Full text of DOI:10.1038/sj.bjp.0704081

British Journal of Pharmacology (2001) 133, 267 ± 274
ã
2001 Nature Publishing Group All rights reserved 0007 ± 1188/01 $15.00
www.nature.com/bjp
Di€erential prevention of morphine amnesia by antisense
oligodeoxynucleotides directed against various Gi-protein a
subunits
1
1Nicoletta Galeotti, *,1Carla Ghelardini & Alessandro Bartolini
1Department of Preclinical and Clinical Pharmacology, Viale G. Pieraccini 6, I-50139 Florence, Italy
1
The e€ect of the i.c.v. administration of pertussis toxin (PTX) and antisense oligodeoxynucleo-
tide directed against the a subunit of di€erent Gi-proteins (anti-Gia1, anti-Gia2, anti-Gia3) on
amnesia induced by morphine was evaluated in the mouse passive avoidance test.
2
The administration of morphine (6 ± 10 mg kg71 i.p.) immediately after the training session
produced amnesia that was prevented by PTX (0.25 mg per mouse i.c.v.) administered 7 days before
the passive avoidance test.
3
Anti-Gia1 (6.25 mg per mouse i.c.v.) and anti-Gia3 (12.5 mg per mouse i.c.v.), administered 18
and 24 h before the training session, prevented the morphine amnesia. By contrast, pretreatment
with anti-Gia2 (3.12 ± 25 mg per mouse i.c.v.) never modi®ed the impairment of memory processes
induced by morphine.
4
At the highest e€ective doses, none of the compounds used impaired motor coordination, as
revealed by the rota rod test, nor modi®ed spontaneous motility and inspection activity, as revealed
by the hole board test.
5 These results suggest the important role played by Gi1 and Gi3 protein subtypes in the
transduction mechanism involved in the impairment of memory processes produced by morphine.
British Journal of Pharmacology (2001) 133, 267 ± 274
Keywords: Morphine; amnesia; learning and memory; pertussis toxin; Gi-proteins; antisense oligodeoxynucleotides
Abbreviations: aODN, antisense oligodeoxyribonucleotide; dODN, degenerate oligodeoxyribonucleotide; i.c.v., intracerebro-
ventricular; i.p. intraperitoneal; PTX, pertussis toxin
Introduction
Learning and memory in animals are well known to be
a€ected by opioids and opioid antagonists. Generally it is
reported that morphine and opioid peptides tend to interfere
with these processes and to produce amnesia. The post-
training administration of b-endorphin or enkephalins
produces memory impairment in di€erent behavioural tasks
(Izquierdo, 1980; Castellano & Pavone, 1985; Schulteis et al.,
1988). The retrograde amnesia induced by b-endorphin can
be antagonized by pretreatment with a relatively low dose of
naloxone (Introini & Baratti, 1984; Del Cerro & Borrell,
1987). Furthermore, the systemic administration of morphine,
when injected shortly after training, has been reported to
impair memory processes in mice and rats (Castellano, 1975;
Izquierdo, 1979; Messing et al., 1981). The morphine-induced
impairment of memory functions can be antagonized by
naloxone (Izquierdo, 1979). Several studies have also
reported facilitation of retention by opiate antagonists,
primarily naloxone, in normal animals in a variety of tasks
such as passive avoidance (Messing et al., 1979; Rush, 1986),
active avoidance, habituation (Izquierdo, 1979) and radial
arm maze (Gallagher et al., 1983).
Pharmacological studies have provided arguments for the
existence of several subsites for each m (m1, m2 and m3), d (d1
and d 2), or k (k1, k2 and k3) receptor class (Kie€er, 1999).
At present, only three homologous genes have been cloned,
and the correlation between the biological activity of their
encoded proteins and the pharmacological subtypes remains
to be established (Law et al., 2000). Morphine, in contrast to
many synthetic opioids, is an opioid compound with low
receptor selectivity. Binding studies performed on rodent
brain membranes (Matthes et al., 1998) or recombinant
receptor preparation (Raynor et al., 1994) have shown that
morphine exhibits a preference for m receptors, with Ki values
in the nanomolar range, but also binds the d and k receptors
with submicromolar anities.
The m, d and k opioid receptors have all been observed to
be involved in memory processes. In laboratory animals, the
administration of selective m and d opioid receptors tends to
interfere with learning and memory and to produce retro-
grade amnesia (Itoh et al., 1994; Ukai et al., 1997a).
Although k-opioid receptor agonists such as dynorphine A-
(1 ± 13) and U-50,488H alone fail to in¯uence learning
behaviour in normal animals, such drugs have been reported
to both ameliorate pharmacologically-induced amnesia (Ukai
et al., 1995; 1997b) and to impair memory (Tilson et al.,
1986; Colombo et al., 1992). Furthermore, selective changes
in m, d and k opioid receptor binding have been reported in
certain limbic regions of the brain in Alzheimer's disease
Opioids produce their principal e€ects by binding to at
least three di€erent types of receptors, the m, d and k opioid
receptors (Loh
& Smith, 1990; Knapp et al., 1995).
