
synaptic transmission. Because presynaptic modulation can be associated
with changes in spike properties (see Klein 1995), the
effects of CGRP were examined on reticulospinal axon action potentials.
In ~60% of experiments (n = 10 of 18), CGRP reduced the spike amplitude (84 ± 5%) and duration (77 ± 8%) and
increased the amplitude of the AHP (181 ± 25%). When these
effects occurred, they occurred together and were significant
(n = 10; P < 0.05; see Fig.
2A2). However, when all experiments were included in the
analysis, the effect was not statistically significant
(n = 18; P > 0.05). In experiments in
which the reticulospinal axon potential was modulated, the amplitude of
the electrical component of the EPSP was reduced by CGRP in four of the
five experiments in which a clear electrical EPSP was obtained (mean
reduction 81 ± 7% of control; data not shown). Although
modulation of the reticulospinal action potential could account for the
presynaptic depression of transmitter release, it was not necessary for
the synaptic modulation, because the chemical component of the EPSP could be reduced in the absence of an observable effect on the spike or
electrical EPSP (n = 4, mean reduction 78 ± 9%
of control).
CCK, PYY, and NPY mimicked the effects of CGRP (see Table 1), each failing to significantly affect the postsynaptic input resistance, membrane potential, electrical component of the EPSP, responses to exogenous glutamate application, or mEPSP amplitude (see Table 1). These peptides thus also appear to act presynaptically. The magnitude of the synaptic depression evoked by the four peptides at 100 nM was not significantly different (P > 0.1, 1-way ANOVA), and in each case partial recovery of the effects occurred after washing for between 60 and 100 min. However, unlike CGRP, CCK, PPY, and NPY never affected the reticulospinal action potential.
Peptidergic modulation of network activity
The above analysis suggests that the peptides act presynaptically to modulate reticulospinal synaptic transmission. As a final step in the individual analysis of the peptides, their effects were examined on NMDA-evoked locomotor activity. Reticulospinal neurons do not contribute to NMDA-evoked network activity in the isolated spinal cord. Thus peptidergic modulation limited to presynaptic effects on reticulospinal synaptic transmission should not significantly affect NMDA-evoked network activity. If an effect was observed, it would suggest either that postsynaptic changes occurred that went undetected, or that synaptic transmission from network neurons can also be presynaptically modulated. At the concentration used above to inhibit reticulospinal synaptic inputs (100 nM), neither PYY (n = 4 of 5), CCK (n = 6 of 8), NPY (n = 4 of 5), or CGRP (n = 5 of 5) significantly (P > 0.1) affected the frequency or regularity of network activity across different NMDA concentrations (50-200 µM) and thus different burst frequencies (data not shown). Where an effect was seen with NPY and PYY, it was a nonsignificant reduction of the burst frequency. CCK could reversibly increase the burst frequency (n = 2 of 8), an effect that was greatest when the initial frequency was low (<1 Hz; data not shown). The failure of the peptides to significantly or consistently affect the network output suggests that they do not act on network neurons or synaptic transmission, further supporting a presynaptic locus for the synaptic modulation. The lack of an effect on network synaptic transmission was supported by preliminary experiments using paired recordings to examine synaptic transmission from excitatory network interneurons to motor neurons. In these experiments, the monosynaptic excitatory interneuron (EIN)-evoked EPSP was not significantly affected (n = 2 CCK; n = 1 PYY; n = 2 NPY; n = 3 CGRP; data not shown; P > 0.05, n = 8).
