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Brain Research 1030
Research report
Involvement of n-opioid receptors and j receptors in memory function
demonstrated using an antisense strategy
Masayuki Hiramatsua,b,*, Takashi Hoshinob
aLaboratory of Neuropsychopharmacology, Graduate School of Environmental and Human Sciences, Meijo University, Tenpaku-ku, Nagoya 468-8503, JapanbDepartment of Chemical Pharmacology, Faculty of Pharmaceutical Sciences, Meijo University, Nagoya 468-8503, Japan
Accepted 17 October 2004
Available online 11 November 2004
Abstract
Although antinociceptive effects of U-50,488H (trans-(F)-3,4-dichloro-N-methyl-N-(2-[1-pyrrolidinyl] cyclohexyl) benzeneacetamide
methanesulfonate and (�)-pentazocine have been reported to influence n-opioid receptors, the involvement of n-opioid receptors in learning
and/or memory is still controversial. We have recently reported that the memory improving effect of (�)-pentazocine was antagonized by j1receptor antagonist. In this study, we examined the effects of several antisense oligodeoxynucleotides (antisenses) to n1-opioid receptors and
j1 receptor on memory and nociceptive function. Male ddY mice were treated subcutaneously (s.c.) with scopolamine (1.65 Amol/kg) and/or
test drugs 30 min before a Y-maze test. U-50,488H significantly improved the scopolamine-induced impairment of spontaneous alternation
behavior. Twenty micrograms of antisense targeting exons 2 and 3 of the n1-opioid receptor significantly reversed the effects of U-50,488H,
but antisense targeting exon 1 and mismatch sense did not. The antisense targeting exon 3 was most effective. These antisenses themselves
did not affect normal mice, indicating that n1-opioid receptors do not tonically regulate memory function. All three antisenses equally
prevented U-50,488H-induced antinociceptive effects in the acetic-acid-induced writhing test. Pretreatment with antisense targeting j1receptors (AS-j1) completely prevented the memory-improving effects of (�)- and (+)-pentazocine, although U-50,488H ameliorated the
scopolamine-induced impairment of spontaneous alternation behavior in AS-j1-treated mice. These results suggest that n1-opioid receptors
containing different exons have a distinct function in memory and nociceptive functions. Furthermore, n-opioid receptors agonist showing
analgesic effects act on n-opioid receptors or j receptors and play important roles only when memory function is impaired, but the two
neuronal systems regulate memory function independently.
D 2004 Elsevier B.V. All rights reserved.
Theme: Neural basis of behavior
Topic: Learning and memory: pharmacology
Keywords: n-Opioid receptor; U-50,488H; j Receptor; Antisense; Learning and memory
1. Introduction
Cholinergic neuronal systems play an important role in
the cognitive deficits associated with aging and neuro-
degenerative diseases [1,2,6,28]. Although investigations of
0006-8993/$ - see front matter D 2004 Elsevier B.V. All rights reserved.
doi:10.1016/j.brainres.2004.10.020
* Corresponding author. Laboratory of Neuropsychopharmacology,
Graduate School of Environmental and Human Sciences, Meijo University,
Tenpaku-ku, Nagoya 468-8503, Japan. Tel.: +81 52 832 1781x342; fax:
+81 52 834 8780.
E-mail address: [email protected] (M. Hiramatsu).
learning and memory have focused primarily on cholinergic
neurotransmission, reports of increased numbers of n-opioidreceptors in the limbic sysytem [8] and in the putamen and
cerebellar cortex of postmortem brains of Alzheimer’s
patients [22] suggest that disruption of opioidergic neuro-
transmission is also involved in the cognitive deficits
associated with Alzheimer’s disease and aging.
