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Centre for Neuroscience and Department of Pharmacology, University of Alberta, Edmonton, Alberta T6G 2H7, Canada
Submitted 2 February 2004; accepted in final form 30 July 2004
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ABSTRACT |
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INTRODUCTION |
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-opioid receptors in the spinal dorsal horn (de Groot et al. 1999
; Zhang et al. 1998). Furthermore, the effect of morphine on N-type Ca2+ current (ICa,N) in DRG cell bodies is reduced after sciatic nerve axotomy (Abdulla and Smith 1998).
By contrast, nerve injury upregulates NPY Y2 receptor expression and increases the ability of Y2 agonists to suppress ICa,N in DRG (Abdulla and Smith 1999; Mantyh et al. 1994; Zhang et al. 1994b). Moreover, expression of NPY and its mRNA increase in DRG and dorsal horn after axotomy or chronic constriction injury (Munglani et al. 1996; Wakisaka et al. 1991). Because NPY acts as an analgesic when applied intrathecally, its receptors represent an attractive target for analgesics that may retain their effectiveness in neuropathic pain states (Hua et al. 1991; Xu et al. 1994). Furthermore, the cellular actions of opioids, which involve suppression of calcium currents, decreased transmitter release, and activation of G-protein-coupled, inwardly-rectifying K+ (GIRK) currents (Grudt and Henderson 1998; Grudt and Williams 1993, 1994; Hori et al. 1992; Yoshimura and North 1983), are shared by NPY (Bleakman et al. 1991; Colmers and Bleakman 1994; Colmers et al. 1991; Qian et al. 1997; Toth et al. 1993; Zidichouski et al. 1990). We have therefore examined the effects of NPY and NPY receptor-selective ligands on dorsal horn neurons of adult rats. Actions of NPY were also compared with those of the µ-opioid agonist, [D-Ala2, NMe-Phe4, Gly-ol5]-enkephalin (DAMGO). We find that NPY acts on a presynaptic Y2 receptor to attenuate excitatory transmission and on a presynaptic Y1 receptor to attenuate both GABAergic and glycinergic inhibitory transmission. Postsynaptic mechanisms may also contribute to the attenuation of inhibition. The majority of excitatory synapses affected by NPY are affected similarly by DAMGO. Both drugs also have similar effects at inhibitory synapses. Because Y1 agonists produce analgesia when administered intrathecally, (Taiwo and Taylor 2002), selective suppression of inhibition in the substantia gelatinosa (SG) may produce an anti-nociceptive effect that is shared by other agonists including opioids (Grudt and Henderson 1998).
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METHODS |
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1–2 ml/min) at room temperature (22°C) with 95% O2-5% CO2 saturated artificial cerebrospinal fluid (ACSF, in mM) 127 NaCl, 2.5 KCl, 1.2 NaH2PO4, 26 NaHCO3, 1.3 MgSO4, 2.5 CaCl2, and 25 D-glucose, pH 7.4. The SGs appeared as translucent bands under infrared differential-interference optics, and cells were patched under visual control. For recording excitatory evoked postsynaptic currents (eEPSCs), bicuculline (10 µM) and strychnine (1 µM) were included to block inhibitory synaptic inputs. For recording evoked inhibitory postsynaptic currents (eIPSCs), 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; 10 µM) and D-2-amino-5-phosphopentanoic acid (D-AP5; 50 µM) were included to block excitatory synaptic inputs. Tetrodotoxin (TTX; 1 µM) was included when recording miniature EPSCs and IPSCs (mEPSC and mIPSC). Pipettes for recording postsynaptic K+ currents had resistances of 5–10 M
when filled with an internal solution containing (in mM) 130 potassium gluconate, 1 MgCl2, 2 CaCl2, 10 HEPES, 10 EGTA, 4 Mg-ATP, and 0.3 Na-GTP, pH 7.2, 290–300 mosM. For recording synaptic currents, a Cs+-based internal solution containing (in mM) 140 CsCl, 5 HEPES, 10 EGTA, 2 CaCl2, 2 Mg-ATP, and 0.3 Na-GTP, pH 7.2, 290–300 mosM was used.
