Department of Cell Biology and Neuroscience, Rutgers University,
Piscataway, New Jersey
To characterize the effects of morphine on serotonin (5-HT) in the
central nervous system, we used microdialysis in freely behaving
rats. Subcutaneous injection of morphine sulfate produced a
dose-dependent increase in extracellular 5-HT in the dorsal raphe
nucleus (DRN) and a forebrain site, the nucleus accumbens (NAcc). To
determine the site of action for this effect, the opioid receptor
antagonist naltrexone was infused into either the DRN or NAcc.
Naltrexone infusion (300 µM) into the DRN but not the NAcc attenuated
the increase in 5-HT elicited by systemic morphine (20 mg/kg). This
suggests that morphine acts in the DRN to alter the activity of 5-HT
neurons that project to NAcc. Consistent with this conclusion, infusion
of the GABAA receptor antagonist bicuculline (100 µM)
into the DRN but not the NAcc also blocked the effect of systemic
morphine. Similarly, the effect of systemic morphine was blocked by
infusion into the DRN of the GABAA receptor agonist
muscimol (30 µM) and attenuated by the GABAB receptor agonist (±)-baclofen (100 µM). This provides evidence that morphine indirectly influences 5-HT release via opioid receptors on GABAergic neurons in the DRN. A new finding is that ionotropic glutamate receptor
antagonists [kynurenate or a mixture of
(±)-2-amino-5-phosphonopentanoic acid and
6,7-dinitro-quinoxaline-2,3-dione] infused in the DRN also attenuated
the effect of systemic morphine. These results suggest that morphine
acts on GABAergic and glutamatergic afferents to indirectly influence
the activity of 5-HT neurons in the DRN. Understanding the details of
this neural circuitry may provide new leads for treatment of opiate addiction.
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Introduction |
The
dorsal raphe nucleus (DRN) is the main source of serotonergic (5-HT)
projections to the nucleus accumbens (NAcc) (Tork, 1990
) and is
implicated in behavioral effects of opioids (Sutton et al., 1997).
Endogenous opioids and opioid receptors are present in the DRN and
surrounding periaqueductal gray (Mansour et al., 1995; Kalyuzhny and
Wessendorf, 1998; Martin-Schild et al., 1999). Opioids increase
extracellular 5-HT in the NAcc and other areas innervated by the DRN
(Grauer et al., 1992; Tao and Auerbach, 1995). In turn, 5-HT stimulates
dopamine release in the NAcc (Guan and McBride, 1989; Benloucif et al.,
1993; Parsons and Justice, 1993). Moreover, the behavior of recombinant
mice lacking 5-HT and dopamine reuptake transporters or
5-HT1B receptors suggests that 5-HT interacts
with dopamine in the NAcc to influence drug self-administration (Rocha
et al., 1998; Uhl et al., 2002). Both the reinforcing property of
morphine and withdrawal syndrome are affected by 5-HT receptor ligands,
supporting the hypothesis that 5-HT plays a role in opiate addiction
(Cervo et al., 1983; Carboni et al., 1989; Harris and Aston-Jones,
2001).
Opioids modulate 5-HT release by several mechanisms. Morphine does not
stimulate 5-HT neuronal discharge (Haigler, 1978). Instead, µ-opioids
may increase 5-HT release by inhibiting GABAergic afferents to the DRN
(Jolas and Aghajanian, 1997). Consistent with this hypothesis,
pharmacological manipulation of GABA transmission attenuated increases
in 5-HT efflux produced by morphine (Tao and Auerbach, 1994). The
influence of excitatory afferents is inhibited by GABAergic afferents
in the DRN (Tao and Auerbach, 2000). Thus, inhibition of GABA release
by morphine may facilitate excitatory transmission as well as
disinhibit 5-HT neurons. Opioids also directly inhibit some 5-HT
neurons in the DRN and inhibit glutamatergic afferents to 5-HT neurons
in the DRN (Jolas and Aghajanian, 1997). In summary, the net effect of
systemic administration of morphine may be determined by at least four
sites of action. Inhibition of GABA and disinhibition of excitatory
afferents would tend to increase 5-HT efflux. Conversely, direct
inhibition of 5-HT neurons and excitatory afferents by morphine would
tend to decrease 5-HT efflux.