We previously reported that n-opioid receptor agonists,
dynorphin A (1–13) and U-50,488H (trans-(F)-3,4-
dichloro-N-methyl-N-(2-[1-pyrrolidinyl] cyclohexyl) ben-
zene-acetamide methanesulfonate, improved scopolamine-
(2004) 247–255
M. Hiramatsu, T. Hoshino / Brain Research 1030 (2004) 247–255248
induced impairment of spontaneous alternation performance
in mice [14,16,18]. Dynorphin A (1–13) and U-50,488H
reversed the decrease in acetylcholine release caused by
carbachol and mecamylamine [13,14]. The j receptor was
initially considered a member of the opioid receptor family
[21], but is now considered a distinct group of receptors
[35]. Interestingly, we demonstrated that a prototype of jreceptor agonist, (+)-N-allylnormetazocine [(+)-SKF-
10,047], and (+)-pentazocine improved impairment of
learning and memory in mice [27]. Moreover, we have
recently reported that (�)-pentazocine acting on n-opioidreceptors and showing an analgesic effect, improved
impairment of memory in mice via the j receptor [17]. It
has been shown that (F)-SKF-10,047 enhanced stimulation-
evoked acetylcholine release in guinea pig cerebral slices
[36], and other j receptor agonists, (+)-SKF-10,047, (F)-
pentazocine, 1,3,-di-o-tolylguanidine (DTG), and R(+)-3-
(3-hydroxyphenyl)-N -propylpiperidine [(+)-3-PPP],
increased extracellular acetylcholine levels in the rat frontal
cortex [23,24] and hippocampus [25]. The activating effects
of (+)-SKF-10,047 on the central cholinergic system were
antagonized by haloperidol at a dose range compatible with
its j1 receptor antagonistic action [24,25]. Therefore, the n-opioidergic system and j receptors in the brain may play an
important role in modulating learning and memory func-
tions, although interaction between the n-opioidergic systemand j receptors and the cholinergic systems in the central
nervous system has not been elucidated. Recently, we
proposed using selective antagonists that the ameliorating
effects on memory impairment are independent and no
direct link exists between the n-opioid and j receptor-
mediated mechanisms [16,17].
The antisense strategy, i.e., the use of synthetic oligo-
deoxynucleotides to inhibit gene expression through
sequence-specific hybridization, is currently used in in vitro
and in vivo studies for the characterization of molecular
sites of action in neurobiology. Antisense strategies have
been used in vivo to alter the expression of numerous
neurotransmitter and neuromodulator receptors and this
technique allows for the discovery of precise receptor
mechanisms mediating behavioral actions of the neuro-
peptide system in the brain. Antisense mapping studies
showed that individual exons affected analgesic responses
of several opioidergic drugs [30,34,38]. These findings
suggest that proteins generated from mRNAs containing
each exon of a gene have different roles in different
functions, i.e., memory and analgesia.
The purpose of this study was to examine the molecular
basis of the involvement of n-opioid receptors and jreceptors in memory and analgesic processes in the mouse,
using an in vivo antisense strategy. The effects of antisense
oligodeoxynucleotide targeting the n1-opioid receptor on theantiamnesic properties of a selective n1-opioid receptor
agonist, U-50,488H, were examined using pharmacological
models of memory impairment induced by scopolamine and
nociception induced by acetic acid. Moreover, we examined
the effects of antisense to the j1 receptor using (+)- and (�)-
pentazocine at the same schedules, because at low doses,
pentazocine or related compounds may be candidates for
antiamnesic drugs. The behavioral test, spontaneous alter-
nation in the Y-maze, was used as a first-intent test for the
antiamnesic effects of n1-opioid receptor agonists and jreceptor agonists, since it is pharmacologically predictive,
does not constrain the animals, and we have several data
using this method accompanied with a passive avoidance test.
2. Materials and methods
2.1. Animals
Seven-week-old male ddY mice (Japan SLC, Japan) were
kept in a controlled environment, with controlled lighting
(12-h light/dark cycle, lights on; 8 a.m. to 8 p.m.) and
temperature (23F2 8C) for at least 5 days before the
experiments, and given free access to food and water. The
experiment was started at 7 weeks and finished by 9 weeks,
and body weight was raised from 29 to 40 g during the
experimental period. Experimental protocols concerning the
use of laboratory animals were approved by the committee of
Meijo University and were performed in accordance with the
guidelines of the Japanese Pharmacological Society (Folia
Pharmacol. Japon, 1992, 99: 35A) and the interministerial
decree of May 25th, 1987 (The Ministry of Education).
