www.elsevier.com/locate/brainres

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: mhiramt@ccmfs.meijo-u.ac.jp (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.

References

[1] R.T. Bartus, R.L. Dean, B. Beer, A.S. Lippa, The cholinergic

hypothesis of geriatric memory dysfunction, Science 217 (1982)

408–414.

[2] R.J. Beninger, B.A. Wirsching, K. Jhamands, R.J. Boegman, Animal

studies of brain acetylcholine and memory, Arch. Gerontol. Geriatr.,

Suppl. 1 (1989) 71–89.

[3] W.D. Bowen, S.B. Hellewell, K.A. McGarry, Evidence for a multi-site

model of the rat brain sigma receptor, Eur. J. Pharmacol. 163 (1989)

309–318.

[4] C.-C. Chien, G.W. Pasternak, Functional antagonism of morphine

analgesia by (+)-pentazocine: evidence for an anti-opioid n1 system,

Eur. J. Pharmacol. 250 (1993) R7–R8.

[5] C.-C. Chien, G.W. Pasternak, Selective antagonism of opioid

analgesia by a sigma system, J. Pharmacol. Exp. Ther. 271 (1994)

1583–1590.

[6] J.T. Coyle, D.L. Price, M.R. DeLong, Alzheimer’s disease: a disorder

of cortical cholinergic innervation, Science 219 (1983) 1184–1190.

[7] T.J. Haley, W.G. McCormick, Pharmacological effects produced by

intracerebral injection of drugs in the conscious mouse, Br. J.

Pharmacol. 12 (1957) 12–15.

[8] J.M. Hiller, Y. Itzhak, E.J. Simon, Selective changes in mu, delta and

kappa opioid receptor binding in certain limbic regions of the brain in

Alzheimer’s disease patients, Brain Res. 406 (1987) 17–23.

[9] M. Hiramatsu, K. Inoue, Improvement by low doses of nociceptin on

scopolamine-induced impairment of learning and/or memory, Eur. J.

Pharmacol. 395 (2000) 149–156.

[10] M. Hiramatsu, M. Sasaki, T. Kameyama, Effects of dynorphin A-(1–

13) on carbon monoxide-induced delayed amnesia in mice studied in a

step-down type passive avoidance task, Eur. J. Pharmacol. 282 (1995)

185–189.

[11] M. Hiramatsu, T. Hyodo, T. Kameyama, U-50,488H, a selective n-opioid receptor agonist, improves carbon monoxide-induced delayed

amnesia in mice, Eur. J. Pharmacol. 315 (1996) 119–126.

[12] M. Hiramatsu, M. Sasaki, T. Nabeshima, T. Kameyama, Effects of

dynorphin A-(1–13) on carbon monoxide-induced delayed amnesia in

mice, Pharmacol. Biochem. Behav. 56 (1997) 73–79.

[13] M. Hiramatsu, H. Murasawa, H. Mori, T. Kameyama, Reversion of

muscarinic autoreceptor agonist-induced acetylcholine decrease and

learning impairment by dynorphin A (1–13), an endogenous kappa-

opioid receptor agonist, Br. J. Pharmacol. 123 (1998) 920–926.

[14] M. Hiramatsu, H. Murasawa, T. Nabeshima, T. Kameyama, Effects of

U-50,488H on scopolamine-, mecamylamine- and dizocilpine-induced

learning and memory impairment in rats, J. Pharmacol. Exp. Ther. 284

(1998) 858–867.

M. Hiramatsu, T. Hoshino / Brain Research 1030 (2004) 247–255 255

[15] M. Hiramatsu, K. Inoue, T. Kameyama, Dynorphin A-(1–13) and (2–

13) improve beta-amyloid peptide-induced amnesia in mice, Neuro-

Report 11 (2000) 431–435.

[16] M. Hiramatsu, T. Hoshino, T. Kameyama, T. Nabeshima, Involvement

of n-opioid and j receptors in short-term memory in mice, Eur. J.

Pharmacol. 453 (2002) 91–96.

[17] T. Hoshino, M. Hiramatsu, T. Kameyama, T. Nabeshima, Improve-

ment of memory impairment by (+)- and (�)-pentazocine via kappa

opioid and/or sigma receptors in mice, Neurosci. Abstr. 30 (2000)

2015.

[18] J. Itoh, M. Ukai, T. Kameyama, Dynorphin A-(1–13) markedly

improves scopolamine-induced impairment of spontaneous alternation

performance in mice, Eur. J. Pharmacol. 236 (1993) 341–345.

[19] M. King, Y.-X. Pan, J. Mei, A. Chang, J. Xu, G.W. Pasternak,

Enhanced n�opioid receptor-mediated analgesia by antisense target-

ing the j 1 receptor, Eur. J. Pharmacol. 331 (1997) R5–R6.