In this study, we used microdialysis to characterize the effects of
systemic morphine on 5-HT in the central nervous system of
unanesthetized rats. The first aim was to test the hypothesis that
opioid receptors in the DRN and not in the forebrain mediate effects of
systemic morphine on 5-HT efflux in the NAcc. The second aim was to
examine the role of GABA in mediating morphine-induced increases in
5-HT efflux. Thus, some acute effects of opioids were ascribed to
reductions in GABA-mediated inhibition of 5-HT neurons (Tao and
Auerbach, 1994; Jolas and Aghajanian, 1997; Sutton et al., 1997).
Finally, we examined the role of glutamate in mediating responses to
systemic morphine. These experiments tested the hypothesis that
morphine, by inhibiting GABA transmission, might enhance the influence
of excitatory afferents in the DRN. This is important in view of
evidence that behavioral sensitization to morphine and the abstinence
syndrome involve changes in glutamatergic transmission (Trujillo and
Akil, 1991; Vanderschuren and Kalivas, 2000).
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Materials and Methods |
Animals.
Male Sprague-Dawley rats (Harlan, Indianapolis, IN)
were individually housed with food and water available ad libitum. The animals were kept on a reversed light/dark cycle (lights off, 9:30
AM-9:30 PM), and all experiments were performed during the lights-off
period. All animal use procedures were in strict accordance with the
National Institutes of Health Guide for the Care and Use of Laboratory
Animals and were approved by the Rutgers University Institutional
Review Board. Rats weighing 300 to 350 g were anesthetized with a
combination of xylazine (4 mg/kg i.p.) and ketamine (80 mg/kg i.p.),
and guide cannulae (21-gauge stainless steel tubing) were implanted as
described previously in detail using standard techniques for
stereotaxic surgery. The coordinates for guide cannulae in the DRN were
AP 1.2 relative to interaural zero, ML 4.0, and DV 1.0 below the
skull surface at a 32° angle lateral to midline; and in the NAcc, AP
10.7, ML 1.4, and DV 1.0 below the skull surface (Paxinos and Watson,
1986). After implantation, the guide cannulae were plugged with
obturators, and the animals were allowed a recovery period of at least
1 week.
Microdialysis.
Microdialysis was performed with an I-shaped
probe constructed from 26-gauge stainless steel tubing and glass
silica. The dialysis tubing was hollow nitrocellulose fiber (200 µm
i.d.; 13,000-mol.wt. cutoff; Spectrum Medical Industries, Los Angeles, CA). The length of the exchange surface of dialysis membrane was adjusted to 1.0 mm for the DRN and 2.5 mm for the NAcc.
The evening before an experiment, rats were briefly anesthetized with
ether, and aseptic dialysis probes were inserted through the guide
cannulae. The target coordinates for the tip of the probe were as
follows: in the DRN, AP 1.2 mm, ML 0.6 mm, and DV 5.5-6.4 mm; and in
the NAcc, AP 10.7 mm, ML 1.4 mm, and DV 6.0-8.5 mm. Rats were then
placed in the test chamber and attached to a fluid swivel that allowed
animals to move freely. Food and water were available ad libitum. The
dialysis probes were perfused overnight with a modified buffered
Ringer's solution (140 mM NaCl, 3.0 mM KCl, 1.5 mM
CaCl2, 1.0 mM MgCl2, 0.27 mM NaH2PO4, 1.2 mM
Na2HPO4, and 1 µM
citalopram, pH 7.4). This Ringer's solution (artificial cerebrospinal
fluid; aCSF) was pumped at a rate of 1.0 µl/min. Sample collection
began at the beginning of the lights-off period under dim red light conditions.
