![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
|
|
|
|
|
||
Vol. 303, Issue 2, 549-556, November 2002
Department of Cell Biology and Neuroscience, Rutgers University, Piscataway, New Jersey
 |
Abstract |
|---|
 Top
 Abstract
 Introduction
Materials and Methods
Results
Discussion
References
|
|---|
Opioid receptor subtypes may have site-specific effects and
play different roles in modulating serotonergic neurotransmission in
the mammalian central nervous system. To test this hypothesis, we used in vivo microdialysis to measure changes in extracellular serotonin (5-hydroxytryptamine; 5-HT) in response to local infusion of
µ-, 
-, and 
-opioid receptor ligands into the dorsal raphe nucleus (DRN), median raphe nucleus (MRN), and nucleus accumbens (NAcc)
of freely behaving rats. The µ-opioids
[D-Ala2-N-Me-Phe4,Gly5-ol]enkephalin
(DAMGO), endomorphin-1, and endomorphin-2 were administered by reverse
dialysis infusion into the DRN. In response, extracellular 5-HT was
increased in the DRN, an effect that was blocked by the selective
µ-receptor antagonist 
-funaltrexamine, but not by the
-receptor antagonist
N,N-diallyl-Tyr-Aib-Aib-Phe-Leu-OH (ICI-174,864). Infusion of -receptor agonists,
[D-Ala2,D-Len5]enkephalin
(DADLE), [D-Pen2,5]enkephalin (DPDPE), and
deltophin-II into the DRN also increased extracellular 5-HT, an effect
that was blocked by selective -receptor antagonists. In contrast to
the DRN, local infusion of µ- and -opioids had no effect on 5-HT
in the MRN or NAcc. These data indicate that µ- and -opioid
ligands have a selective influence on serotonergic neurons in the
DRN. Finally, the -receptor agonist U-50,488
[trans-(±)-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)cyclohexyl]benzeneacetamide] produced similar decreases in 5-HT during local infusion into the DRN,
MRN, and NAcc. These results provide evidence of differential regulation of 5-HT release by opioid receptor subtypes in the midbrain
raphe and forebrain.
| |
Introduction |
|---|
|
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
|
|---|
The
largest population of serotonergic cell bodies is located in the dorsal
raphe nucleus (DRN) within the ventral portion of periaqueductal gray
(PAG), an area richly endowed with opioids and involved in integrating
responses to stress (Basbaum and Fields, 1984
). Pain and stressful
stimuli activate opioidergic neurons in the PAG, which in turn may
modulate the activity of serotonergic neurons with projections to sites
involved in arousal and emotional state (Ma and Han, 1992; Grahn et
al., 1999). Immunocytochemical (Kalyuzhny et al., 1996) and
neurochemical (Tao and Auerbach, 1995) studies provide further evidence
that opioids modulate serotonergic neuronal activity. However, single
unit recording data suggest that the opioid receptor agonist morphine
does not directly stimulate serotonergic neurons (Haigler, 1978).
Instead, opioids may inhibit both inhibitory and excitatory afferents
to the DRN (Jolas and Aghajanian, 1997) and thus could indirectly
affect the pattern of serotonergic neuronal discharge.
Four types of opioid receptors, µ, , , and ORL-1, have been
identified on the basis of pharmacological and molecular criteria (Knapp et al., 1995; Neal et al., 1999). Endogenous ligands for opioid
receptors have been determined, and these have distinct, albeit
overlapping, patterns of distribution in the CNS (Mansour et al., 1995;
Martin-Schild et al., 1999; Neal et al., 1999). In particular, the
ventral PAG, which encompasses the DRN, has moderate to high densities
of each of the endogenous opioids and corresponding opioid receptor
types (Mansour et al., 1995; Neal et al., 1999). Moreover, there are
distinctive and in some instances opposing physiological effects of
selective agonists of the four opioid receptor types. For example,
selective µ-opioid receptor agonists are strong analgesics but
produce greater physical dependence relative to selective -opioid
receptor agonists (Maldonado et al., 1990). In contrast, - and ORL-1
receptor agonists induce hyperalgesia (Lutfy and Maidment, 2000) and
can reverse the analgesic effects of µ-opioid receptor activation
(Pan et al., 1997).
