Download Neuropeptide-Mediated Facilitation and Inhibition of Sensory Inputs

Neuropeptide-Mediated Facilitation and Inhibition of Sensory Inputs
and Spinal Cord Reflexes in the Lamprey
MARIA ULLSTRÖM, DAVID PARKER, ERIK SVENSSON, AND STEN GRILLNER
Nobel Institute for Neurophysiology, Department of Neuroscience, Karolinska Institute, S-171 77 Stockholm, Sweden
INTRODUCTION
Although it is important to know the effects of neuromodulators at the cellular and synaptic levels, it is also important to
know the network or behavioral relevance of these effects.
Because extrapolations between different levels in the nervous
system must be made with caution, it is necessary to examine
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effects directly at each of these levels. Spinal reflexes were
used to investigate the behavioral effects of sensory modulation in mammals (see Wiesenfeld-Hallin 1995). However, in
these preparations, it is difficult to obtain detailed mechanistic
explanations at the cellular and synaptic levels. Conversely,
although detailed cellular information was obtained with dissociated cells or tissue slices (see Murase et al. 1989), the
network or behavioral relevance of these results must be extrapolated.
In the lamprey, a lower vertebrate, the effects of neuromodulators on sensory circuitry can be examined on identified neurons and monosynaptic connections in the intact spinal cord
(see Christenson and Grillner 1991; El Manira et al. 1997;
Parker and Grillner 1996). These neurons include the cutaneous touch and pressure-sensitive dorsal cells, which have large
intraspinal cell bodies, and large spinobulbar neurons, which
receive monosynaptic glutamatergic inputs from dorsal cells
and other primary afferents (Brodin et al. 1987; Rovainen
1967). We examined the relevance of the modulation of sensory circuitry with reflex responses evoked by electrical stimulation of the attached tail fin in an isolated spinal cord preparation (McClellan and Grillner 1983). Reflex responses can be
monitored by recording extracellularly from spinal ventral
roots the activity corresponding to that recorded myographically in intact animals (McClellan and Grillner 1983). In addition, the effects of neuromodulators on sensory inputs can be
examined independently of their direct effects on motor or
premotor neurons (see Parker and Grillner 1998) by dividing
the spinal cord into separate pools, thus allowing the relevance
of sensory modulation to be determined unequivocally.
The spinal dorsal horn contains a large number of neuropeptides (see Nyberg et al. 1995), although in most cases their
effects are unclear (see DISCUSSION). Many of these peptide
systems were conserved throughout vertebrate evolution (see
Brodin et al. 1995), and thus the lamprey is a relevant model
system in which to examine neuropeptide-mediated sensory
modulation. Tachykinin immunoreactivity in the lamprey is
found in the dorsal root, dorsal column, and dorsal horn (Van
Dongen et al. 1986) and in close apposition to sensory dorsal
column axons (Svensson and Grillner, unpublished observations). Tachykinins have excitatory effects on the sensory
circuitry in that they depolarize mechanosensory dorsal cells,
increase the excitability of dorsal cells and second-order spinobulbar neurons, and presynaptically potentiate excitatory but
reduce inhibitory, synaptic inputs (Parker and Grillner 1996).
Certain of these effects are mediated through protein kinase C
(Parker et al. 1997). Neuropeptide Y (NPY) immunoreactivity
is found in small bipolar interneurons, the axons of which are
0022-3077/99 $5.00 Copyright © 1999 The American Physiological Society
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Ullström, Maria, David Parker, Erik Svensson, and Sten Grillner.
Neuropeptide-mediated facilitation and inhibition of sensory inputs
and spinal cord reflexes in the lamprey. J. Neurophysiol. 81:
1730 –1740, 1999. The effects of neuromodulators present in the
dorsal horn [tachykinins, neuropeptide Y (NPY), bombesin, and
GABAB agonists] were studied on reflex responses evoked by cutaneous stimulation in the lamprey. Reflex responses were elicited in an
isolated spinal cord preparation by electrical stimulation of the attached tail fin. To be able to separate modulator-induced effects at the
sensory level from that at the motor or premotor level, the spinal cord
was separated into three pools with Vaseline barriers. The caudal pool
contained the tail fin. Neuromodulators were added to this pool to
modulate sensory inputs evoked by tail fin stimulation. The middle
pool contained high divalent cation or low calcium Ringer to block
polysynaptic transmission and thus limit the input to the rostral pool
to that from ascending axons that project through the middle pool.