*Author for correspondence; E-mail: ghelard@server1.pharm.uni®.it

268
N. Galeotti et al
Gi-proteins and morphine amnesia
patients with memory dysfunction (Hiller et al., 1987). These
data further support the hypothesis of a role of opioid
receptors in memory processes.
consisted of a two-compartment acrylic box with a lighted
compartment connected to a darkened one by a guillotine
door. As soon as the mouse entered the dark compartment, it
received a punishing electrical shock (0.5 mA, 1 s). The
latency times for entering the dark compartment were
measured in the training test and after 24 h in the retention
test. The maximum entry latency allowed in the training and
retention sessions was, respectively, 60 and 180 s. In this test,
morphine (6 ± 10 mg kg71) was i.p. injected immediately after
the training session, PTX (0.25 mg per mouse) was i.c.v.
injected 7 days prior to the training session whereas aODNs
(1.56 ± 25 mg per mouse) were i.c.v. injected 24 and 18 h
before training. Between 11 and 25 mice were tested.
By considering the regulation of receptor signalling, m, d
and k opioid receptors could regulate the same spectrum of
e€ectors. All subtypes of opioid receptors are prototypical
Gi-coupled receptors and they have long been known to
inhibit adenylyl cyclase and Ca2+ channels, as well as to
stimulate K+ channels and to increase intracellular Ca2+
levels (Law et al., 2000). The post-receptorial mechanism
involved in morphine-induced amnesia has not yet been
established. By taking into account that opioid receptors are
Gi-protein coupled receptors, we investigated the involvement
of Gi proteins into the mechanism of action of morphine by
means of an antisense strategy. In particular, we used
antisense oligonucleotides (aODN) against the a subunits of
the Gi1, Gi2 and Gi3 proteins in order to determine the role
of each subtype in the memory impairment induced by
morphine in a mouse passive avoidance paradigm.
Hole-board test
The hole board test consisted of a 40 cm square plane with
16 ¯ush mounted cylindrical holes (3 cm diameter)
distributed four by four in an equidistant, grid-like manner.
Mice were placed on the centre of the board one by one and
allowed to move about freely for a period of 10 min each.
Two electric eyes, crossing the plane from mid-point to mid-
point of opposite sides, thus dividing the plane into four
equal quadrants, automatically signalled the movement of the
animal (counts in 5 min) on the surface of the plane
(locomotor activity). Miniature photoelectric cells, in each
of the 16 holes, recorded (counts in 5 min) the exploration of
the holes (exploratory activity) by the mice. The test was
performed 18 ± 24 h after the i.c.v. injection of degenerate
ODN (dODN; 25 mg per mouse) or aODN (12.5 ± 25 mg per
mouse). Twelve mice per group were tested.
In order to exclude that the e€ects produced by aODN
treatments were due to the induction of side e€ects, some
additional behavioural tests (rota rod, hole board) were
performed.
Methods
Animals
Male Swiss albino mice (23 ± 30 g) from the Morini (San Polo
d'Enza, Italy) breeding farm were used. Fifteen mice were
housed per cage (26641 cm). The cages were placed in the
experimental room 24 h before the test for acclimatization.
The animals were fed a standard laboratory diet and tap
water ad libitum and kept at 23+18C with a 12 h light/dark
cycle, light on at 0700 h. All experiments were carried out
according to the guidelines of the European Community
Council for experimental animal care.
Rota-rod test
The apparatus consisted of a base platform and a rotating
rod with a diameter of 3 cm and a non-slippery surface. The
rod was placed at a height of 15 cm from the base. The rod,
30 cm in length, was divided into ®ve equal sections by six
disks. Thus, up to ®ve mice were tested simultaneously on the
Intracerebroventricular injection technique
apparatus, with a rod-rotating speed of 16 r.p.m. The
integrity of motor coordination was assessed on the basis
of the number of falls from the rod in 30 s according to
Vaught et al. (1985). Those mice scoring less than three and
more than six falls in the pretest were rejected (20%). The
performance time was measured before (pretest) and 15, 30
and 45 min after i.p. administration of saline solution.
Animals were i.c.v. pretreated 18 ± 24 h prior to the test with
degenerate ODN (dODN; 25 mg per mouse) or aODN (12.5 ±
25 mg per mouse). Twelve mice per group were tested.
Intracerebroventricular (i.c.v.) administration was performed
under ether anaesthesia, according to the method described
by Haley & McCormick (1957). Brie¯y, during anaesthesia,
mice were grasped ®rmly by the loose skin behind the head.
A 0.4 mm external diameter, hypodermic needle attached to a
10 ml syringe was inserted perpendicularly through the skull
and no more than 2 mm into the brain of the mouse, where
5 ml were then administered. The injection site was 1 mm to
the right or left from the midpoint on a line drawn through
to the anterior base of the ears. Injections were performed
into the right or left ventricle randomly. To ascertain that the
drugs were administered exactly into the cerebral ventricle,
some mice (20%) were injected with 5 ml of diluted 1 : 10
India ink and their brains examined macroscopically after
sectioning. The accuracy of the injection technique was
evaluated and the percentage of correct injections was 95.