Samples were collected every 30 min and analyzed by high-performance
liquid chromatography with electrochemical detection. Separation of
5-HT was achieved on a column (10 cm × 3.2 mm) with ODS 3-µm
packing (BAS, Inc., West Lafayette, IN). The mobile phase composition
was 0.12 M NaOH, 0.18 mM EDTA, 0.15 M monochloroacetic acid, 1.0 mM
sodium octane sulfonic acid, and 56 ml/l acetonitrile, pH 3.4, and was
pumped at a rate of 0.90 ml/min. Levels of 5-HT in the aCSF were
measured using a dual potentiostat electrochemical detector (EG&G PARC,
Oak Ridge, TN) and dual glassy carbon electrodes (BAS Inc.) in the
parallel configuration. Applied potentials, relative to a Ag/AgCl
electrode were set at approximately maximal and half-maximal for
oxidation of 5-HT. These values were checked frequently and were
usually about 590 and 530 mV. The detection limit for 5-HT was
approximately 0.3 pg/sample based on a signal-to-noise ratio of 3:1.
Experimental Design and Data Analysis.
To study the
interactions among GABA, glutamate, and opioids in regulating
extracellular levels of 5-HT, receptor agonists and antagonists were
added to the aCSF and locally infused into the DRN and NAcc by reverse
microdialysis. Infusion of receptor ligands started 3 h before
systemic administration of morphine sulfate. Mean baseline 5-HT levels
were calculated as the average of the four successive samples before
systemic morphine administration and reported in Table
1 as picograms per sample, uncorrected for probe recovery. The data presented in figures are expressed as mean
(± S.E.M.) percentage of change from the averaged baseline measurements. This normalizes for baseline variability and the effects
of ligand infusion before systemic morphine injection. Thus, the
figures illustrate the influence of GABA and glutamate receptor ligands
on the effect of morphine. Significance (P < 0.05) was
determined using repeated measures ANOVA followed by Scheffè's
post hoc test except for the data shown in Table 1, which were analyzed
using factorial ANOVA followed by Fisher's protected least significant
difference test.
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TABLE 1
Extracellular 5-HT levels in the DRN and NAcc in response to
pretreatment with opioidergic, GABAergic, and glutamatergic receptor
ligands
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Materials.
Morphine sulfate (National Institutes of Health)
was dissolved in physiological saline (0.9% NaCl) and administered
systemically. Doses of morphine refer to the salt form. Other drugs
were dissolved in the aCSF for reverse dialysis infusion into the DRN
or NAcc. Bicuculline methiodide, (±)-2-amino-5-phosphonopentanoic acid (AP-5), 6,7-dinitro-quinoxaline-2,3-dione (DNQX), and phaclofen were
purchased from Sigma/RBI (Natick, MA). Naltrexone hydrochloride, kynurenic acid, and (±)-baclofen were purchased from Sigma-Aldrich (St. Louis, MO).
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Results |
Effect of Morphine on 5-HT.
Subcutaneous injection of morphine
produced a dose-dependent increase in extracellular 5-HT in the DRN. As
shown in Fig. 1A, morphine sulfate at a
dose of 5 mg/kg (equivalent to 4.2 mg/kg free base) elicited a small
but significant increase in extracellular 5-HT in the DRN. In response
to 10 mg/kg morphine sulfate (8.5 mg/kg free base), 5-HT increased to a
maximum of ~60% above baseline. At 20 mg/kg (17.1 mg/kg free base),
morphine sulfate produced a sustained ~100% increase in 5-HT in the
DRN. Systemic administration of the opioid receptor antagonist
naltrexone (10 mg/kg s.c.) 30 min before morphine sulfate (20 mg/kg
s.c.) completely blocked the increase in extracellular 5-HT in the DRN
(Fig. 1B).
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Fig. 1.
Effect of morphine sulfate on extracellular 5-HT in
the DRN. Mean baseline level of 5-HT was 6.8 ± 1.0 pg/sample
(n = 30). The arrows indicate the injection of
morphine sulfate (20 mg/kg s.c.) and naltrexone (10 mg/kg s.c.). A,
morphine induced a dose-dependent increase in extracellular 5-HT in the
DRN: F(3,19) = 9.36, P < 0.0005. All three doses induced significant increases compared with the
vehicle control [5 mg/kg, F(1,10) = 10.09, P < 0.01; 10 mg/kg, F(1,10) = 47.56, P < 0.0001; and 20 mg/kg,
F(1,9) = 14.34, P < 0.01].