2.2. Design and administration of oligodeoxynucleotides
The sequences of the antisense oligodeoxynucleotides
(antisenses) for the n1-opioid receptor, n3-opioid receptor,
and j1 receptor were published by Pasternak et al. [34], Pan
et al. [30], and King et al. [19] and based on the cDNA
sequence for the mouse n1-opioid receptor, n3-opioidreceptor, and j1 receptor, respectively. The 21-mer phos-
phorothioate-modified antisenses, except AS-n3 which was
20 mer, targeted an area distant from the initiation codon, 5V-GCTGCTGATCCTCTGAGCCCA-3V (=AS-n1-1, exon 1
location 310–330), 5V-TGGCACACAGCAATGTAGCGG-3V (=AS-n1-2, exon 2 location 650–670), 5V-GCTTCCCA-GAGCCTCCACCAG-3V (=AS-n1-3, exon 3 location 1068–1088), 5V-GGGCTGTGCAGAAGCCGAGA-3V (=AS-n3,location 1189–1208), 5V-GAGTGCCCAGCCACAAC-
CAGG-3V (=AS-j1 location 77–97). The mismatched ana-
logue for the n1-opioid receptor including two switched
pairs was based upon AS-n1-1, 5V-GCTCGTGAT
CCTCTGGACCCA-3V (=mismatch sense). The oligodeox-
ynucleotides were synthesized and purified using high-
pressure liquid chromatography (Rikaken, Nagoya, Japan).
They were dissolved in artificial cerebrospinal fluid (aCSF),
which contained 128.5 mM NaCl, 3.0 mM KCl, 1.15 mM
CaCl2, 0.8 mM MgCl2, 21.0 mM NaHCO3, 0.25 mM
Na2HPO4, and 3.4 mM glucose. Each antisense was
administered unilaterally before the test session into the
M. Hiramatsu, T. Hoshino / Brain Research 1030 (2004) 247–255 249
lateral ventricular (i.c.v.) region of the mouse brain according
to the methods of Haley and McCormick [7] in a volume of 5
Al/mouse using a microinfusion pump (Kd Scientific Model
270, speed at 5 Al/30 s) under brief ether anesthesia.
Antisenses (5–20 Ag) were administered on days 1, 3, and
5, and the behavioral analysis was conducted on day 6. This
time course of treatment both downregulates the synthesis of
new receptors and permits turnover of existing receptors
[33]. Antisenses were administered again on day 7, and the
antinociceptive test was conducted on day 8.
2.3. Drugs
The following drugs were used: U-50,488H (trans-(F)-
3,4-dichloro-N-methyl-N-(2-[1-pyrrolidinyl] cyclohexyl)
benzeneacetamide methanesulfonate (Sigma, St. Louis,
MO); (+)- and (�)-pentazocine (Santen Pharmaceuticals,
Osaka, Japan); and scopolamine hydrobromide (scopol-
amine, Tokyo Chemical Industry, Tokyo, Japan). All doses
were calculated as those of the bases. Drugs were dissolved
in an isotonic saline solution (Otsuka Pharmaceuticals,
Tokyo, Japan) at a volume of 0.1 ml/10 g body weight.
Scopolamine was administered subcutaneouly (s.c.) around
the abdominal area 30 min before the behavioral tests. U-
50,488H, (+)-pentazocine or (�)-pentazocine was adminis-
tered s.c. under the skin of the dorsal neck immediately
after scopolamine injection. Control mice received aCSF
i.c.v. at a volume of 5 Al/side in mouse brain, and saline s.c.
and/or intraperitoneally (i.p.) at a volume of 0.1 ml/10 g
body weight.
2.4. Spontaneous alternation behavior
Immediate working memory performance was assessed
by recording spontaneous alternation behavior during a
single session in a Y-maze with minor modification [12,36].
Each Y-maze arm was 40 cm long, 12 cm high, with a width
of 3 cm at the bottom and 10 cm at the top. The arms
converged in an equilateral triangular central area. Each
mouse, new to the maze, was placed at the end of one arm
and allowed to move freely through the maze. The series of
arm entries was recorded visually during an 8-min session,
and the total number of arm entries was counted. Arm entry
was considered to be completed when the hind paws of the
mouse passed completely over the threshold of the arm. The
observer did not know which drugs were administered
because the treatments were randomly assigned. Alternation
events were defined as successive entries into the three
arms, in overlapping triplet sets, and the number of these
events was counted. The effect was calculated as percent
alternation according to the following formula:
percent alternation ¼ number of alternation events½ �
= total number of arm entries½ � � 2ð Þ
� 100%
2.5. Acetic-acid-induced writhing test
The writhing test was conducted 30 min after the s.c.
injection of each drug. Mice were treated with a 0.7% acetic
acid solution (i.p.) at a volume of 0.1 ml/10 g body weight
10 min before the writhing test, and then writhing responses
were enumerated for 10 min.