When recording eEPSCs and eIPSCs, the voltage-gated ion channel blocker QX-314 (5 mM) (Strichartz 1973) was included in the internal solution to prevent action potential discharge. QX-314 was omitted from the internal solution when recording postsynaptic K+ currents. Membrane potential was clamped at –60 or –70 mV for recording eEPSCs and eIPSCs when a CsCl-based internal was used. The ECl was therefore 0 mV, and eIPSCs appeared as inward currents at a holding potential of –60 or –70 mV. When a K+-gluconate-based internal solution was used, membrane potential was clamped at –60 or –70 mV for recording eEPSCs and postsynaptic K+ currents and 0 mV for recording eIPSCs. Under these conditions, ECl = –78mV, so eIPSCs appear as outward currents. eEPSCs were evoked at 0.05 Hz with a bipolar concentric stimulating electrode (FHC, Bowhoidon, ME) or a custom-made bipolar, Teflon-coated nichrome stimulating electrode. These were placed on the dorsal root or near the dorsal root entry zone to activate primary afferent fibers. eIPSCs were generated at 0.05 Hz by focal stimulation (positioned 50–100 µm from the cell body) with a patch pipette containing 2 M NaCl. The stimulating electrode was repositioned until a reliable synaptic input to the cell was found. The orientation of the stimulating electrode, relative to the cell body, varied in the dorsoventral and lateromedial axes. Stimulus intensity was between 2 and 30 V for eEPSCs and eIPSCs. Stimulus duration was 100 or 400 µs for both eEPSCs and eIPSCs. Monosynaptic eEPSCs and eIPSCs were identified by their ability to follow high-frequency stimulation (10–20 Hz) with constant latency and the absence of failures. To examine the effect of NPY or DAMGO, eEPSCs or eIPSCs were generated once per 20 s and three to six events were averaged electronically. Averaged events were acquired for 
10 min prior to superfusion of the drugs. Stimulation continued throughout drug superfusion and washout. NPY and/or DAMGO were deemed effective at a particular synapse when they produced >20% reversible or partly reversible depression of the evoked synaptic responses. For statistical analysis of drug effects, mean percentage suppression of eIPSCs or eEPSCs was calculated only from responsive cells (i.e., nonresponsive cells that displayed 0 to <20% change in synaptic events during drug exposure were excluded from the calculation of the mean effect). Because relatively high concentrations of peptides were used (300 –1000 nM), bona fide responses were easily distinguished form alterations in recording conditions. This was because most peptide responses involved a clear 40–60% suppression of the eEPSC or eIPSC. To avoid possible consequences of receptor desensitization after multiple drug applications, only one neuron per slice was exposed to NPY or its analogues.
Data were acquired and analyzed using pCLAMP 8.0 (Axon Instruments, Burlingame, CA). Figures were produced with Origin 5.0, 6.1 or 7.0 (OriginLab, Northampton, MA) and Adobe Illustrator 10 (Adobe Software, San Jose, CA). Statistical comparisons were made with paired t-test, unless otherwise specified, using GraphPad InStat 3.05 (GraphPad Software, San Diego, CA). Statistical significance was taken as P < 0.05.
Mini Analysis Program software (Synaptosoft, Decatur, GA) was used to distinguish mEPSCs and mIPSCs from baseline noise. Details of this program and its implementation may be found at www.synaptosoft.com. Miniature postsynaptic currents were detected automatically using an amplitude threshold of 10 pA and an area threshold of 15 fC. All detected events were then re-examined and visually accepted or rejected based on subjective visual examination. Mini Analysis Program was used to further analyze the data and to generate figures.
Cumulative distribution plots were generated to compare the effects of various drugs on the amplitude and interevent intervals of mEPSCs and mIPSCs. Cumulative distribution plots ranked individual amplitudes or interevent intervals in order of increasing size and plotted this rank value against the amplitude or interevent interval size. The Kolmogorov-Smirnov two-sample test was used to compare control and drug distributions of amplitudes and interevent intervals. The Kolmogorov-Smirnov method tested the null hypothesis that two independent samples come from populations that are identical with respect to location and distribution. The Kolmogorov-Smirnov test was used because it compared the entire distribution of amplitudes or interevent intervals rather than mean amplitude or mean interevent intervals (Van der Kloot 1991). Distributions were considered statistically significant if P < 0.05. For each cell, typically 500 to 2000 events were analyzed. Ten minutes was allowed for stabilization of the cell after patch-rupture. Baseline miniature events were recorded for a further 10–15 min prior to application of NPY (or NPY analogues) for 5–6 min. An obvious effect of the peptide on mEPSC or mIPSC frequency could usually be seen 3 min after the start of wash-in. Miniature events were recorded for a further 5–10 min in the presence of NPY (or NPY analogue).
