The Journal of Neuroscience, August 13, 2003, 23(19):7262-7268
Previous Article | Next Article 
Presynaptic Mechanism for Anti-Analgesic and Anti-Hyperalgesic Actions of 
-Opioid Receptors
Bihua Bie and
Zhizhong Z. Pan
Departments of Symptom Research and Biochemistry and Molecular Biology,
The University of Texas-MD Anderson Cancer Center, Houston, Texas 77030
 |
Abstract
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Glutamate neurotransmission plays an important role in the processing of
pain and in chronic opioid-induced neural and behavioral plasticity, such as
opioid withdrawal and opioid dependence. 
-Opioid receptors also have
been implicated in acute opioid modulation of pain and chronic opioid-induced
plasticity, both of which are primarily mediated by µ-opioid receptors.
Using whole-cell patch clamp recordings in brain slices in vitro and
system analysis of pain behaviors in rats in vivo, this study
investigated the functional role of glutamate synaptic transmission
and-opioid receptors in two behavioral pain
conditions:µ-opioid-induced analgesia (decreased pain) and µ-opioid
withdrawal-induced hyperalgesia (increased pain). In the nucleus raphe magnus
(NRM), a brainstem structure that controls spinal pain transmission, we found
that-receptor agonists presynaptically inhibited glutamate synaptic
currents in both of the two cell types that are thought to respectively
inhibit or facilitate spinal pain transmission. In rats, both glutamate
receptor antagonists and the agonist microinjected into the NRM
attenuated µ-opioid-induced analgesia, which is most likely mediated
through activation of such pain-inhibiting neurons. However, during opioid
abstinence-induced withdrawal, the same doses of glutamate receptor
antagonists and the agonist administered in the NRM suppressed the
withdrawal-induced hyperalgesia, which is thought to be mediated by activation
of those pain-facilitating neurons during opioid withdrawal. These results
demonstrate that -opioid receptors antagonize µ-receptor-induced
effects in both analgesic and hyperalgesic states, and suggest inhibition of
glutamate synaptic transmission as a presynaptic mechanism for the
antagonism of these two µ receptor-mediated actions.
Key words: receptors; µ receptors; opioid; glutamate; analgesia; hyperalgesia; pain
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Introduction
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Glutamate and other excitatory amino acids (EAAs) are principal excitatory
neurotransmitters in neuronal circuits involved in a variety of CNS functions,
including pain modulation (Fundytus,
2001
). Glutamate and other EAAs can produce potent inhibition of
pain when applied locally into such key structures of the endogenous
pain-modulating system as the periaqueductal gray (PAG) in the midbrain, and
the main projection target of the PAG, the nucleus raphe magnus (NRM) in the
medulla (Aimone and Gebhart,
1986; Jacquet,
1988). Systemic application or local administration of glutamate
receptor antagonists into the PAG or the NRM blocks morphine-induced analgesia
(Aimone and Gebhart, 1986;
Jacquet, 1988;
Lipa and Kavaliers, 1990;
Heinricher et al., 2001),
indicating that glutamate-mediated synaptic transmission is required for
opioid analgesia. The glutamate neurotransmission system, and in particular,
the NMDA receptor, is also critically involved in chronic opioid-induced
neural adaptations, such as opioid dependence and withdrawal (Trujillo and
Akil, 1991,
1995;
Nestler, 1996), and in chronic
pain-induced hyperalgesia (increased pain sensitivity) involving the NRM
(Coderre et al., 1993;
Urban and Gebhart, 1998;
Porreca et al., 2002).
Despite the importance of glutamate neurotransmission in these acute and
chronic pain states, how it functions in the pain-modulating circuits
activated by acute opioids remains unclear, hampering further attempts to
understand the role of glutamate synaptic transmission and its plastic changes
in chronic opioid-induced or chronic pain-induced behavioral conditions. In
our previous studies of the NRM, the main component of the descending
pain-inhibiting system (Fields and Basbaum,
1999), we characterized a disinhibition mechanism for the
activation of this system by local µ-opioid receptor agonists, without
involving glutamate synaptic transmission (Pan et al.,
1990,
1997). Nevertheless, other
studies have indicated that systemically applied µ-opioids
activate glutamate inputs and those neurons thought to inhibit spinal pain
transmission in the rostral ventromedial medullar (RVM), which includes the
NRM (Spinella et al., 1996;
Fields and Basbaum, 1999;
Heinricher et al., 2001).