Asterisks indicating significant differences were omitted from graph
for the sake of clarity. B, systemic naltrexone (10 mg/kg s.c.) blocked
the effect of morphine on extracellular 5-HT in the DRN:
F(1,10) = 18.01, P < 0.01. Data for the effect of morphine sulfate (20 mg/kg s.c.) alone are
replotted from Fig. 1A and are shown without error bars.  ,
P < 0.05, ANOVA followed by Scheffé's post
hoc test for the comparison to morphine alone.
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To investigate the location of the opioid receptors involved in the
effect of morphine on 5-HT, naltrexone was infused by reverse dialysis
into the DRN. Naltrexone alone had no significant effect on baseline
levels of 5-HT in the DRN (Table 1). However, the effect of systemic
administration of morphine sulfate (20 mg/kg s.c.) on extracellular
5-HT in the DRN was significantly attenuated by local infusion of
naltrexone at concentrations of 300 and 1000 µM in the aCSF (Fig.
2A).
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Fig. 2.
Effect of naltrexone infusion into the DRN on
systemic morphine-induced increases in extracellular 5-HT. The
horizontal bar indicates the infusion of naltrexone, and the arrow
indicates the injection of morphine sulfate (20 mg/kg s.c.). A,
infusion of naltrexone (100 µM in the aCSF) into the DRN did not
attenuate the effect of morphine: F(1,9) = 0.31, P = 0.59. The effect of morphine on extracellular
5-HT in the DRN was significantly attenuated at higher concentrations
of naltrexone: 300 µM, F(1,14) = 11.32, P < 0.005; and 1000 µM,
F(1,8) = 9.87, P < 0.05. *,
P < 0.05, ANOVA followed by Scheffé's post
hoc test for the comparison to morphine alone. B, infusing naltrexone
(300 µM) into the NAcc did not alter the effect of systemic morphine
on extracellular 5-HT in the NAcc: F(1,12) = 0.027, P = 0.87.
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Systemic administration of morphine sulfate (20 mg/kg s.c.) also
increased 5-HT in the NAcc (Fig. 2B). Previous results provide evidence
that morphine acts in the DRN to increase 5-HT efflux in forebrain
projection sites such as the NAcc (Tao and Auerbach, 1995).
Nevertheless, it is possible that morphine might act additionally at
the site of nerve endings in the NAcc to modulate 5-HT release. If this
hypothesis is correct, local infusion of naltrexone into the NAcc
should alter the effect of systemic morphine. However, as shown in Fig.
2B, infusion of naltrexone (300 µM) into the NAcc failed to block the
effect of morphine sulfate (20 mg/kg s.c.) on 5-HT in the NAcc.
Effect of GABA Receptor Ligands on Morphine-Induced Increases in
5-HT.
Morphine does not directly stimulate 5-HT neurons. Instead,
opioids may inhibit GABAergic afferents and thus have a disinhibitory influence on 5-HT neurons (Tao and Auerbach, 1994; Jolas and
Aghajanian, 1997). One prediction of this hypothesis is that
administration of an exogenous GABA receptor agonist should block
morphine-induced increases in 5-HT efflux by offsetting the effect of
decreased release of endogenous GABA. To test this prediction, the
GABAA receptor agonist muscimol was infused into
the DRN or the NAcc by reverse microdialysis. Infusion of muscimol (30 µM) had no significant effect on baseline levels of 5-HT in the DRN
or the NAcc (Table 1). However, muscimol in the DRN but not in the NAcc blocked the effect of morphine sulfate (20 mg/kg s.c.) on 5-HT efflux
(Fig. 3A).
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Fig. 3.
Effect of the GABAA receptor agonist
muscimol on morphine-induced increases in extracellular 5-HT in the DRN
and the NAcc. The horizontal bar indicates the infusion of muscimol (30 µM) or baclofen (100 µM), and the arrow indicates the injection of
morphine sulfate (20 mg/kg s.c.). A, muscimol infusion into the DRN
blocked the effect of morphine on extracellular 5-HT in the DRN
[F(1,9) = 12.24, P < 0.01].
In contrast, muscimol in the NAcc did not block the effect of systemic
morphine on extracellular 5-HT in the NAcc
[F(1,11) = 1.55, P = 0.24].
