Synaptic Actions of Neuropeptide FF in the Rat Parabrachial Nucleus: Interactions With Opioid Receptors

Xihua Chen,1 Jeffrey A. Zidichouski,2 Kim H. Harris,2 and Jack H. Jhamandas2

 1Division of Basic Medical Sciences, Memorial University of Newfoundland, St. John's, Newfoundland A1B 3V6; and  2Department of Medicine (Neurology) and Division of Neuroscience, University of Alberta, Edmonton, Alberta T6G 2B7, Canada


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
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DISCUSSION
REFERENCES

Chen, Xihua, Jeffrey A. Zidichouski, Kim H. Harris, and Jack H. Jhamandas. Synaptic Actions of Neuropeptide FF in the Rat Parabrachial Nucleus: Interactions With Opioid Receptors. J. Neurophysiol. 84: 744-751, 2000. The pontine parabrachial nucleus (PBN) receives both opioid and Neuropeptide FF (NPFF) projections from the lower brain stem and/or the spinal cord. Because of this anatomical convergence and previous evidence that NPFF displays both pro- and anti-opioid activities, this study examined the synaptic effects of NPFF in the PBN and the mechanisms underlying these effects using an in vitro brain slice preparation and the nystatin-perforated patch-clamp recording technique. Under voltage-clamp conditions, NPFF reversibly reduced the evoked excitatory postsynaptic currents (EPSCs) in a dose-dependent fashion. This effect was not accompanied by apparent changes in the holding current, the current-voltage relationship or alpha -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid-induced inward currents in the PBN cells. When a paired-pulse protocol was used, NPFF increased the ratio of these synaptic currents. Analysis of miniature EPSCs showed that NPFF caused a rightward shift in the frequency-distribution curve, whereas the amplitude-distribution curve remained unchanged. Collectively, these experiments indicate that NPFF reduces the evoked EPSCs through a presynaptic mechanism of action. The synaptic effects induced by NPFF (5 µM) could not be blocked by the specific µ-opioid receptor antagonist, D-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH2 (1 µM), but application of delta -opioid receptor antagonist Tyr-Tic-Phe-Phe (5 µM) almost completely prevented effects of NPFF. Moreover, the delta -opioid receptor agonist, Deltorphin (1 µM), mimicked the effects as NPFF and also occluded NPFF's actions on synaptic currents. These results indicate that NPFF modulates excitatory synaptic transmission in the PBN through an interaction with presynaptic delta -opioid receptors. These observations provide a cellular basis for NPFF enhancement of the antinociceptive effects consequent to central activation of delta -opioid receptors.


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The octapeptide termed Neuropeptide FF (NPFF) has been designated as morphine modulatory peptide on the basis of its ability to influence the actions of opioid peptides within the spinal cord and brain (Panula et al. 1996). In earlier studies, NPFF was reported to inhibit opioid antinociception (Yang et al. 1985), precipitate opioid withdrawal syndrome (Malin et al. 1990), and block inhibitory effects of morphine on the firing rate of dorsal horn neurons (Magnuson et al. 1990), actions that are consistent with an anti-opioid profile of activity. However, in other studies, NPFF has been reported to exert opioid-like effects. Thus like morphine, it inhibits colonic motor activity (Raffa and Jacoby 1989) and, when injected spinally in conscious rats, it produces antinociceptive effects and enhances morphine's effects (Gouarderes et al. 1993). The pharmacologic effects of NPFF are not considered to be mediated by a direct action at the opioid receptors, since receptor binding studies in the spinal cord have shown this peptide to have a poor affinity for opioid receptors (Allard et al. 1989). These studies show that NPFF binds to distinct sites that are distributed in the spinal cord and brain regions (Allard et al. 1989).

