Download involvement of the bed nucleus of the stria terminalis activated by

Neuroscience 134 (2005) 9 –19
INVOLVEMENT OF THE BED NUCLEUS OF THE STRIA TERMINALIS
ACTIVATED BY THE CENTRAL NUCLEUS OF THE AMYGDALA IN
THE NEGATIVE AFFECTIVE COMPONENT OF MORPHINE
WITHDRAWAL IN RATS
T. NAKAGAWA,a* R. YAMAMOTO,a M. FUJIO,a
Y. SUZUKI,a M. MINAMI,a M. SATOHa,b AND S. KANEKOa
Key words: conditioned place aversion, drug dependence,
extended amygdala, c-Fos, immunohistochemistry, lesion.
a
Department of Molecular Pharmacology, Graduate School of Pharmaceutical Sciences, Kyoto University, Kyoto 606-8501, Japan
b
Chronic use of morphine is well known to lead to physical
and psychological dependence, characterized by the expression of withdrawal symptoms including both somatic
and affective components, upon cessation of drug administration. In animals, morphine withdrawal produces various characteristic somatic signs, as well as disruption of
schedule-controlled operant responses for food (Higgins
and Sellers, 1994; Koob et al., 1989), elevation of intracranial self-stimulation thresholds (Schaefer and Michael,
1986) and aversive avoidance behavior from the environment previously associated with morphine withdrawal
(conditioned place aversion; CPA) (Mucha, 1987; Stinus et
al., 1990). These behavioral changes are thought to reflect
the negative affective component of morphine withdrawal,
such as dysphoria, irritability and anxiety, which might
contribute to aversively motivated drug seeking.
The extended amygdala is a neuronal continuum within
the basal forebrain with considerable morphological, neurochemical and neuroanatomical similarities, and seems to
contribute to motivational and emotional responses. The extended amygdala is divided into medial and central components. The central extended amygdala includes the central
nucleus of the amygdala (Ce), the lateral bed nucleus of
the stria terminalis (BST), and the central sublenticular
extended amygdala, which is a part of the dorsal substantia innominata, while the medial extended amygdala includes the medial nucleus of the amygdala and the medial
BST (Alheid et al., 1995). Recently, it has been suggested
that the extended amygdala is involved in both the acute
positive reinforcing effects of drugs of abuse and the negative reinforcement associated with drug withdrawal
(Koob, 1999; Koob and Le Moal, 2001). A number of
studies have shown that morphine withdrawal increases
the expression of a transcription factor, c-Fos protein, as a
marker of neural activation (Kovacs, 1998), in several components of the extended amygdala, including the Ce and
the BST (Frenois et al., 2002; Gracy et al., 2001; Le Guen
et al., 2001; Stornetta et al., 1993; Veinante et al., 2003).
The c-Fos induction in them was reported to correlate with
the negative affective, rather than somatic, component of
morphine withdrawal (Frenois et al., 2002; Gracy et al.,
2001). Indeed, it was shown that microinjection of opioid
antagonists into the amygdala, as well as the nucleus
accumbens, of morphine-dependent rats produced mor-
Nara Institute of Science and Technology, Ikoma 630-0101, Japan
Abstract—The central nucleus of the amygdala (Ce) and the
bed nucleus of the stria terminalis (BST) are key structures of
the extended amygdala, which is suggested to be involved in
drug addiction and reward. We have previously reported that
the Ce plays a crucial role in the negative affective component
of morphine withdrawal. In the present study, we examined the
involvement of the neural pathway between the Ce and the BST
in the negative affective component of morphine withdrawal in
rats. Rats were rendered morphine dependent by s.c. implantation of a 75-mg morphine pellet for 3 days, and morphine withdrawal was precipitated by an i.p. injection of naloxone (0.3 mg/
kg). In the place-conditioning paradigm, discrete bilateral excitotoxic lesions of the Ce or the BST significantly reduced
naloxone-precipitated morphine withdrawal-induced conditioned place aversion. On the other hand, they had little effect
on morphine withdrawal-induced somatic signs. In an immunohistochemical study for c-Fos protein, naloxone-precipitated
morphine withdrawal dramatically induced c-Fos-immunoreactive neurons in the capsular part of the Ce, and the lateral and
medial divisions of the BST. Bilateral excitotoxic lesion of the
Ce reduced the number of morphine withdrawal-induced c-Fosimmunoreactive neurons in the lateral and medial BST, with
significant decreases in the posterior, ventral and juxtacapsular
parts of lateral division, and anterior part of the medial division,
but not in the ventral part of the medial division of the BST. On
the other hand, bilateral excitotoxic lesion of the BST had no
effect on such c-Fos induction within the capsular part, nor the
ventral and medial divisions of the Ce. These results suggest
that activation of the BST mediated through the neural pathway
from the Ce contributes to the negative affective component of
morphine withdrawal. © 2005 IBRO. Published by Elsevier Ltd.