In contrast to the evident role of µ-receptors in opioid analgesia and
dependence (Matthes et al.,
1996), the function of -opioid receptors is less clear. We
have shown a µ-opposing effect of -receptors through a postsynaptic
hyperpolarization in opioid analgesia (Pan
et al., 1997), representing an emerging anti-µ function of
-receptors in many opioid effects in the brain
(Pan, 1998). Under normal
conditions, agonists can have an analgesic action by inhibiting
glutamate synaptic currents (Randic et
al., 1995; Ackley et al.,
2001). Under conditions of chronic opioids or chronic pain,
-receptors interfere with the development of µ-opioid tolerance and
dependence (Takahashi et al.,
1991; Takemori et al.,
1992; Tao et al.,
1994), and are implicated in several forms of chronic pain
behaviors (Cheng et al., 2002).
However, the underlying mechanisms for these actions remain
unknown.
In the current study of NRM neurons, we used both slice preparations in
vitro and intact rats in vivo to investigate the functional role
of glutamate synaptic transmission and its modulation by -receptors in
both µ-opioid-induced analgesic and µ-opioid withdrawal-induced
hyperalgesic states.
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Materials and Methods
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Brain slice preparations. The brain of a male, neonatal (9-14 d
old) Wistar rat was cut in a vibratome in cold (4°C) physiological saline
to obtain brainstem slices (200-µm-thick) containing the NRM. A single
slice was submerged in a shallow recording chamber and perfused with preheated
(35°C) physiological saline (in mM: NaCl, 126; KCl, 2.5;
NaH2PO4, 1.2; MgCl2, 1.2; CaCl2,
2.4; glucose, 11; NaHCO3, 25, saturated with 95% O2 and
5% CO2, pH 7.2-7.4). Slices were maintained at 
35°C
throughout the recording experiment. Neonatal rats were used for better
visualization of neurons in brain slices with an infrared Nomarski microscope.
It has been demonstrated that the physiological and pharmacological properties
of neurons from these young rats are indistinguishable from those of adult
rats (Pan et al., 1997).
Whole-cell recordings and data analyses. Visualized whole-cell
patch clamp recordings were made from identified neurons with a glass pipette
(resistance 3-5 M
) filled with a solution containing (in
mM): potassium gluconate, 126; NaCl, 10; MgCl2, 1; EGTA,
11; HEPES, 10; ATP, 2; GTP, 0.25; pH adjusted to 7.3 with KOH; osmolarity
280-290 mOsmol/l. An AxoPatch 1-D amplifier and AxoGraph software (Axon
Instruments, Foster City, CA) were used for data acquisition and
on-line/off-line data analyses. A seal resistance of 
2G and an
access resistance of 
15 M were considered acceptable. Series
resistance was optimally compensated. The access resistance was monitored
throughout the experiment. Electrical stimuli of constant current (0.25 msec,
0.2-0.4 mA) were used to evoke EPSCs with bipolar stimulating electrodes
placed lateral (200-400 µM) to the recording electrode within
the NRM. A pair of EPSCs was evoked by two stimuli with an interval of 40
msec. The pair-pulse ratio was determined by the ratio of the second EPSC
amplitude over the first one. Spontaneous EPSCs (sEPSCs) or miniature EPSCs
(mEPSCs) were obtained in 60 sec epochs in control or in the presence of
drugs. The AxoGraph 4.7 was used to detect and measure the amplitude and
intervals of the synaptic events and analyze their distribution data.
Statistic analyses of sEPSCs and mEPSCs were performed using Statview software
with either the Kolmogorov-Smirnov, or the Mann-Whitney U test. Other
numeral data were statistically analyzed by Student's t tests and
presented as mean ± SEM.
