Pain Mechanisms

Section Editors: Tony L. Yaksh/Quinn H. Hogan

Central Administration of Minocycline and Riluzole

Prevents Morphine-Induced Tolerance in Rats
Bohlool Habibi-Asl, PharmD, PhD
Kambiz Hassanzadeh, PharmD
Mohammad Charkhpour, PharmD,
PhD
BACKGROUND: Long-term exposure to opiates induces tolerance to the analgesic
effect. The neurobiological mechanism of this phenomenon is not completely clear.
In this study, we evaluated the effects of central administration of minocycline (a
tetracycline derivative) and riluzole (an antiglutamatergic drug) on morphine-
induced tolerance in rats.
METHODS: Groups of rats received daily morphine (10 mg/kg, IP) in combination
with saline (10 ␮L/rat, intracerebroventricular [ICV]) or 1% Tween 80 (10 ␮L/rat,
ICV) or minocycline (60, 120, and 240 ␮g/10 ␮L per rat, ICV) or riluzole (20, 40, 80
␮g/10 ␮L per rat, ICV). Nociception was assessed using hotplate apparatus
(55°C ⫾ 0.5°C). Hotplate latency was recorded when the rat licked its hindpaw.
Baseline latencies were determined once per day for each rat, then morphine (10
mg/kg) was injected. After 20 min, the above-mentioned drugs were administered
and postdrug latency was measured 10 min after the injection of drugs or vehicles.
RESULTS: Results showed that ICV administration of minocycline and riluzole
delayed morphine-induced tolerance. Morphine tolerance was complete after 8
days in the control groups but was complete in the groups treated with minocycline
(120 ␮g/10 ␮L per rat) and riluzole (80 ␮g/10 ␮L per rat) on the 13th day. In
addition, our results showed that minocycline and riluzole increased the total
analgesic effect of morphine (area under the curve of the percentage of maximal
possible effect values).
CONCLUSION: The effects of minocycline on nitric oxide and the glutamatergic system
and the effect of riluzole on the glutamate system are potentially important
mechanisms in delaying morphine-induced tolerance.
(Anesth Analg 2009;109:936 –42)

O pioids such as morphine are the most widely used

drugs for the alleviation of moderate to severe chronic
pain. Systemically administered morphine produces
antinociception via actions at both spinal and su-
praspinal sites.1
Repeated use of opioids induces tolerance that results
in a loss of drug effect or the requirement for escalating
doses to produce pain relief. The neurobiological mecha-
nisms of the development of opioid tolerance are multi-
faceted and only partially understood.
There are several lines of evidence that suggest that
N-methyl-d-aspartate (NMDA) glutamate receptors
(NMDARs) are involved in the plasticity that arises
from long-term administration of morphine.2–5 This
idea was suggested by Trujillo and Akil2 who reported
that the NMDA receptor antagonist, MK-801 (dizo-
cilpine), inhibited the development of tolerance to the
From the Department of Pharmacology and Toxicology, Faculty
of Pharmacy, Tabriz University of Medical Sciences, Tabriz, Iran.
Accepted for publication April 13, 2009.
Address correspondence and reprint requests to Kambiz Has-
sanzadeh, Department of Pharmacology and Toxicology, Faculty of
Pharmacy, Tabriz University (Medical Sciences), Tabriz 51664-
14766, Iran. Address e-mail to hassanzadehk@tbzmed.ac.ir.
Copyright © 2009 International Anesthesia Research Society
DOI: 10.1213/ane.0b013e3181ae5f13
936
antinociceptive effect of morphine and morphine
physical dependence.
Using behavioral studies, we and others have
shown that a variety of NMDA receptor antagonists
have the ability to inhibit the development of opiate
tolerance and dependence.2– 8
Other studies have shown that activation of
NMDARs can lead to neurotoxicity under many cir-
cumstances.9 For instance, peripheral nerve injury has
been shown to activate spinal cord NMDARs, which
results in not only intractable neuropathic pain but
also neuronal cell death because of apoptosis.10 –12
There are also several lines of evidence which
suggest that activation of NMDARs leads to toxic
calcium influx, which activates numerous enzymes,
including neuronal nitric oxide (NO) synthase (NOS).