Antisense oligonucleotides
Phosphodiester oligonucleotides (ODNs) protected from
terminal phosphorothioate double substitution (capped ODNs)
against possible exonuclease-mediated degradation were pur-
chased by Tib Molbiol. The sequences of the 33-mer antisense
oligonucleotides used in the present study were the following:
Passive-avoidance test
aODN
sequence
anti-Gia1
anti-Gia2
anti-Gia3
5'-G*C*T GTC CTT CCA CAG TCT CTT TAT GAC GCC G*G*C-3'
5'-A*T*G GTC AGC CCA GAG CCT CCG GAT GAC GCC C*G*A-3'
5'-G*C*C ATC TCG CCA TAA ACG TTT AAT CAC GCC T*G*C-3'
The test was performed according to the step-through
method described by Jarvik & Kopp (1967). The apparatus
British Journal of Pharmacology vol 133 (2)

N. Galeotti et al
Gi-proteins and morphine amnesia
269
All oligos were previously characterized by in vitro
(immunoblotting) and in vivo (tail ¯ick) experiments (Ra€a
et al., 1994; Sanchez-Blazquez et al., 1995; Sanchez-Blazquez
& Garzon, 1998). A 33-mer fully degenerated ODN (dODN)
5'-N*N*N NNN NNN NNN NNN NNN NNN NNN
NNN NNN N*N*N -3' (where N is G, or C, or A, or T)
was used as a control. ODNs were vehiculated intracellularly
by an arti®cial cationic lipid (DOTAP, Sigma) to enhance
both uptake and stability, as described previously (Capac-
cioli et al., 1993). aODN or dODN were preincubated at
378C for 30 min with 13 mM DOTAP and supplied to mice
by i.c.v. injection of 5 ml solution 18 and 24 h prior to the
behavioural tests.
Drugs
The following drugs were used: pertussis toxin (RBI);
morphine hydrochloride (S.A.L.A.R.); D-amphetamine hy-
drochloride (De Angeli); scopolamine hydrobromide, DO-
TAP (Sigma).
All drugs were dissolved in isotonic (NaCl 0.9%) saline
solution immediately before use, except for pertussis toxin
which was dissolved in a water solution containing 0.01 M
sodium phosphate bu€er, pH=7.0, with 0.05 M sodium
chloride. Drug concentrations were prepared in such a way
that the necessary dose could be administered in a volume of
10 ml kg71 by i.p. injection or 5 ml per mouse by i.c.v.
injection.
Figure 1 Dose-response curve of morphine in the mouse passive
avoidance test. Morphine (6 ± 10 mg kg71 i.p.) and scopolamine
(1.5 mg kg71 i.p.) were administered immediately after the training
session. Vertical lines represent s.e.mean; between 19 and 25 mice
were tested; the dose administered is reported in each column.
*P50.05 in comparison with saline-treated mice.
Statistical analysis
All experimental results are given as the mean+s.e.mean.
Analysis of variance (ANOVA), followed by Fisher's
Protected Least Signi®cant Di€erence (PLSD) procedure for
post-hoc comparison, was used to verify signi®cance between
two means. Data were analysed with the StatView software
for the Macintosh (1992). P values of less than 0.05 were
considered signi®cant.
Results
Effect of pertussis toxin on morphine amnesia
Morphine, injected immediately after the training session,
induced amnesia in the mouse passive avoidance test. A
statistically signi®cant amnesic e€ect of morphine was
reached at the dose of 10 mg kg71 i.p. whereas the dose
of 6 mg kg71 i.p. was completely ine€ective (Figure 1).
The amnesia induced by morphine was of the same
intensity as that produced by the well known amnesic
drug scopolamine (1.5 mg kg71 i.p.) used as the reference
drug (Figure 1).
Pretreatment with pertussis tossin (PTX), injected i.c.v. at
the dose of 0.25 mg per mouse 7 days before the test,
completely prevented the morphine induced amnesia (Figure
2). PTX, when injected alone, was devoid of any e€ect on
memory processes in comparison with vehicle+saline-treated
mice (Figure 2).
Figure 2 Prevention by pertussis toxin (PTX; 0.25 mg per mouse
i.c.v.) pretreatment on morphine-induced (10 mg kg71 i.p.) amnesia
in the mouse passive avoidance test. The test was performed 7 days
after
a single i.c.v. injection of vehicle or PTX. Vertical lines
represent s.e.mean; between 11 and 16 mice were tested. *P50.05 in
comparison with vehicle-saline treated mice; 6P50.05 in comparison
with vehicle-morphine treated mice.