Cell classification and drug application. All NRM cells recorded
were classified into either a primary or secondary cell type according to the
criteria described in our previous study
(Pan et al., 1990). Briefly,
primary cells have a wider action potential, have a more negative resting
membrane potential, and are insensitive to µ agonists. Secondary cells have
a narrower action potential, often display spontaneous firing, and are
hyperpolarized by µ agonists. Cells that could not be clearly classified
were not included in the results. Drugs were applied through the perfusing
solution.
Behavioral experiments and microinjection. In behavioral
experiments, male Wistar rats (250-300 gm) were maintained lightly
anesthetized in a stereotaxic apparatus with a constant intravenous infusion
of methohexital (10 mg/ml at 0.8 ml/hr). A 26 gauge single-guide cannula was
aimed at the ventrolateral PAG [anteroposterior (AP): -7.8; lateral (L):
±0.8; ventral (V): - 6.0] and a second, double-guide cannula was aimed
at the NRM (AP: -11.0; L: 0; V: -10.7). Tail-flick latency to a radiant heat
stimulus was measured every 2 min. The heat intensity was set to elicit stable
baseline latencies with a cutoff time of 12 sec. After six baseline trials,
drugs were delivered through a 33 gauge cannula with an infusion pump at a
rate of 0.5 µl/min. All cannula placements for both the PAG and the NRM
were histologically verified afterward. Drug effects were statistically
analyzed by an ANOVA for repeated measures and either the Tukey-Kramer or the
Newman-Keuls test of post hoc analysis using GB-Stat software. All
drugs were purchased from Research Biochemicals International (Natick, MA) or
Sigma-Aldrich.
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Results
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agonists inhibit glutamate synaptic transmission
NRM cells recorded in brain slices were classified into either a primary
cell type lacking µ receptors or a secondary cell type containing µ
receptors according to the criteria described in our previous studies (Pan et
al., 1990,
1997). Primary cells had an
average resting membrane potential of -60.6 ± 0.7 mV and an action
potential of 51.5 ± 0.7 mV (n = 66). Secondary cells had a
less negative resting potential (-54.1 ± 0.5 mV) and an action
potential of 49.5 ± 0.7 mV (n = 85). EPSCs were evoked through
local stimulation in cells under whole-cell voltage clamp with a holding
potential of -60 mV. Bicuculline (10 µM) was included in all
experiments to block the GABAA receptor-mediated synaptic
transmission present in these cells. The average amplitude of the evoked EPSC
(eEPSC) was 114 ± 6 pA in all cells recorded (n = 151). The
EPSC was completely blocked by the combination of AP-5 (10 µM)
and CNQX (10 µM), antagonists of NMDA and non-NMDA receptors,
respectively (n = 8). Application of the -opioid receptor
agonist U69593
[GenBank]
(300 nM) significantly inhibited the EPSC amplitude
in every cell tested. In primary cells (n = 12), the average
inhibition was 43.3 ± 7.4%, and the EPSC was recovered in minutes after
wash of U69593
[GenBank]
(Fig.
1A). No significant difference was observed in the
inhibition between primary cells that were hyperpolarized by the
agonist (44.7 ± 3.9%; n = 5) and primary cells that
lacked the postsynaptic response (42.3 ± 4.5%; n = 7).
Similar U69593
[GenBank]
-mediated inhibition of eEPSCs was also found in the
µ-sensitive secondary cells with an average inhibition of 44.7 ±
5.7% (n = 15) (Fig.
1B). U69593
[GenBank]
at a lower concentration (10 nM)
caused a smaller inhibition in the EPSC amplitude in both cell types (control,
109.0 ± 18.1 pA; U69593
[GenBank]
, 82.5 ± 16.2 pA; wash, 101.8 ±
17.0 pA; n = 6). In the presence of the -receptor antagonist
nor-BNI (100 nM), the inhibition induced by U69593
[GenBank]
(300
nM) was completely blocked in both cell types (n = 11 in
each type), confirming the action of -receptors
(Fig. 1). Nor-BNI itself had no
effect on the EPSC in all these cells of both types (control vs nor-BNI: 95
± 13.4 pA vs 97 ± 13.7 pA in primary cells; n = 11 and
111 ± 12.6 pA vs 108 ± 17.6 pA in secondary cells, n =
11).