Neuropeptide Y was from Tocris (Ballwin, MO) or from Peptidec Technologies (Montreal, PQ, Canada). The Y1-agonist [Leu31, Pro34] NPY and the Y2-agonist NPY 13–36 were from Tocris. The Y1-selective agonist [F7, P34] NPY and the Y2-selective agonist [ahx5–24] NPY were kindly provided by Dr. Annette Beck-Sickinger (Institute for Biochemistry, University of Leipzig, Leipzig, Germany). CNQX, 2-amino-5-phosphonovaleric acid (D-AP5), bicuculline and ruthenium red were from Tocris. QX-314 was from Astra Zeneca. Strychnine Hydrochloride was from RBI. All other chemicals from Sigma (Oakville, ON, Canada).
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RESULTS |
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eEPSCs were recorded at a holding potential (Vh) of –70 mV after stimulation of the dorsal root or dorsal root entry zone in the presence of bicuculline (10 µM) and strychnine (1 µM). NPY (300 nM or 1 µM) reduced eEPSC amplitude by 45.5 ± 4.6% in 17/24 cells tested (n = 17; P < 0.0001, paired t-test). In the cell illustrated in Fig. 1A, left, the effect of NPY took 10 min to develop and recovered over a period of 10 min (Fig. 1A, right). However, the time course of action of NPY varied from cell to cell; wash in times ranged from 5 to 10 min and washout times ranged from 10 to 60 min or longer.
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) did not affect eEPSC amplitude in any of the six cells tested (n = 6, P > 0.05, paired t-test; Fig. 1B). By contrast, the mixed Y2/Y5 agonist, NPY 13–36 (300 nM) reduced eEPSC amplitude by 45.5 ± 5.9% (n = 8/14 cells tested, P < 0.005; Fig. 1C). Because NPY 13–36 has been shown to bind to Y5 receptors (Gerald et al. 1996), a more selective Y2 agonist, [ahx5–24]NPY (Rist et al. 1996) was also tested. [ahx5–24]NPY (500 nM or 1 µM) reduced the amplitude of the eEPSC by 38.6 ± 6.1% (n = 9/13 cells tested, P < 0.005, paired t-test; Fig. 1D). The effects of NPY agonists on eEPSCs are summarized in Fig. 1E.
Although we always attempted to obtain a steady baseline level for eEPSC amplitude for 10min prior to drug application, some recordings were associated with rundown, and recordings that displayed >40% rundown during 40 min wererejected. In other cells, rundown was quite variable, and because data were collected from cells that displayed 0 to 33% rundown during 40 min of recording, the reported values of the effectiveness of NPY and Y-2 agonists likely reflect an overestimate of their ability to attenuate eEPSC amplitude.
Presynaptic Y2 receptors are involved in NPY suppression of excitatory synaptic transmission
The effect of NPY on paired eEPSCs was examined to characterize further the mechanism by which it reduced eEPSC amplitude. Paired-pulse stimulation can produce either facilitation or depression. Facilitation is thought to reflect an enhancement in neurotransmitter release due to the transient accumulation of Ca2+ close to release sites (Zucker and Regehr 2002). By contrast, short-term paired-pulse depression is thought to reflect decreased transmitter release from the presynaptic terminal. This may involve depletion of transmitter stores, failure of the action potential to invade the presynaptic terminal, activation of presynaptic autoreceptors, a reduction in activity-dependent ICa responsible for transmitter release (Zucker and Regehr 2002) or postsynaptic receptor desensitization (Mennerick and Zorumski 1996; Trussell et al. 1993). A postsynaptic effect of NPY would be assumed to affect the first and second synaptic responses equally, whereas a presynaptic effect would be expected to alter the ratio of synaptic current amplitudes.