Effect of aODN against Gia subunits on morphine
amnesia
The amnesia induced by morphine (10 mg kg71 i.p.) was
prevented, in the mouse passive avoidance test, by pretreat-
ment with the aODN against the a subunit of the Gi1
proteins (Figure 3). Anti-Gia1 (1.56 ± 12.5 mg per mouse i.c.v.)
produced a dose-dependent antagonism of the morphine-
induced amnesia. The dose of 1.56 mg per mouse i.c.v. was
completely ine€ective; 3.12 mg per mouse i.c.v., partially
No di€erence between the entrance latencies of each group
in the training session of the passive avoidance test was
observed (Figures 1 and 2).
British Journal of Pharmacology vol 133 (2)

270
N. Galeotti et al
Gi-proteins and morphine amnesia
Figure 3 Prevention by pretreatment with an antisense oligonucleotide (aODN) to the a subunit of Gi1-protein gene (1.56 ± 12.5 mg
per mouse i.c.v.) of morphine-induced (10 mg kg71 i.p.) amnesia in the mouse passive avoidance test. The test was performed 18 ±
24 h after the i.c.v. injection of degenerate ODN (dODN; 12.5 mg per mouse i.c.v.) or aODN. Vertical lines represent s.e.mean;
between 12 and 16 mice were tested; the dose administered is reported in each column. **P50.01 in comparison with
dODN+morphine group.
prevented the morphine amnesic e€ect, even if the statistical
signi®cance was not reached, while the doses of 6.25 and
12.5 mg per mouse i.c.v. enhanced the entrance latency in the
retention session up to a value comparable to that produced
by control animals (Figure 3). Anti-Gia1 (12.5 mg per mouse
i.c.v.) did not produce any e€ect in the mouse passive
avoidance test in comparison with saline- and dODN-treated
mice when given alone (Figure 3).
The administration of an aODN against the a subunit of
the Gi2 proteins (3.12 ± 25 mg per mouse i.c.v.), in contrast to
anti-Gia1, was unable to prevent morphine-induced amnesia
(Figure 4). At the highest dose employed, anti-Gia2 did not
modify the entrance latency in mice in comparison with the
control groups (Figure 4).
behaviour or motor coordination as revealed by the rota rod
test (Figure 6). None of the aODNs, administered at the
highest active doses, increased the number of falls from the
rotating rod in comparison with dODN-treated mice (Figure
6). The number of falls in the rota rod test progressively
decreased since mice learned how to balance on the rotating
rod.
The spontaneous motility and exploratory activity of mice
was not modi®ed by administration of the above-mentioned
aODNs as revealed by the hole-board test in comparison with
saline-, vehicle- and dODN-treated mice (Figure 7). In the
same experimental conditions D-amphetamine (1 mg kg71
i.p.), used as the reference drug, increased both parameters
evaluated.
The administration of an aODN against the a subunit of
the Gi3 proteins (12.5 ± 25 mg per mouse i.c.v.) antagonized
the memory disruption produced by morphine without
showing any memory facilitating activity when given alone
(Figure 5). Anti-Gia3 was inactive in preventing morphine-
induced amnesia at lower doses (3.12 ± 6.25 mg per mouse
i.c.v.) as illustrated in Figure 5.
Discussion
Present results indicate that the activation of Gi proteins is
required for the induction of the amnesia produced by
morphine in the mouse passive avoidance test.
No di€erence between the entrance latencies of each group
in the training session of the passive avoidance test was
observed (Figures 3 ± 5).
In the present experimental conditions, post-training i.p.
administration of morphine produced an impairment of
memory functions of intensity comparable to that exerted
by the amnesic drugs scopolamine. The morphine-induced
amnesia was completely prevented by pretreatment with
Effect of aODN against Gia subunits on mouse rota rod
and hole board tests
pertussis toxin (PTX), a bacterial toxin produced by
Bordetella pertussis that ADP-ribosylates and inactivates the
a subunit of Gi proteins (Katada & Ui, 1982), indicating the
important role played by the Gi proteins in the signal
transduction mechanism activated by morphine.
The administration of the aODN against the a subunits of
the Gi proteins used in the present investigation elicited their
e€ect on cognitive processes without changing either gross
British Journal of Pharmacology vol 133 (2)

N. Galeotti et al
Gi-proteins and morphine amnesia
271
Figure 4 Lack of e€ect of pretreatment with an antisense oligonucleotide (aODN) to the a subunit of Gi2-protein gene (3.12 ±
25 mg per mouse i.c.v.) on morphine-induced (10 mg kg71 i.p.) amnesia in the mouse passive avoidance test. The test was performed
18 ± 24 h after the i.c.v. injection of degenerate ODN (dODN; 25 mg per mouse i.c.v.) or aODN. Vertical lines represent s.e.mean;
between 11 and 16 mice were tested; the dose administered is reported in each column. *P50.05 in comparison with dODN+saline
group.
Figure 5 Prevention by pretreatment with an antisense oligonucleotide (aODN) to the a subunit of Gi3-protein gene (3.12 ± 25 mg
per mouse i.c.v.) of morphine-induced (10 mg kg71 i.p.) amnesia in the mouse passive avoidance test. The test was performed 18 ±
24 h after the i.c.v. injection of degenerate ODN (dODN; 25 mg per mouse i.c.v.) or aODN. Vertical lines represent s.e.mean;
between 13 and 18 mice were tested; the dose administered is reported in each column. *P50.05 in comparison with dODN+saline
group; 6P50.05 in comparison with dODN+morphine-treated mice.