In this study, we used in vivo microdialysis to test the hypothesis
that different opioid receptor subtypes have distinct and site-specific
roles in regulation of serotonergic neurons. We compared the effect of
µ-, - and -opioid-receptor ligands on extracellular levels of
5-HT in the CNS. Anesthetics that alter glutamate or GABA transmission
attenuate the effect of morphine on analgesia (Banks et al., 1988) and
5-HT turnover and release (Rivot et al., 1988; Tao and Auerbach,
1994a). Thus, we used freely behaving rats to avoid interference by
anesthetics with the afferent neurons that may indirectly mediate the
effects of opioids. Opioids were infused into the DRN by reverse
microdialysis. To determine whether there are regionally selective
effects on 5-HT, opioids were also infused into the median raphe
nucleus (MRN), which contains the second largest cluster of
serotonergic cell bodies, and the nucleus accumbens (NAcc), a forebrain
site innervated by projections from the DRN and implicated in
addiction, sensitization, and other behavioral consequences of opiate drugs.
| |
Materials and Methods |
|---|
|
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
|
|---|
Animal Preparation. Male Sprague-Dawley rats purchased from 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 from 9:30 AM to 9:30 PM), and all experiments were performed during the dark phase. 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 cannulas (21-gauge stainless steel tubing) were implanted using standard stereotaxic techniques. After surgery, the guide cannulas were plugged with stylets and the rats were allowed a recovery period of at least 1 week.
Microdialysis Procedures.
Microdialysis was performed with a
concentric design probe constructed from 26-gauge stainless steel
tubing and glass silica. The dialysis tubing was hollow nitrocellulose
fiber (0.25 mm o.d., 13,000 mol. wt. cut-off; Spectrum Medical
Industries, Los Angeles, CA). The dialysis membrane exchange surface
was 1 mm for probes in the DRN and MRN, and 2.5 mm in the NAcc.
Stereotaxic coordinates for the DRN were: AP 1.2, DV 6.1 to 6.7, and ML
±0.4 at a 32° angle lateral to the midline; in the MRN: AP 1.2, DV
8.2 to 8.6, and ML ±0.5 at a 26°angle lateral to the midline; in
NAcc: AP 10.7, DV 6.0 to 8.5, and ML 1.4 (Paxinos and Watson, 1986).
Most experiments used conventional microdialysis techniques with a single probe implanted in the DRN, MRN or NAcc. In some "dual probe" experiments, one dialysis probe in the NAcc was used to sample
changes in extracellular 5-HT in response to opioid infusion through a
second dialysis probe implanted in the area of serotonergic cell bodies
in the DRN.
). Moreover, citalopram did not
qualitatively alter the effects of experimental manipulations such as
blocking GABAA receptors or systemic
administration of opioids. Thus, local infusion of bicuculline into the
DRN produced a 3-fold increase in extracellular 5-HT irrespective of
the presence of a 5-HT reuptake inhibitor (Tao and Auerbach, 2000).
Also, systemic morphine produced an ~50% increase in forebrain
levels of extracellular 5-HT with or without citalopram in the aCSF,
and this effect was less variable with reuptake blocked (Tao and
Auerbach, 1994b).
Samples were collected every 30 min during the lights-off period and
analyzed by high-performance liquid chromatography with electrochemical
detection. Separation of 5-HT was achieved on a 10 cm × 3.2 mm
column with ODS 3 µm packing (BAS Bioanalytical Systems 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. Monoamines were measured using a dual potentiostat
electrochemical detector (PerkinElmer Life Sciences, Boston, MA) and
dual glassy carbon electrode in the parallel configuration. Applied
potentials relative to an 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 540 mV. The detection
limit for 5-HT was approximately 0.3 pg/sample based on a
signal-to-noise ratio of 3:1.
After extracellular levels of 5-HT were stable in four consecutive
samples (less than ±10% variation), opioid receptor ligands were
administered by reverse dialysis infusion. Ligands were dissolved in
the aCSF at concentrations based on microdialysis studies of the
effects of opioids on extracellular dopamine in the rat CNS (Spanagel
et al., 1990). Also, we used analgesiometry to evaluate the
physiological significance of the concentrations that we used (see below).