Within the brain stem, NPFF immunoreactivity has been identified in discrete autonomic sites including the nucleus tractus solitarius (NTS) and the pontine parabrachial nucleus (PBN) (Kivipelto and Panula 1991; Kivipelto et al. 1989). The PBN is an important central structure for the regulation of a wide variety of physiological functions (Saper 1995). In particular, the PBN is a major site for the modulation of nociception and receives ascending opioid-containing inputs directly from the spinal cord as well as through relay sites in the caudal brain stem (Riche et al. 1990; Standaert et al. 1986). NPFF-positive neurons in the NTS, which are activated in response to cardiovascular stimulation, project densely to the lateral subnuclei of the PBN (Jhamandas et al. 1998). There is thus a strong convergence of NPFF input to those areas of the PBN that also receive opioid projections. Although such anatomical studies suggest an interaction of NPFF and opioid peptides at a cellular level within the PBN, direct evidence for this interaction is lacking.

Cellular mechanisms underlying the actions of NPFF are less well understood. Exposure of cultured mouse spinal neurons to NPFF caused a transient hyperpolarization (increase in potassium conductance) followed by a depolarization (due to probable closure of Ca2+-dependent K+ channels) (Guzman et al. 1989). In freshly dissociated mouse dorsal root ganglion neurons, NPFF and its analogues suppressed depolarization-induced rise in intracellular Ca2+ through L- and N-type voltage-sensitive Ca2+ channels (Rebeyrolles et al. 1996). In the hippocampus, NPFF has no direct effects on CA1 neurons or on the GABA-mediated inhibitory synaptic transmission (Miller and Lupica 1997). However, in the same study, NPFF was shown to attenuate the morphine-evoked decrease in the amplitude of inhibitory synaptic potentials, possibly via a presynaptic mechanism. The range of the actions of NPFF may be related to its different physiological roles in the spinal cord and the brain.

The present study was undertaken to characterize, at a cellular level, the pre- and postsynaptic actions of NPFF in the PBN using a brain stem slice preparation and the perforated patch-clamp recording technique. In the lateral PBN, application of enkephalin, an endogenous opioid ligand, evokes hyperpolarizing responses that are mediated via activation of K+ conductance (Christie and North 1988). We therefore also investigated the ability of specific opioid receptor antagonists to modulate the synaptic actions of NPFF.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Slice preparation

Male Sprague-Dawley rats (150-250 g) were anesthetized with halothane. The brain was quickly removed and placed into ice-cold, carbogenated (95% O2-5% CO2) artificial cerebrospinal fluid (ACSF; pH 7.3-7.4). Coronal slices (400 µm thick) were cut from a block of brain stem tissue containing the PBN (bregma -9.1 to -9.8 mm) (Paxinos and Watson 1997) in cold (4°C) carbogenated ACSF using a vibratome. Slices were hemisected and incubated in ACSF at room temperature (22°C) for at least 1 h prior to recording. A slice was then transferred into a 500-µl recording chamber where it was submerged and continuously perfused with prewarmed ACSF (28-30°C) at a rate of 2-3 ml/min. The composition of the ACSF was (in mM) 126 NaCl, 2.5 KCl, 1.2 NaH2PO4, 1.2 MgCl2, 2.4 CaCl2, 18 NaHCO3, and 11 glucose. To eliminate possible GABAergic contamination of excitatory synaptic responses, 50 µM picrotoxin was present in the ACSF at all times.

Nystatin-patch recording

Nystatin patch recordings from PBN neurons were made with glass micropipettes (Garner Glass; tip resistance 4-10 MOmega ) first back-filled at the tip with a solution containing (in mM) 120 K-Acetate, 40 HEPES, 5 MgCl2, and 10 EGTA and then filled with the same solution containing 450 µg/ml nystatin and Pluronic F127 (dissolved in dimethyl sulfoxide, DMSO). High resistance seals (1-3 GOmega ) were made using an Axoclamp 2A or an Axopatch 1D amplifier. A small voltage step was applied to monitor the partitioning of nystatin into the membrane, and maximum access to the cell (series resistance of 15-30 MOmega ) was usually attained within 30 min after seal formation.