British Journal of Pharmacology vol 133 (2)

272
N. Galeotti et al
Gi-proteins and morphine amnesia
Figure 6 Lack of e€ect of pretreatment with an antisense
oligonucleotide (aODN) to the a subunit of Gi1- (12.5 mg per mouse
i.c.v.), Gi2- (25 mg per mouse i.c.v.) and Gi3- (25 mg per mouse i.c.v.)
protein gene on motor coordination in the mouse rota rod test. The
test was performed 18 ± 24 h after the i.c.v. injection of degenerate
ODN (dODN; 25 mg per mouse) or aODN. Vertical lines represent
s.e.mean; 12 mice per group were tested.
Figure 7 Lack of e€ect of pretreatment with an antisense
oligonucleotide (aODN) to the a subunit of Gi1- (12.5 mg per mouse
i.c.v.), Gi2- (25 mg per mouse i.c.v.) and Gi3- (25 mg per mouse i.c.v.)
protein gene on spontaneous motility (A) and inspection activity (B)
in the mouse hole board test. The test was performed 18 ± 24 h after
the i.c.v. injection of degenerate ODN (dODN; 25 mg per mouse) or
aODN. Vertical lines represent s.e.mean; 12 mice per group were
tested. Veh: vehicle (DOTAP 13 mM); amphet: D-amphetamine
1 mg kg71 i.p. *P50.05 in comparison with saline group.
The Gi protein subfamily is composed of di€erent
members, Gi1, Gi2 and Gi3 (Simon et al., 1991). Since PTX
inactivates all members of the Gi protein family, the role of
each subtype was investigated by pretreating animals with
aODNs against the a subunits of these Gi protein subtypes.
The inhibition of the expression of Gia1 and Gia3 produced a
dose-dependent prevention of morphine-induced amnesia
whereas the administration of an aODN against Gia2 never
exerted any modi®cation of morphine activity. These results
indicate a di€erential involvement of the Gi protein subtypes
in the mechanism of action of the investigated opioid agonist.
In particular, the integrity and functionality of Gi1 and Gi3
proteins appears essential to produce memory impairment
after activation of opioid receptors by morphine. By contrast,
the Gi2 subtype, in these experimental conditions, appears
not to be involved.
Morphine is able to activate Gia1, Gia2 and Gia3 to a
similar extent (Burford et al., 1998). However, the stimulation
of m opioid receptors has been reported to produce a
preferential activation of the Gia3 subtypes (Chakrabarti et
al., 1995; Connor & Christie, 1999). By taking into account
that morphine has a preferential interaction with m receptors,
the above-mentioned data can, at least in part, explain the
involvement of Gia3 into the mechanism of amnesic action of
morphine. Although m receptors show some preference for
Gi3, the m, d and k receptors appear to preferentially activate
the Gi2 over PTX-sensitive G-proteins (Connor & Christie,
1999). In the present study we observed the lack of e€ect of
an aODN against Gia2 that implies that this subunit is not a
major component of transduction mechanisms leading to
amnesia. The lack of involvement of Gia2 has also been
observed for other pharmacological activities of morphine,
such as constipation or acute dependence, whereas some
others, such as analgesia, are mediated only by activation of
the Gi2 protein subtype (Ra€a et al., 1994; 1996). It is
possible that multiple morphine-induced e€ects are mediated
by di€erent Gi-protein subunits and that morphine could
have di€erent intrinsic activity for each e€ect.
All three Gia subtypes were shown to inhibit the adenylyl
cyclase activity with a consequent reduction of intracellular
cyclic AMP levels (Wong et al., 1992). The role of the cyclic
AMP cascade in memory processes has been recently
evidenced. Genetic and pharmacological studies in mice and
rats demonstrated that the cyclic AMP responsive element
binding protein (CREB) is required for a variety of complex
forms of memory, including spatial and social learning (Silva
et al., 1998). The activation of the cyclic AMP-dependent
protein kinase (PKA) induces long-term memory (Muller,
2000) and the inhibition of cyclic AMP phosphodiesterase
reverses memory de®cits in the radial arm maze task (Zhang
et al., 2000). Several studies have also shown that the
regulation of adenylyl cyclase activity is disrupted in
Alzheimer's disease patients. The alteration of adenylyl
cyclase activity in post-mortem brain is related to an
impairment of the stimulatory G-proteins (Gs) whereas the
British Journal of Pharmacology vol 133 (2)

N. Galeotti et al
Gi-proteins and morphine amnesia
273
Gi-protein-mediated inhibition of the enzyme is unaltered
(Schnecko et al., 1994; Fowler et al., 1995). Furthermore,
lower Gsa levels and unmodi®ed Gia levels were observed in
®broblasts from familial Alzheimer's disease patients (Shana-
han et al., 1997). Recently, it has been reported that amyloid
b-peptides cause toxicity through activation of Gi proteins
(Soomets et al., 1999; Rymer & Good, 2001). We can
hypothesize that an intact Gi-protein functionality is essential
for the induction of amnesia. Our results on morphine
amnesia con®rm the hypothesis of a Gi-protein involvement
in the induction of memory impairment. By considering that
high cyclic AMP levels are required for mnemonic integrity,
we can also suppose that morphine induces amnesia through
These results have been obtained by using a working
memory test. In future research, these data should be
validated in spatial memory paradigms in order to further
elucidate the intracellular processes involved in opioid-
induced amnesia.