All rights reserved.
*Corresponding author. Tel: ⫹81-75-753-4548; fax: ⫹81-75-753-4586.
E-mail address: [email protected] (T. Nakagawa).
Abbreviations: BST, bed nucleus of the stria terminalis; BSTLD, dorsal
part of the lateral division of the bed nucleus of the stria terminalis;
BSTLJ, juxtacapsular part of the lateral division of the bed nucleus of
the stria terminalis; BSTLP, posterior part of the lateral division of the
bed nucleus of the stria terminalis; BSTLV, ventral part of the lateral
division of the bed nucleus of the stria terminalis; BSTMA, anterior part
of medial division of the bed nucleus of the stria terminalis; BSTMV,
ventral part of medial division of the bed nucleus of the stria terminalis;
Ce, central nucleus of the amygdala; CeC, capsular part of the central
nucleus of the amygdala; CeL, lateral division of the central nucleus of
the amygdala; CeM, medial division of the central nucleus of the
amygdala; CPA, conditioned place aversion; CRF, corticotropinreleasing factor; NMDA, N-methyl-D-aspartic acid; PBS, phosphatebuffered saline.
0306-4522/05$30.00⫹0.00 © 2005 IBRO. Published by Elsevier Ltd. All rights reserved.
doi:10.1016/j.neuroscience.2005.03.029
9
10
T. Nakagawa et al. / Neuroscience 134 (2005) 9 –19
phine withdrawal-induced CPA (Maldonado et al., 1992;
Stinus et al., 1990). Furthermore, we and other groups
reported that excitotoxic lesion of the Ce (Kelsey and
Arnold, 1994; Watanabe et al., 2002b), or microinjection of
␤-adrenoceptor antagonists into the Ce (Watanabe et al.,
2003) or the BST (Delfs et al., 2000) attenuated the morphine withdrawal-induced CPA, while they had no or
weaker effects on the morphine withdrawal-induced somatic signs. These findings support the notion that the Ce
and the BST play an important role in the negative affective, rather than somatic, component of morphine withdrawal.
It has been reported that the Ce and the BST are
reciprocally connected through the stria terminalis (Dong
and Swanson, 2004; Han and Ju, 1989; Ju and Swanson,
1989; Krettek and Price, 1978; Sun et al., 1991; Sun and
Cassell, 1993; Veinante and Freund-Mercier, 1998). Furthermore, it has been suggested that they have common
physiological roles in autonomic and behavioral responses
associated with motivational and emotional responses
(Delfs et al., 2000; Dunn, 1987; Dunn and Whitener, 1986;
Watanabe et al., 2003). However, whether the neural pathway between them is involved in the negative affective
component of morphine withdrawal has not been established. In this study, we examined the effects of bilateral
excitotoxic lesions of the Ce and the BST on the naloxoneprecipitated morphine withdrawal-induced CPA, somatic
signs, and c-Fos expression in the subdivisions of the BST
and the Ce in rats. We show here that activation of the
neural pathway from the Ce to the BST plays a crucial role
in the negative affective component of morphine withdrawal.