Ascending inputs and reflex responses were monitored by making
intracellular recordings from motor neurons and extracellular recordings from ventral roots in the rostral pool. The tachykinin neuropeptide substance P, which has previously been shown to potentiate
sensory input at the cellular and synaptic levels, facilitated tail finevoked synaptic inputs to neurons in the rostral pool and concentration dependently facilitated rostral ventral root activity. Substance P
also facilitated the modulatory effects of tail fin stimulation on ongoing locomotor activity in the rostral pool. In contrast, NPY and the
GABAB receptor agonist baclofen, both of which have presynaptic
inhibitory effects on sensory afferents, reduced the strength of ascending inputs and rostral ventral root responses. We also examined the
effects of the neuropeptide bombesin, which is present in sensory
axons, at the cellular, synaptic, and reflex levels. As with substance P,
bombesin increased tail fin stimulation-evoked inputs and ventral root
responses in the rostral pool. These effects were associated with the
increased excitability of slowly adapting mechanosensory neurons
and the potentiation of glutamatergic synaptic inputs to spinobulbar
neurons. These results show the possible behavioral relevance of
neuropeptide-mediated modulation of sensory inputs at the cellular
and synaptic levels. Given that the types and locations of neuropeptides in the dorsal spinal cord of the lamprey show strong homologies
to that of higher vertebrates, these results are presumably relevant to
other vertebrate systems.
MODULATION OF SPINAL REFLEXES IN LAMPREY
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found in close apposition to axons in the dorsal column and
dorsal horn (Bongianni et al. 1990). NPY has the opposite
effect of tachykinins in that it reduces the excitability of
spinobulbar interneurons and presynaptically reduces dorsal
cell-mediated glutamatergic synaptic transmission (Parker et
al. 1998b). GABA is colocalized with NPY in bipolar neurons
in the dorsal column (Parker et al. 1998b). The GABAB receptor agonist baclofen has complementary effects to NPY in
depressing sensory synaptic transmission (Christenson et al.
1991; Parker et al. 1998b).
Because the effects of neuropeptides were characterized at
the cellular and synaptic levels, the aim of this study was to
examine their actions at the network level by investigating their
effects on spinal reflexes. In addition, we examined the effects
of the neuropeptide bombesin, which is colocalized with 5-hydroxytryptamine (5-HT) and calcitonin gene-related peptide
(CGRP) in primary afferents (Brodin et al. 1988) at the cellular, synaptic, and reflex levels.
METHODS
Adult male and female lampreys (Lampetra fluviatilis) were anaesthetised with tricaine methane sulfonate (MS-222; Sandoz, Switzerland), and the spinal cord and notochord were dissected from the
mid-trunk region to the tail fin ( ;50 segments). The tail fin was left
attached, and the preparation was placed dorsal side up in a Sylgard
(Dow Corning)-lined chamber 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. High divalent Ringer contained (in mM)
119.8 NaCl, 2.1 KCl, 10.8 CaCl2, 7.2 MgCl2, 4 glucose, 2 HEPES,
and 0.5 L-glutamine. Low calcium– high magnesium Ringer contained
(in mM) 119.8 NaCl, 2.1 KCl, 0.1 CaCl2, 7.2 MgCl2, 4 glucose, 2
HEPES, and 0.5 L-glutamine. All solutions were bubbled with O2, and
the pH was adjusted to 7.4 with 1 M NaOH. The experimental
chamber was kept at a temperature of 10 –12°C.
The connective tissue and meninx primitiva were removed from the
dorsal surface of the spinal cord. The spinal cord–notochord preparation was split into three pools by placing Vaseline barriers at
different positions (see Fig. 1A). One barrier was placed ;15 segments rostral to the tip of the tail fin, with a second barrier placed $20
segments more rostral to this barrier. High divalent cation or low
calcium/high magnesium Ringer was placed in the middle pool between these two barriers to reduce the influence of polysynaptic
pathways through the middle pool either by raising the threshold for
excitation or by reducing synaptic transmission, respectively (Berry
and Pentreath 1976). Both Ringers were effective in reducing ascending inputs, shown by the reduction of ventral root activity recorded in
the rostral pool (see Fig. 1, Bi and Bii). Thus inputs to the rostral pool
were limited to neurons with long axons that projected rostrally
beyond the middle pool, i.e., afferent and spinobulbar axons (Dubuc
et al. 1992; Rovainen 1967). The caudal and rostral pools contained
normal Ringer. The barriers were checked to ensure that they did not
allow the solutions in the different pools to mix. The preparation was
usually stable for ;4 h after stimulation started, but beyond this time
the responses could begin to deteriorate.
The rostral most pool was continuously superfused with Ringer
with a peristaltic pump. The high divalent or low calcium Ringer
in the middle pool was changed regularly with a Pasteur pipette.
Drugs were dissolved in Ringer to the appropriate concentration
and added to the tail fin pool with a Pasteur pipette. Drugs were
applied for 10 min.