NO is able to further increase excitotoxicity by enhanc-
ing glutamate release from presynaptic neurons and
inhibiting glial glutamate transporters.13–15
Minocycline, a semisynthetic tetracycline deriva-
tive, is able to provide neuroprotection against global
ischemia in gerbils and focal brain ischemia in rats.16,17
Another study strongly suggested that minocycline
acts at an earlier plasmalemmal step by limiting
glutamate release and the ensuing [Ca2⫹] elevation in
target neurons. Minocycline may prevent the activa-
tion of this [Ca2⫹]-dependent intracellular pathway,
Vol. 109, No. 3, September 2009
thus inhibiting neuronal death. A decrease in neuronal
excitability, together with a marked decrease in gluta-
mate release, may explain the cytoprotective proper-
ties of minocycline.18 –20
Many studies have indicated that the protective effect
of minocycline was associated with reduced activation of
inducible NOS and interleukin-1b-converting enzyme,
which are mainly expressed by microglia.19
Riluzole is the only proven effective medicine for
amyotrophic lateral sclerosis (ALS) because it has been
demonstrated to delay the time of death in ALS
patients.20 Riluzole is an antiglutamatergic drug,
which interferes with responses mediated by excita-
tory amino acids, even though it does not interact with
any known binding site on NMDA, kainate, or
␣-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid
(AMPA) glutamate receptors.20 In addition, it has been
shown that coadministration of riluzole with morphine
decreased the intensity of the withdrawal syndrome,
reflecting a reduction in physical dependence.21
In view of these data, both minocycline and riluzole
have a common mechanism of action on the glutama-
tergic system; therefore, in this study, we evaluated
the effect of intracerebroventricular (ICV) administra-
tion of minocycline and riluzole on morphine toler-
ance in rats.
METHODS
Animals
Male Wistar rats (Razi Institute, Tehran, Iran) weigh-
ing 250 –300 g (eight animals in each group) were used in
this study. They were kept in a temperature-controlled
room (24°C ⫾ 0.5°C) and maintained on a 12-h light/
dark cycle (light on 08:00 am) with free access to food
and water. All experiments were executed in accordance
with the Guide for the Care and Use of Laboratory
Animals (National Institutes of Health Publication No.
85-23, revised 1985) and were approved by the research
and ethics committee of Tabriz University of Medical
Sciences.
Intracerebroventricular Cannula Implantation
Rats were anesthetized with sodium pentobarbital
(50 mg/kg, IP) (Merck, Germany) and a stainless steel
guide cannula (23 gauge) was implanted stereotaxi-
cally into the lateral cerebral ventricle (coordinates:
⫺0.8 mm posterior, ⫺1.3 mm midline to lateral, and
3.5 mm ventral) with respect to bregma.22
A stainless steel guide (30 gauge) was placed into
the guide cannula as a dummy cannula to maintain
patency. After surgery, a recovery period of 7 days
was allowed before experiments. During the recovery
period, animals were habituated to the testing envi-
ronment including transfer to the experimental room
and twice daily handling, weighing, restraining on the
platform for 1 min, and gently removing and replac-
ing the dummy cannula. Animals were also habitu-
ated to the hotplate apparatus and testing started after
the recovery period of 7 days in all groups.
Vol. 109, No. 3, September 2009
Verification of Cannula Placement
At the end of all experiments, methylene blue
solution (5 ␮L/rat, ICV) was injected into the cannula
and the animals were killed by an overdose of ether
followed by decapitation. The brain of each animal
was dissected out and cut in the coronal plane to
verify the placement of the guide cannula and distri-
bution of methylene blue in the ventricles. Only data
from animals that showed uniform distribution of
methylene blue in the ventricles were considered for
statistical analysis. In all, six animals were discarded
because the placement of the guide cannula was
incorrect.