2.6. Data analysis
The behavioral data are expressed in terms of the
meanFS.E.M for the writhing test, and the median and
interquartile range for the Y-maze test because the data for
the Y-maze test did not always show Gaussian distributions.
The significance of differences was evaluated using
Student’s t-test or the one-way analysis of variance followed
by Bonferroni’s test for the writhing test, and using the
Mann–Whitney U-test or the Kruskal–Wallis test followed
by Bonferroni’s test for nonparametiric type multiple
comparisons for the Y-maze test. The criterion for signifi-
cance was pb0.05 in all statistical evaluations.
3. Results
3.1. Effects of antisense oligodeoxynucleotide to j1-opioidreceptor on U-50,488H-induced analgesia in the
acetic-acid-induced writhing test
In the acetic-acid-induced writhing test, saline-treated
control mice showed approximately 20 writhing responses
during the 10-min observation period starting from 10 min
after injection of the 0.7% acetic acid solution. A
significant antinociceptive effect was observed after s.c.
injection of U-50,488H (2.15 Amol/kg, s.c.) 30 min before
the writhing test ( pb0.01). Antisense targeting exon 3
(AS-n1-3) dose-dependently antagonized the U-50,488H-
induced antinociceptive effect and 10 and 20 Ag of
antisense completely antagonized the antinociceptive effect
(Fig. 1A, pb0.01 and pb0.05, respectively). All antisenses
targeting exon 1 (AS-n1-1, pb0.01), 2 (AS-n1-2, pb0.05),or 3 (AS-n1-3, pb0.05) of the n1-opioid receptor
completely blocked the U-50,488H-induced antinocicep-
tive effect, but the mismatch sense did not have any effect
(Fig. 1B).
3.2. Effects of antisense oligodeoxynucleotide to j1-opioidreceptor on spontaneous alternation behavior and number
of arm entries in the Y-maze test
In the next experiment, we examined whether antisense
showed some effect on spontaneous alternation behavior
and total number of arm entries during an 8-min session
in the Y-maze test. None of the antisenses to the n1-opioid receptor had an effect compared with the control
(Fig. 2).
Fig. 2. Effects of antisense oligodeoxynucleotide to n1-opioid receptor on
spontaneous alternation (A) and total arm entries (B) in the Y-maze test.
Antisense oligodeoxynucleotides (20 Ag/mouse, i.c.v.) to the n1-opioidreceptor were administered on days 1, 3, and 5, and a behavioral analysis
was conducted on day 6. Data are shown as the median (vertical column)
and first and third quartiles (vertical line). The number of mice used is
shown in parentheses.
Fig. 1. Effects of antisense oligodeoxynucleotide to n1-opioid receptor on
U-50,488H-induced analgesia in the acetic-acid-induced writhing test.
Antisense oligodeoxynucleotides were administered on days 1, 3, 5, and 7,
and a behavioral analysis was conducted on day 8. Mice were treated with
U-50,488H s.c. 30 min before testing. Acetic acid (0.7%) was injected i.p.
10 min before testing and writhing responses were enumerated for 10 min.
Data are shown as the meanFS.E.M. The number of mice used is shown in
parentheses. Levels of significance: **pb0.01 vs. control (Student’s t-test),#pb0.05, ##pb0.01 vs. U-50,488H alone (Bonferroni’s test).
M. Hiramatsu, T. Hoshino / Brain Research 1030 (2004) 247–255250
3.3. Effects of antisense oligodeoxynucleotide to j1-opioidreceptor on U-50,488H-induced amelioration of memory
impairment in the Y-maze test
In this experiment, we first reconfirmed whether scopol-
amine impaired spontaneous alternation behavior and U-
50,488H ameliorated scopolamine-induced impairment (Fig.