EXPERIMENTAL PROCEDURES
The present study was conducted in accordance with the ethical
guidelines of the Kyoto University Animal Experimentation Committee and the Japanese Pharmacological Society and was in
complete compliance with the National Institutes of Health Guide
for the Care and Use of Laboratory Animals. All efforts were made
to minimize the number of animals used and limit experimentation
to what was necessary to produce reliable scientific information.
Animals
Male Sprague–Dawley rats (Japan SLC, Hamamatsu, Japan) initially weighing 180 –220 g were used. They were kept at a constant ambient temperature of 24⫾1 °C under a 12-h light/dark
cycle with free access to food and water. After arrival, rats were
individually housed in plastic cages with woodchip bedding for at
least 1 day before surgery.
Materials
Morphine hydrochloride was purchased from Takeda Chemical
Industries (Osaka, Japan). Morphine pellets each containing 75
mg of morphine base were prepared according to the method of
Gibson and Tingstad (1970). N-methyl-D-aspartic acid (NMDA)
and naloxone hydrochloride were purchased from Sigma (St.
Louis, MO, USA).
Surgery
Bilateral excitotoxic lesions of the Ce or the BST were made as
previously described (Watanabe et al., 2002b) with slight modifica-
tions. Briefly, under thiamylal sodium anesthesia (50 mg/kg, i.p.),
0.3 M NMDA dissolved in phosphate-buffered saline (PBS) was
bilaterally infused into the Ce (1.8 mm caudal to bregma, 3.8 mm
lateral to the midline, and 8.0 mm below the surface of the skull) or
the BST (0.3 mm caudal to bregma, 1.6 mm lateral to the midline,
and 7.0 mm below the surface of the skull) according to the atlas of
Paxinos and Watson (1998) in a volume of 0.3 ␮l/side over 3 min via
stainless steel injection cannulas (33 gauge, o.d. 0.2 mm). The
injection cannulas were left in place for an additional 2 min to prevent
backflow. Sham-operated rats received identical surgical treatment
except that PBS alone was infused. Then, the rats were individually
returned to their respective cages and left for at least 1 week before
the experiment. A total of 140 rats were divided five groups, i.e. two
non-operated, 24 Ce-sham-operated, 46 Ce-lesioned, 24 BSTsham-operated and 44 BST-lesioned animals.
Induction of morphine dependence and precipitation
of morphine withdrawal
Rats were rendered morphine dependent by s.c. implantation of a
morphine pellet as previously described (Watanabe et al., 2002a).
Under light ether anesthesia, rats had either a morphine or placebo pellet implanted s.c. in the back of the neck. Three days
later, the animals received a single i.p. injection of naloxone
(0.3 mg/kg) dissolved in saline without removing the implanted
pellet.
CPA paradigm
Twelve Ce-sham-operated, 20 Ce-lesioned, 12 BST-sham-operated and 20 BST-lesioned animals, which were implanted with a
morphine pellet, were used in the CPA experiments.
Apparatus. CPA was conducted as previously described
(Watanabe et al., 2002a). The place conditioning apparatus consisted of a shuttle box (30⫻60⫻30 cm: width⫻length⫻height)
divided into two equal-sized compartments. The inner surface of
one compartment was black with a smooth floor; and the other
was white with a textured floor. The time spent in each compartment during a period of 15 min (900 s) was measured automatically in a blind fashion using a computer system (KN-80; Natsume
Seisakusho, Tokyo, Japan). The apparatus was enclosed by a
sound- and light-attenuated box under conditions of dim illumination and masking white noise.
Preconditioning session. On the first day (day 1), the partition
separating the two compartments was raised 12 cm above the floor,
and a metal neutral platform (5⫻2⫻12 cm) was inserted along the
seam separating the compartments. The rats were individually
placed on the platform and allowed to climb down and freely explore
the two compartments for 900 s, so that they could habituate themselves to the apparatus. On day 2 (preconditioning session), the
same trial was performed, and the time spent in each compartment
for 900 s was measured. There was no significant difference between time spent in the black compartment with a smooth floor
(455⫾19.9 s, n⫽36) and time in the white compartment with a
textured floor (444⫾19.9 s, n⫽36), indicating that there was no
preference bias before conditioning in the apparatus. We determined
the preferred compartment for each rat in which the rats spent
greater than 50% of the total time (i.e. 450 s). Four Ce-shamoperated rats, a Ce-lesioned and a BST-sham-operated rat that
spent more than 80% of the time (i.e. 720 s) in one side on day 3, or
that spent more than 600 s in one side on day 2 and more than 600 s
on the other side on day 3 were excluded from further analysis.