The endogenous agonist dynorphin also produced a reversible and
nor-BNI-sensitive inhibition of the eEPSC in these cells. Dynorphin at 300
nM inhibited the EPSC amplitude by 41.8 ± 5.5% in primary
cells (n = 9) and by 44.2 ± 4.9% in secondary cells
(n = 11) (Fig.
2A,B). The dynorphin effect was dose-dependent with an
estimated dose range of 1-300 nM. The EC50 estimated
from dose-response curves was 14.5 nM in primary cells and 16.7
nM in secondary cells (Fig.
2C).
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Figure 2. Endogenous -receptor agonist dynorphin inhibits glutamate EPSCs.
A, EPSCs from a secondary cell in control, in dynorphin (300
nM), and after washout of dynorphin. B, A plot of
normalized amplitudes of pooled EPSCs from secondary cells in control (open
circles, n = 11) and in nor-BNI (100 nM, filled circles,
n = 10). C, A dose-response plot of EPSC inhibition by
dynorphin in secondary cells (n = 7-8 cells at each
concentration).
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Together, these results suggest that activation of -opioid receptors
inhibits glutamate synaptic transmission in both primary and secondary cell
types.
The inhibition is presynaptic
To determine the synaptic site of the -receptor action, we first
used the paradigm of paired-pulse ratio (PPR), whose change is attributed to
an altered transmitter release through a presynaptic mechanism
(Manabe et al., 1993). Thus,
an increase in the PPR (the ratio of the second EPSC amplitude over the first
one) suggests presynaptic inhibition of transmitter release and vice versa. In
the present study, primary cells had a PPR of 1.39 ± 0.12 in control
(n = 14), indicating a synaptic facilitation. The PPR increased to
2.0 ± 0.24 in the presence of U69593
[GenBank]
(p < 0.05; n
= 14) (Fig. 3). PPR in
secondary cells was similarly increased by U69593
[GenBank]
(control, 1.96 ±
0.15; U69593
[GenBank]
, 2.46 ± 0.21; p < 0.01; n = 23).
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Figure 3. U69593 increases the paired-pulse ratio (PPR) of glutamate EPSCs. Pairs of
EPSCs from a primary cell in control and in U69593
[GenBank]
. Note the increase by
U69593
[GenBank]
(300 nM) in the PPR (ratio of the second EPSC amplitude over
the first).
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We next examined the effect on glutamatergic spontaneous EPSCs
(sEPSCs). Primary cells displayed sEPSCs with an average amplitude of 24 pA
(n = 7) (Fig.
4A). U69593
[GenBank]
(300 nM) significantly inhibited
the frequency of the synaptic events (control, 5.2 ± 1.0 Hz; U69593
[GenBank]
,
2.3 ± 1.0 Hz; p < 0.01; Kolmogorov-Smirnov test, n
= 7) (Fig. 4B), but
had no effect on the amplitude (control, 24.3 ± 0.9 pA; U69593
[GenBank]
, 23.4
± 1.0 pA; p > 0.05; Kolmogorov-Smirnov test, n =
7) (Fig. 4C). Similar
U69593
[GenBank]
effects were observed in secondary cells (control vs U69593
[GenBank]
: 7.2
± 1.7 Hz vs 4.1 ± 1.2 Hz, p < 0.01; 27.0 ±
1.7 pA vs 27.4 ± 2.5 pA; p > 0.05; Kolmogorov-Smirnov test,
n = 8) (Fig.
4D,E). In additional four cells (two in each type), we
tested U69593
[GenBank]
effect on eEPSCs and on sEPSCs in the same cell. In all four
cells tested, U69593
[GenBank]
(300 nM) inhibited the eEPSC with an increase
in the PPR (from 1.65 ± 0.34 to 2.35 ± 0.26; p <
0.05), and reduced the frequency of sEPSCs (control, 6.34 ± 0.87 Hz;
U69593
[GenBank]
, 4.08 ± 0.91 Hz; p < 0.05) with no change in the
sEPSC amplitude (control, 22.0 ± 1.3 pA; U69593
[GenBank]
, 21.5 ± 1.3 pA;
p > 0.05).