3A and B). In the next test, we tested whether the antisense at
different doses and different antisenses antagonized the
improving effect of U-50,488H (scopolamine+U-50,488H
group as a control group). Scopolamine (1.65 Amol/kg, s.c.)
markedly impaired the spontaneous alternation behavior as
indicated by a decrease in percent alternation, and increased
the total number of arm entries (Fig. 3A and B, pb0.01). U-
50,488H (0.64 Amol/kg, s.c.) attenuated the impairment of
spontaneous alternation behavior induced by scopolamine
(Fig. 3A, pb0.01) consistent with a previous report [16].
Antisense targeting exon 3 (AS-n1-3) blocked the amelio-
rating effect of U-50,488H, and 10 and 20 Ag of antisense
had a significant effect (Fig. 3C, pb0.01). None of the
antisenses to the n1-opioid receptor had an effect on the totalnumber of arm entries compared with the (scopolamine+U-
50,488H)-treated group during 8-min sessions in the Y-maze
test (Fig. 3D and F).
Twenty micrograms of each antisense targeting exons
2 (AS-n1-2, pb0.05) and 3 (AS-n1-3, pb0.01) of the
n1-opioid receptor significantly reversed the effects of
U-50,488H, but antisense targeting exons 1 (AS-n1-1).The control mismatch probe that differed from the
effective n1-opioid receptor exon antisense probe by
the sequence reversal of only two pairs of bases was
ineffective in altering U-50,488H-induced improvement
of memory impairment in our behavioral paradigms [%
alternation: 60.9 (51.5–65.2), total arm entries: 28.0
(24.0–35.5)].
3.4. Effects of AS-r1 and AS-j3 on U-50,488H-induced
analgesia in the acetic-acid-induced writhing test
To determine whether j1 and n3-opioid receptors are
involved in the antinociceptive effects of U-50,488H-
induced analgesia, antisenses targeting j1 and n3-opioidreceptors were tested. It has been reported that antisense to
the j1 receptor enhanced the analgesic activities of U-
Fig. 3. Effects of antisense oligodeoxynucleotide to n1-opioid receptor on U-50,488H-induced memory improving effects in the Y-maze test. Antisense
oligodeoxynucleotides were administered on days 1, 3, and 5, and a behavioral analysis was conducted on day 6. Scopolamine (1.65 Amol/kg) was
administered s.c. around the abdominal area 30 min before the behavioral tests. U-50,488H (0.64 Amol/kg) was administered s.c. under the skin of the dorsal
neck immediately after scopolamine injection. In this experiment, we first reconfirmed whether scopolamine impaired the spontaneous alternation behavior and
U-50,488H ameliorated scopolamine-induced impairment (Panels A and B). In the next test, we tested whether the antisense at different doses and different
antisenses antagonized the improving effect of U-50,488H (scopolamine+U-50,488H group as a control group). Data are shown as the median (vertical
column) and first and third quartiles (vertical line). The number of mice used is shown in parentheses. Levels of significance: **pb0.01 vs. control (Mann–
Whitney U-test). $$pb0.01 vs. scopolamine (Mann–Whitney U-test). #pb0.05, ##pb0.01 vs. scopolamine+U-50,488H (Bonferroni’s test).
M. Hiramatsu, T. Hoshino / Brain Research 1030 (2004) 247–255 251
50,488H [19]. However, pretreatment with the antisense
(AS-j1) had no effect on the U-50,488H-induced analgesia
as shown in Fig. 4A. Pretreatment with antisense to the n3-opioid receptor did not have any effect either (Fig. 4B).
3.5. Effects of AS-r1 on U-50,488H-induced ameliorating
effects in the Y-maze test
We have previously reported that the ameliorating
effects of n-opioid and j receptors on memory impairment
are independent and no direct modulation exists in these
neuronal systems [16]. To confirm this, we examined
whether n-opioid receptors and j receptors interact using
an antisense strategy. Pretreatment with antisense to the
j1 receptor did not affect the U-50,488H-induced
improvement in the scopolamine-induced impairment of
spontaneous alternation behavior (Fig. 5). In other words,
U-50,488H significantly improved the scopolamine-
induced impairment of spontaneous alternation behavior
even when the j1 receptor malfunctioned ( pb0.01),
indicating that the j1 receptor was not necessary for the
effect of U-50,488H.