Numerous hormones and neurotransmitters activate the
Gi-protein system. The administration of PTX and aODN
against the a subunits of Gi proteins could induce side e€ects
that make the interpretation of the results obtained dicult.
PTX, at the dose used, did not modify the motor
coordination of the animal (Galeotti et al., 1996). Moreover,
aODN did not impair motor coordination as revealed by the
rota rod test nor modify spontaneous motility and inspection
activity as indicated by the hole board test. We can, thus,
suppose that the e€ects produced by these treatments were
not due to compromised behavioural paradigms.
In conclusion, our results evidence the important role
played by Gia1 and Gia3, but not by Gia2, in morphine-
induced amnesia. The elucidation of the post-receptorial
mechanisms involved in memory processes can not only help
unravel the complex systems mediating cognitive functions,
but also the manipulation of Gi-protein activity may lead to
novel treatments for pathologies characterized by memory
dysfunction.
a reduction of intracellular cyclic AMP levels via the
activation of Gi1 and Gi3 proteins.
Pretreatment with PTX did not produce any ameliorative
e€ect on memory processes in the mouse passive avoidance
test, in agreement with previous results obtained in the rat
(Chou & Lee, 1995). Similarly, aODN against the a subunits
of Gi proteins never modi®ed the entrance latency in
comparison with control animals. We can, therefore exclude
that the prevention of morphine amnesia is due to a
procognitve e€ect exerted by these treatments. Furthermore,
pretreatment with dODN, used as the reference ODN, never
modi®ed the amnesia induced by morphine in comparison
with saline-treated animals, excluding the possibility of a
sequence-independent e€ect induced by the aODNs.
This work was supported by grants from MURST and CNR.
References
BURFORD, N.T., TOLBERT, L.M. & SADEE, W. (1998). Speci®c G
protein activation and m-opioid receptor internalization caused
by morphine, DAMGO and endomorphin I. Eur. J. Pharmacol.,
342, 123 ± 126.
GALEOTTI, N., GHELARDINI, C. & BARTOLINI, A. (1996). E€ect of
pertussis toxin on morphine, diphenhydramine, baclofen,
clomipramine and physostigmine antinociception. Eur. J.
Pharmacol., 308, 125 ± 133.
CAPACCIOLI, S., DI PASQUALE, G., MINI, E., MAZZEI, T.
&
GALLAGHER, M., KING, R.A. & YOUNG, N.B. (1983). Opiate
QUATTRONE, A. (1993). Cationic lipids improve antisense
oligonucleotide uptake and prevent degradation in cultured cells
and in human serum. Biochem. Biophys. Res. Comm., 107, 818 ±
825.
antagonists improve spatial memory. Science, 221, 975 ± 976.
HALEY, T.J. & MCCORMICK, W.G. (1957). Pharmacological e€ects
produced by intracerebral injection of drugs in the conscious
mouse. Br. J. Pharmacol. Chemother., 12, 12 ± 15.
CASTELLANO, C. (1975). E€ects of morphine and heroin on
discrimination, learning, and consolidation in mice. Psychophar-
macologia, 42, 235 ± 242.
HILLER, J.M., ITZHAK, Y. & SIMON, E.J. (1987). Selective changes in
m, d and k opioid receptor binding in certain limbic regions of the
brain in Alzheimer s disease patients. Brain Res., 106, 17 ± 23.
INTROINI, I.B. & BARATTI, C.B. (1984). The impairment of retention
induced by b-endorphin in mice may be mediated by a reduction
of central cholinergic activity. Behav. Neural Biol., 41, 152 ± 163.
ITOH, J., UKAI, M. & KAMEYAMA, T. (1994). Dynorphin A(1-13)
potently improves the impairment of spontaneous alternation
performance induced by the m-selective opioid receptor agonist
DAMGO in mice. J. Pharmacol. Exp. Ther., 269, 15 ± 21.
IZQUIERDO, I. (1979). E€ect of naloxone and morphine on various
forms of memory in the rat: possible role of endogenous opiate
mechanisms in memory consolidation. Psychopharmacology, 66,
199 ± 203.
CASTELLANO, C. & PAVONE, F. (1985). Dose- and strain-dependent
e€ects of dermorphin and [D-Ala2-D-Leu5]enkephalin on passive
avoidance behavior in mice. Behav. Neurosci., 99, 1120 ± 1127.
CHAKRABARTI, S., PRATHER, P.L., YU, L., LAW, P.Y. & LOH, H.H.