Data acquisition and analysis

After adequate access was attained, resting membrane potential was measured and current-voltage (I-V) characteristics were obtained by current injections (in steps of 10 pA) in the current-clamp mode. All responses were filtered at 1 or 3 kHz. Cells were then voltage clamped near the resting membrane potential at -65 mV (Zidichouski et al. 1996). Synaptic currents were evoked by applying single or paired pulses via a bipolar stimulating electrode placed close to the ventral tip of the cerebellar peduncle. A stimulus intensity that yielded a response 50-60% of the maximum synaptic response was used for the remainder of the experiment. Three successive synaptic responses were taken 10 s apart, digitally averaged and stored for off-line analysis. When paired pulses were given, they were identical in intensity and were separated by a fixed interval of 50 ms. In all experiments examining synaptic currents, a -20-mV, 100-ms duration square pulse was applied 150 ms after synaptic stimulation to monitor input and series/access resistance. In addition to the computer-assisted data acquisition, continuous records of membrane potentials and currents were made on a pen chart recorder (Gould Instruments).

Miniature excitatory postsynaptic currents (mEPSCs) were acquired using pClamp software in the presence of 1 µM tetrodotoxin (TTX) and 50 µM picrotoxin at a sampling rate of 2 kHz. The cells were held at -80 mV. A stretch of about 3 min of mEPSC activity was collected, and then NPFF (5 µM) was bath-applied for 2-3 min (approximate time for its synaptic effects to occur) and mEPSCs were further collected for about 3 min. The amplitude and the inter-event interval were measured and plotted to generate amplitude- and frequency-distribution curves. The mean frequencies of mEPSCs in control and in NPFF were also calculated for statistical comparisons.

Current-voltage relationships (I-V curves) were generated by applying slow voltage ramps (-120 to -30 mV over 20 s) and recording the corresponding steady-state current. alpha -Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-induced inward currents were achieved by bath applying 5 µM AMPA for 30 s, and currents were recorded on a pen chart recorder.

All acquired data were analyzed off-line using Clampfit (Axon Instruments, Foster City, CA). Data are expressed as means ± SE in either absolute values or normalized percentages. Statistical comparisons were performed using paired or unpaired Student's t-test, or ANOVA where appropriate. Significance was accepted at the 0.05 level.

All drugs were bath applied by perfusion with ACSF containing the final concentration of the drug. NPFF and Tyr-D-Ala-Gly-N-Me-Phe-Gly-ol (DAGO) were from Bachem, Deltorphin (Peninsula Laboratories); D-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH2 (CTAP, Peninsula), 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), and alpha -amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) were from RBI; and naloxone, nystatin, TTX, picrotoxin, and ACSF components were from Sigma. Tyr-Tic-Phe-Phe (TIPP) was a kind gift from Dr. P. Schiller (Clinical Research Institute of Montreal). Stock solutions of CNQX, AMPA, NPFF, DAGO, Deltorphin, CTAP, and TIPP were aliquoted and frozen at -70°C and diluted into ACSF immediately before the experiment.


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Whole cell patch-clamp recordings were made from cells located in both mesencephalic and pontine divisions of the lateral PBN, and predominantly from the dorsal lateral and external lateral subnuclei. Cell characteristics were comparable to those reported previously (Chen et al. 1999; Saleh et al. 1997; Zidichouski et al. 1996) and CNQX-sensitive synaptic current could be evoked by stimulating afferent fibers near the ventral tip of the cerebellar peduncle.

NPFF reversibly and dose-dependently reduces the evoked EPSC

In voltage-clamp mode when cells were held at -65 mV (close to their resting membrane potential), NPFF consistently and reversibly reduced the evoked EPSC without changing the holding current (Fig. 1A1). Figure 1A2 shows the time course of the NPFF effect on the evoked synaptic response. At the dose of 5 µM, NPFF caused an average reduction of 34.5 ± 5.8% (mean ± SE, n = 11, Fig. 1A2) of the EPSC amplitude. The depressant effect of NPFF on the EPSC was also dependent on the intensity of stimulus as shown in the input/output relationships (Fig. 1, B1 and B2). In six cells that were treated with cumulative doses of NPFF ranging from 0.1 to 10 µM (3-4 min exposure at each dose followed by the next higher dose without wash out), the responses were clearly dose dependent (Fig. 4D).