Tail fin stimulation was performed with square silver electrodes
(3 3 3 mm) placed on either side of the tail fin (see Fig. 1A) (Brodin
and Grillner, 1985). The tail fin was rotated to one side to facilitate
stimulation, which consisted of a train of 5–50 1-ms pulses of 1–5 mA
at a frequency of 100 Hz. The stimulation typically evoked significant
activity on only one side of the spinal cord, the activated side remaining constant throughout the experiment. Cases in which the activated
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FIG. 1. A: diagram of the isolated spinal
cord-tail fin preparation. The spinal cord is
separated by Vaseline barriers into a tail fin
pool (;15 segments), a middle pool (;20
segments), and a rostral pool. The tail fin is
stimulated electrically by square silver electrodes placed on either side of the tail. Neuromodulators are added to the tail fin bath.
High divalent cation or low calcium/high
magnesium Ringer is placed in the middle
pool to block polysynaptic transmission
through this pool. Thus the input to the rostral pool is due to axons from the caudal pool
that project beyond the middle pool. Extracellular ventral root and intracellular recordings are made from the rostral pool. Graph of
the ventral root burst area (Bi) and traces
showing raw and rectified and integrated
ventral root bursts (Bii) showing that high
divalent cation Ringer reduces ventral root
activity recorded in the rostral pool, presumably because of the reduction of polysynaptic
inputs. The bar underneath the trace indicates the onset and duration of the tail fin
stimulation (see METHODS).
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¨ M, D. PARKER, E. SVENSSON, AND S. GRILLNER
M. ULLSTRO
RESULTS
Effects of substance P on reflex responses
The tachykinin substance P depolarizes mechanosensory
dorsal cells, increases the excitability of dorsal cells and
spinobulbar neurons, and increases dorsal root and dorsal
column-evoked glutamatergic inputs but reduces dorsal column-evoked inhibitory inputs (Parker and Grillner 1996).
The behavioral effect of this sensory modulation was examined by stimulating the tail fin in the isolated spinal cord–
tail fin preparation (see Fig. 2A, inset). Application of substance P (100 nM) to the tail fin pool increased the intensity
of the tail fin stimulation-evoked ventral root activity in the
rostral pool (Fig. 2, A and B; n 5 8/10; P , 0.05). In the
remaining two preparations, substance P had no effect on
the ventral root response. Substance P also increased the tail
fin stimulation-evoked synaptic response in motor neurons
(n 5 3) and unidentified gray matter neurons (n 5 1) in the
rostral pool (Fig. 2D; P , 0.01). In cells that spiked in
control, the number of spikes evoked by the stimulation was
increased (n 5 3; see Fig. 2A). These effects of substance P
partly recovered after washing for 1 h (Fig. 2, B–D).
Substance P has a biphasic concentration-dependent effect
on sensory neurons, the maximal effect occurring with 100 nM
substance P, but a reduced or even opposite effect occurring
with 1 mM (Parker et al. 1997). The modulation of tail finevoked responses also exhibited this concentration-dependent
trend (Fig. 2C); 100 nM substance P usually increased tail fin
stimulation-evoked ventral root responses (n 5 8/10; P ,
0.05), whereas 1 mM substance P had a smaller (n 5 3) or even
opposite (n 5 2) effect.
The tachykinin antagonist Spantide II (4 mM) (Parker and
Grillner 1996) blocked the facilitating effect of 100 nM substance P on tail fin stimulation-evoked ventral root responses
(Fig. 2E; n 5 4/4; P . 0.1), suggesting that the effect was
mediated through tachykinin receptors. A second application
of substance P after washing off Spantide II for $1 h could
result in an increase in the ventral root response (n 5 2/4;
Fig. 2E).
Effects of substance P on tail fin stimulation-evoked
modulation of locomotor activity
Locomotor activity can be elicited in the lamprey spinal cord
by bath application of excitatory amino acids (Cohen and
Wallén 1980; Poon 1980). To examine the effects of sensory
input and its modulation on ongoing locomotor activity,
NMDA was applied to the rostral pool of the isolated spinal
cord-tail fin preparation (Fig. 3Aii). Stimulating the tail fin
increased the frequency of rostral ventral root bursts (Fig. 3;
n 5 4/4; P , 0.05). The addition of substance P (100 nM) to
the tail fin pool had two effects. First, it increased the basal
frequency of NMDA-evoked ventral root responses, presumably because of the tonic activation of ascending neurons
(Parker and Grillner 1998). In addition, substance P facilitated
the increase in burst frequency evoked by tail fin stimulation
(Fig. 3; n 5 4/4; P , 0.05).