Conditioning session. On day 3, place conditioning was
performed as follows: each rat was injected i.p. with saline and
confined to its non-preferred compartment for 1 h. After at least
3 h, the rat was injected i.p. with naloxone (0.3 mg/kg) and then
confined to its preferred compartment for 1 h.
T. Nakagawa et al. / Neuroscience 134 (2005) 9 –19
Test session. On day 4, the partition separating the two
compartments was raised, and the neutral platform was inserted
again. The rats were individually placed on the platform and
allowed to freely explore the two compartments, and the time
spent in each compartment during 900 s was then measured
without any injection. The CPA scores represent the time spent in
the naloxone-paired compartment in the test session minus the
time spent in the same compartment in the preconditioning session, and are expressed as means⫾S.E.M.
Measurement of naloxone-precipitated morphine
withdrawal-induced somatic signs
Twelve Ce-sham-operated, 26 Ce-lesioned, 12 BST-sham-operated and 24 BST-lesioned animals, which were implanted with a
morphine pellet, were used in the experiments for somatic signs
assessment. Measurement of naloxone-precipitated morphine
withdrawal-induced somatic signs was performed as previously
described (Watanabe et al., 2002a). Briefly, each morphine-dependent rat was placed in a Plexiglas cylinder to acclimate it to the
experimental environment. After a 60-min habituation period, naloxone (0.3 mg/kg) was injected i.p., and then behavior was observed for 60 min. Body weight was measured just before and 1 h
after naloxone injection, and is presented as the means⫾S.E.M.
of percentage body weight loss. The number of occurrences of
stretching, wet-dog shaking, teeth chattering, jumping, paw shaking, head shaking, ejaculation, penile grooming and grooming was
counted, and data are presented as means⫾S.E.M. of total numbers. The occurrence of diarrhea, salivation, lacrimation, rhinorrhea and ptosis was monitored and is presented as the number of
rats showing positive signs divided by the total number of rats
tested.
Immunohistochemistry for c-Fos
Four Ce-sham-operated, four Ce-lesioned, four BST-sham-operated and four BST-lesioned rats, which were used for measurement of morphine withdrawal-induced somatic signs, and two
non-operated and placebo pellet-implanted rats were used in the
immunohistochemical experiments. Ninety minutes after the i.p.
injection of naloxone (0.3 mg/kg), the animals were deeply anesthetized with thiamylal sodium and perfused transcardially through
the ascending aorta with 0.1 M PBS, immediately followed by 4%
paraformaldehyde in PBS. Brains were removed, post-fixed in the
same fixative for 3– 4 h, cryoprotected with 15% sucrose in PBS
for 48 h at 4 °C, and then frozen in powdered dry-ice. Coronal
sections (25 ␮m) including the Ce or the BST were prepared on a
cryostat and collected in PBS at 4 °C to be processed immunohistochemically as free-floating sections. The atlas of Paxinos and
Watson (1998) was used to identify the brain regions.
The rat brain sections were incubated for 10 min in 1.0% H2O2
in phosphate-buffer (pH 7.4) and gently washed three times (5 min
each) in PBS. The sections were permeabilized and blocked for
1 h in 4% normal goat serum in PBS containing 0.5% Triton X-100
at room temperature. They were then incubated overnight with
primary polyclonal rabbit anti c-Fos antibody (SC-253; Santa Cruz
Biotechnology, Santa Cruz, CA, USA, 1:10,000 dilution) in PBS
containing 0.5% Triton X-100 and 4% normal goat serum at 4 °C.