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Fig. 1. Neuropeptide FF (NPFF) reduces the evoked excitatory postsynaptic current (EPSC). A1: traces from a representative cell showing NPFF reversibly reduces the evoked EPSC without any changes in the holding current. A2: time course of NPFF response. EPSCs are normalized and averaged (n = 11). B1: traces from a representative cell showing EPSC dependency on stimulation intensity in control and 5 µM NPFF. B2: input-output relationship plotted using data from B1.

NPFF acts presynaptically to depress the EPSC

The lack of NPFF effects on the holding current suggested a presynaptic locus for NPFF modulation of synaptic currents. To exclude postsynaptic involvement, we first used a steady-state ramp protocol to reveal voltage-current relationship under control conditions and in the presence of 5 µM NPFF. In contrast to the µ-opioid receptor agonists that induce an outward current (data not shown) (see Christie and North 1988), 5 µM NPFF did not change the I-V relationship over the full range of voltages tested (-110 to -30 mV, Fig. 2A). Second, we sought to determine whether NPFF changed the sensitivity of AMPA receptors that could account for the observed reduction in the EPSC. Cells were voltage clamped near their resting membrane potential and AMPA (5 µM for 30 s) was bath-applied briefly to induce an inward current. The AMPA-generated currents were not affected by 5 µM NPFF (control: 66 ± 4 pA, in NPFF, 62 ± 2 pA, P > 0.05, paired t-test n = 5, Fig. 2B).



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Fig. 2. NPFF depresses the EPSC presynaptically. A: NPFF does not change the current-voltage relationship obtained by a slow ramp from -120 to -35 mV over 20 s. B: AMPA-induced inward currents (5 µM applied for 30 s) are unchanged after 5 µM NPFF. C1: NPFF increases paired-pulse ratio, the average increase in paired-pulse ratio (peak amplitude of the 2nd evoked EPSC divided by the 1st) is plotted in C2 (n = 8). Inter-pulse intervals are 50 ms.

Our approach to confirm a presynaptic mechanism of action included the use of a paired-pulse protocol and an analysis of mEPSCs. Paired-pulse protocol employs two closely applied pulses (50 ms in this study) to evoke two synaptic responses where a change in the ratio of these two synaptic responses points to a change in transmitter release from the presynaptic terminal. In cells from which paired-pulse facilitation could be elicited, NPFF (5 µM) decreased the first response to a much greater extent than the second response (Fig. 2C1), thus increasing the paired-pulse ratio (control: 1.26 ± 0.07, in NPFF: 1.65 ± 0.14, P < 0.05, paired t-test, n = 8, Fig. 2C2).

Second, we used mEPSC analysis to establish that the release probability was altered by NPFF, hence providing further evidence for a presynaptic site of action. mEPSCs were collected in the presence of 50 µM picrotoxin and 1 µM tetrodotoxin, and, under these conditions, NPFF reduced the number of mEPSCs but had no effect on the amplitude of mEPSCs (Fig. 3, A1 and A2). This is reflected in distribution curves that NPFF cause a rightward shift in the frequency distribution curve, whereas the amplitude distribution remained unaltered (Fig. 3, B and C). The mean frequency of the mEPSCs was reduced by NPFF (control: 0.7 ± 0.15 Hz; NPFF: 0.33 ± 0.06, P < 0.05, paired t-test, n = 6). These results are inconsistent with a presynaptic locus of action for NPFF.