In the experiment illustrated in Fig. 2A, two stimuli separated by an interstimulus interval (ISI; 50 ms) resulted in slight paired-pulse facilitation of eEPSCs. In the presence of NPY, however, the amplitude of the second eEPSC was considerably smaller than the first. In Fig. 2B, the eEPSCs have been normalized to the amplitude of eEPSC1 to better illustrate the change in the paired-pulse ratio. For all neurons studied, the mean ratio of the amplitude of the paired eEPSCs with ISIs of 30–100 ms was 1.6 ± 0.3 (eEPSC2/eEPSC1; n = 7). In 7 of 13 cells, NPY (300 nM) decreased the mean ratio of eEPSC2/eEPSC1 to 0.8 ± 0.2 (n = 7, P < 0.05, paired t-test, Fig. 2C). This is consistent with a presynaptic effect of NPY.
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Effects of NPY on evoked inhibitory synaptic transmission
Unless stated otherwise, eIPSCs were recorded at –70 mV after focal stimulation with a patch electrode in the presence of AP5 (50 µM) and CNQX (10 µM). Effects of NPY and Y1- and Y2-selective agonists are illustrated in Fig. 3. Representative recordings of three to six digitally averaged traces are illustrated in the left panels and the time course of effect of each agonist on eIPSCs in each individual neuron is illustrated in the right panels.
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) (1 µM) reduced eIPSC amplitude by 52.0 ± 4.7% in 6/12 cells tested (n = 6; P < 0.0002, paired t-test; Fig. 3B). Similarly, the Y1/Y5 agonist [Leu31,Pro34]NPY (500 nM) (Gerald et al. 1996) reduced eIPSC amplitude by 48.1 ± 13.3% in 5/9 cells tested (n = 5; P < 0.01, paired t-test, data not shown). By contrast, the Y2/Y5 agonist, NPY 13–36 (300 nM; n = 14, Fig. 3C) and the selective Y2 agonist [ahx5–24]NPY (n = 4, Fig. 3D) did not affect eIPSC amplitude. Figure 3E summarizes the effects of NPY agonists on eIPSCs. The amplitude of eIPSC displayed a certain amount of rundown, but this was generally less than that seen with eEPSCs. Because data were collected from cells in which eIPSC rundown ranged from 0 to 25% in 40 min, our measurements of the effectiveness of NPY and Y1 agonists represent a slight overestimate.
Mechanism of Y1 receptor suppression of inhibitory synaptic transmission
Paired-pulse experiments with eIPSCs were complicated by the duration of the events studied. With interevent intervals >100 ms, little or no paired-pulse facilitation or suppression could be seen. It was therefore necessary to use shorter intervals but this meant that the second eIPSC occurred before the first had completely abated. The amplitudes of the first and second eIPSCs were therefore measured from "foot to peak." Even with short interevent intervals, responses were associated with considerable inter-trial variability. For example, in a trial where the first pair of stimuli exhibited paired-pulse facilitation, the next pair of stimuli exhibited either paired-pulse depression or facilitation. When 10–20 successive paired eIPSCs were averaged, the response usually exhibited overall paired-pulse facilitation, but the mean ratio of the amplitude of the paired eIPSCs was only 1.04 ± 0.11 (eIPSC2/eIPSC1; n = 5). In 5/8 cells, NPY (300 nM) decreased the mean ratio of eIPSC2/eIPSC1 to 0.79 ± 0.11 (n = 5, P < 0.003, paired t-test). These data are consistent with a presynaptic action of NPY. In the cell illustrated in Fig. 4A, there is an initial pronounced paired-pulse facilitation that is clearly attenuated in the presence of NPY. In Fig. 4B, the eIPSCs have been normalized to the amplitude of eIPSC1 to better illustrate the change in the paired-pulse ratio. Figure 4C summarizes the effect of NPY on the paired-pulse ratio of eIPSCs.
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The Y1 agonist [F7, P34]NPY (1 µM) reduced both mIPSC frequency (n = 4/7 cells tested; Kolmogorov-Smirnov test, P < 0.05 for all 4 cells, Fig. 4, G and H), and mIPSC amplitude (n = 4/7 cells tested; Kolmogorov-Smirnov test, P < 0.05 for all 4 cells, Fig. 4I). This is consistent with the presence of Y1 receptors on presynaptic inhibitory terminals and likely also postsynaptically on SG neurons. (Zhang et al. 1994a, 1999).