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Figure 4. U69593 reduces the frequency of spontaneous glutamate EPSCs. A,
Current traces showing spontaneous EPSCs from a primary cell in control and in
U69593
[GenBank]
(300 nM). B, C, Plots of cumulative distribution of
interevent intervals and EPSC amplitudes in control and in U69593
[GenBank]
for the cell
in A. D, E, Distribution plots for a secondary cell.
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To further verify the action site, we then examined the effect of
the agonist on activity-independent mEPSCs in the presence of TTX (1
µM). In primary cells (n = 5), U69593
[GenBank]
significantly
decreased the frequency of mEPSCs (control, 5.8 ± 0.9 Hz; U69593
[GenBank]
, 3.6
± 0.5 Hz; p < 0.05), but it did not alter the mEPSC
amplitude (control, 24.9 ± 2.0 pA; U69593
[GenBank]
, 25.2 ± 2.3 pA;
p > 0.05). As shown in Figure
5, the agonist also inhibited the frequency but not the
amplitude of mEPSCs in secondary cells (control vs U69593
[GenBank]
: 10.9 ± 2.0
Hz vs 6.4 ± 1.3 Hz; p < 0.05; 24.5 ± 2.4 pA vs 23.7
± 2.4 pA; p > 0.05; n = 6).
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Figure 5. U69593 inhibits the frequency of miniature glutamate EPSCs. Data from a
secondary cell. A, Current traces showing miniature EPSCs in control
and in U69593
[GenBank]
(300 nM). B, C, Plots of cumulative
distribution of interevent intervals and miniature EPSC amplitudes in control
and in U69593
[GenBank]
.
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These findings suggest that activation of -receptors on presynaptic
sites inhibits glutamate release onto both NRM primary and secondary cell
types.
agonist inhibits glutamate-mediated opioid analgesia
We have shown previously that µ-opioids produce analgesia by
disinhibiting NRM primary cells and by inhibiting µ-receptor-containing
secondary cells in rats (Pan et al.,
1990,
1997). To investigate the role
of glutamate synaptic transmission in the excitation of primary cells during
opioid analgesia, we next performed NRM microinjections in lightly
anesthetized rats in vivo while monitoring changes in pain threshold
with the tail-flick test. Microinjection of the µ-receptor agonist
D-Ala2-N-Me-Phe4-Glycol5]-enkephalin
(DAMGO) (0.05 µg/0.25 µl) into the PAG produced an immediate increase in
the tail-flick latency to the cutoff time (12 sec), demonstrating a potent
antinociceptive effect (n = 5 rats)
(Fig. 6). When the glutamate
receptor antagonists AP-5 (0.197 µg/1 µl) and CNQX (0.232 µg/1 µl)
were microinjected into the NRM just before the DAMGO application in PAG, the
DAMGO-induced antinociceptive effect was blocked (n = 5 rats). NRM
application of the glutamate antagonists alone did not change the pain
threshold (n = 5 rats) (Fig.
6). These results indicate that during the PAG opioid-induced
analgesia, the activation of NRM primary cells is mediated by glutamate
synaptic inputs activated through the PAG.
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Figure 6. Glutamate receptor antagonists in the NRM block local µ-opioid-induced
analgesia. Tail-flick latencies were measured every 2 min before (BL,
baseline, average of 6 trials) and after drug microinjections into the NRM and
then into the PAG (arrow, time = 0). The cutoff time was 12 sec. Group 1 (open
circles): saline in NRM and DAMGO in PAG; group 2 (filled circles): AP-5 +
CNQX in NRM and DAMGO in PAG; group 3 (open squares): AP-5 + CNQX in NRM and
saline in PAG, n = 5 rats in each group, **p <
0.01 (ANOVA for repeated measures and the Tukey-Kramer test of post
hoc analysis).
