Nobel Institute for Neurophysiology, Department of Neuroscience, Karolinska Institute, S-171 77 Stockholm, Sweden
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Ullström, Maria, David Parker, Erik Svensson, and Sten Grillner. Neuropeptide-mediated facilitation and inhibition of sensory inputs and spinal cord reflexes in the lamprey. 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 fin-evoked 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.
![]() |
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 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 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 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 side changed during the
experiment (n = 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 M. 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 µM)
(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 ± SE; n numbers given in the text refer to the number of animals studied.
![]() |
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 = 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 = 3) and
unidentified gray matter neurons (n = 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 = 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 µM (Parker et
al. 1997). The modulation of tail fin-evoked responses also exhibited this concentration-dependent trend (Fig. 2C); 100 nM substance P usually increased tail fin stimulation-evoked ventral root responses (n = 8/10; P < 0.05),
whereas 1 µM substance P had a smaller (n = 3) or
even opposite (n = 2) effect.
The tachykinin antagonist Spantide II (4 µM) (Parker and
Grillner 1996) blocked the facilitating effect of 100 nM
substance P on tail fin stimulation-evoked ventral root responses (Fig. 2E; n = 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 = 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 = 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 = 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
apposition 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 = 6/7; P < 0.05).
This effect was associated with a reduction in the peak amplitude of
ascending synaptic inputs to motor neurons (n = 2) and
unidentified gray matter neurons (n = 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 = 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 ± 11.2 compared with 35.3 ± 9 when it was applied to a control cord
(n = 3; Fig. 4D). These effects of NPY
recovered partially after washing for 1 h (Fig. 4, B
and C).
|
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 µM) (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 = 4/4;
P < 0.01) and also reduced the peak amplitude of
ascending synaptic inputs or spiking in motor neurons (n = 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 ± 4.6) than NPY (35.3 ± 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 ± 4; NPY 16 ± 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 = 5/6; P < 0.01) and increased
synaptic input and spiking in motor neurons (Fig. 6, A and
C; n = 3) and unidentified gray matter neurons (n = 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 = 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 = 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 = 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).
|
![]() |
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 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 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
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 transmission 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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
These results and those of previous papers (Christenson and
Grillner 1991; El Manira et al. 1997
;
Parker and Grillner 1996
; Parker et al.
1998b
) show that the modulation of sensory inputs can
be examined directly at the behavioral, cellular, and synaptic levels
in the intact lamprey spinal cord. In mammalian preparations, the roles
of many neuropeptides are unclear, with sometimes contradictory effects
being reported. For example, bombesin depresses sensory synaptic inputs
to nociceptive neurons in the superficial dorsal horn (De
Koninck and Henry 1989
) but induces or facilitates nociceptive reflex responses (Cridland and Henry 1992
; Mao et
al. 1992
). Somatostatin inhibits responses of dorsal horn
neurons to thermal stimulation (Sandkühler et al.
1990
) but facilitates thermal stimulation-evoked reflexes
(Wiesenfeld-Hallin 1986
). In addition, whereas
somatostatin inhibits nociceptive inputs (Randic and Miletic
1978
), it facilitates nociceptive behaviors, including the
hindlimb flexion reflex and scratching (Wiesenfeld-Hallin
1985
). In this study, it was possible to separate the sensory
effects of neuromodulators from those exerted on premotor interneurons
and motor neurons. This is of crucial importance. For example, in
addition to its sensory effects (Parker and Grillner
1996
), substance P also has specific effects on all components
of the locomotor network (motor neurons and premotor interneurons)
(Parker and Grillner 1998
). Effects of intrathecally
administered modulators at the motor or premotor level could thus
contribute to the variability in the reported effects in mammalian
preparations and the lack of consistency between results at the
cellular and network levels, thus illustrating the difficulties
involved in extrapolating functional effects among different levels in
the nervous system.
Because the types and spinal locations of the neuropeptides examined
here correspond to those in higher vertebrates (Brodin and
Grillner 1990; Brodin et al. 1995
), these
results are presumably also relevant to other vertebrate systems. Given
the possibility to examine the effects of these modulators in the
intact spinal cord at the cellular, synaptic, and reflex levels, the
lamprey provides a useful model system in which to examine the role and underlying mechanisms of neuropeptides in modulating sensory inputs.
![]() |
ACKNOWLEDGMENTS |
---|
This work was supported by Swedish Medical Research Council Grants 3026 and 12589, the Karolinska Institute, the Wellcome Trust, and the Swedish Brain Foundation.
![]() |
FOOTNOTES |
---|
Address for reprint requests: D. Parker, Nobel Institute of Neurophysiology, Dept. of Neuroscience, Karolinska Institute, S-171 77 Stockholm, Sweden.
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 14 September 1998; accepted in final form 25 November 1998.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|