Drug Treatment
Morphine sulfate (Sigma-Aldrich; Sigma-Aldrich,
Germany) (10 mg/kg, daily) was dissolved in double
distilled water and was injected IP using 1-mL insulin
syringes. Minocycline (Sigma-Aldrich) (60, 120, and 240
␮g/10 ␮L per rat) was dissolved in double distilled
water and infused ICV using a Hamilton microsy-
ringe. Riluzole (Sigma-Aldrich) (20, 40, and 80 ␮g/10
␮L per rat) was dissolved (1% Tween 80 in sterile 0.9%
normal saline) and infused ICV using a Hamilton
syringe. Dizocilpine (Sigma-Aldrich) (1 ␮g/10 ␮L per
rat) was dissolved in double distilled water and
infused ICV using a Hamilton microsyringe. Two
control groups were included, which received either
morphine, IP ⫹ 1% Tween 80 in saline normal 0.9%,
ICV or morphine, IP ⫹ distilled water 0.9%, ICV.
Volume of infusion was 10 ␮L at a rate of 10 ␮L/min
in each rat.
Assessment of Nociception
Nociception was assessed using the hotplate appa-
ratus (55°C ⫾ 0.5°C).23 Hotplate latency was recorded
when the rat licked its hindpaw. A cut-off time (40 s)
was imposed to prevent tissue damage.24 Hotplate
response latencies (s) are expressed as the percentage
of maximal possible effect (%MPE) using the equation
below:
Post-drug latency (s) ⫺ Baseline latency (s)
%MPE ⫽ ⫻ 100
Cut-off value (s) ⫺ Baseline latency (s)
Baseline latency was determined once per day for
each rat, before daily injection of morphine (10 mg/kg).
After 20 min, the drugs were administered and the
postdrug latency was measured 10 min after the injec-
tion of drugs or vehicles (30 min after the injection of
morphine). The %MPE was then calculated for that day.
The experiments continued until there was no significant
difference in %MPE between the vehicle- or drug-treated
groups and the saline group.
Evaluation of the Global Analgesic Effect
To evaluate the global analgesic effect and to allow
a comparison of the effects from different behavioral
tests, the area under the curve (AUC) of the %MPE
was calculated. To calculate the AUC, the trapezoidal
© 2009 International Anesthesia Research Society 937

Figure 1. Analgesic effect of daily administration of mor-
phine (10 mg/kg, IP) in combination with distilled water (10
␮L/rat) or 1% Tween 80 in normal saline (10 ␮L/rat).
Developed tolerance to the analgesic effect of morphine was
complete on the 8th day when there were no significant
differences in percentage of maximal possible effect (%MPE)
between the control groups and the saline group. Each bar
represents the mean of %MPE ⫾ sem for eight rats. M ⫽
morphine; DW ⫽ distilled water; Mino ⫽ minocycline;
Rilu ⫽ riluzole.
rule was used. The AUC (1–13 days) was calculated
using the trapezoidal rule from the observed values.
Evaluation of Tolerance Induction
To evaluate the induction of tolerance, groups of
rats received either saline or morphine (10 mg/kg, IP)
⫹ saline or morphine (10 mg/kg, IP) ⫹ minocycline
(the most effective dose) or morphine (10 mg/kg, IP)
⫹ riluzole (the most effective dose) for 8 days, then on
the 9th day (1 day after morphine tolerance in the
control group) several doses of morphine (10, 25, 50,
and 100 mg/kg, IP) were administered to generate
analgesic dose-response curves. Morphine antinoci-
ceptive 50% effective dose (ED50) values for each of the
drug groups were derived using linear regression of
%MPE of the morphine dose. Differences in the ED50
estimations were determined using the confidence
interval method at P ⫽ 0.05.25
Data Analysis
Data are expressed as the mean of %MPE ⫾ sem of
eight rats per group. One-way analysis of variance
followed by Tukey’s test were used to analyze statis-
tical significance in multiple comparisons. P values
⬍0.05 were considered to be significant in all analyses.
*P ⬍ 0.05, **P ⬍ 0.01, and ***P ⬍ 0.001 indicate a
significant difference as compared with the saline
group for that day. The AUC1–13 data were analyzed
by one-way analysis of variance.