[20] R. Leslie, D. Jones, Antisense gene knockdown in the nervous

system revisited: optimism for the future, Trends Neurosci. 20 (1997)

321–322.

[21] W.R. Martin, C.G. Eades, J.A. Thompson, R.E. Huppler, P.E. Gilbert,

The effect of morphine and morphine-like drugs in the non-dependent

and morphine-dependent chronic spinal dog, J. Pharmacol. Exp. Ther.

197 (1976) 517–532.

[22] A.M. Mathieu-Kia, L.Q. Fan, M.J. Kreek, E.J. Simon, J.M. Hiller,

Mu-, delta- and kappa-opioid receptor populations are differentially

altered in distinct areas of postmortem brains of Alzheimer’s disease

patients, Brain Res. 893 (2001) 121–134.

[23] K. Matsuno, K. Matsunaga, S. Mita, Increase of extracellular

acetylcholine level in rat frontal cortex induced by (+)-N-allylnorme-

tazocine as measured by brain microdialysis, Brain Res. 575 (1992)

315–319.

[24] K. Matsuno, K. Matsunaga, T. Senda, S. Mita, Increase in

extracellular acetylcholine level by sigma ligands in rat frontal cortex,

J. Pharmacol. Exp. Ther. 265 (1993) 851–859.

[25] K. Matsuno, T. Senda, T. Kobayashi, S. Mita, Involvement of j1receptor in (+)-N-allylnormetazocine-stimulated hippocampal choli-

nergic functions in rats, Brain Res. 690 (1995) 200–206.

[26] K. Matsuno, M. Nakazawa, K. Okamoto, Y. Kawashima, S. Mita,

Binding properties of SA4503, a novel and selective j1 receptor

agonist, Eur. J. Pharmacol. 306 (1996) 271–279.

[27] T. Maurice, M. Hiramatsu, J. Itoh, T. Kameyama, T. Hasegawa, T.

Nabeshima, Behavioral evidence for a modulating role of sigma

ligands in memory processes: I. Attenuation of dizocilpine (MK-801)-

induced amnesia, Brain Res. 647 (1994) 44–56.

[28] A. Newhouse, Cholinergic drug studies in dementia and depression,

Adv. Exp. Med. Ther. 282 (1990) 65–76.

[29] S. Okuyama, S. Chaki, T. Yae, A. Nakazoto, M. Muramatsu,

Autoradiographic characterization of binding sites for [3H] NE-100

in guinea pig brain, Life Sci. 57 (1995) 333–337.

[30] Y.X. Pan, J. Cheng, J. Xu, G. Rossi, E. Jacobson, J. Ryan-Moro, A.I.

Brooks, G.E. Dean, K.M. Standifer, G.W. Pasternak, Cloning and

functional characterization through antisense mapping of a kappa 3-

related opioid receptor, Mol. Pharmacol. 47 (1995) 1180–1188.

[31] G.W. Pasternak, Multiple morphine and enkephalin receptors and the

relief of pain, JAMA 259 (1988) 1362–1367.

[32] G.W. Pasternak, Pharmacological mechanisms of opioid analgesia,

Clin. Neuropharmacol. 16 (1993) 1–18.

[33] K.R. Pasternak, K.M. Standifer, Mapping of opioid receptors using

antisense oligodeoxynucleotides: correlating their molecular biology

and pharmacology, Trends Pharmacol. Sci. 16 (1995) 344–350.

[34] K.R. Pasternak, G.C. Rossi, A. Zuckerman, G.W. Pasternak,

Antisense mapping KOR-1: evidence for multiple kappa analgesic

mechanisms, Brain Res. 826 (1999) 289–292.

[35] R. Quirion, W.D. Bowen, Y. Itzhak, J.L. Junien, J.M. Musacchio, R.B.

Rothman, T.-P. Su, S.W. Tam, D.P. Taylor, A proposal for the

classification of sigma binding sites, Trends Pharmacol. Sci. 13 (1992)

85–86.

[36] M. Sarter, G. Bodewitz, D.N. Stephens, Attenuation of scopolamine-

induced impairment of spontaneous alternation behavior by antagonist

but not inverse agonist and agonist h-carbolines, Psychopharmacol-

ogy 94 (1988) 491–495.

[37] A. Siniscalchi, P. Cristofori, E. Veratti, Influence of N-allyl-

normetazocine on acetylcholine release from brain slices: involvement

of muscarinic receptors, Naunyn-Schmiedeberg’s Arch. Pharmacol.

336 (1987) 425–429.

[38] B. Weiss, G. Davidkova, S.-P. Zhang, Antisense strategies in

neurobiology, Neurochem. Int. 31 (1997) 321–348.