Effects of NPY and baclofen on reflex responses
NPY immunoreactivity is found in the lamprey dorsal column and dorsal horn (Parker et al. 1998b) and in close appo-
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side changed during the experiment (n 5 2) were not included in the
analysis.
Extracellular recordings were made from ventral roots in the rostral
pool with glass suction electrodes. Intracellular recordings were also
made from motor neurons or unidentified gray matter neurons in the
rostral pool with thin-walled glass micropipettes filled with 4 M K
acetate and with resistances of 40 – 60 MV. To facilitate intracellular
recordings, in some experiments approximately three segments of the
rostral end of the spinal cord were isolated from the notochord. This
region of the spinal cord was stabilized by placing a plastic net over
it, which was secured by pinning it onto the Sylgard. Care was taken
not to stretch the cord where it was still attached to the notochord.
Motor neurons were identified by recording 1:1 orthodromic spikes in
the adjacent ventral root after current injection into the soma. An
Axoclamp 2A amplifier was used for amplification and in discontinuous current-clamp mode for current injection.
To quantify the effects of neuromodulators on ventral root responses, ventral root activity was analyzed off-line with pClamp
software (version 6, Axon Instruments). The burst was first rectified
and then integrated over time (see Fig. 1Bii). The area of the burst
(i.e., the train of spikes evoked by tail fin stimulation) was measured
as the peak of the integrated trace. The area was measured for as long
as the burst evoked by tail fin stimulation occurred, typically ,500
ms. Three trials were performed in control, in the presence of the drug,
and after wash-off. Each trial was separated by a 2-min interval. This
delay between trials ensured that the stimulation trials themselves did
not cause any changes in the intensity of the ventral root response
(data not shown). The effect of tail fin stimulation on ongoing locomotor activity and its modulation was examined by eliciting fictive
locomotion in the rostral most pool by applying N-methyl-D-aspartate
(NMDA; 150 mM) (Brodin et al. 1985). In this case, the frequency
was measured in 2-s bins, before and after substance P application.
To investigate the cellular and synaptic effects of bombesin, a piece
of the caudal region of the spinal cord (;10 segments) was isolated
from the notochord. Action potentials were elicited in dorsal cells and
spinobulbar neurons by the injection of 1-ms depolarizing current
pulses of 10 –20 nA. Four action potentials were elicited at 1 Hz in
control and then in the presence of bombesin. Stimulation at this
frequency did not cause any activity-dependent changes in the action
potential (data not shown). The spikes in each trial were averaged for
analysis. The spike amplitude was measured from the baseline preceding the spike to the peak of the spike, and the AHP amplitude was
measured from the baseline preceding the spike to the maximum
hyperpolarized potential reached after the spike. The spike duration
was measured at one-half height. Excitability and input resistance
were examined by injecting 100-ms depolarizing or hyperpolarizing
current pulses, respectively, of 1–5 nA into the dorsal cell or spinobulbar neuron somata. Dorsal cells and spinobulbar neurons were
identified by their characteristic shapes and positions in the spinal
cord (Rovainen 1967). Synaptic inputs to spinobulbar neurons were
evoked by stimulating the dorsal column caudal to the spinobulbar
neuron in an isolated piece of spinal cord (;10 segments). High
divalent cation Ringer was used to block polysynaptic inputs when
stimulating the dorsal column. The lateral tracts and gray matter were
lesioned rostral to the stimulating electrode to prevent inputs to
spinobulbar interneurons from neurons in these areas. Axon Instruments software (Axotape and pClamp) was used for data acquisition,
and analysis was performed on a 486 PC computer equipped with an
A/D interface (Digidata 1200, Axon Instruments, CA).
Substance P acetate, bombesin acetate, and porcine NPY were
obtained from Sigma. Baclofen was obtained from Tocris (Bristol,
UK). Unless stated otherwise, statistical significance was examined
with two-tailed, paired t-tests. The statistical values refer to the pooled
data from the number of experiments indicated in each case. Results
are expressed as means 6 SE; n numbers given in the text refer to the
number of animals studied.
MODULATION OF SPINAL REFLEXES IN LAMPREY
1733
sition to dorsal column axons, including the axons of mechanosensory dorsal cells (Bongianni et al. 1990). NPY
presynaptically reduces glutamatergic inputs from the dorsal
cells and reduces the excitability of spinobulbar neurons
(Parker et al. 1998b). Application of NPY (100 nM) (Parker et
al. 1998b) to the tail fin pool of the isolated spinal cord-tail fin
preparation reduced the ventral root activity recorded in the
rostral pool (Fig. 4, A and B; n 5 6/7; P , 0.05). This effect
was associated with a reduction in the peak amplitude of
ascending synaptic inputs to motor neurons (n 5 2) and un-
identified gray matter neurons (n 5 1) in the rostral pool (Fig.