B, (±)-baclofen infusion into the DRN significantly attenuated the
effect of morphine on extracellular 5-HT in the DRN:
F(1,10) = 6.98, P < 0.05. Data
for the effect of morphine alone are replotted from Fig. 2A and are
shown without error bars. , P < 0.05, ANOVA
followed by Scheffé's post hoc test for the comparison with
morphine alone.
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Activation of either GABAA or
GABAB receptors in the DRN inhibits the discharge
of 5-HT neurons (Innis and Aghajanian, 1987). Thus,
GABAB receptor agonists might similarly interfere
with the effects of morphine on 5-HT. To test this hypothesis,
(±)-baclofen (100 µM) was infused into the DRN. At this
concentration, (±)-baclofen had no significant effect on baseline
levels of 5-HT (Table 1) but significantly attenuated the effect of
morphine sulfate (20 mg/kg s.c.) on 5-HT efflux (Fig. 3B).
Infusion of a GABAA receptor antagonist,
bicuculline (100 µM), into the DRN produced a significant 3-fold
increase in baseline levels of extracellular 5-HT (Fig.
4A; Table 1). This results from blockade
of tonic GABA-mediated inhibition of 5-HT efflux (Tao et al., 1996).
Thus, if morphine increases 5-HT efflux by inhibiting GABA release,
pretreatment with bicuculline should attenuate this effect because 5-HT
neurons would already be disinhibited. Consistent with this prediction,
local infusion of bicuculline into the DRN blocked the effect of
morphine sulfate (20 mg/kg s.c.) on 5-HT efflux in the DRN (Fig. 4B).
In contrast, infusion of bicuculline into the NAcc had no significant
effect on baseline levels of 5-HT in the NAcc (Table 1) and did not
attenuate morphine-induced increases in 5-HT efflux in the NAcc (Fig.
4C).
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Fig. 4.
Effect of infusing the GABAA receptor
antagonist bicuculline into the DRN or NAcc on morphine-induced
increases in extracellular 5-HT. The horizontal bars indicate the
infusion of bicuculline (100 µM), and arrows indicate the injection
of morphine sulfate (20 mg/kg s.c.). A, bicuculline infusion into the
DRN significantly elevated baseline levels of extracellular 5-HT in the
DRN. B, changes in 5-HT are plotted as a percentage of normalized
baseline levels to clearly illustrate the influence of bicuculline on
the effect of morphine. Infusion of bicuculline into the DRN blocked
the effect of morphine on 5-HT in the DRN: F(1,9) = 14.58, P = 0.01. Data for the effect of morphine
alone are replotted from Fig. 2A and are shown without error bars. ,
P < 0.05, ANOVA followed by Scheffé's post
hoc test for the comparison with morphine alone. C, bicuculline
infusion into the NAcc did not alter the effect of systemic morphine on
5-HT in the NAcc: F(1,15) = 0.32, P = 0.58. Data for the effect of morphine alone are
replotted from Fig. 2B and are shown without error bars.
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Phaclofen (100 µM), a selective GABAB receptor
antagonist, was infused into the DRN. At this concentration, phaclofen
blocked the effect of GABAB agonists in the DRN
(Tao et al., 1996) but did not significantly influence baseline levels
of 5-HT in the DRN (Table 1) or block the effect of systemic morphine
sulfate (Fig. 5A). Similarly, phaclofen
in the NAcc had no influence on either baseline levels of 5-HT (Table
1) or morphine-induced increases in 5-HT efflux in the NAcc (Fig. 5B).
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Fig. 5.
Effect of the GABAB receptor antagonist
phaclofen on morphine-induced increases in extracellular 5-HT. The
horizontal bars indicate the infusion of phaclofen (100 µM) and
arrows indicate the injection of morphine sulfate (20 mg/kg s.c.). A,
phaclofen infusion into the DRN did not alter the effect of morphine on
5-HT in the DRN [F(1,8) = 0.0004, P = 0.98]. Data for the effect of morphine alone
are replotted from Fig. 2A and are shown without error bars. B,
phaclofen infusion into the NAcc did not alter the effect of morphine
on 5-HT in the NAcc: F(1,12) = 0.34, P = 0.86. Data for the effect of morphine alone are
replotted from Fig. 2B and are shown without error bars.
