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
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ABSTRACT |
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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 -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
-opioid receptor antagonist
Tyr-Tic-Phe-Phe (5 µM) almost completely prevented effects of NPFF.
Moreover, the
-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
-opioid receptors. These observations provide a cellular
basis for NPFF enhancement of the antinociceptive effects consequent to
central activation of
-opioid receptors.
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INTRODUCTION |
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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.
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METHODS |
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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 M) 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 G
) 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 M
) 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.
-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
-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.
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RESULTS |
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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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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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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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NPFF recruits -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
-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 -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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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 -opioid receptor. Finally, we used
Deltorphin (1 µM), a selective agonist for the
-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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DISCUSSION |
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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 -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,
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
-opioid receptor
activation is virtually identical. It is possible that a decreased
calcium influx into the terminals consequent to the activation of
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
-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
-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
-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
-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
-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
-opioid receptors and depress
excitatory synaptic transmission. Such a scheme would explain the
synaptic effects of both exogenously applied NPFF and the
-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
-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
-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.
|
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
-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
-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
- but not µ-opioid receptor mediated analgesia
(Desprat and Zajac 1997
).
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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.
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FOOTNOTES |
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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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REFERENCES |
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