RESULTS
Induction of Tolerance to the Antinociceptive Effect
of Morphine
As shown in Figure 1, daily administration of
morphine (10 mg/kg, IP) induced tolerance to the
antinociceptive effect of this drug in both the control
groups which received 1% Tween 80 in saline normal
938 Minocycline and Riluzole Decreased Morphine Tolerance
Figure 2. Effect of daily ICV injections of minocycline (60,
120, and 240 ␮g/10 ␮L per rat) on tolerance to the analgesic
effect of morphine. Each bar represents the mean of percent-
age of maximal possible effect (%MPE) ⫾ sem for eight rats.
One-way analysis of variance (ANOVA) followed by
Tukey’s test were used to analyze the statistical signifi-
cances. P values ⬍0.05 were considered to be significant in
all analyses. *P ⬍ 0.05; **P ⬍ 0.01; ***P ⬍ 0.001 when
compared with the control group. Arrow represents the day
of morphine tolerance. M ⫽ morphine; DW ⫽ distilled
water; Mino ⫽ minocycline.
0.9% or distilled water. The analgesic effect of mor-
phine decreased on the 8th day compared with the 1st
day and, because there were no significant differences
between the control groups and the saline-treated
animals on Day 8, this was considered the day of
morphine tolerance initiation.
Evaluation of the Effect of Minocycline on Morphine-
Induced Tolerance to the Analgesic Effect
Minocycline delayed the onset of morphine-
induced tolerance. Minocycline (60, 240, and 120
␮g/10 ␮L per rat) delayed morphine tolerance for 4, 4
and 5 days, respectively (Fig. 2). Analysis of the AUC
of hotplate latency (Table 1), which allowed evalua-
tion of the global effect, showed that repeated treat-
ment with minocycline before morphine enhanced the
effectiveness of morphine. On the other hand, mino-
cycline (120 ␮g/10 ␮L per rat) had the greatest AUC of
%MPE (289.3) compared with the control group
(177.5) or minocycline (60 ␮g/10 ␮L per rat) (264.7) or
minocycline (240 ␮g/10 ␮L per rat) (234.7) (Table 1).
In addition, the results in Figure 3 show a significant
shift to the right in the dose-response curve for
animals who received morphine (10 mg/kg) ⫹ saline
(10 ␮L/rat) compared with those receiving saline (10
␮L/rat) or morphine (10 mg/kg) ⫹ minocycline (120
␮g/10 ␮L per rat). A significant shift to the right in
ED50 in the control group (88.64) compared with saline
(29.5) or morphine (10 mg/kg) ⫹ minocycline (120
␮g/10 ␮L per rat) (61.1) treated animals was also seen.
Evaluation of the Effect of Riluzole on Morphine-Induced
Tolerance to the Analgesic Effect
Riluzole (20, 40, and 80 ␮g/10 ␮L per rat) also
delayed morphine tolerance. Riluzole (20 and 40
␮g/10 ␮L per rat) delayed morphine tolerance for 4
days; however, the results indicated that riluzole (80
␮g/10 ␮L per rat) decreased the development of
ANESTHESIA & ANALGESIA
Table 1. The Global Analgesic Effect of Morphine in the Control and Treatment Groups During 13 Days of Experimentation
Minocycline AUC
Control mino 177.5
60 ␮g/10 ␮L per rat 264.7*
120 ␮g/10 ␮L per rat 289.3*
240 ␮g/10 ␮L per rat 234.7*
Area under the curve (AUC) of percentage of maximal possible effect (%MPE) was calculated for each group for 13 days. To calculate the AUC, the trapezoidal rule was used. One-way analysis
of variance followed by Tukey’s test was used to analyze the differences between the control and treatment groups.
Mino ⫽ Minocycline; Rilu ⫽ Riluzol; Dizo ⫽ Dizocilpine.
P values less than 0.05 were considered to be significant in all analyses. *P ⬍ 0.001; †P ⬍ 0.01; ‡P ⬍ 0.05 when compared with the control group.