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Because agonists presynaptically inhibit glutamate synaptic
transmission in primary cells, as described above, agonists should
also reduce the DAMGO-induced antinociception. In fact, we have shown
previously that microinjection of the agonist U69593
[GenBank]
(0.178 µg/1
µl) into the NRM significantly attenuates this PAG DAMGO-induced
antinociception and that the U69593
[GenBank]
effect can be completely blocked by NRM
comicroinjection of nor-BNI (Pan et al.,
1997). The same results were obtained in the present study
(n = 2 rats). These results suggest that activation of presynaptic
-receptors inhibits the synaptic release of glutamate onto primary
cells and thereby decreases the PAG DAMGO-induced analgesia, which requires
the activation of NRM primary cells by glutamate inputs.
agonist attenuates glutamate-mediated hyperalgesia
NRM secondary cells are directly hyperpolarized by µ-opioids during
opioid analgesia and therefore, their glutamate inputs are unlikely to be
activated in a behavioral condition of analgesia
(Fields and Basbaum, 1999).
However, these cells are activated during opioid abstinence-induced
withdrawal, and their activation is implicated in mediating the state of
increased pain sensitivity (hyperalgesia) during opioid withdrawal
(Kaplan and Fields, 1991;
Pan et al., 2000). The
following experiments were then conducted to test our hypothesis that
glutamate synaptic inputs mediated the activation of secondary cells during
opioid withdrawal.
Morphine (2 mg/kg), mainly a µ-receptor agonist, was injected
intraperitoneally in rats to induce opioid analgesia. Rats were then injected
with naloxone (1 mg/kg, i.p.) to induce acute opioid withdrawal. As shown in
Figure 7A,
systemically applied morphine produced potent antinociception with a marked
increase in tail-flick latency. Application of naloxone 26 min later quickly
decreased the rat's pain threshold from the cutoff time (12 sec) to levels
below the pre-morphine baseline, indicating opioid abstinence-induced
hyperalgesia (n = 5 rats). When the same doses of AP-5 (0.197 µg/1
µl) and CNQX (0.232 µg/1 µl), instead of saline, were microinjected
into the NRM immediately after naloxone injection, this hyperalgesia was
significantly attenuated (n = 6 rats)
(Fig. 7A). This
demonstrates an important role of glutamate synaptic transmission in the NRM
in mediating the hyperalgesic condition following acute opioid withdrawal.
Based on above findings that the agonist inhibited glutamate EPSCs in
secondary cells, we predicted that the agonist would also decrease
opioid withdrawal-induced hyperalgesia. Indeed, the same dose of
agonist U69593
[GenBank]
(0.178 µg/1 µl) microinjected into the NRM immediately
after intraperitoneal injection of naloxone significantly reduced the
hyperalgesia (n = 6 rats) (Fig.
7B). Comicroinjection of U69593
[GenBank]
with the
antagonist nor-BNI (0.367 ng/1 µl) into the NRM completely reversed the
U69593
[GenBank]
effect, confirming a specific -receptor-mediated effect
(n = 5 rats) (Fig.
7B).
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Figure 7. Glutamate receptor antagonists and -receptor agonist attenuate
opioid abstinence-induced hyperalgesia. After measurements of baseline
tail-flick latencies, rats were injected with morphine (2 mg/kg, i.p.) and
followed by naloxone (1 mg/kg, i.p.) 26 min later to induce opioid abstinence
(withdrawal)-induced hyperalgesia. NRM microinjections were made immediately
after the naloxone injection. The dashed line indicates pre-morphine baseline.
A, Open circles: saline in NRM (n = 5 rats). Filled circles:
AP-5 + CNQX in NRM (n = 6 rats). B, Open circles: same as in
A. Filled circles: U69593
[GenBank]
in NRM (n = 6 rats). Filled
squares: U69593
[GenBank]
+ nor-BNI in NRM (n = 5 rats). *p
< 0.05, **p < 0.01 (ANOVA for repeated measures and the
Tukey-Kramer test of post hoc analysis).