4C; P , 0.05) or with a reduction in the number of spikes
evoked in neurons that spiked in control (n 5 2; Fig. 4A). The
effects of NPY on ventral root activity were greater when the
initial activity level was high. For example, after potentiating
reflex activity with 100 nM substance P, NPY reduced the
response by 61.5 6 11.2 compared with 35.3 6 9 when it was
applied to a control cord (n 5 3; Fig. 4D). These effects
of NPY recovered partially after washing for 1 h (Fig. 4, B
and C).
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FIG. 2. The effects of substance P (100 nM) on ascending inputs and reflex activity. A: activity recorded extracellularly from
a ventral root and intracellularly from a motor neuron in the rostral pool. Substance P increased the intracellular response in the
motor neuron and potentiated the ventral root activity. The spikes are clipped in this and subsequent figures. Inset in this and
subsequent figures shows a schematic diagram of the experimental preparation. B: graph showing data from a single experiment
in which the 3 trials in control, in the presence of 100 nM substance P, and after wash-off are shown. C: graph showing the effect
of 100 nM and 1 mM substance P on the area of the ventral root burst compared with control. Data from 10 experiments is shown
with 100 nM and 5 experiments with 1 mM. D: graph with data from 4 cells in which spikes were not elicited by tail fin stimulation,
showing the effect of substance P (100 nM) on the peak amplitude of the tail fin stimulation-evoked depolarization. E: effects of
substance P (100 nM) on the area of the ventral root burst are blocked by the tachykinin antagonist spantide II (4 mM). Substance
P could result in an increase in ventral root area when spantide II was washed off. Data from 2 experiments are shown in E.
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¨ M, D. PARKER, E. SVENSSON, AND S. GRILLNER
M. ULLSTRO
As in higher vertebrates (see Todd and Spike 1993), NPY
colocalizes with GABA in bipolar cells in the dorsal column
(Parker et al. 1998b). The GABAB receptor agonist baclofen
has complementary but not identical effects to NPY in
presynaptically modulating glutamatergic synaptic transmission from sensory neurons to spinobulbar neurons
(Parker et al. 1998b). The effect of baclofen (10 mM)
(Parker et al. 1998b) on reflex responses was thus also
investigated. Baclofen reduced the ventral root activity in
the rostral pool (Fig. 5, A and B; n 5 4/4; P , 0.01) and also
reduced the peak amplitude of ascending synaptic inputs or
spiking in motor neurons (n 5 4; Fig. 5, A and C). These
effects of baclofen largely recovered after washing for 1 h
(Fig. 5, B and C).
Although baclofen and NPY have qualitatively similar effects on glutamatergic synaptic transmission to spinobulbar
neurons, their effects are quantitatively different in that baclofen has a larger maximal effect that recovers faster than the
effect of NPY (Parker et al. 1998b). A similar quantitative
difference was seen in the effects of baclofen and NPY on tail
fin-evoked responses. Baclofen resulted in a larger reduction of
the ventral root burst area (71 6 4.6) than NPY (35.3 6 9; P ,
0.05, two-tailed, independent t-test). In addition, the extent of
the recovery after washing for 1 h was greater for baclofen than
for NPY (baclofen 43.1 6 4; NPY 16 6 10.3; compare Figs.
4, B and C, and 5, B and C).
Effects of bombesin on cellular, synaptic, and reflex
properties
The previous experiments examined the reflex effects of
neuromodulators previously studied with regard to cellular
and synaptic sensory modulation. In addition, we examined
the effects of the neuropeptide bombesin, which was not
previously examined at any level in the lamprey. Bombesin
colocalizes with 5-HT and CGRP in afferent axons (Brodin
et al. 1988). At the reflex level bombesin (100 nM) mimicked the effects of substance P in that it increased the
ventral root activity in the rostral pool (Fig. 6, A and B; n 5
5/6; P , 0.01) and increased synaptic input and spiking in
motor neurons (Fig. 6, A and C; n 5 3) and unidentified gray
matter neurons (n 5 1; P , 0.05). However, in contrast
to the effects of substance P, no appreciable recovery of
the effects of bombesin were seen after washing for 2 h
(Fig. 6C).
As with substance P (Parker and Grillner 1996), bombesin
(100 –500 nM) did not significantly modulate the mechanosensory dorsal cell input resistance (data not shown), and in
contrast to substance P bombesin did not affect the action
potential properties of any dorsal cell examined (Fig. 7, A and
B; n 5 11). Bombesin (100 nM) had variable effects on the
excitability of mechanosensory neurons, an increase being seen
in 5 of 11 dorsal cells. However, a consistent effect on excitability was seen when the dorsal cells were divided into those
that responded to a 100 –ms depolarizing current pulse with a
train of spikes and those that responded with only one or two
spikes, putatively slowly adapting pressure and rapidly adapting touch-sensitive dorsal cells, respectively (Christenson et al.