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Fig. 3. NPFF decreases miniature EPSC (mEPSC) frequency. mEPSCs were acquired in the presence of 50 µM picrotoxin and 1 µM tetrodotoxin. A1 and A2: consecutive traces of mEPSC recordings in control and 5 µM NPFF. B: amplitude-distribution curve is not altered by NPFF. C: NPFF shifts frequency-distribution curve to the right.

NPFF recruits delta -opioid receptors to depress EPSC

DAGO, a µ-receptor opioid agonist, reduced the evoked EPSC in the PBN, an effect that was accompanied by an outward current, and both the direct and synaptic effects of DAGO were reversibly blocked by naloxone (10 µM, data not shown). Despite a lack of postsynaptic effect, the NPFF-induced reduction in the EPSC was also sensitive to naloxone blockade. This observation suggested that NPFF may modulate the synaptic response through its interactions with opioid receptors. Since naloxone does not allow for a differentiation of opioid receptor subtypes involved in the NPFF effect, we used specific µ- and delta -receptor opioid antagonists to address this issue.

In 13 synaptic responses, we examined the following sequential application of pharmacological agents: NPFF (5 µM), a µ-receptor opioid antagonist (CTAP, 1 µM) or a specific delta -receptor opioid antagonist (TIPP, 5 µM) followed by the same dose of NPFF in the presence of the antagonist being tested. Under these conditions, CTAP was ineffective in blocking NPFF's response, whereas TIPP blocked most of the response (Fig. 4, A and B). NPFF alone on average produced a reduction in the EPSC amplitude of 32.1 ± 3.7% (n = 13). In the presence of CTAP, the same dose of NPFF decreased EPSC by 25.2 ± 4.1% (n = 6), whereas the EPSC reduction was only 10.0 ± 2.9% (n = 9) when the cells were perfused with TIPP prior to and during a challenge with NPFF (Fig. 4C). One-way ANOVA comparisons revealed that NPFF caused similar EPSC reductions when applied alone or with CTAP, but a significantly smaller reduction in the presence of TIPP (P < 0.05).



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Fig. 4. NPFF's synaptic effects are mediated through delta -opioid receptors. A: traces from a representative cell showing a sequential application of NPFF (5 µM) alone and in the presence of a µ-receptor antagonist [D-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH2 (CTAP), 1 µM] or a delta -receptor antagonist [Tyr-Tic-Phe-Phe (TIPP), 5 µM]. B: EPSC responses to NPFF alone and in the presence of CTAP or TIPP in a representative cell. C: averaged decrease in the EPSC in response to 5 µM NPFF. Control denotes NPFF alone, CTAP and TIPP represent NPFF-induced EPSC reduction in the presence of CTAP (1 µM) or TIPP (5 µM). Number in brackets are number of cells. *P < 0.05 vs. control and CTAP groups. D: NPFF-induced EPSC decrease is dose dependent, and the dose-response curve is shifted to the right by 5 µM TIPP. E: traces from a representative cell showing Tyr-D-Ala-Gly-N-Me-Phe-Gly-ol (DAGO; 5 µM), a µ-opioid receptor agonist, induces an outward current and a decrease in the EPSC. These responses are blocked by 1 µM CTAP.

To rule out the possibility that the smaller response to NPFF in the presence of TIPP may have occurred as a result of desensitization, a phenomenon that frequently occurs with the application of opioid agonists over a prolonged period, we performed a cumulative dose-response curve of NPFF alone and with a fixed TIPP concentration (5 µM). Each dose of NPFF was applied for 3-4 min and then followed by a higher dose without wash out. As shown in Fig. 4D, NPFF caused a nonlinear, dose-dependent, reduction in the EPSC over the dose range of 0.1 to 10 µM (n = 6). In the presence of 5 µM TIPP, the dose-response curve was considerably shifted to the right (n = 4).