3.6. Effects of antisense oligodeoxynucleotide to r1 receptoron (�)-pentazocine-induced analgesia in the acetic-acid-
induced writhing test and pentazocine-induced effects in the
Y-maze test
(�)-Pentazocine exhibits an analgesic effect by acting on
n-opioid receptors in mice and humans [31,32], while (+)-
pentazocine improves learning and memory impairment in
mice, acting on j receptors [17,27]. The antinociceptive
effect of (�)-pentazocine (3.50 Amol/kg, s.c.) was not
antagonized by pretreatment with AS-j1 (Fig. 6).
We previously reported that (�)-pentazocine (3.50 Amol/
kg) and (+)-pentazocine (0.35 Amol/kg) improved the
scopolamine-induced impairment of spontaneous alternation
performance in mice at this dose via j1 receptors in the Y-
maze test [17]. Pretreatment with AS-j1 completely
prevented these effects of (�)- and (+)-pentazocine as
shown in Fig. 7, although U-50,488H ameliorated the
scopolamine-induced impairment of spontaneous alternation
behavior (Fig. 5). Preliminary experiment showed that AS-
j1 had no significant effect compared with the saline-treated
group (data not shown). In this study, mice pretreated with
Fig. 4. Effects of AS-j1 and AS-n3 on U-50,488H-induced analgesia in the
acetic-acid-induced writhing test. Antisense oligodeoxynucleotide, AS-j1(A) or AS-n3 (B), was administered on days 1, 3, 5 and 7, and a behavioral
analysis was conducted on day 8. Mice were treated with U-50,488H s.c. 30
min before testing. Acetic acid (0.7%) was injected i.p. 10 min before
testing and writhing responses were enumerated for 10 min. Data are shown
as the meanFS.E.M. The number of mice used is shown in parentheses.
Levels of significance: **pb0.01 vs. control (Bonferroni’s test).
Fig. 5. Effects of AS-j1 on U-50,488H-induced effects in the Y-maze test.
Antisense oligodeoxynucleotide, AS-j1, was administered on days 1, 3,
and 5, and a behavioral analysis was conducted on day 6. Scopolamine
(1.65 Amol/kg) was administered s.c. around the abdominal area 30 min
before the behavioral tests. U-50,488H (0.64 Amol/kg) was administered
s.c. under the skin of the dorsal neck immediately after scopolamine
injection. Data are shown as the median (vertical column) and first and third
quartiles (vertical line). The number of mice used is shown in parentheses.
Levels of significance: **pb0.01 vs. AS-j1 alone, ##pb0.01 vs. AS-
j1+scopolamine (Mann–Whitney U-test).
M. Hiramatsu, T. Hoshino / Brain Research 1030 (2004) 247–255252
AS-j1 itself also showed about 70% alternation, which was
similar to the percent alternation exhibited by control mice
(see Fig. 2).
Fig. 6. Effects of antisense oligodeoxynucleotide to j1 receptor on (�)-
pentazocine-induced analgesia in the acetic-acid-induced writhing test.
Antisense oligodeoxynucleotide, AS-j1, was administered on days 1, 3, 5
and 7, and a behavioral analysis was conducted on day 8. Mice were treated
s.c. with (�)-pentazocine 30 min before testing. Acetic acid (0.7%) was
injected i.p. 10 min before testing and writhing responses were enumerated
for 10 min. Data are shown as the meanFS.E.M. The number of mice used
is shown in parentheses. Levels of significance: *pb0.05 vs. control
(Student’s t-test).
4. Discussion
(F)-Pentazocine is widely used clinically to treat mild to
moderate pain as a racemic compound. It has been reported
that each enantiomer of pentazocine acts on different
receptors and has a distinct pharmacology [3,32]. (�)-
Pentazocine shows analgesic effects by acting on n-opioidreceptors in mice and humans [31,32], while (+)-pentazo-
cine improves learning and memory impairments in mice,
acting on j receptors [17,27]. We further reported that (�)-
pentazocine improved the scopolamine-induced impairment
of spontaneous alternation behavior in the Y-maze test [17].