1988). Bombesin had an effect on the excitability of only one
of six touch-sensitive cells (Fig. 7C) but increased the excitability in four of five pressure-sensitive cells (Fig. 7D). Increasing the bombesin concentration to 500 nM made the effect
on the excitability of putative pressure-sensitive cells less
consistent, an increase occurring in two of five cells. The
excitability of putative touch-sensitive neurons was again not
affected by higher bombesin concentrations (n 5 6; data not
shown).
Finally, the synaptic effects of bombesin were examined by
evoking depolarizing synaptic potentials in spinobulbar interneurons by extracellular stimulation of the dorsal column,
activating the axons of dorsal cells and other sensory axons.
Bombesin (100 nM) increased the amplitude of the synaptic
potential in spinobulbar neurons (n 5 4/5; P , 0.05) independently of an effect on the input resistance of membrane potential of the spinobulbar neuron. These effects were also long
lasting, recovery occurring after washing in excess of 2 h (Fig.
8, Ai and Aii).
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FIG. 3. The effects of substance P on tail fin stimulation-evoked modulation of locomotor activity. The tail fin was stimulated
at the start of a ventral root burst. This resulted in an increase in the frequency of ventral root activity elicited by N-methyl-Daspartate (150 mM) in the rostral pool (Ai). Each bar represents the frequency in 2-s time bins. Substance P increased the frequency
of ventral root bursts in the absence of tail fin stimulation and potentiated the effects of tail fin stimulation on burst frequency. Data
from four experiments is shown. Aii: traces showing the effects of tail fin stimulation on ventral root activity in control and in the
presence of substance P. The bar under the trace indicates the onset and duration of tail fin stimulation.
MODULATION OF SPINAL REFLEXES IN LAMPREY
1735
DISCUSSION
The possible network relevance of neuropeptide-mediated
sensory modulation, as monitored by reflex responses after
cutaneous stimulation, was examined in this study. The results
suggest facilitatory and inhibitory roles, respectively, for substance P and NPY/baclofen in modulating cutaneous stimulation-evoked responses, in agreement with previous studies at
the cellular and synaptic levels (Parker and Grillner 1996;
Parker et al. 1998b). We have also shown that bombesin
potentiates reflex activity and have examined the cellular and
synaptic mechanisms underlying this effect.
Lampetra NPY was sequenced and has strong homology to
mammalian NPY (Söderberg et al. 1994). In a previous electrophysiological study (Parker et al. 1998b) no significant
difference in the cellular and synaptic effects of synthetic
Lampetra NPY, porcine NPY, and mammalian NPY were
found, suggesting that this peptide is also functionally conserved. Lampetra tachykinins were also sequenced, the functionally important C terminal having strong homology to
higher vertebrates (Waugh et al. 1995). Molluscan, amphibian,
and mammalian tachykinins also had quantitatively similar
effects at the sensory and network levels (Parker and Grillner
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FIG. 4. Effects of neuropeptide Y (NPY; 100 nM) on ascending inputs and reflex activity. A: activity recorded extracellularly
from a ventral root and intracellularly from a motor neuron in the rostral pool. B: graph showing the effect of NPY on the ventral
root burst area. Data from 7 experiments are shown. C: graph with data from 3 cells that did not spike in response to tail fin
stimulation, showing the effect of NPY on the amplitude of the tail fin stimulation-evoked depolarization. D: graph showing the
increased effect of NPY (100 nM) on the ventral root burst area after it was first potentiated by substance P (100 nM). Data from
3 experiments are shown.
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¨ M, D. PARKER, E. SVENSSON, AND S. GRILLNER
M. ULLSTRO
1996; Parker et al. 1998a), again suggesting functional conservation of this peptide. Although the structure of Lampetra
bombesin is currently unknown and thus it is possible that the
commercial peptide used here does not fully match the effects
of endogenous bombesin, the conservation of the functional
effects of NPY and tachykinins suggests that the effects of
exogenous bombesin shown here will resemble those of the
endogenous peptide.