Analgesia Measurements. The analgesic effect of reverse dialysis infusion of DAMGO in the DRN was determined using the tail-flick test. For this experiment, a separate group of rats was used with the major aim of determining whether the concentration of DAMGO was in a physiologically relevant range. A secondary aim was to determine whether analgesia was correlated with opioid-induced changes in 5-HT. The rats were kept in the dialysis chamber overnight and aCSF (containing 1 µM citalopram) was infused into the DRN via a probe. Analgesia measurements were carried out the next day during the lights-off period under dim red light illumination. The room temperature (22 ± 0.5°C) and humidity were thermostatically controlled. Once every 15 min, rats were transferred from the dialysis chamber to an analgesiometer (Ugo Basile, Varese, Italy) to determine tail-flick latencies during continuous infusion of aCSF into the DRN. The radiant intensity was set to maintain basal flick latencies in the range of 3.9 to 4.9 s with the cut-off time set at 12 s to avoid cumulative damage to tissue. After three trials averaged as the baseline, DAMGO was added to the aCSF and infused into the DRN for 2 h. Latency measurements were carried out every 15 min during the period of drug infusion, and one final measurement was taken 2 h after the end of the drug infusion period.
Data Analysis. For determining changes in extracellular 5-HT, the four consecutive samples before drug administration were averaged to obtain mean basal levels expressed as picograms per sample. Baseline levels were also normalized to 100% with changes induced by drug treatments expressed as a percentage of mean basal levels and plotted against sample time in the figures. Absolute amounts of baseline 5-HT (picograms per sample) are presented in the figure legends. For analgesic experiments, the data were expressed as the latency of tail withdrawal in response to radiant heat stimulation. Significance of differences (p < 0.05) was determined using repeated measures analysis of variance, followed by Scheffé's post hoc test.
DAMGO ([D-Ala2-N-Me-Phe4,Gly5-ol]enkephalin) and DPDPE ([D-Pen2,5]enkephalin) were obtained from Sigma-Aldrich (St. Louis, MO). U-50,488, nor-BNI (nor-binaltorphimine), DADLE ([D-Ala2, D-Len5]enkephalin) and-FNA were purchased from Sigma/RBI (Natick, MA).
[D-Ala]2-Deltorphin II,
ICI-174,864 (N,N-diallyl-Tyr-Aib-Aib-Phe-Leu-OH), endomorphin-1 (Tyr-Pro-Trp-Phe-NH2), and
endomorphin-2 (Tyr-Pro-Phe-Phe-NH2) were
generously provided by the National Institute on Drug Abuse (Bethesda,
MD). Reagents were dissolved in aCSF (containing 1 µM citalopram) and
administered by reverse dialysis.
| |
Results |
|---|
|
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
|
|---|
Effect of µ-Opioid Receptor Ligands on 5-HT.
DAMGO is a
synthetic peptide selective for µ-opioid receptors (Knapp et al.,
1995). Reverse dialysis infusion of DAMGO (100-1000 µM) into the DRN
for 2 h elicited dose-dependent increases in extracellular levels
of 5-HT in the DRN (Fig. 1A). The mean
maximal effect was an ~80% increase above baseline, and levels
returned to baseline ~2 h after the end of infusion. Two endogenous
µ-opioid peptides, endomorphin-1 and endomorphin-2 (Zadina et al.,
1997) were also infused for 2 h into the DRN. Endomorphin-1 (300 µM) produced an ~70% increase in 5-HT in the DRN (Fig. 1B). At the same concentration, endomorphin-2 produced an ~40% increase in extracellular 5-HT. The difference between the effects of endomorphin-1 and endomorphin-2 was not statistically significant. Extracellular 5-HT
returned to baseline levels within 1 h of the end of endomorphin infusion.
|
-FNA is a selective antagonist of µ-opioid receptors (Recht
and Pasternak, 1987). Reverse dialysis infusion of -FNA alone at the
concentration of 300 µM had no effect on 5-HT in the DRN, but blocked
DAMGO-induced increases in 5-HT (Fig.
2A). In contrast, infusion of a
-opioid selective receptor antagonist, ICI-174,864, had no effect on
DAMGO-induced increases in 5-HT (Fig. 4).
In a separate dual-probe experiment, DAMGO was infused into the DRN to
determine whether this elicited parallel changes in 5-HT efflux in the
DRN and NAcc. As shown in Fig. 2B, DAMGO infusion into the DRN produced
significant increases in extracellular 5-HT in both the DRN and the
NAcc. In contrast, infusion of aCSF in the DRN had no effect on 5-HT in
the NAcc.
|
Effect of -Opioid Receptor Ligands on 5-HT.