U-50,488H, a selective n-opioid receptor agonist, and
dynorphin A (1–13) also improve learning and memory
Fig. 7. Effects of antisense oligodeoxynucleotide to j1 receptor on (�)- and
(+)-pentazocine-induced memory improving effects in the Y-maze test.
Antisense oligodeoxynucleotide, AS-j1, was administered on days 1, 3,
and 5, and a behavioral analysis was conducted on day 6. Scopolamine
(1.65 Amol/kg, s.c.) was injected 30 min before testing. Immediately after
scopolamine injection, mice were treated s.c. with (�)- and (+)-pentazocine
at doses of 3.50 and 0.35 Amol/kg, s.c., respectively. Data are shown as the
median (vertical column) and first to third quartiles (vertical line). The
number of mice used is shown in parentheses. Levels of significance:
**pb0.01 vs. AS-j1 alone (Bonferroni’s test).
M. Hiramatsu, T. Hoshino / Brain Research 1030 (2004) 247–255 253
impairment in various animal models, such as carbon
monoxide (CO)-induced delayed amnesia in mice [10,11],
and h�amyloid peptide (25–35)- and carbachol-induced
impairment of learning and memory in mice and rats,
respectively [13,15]. Dynorphin A (1–13) and U-50,488H
reversed the decrease in acetylcholine release caused by
carbachol and mecamylamine [13,14]. Therefore, the n-opioidergic system in the brain may play an important role
in modulating learning and memory functions, although the
mechanism linking the n-opioidergic and cholinergic
systems in the central nervous system has not been
elucidated.
j Receptor agonists, (+)-SKF-10,047, 1-(3,4-dimethox-
yphenethyl)-4-(3-phenylpropyl) piperazine dihydrochloride
(SA-4503) and (+)-pentazocine, improved impairments of
learning and memory in mice [26,27]. Interestingly, we have
reported that (�)-pentazocine acted on n-opioid receptors to
exert an analgesic effect, and also improved impairment of
memory in mice by acting on the j receptor [17]. Acting
doses on the memory of (+)-pentazocine and (�)-pentazo-
cine were 10 times different, thus the improving effect of
(�)-pentazocine was about 10 times weaker than (+)-
pentazocine. This may be consistent with the affinity of
(+)- and (�)-pentazocine for j receptors in that (�)-
pentazocine was about 10 times weaker than (+)-pentazo-
cine [29]. (F)-SKF-10,047 enhanced stimulation-evoked
acetylcholine release in guinea pig cerebral slices [36], and
other j receptor agonists, (+)-SKF-10,047, (F)-pentazo-
cine, DTG, and (+)-3-PPP, increased extracellular acetylcho-
line levels in the rat frontal cortex [23,24] and hippocampus
[25]. The activating effect of (+)-SKF-10,047 on the central
cholinergic system was antagonized by haloperidol at a dose
range compatible with its j1 receptor antagonist action
[24,25]. Therefore, j receptor agonists may also be effective
in improving memory impairment involving the cholinergic
systems.
The in vivo antisense strategy appears to be a highly
sensitive and selective approach to identifying the proteins
involved in particular behaviors and/or to firmly establish
the physiological roles of proteins with recently cloned
cDNA [20,37]. The existence of n1-opioid receptors splice
variants has been suggested in a recent report [34]. In the
present study, the analgesic actions of U-50,488H in mice
were blocked by antisenses targeting exons 1, 2, and 3, but
the improving of memory impairment was not antagonized
by antisenses targeting exon 1 of the n1-opioid receptors.
In addition, we previously found that nor-binaltorphimine
antagonized the effect of U-50,488H [14], the combined
use of selective antagonists and additional modern
molecular tools such as antisense technique allow for the
discovery of precise receptor mechanisms mediating
memory and other behavioral actions of the endogenous
opioid system. Furthermore, using an in vivo antisense
strategy, a role for the j1 receptor in the modulation of n-opioid receptor-mediated analgesia was recently demon-
strated in the mouse by King et al. [19]. This group
reported that the analgesic activity of either the n1-opioidreceptor agonist U-50,488H or the n3-opioid receptor
agonist naloxone benzoylhydrazone was selectively
enhanced after antisense treatment. The same group found
that opioid analgesia was antagonized by a j system [4,5].