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Influence of Ionotropic Glutamate Receptors on Morphine-Induced
Increases in 5-HT.
Glutamatergic neurons have a weak tonic
excitatory influence on 5-HT neurons in the DRN (Tao and Auerbach,
2000) and may also be involved in the effects of opioids on 5-HT (Jolas
and Aghajanian, 1997). To test this, ionotropic glutamate receptor
antagonists were infused into the DRN before systemic administration of
morphine sulfate (20 mg/kg s.c.). Infusion of kynurenic acid (1 mM), a nonselective ionotropic glutamate receptor antagonist, or the combination of AP-5 (1000 µM) + DNQX (300 µM), to block
N-methyl-D-aspartate and
-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid/kainate receptors, decreased 5-HT in the DRN to ~50% of control levels. As
shown in Table 1, this change was statistically significant for
kynurenate (P < 0.05), but because of variability in
absolute levels of 5-HT in baseline samples was not quite significant
for AP-5 + DNQX (P < 0.06). However, combined infusion
of AP-5 and DNQX into the DRN significantly attenuated the effect of
morphine on 5-HT (Fig. 6A). Similarly,
kynurenic acid in the DRN significantly attenuated the effect of
morphine on 5-HT (Fig. 6B).
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Fig. 6.
Effect of glutamate receptor antagonists on
morphine-induced increases in extracellular 5-HT in the DRN. The
horizontal bars indicate the infusion of glutamate receptor
antagonists, and arrows indicate the injection of morphine sulfate (20 mg/kg s.c.). A, combined infusion of AP-5 with DNQX into the DRN
significantly attenuated the effect of morphine on 5-HT in the DRN:
F(1,10) = 8.83, P < 0.05. Data
for the effect of morphine alone are replotted from Fig. 2A and are
shown without error bars. B, kynurenic acid infusion into the DRN
attenuated the effect of morphine on 5-HT in the DRN:
F(1,8) = 5.65, P < 0.05. Data
for the effect of morphine alone are replotted from Fig. 2A and are
shown without error bars. , P < 0.05, ANOVA
followed by Scheffé's post hoc test for the comparison to
morphine alone.
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Discussion |
These results demonstrate that extracellular 5-HT in the DRN and
NAcc increases in response to systemic administration of morphine. This
confirms reports that opioids increase 5-HT turnover and efflux in the
DRN and forebrain regions innervated by the DRN (Snelgar and Vogt,
1981; Spampinato et al., 1985; Grauer et al., 1992; Tao and Auerbach,
1995). Increased 5-HT efflux was mediated at least in part by opioid
receptors on GABAergic neurons in the DRN. Consistent with other
studies (Tao and Auerbach, 1994; Jolas and Aghajanian, 1997), the
results indicate that morphine inhibits GABAergic afferents and thus
disinhibits 5-HT neurons. A major new finding is that an enhanced
influence of excitatory afferents also contributes to increased 5-HT
efflux in response to systemic morphine.
In contrast to systemic administration, local infusion of naltrexone
into the DRN did not fully block increased 5-HT in response to systemic
morphine. The interstitial concentration of substances administered by
reverse dialysis drops steeply and approaches zero at a distance of
~1 mm from the probe (Dykstra et al., 1992). Thus, the residual
increase in 5-HT in response to systemic morphine could be explained by
binding to opioid receptors outside the area perfused with naltrexone
during local infusion of the antagonist. Opioids might act also in
forebrain sites to modulate 5-HT release. However, in contradiction of
this possibility, infusing naltrexone into the NAcc did not block 5-HT
efflux induced by morphine. This supports the conclusion that the
effect of systemic morphine on 5-HT efflux in the forebrain is mediated
in the DRN and nearby periaqueductal gray.
Role of GABAergic Neurons.
Opioids do not stimulate 5-HT
neuronal discharge (Haigler, 1978) and have direct inhibitory effects
on the excitability of some 5-HT neurons in the DRN (Jolas and
Aghajanian, 1997). Instead, because µ-opioids reduce GABA-mediated
postsynaptic currents in 5-HT neurons (Jolas and Aghajanian, 1997),
morphine might increase 5-HT efflux by inhibiting GABAergic afferents.