The
enkephalin analog DADLE (300-1000 µM) was infused by reverse
dialysis into the DRN and induced a significant, ~40% increase in
5-HT in the DRN (Fig. 3A). DADLE binds to
both the putative 1- and
2-opioid receptor subtypes (Xu et al., 1998),
which may have differential functions in regulating neuronal activity
(Acosta and Lopez, 1999). Hence, we examined the role of ligands
selective for either the 1- or
2-opioid receptor subtypes. The enkephalin analog, DPDPE, preferentially binds to the putative
1-opioid receptor subtype (Acosta and Lopez,
1999). Infusion of DPDPE (1000 µM) into the DRN produced a
significant, ~40% increase in DRN 5-HT (Fig. 3B). Another enkephalin
analog, deltorphin II, is selective for the putative
2-opioid receptor subtype (Acosta and Lopez, 1999). Reverse dialysis infusion of deltorphin II (1000 µM) into the
DRN also produced an ~40% increase in DRN 5-HT (Fig. 3B). Thus,
1- and 2-receptor
ligands had similar effects on 5-HT, and the effect of a nonselective
-opioid agonist was not the sum of the effects of the
1- and 2-opioid
receptor agonists.
|
-opioid antagonist ICI-174,864 (300 µM) was infused into the DRN
beginning 30 min before DADLE. Reverse dialysis infusion of ICI-174,864
alone at a concentration of 300 µM had no effect on DRN 5-HT, but
blocked the effect of DADLE (Fig. 4). ICI-174,864 may also act as an
inverse agonist for -opioid receptors (Chiu et al., 1996; Neilan et
al., 1999), and consistent with this possibility, at a concentration of
1000 µM in the aCSF, ICI-174,864 induced a significant decrease in
DRN 5-HT (Fig. 5A). Naltrindole, a
putatively pure antagonist of -opioid receptors (Chiu et al., 1996;
Neilan et al., 1999) also induced a reduction in 5-HT in the DRN, and this effect was dose-dependent (Fig. 5B). In contrast, 5-HT in the DRN
was not reduced during infusion of the µ-opioid receptor antagonist
-FNA at a concentration of 1000 µM in the aCSF (data not shown).
|
|
Effect of a -Opioid Receptor Agonist on 5-HT in the DRN.
In
contrast to µ- and -opioid receptor agonists, a -opioid
elicited decreases in extracellular 5-HT. Thus, reverse dialysis infusion of the nonpeptide -agonist U-50,488 (100, 300, and 1000 µM) into the DRN produced a dose-dependent decrease in extracellular 5-HT in the DRN (Fig. 6A). Decreases were
significant beginning 1 h after the start of U-50,488 infusion.
The -receptor antagonist nor-BNI (300 µM) alone had no
effect on DRN 5-HT, but blocked the U-50,488-induced decrease in 5-HT
(Fig. 6B).
|
Effect of a -Opioid on 5-HT in the MRN and NAcc:
Comparison with µ- and -Opioids.
The results of infusing
µ-, -, and -opioid receptor agonists into the MRN and NAcc were
compared with effects in the DRN. As shown in Fig.
7, the -agonist, U-50,488 produced
decreases in extracellular 5-HT during reverse dialysis infusion into
the MRN and NAcc. Similar to the effect of U-50,488 in the DRN, the decreases in 5-HT in the MRN and the NAcc were significant, beginning 1 h after the start of drug infusion (Fig. 7, A and B). In
contrast, infusion of the µ-opioid agonist DAMGO or the
-opioid agonist DPDPE into the MRN and the NAcc did not
affect 5-HT in these sites (Fig. 7, A, C, and D). Although there was a
small, transient increase in 5-HT during infusion of DPDPE (1000 µM)
into the NAcc, this effect was not significant (Fig. 7D).
|
Nociceptive Response to DAMGO during Microdialysis Infusion.
The microdialysis membrane is a barrier to free diffusion of
substances into and out of the microdialysis probe. Thus, during reverse dialysis infusion of opioids, the concentration in
extracellular space is much lower than in the perfusion medium. To
determine whether the concentrations that we used resulted in
physiologically relevant levels, a separate group of rats was used for
measurement of changes in tail-flick latency in response to infusion of
DAMGO through a dialysis probe in the DRN. DAMGO at a concentration of
100 µM in the infusion medium had no significant effect on tail-flick
latency compared with the baseline measurement of 4.5 s or
controls infused with aCSF alone (Fig.