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Discussion
|
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The present study illustrates that activation of -opioid receptors
presynaptically inhibits synaptic release of glutamate or other EAAs onto both
primary cells and µ-expressing secondary cells in the NRM. Our behavioral
data further show that both glutamate receptor antagonists and
receptor agonists in the NRM antagonize the PAG opioid-induced analgesia or
attenuate opioid withdrawal-induced hyperalgesia. These results suggest that
the excitatory glutamate synaptic inputs are required to mediate both the
activation of NRM primary cells for the µ-opioid analgesia and the
activation of secondary cells for opioid withdrawal-induced hyperalgesia.
Therefore, presynaptic inhibition of glutamate synaptic transmission by
-opioid receptors could function as one of the mechanisms for the
anti-analgesic and anti-hyperalgesic actions of -receptors in the two
opposite pain states induced through µ-opioid receptors. Several
observations in the current study suggest that -opioid receptor
agonists inhibit glutamate EPSCs by acting on presynaptic sites in NRM cells.
First, U69593
[GenBank]
significantly and consistently increased the paired pulse ratio
of the eEPSC, indicating a change in presynaptic release rather than a
postsynaptic effect. This method often has been used to determine a
presynaptic effect (Manabe et al.,
1993; Manzoni and Williams,
1999; Hjelmstad and Fields,
2001). Second, the agonist reduced the frequency but not
the amplitude of sEPSCs, indicating an action on presynaptic sites. Third,
although U69593
[GenBank]
postsynaptically hyperpolarized a subpopulation of primary
cells, the amount of its inhibition of EPSCs was not statistically different
in the primary cells hyperpolarized by the agonist and in those
without the postsynaptic response. It indicates that the action on
presynaptic sites is dominant for the inhibition of EPSCs in these cells.
Last, U69593
[GenBank]
inhibited mEPSC frequency, but not its amplitude, suggesting that
the agonist inhibits glutamate release from terminals. Anatomically,
-opioid receptor immunoreactivity is present on both processes and cell
bodies in the NRM (Kalyuzhny and
Wessendorf, 1999).
Mounting evidence indicates that receptors can oppose several µ
receptor-mediated actions of acute and chronic opioids in the brain, including
opioid analgesia (Pan, 1998;
Ghozland et al., 2002;
Schmidt et al., 2002).
Previous research in the RVM or the NRM has shown that µ-sensitive cells
are inhibited during opioid analgesia, and it is the activation of RVM
off-cells lacking µ receptors (NRM primary cells) that inhibits spinal pain
transmission through their spinal projections
(Pan et al., 1997;
Fields and Basbaum, 1999;
Heinricher et al., 2001). The
data from this study suggest that the activation of these primary cells is
primarily mediated by their excitatory glutamate inputs originating most
likely from the PAG (Aimone and Gebhart,
1986). Inhibition of glutamate EPSCs by agonists has been
reported in the neurons of several brain areas
(Wagner et al., 1993;
Weisskopf et al., 1993;
Randic et al., 1995;
Hjelmstad and Fields, 2001).
Using the defined NRM circuit for opioid analgesia, the current study suggests
that inhibition of glutamate synaptic transmission in primary cells
contributes to the anti-analgesic action of agonists by reducing
glutamate-induced excitation of primary cells. In a previous study, we
described how -receptors opposed the µ-opioid analgesia through a
postsynaptic hyperpolarization in a subpopulation of NRM primary cells
(Pan et al., 1997). Compared
with the EC50 of 7.4 nM for its postsynaptic effect of
membrane hyperpolarization (Pan et al.,
1997), dynorphin had a higher EC50 value (14.5
nM) at the presynaptic site for EPSC inhibition in primary cells,
indicating that dynorphin is more potent at the postsynaptic -receptors
than at those on glutamatergic terminals. However, while only a subpopulation
of primary cells are hyperpolarized by agonists, inhibition of
glutamate EPSCs has been observed in all primary cells tested in the current
study. Thus, both of the presynaptic and postsynaptic mechanisms could reduce
µ opioid-induced excitation of primary cells and account for the behavioral
anti-analgesic action of agonists during opioid analgesia.
Hyperalgesia is a common symptom associated with opioid withdrawal
(Kaplan and Fields, 1991).