Consistent with this hypothesis, our results show that the effect of
morphine was attenuated during infusion into the DRN of a
GABAA receptor agonist, muscimol; a
GABAA receptor antagonist, bicuculline; or a
GABAB receptor agonist, baclofen. Similarly,
pentobarbital, which binds with high affinity to
GABAA receptors, prevented the effects of
morphine on 5-HT metabolism and efflux in the central nervous system
(Rivot et al., 1988; Tao and Auerbach, 1994). In contrast, infusion of
muscimol and baclofen into the NAcc did not block the effect of
morphine. Thus, our results confirm and extend previous evidence that
opioids act in the area of the DRN to increase 5-HT release by a
disinhibitory mechanism.
GABAergic afferents synapse with and have a strong influence on the
activity of 5-HT neurons in the DRN (Wang et al., 1992; Gervasoni et
al., 2000). Infusion of the GABAA agonist
muscimol, at concentrations higher than used in the present study,
greatly reduced 5-HT efflux (Tao et al., 1996). Conversely, when
GABAA antagonists are infused, we observed large
increases in extracellular 5-HT, indicating that GABA tonically
inhibits 5-HT transmission via GABAA receptors
under our experimental conditions. Stimulation of
GABAB receptors inhibits 5-HT neurons (Innis and
Aghajanian, 1987), but GABAB receptors also serve
as autoreceptors for GABAergic neurons (Waldmeier et al., 1988). Using
(±)-baclofen, we saw only a small, nonsignificant decrease in baseline
5-HT and suggested that this represented a balance between direct
inhibition of 5-HT neuronal activity and the disinhibitory influence of
blocking GABA release (Tao et al., 1996). In contrast, Abellan et al.
(2000) observed an increase in extracellular 5-HT in response to
(+)-baclofen, indicating that the predominant effect of the active
enantiomer is autoreceptor-mediated reduction of GABA release and thus
disinhibition of 5-HT neurons in the DRN. However, in contrast to
bicuculline, the GABAB antagonist phaclofen had
no significant influence on baseline levels. This suggests that
GABAB receptors do not have a net tonic influence
on 5-HT efflux in the DRN under our baseline experimental conditions.
Muscimol, bicuculline, and (±)-baclofen all attenuated the effect of
morphine, presumably by different mechanisms. We suggest that the GABA
agonist muscimol restrains the increase in 5-HT efflux that otherwise
results from morphine-induced decreases in release of endogenous GABA.
At the relatively low concentration that we used, muscimol did not
significantly reduce baseline levels of 5-HT in the DRN. This suggests
that muscimol did not block the effect of morphine simply as a
consequence of supramaximal inhibition of 5-HT neuronal excitability.
Conversely, bicuculline infusion into the DRN presumably blocked
GABAA receptors, and thus 5-HT neurons were
already disinhibited before morphine administration. Bicuculline
produced a very large increase in baseline levels in the DRN, and the
inability of morphine to elicit a further increase in 5-HT might thus
represent a "ceiling effect". Contrary to this possibility, forced
treadmill running and bicuculline at the same concentration used in the
present study had additive effects on 5-HT in the DRN (R. Tao and
S. B. Auerbach, unpublished observations).
Because baclofen inhibits 5-HT neuronal discharge and activates
autoreceptors to inhibit GABA release, both direct inhibition and
disinhibition may have contributed to the attenuation of
morphine-induced 5-HT efflux that we observed. In contrast, the
GABAB receptor antagonist phaclofen did not
attenuate morphine-induced increases in 5-HT. This suggests that opiate
effects are not mediated by endogenous GABA acting at
GABAB receptors in the DRN. Nevertheless, it is
interesting to note that, in combination with clonidine and sedatives,
baclofen has been used as a centrally acting muscle relaxant to assist
in opiate detoxification (Gerra et al., 2000). It is conceivable that
effects of baclofen on 5-HT release might play a role in the clinical
efficacy of this treatment for withdrawal symptoms after long-term
administration of opiates.
Role of Glutamatergic Neurons.