NPY actions on GABA- and glycine-mediated synaptic currents
Because GABA- and glycine-mediated events are generated by focal stimulation in dorsal horn (Chery and de Koninck 1999; Grudt and Henderson 1998), it is possible NPY may have differentially affected GABA- and glycine-mediated eIPSCs. We therefore compared its effect on pharmacologically isolated GABA-mediated eIPSCs with its effect on glycine-mediated eIPSCs.
Focal stimulation in the presence of strychnine (1 µM), AP5, and CNQX generated eIPSCs that were GABAergic as they were antagonized by bicuculline (Moran 2003). NPY (300 nM) reduced the amplitude of these GABAergic eIPSCs in 3/6 cells tested by 36.5 ± 2.0% (n = 3; P < 0.001, paired t-test; Fig. 5A). Focal stimulation in the presence of bicuculline (10 µM), AP5, and CNQX generated glycinergic eIPSCs that were antagonized by strychnine (Moran 2003). NPY (300 nM) reduced the amplitude of the glycinergic eIPSC in 3/6 cells tested by 38.3 ± 4.2% (n = 3; P < 0.005, paired t-test; Fig. 5B). The effect of NPY on GABA- and glycine-mediated eIPSCs is consistent with the observation that GABA and glycine are often co-released from interneurons in the spinal cord (Chery and de Koninck 1999; Jonas et al. 1998; Li et al. 1999).
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Because intrathecal application of NPY produces analgesic effects (Naveilhan et al. 2001; Taiwo and Taylor 2002), we compared its actions with those of the µ-opioid agonist, DAMGO.
The effects of NPY and DAMGO on the eEPSC were similar. NPY reduced the amplitude of the eEPSC by 45.5 ± 4.6% (n = 17/24; P < 0.0001, paired t-test) and DAMGO reduced it by 59.4 ± 7.4% (n = 9/9; P < 0.0005, paired t-test). Figure 6A illustrates superimposed recordings of digitally averaged eEPSCs before, during and after application of 1 µM NPY, and B illustrates the effect of 1 µM DAMGO on eEPSCs in the same cell. Time course of effects of the two agonists on this cell are illustrated in Fig. 6C.
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Postsynaptic actions of NPY
NPY activates an inwardly-rectifying K+ conductance in a variety of neuron types (Sun and Miller 1999; Sun et al. 2001; Zidichouski et al. 1990), and a similar effect was seen in some SG neurons. NPY (300 nM-1 µM) activated an inwardly rectifying conductance in 14/53 cells tested (26.4%). This was reflected by a 49.0 ± 6.0 pA increase in current at –140 mV (Fig. 6, D–F). Because the postsynaptic response to NPY was observed infrequently, it was not feasible to investigate the receptor subtype involved. Similarly and in confirmation of previous reports, DAMGO (1 µM) activated an inwardly rectifying K+ current of 60.5 pA ±10.0 pA at –140 mV in 8/32 (25%) cells tested (Fig. 6, G–I) (Grudt and Williams 1994; Schneider et al. 1998). The magnitude of the current activated by NPY was not statistically different from that activated by DAMGO and the number of cells that responded to either ligand was similar. However, only 2/27 cells exhibited postsynaptic responses to both NPY and DAMGO, suggesting that there may be differential expression of NPY receptors and µ-opioid receptors on the cell bodies of SG neurons.
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DISCUSSION |
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Y2 suppression of excitatory synaptic Transmission
The finding that presynaptic Y2-receptor activation suppressed EPSCs is supported by the paired-pulse data with NPY and by the effect of NPY and/or the Y2-selective agonist, [ahx5–24]NPY, on mEPSC frequency but not amplitude distribution. Although the decrease in eEPSC2: eEPSC1 ratio seen with in paired-pulse experiments NPY could reflect rapid desensitization of AMPA and/or N-methyl-D-aspartate receptors (Trussell et al. 1993; Mennerick and Zorumski 1996), the mEPSC data are less equivocal. Moreover, a putative presynaptic Y2 effect is consistent with binding and in situ hybridization studies that show Y2 receptor expression on primary afferent terminals in lamina II (Mantyh et al. 1994; Zhang et al. 1995) and a report of Y2 suppression of glutamate release from spinal cord synaptosomes (Martire et al. 2000). Because the effects of NPY were slow to develop and very slow to wash out, it was difficult to obtain more than two or three responses in a given neuron. This precluded determination of concentration dependence of NPY effects and complicated studies with antagonists.