Interactive effects of neuropeptides on reticulospinal synaptic transmission
Due to the abundance of neuromodulators, and the relatively slow
time course of their effects, interactions between modulators, even if
they are not co- or simultaneously released, can occur (see
INTRODUCTION). The analysis of the individual effects of the four peptides provided a basis on which to examine their
interactive effects on reticulospinal synaptic transmission. Not all
combinations of peptide application were examined (see Table
2). However, because the peptides
originate either from the brain stem or from primary afferents or
sensory interneurons, the analysis was simplified by initially
examining the interactions between the brain stem peptides CCK and PYY
(n = 4) (Brodin et al. 1988a,b
,
1989
), or between the sensory peptides CGRP and NPY
(n = 6) (Brodin et al. 1988a
,
1989
). For the brain stem peptides, application of CCK (100 nM) after PYY (100 nM) resulted in a significant additive inhibitory effect on the amplitude of reticulospinal synaptic inputs
(n = 4; P < 0.05; Fig.
3, A1 and A2).
Similarly, for the sensory peptides, application of CGRP (100 nM) after
NPY (100 nM; n = 3), or NPY (100 nM) after CGRP (100 nM; n = 2) resulted in significant additive inhibition
(Fig. 3B; P < 0.05). In both cases,
however, the effects were not arithmetically additive, because the
inhibition evoked by the second peptide was on average less (sensory
peptides 12.1 ± 3.2%; reticulospinal peptides 15.2 ± 5.7%) than that evoked when it was applied individually (~25%; see
above).
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Interactions between brain stem and sensory peptides were also investigated. Initially, the effects of brain stem peptides applied in the presence of sensory peptides were examined (n = 8). Application of CCK (100 nM) after either CGRP (100 nM; n = 3; Fig. 4A) or CGRP and NPY (100 nM; n = 2), also resulted in additive inhibition of reticulospinal inputs, as did application of PYY (100 nM) after NPY (100 nM; n = 2; Fig. 4B). Again, the interactive inhibition evoked by CCK or PYY was less than that evoked when they were applied separately (11.1 ± 2.1%; P < 0.05; n = 8). In the converse case, however, when sensory peptides were applied after brain stem peptides, additive inhibitory effects were never seen (n = 9). Instead, after the initial application of PYY (100 nM), NPY (100 nM) either had no effect (n = 1 of 3), or it increased the amplitude of the reticulospinal EPSP (n = 2 of 3; data not shown), thus reducing the depression evoked by PYY. Similarly, in the presence of CCK (100 nM), CGRP either had no effect (n = 3 of 6) or increased the EPSP amplitude (n = 3 of 6), again reducing the CCK-mediated depression of the EPSP (Fig. 4, C1 and C2). CGRP did not modulate the reticulospinal action potential in any experiment when it was applied in the presence of CCK (n = 6; see Fig. 4C2). The sensory peptide-mediated increase in the EPSP amplitude was significant (P < 0.05; n = 5), the EPSP typically approaching the amplitude obtained in control before the brain stem peptides were applied (see Fig. 4, C1 and C2). Brain stem peptides, sensory peptides, and brain stem peptides in the presence of sensory peptides thus have additive inhibitory effects, whereas sensory peptide effects were either occluded (n = 4 of 9), or they now increased reticulospinal EPSP amplitudes (n = 5 of 9). This is in contrast to the effects of CGRP or NPY alone, in which the EPSP amplitude was usually reduced (n = 21 of 29), an increase occurring in only a small proportion of experiments (n = 3 of 29; see Table 1).
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Although the mechanisms have not yet been examined in detail, the
interactions between sensory and brain stem peptides also appear to be
mediated presynaptically. For example, applying CGRP (n = 4) or NPY (n = 3) in the presence of PYY or CCK did
not affect the amplitude of postsynaptic responses to glutamate (1 mM;
Fig. 5A), or the amplitude of
mEPSPs [QKS() > 0.05;
n = 5; Fig. 5B], suggesting that
postsynaptic mechanisms do not underlie the interactive effects of
sensory peptides in the presence of brain stem peptides. PYY or CCK
applied in the presence of NPY or CGRP also failed to affect the
amplitude of glutamate-evoked depolarizations (n = 4)
or mEPSPs [QKS(
) > 0.05;
n = 4; data not shown], suggesting that additive
inhibitory effects were also mediated presynaptically. Finally,
applying CGRP after PYY (n = 4) or CCK
(n = 3) did not affect the frequency of NMDA-evoked
network activity (data not shown), again suggesting a lack of effect on
postsynaptic locomotor network neurons.