The preparation used (see Fig. 1) was designed to allow the
effects of neuromodulators to be examined on the sensory level
in the absence of modulator-induced effects on motor and
premotor interneurons. Drugs were applied to the caudal-most
region of the spinal cord, where the axons of sensory neurons
activated by tail fin stimulation enter the spinal cord. Low
calcium/high magnesium Ringer or high divalent cation Ringer
was applied to the middle pool to reduce synaptic transmission
or raise the threshold for activation of neurons in the middle
pool (Berry and Pentreath 1976). This will prevent rostral pool
neurons from being activated through a chain of ascending
local interactions, thus limiting the stimulation-induced input
to the rostral pool to caudal pool neurons with ascending axons
that project directly to the rostral pool. This will include dorsal
root and dorsal column axons (Dubuc et al. 1992), including
those of mechanosensory dorsal cells and spinobulbar neurons
(Rovainen 1967, 1974) and propriospinal neurons with long
ascending axons in the lateral column (Vinay et al. 1998).
Dorsal cell, dorsal root, and dorsal column axons make monosynaptic connections with spinobulbar neurons and polysynaptic excitatory and inhibitory connections to motor and premotor
neurons (Birnberger and Rovainen 1971; Rovainen 1967). Although intracellular stimulation of single spinobulbar neurons
was not sufficient to elicit reflex responses (Teräväinen and
Rovainen 1971), the combined activation of sensory dorsal
column axons and spinobulbar neurons appears to mediate tail
fin stimulation-evoked reflex responses (McClellan and Grillner 1983). Thus the neurons examined in the previous cellular
and synaptic studies contribute to the input to motor and
premotor neurons in the rostral pool and thus influence the
ventral root activity elicited in response to tail fin stimulation.
Comparison of the effects of substance P, NPY, and baclofen
at the cellular/synaptic and reflex levels
Substance P has excitatory effects on sensory dorsal cells
and spinobulbar neurons and potentiates dorsal column and
dorsal root-evoked synaptic inputs to spinobulbar neurons
(Parker and Grillner 1996). In the experiments presented here,
substance P was shown to potentiate the effects of ascending
synaptic inputs and increase ventral root activity in the rostral
pool. In addition, the concentration dependence of the effects
on ventral root responses in this study matched the concentration dependence previously found at the cellular and synaptic
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FIG. 5. The effects of baclofen (10 mM) on ascending inputs and reflex activity. A: activity recorded extracellularly from a
ventral root and intracellularly from a motor neuron in the rostral pool. B: graph showing the effect of baclofen on the area of the
ventral root burst. Data from 4 experiments are shown. C: graph with data from 2 cells in which spikes were not elicited by tail
fin stimulation, showing the effect of baclofen on the amplitude of the tail fin stimulation-evoked depolarization and the recovery
after washing for 1 h.
MODULATION OF SPINAL REFLEXES IN LAMPREY
1737
levels (Parker et al. 1997), maximal effects being obtained with
100 nM substance P, but reduced or opposite effects being seen
with higher concentrations.
NPY has opposite cellular and synaptic effects to substance
P in that it reduces the excitability of spinobulbar neurons and
presynaptically depresses dorsal cell, dorsal root, and dorsal
column-evoked inputs to spinobulbar neurons (Parker et al.
1998b). NPY colocalizes with GABA in bipolar neurons in the
dorsal column (Parker et al. 1998b). The GABAB receptor
agonist baclofen qualitatively mimics the effects of NPY in
presynaptically reducing glutamatergic afferent-evoked inputs
to spinobulbar neurons. However, baclofen and NPY have
quantitatively different effects at the synaptic level in that
baclofen has a larger effect that recovers more rapidly than that
of NPY. In this paper, both NPY and baclofen reduced ascending inputs to rostral locomotor network neurons and reduced
rostral ventral root activity. Quantitative differences between
the effects of NPY and baclofen were also seen, baclofen again
having a larger effect than NPY, which recovers to a greater
extent after washing for 1 h. The modulation of reflex activity
is thus consistent with the effects of substance P, NPY, and
baclofen at the cellular and synaptic levels.
Role of bombesin in modulating sensory input
Bombesin (100 nM) increased tail fin stimulation-evoked
ascending inputs and ventral root activity, thus mimicking the
effects of substance P. The cellular and synaptic effects of
bombesin in the lamprey were not previously examined, but
the effects on ascending inputs and reflex responses suggested
that bombesin would have excitatory effects at the cellular and
synaptic levels. As with substance P, bombesin increased the
excitability of the dorsal cells. Unlike substance P, however,
this effect was only seen on putative pressure-sensitive dorsal
cells, i.e., those that responded to a depolarizing current pulse
with a train of spikes (Christenson et al. 1988). Bombesin also
potentiated dorsal column stimulation-evoked inputs to spinobulbar neurons, thus mimicking the effects of substance P on
sensory synaptic transmission. Unlike substance P, however,
bombesin did not affect the membrane potential or action
potential properties of the dorsal cells. Because bombesin did
not affect the resting input resistance or the properties of dorsal
cell action potentials, it may increase the excitability of the
dorsal cells by acting on voltage-activated conductances that
do not contribute significantly to the depolarizing or repolarizing phases of the action potential. The effect of bombesin on
the excitability of putative pressure-sensitive dorsal cells appears to be concentration dependent because, in contrast to 100
nM, 500 nM bombesin had no consistent effect on the excitability of pressure-sensitive dorsal cells (data not shown).