8). Tail-flick latency was significantly
increased to ~8 s during infusion of DAMGO at a concentration of 300 µM in the aCSF, and DAMGO at 1000 µM increased latency nearly to
the cut-off time of 12 s. However, in contrast to the sustained
effect on extracellular 5-HT, tail-flick latency decreased markedly
during the second hour of DAMGO infusion, and baseline values were
obtained within 2 h after the end of infusion. Thus, the
dose-response curves for DAMGO-induced increases in tail-flick latency
and extracellular levels of 5-HT were similar, but there was a
dissociation between the time course of the two effects.
|
| |
Discussion |
|---|
|
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
|
|---|
These results demonstrate that µ-opioids in the DRN can elicit
increases in 5-HT efflux in the DRN and the NAcc of unanesthetized rats. -Opioids in the DRN also produced increases in 5-HT efflux. In
contrast, µ- and -opioids had no effect on 5-HT when infused into
the MRN or NAcc. These results are consistent with other reports that
systemic administration of morphine elicits selective increases in 5-HT
in forebrain sites innervated by the DRN (Spampinato et al., 1985; Tao
and Auerbach, 1995). In contrast, -receptor agonists decreased 5-HT
during infusion into the DRN, MRN, or NAcc. Thus, µ-, -, and
-opioid receptors may have different roles and sites of action in
regulation of 5-HT neurotransmission in the CNS.
µ-Opioids.
DAMGO infusion into the DRN increased
extracellular 5-HT. -FNA, a µ-receptor antagonist, but not
ICI-174,864, a -opioid antagonist, blocked this effect, indicating
that µ-opioid receptors mediated DAMGO-induced increases in 5-HT.
Because concentrations of DAMGO were high, this may be particularly
relevant to the pharmacological effects of µ-opioids. However,
endogenous opioids are abundant in the ventral PAG/DRN and mediate
responses to stress (Grahn et al., 1999). Moreover, we found that the
endogenous peptides, endomorphin-1 and endomorphin-2, also induced
increases in 5-HT in the DRN. Compared with the synthetic opioid DAMGO,
the increase in response to endomorphin-2 was somewhat smaller, and
both endomorphin-1 and endomorphin-2 had shorter-lasting effects. This
may be attributable to faster metabolic clearance of the endomorphins,
although we cannot exclude other possibilities such as differences in
efficacy without data concerning the effects of higher doses of these
two endogenous peptides.
). Conversely, tetrodotoxin infusion
into the DRN decreased extracellular 5-HT in the DRN and NAcc (Tao and
Auerbach, 2000). This indicates that 5-HT release in the raphe (from
axon collaterals or dendrites) parallels changes in forebrain
projection sites and depends on action potential propagation from
serotonergic cell bodies. Together, these data suggest that
extracellular levels in the raphe can be used as an indicator of
neuronal activity and 5-HT release in the forebrain.
The DRN and surrounding ventrolateral PAG are involved in the analgesic
effects of opioids (Basbaum and Fields, 1984). Thus, to determine
whether opioids were administered in a physiological concentration
range, we measured the analgesic response to reverse dialysis infusion
of DAMGO in the DRN. Similar to the effect on extracellular 5-HT, we
observed elevated tail-flick latencies only at concentrations of DAMGO
above 100 µM in the aCSF. In comparison, direct microinjection of 50 ng of DAMGO into the ventral PAG elicited analgesia (Pan et al., 2000).
This suggests that reverse dialysis of DAMGO at concentrations of 100 to 1000 µM in the aCSF bracketed the physiologically effective range
for this peptide. The concentration of a substance added to the aCSF
falls steeply after crossing out of the dialysis probe and approaches
zero at a distance of ~1 mm (Dykstra et al., 1992). Thus, compared
with microinjection, administration by reverse dialysis requires high
drug concentrations for effective perfusion of surrounding tissue.
Moreover, in contrast to the effect of infusion into the DRN, morphine
infusion into the MRN at a site ~1 mm below the DRN did not increase
5-HT in the NAcc (Tao and Auerbach, 1995). The NAcc is innervated by
serotonergic projections from the DRN but not the MRN. Thus, these data
support the conclusion that drug diffusion was limited to a small area around the dialysis probe.