Several studies (Kim et al.,
1990; Kaplan and Fields,
1991; Pan et al.,
2000) have suggested that this sensitized pain state results from
the excessive activity of µ-sensitive neurons in the RVM, or NRM secondary
cells, which are thought to facilitate spinal pain transmission through their
direct spinal projections (Fields and
Basbaum, 1999; Ackley et al.,
2001). In fact, recent research has provided convincing evidence
establishing the importance of the descending pain-facilitating actions of the
RVM in several hyperalgesic states of chronic pain, including neuropathic and
inflammatory pain states (Zhuo and
Gebhart, 1997; Porreca et al.,
2002). Interestingly, both µ-receptor-containing neurons
(Porreca et al., 2001) and
glutamate receptors (Urban and Gebhart,
1999) in the RVM have been implicated in mediating these abnormal
pain states. However, what drives the excessive activity of these
pain-facilitating neurons in the hyperalgesic states has remained unclear. The
present finding that glutamate receptor antagonists in the NRM attenuate the
hyperalgesia suggests that glutamate synaptic transmission mediates the
excitation of these cells in opioid withdrawal-induced hyperalgesia, and
perhaps in the hyperalgesia of those chronic pain states as well.
Although agonists can antagonize both analgesia and hyperalgesia
under different behavioral conditions, differential actions on pain
threshold in normal conditions have been reported. Application of
agonists into the RVM has no effect on pain threshold in male rats
(Pan et al., 1997;
Tershner et al., 2000), but
has an analgesic effect in female rats, as assessed by the tail-flick test
(Tershner et al., 2000). A
recent study has also shown an analgesic action of agonists in the rat
RVM with the paw-withdrawal test, but no effect with the tail-flick test, and
the analgesic action has been attributed to inhibition of glutamate
EPSCs in secondary cells (Ackley et al.,
2001). Because results of this type of experiment are primarily
determined by effects on spontaneous and test-elicited activity of
neurons and their glutamate synapses in the RVM, factors that alter
spontaneous activity or different pain tests could influence the behavioral
effects of agonists. Such influential experimental conditions include
anesthesia levels, animal sex, and analgesia tests. The current study
demonstrates the anti-analgesic and anti-hyperalgesic actions of the
agonist by examining its effect on glutamate synaptic transmission that has
been activated differentially in the two NRM cell types under the two
behavioral conditions, opioid analgesia and opioid withdrawal-induced
hyperalgesia.
In addition to the pain states in neuropathic conditions and tissue
inflammation, glutamate synaptic transmission has also been implicated in the
effects of chronic opioids. NMDA receptor antagonists block the development of
chronic morphine-induced analgesic tolerance and physical dependence
(Trujillo and Akil, 1991).
Application of -receptor agonists also suppresses morphine tolerance
and dependence (Takahashi et al.,
1991; Takemori et al.,
1992; Tao et al.,
1994). In view of the important involvement of glutamate receptors
in these pain states, -receptor-mediated inhibition of glutamate
synaptic transmission, as a presynaptic mechanism for regulation of glutamate
release, could play an important role in mediating the behavioral states.
Although glutamate receptor antagonists could be useful in the clinical
treatment of chronic pain and opioid dependence
(Trujillo and Akil, 1995;
Fundytus, 2001), their
therapeutic use may be limited because of the wide distribution and important
role of glutamate synapses in many brain functions, such as learning, memory,
and cognition. The current results may suggest agonists as a potential
alternative in the treatment of opioid withdrawal-related pain and other
chronic pain problems.
|
Footnotes
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|---|
Received Feb. 21, 2003;
revised May. 13, 2003;
accepted May. 20, 2003.
This work was supported by a grant from the National Institute on Drug
Abuse, National Institutes of Health. We thank Jeanie Woodruff for editing
this manuscript.
Correspondence should be addressed to Dr. Zhizhong Z. Pan, Department of
Symptom Research, Box 110, UT-MD Anderson Cancer Center, 1515 Holcombe
Boulevard, Houston, TX 77030. E-mail:
zzpan{at}mdanderson.org.
Copyright © 2003 Society for Neuroscience
0270-6474/03/237262-07$15.00/0
|
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Top
Abstract
Introduction