The main novel finding of this
article is that infusion of glutamate receptor antagonists into the DRN
significantly attenuated the effect of morphine. Kynurenic acid, a
nonselective glutamate receptor antagonist, and combined infusion of
the N-methyl-D-aspartate antagonist
AP-5 with the -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid/kainate antagonist DNQX were equally effective. These results suggest that glutamate-mediated simulation of 5-HT neurons increased in
response to morphine, perhaps as a consequence of decreased GABAergic
transmission, and thus disinhibition of excitatory afferents. Consistent with this hypothesis, GABA tonically restrains the stimulatory influence of glutamate on 5-HT efflux (Tao and
Auerbach, 2000). However, it is important to note that µ-opioids also
directly inhibit glutamatergic afferents to 5-HT neurons in the DRN
(Jolas and Aghajanian, 1997). Thus, opioids may have competing effects on glutamatergic neurons: an indirect disinhibitory influence offset by
direct inhibition. The apparent balance between these two effects is a
net increase in excitatory input to 5-HT neurons under our experimental
conditions. Based on single unit recording of 5-HT neurons in the cat
DRN, ionotropic glutamate receptors do not mediate large increases in
discharge rate but instead may be involved in synchronization of
activity in response to phasic sensory stimuli (Levine and Jacobs,
1992). Thus, we suggest that opioid-induced disinhibition of
glutamatergic as well as GABAergic inputs contribute to increased 5-HT
efflux without stimulation of 5-HT neuronal discharge. The
glutamatergic cell bodies responsive to µ-opioids are located outside
of the DRN (Jolas and Aghajanian, 1997). Hence, the attenuation of
morphine's effect by glutamate receptor blockers might involve
axo-axonic connections of GABAergic neurons with glutamatergic
terminals in the DRN. Alternatively, the interaction might be mediated
postsynaptically with morphine-induced inhibition of GABA release
facilitating excitatory afferent influences on 5-HT neurons.
Some behavioral effects of opioids depend on glutamatergic synaptic
transmission. For example, glutamate receptor antagonists attenuated
the development of behavioral sensitization and tolerance to repeated
administration of opioids (Trujillo and Akil, 1991; Jeziorski et al.,
1994). Synaptic plasticity mediated by
N-methyl-D-aspartate receptors could
be involved in adaptations to prolonged administration of morphine.
Consistent with this suggestion, endogenous opioids, acting via
µ-receptors to inhibit GABA release, enhanced long-term potentiation
in the dentate gyrus (Bramham and Sarvey, 1996). Our data provide novel
evidence that disinhibition of glutamatergic transmission contributes
to morphine-induced increases in 5-HT efflux and support the
possibility that plasticity in the strength of afferent inputs to 5-HT
neurons could be involved in some consequences of long-term
administration of opioids (Tao et al., 1998; Jolas et al., 2000).
In summary, morphine in the DRN increases 5-HT efflux in forebrain
projections sites such as the NAcc. Morphine acts by inhibiting GABAergic afferents, and a novel finding of this study is that glutamatergic inputs also contribute to increased 5-HT efflux. The
effect of morphine on 5-HT is relatively small compared with the
~3-fold increase produced by GABAA receptor
antagonists. This could be explained by the direct inhibitory influence
of opioids on glutamatergic and 5-HT neurons in the DRN (Jolas and
Aghajanian, 1997). Thus, the response to morphine may represent a
balance between direct inhibitory and indirect disinhibitory effects on 5-HT neurons. Because self-administration of opioids is influenced by
5-HT (Harris and Aston-Jones, 2001), understanding the neural circuitry
that regulates 5-HT release could help in developing new treatments for
opioid addiction.
This research was supported by National Institutes of Health
Grants MH51080 (to S.B.A.) and DA14541 (to R.T.).
DRN, dorsal raphe nucleus;
5-HT, 5-hydroxytryptamine, serotonin;
NAcc, nucleus accumbens;
aCSF, artificial cerebrospinal fluid;
ANOVA, analysis of variance;
AP-5, (±)-2-amino-5-phosphonopentanoic acid;
DNQX, 6,7-dinitro-quinoxaline-2,3-dione;
AP, anteroposterior;
ML, mediolateral;
DV, dorsoventral.
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Abstract
Introduction
-1 opioid receptors is required for long-term potentiation induction in the lateral perforant path: dependence on GABAergic inhibition.
J Neurosci
16:
8123-8131