Although the dose-response curves for elevations in 5-HT and tail-flick
latency were similar, the time courses were different. Despite
sustained increases in extracellular 5-HT, tail-flick latencies
returned toward baseline during DAMGO infusion. This suggests that
enhanced activity of serotonergic neurons in the DRN is not sufficient
in mediating the analgesic effects of opioids. Because changes in
extracellular 5-HT provide only an indirect and summed measure of
release, it is possible that a subpopulation of serotonergic neurons is
involved in modulation of nociception (Mason, 1999). However, the
elevation in tail-flick latency produced by opioids in the ventral PAG
has been more definitively linked to activation of glutamatergic
neurons with projections to the medulla (Fields et al., 1991).
The observation that DAMGO and endomorphins enhanced extracellular 5-HT
contrasts with electrophysiological evidence that opioids have
inhibitory effects (North, 1986) and do not increase serotonergic
neuronal discharge in the DRN (Haigler, 1978). This may be explained by
evidence that the effects of µ-opioids on 5-HT are indirectly
mediated by inhibition of GABAergic neurons in the ventral PAG
(Kalyuzhny et al., 1996; Jolas and Aghajanian, 1997). Thus, µ-opioids
could have a disinhibitory influence similar to their effect on
dopaminergic neurons (Johnson and North, 1992). However, µ-opioids
also inhibit glutamatergic afferents (Jolas and Aghajanian, 1997). By
inhibiting both GABAergic and glutamatergic input, µ-opioids might
regularize serotonergic neuronal discharge. Although GABA-mediated
decreases in discharge would be blocked, the maximal instantaneous rate
of serotonergic neuronal discharge could be unchanged or even decreased
because the excitatory influence of glutamate is also inhibited. The
net effect of µ-opioids would be an increase in extracellular 5-HT
because GABA but not glutamate has a strong tonic influence on
serotonergic neurons in the DRN under our experimental conditions (Tao
et al., 1996; Tao and Auerbach, 2000). This hypothesis is supported by
evidence that blocking GABA receptors in the DRN abolishes increases in
5-HT elicited by morphine and DAMGO (Tao and Auerbach, 1994a;
unpublished results). In contrast, GABA does not have a strong tonic
influence on 5-HT in the MRN (Tao et al., 1996; Tao and Auerbach,
2000). This could explain the observation that infusion of µ-opioids
into the MRN had no effect on extracellular 5-HT. The physiological
significance of the effect of opioids on DRN but not MRN serotonergic
neurons is difficult to assess. However, based in part on established functions of serotonergic neurons, this may contribute to the changes
in behavioral state such as interruption of normal sleep cycles
produced by opioids (Jacobs and Fornal, 1991).
-Opioids.
-Opioid receptor agonists also produced
increases in 5-HT in the DRN but not in the MRN or NAcc. In addition,
the -receptor antagonist ICI-174,864 blocked the effect of DADLE,
but not DAMGO. These results support the conclusion that the effects of
-agonists on 5-HT were mediated by -opioid receptors. A common
pool of Gi/o proteins may transduce the effects
of µ- and -opioids (Alt et al., 2002), and it is possible that
both receptor subtypes increase 5-HT by a similar disinhibitory
mechanism. Our observation that -opioid receptor agonists had
smaller effects on 5-HT efflux may be related to the much weaker
inhibition of GABAergic afferents by DADLE compared with DAMGO (Jolas
and Aghajanian, 1997).
-FNA, naltrindole and ICI-174,864
by themselves at high concentrations reduced 5-HT efflux in the DRN.
This is interesting in relation to in vitro evidence that
-receptors, unlike other opioid receptor subtypes, can be active in
the absence of agonist. Moreover, ICI-174,864 acted as an inverse
agonist to reduce constitutive activity of cloned -opioid receptors
expressed in kidney and glioma cell lines (Chiu et al., 1996; Neilan et
al., 1999). In contrast, naltrindole acted in vitro on cloned
-receptors as a pure antagonist (Chiu et al., 1996; Neilan et al.,
1999). It is conceivable that both naltrindole and ICI-174,864 inhibit
constitutive activity of -opioid receptors in vivo and, thus, reduce
5-HT efflux. Alternatively, endogenous -opioids may have a tonic
excitatory influence on serotonergic neurons under our experimental
conditions. Further experiments are necessary to evaluate these
hypotheses and exclude the possibility that ICI-174,864 and naltrindole
at high concentrations had nonselective effects on 5-HT efflux.
We also investigated the role of putative -opioid receptor
subtypes in regulation of 5-HT release. DPDPE and deltorphin II have
been used as selective 1- and
2-receptor agonists, respectively (Acosta and
Lopez, 1999). However, the two agonists produced similar increases in
extracellular 5-HT. Conceivably, the two -receptor subtypes
contribute independently to modulation of 5-HT release, but the effect
of the nonselective -agonist, DADLE, did not have a greater effect
than DPDPE or deltorphin II. Thus, the increase in response to DADLE
cannot be ascribed to the sum of 1- and 2-receptor effects.