Figure 3. Hotplate responses and percent of maximal pos-
sible effect (%MPE) to various morphine doses (10, 25, 50,
and100 mg/kg, IP) administered on Day 9 after 8 days of
continuous ICV drug infusion. Each point represents the
mean ⫾ sem of eight rats. Different morphine doses admin-
istered on Day 9 demonstrated a significant shift to the right
in the dose-response curve and antinociceptive ED50 values
in animals treated with morphine ⫹ saline compared with
those receiving saline or morphine ⫹ minocycline (120
␮g/10 ␮L per rat) or morphine ⫹ riluzole (80 ␮g/10 ␮L per
rat).
morphine tolerance and could delay morphine toler-
ance for 5 days (Fig. 4). Riluzole (80 ␮g/10 ␮L per rat)
had the greatest AUC of %MPE (261) compared with
the control group (156) or riluzole (20 ␮g/10 ␮L per
rat) (216) or riluzole (40 ␮g/10 ␮L per rat) (197) (Table
1). Figure 3 shows a significant shift to the right in the
dose-response curve for animals that received mor-
phine (10 mg/kg) ⫹ saline (10 ␮L/rat) compared with
those receiving saline (10 ␮L/rat) or morphine (10
mg/kg) ⫹ riluzole (80 ␮g/10 ␮L per rat). Further-
more, a significant shift to the right in ED50 in the
control group (88.64) was observed when compared
with saline (29.5) and morphine (10 mg/kg) ⫹ riluzole
(80 ␮g/10 ␮L per rat) (66.8) treated animals.
Evaluation of the Analgesic Effects of Minocycline
and Riluzole
Administration of the most effective doses of mino-
cycline (120 ␮g/10 ␮L per rat) or riluzole (80 ␮g/10
␮L per rat) on morphine-induced tolerance did not
have a significant analgesic effect and there were
significant differences between the administration of
these drugs and saline (Fig. 5).
Vol. 109, No. 3, September 2009
sem Riluzole AUC sem
4.2 Control rilu 156 3.4
3.7 20 ␮g/10 ␮L per rat 216† 3.1
2.6 40 ␮g/10 ␮L per rat 197‡ 2.4
2.9 80 ␮g/10 ␮L per rat 261* 3.4
Dizo 1 ␮g/10 ␮L per rat 363.8* 4.1
Figure 4. Effect of daily ICV injections of riluzole (20, 40, and
80 ␮g/10 ␮L per rat) on tolerance to the analgesic effect of
morphine. Each bar represents the mean of percentage of
maximal possible effect (%MPE) ⫾ sem for eight rats.
One-way analysis of variance (ANOVA) followed by
Tukey’s test were used to analyze the statistical signifi-
cances. P values ⬍0.05 were considered to be significant in
all analyses. *P ⬍ 0.05; **P ⬍ 0.01; ***P ⬍ 0.001 when
compared with the control group. Arrow represents the day
of morphine tolerance. M ⫽ morphine; Rilu ⫽ riluzole.
Comparison of the Effect of Minocycline or Riluzole
with Dizocilpine on Attenuation of Morphine
Tolerance Development
To examine the possible mechanism involved in
morphine-induced tolerance, we tested the effect of
ICV administration of dizocilpine (1 ␮g/10 ␮L per rat)
(a noncompetitive NMDA receptor antagonist), be-
cause this agent has well-established effects on opioid
tolerance and was used as a positive control in this
study. As shown in Figure 6, dizocilpine attenuated
morphine tolerance and delayed tolerance initiation
for 6 days at this dose. Dizocilpine (1 ␮g/10 ␮L per
rat) did not have a significant analgesic effect, thus the
effects of this drug are not related to analgesia.
DISCUSSION
Tolerance is a behavioral adaptation to the pro-
longed use of opioid drugs, such as morphine. The
cellular mechanism underlying the development of
morphine tolerance remains controversial. In the cur-
rent study, we investigated the effect of minocycline
and riluzole on morphine-induced tolerance to the
analgesic effect. The main findings of this study indi-
cate that ICV administration of minocycline and ri-
luzole can prevent the development of morphine
tolerance.