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Functional effect of neuropeptide-mediated modulation of descending inputs
To determine the functional consequences of the modulation of
reticulospinal synaptic transmission, the effects of two of the
peptides, the brain stem peptide CCK and the sensory peptide CGRP, were
examined on ventral root activity evoked by stimulation of the brain
stem to directly activate reticulospinal cell bodies (n = 18; see METHODS). Whereas brain stem stimulation in the
lamprey can evoke locomotor activity (McClellan and Grillner
1984), prolonged periods of regular alternating were rarely
evoked (n = 2 of 18). However, brain stem stimulation
reliably evoked ventral root activity. CCK (n = 8) and
CGRP (n = 10) significantly (P < 0.05)
reduced brain stem-evoked ventral root responses and the amplitude of brain stem-evoked compound EPSPs in motor neurons and unidentified spinal cord neurons (CCK n = 4; CGRP n = 5), effects that are consistent with their inhibition of
reticulospinal synaptic transmission (Fig.
6, A and B). In
addition, application of CGRP in the presence of CCK resulted in a
significant increase in the amplitude and duration of the ventral root
response (P < 0.05; n = 8; Fig. 6, A and C), an effect that is also consistent with
the interactive potentiation of reticulospinal inputs by these
peptides. As with the reticulospinal EPSP, CGRP potentiated the
ventral root activity to the level that occurred in control. However,
CCK also significantly increased the integrated ventral root response
in the presence of CGRP (P < 0.05; n = 7; Fig. 6A), an effect that does not fit with the
interactive modulation of reticulospinal synaptic transmission. The
reason for this discrepancy is not known at present but could be due to
differential peptidergic modulation of populations of reticulospinal
axons activated by the brain stem stimulation.
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DISCUSSION |
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This study provides the first detailed examination of the effects of endogenous neuropeptides on reticulospinal synaptic transmission. PYY, CCK, NPY, and CGRP all appear to presynaptically reduce monosynaptic reticulospinal EPSPs. Interactions between the peptides have also been examined, additive or novel interactive effects occurring, depending on the peptides used and the order of their application. Functionally, the modulation of reticulospinal synaptic inputs affects the descending activation of spinal networks, shown by their effects on ventral root activity in response to brain stem stimulation.
Paired recording were used to examine en passant synapses made onto
postsynaptic spinal cord neurons from reticulospinal axons in the
ventromedial column. PYY immunoreactivity is found in the dorsal part
of the lateral column, where appositions onto motor neuron dendrites
are found, and to a lesser extent in the ventromedial column
(Brodin et al. 1989). CCK immunoreactivity is found in the lateral and ventral columns, and in close apposition to the dendrites of motor neurons in the ventromedial column (Ohta et al. 1988
). NPY co-localizes with GABA in small bipolar cell
bodies dorsal to the central canal, immunoreactive fibers being found in the dorsal horn, and the lateral and dorsal columns (Brodin et al. 1989
; Parker et al. 1998a
). CGRP is
contained in primary afferents, immunoreactive fibers mainly being
located in the dorsal horn and dorsal column (Brodin et al.
1988a
). Thus, of these peptides, only CCK is present to a
significant extent in the ventromedial region of the spinal cord where
the reticulospinal synapses examined here are located. However,
immunoreactivity only shows potential release sites, and not the
location of neuropeptide receptors. Because neuropeptides lack
high-affinity uptake systems and are degraded slowly, they can activate
receptors at some distance from their release sites (see Duggan
1995
; Fuxe and Agnati 1991
). This effect will be
facilitated by the ease of diffusion of even large molecules in the
lamprey spinal cord (see Wald and Selzer 1981
). Because
the peptide effects were consistent, it thus seems likely that they can
act at some distance from their release sites (volume transmission)
(see Fuxe and Agnati 1991
).