-Opioids.
In contrast to µ- and -opioids, -opioids
reduced extracellular 5-HT in all brain areas examined. -Opioids
inhibited 5-HT efflux from spinal cord synaptosomes, suggesting a
direct effect on nerve endings (Monroe et al., 1995). -Opioids also
decreased, whereas µ and agonists enhanced, extracellular
dopamine (Spanagel et al., 1990). It is interesting to note that the
effects of -opioids on both 5-HT and dopamine were delayed. For
dopamine, this has been ascribed to delayed enhancement of dopamine
reuptake rather than inhibition of release (Thompson et al., 2000). The
possibility that -opioids act at the site of nerve endings to either
enhance 5-HT clearance or inhibit exocytosis could explain the
observation that -opioids had no effect on electrophysiological
properties of serotonergic neurons (Jolas and Aghajanian, 1997).
-Opioid receptor agonists in the DRN but
not MRN enhanced 5-HT efflux. In contrast, -agonists acted in the
DRN, MRN, and forebrain to decrease extracellular 5-HT. Thus,
-opioids may act directly on serotonergic nerve endings, whereas
µ- and -opioids presumably act indirectly by inhibiting afferents
to serotonergic neurons in the DRN (Tao and Auerbach, 1994a; Jolas and
Aghajanian, 1997). These results support the conclusion that µ-,
-, and -opioids differentially modulate serotonergic
neurotransmission in specific CNS sites implicated in general control
of arousal state and specific behaviors such as drug
self-administration.
| |
Acknowledgments |
|---|
We thank Zhiyuan Ma for excellent technical assistance.
| |
Footnotes |
|---|
Accepted for publication July 15, 2002.
Received for publication April 23, 2002.
1 Present Address: Research, 151-C, Harvard Medical School VA Medical Center, 940 Belmont Street, Brockton, MA 02301-559
This research was supported by U.S. Public Health Service Grants MH51080 (S.B.A.) and DA14541 (R.T.).
DOI: 10.1124/jpet.102.037861
Address correspondence to: Dr. Sidney B. Auerbach, Rutgers, The State University of New Jersey, Department of Cell Biology and Neuroscience, 604 Allison Road, Piscataway, NJ 08854. E-mail: auerbach{at}biology.rutgers.edu
| |
Abbreviations |
|---|
5-HT, 5-hydroxytryptamine (serotonin);
DRN, dorsal raphe nucleus;
PAG, periaqueductal gray;
ORL-1, opioid
receptor-like receptor 1;
CNS, central nervous system;
MRN, median
raphe nucleus;
NAcc, nucleus accumbens;
AP, anteroposterior;
DV, dorsoventricular;
ML, mediolateral;
aCSF, artificial cerebrospinal
fluid;
DAMGO, [D-Ala2-N-Me-Phe4,Gly5-ol]enkephalin;
DPDPE, [D-Pen2,5]enkephalin;
U-50,488, trans-(±)-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)cyclohexyl]benzeneacetamide;
nor-BNI, nor-binaltorphimine;
DADLE, [D-Ala2,D-Len5]enkephalin;
-FNA, -funaltrexamine;
ICI-174,864, N,N-diallyl-Tyr-Aib-Aib-Phe-Leu-OH.
| |
References |
|---|
|
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
|
|---|
This article has been cited by other articles:
|
|
 |
|
  L. Leggio and G. Addolorato Invited Commentary * Serotonin transporter (SERT) brain density and neurobiological cloninger subtypes model: a lesson by human autoradiography studies Alcohol Alcohol., March 1, 2008; 43(2): 148 - 150. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B. B. Land, M. R. Bruchas, J. C. Lemos, M. Xu, E. J. Melief, and C. Chavkin The Dysphoric Component of Stress Is Encoded by Activation of the Dynorphin {kappa}-Opioid System J. Neurosci., January 9, 2008; 28(2): 407 - 414. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Fichna, A. Janecka, J. Costentin, and J.-C. Do Rego The Endomorphin System and Its Evolving Neurophysiological Role Pharmacol. Rev., March 1, 2007; 59(1): 88 - 123. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||
, p < 0.05 compared with 