© 2009 International Anesthesia Research Society 939
Figure 5. Analgesic effects of daily ICV injections of dizo-
cilpine (1 ␮g/10 ␮L per rat), minocycline (120 ␮g/10 ␮L per
rat), riluzole (80 ␮g/10 ␮L per rat), and saline (10 ␮L/rat).
Each bar represents the mean of percentage of maximal
possible effect (%MPE) ⫾ sem for eight rats. One-way
analysis of variance (ANOVA) followed by Tukey’s test
were used to analyze the differences between the saline and
treatment groups. P values ⬍0.05 were considered to be
significant in all analyses. Mino ⫽ minocycline; Rilu ⫽
riluzole; Dizo ⫽ dizocilpine.
Several studies have indicated that repeated admin-
istration of opiates can activate the NMDA receptor
through the G-protein associated with the opioid
receptor and/or intracellular mechanisms.26,27 This
opiate-related activation of NMDARs may initiate
subsequent intracellular changes, such as the produc-
tion of NO and/or the activation of protein kinase C
(PKC). Both NO and PKC have been shown to be
critical in the development of morphine tolerance.28,29
Previous studies have shown that minocycline, a
semisynthetic tetracycline derivative, exhibits neuro-
protective effects against neuronal damage in animal
disease models.18,30 –33 Our results showed that mino-
cycline (120 ␮g/10 ␮L per rat) postponed morphine
tolerance for 5 days and minocycline (60 and 240
␮g/10 ␮L per rat) shifted morphine tolerance from the
8th to the 12th day (Fig. 2). On the other hand, the total
analgesic effect of morphine (AUC of %MPE) signifi-
cantly increased in all treatment groups (Table 1).
Although minocycline (120 ␮g/10 ␮L per rat) delayed
morphine tolerance more than other doses, there was
no significant difference among the three doses in
their effects on morphine tolerance. Furthermore, the
results shown in Figure 5 demonstrate that the doses
of minocycline administered in this study did not
have an analgesic effect. Therefore, the action of
minocyline in preventing morphine tolerance is not
related to its analgesic effect. Several reports attribute
the neuroprotective effects of minocycline to various
intracellular signaling pathways, including antioxi-
dant systems,34 inhibition of NOS19 blockade of in-
flammatory responses,35 prevention of the activation
of Ca2⫹-dependent intracellular pathways, and a
marked decrease in glutamate release.20
In addition, another study showed that NMDA-
induced neuronal death involved proliferation and
activation of microglial cells and that minocycline
940 Minocycline and Riluzole Decreased Morphine Tolerance
Figure 6. Effects of daily ICV injections of the most effective
doses of minocycline (120 ␮g/10 ␮L per rat) and riluzole (80
␮g/10 ␮L per rat) were compared with dizocilpine (1 ␮g/10
␮L per rat) used as a positive control. Each bar represents the
mean of percentage of maximal possible effect (%MPE) ⫾ sem
for eight rats. M ⫽ morphine; DW ⫽ distilled water; Mino ⫽
minocycline; Rilu ⫽ riluzole; Dizo ⫽ dizocilpine.
completely prevented NMDA toxicity and the preced-
ing activation and proliferation of microglial cells.
These results support the notion that microglial activa-
tion contributes to excitotoxic neuronal death, which can
be inhibited by antiinflammatory compounds, such as
minocycline.18 The mechanism underlying the role of
glial cells in the effects of morphine on naive mice is
unclear. It is possible that morphine acts directly on
microglia, triggering alterations in their morphology,
metabolism, and function.36 Furthermore, a recent
study indicated that systemic administration of mino-
cycline attenuated morphine antinociceptive toler-
ance.37 Mika et al.37 concluded that the effect of
minocycline on morphine tolerance is related to mi-
croglia. Their results provide evidence that systemic
administration of minocycline in mice influences mor-
phine’s effectiveness and delays the development of
morphine tolerance by attenuating microglial activa-
tion and its markers.