NPY and PYY are members of the pancreatic polypeptide family of
peptides (Sundler et al. 1993). Although PYY and NPY had
additive inhibitory effects, this could have been due to the use of
nonsaturating concentrations. However, applying NPY in the presence of
PYY could increase the EPSP amplitude, an effect that was never seen
with PYY, thus suggesting that NPY and PYY activate separate receptors and do not simply cross-react at a single receptor.
Neuropeptide-mediated inhibition of reticulospinal inputs is mediated presynaptically
The evidence suggests that the peptides presynaptically inhibited
reticulospinal inputs. First, the peptides did not significantly affect
postsynaptic input resistance or resting membrane potential, or the
amplitude of the electrical component of the EPSP. However, these
results provide only limited support for a presynaptic locus, because
input resistance or membrane potential changes may not be seen at
somatic recording sites, and the amplitude of the electrical component
of the EPSP will not reveal modulation of ligand-activated conductances
underlying the chemical component of the EPSP. However, the peptides
also reduced the coefficient of variation of the EPSPs, suggesting a
presynaptic reduction in release probability (Selig et al.
1995; but see Faber and Korn 1991
). In addition, postsynaptic responses to exogenous glutamate and the amplitude of
mEPSPs were unaffected by the peptides. These results provide stronger
support for presynaptic modulation, the failure to affect mEPSP
amplitudes demonstrating that postsynaptic membrane, synaptic, and
dendritic properties are unaffected. The peptides also failed to affect
NMDA-evoked locomotor activity in the isolated spinal cord. Although
reticulospinal neurons contribute to the initiation and modulation of
locomotor activity in the intact animal, they have little role in
NMDA-induced activity in the isolated spinal cord. Thus the absence of
an effect on network activity further supports the presynaptic
modulation of reticulospinal inputs.
Although the lack of an effect on mEPSP amplitudes is consistent with
presynaptic modulation, the mEPSP frequency, which should be reduced if
the modulation was presynaptic (Katz 1966), was also
unaffected. The failure to affect mEPSP frequency may reflect a
difference in the modulation of spontaneous and evoked transmitter release. In addition, mEPSPs can also originate from cutaneous afferents (Brodin et al. 1987
; Rovainen
1967
), intraspinal stretch receptors (Viani Di Prisco et
al. 1990
), and network interneurons (Buchanan
1982
; Buchanan and Grillner 1987
). A significant
effect on the frequency of mEPSPs from reticulospinal axons could be occluded if these inputs are not also modulated presynaptically, as
seems likely from the failure of the peptides to modulate NMDA-evoked network activity, and in preliminary experiments, synaptic transmission from excitatory network interneurons.
Although presynaptic modulation thus seems likely, only CGRP affected
the presynaptic action potential. This modulation occurred in ~60%
of experiments. Because reticulospinal axons were not uniquely
identified, the variability in the effects of CGRP and the lack of
effect of the other peptides may reflect differences in the modulation
of particular reticulospinal axons. Alternatively, the peptides may
only modulate the action potential at or near release sites. However,
because reticulospinal axons make en passant synapses along the length
of the spinal cord, the failure to see a consistent or significant
effect on the action potential suggests that if modulation occurs
consistently, it can only occur directly at the release sites.
Alternatively, the peptides may act directly on the release machinery
(see Miller 1998), as shown for the
5-HT-mediated presynaptic inhibition of reticulospinal inputs
(Shupliakov et al. 1995
).
Interactive effects of the neuropeptides
The nervous system contains an abundance of neurotransmitters and
neuromodulators. Several possibilities have been suggested for the
complexity of transmitter/modulator systems (see Bowers 1994; Kupfermann 1991
). Neuromodulation is
typically slow. This will allow modulators to interact even when they
are not co- or simultaneously released. Such interactions occur in
invertebrate (e.g., Dickinson et al. 1997
; Wood
1995
) and vertebrate systems (see Strand et al.