Although the precise mechanism of this presynaptic Y2 effect remains to be determined, it is noteworthy that Y2 receptors are found on the cell bodies of small DRG neurons and these receptors are negatively coupled, via G proteins, to N-type calcium channels (Abdulla and Smith 1999; Bleakman et al. 1991; Walker et al. 1988). Therefore it is plausible that these receptors are transported to the central terminals of primary afferents where they couple to N-type calcium channels and thereby regulate transmitter release. NPY decreases Ca2+ influx through N-type calcium channels in the terminals of sympathetic neurons (Toth et al. 1993). Because NPY and Y2 agonists decreased the frequency of TTX-R mEPSCs (Fig. 2, D and E), they also likely modulate the vesicle release machinery. A similar effect of NPY has been described in the arcuate nucleus of the hypothalamus (Rhim et al. 1997). It should also be mentioned that mEPSCs recorded in our experiments may have originated from local interneurons or descending inputs (Holets et al. 1988) as well as from primary afferent terminals. We therefore cannot rule out the possibility that NPY affects presynaptic effects on terminals of local interneurons, terminals of descending inputs as well as primary afferent terminals via actions on presynaptic Ca2+ channels and/or release machinery.
Y1 Suppression of inhibitory synaptic transmission
The finding that presynaptic Y1 receptors suppressed inhibitory synaptic transmission is supported by the paired-pulse experiments with NPY. Because the ratio of eIPSC2 to eIPSC1 was decreased, there is again a concern about possibility of rapid GABA/glycine receptor desensitization in the presence of NPY. It was also necessary to evoke the second eIPSP before the first had completely abated (see Fig. 4A) as paired-pulse facilitation could not be reliably attained with longer intervals. Because the currents are additive and eIPSCs were measured from foot to peak, the measurements likely give a reasonable measure of how much transmitter is released. Because the ratio of eIPSC2 to eIPSC1 always changed in the presence of NPY, its effects are best explained in terms of a presynaptic site of action. Moreover, NPY and the Y1-selective agonist, [F7, P34]NPY also affected mIPSC frequency. The mIPSC frequency decrease caused by [F7, P34]NPY is consistent with a report of presynaptic Y1 receptors in the dorsal horn (Brumovsky et al. 2002). It is also consistent with the possibility that a direct effect on release process contributes to Y1 inhibition of neurotransmitter release.
In some cells, NPY and [F7, P34]NPY also decreased mIPSC amplitude distributions, suggesting an additional postsynaptic action. This is consistent with reports that Y1 receptors are localized to the dendrites of somatostatin (SST)-positive dorsal horn neurons (Zhang et al. 1994a, 1999). SST-containing neurons in the dorsal horn represent a morphologically heterogeneous population that is composed of both stalked and islet cells (Ribeiro-da-Silva and Cuello 1990; Todd and Spike 1993), some of which are thought to be excitatory interneurons (Todd and McKenzie 1989; Spike and Todd 1992). Although the postsynaptic mechanism by which NPY or [F7, P34]NPY affect mIPSC amplitude remains to be elucidated, these agonists may have activated an inwardly rectifying K+ conductance in the dendrites of SG neurons. This mechanism has been reported to modulate dendritic excitability in both hippocampal (Drake et al. 1997; Ponce et al. 1996) and cortical neurons (Takigawa and Alzheimer 1999). Another possibility may involve cross-talk between the Y1 receptor and postsynaptic GABAA and/or glycine receptors, as has been described for dopamine G-protein-coupled receptors and GABAA receptors (Liu et al. 2000).
It is not known if NPY receptors are differentially expressed on GABAergic or glycinergic terminals, but the ability of NPY to suppress both GABA- and glycine-mediated currents is consistent with studies that show these neurotransmitters are often co-localized and -released from synapses in the superficial dorsal horn (Chery and de Koninck 1999; Li et al. 1999; Todd et al. 1996).