Bombesin facilitates the tail flick reflex in the rat (Cridland and Henry 1992), and the bombesin agonist neuromedin C enhances mechanical nociception in the rat paw
pressure test (Onogi et al. 1995). However, bombesin and
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FIG. 6. The effects of bombesin (100 nM) on ascending inputs and reflex activity. A: activity recorded extracellularly from a
ventral root and intracellularly from a motor neuron in the rostral pool. B: graph showing the effect of bombesin on the area of the
ventral root burst. Data from 6 experiments are shown. C: graph with data from 2 cells in which spikes were not elicited by tail
fin stimulation, showing the effect of bombesin on the amplitude of the tail fin stimulation-evoked depolarization.
1738
¨ M, D. PARKER, E. SVENSSON, AND S. GRILLNER
M. ULLSTRO
neuromedin B and C reduced spontaneous and evoked synaptic inputs to superficial dorsal horn neurons (De Koninck
and Henry 1989), an effect that is not consistent with its
effects on reflex activity. The concentration-dependent effect of bombesin, shown in this study, could contribute to
the inconsistent effects of bombesin at the reflex and cellular
levels (Cridland and Henry 1992; De Koninck and Henry
1989) because the cellular study used ionophoresis of 1 mM
bombesin, and the latter study used intrathecal administration of low nM concentrations. The concentration-dependent effects of bombesin at the reflex and synaptic levels in
the lamprey remain to be investigated.
Role of the sensory modulation
Substance P and bombesin are found in separate populations of
primary afferents (Brodin et al. 1988; Van Dongen et al. 1986),
whereas NPY and GABA are colocalized in bipolar interneurons
in the dorsal column (Parker et al. 1998b). The results of this and
previous studies (Parker and Grillner 1996; Parker et al. 1997)
suggest that activation of substance P and bombesin-containing
afferents will facilitate sensory inputs and thus sensory-evoked
responses through cellular and synaptic effects on other primary
afferents and sensory interneurons. Conversely, activation of
NPY/GABAergic interneurons will inhibit sensory-evoked responses through presynaptic inhibition of afferent synaptic trans-
FIG. 8. Bombesin potentiates afferent synaptic transmission. Ai: effects of bombesin (100 nM) on the excitatory postsynaptic potential (EPSP) elicited in spinobulbar neurons by stimulation of the dorsal column. The
bar indicates the onset and duration of bombesin application. Aii: traces showing averaged EPSPs (n 5 20) in
control and in the presence of bombesin.
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FIG. 7. The effects of bombesin (100 nM) on the cellular properties of the mechanosensory dorsal cells. A: overlaid averaged
(n 5 4) dorsal cell action potentials in control and after application of bombesin, showing the lack of effect of bombesin. B: graphs
showing the lack of effect of bombesin on dorsal cell action potential properties (n 5 11). C: bombesin did not affect the excitability
of dorsal cells that spiked once in response to a 100-ms depolarizing current pulse (rapidly adapting or touch-sensitive cells; n 5
6) but did increase the excitability of neurons that spiked several times in response to the current pulse (slowly adapting or
pressure-sensitive cells; n 5 5) D: insets in C and D show responses to dorsal cells of the 2 types in response to a 100-ms
depolarizing current pulse in control (top) and after the application of 100 nM bombesin for 10 min (bottom).
MODULATION OF SPINAL REFLEXES IN LAMPREY
mission and a reduction in the excitability of sensory interneurons
(Parker et al. 1998b). 5-HT also presynaptically inhibits afferent
inputs (El Manira et al. 1997) and colocalizes with bombesin and
CGRP in primary afferents (Brodin et al. 1988). These afferents
would thus be able to facilitate or inhibit sensory inputs if a
mechanism exists for the selective release of bombesin or 5-HT.
Sensory input to the spinal cord is thus subject to presynaptic
and/or postsynaptic facilitation or inhibition by different spinal or
afferent systems.
CONCLUSION
This work was supported by Swedish Medical Research Council Grants
3026 and 12589, the Karolinska Institute, the Wellcome Trust, and the Swedish
Brain Foundation.
Address for reprint requests: D. Parker, Nobel Institute of Neurophysiology,
Dept. of Neuroscience, Karolinska Institute, S-171 77 Stockholm, Sweden.
Received 14 September 1998; accepted in final form 25 November 1998.
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