Such findings imply that j1 receptors are potent modu-
lators of the n-opioid receptor-mediated effects on analge-
sia. However, it is also reported that j systems are
responsible for some strain differences in n-opioid receptor
sensitivity. Unlike CD-1 mice, BALB-C mice are relatively
insensitive toward the n1 agent U-50,488H [5]. Further-
more, not all opioid actions were linked with j systems,
such as gastrointestinal tansit or lethality [5]. We pre-
viously reported that not only the antinociceptive effects,
but also the ameliorating effects on memory impairment,
are independent and no direct modulation exists in the n-opioid and j receptor-mediated mechanisms in ddY mice
[16]. In the present study, we used these antisense
oligodeoxynucleotide probes in different experiments to
examine the molecular basis of the involvement of n-opioid receptors and j receptors in memory and analgesic
processes.
M. Hiramatsu, T. Hoshino / Brain Research 1030 (2004) 247–255254
Consistent with our previous results [16], the U-
50,488H-induced improvement in memory impairment
was reversed by the antisenses targeting exons of the
n1-opioid receptor, but not antisense targeting the j1receptor. In addition, the U-50,488H-induced antinocicep-
tive effect was prevented by the antisenses targeting exons
of the n1-opioid receptor, but not by the antisenses
targeting the j1 receptor in the acetic-acid-induced
writhing test. Interestingly, not only the (+)-pentazocine-
but also the (�)-pentazocine-induced ameliorating effects
were blocked when antisense targeting exons of the j1receptor was used in the memory test. These results
suggest that the ameliorating effects of U-50,488H and
(�)-pentazocine on memory impairment are mediated in
different mechanisms and each n-opioid and j system
independently plays a significant role in ddY mice toward
memory function.
The antisenses used in the present experiment did not
themselves affect normal mice in the Y-maze test. More-
over, 20 Ag of antisense targeting exons 2 and 3 of the n1-opioid receptor significantly reversed the effects of U-
50,488H but antisense targeting exon 1 did not. The
antisense targeting exon 3 was most effective as reported
by Pasternak et al. [34]. However, all these antisenses
equally prevented U-50,488H-induced antinociceptive
effects in the acetic-acid-induced writhing test. These
results suggest that n1-opioid receptors containing different
exons have a distinct function in memory and nociceptive
functions. The ability of the antisense probe AS-n1-3 to
block the effects of U-50,488H is dependent upon its
relationship to the third exon located in the sixth trans-
membrane region (third extracellular loop), indicating that
this region may be most important to the pharmacological
effects of U-50,488H on n1-opioid receptors. On the
contrary, pretreatment with antisense to the n3-opioidreceptor did not block the effects of U-50,488H, indicating
that this effect was not mediated by n3-opioid receptors
and U-50,488H mainly acting as n1-opioid receptors. This
is supported by our previous report that noloxone
benzoylhydrazone, a n3-opioid receptor agonist, did not
show significant changes in normal mice and scopolamine-
induced impairment of spontaneous alternation indicating
that n3-opioid receptors do not play an important role in
memory function [9].
In conclusion, the present study confirms and extends
our prior observations that n-opioid receptors agonists
showing analgesic effects act as n-opioid receptors or jreceptors and play important roles only when memory
function is impaired, but the two neuronal systems regulate
memory function independently. Furthermore, our results
suggest that n1-opioid receptors containing different exons
have a distinct function in memory and nociceptive
functions, but the mechanisms behind the differences
remain unknown. Although the antinociceptive effects of
pentazocine were mediated via n-opioid receptors, our
results suggest that the ameliorating memory effects of not
only (+)- but also (�)-pentazocine are mediated mainly via
j receptors, not via n-opioid receptors. At low doses,
pentazocine or related compounds may be candidates for
antiamnesic drugs.
Acknowledgements
We are grateful to Santen Pharmaceuticals (Osaka) for
supplying the (�)- and (+)-pentazocine. This study was
supported in part by Grants-in-Aid for Scientific Research
(No. 16590442 for M.H., High-Tech Research Center
Project and Scientific Frontier Research Project of Meijo
University) from the Ministry of Education, Science, Sports
and Culture, Japan.
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