(1995). Expression of the mu-opioid receptor in CHO cells:
ability of mu-opioid ligands to promote alpha-azidoanili-
do[32P]GTP labeling of multiple G protein alpha subunits. Eur.
J. Pharmacol., 64, 2534 ± 2543.
CHOU, J.C.
& LEE, E.H. (1995). Di€erential involvement of
hippocampal G-protein subtypes in the memory process of rats.
Neuroscience, 64, 5 ± 15.
COLOMBO, P.J., MARTINEZ JR, J.L., BENNET, E.L. & ROSENZWEIG,
M.R. (1992). k-opioid receptor activity modulates memory for
peck-avoidance training in the 2-day-old chick. Psychopharma-
cology, 108, 235 ± 240.
CONNOR, M. & CHRISTIE, M.D. (1999). Opioid receptor signalling
mechanisms. Clin. Exp. Pharmacol. Physiol., 26, 493 ± 499.
DEL CERRO, S. & BORRELL, J. (1987). b-Endorphin impairs forced
extinction of an inhibitory avoidance response in rats. Life Sci.,
41, 579 ± 584.
IZQUIERDO, I. (1980). E€ect of b-endorphin and naloxone on
acquisition, memory and retrieval of shuttle avoidance and
habituation learning in rats. Psychopharmacology, 69, 111 ± 115.
JARVIK, M.E. & KOPP, R. (1967). An improved one-trial passive
avoidance learning situation. Psychol. Rep., 21, 221 ± 224.
KATADA, T. & UI, M. (1982). Direct modi®cation of the membrane
adenylate cyclase system by islet-activating protein due to ADP
ribosilation of a membrane protein. Proc. Natl. Acad. Sci.
U.S.A., 79, 3129 ± 3133.
FOWLER, C.J., COWBURN, R.F., GARLIND, A., WINBLAND, B. &
O'NEILL, C. (1995). Disturbances in signal transduction mechan-
isms in Alzheimer's disease. Mol. Cell Biochem., 149 ± 150, 287 ±
292.
KIEFFER, B.L. (1999). Opioids: ®rst lessons from knockout mice.
Trends Pharmacol. Sci., 20, 19 ± 25.
British Journal of Pharmacology vol 133 (2)

274
N. Galeotti et al
Gi-proteins and morphine amnesia
KNAPP, R.J., MALATYNSKA, E., COLLINS, N., FANG, L., WANG, J.,
HRUBY, V.J., ROESKE, W.R. & YAMAMU, H.I. (1995). Molecular
biology and pharmacology of cloned opioid receptors. FASEB J.,
9, 516 ± 525.
SCHNECKO, A., WITTE, K., BOHL, J., OHM, T. & LEMMER, B. (1994).
Adenylyl cyclase activity in Alzheimer's disease brain: stimula-
tory and inhibitory signal transduction pathways are di€erently
a€ected. Brain Res., 644, 291 ± 296.
LAW, P.Y., WONG, Y.H. & LOH, H.H. (2000). Molecular mechanisms
and regulation of opioid receptor signaling. Annu. Rev.
Pharmacol. Toxicol., 40, 389 ± 430.
SCHULTEIS, G., MARTINEZ, J.L. & HRUBY, V.J. (1988). Stimulation
and antagonism of opioid delta-receptors produce opposite
e€ects on active avoidance conditioning in mice. Behav.
Neurosci., 102, 678 ± 686.
SHANAHAN, C., GIBSON, G.E., COWBURN, R.F., JOHNSTON, J.A.,
WIEHAGER, B., LANNFELT, L. & O'NEILL, C. (1997). G protein
subunit levels in ®broblasts from familial Alzheimer's disease
patients: lower levels of high molecular weight Gs alpha isoform
in patients with decreased b-adrenergic receptor stimulated
cAMP formation. Neurosci. Lett., 232, 33 ± 36.
SILVA, A.J., KOGAN, J.H., FRANKLAND, P.W. & KIDA, S. (1998).
CREB and memory. Annu. Rev. Neurosci., 21, 127 ± 148.
SIMON, M.I., STRATHMAN, M.P. & GAUTMAN, N. (1991). Diversity
of G proteins in signal transduction. Science, 252, 802 ± 808.
SOOMETS, U., MAHLAPUU, R., TEHRANIAN, R., JARVET, J.,
KARELSON, E., ZILMER, M., IVERFELDT, K., ZORKO, M.,
GRASLUND, A. & LANGEL, U. (1999). Regulation of GTPase
and adenylate cyclase activity by amyloid b-peptide and its
fragments in rat brain tissue. Brain Res., 850, 179 ± 188.
TILSON, H., MCLAMB, R. & HONG, J. (1986). Behavioral e€ects of
centrally administered dynorphin and (D-Ala2-D-leu)enkephalin
(DADLE) in rats. Neuropeptides, 8, 193 ± 206.
UKAI, M., ITOH, J., KOBAYASHI, T., SHINKAI, N. & KAMEYAMA, T.