Comparison of NPY and DAMGO
The actions of NPY and Y2 agonists on excitatory synaptic transmission were quantitatively very similar to those of the µ-opioid, DAMGO (Kohno et al. 1999). Because synapses affected by NPY were also affected by DAMGO, this is consistent with the involvement of (presynaptic) Y2 receptors in the modulation of nociceptive information. Several other lines of evidence support this hypothesis. First, NPY and opioid receptor binding sites in the superficial dorsal horn are reduced after dorsal rhizotomy or by capsaicin treatment (Gouarderes et al. 1985; Kar and Quirion 1992). Second, NPY and morphine inhibit the release of substance P from nociceptive primary afferents (Duggan et al. 1991; Jessell and Iversen 1977). Third, NPY and opioid agonists both produce analgesia when applied intrathecally or intracerebroventricularly (Broqua et al. 1996; Hua et al. 1991; Xu et al. 1994).
NPY and DAMGO activated a GIRK conductance of similar magnitude in 25% of the cells tested. However, unlike the effects on excitatory synaptic transmission, cells that expressed postsynaptic NPY receptors did not respond to DAMGO and vice versa, suggesting that NPY and µ-opioid receptors are not co-localized on the same postsynaptic membrane. Given the rather low proportion of NPY-sensitive neurons, it was not feasible to carry out a detailed analysis of concentration dependence or receptor subtype dependence of the postsynaptic effect.
The similarity of Y2-mediated suppression of excitatory transmission to µ-opioids provides a mechanistic explanation for the analgesic effect of NPY. However, the observation that Y1 agonists selectively suppress inhibitory transmission but still produce analgesia (Broqua et al. 1996; Naveilhan et al. 2001; Taiwo and Taylor 2002) is difficult to reconcile. DAMGO and Y1 agonists have similar effects in that they both suppress eIPSCs in 50–60% of dorsal horn cells (Grudt and Henderson 1998; Marinelli et al. 2002; Moran and Smith 2002). By contrast, high concentrations of the morphine metabolite, morphine 3 glucuronide (M3G), suppresses all inhibitory synapses in the SG (Moran and Smith 2002) and cause hyperalgesia rather than analgesia (Woolf 1981). This implies that, regardless of the exact circuitry involved, suppression of a fraction of inhibitory synapses, as is seen with opioids or Y1 agonists, leads to an analgesic effect, whereas suppression of all synapses, as seen with M3G can produce hyperalgesia.
Comparison with in vivo studies
The overall spinal actions of NPY are complex (Broqua et al. 1996; Hua et al. 1991; Taiwo and Taylor 2002; Xu et al. 1994) as it has a biphasic dose-effect curve with pro-nociceptive actions at low doses and anti-nociceptive actions at high doses (Xu et al. 1999; Xu et al. 1994). Despite this, the analgesic effect of NPY appears to be mediated via Y1 receptors in intact rats, while Y1 and Y2 mediate its anti-nociceptive actions after axotomy (Xu et al. 1999). Further support for Y1-mediated anti-nociception is provided by Y1 receptor knockout mice, which have increased pain responses to a variety of modalities before and after nerve injury (Naveilhan et al. 2001).
In summary, NPY and receptor-specific agonists potently modulate synaptic transmission in the SG. Y1 receptors selectively suppress inhibitory transmission by pre- and postsynaptic mechanisms, whereas Y2 receptors suppress excitatory transmission by a presynaptic mechanism. NPY also activates an inwardly rectifying conductance in a population of SG neurons. These effects of NPY are comparable to the actions of µ-opioids and suggest NPY has promise as an analgesic for the treatment of neuropathic pain.
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GRANTS |
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ACKNOWLEDGMENTS |
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Present address of T. D. Moran: Programmes in Brain and Behaviour, The Hospital for Sick Children, Toronto, Ontario M5G 1X8, Canada.
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FOOTNOTES |
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Address for reprint requests and other correspondence: P. A. Smith, Dept. of Pharmacology, University of Alberta, 9-75 Medical Sciences Bldg., Edmonton, Alberta, T6G 2H7, Canada (E-mail peter.a.smith{at}ualberta.ca).
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