We validated the adequacy of the CTAP dose by demonstrating that the dose of CTAP used in the NPFF experiments blocked both the DAGO-induced shift in holding current and the reduction in EPSC amplitude (Fig. 4E). The experiments using specific opioid receptor antagonists is consistent with the notion that NPFF's synaptic effects occur via action at the delta -opioid receptor. Finally, we used Deltorphin (1 µM), a selective agonist for the delta -receptor, to assess whether its effects were similar to those of NPFF. As shown in Fig. 5, A and B, Deltorphin caused comparable reduction in the EPSC to that induced by NPFF (5 µM), without an apparent change in holding current. After the synaptic effects of Deltorphin were maximal, addition of 5 µM NPFF did not produce a further reduction in the EPSC. Figure 5C summarizes averaged EPSC reduction in response to Deltorphin, NPFF, and NPFF in the presence of Deltorphin. Deltorphin (1 µM) decreased EPSC by 34.5 ± 2.5% (n = 6), which was not significantly different from that induced by NPFF (5 µM; 29.5 ± 3%, P > 0.05, n = 4). However, NPFF failed to cause further EPSC reduction in addition to that induced by Deltorphin (7.7 ± 2.9%, n = 4; 1-way ANOVA: P < 0.05).



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Fig. 5. NPFF's synaptic effects are occluded by a delta -agonist (Deltorphin, 1 µM). A: traces from a representative cell showing Deltorphin and NPFF reduce the EPSC, and Deltorphin occludes NPFF's effects. B: plots of EPSC amplitude in response to Deltorphin, NPFF, and Deltorphin plus NPFF. C: averaged EPSC decrease in response to Deltorphin (1 µM), NPFF (5 µM), or NPFF after Deltorphin. Numbers in brackets are number of cells. * P < 0.05 vs. Deltophin and NPFF groups.


    DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The results of the present study indicate that, in lateral PBN neurons of the brain stem, NPFF depresses excitatory glutamatergic synaptic transmission in a reversible and dose-dependent manner. These effects of NPFF appear to be mediated at the presynaptic locus and a pharmacological analysis of its actions demonstrated that these effects occur specifically through an action on the delta -opioid receptors and not the µ-receptor subtype.

NPFF's effects are presynaptic

In the present study, three lines of evidence support the notion that NPFF-induced inhibition of excitatory synaptic transmission is attributable to a reduced probability of presynaptic transmitter release rather than to a depression of postsynaptic sensitivity. First, NPFF did not cause any direct changes in the passive conductance of PBN neurons. In addition, there is no evidence for a postsynaptic interaction between NPFF and glutamate receptors since NPFF did not alter AMPA receptor-induced inward currents. Second, NPFF reduced the amplitude of the evoked postsynaptic currents while increasing the paired-pulse facilitation of these evoked postsynaptic currents. An increase in the paired-pulse facilitation (ratio) is associated with decreased transmitter release and is indicative of a presynaptic locus of action (Zucker 1989). Third, NPFF reduced the frequency of spontaneous action potential-independent miniature EPSCs without affecting their amplitude distribution. This rightward shift of the frequency distribution curve by NPFF indicates a decreased likelihood of transmitter release from presynaptic terminals.

The mechanisms underlying the presynaptic inhibitory actions of NPFF could either be due to a reduction in calcium influx or a hyperpolarization of the nerve terminals as a result of an increase in potassium conductance. It is known that opiates negatively regulate synaptic strength through an action on the presynaptic potassium channels (Jiang and North 1992; Rhim et al. 1993), and given our observations that NPFF's synaptic effects could be blocked by opioid antagonists, this would seem a plausible mechanism. On the other hand, in freshly dissociated mouse dorsal root ganglion neurons, NPFF has been shown to reverse rises in intracellular Ca2+ induced by µ-opioid receptor agonist DAGO through its actions on L- and N-type Ca2+ channels (Rebeyrolles et al. 1996). Recently, delta  receptor agonists have also been shown to presynaptically inhibit Ca2+ currents in sensory neurons (Acosta and Lopez 1999). In the present study the profile of presynaptic inhibition caused by NPFF and delta -opioid receptor activation is virtually identical. It is possible that a decreased calcium influx into the terminals consequent to the activation of delta  rather than µ-opioid receptor activation may be the mechanism that underlies the NPFF-induced depression of excitatory synaptic transmission.