According to the above-mentioned studies and our
findings, it is concluded that the effect of minocycline
in this study is possibly related to its neuroprotective
property, its effect on preventing glutamate release,
and its inhibition of microglial cells and NOS.
Figure 4 showed that riluzole decreased tolerance
to the analgesic effect of morphine. Riluzole is the only
proven effective medicine for ALS and was demon-
strated to delay the time of death in these patients.38,39
This is thought to result from the neuroprotective
effect of riluzole, which has a complex mechanism of
action involving several effects: inhibition of voltage-
dependent sodium channels,40,41 high-voltage-activated
calcium and potassium channels,41 and inhibition of
PKC, suggesting involvement in antioxidative pro-
cesses.42 Our results showed that riluzole (20, 40, and 80
␮g/10 ␮L per rat) delayed morphine tolerance. As
shown in Figure 4, low doses of riluzole (20 and 40
␮g/10 ␮L per rat) only delayed morphine tolerance
for 2 days and 1 day, respectively; however, riluzole
(80 ␮g/10 ␮L per rat) delayed morphine tolerance for
ANESTHESIA & ANALGESIA
5 days. On the other hand, the total analgesic effect of
morphine (AUC of %MPE) significantly increased in
animals treated with morphine ⫹ riluzole (80 ␮g/10
␮L per rat). The results shown in Figure 5 indicate that
the most effective dose of riluzole in morphine toler-
ance (80 ␮g/10 ␮L per rat) did not have a significant
analgesic effect. Previous studies have indicated that
the most important mechanism of action for riluzole is
its effect on glutamatergic transmission: riluzole inhib-
ited glutamate release from presynaptic terminals
through a mechanism linked to G-protein signaling.43
Riluzole also affects neurotransmission mediated by
AMPA/kainate receptors and reduces NMDA-evoked
responses.44,45 Finally, riluzole enhances high-affinity
glutamate uptake in rat spinal cord synaptosomes in
vitro and after treatment in vivo.46,47 A recent study
demonstrated that riluzole significantly increased glu-
tamate uptake mediated by transporters, such as
GLAST, GLT1, and EAAC1.48 As previously men-
tioned, it has been shown that the neuroprotectant
riluzole has a direct inhibitory action on PKC, which
has a critical role in the development of morphine
tolerance,49 and it has been confirmed that riluzole at
a concentration ⬍30 mM protects against excitotoxic
neuronal injury induced by NMDA or kainate in
mouse cortical cultures.42 Furthermore, there is evi-
dence that riluzole inhibits the electrophysiological
responses mediated by rat kainate and NMDA recep-
tors expressed in Xenopus oocytes.50 In this study, we
examined the analgesic effect of three other doses of
morphine in combination with saline or minocycline
(120 ␮g/10 ␮L per rat) or riluzole (80 ␮g/10 ␮L per
rat) on the 9th day (the day after morphine tolerance
in the control group). The significant shift to the right
in the dose-response curve and ED50 for animals
receiving morphine (10 mg/kg) ⫹ saline (10 ␮L/rat)
showed that these animals were tolerant to morphine
analgesia. The ED50 in those rats that received mor-
phine ⫹ minocycline or riluzole indicated that these
drugs could prevent tolerance to the analgesic effect of
morphine and that they showed significant differences
in ED50 compared with the control group. Further-
more, our results showed that dizocilpine (1 ␮g/10 ␮L
per rat), a noncompetitive NMDA receptor antagonist,
which has a well-established effect on opioid toler-
ance, could attenuate morphine tolerance and the
effects of minocycline and riluzole were similar to this
drug, thus dizocilpine could help to elucidate the
mechanisms involved in morphine tolerance. Dizo-
cilpine alone did not have a significant analgesic
effect; however, the global analgesic effect of this drug
when combined with morphine was greater than mor-
phine ⫹ minocycline or morphine ⫹ riluzole. Similarly,
dizocilpine delayed morphine tolerance from the 8th day
to the 14th day. In conclusion, our results showed that
ICV administration of minocycline and riluzole could
delay morphine tolerance; however, further studies are
needed to clarify the exact mechanism involved.
Vol. 109, No. 3, September 2009
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