(1997b). E€ects of the k-opioid dynorphin A-(1-13) on learning
and memory in mice. Behav. Brain Res., 83, 169 ± 172.
UKAI, M., SHINKAI, N. & KAMEYAMA, T. (1995). k-opioid receptor
agonists improve pirenzepine-induced disturbance of sponta-
neous alternation performance in the mouse. Eur. J. Pharmacol.,
81, 173 ± 178.
UKAI, M., TAKADA, A., SASAKI, Y. & KAMEYAMA, T. (1997a).
Stimulation of d1- and d2-opioid receptors produces amnesia in
mice. Eur. J. Pharmacol., 338, 1 ± 6.
VAUGHT, J., PELLEY, K., COSTA, L.G., SETHER, P. & ENNA, S.J.
(1985). A comparison of the antinociceptive responses to GABA-
receptor agonists THIP and baclofen. Neuropharmacology, 24,
211 ± 216.
WONG, Y.H., CONKLIN, B.R. & BOURNE, H.R. (1992). Gz-mediated
hormonal inhibition of cyclic AMP accumulation. Science, 255,
339 ± 342.
ZHANG, H.T., CRISSMAN, A.M., DORAIRAJ, N.R., CHANDLER, L.J.
& O'DONNELL, J.M. (2000). Inhibition of cyclic AMP phospho-
diesterase (PDE4) reverses memory de®cits associated with
NMDA receptor antagonism. Neuropsychopharmacology, 23,
198 ± 204.
LOH, H.H. & SMITH, A.P. (1990). Molecular characterization of
opioid receptors. Annu. Rev. Pharmacol. Toxicol., 30, 123 ± 147.
MATTHES, H.W., SMADJA, C., VALVERDE, O., VONESH, J.L.,
FOUTZ, A.S., BOUDINOT, E., DENAVIT-SAUBIE, M., SEVERINI,
C., NEGRI, L., ROQUES, B.P., MALDONADO, R. & KIEFFER, B.L.
(1998). Activity of the d-opioid receptor is partially reduced,
whereas activity of the k-receptor is maintained in mice lacking
the m-receptor. J. Neurosci., 18, 7285 ± 7295.
MESSING, R.B., JENSEN, R.A., MERTINEZ, JR., J.L., SPIEHLER, V.R.,
VASQUEZ, B.J., SOUMIREU-MOURAT, B., LIANG, K.C.
&
MCGAUGH, J.L. (1979). Naloxone enhancement of memory.
Behav. Neural Biol., 27, 266 ± 275.
MESSING, R.B., JENSEN, R.A., VASQUEZ, B.J., MARTINEZ, JR, J.L.,
SPIEHLER, V.R. & MCGAUGH, J.L. (1981). Opiate modulation of
memory. In Endogenous peptides and learning and memory
processes. ed. Martinez JR. J.L., Jensen, R.A., Messing, R.B.,
Rigter H., McGaugh, J.L. pp 431 ± 443. New York: Academic
Press.
MULLER, U. (2000). Prolonged activation of cAMP-dependent
protein kinase during conditioning induces long-term memory
in honeybees. Neuron, 27, 156 ± 168.
RAFFA, R.B., GOODE, T.L., MARTINEZ, R.P. & JACOBY, H.I. (1996).
A Gi2a antisense oligonucleotide di€erentiates morphine anti-
nociception, constipation and acute dependence in mice. Life
Sci., 58, PL73 ± 76.
RAFFA, R.B., MARTINEZ, R.P. & CONNELLY, C.D. (1994). G-protein
antisense oligodeoxynucleotides and m-opioid supraspinal anti-
nociception. Eur. J. Pharmacol., 258, R5 ± 7.
RAYNOR, K., KONG, H., CHEN, Y., YASUDA, K., YU, L., BELL, G.I. &
REISINE, T. (1994). Pharmacological characterization of the
cloned k-, d-, and m-opioid receptors. Mol. Pharmacol., 45, 330 ±
334.
RUSH, D.K. (1986). Reversal of scopolamine-induced amnesia of
passive avoidance by pre- and post-training naloxone. Psycho-
pharmacology, 89, 296 ± 300.
RYMER, D.L. & GOOD, T.A. (2001). The role of G protein activation
in the toxicity of amyloidogenic Ab(1 ± 40), Ab(25 ± 35), and
bovine calcitonin. J. Biol. Chem., 276, 2523 ± 2530.
SANCHEZ-BLAZQUEZ, P., GARCIA-ESPANA, A.
& GARZON, J.
(1995). In vivo injection of oligodeoxynucleotides to Ga subunits
and supraspinal evoked by mu and delta opioid agonists. J.
Pharmacol. Exp. Ther., 275, 1590 ± 1596.
SANCHEZ-BLAZQUEZ, P.
subtypes activates inositol-signaling pathways in the production
&
GARZON, J. (1998). Delta opioid
(Received November 11, 2000
Revised March 6, 2001
Accepted March 14, 2001)
of antinociception. J. Pharmacol. Exp. Ther., 285, 820 ± 827.
British Journal of Pharmacology vol 133 (2)