Presynaptic and Interactive Peptidergic Modulation of Reticulospinal Synaptic Inputs in the Lamprey

David Parker

Nobel Institute for Neurophysiology, Department of Neuroscience, Karolinska Institute, S-17177 Stockholm, Sweden


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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.


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 MOmega ). 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(lambda )] 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.


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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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Fig. 1. Neuropeptide-mediated reduction of the amplitude of reticulsopinal-evoked excitatory postsynaptic potentials (EPSPs). A: graph showing the reduction in the amplitude of the chemical component of the EPSP (EPSPchem) by peptide YY (PYY; n = 16), cholecystokinin (CCK; n = 11), neuropeptide Y (NPY; n = 11), and calcitonin-gene-related peptide (CGRP; n = 18). The peptides did not, however, significantly affect the amplitude of the electrical component of the EPSP (EPSPelect; PYY, n = 5; CCK, n = 5; NPY, n = 6; CGRP, n = 8). B: traces showing the reduction of monosynaptic reticulospinal-evoked EPSPs by the different peptides. Notice that only CGRP affected the presynaptic action potential. C: graph plotting the mean amplitude and 1/CV2 of reticulsopinal-evoked EPSPs. An example from a single experiment with each peptide is shown.

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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Table 1. Summary table showing the inhibitory effects of PYY, CCK, NPY, and CGRP on reticulospinal synaptic transmission to spinal cord neurons

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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Fig. 2. Effects of CGRP on reticulospinal synaptic transmission. A1: graph showing the effects of CGRP (100 nM) on the monosynaptic EPSP from a reticulospinal axon to a motor neuron in a single experiment. A2: traces showing the effects of CGRP on the reticulospinal EPSP. Note that CGRP could reduce the amplitude and duration of the presynaptic reticulospinal action potential, but increase the AHP amplitude, an effect that partially recovered after washing for 1 h. B1: graph showing the lack of effect of CGRP (100 nM) on responses to pressure-applied glutamate (1 mM), again in one experiment. Inset: overlaid averaged (n = 10) responses evoked by glutamate application (indicated by the bar under the traces) in control and in the presence of CGRP (scale 1 mV, 1 s). B2: graph showing the lack of effect of CGRP on the postsynaptic input resistance. Traces on the graph show the effect of CGRP on voltage responses evoked by 1-nA hyperpolarizing current pulses (scale, 100 ms, 12 mV). C1: frequency histogram showing the occurrence of spontaneous miniature EPSPs (mEPSPs) recorded in the presence of TTX (1.5 µM) and strychnine (5 µM) in control and in the presence of CGRP (100 nM) in a single experiment. Inset: cumulative probability plot of the same data shown on the histogram (, control; , CGRP). C2: traces showing spontaneous mEPSPs in control and in the presence of CGRP.

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(lambda ) > 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(lambda ) > 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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Table 2. Interactive effects of reticulospinal and sensory neuropeptides



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Fig. 3. Brain stem peptides or sensory peptides have additive inhibitory effects on reticulospinal synaptic transmission. A1: graph showing the additive inhibitory effect of CCK (100 nM) on the amplitude of monosynaptic reticulospinal inputs when it was applied after, but in the presence of, PYY (100 nM). A2: traces showing the additive inhibitory effects of PYY and CCK on the amplitude of the reticulospinal synaptic input. Notice that the presynaptic action potential and the electrical component of the EPSP were again not affected. B: graph showing the additive inhibitory effect of NPY (100 nM) on the amplitude of monosynaptic reticulospinal inputs when it was applied after, but in the presence of, CGRP (100 nM).

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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Fig. 4. Interactive effects of brain stem and sensory peptides on reticulospinal synaptic transmission. A: graph showing the additive inhibitory effect of CCK (100 nM) when it was applied in the presence of CGRP (100 nM). B: graph showing the additive effect of PYY (100 nM) when it was applied in the presence of NPY (100 nM). C1: graph showing the effect of CGRP (100 nM) when it was applied after, but in the presence of, CCK (100 nM). Note that in this case, CGRP increased the amplitude of the reticulospinal input, reversing the CCK-mediated depression. C2: traces showing the effects of CGRP when it was applied in the presence of CCK. Notice that CGRP increased the amplitude of the EPSP back to the control value, but that there was no effect on the presynaptic action potential or the electrical component of the EPSP.

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(lambda ) > 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(lambda ) > 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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Fig. 5. Interactive facilitation of reticulospinal inputs appears to be mediated presynaptically. A: CGRP (100 nM) did not have any effect on postsynaptic responses to pressure applied glutamate (1 mM) when it is applied in the presence of CCK (100 nM). Traces on the graph show the glutamate-evoked responses in control, in CCK, and in CCK and CGRP. B: CGRP (100 nM) also failed to affect the amplitude of mEPSPs when applied in the presence of CCK (100 nM). Inset: cumulative probability plot from a different experiment to that shown in the histogram. Responses in CCK and in CCK () and CGRP are shown, although the lack of an effect means that the 2 plots overlap entirely.

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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Fig. 6. Neuropeptide-mediated modulation of ventral root responses evoked by brain stem stimulation. A: graph showing single and interactive effects of CCK and CGRP on rectified and integrated ventral root responses and compound EPSP amplitudes after stimulation of the brain stem. The effects of CCK and CGRP are expressed as percent of the control response. Interactive effects of CCK in the presence of CGRP, and CGRP in the presence of CCK, are expressed as the percent change of the response in the presence of the 1st peptide. B: traces showing the effect of CGRP (100 nM) on ventral root responses and the compound EPSP amplitude in a motor neuron after brain stem stimulation. C: traces showing a ventral root response to brain stem stimulation, and its modulation by CCK (100 nM), and by CCK and CGRP (100 nM).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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.


    ACKNOWLEDGMENTS

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).


    FOOTNOTES

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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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