NPFF interactions with opioid receptors in the PBN

The demonstration of both a pro- and an anti-opioid profile of action for NPFF in prior studies prompted us to examine its interactions with opioid receptors in the PBN. Binding studies have shown that NPFF receptors are distinct and that opioid ligands do not compete for these sites (Allard et al. 1989; Gouarderes et al. 1997). These studies have also suggested that NPFF does not directly interact with µ- and delta -opioid receptors. The lateral subnuclei of PBN contains one of the highest densities of NPFF immunoreactive terminals within this region (Kivipelto and Panula 1991; Kivipelto et al. 1989) and is enriched in NPFF binding sites (Allard et al. 1992) although the anatomical distribution of such receptors, i.e., pre- versus postsynaptic localization, is unknown. The lateral PBN is similarly well endowed in opioid immunoreactivity and receptors (Standaert et al. 1986; Xia and Haddad 1991). In general, opioid peptides have been shown to hyperpolarize neurons in a number of brain regions including the PBN, an effect that is mostly attributable to µ-opioid receptor activation (Christie and North 1988; Jiang and North 1992; Rhim et al. 1993). In this study, we determined that activation of µ-opioid receptors with the agonist DAGO results not only in an outward current but is accompanied by a marked reduction of evoked excitatory postsynaptic currents, effects that are both naloxone sensitive. As naloxone also blocked the synaptic effects of NPFF, we explored the possibility that these effects might be mediated through µ-opioid receptors. However, CTAP, a specific µ-opioid receptor antagonist, failed to influence the depressant syn-aptic effects of NPFF at doses where it was effective in blocking DAGO-induced responses. Thus the effects of NPFF in the PBN do not appear to be modulated through µ-opioid receptors as has been observed in dorsal root ganglion and hippocampal neurons (Miller and Lupica 1997; Rebeyrolles et al. 1996). In contrast, TIPP, a specific delta -opioid receptor antagonist, blocked the synaptic effects of NPFF. Deltorphin, in a similar manner to NPFF, did not induce any postsynaptic effects, but effectively and reversibly reduced excitatory synaptic currents. In addition, Deltorphin occluded the effects of NPFF, suggesting that the two peptides share a common final pathway for presynaptic inhibition in the PBN. However, suitable antagonists for NPFF, as yet unavailable, are required to confirm whether NPFF also acts via its own distinct receptors.

Current knowledge indicates that NPFF has very poor affinity for delta -opioid receptors in the spinal cord (Allard et al. 1989; Gouarderes et al. 1997). Since our data reveals that NPFF-induced synaptic effects could be blocked or occluded using specific delta -opioid receptor antagonist and agonist, respectively, we considered the possibility that NPFF's effects may occur through the release of an endogenous opioid peptide as an intermediary (Fig. 6). Enkephalin, which is localized in the lateral PBN and preferentially acts at the delta -opioid receptors is a strong candidate to act as such an intermediary (Goldstein and Naidu 1989). Indeed, recent data in the spinal cord show that NPFF can promote enkephalin release (Ballet et al. 1999). As such, a similar mechanism could also exist in the PBN, whereby NPFF could release enkephalin to activate presynaptically located delta -opioid receptors and depress excitatory synaptic transmission. Such a scheme would explain the synaptic effects of both exogenously applied NPFF and the delta -opioid receptor agonist Deltorphin. However, if NPFF's effects are hypothesized to occur through the release of enkephalin, one might also expect NPFF to also have direct hyperpolarizing effects as enkephalin has via activation of postsynaptic µ-opioid receptors (Christie and North 1988). In this study we observed no direct actions of NPFF, and this finding favors the notion that NPFF acts at presynaptic delta -opioid receptors rather than through an intermediary such as enkephalin. It may thus be prudent to re-examine opioid-NPFF binding studies with newer and more specific delta -opioid receptor ligands that are now available (Schiller et al. 1993). Alternately, one must consider the possibility that NPFF exhibits different affinities for opioid receptors in the brain compared with the spinal cord.