1991
). Here, the brain stem peptides PYY and CCK additively
inhibited reticulospinal inputs, as did the sensory peptides CGRP and
NPY. Brain stem peptides also had additive effects when applied in the
presence of sensory peptides. In contrast, however, CGRP and NPY either
failed to affect or increased the amplitude of reticulospinal inputs
when applied in the presence of brain stem peptides. The mechanisms
underlying these individual and interactive effects have not yet been
examined but could be due to either additive activation of shared
intracellular or effector pathways, or additive or antagonistic effects
on separate pathways and effectors.
Role of the peptidergic reticulospinal modulation
Reticulospinal inputs are thus subject to modulation by endogenous
neuropeptides. Other endogenous modulators, including 5-HT (Buchanan and Grillner 1991), dopamine
(Wikström et al. 1995
), metabotropic glutamate
receptors (Cochilla and Alford 1998
; Krieger et
al. 1996
), and substance P (Parker and
Grillner 1998
) affect reticulospinal inputs. With the exception
of substance P (Parker and Grillner 1998
) and
group 1 mGluR agonists (Cochilla and Alford 1998
), all
modulators presynaptically inhibit reticulospinal inputs. The potential
modulation by several different modulatory systems is presumably
related to the profound effects that reticulospinal inputs can have on
the locomotor network (see Brodin et al. 1988b
; Wannier et al. 1998
). Release of NPY or CGRP, which in
the latter case will occur directly in response to sensory stimulation,
will reduce descending inputs to the spinal cord. NPY, but not CGRP (unpublished observation) also reduces the amplitude of mechanosensory inputs (Parker et al. 1998a
) and thus appears to have a
general depressing effect on spinal synaptic inputs to the spinal cord. CCK and PYY also reduce the amplitude of reticulospinal inputs, with
PYY also reducing the amplitude of mechanosensory inputs (Parker
et al. 1998a
). CCK and PYY are contained in smaller diameter reticulospinal neurons (Brodin et al. 1988b
). Although
these peptides may also act in the brain stem to reduce descending
inputs, possibly through an effect on the excitability of
reticulospinal neurons, a spinal locus is potentially useful because it
allows the potential modulation of Müller cell inputs to
different network neurons (see Ohta and Grillner 1989
).
In addition, the modulation of reticulospinal inputs in different
spinal cord segments could alter the phase lag between segments,
contributing to the modulation of different motor patterns or even the
direction of swimming (see Matsushima and Grillner
1992
). Finally, as suggested in this study, by acting in the
spinal cord the possibility exists for reticulospinal peptides to
interact with spinally located modulators, resulting in additive or
nonlinear interactive effects.
Neuromodulation is typically slow, the effects seen here lasting at
least 1 h. A mechanism to reverse the modulation if required by
changes in internal or external conditions would thus be useful. The
facilitation evoked by CGRP and NPY in the presence of CCK and PYY
could provide such a mechanism. CGRP is contained in small afferents
(Brodin et al. 1988a), suggesting that its release
requires a strong, possibly nociceptive, stimulus, which may elicit
locomotor activity to remove the animal from the source of the
stimulation. The CGRP-mediated potentiation of descending inputs that
have previously been inhibited by brain stem peptides, and the
resulting increase in descending excitatory drive, would be appropriate under these conditions.
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ACKNOWLEDGMENTS |
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I thank E. Svensson and S. Grillner for comments on the manuscript.
This work was supported by grants from the Wellcome Trust, Karolinska Institutes Fonder, the Swedish Brain Foundation, and the Swedish Medical Research Council (12589).
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FOOTNOTES |
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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 12 October 1999; accepted in final form 10 January 2000.
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REFERENCES |
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