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Fig. 6. Schematic depicting possible models of NPFF actions in the pontine parabrachial nucleus (PBN). NPFF terminals originating from the nucleus tractus solitarius (NTS) or the hypothalamus presynaptically depress the release of glutamate onto lateral PBN neurons through 2 potential mechanisms. Under scheme 1, NPFF's presynaptic effects may be mediated directly through its own receptor and/or at the delta -opioid receptor site. A 2nd possibility (2) is that NPFF releases enkephalin (ENK) from terminals arising from the NTS, the spinal cord or the paraventricular nucleus (PVN) of the hypothalamus. The enkephalin in turn also depresses glutamate release through presynaptic delta - (and possibly µ-) opioid receptors. Postsynaptic opioid effects occur through µ-receptor subtypes.

Anatomical and functional perspectives

These observations suggest an important role for NPFF in the modulation of nociceptive responses within the PBN. The PBN receives a dense NPFF input that arises from the NTS in the dorsomedial medulla and a more modest projection from NPFF containing neurons located within the hypothalamus (Harris and Jhamandas 1996; Jhamandas et al. 1998; Kivipelto and Panula 1991). There is also a significant enkephalinergic input to the PBN that arises from three major sources that include the NTS, the spinal cord, and the hypothalamic paraventricular nucleus (Moga et al. 1990; Riche et al. 1990; Standaert et al. 1986). However, in the NTS, where there is a significant representation of both NPFF and opioids, there is no co-localization of the two peptides in PBN-projecting neurons (unpublished observations). There is nonetheless a strong covergence of both peptidergic inputs to the lateral tier of subnuclei within the PBN. Our results suggest a model whereby NPFF and opioid peptide (enkephalin) inputs to the PBN converge on glutamatergic terminals on lateral PBN neurons and negatively modulate the excitatory synaptic transmission (Fig. 6). Of course, the possibility that NPFF may be localized within the same terminals as glutamate cannot be discounted as our previous in vivo studies have shown glutamate-evoked excitation of PBN neurons following NTS stimulation (Jhamandas and Harris 1992).

From a functional perspective, intrathecal administration of NPFF and its analogues have been shown to enhance morphine antinociception at the spinal level through an indirect activation of µ- and delta -opioid receptors (Gouarderes et al. 1996). However, when administered intracerebroventricularly, NPFF's effects are more complex. Behavioral studies have shown NPFF to exert an anti-opioid effect, but there is also evidence for antinociceptive activity (for review see Roumy and Zajac 1998). In part, these conflicting data are due to a lack of model system in which both NPFF and opioid peptides are anatomically represented to permit an analysis of the cellular mechanisms of actions and synaptic interactions of the peptides. Our observation that NPFF presynaptically modulates delta -opioid receptor function provides a cellular basis for understanding the analgesic actions of NPFF at the supraspinal level. In addition, it helps explain why under certain conditions NPFF enhances delta - but not µ-opioid receptor mediated analgesia (Desprat and Zajac 1997).


    ACKNOWLEDGMENTS

We thank Dr. Khem Jhamandas for helpful comments and suggestions, and C. Krys for typing the manuscript. Dr. X. Chen was a recipient of fellowship awards from the Alzheimer's Society of Canada and the Alberta Heritage Foundation for Medical Research.

This work was supported by the Heart and Stroke Foundation of Alberta and Canada.


    FOOTNOTES

Address for reprint requests: J. H. Jhamandas, 530 Heritage Medical Research Building, Dept. of Medicine (Neurology), University of Alberta, Edmonton, Alberta T6G 2S2, Canada (E-mail: jack.jhamandas{at}ualberta.ca).

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 21 January 2000; accepted in final form 31 March 2000.


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