The sections were washed three times in PBS, and then incubated
for 1 h with biotinylated goat anti-rabbit IgG (Jackson ImmunoResearch, West Grove, PA, USA, 1:200 dilution) in PBST with
0.5% Triton X-100 and 4% normal goat serum at room temperature. After three washes in PBS, the sections were incubated for
1 h in a solution of avidin– biotin–peroxidase complex (ABC Elite
kit, Vectastain, Vector Laboratories, Burlingame, CA, USA) at
room temperature. Finally, the sections were washed three times
in PBS and once in 0.05 M Tris buffer (pH 7.6). The peroxidase
reaction product was visualized in Tris buffer containing 0.022%
3–3= diaminobenzidine (Sigma), 0.003% hydrogen peroxidase
11
and 0.07% nickel chloride. The sections were washed in Tris
buffer and mounted on gelatin-coated slides, air dried, dehydrated
through a graded ethanol series, cleared in xylene and coverslipped.
Quantitative analysis of c-Fos immunoreactive
neurons
Sections were examined under a microscope (Nikon MicrophotoFXA; Nikon Co., Tokyo, Japan). c-Fos immunoreactive neurons were
counted in the different subdivisions of the Ce at a level of 2.80 mm
(see Fig. 4 and Table 3) and the BST at a level of 0.26 mm caudal to
bregma (see Fig. 3 and Table 2). Anatomical boundaries were determined according to the atlas of Paxinos and Watson (1998) using
regional landmarks directly on the c-Fos-labeled sections, and/or
adjacent Cresyl-Violet-stained sections. For each structure, the
counting was performed on three adjacent sections from each rat
and used to calculate an intra-animal average value. The
means⫾S.E.M. were then calculated for each structure in each
group.
Histology
To assess the extent of excitotoxic lesions in the Ce and the BST,
histological analyses were performed. The rats used in behavioral
experiments were killed by decapitation and the brain was rapidly
removed and frozen in powdered dry ice. The brains used in immunohistochemical experiments were prepared as above. Then, coronal sections (50 ␮m) were prepared on a cryostat, thaw-mounted
onto gelatin-coated slides and stored at ⫺80 °C until use. The
sections were stained with Cresyl Violet and each section was examined by microscopy (⫻400). Lesion boundaries were identified on
the basis of neuronal loss and reactive gliosis. Only rats with bilateral
excitotoxic lesions of the Ce or the BST were included in the analysis.
Statistical analyses
In the case of CPA experiments, statistical significance was calculated using Student’s t-test. In the case of morphine withdrawal
somatic signs, the data on weight loss and counted signs were
analyzed statistically by one-way analysis of variance, and the
data on checked signs were compared by the chi-square test. The
differences of the numbers of c-Fos-immunoreactive neurons between the sham-operated control and lesioned rats were analyzed
by Student’s t-test. Differences with P⬍0.05 were considered
significant.
RESULTS
Histology of the bilateral excitotoxic lesions of the
Ce and the BST
The neuronal cells were clearly retained in the Ce, BST
and their surroundings of the 20 Ce- and 23 BST-shamoperated control rats.
Illustrations of excitotoxin-induced lesion sites in 19 of
the rats that received bilateral infusion of NMDA into the
Ce are shown in Fig. 1A. The excitotoxic lesion encompassed a significant amount of the capsular part (CeC) and
the lateral (CeL) and medial (CeM) divisions of the Ce,
although the neuronal cells in the surrounding sites were
not damaged. In some animals, there was neuronal damage
in portions of the lateral globus pallidus and substantia innominata. In a few animals, there was neuronal damage in
portions of the lateral or dorsomedial nucleus of the
amygdala. The data from 26 rats were excluded from the
final analysis, i.e. in 19 cases neuronal damage sub-
12
T. Nakagawa et al. / Neuroscience 134 (2005) 9 –19
Fig. 1. Schematic representation of bilateral excitotoxic lesions of the Ce (A) and the BST (B). Shaded areas represent the smallest (black) and largest
(gray) extent of neuronal damage. Outlines represent sections ranging from ⫺1.8 to ⫺2.8 mm (A) and from 0.20 to ⫺1.40 mm (B) from bregma
(Paxinos and Watson, 1998).
stantially extended into the lateral, basolateral and dorsomedial nucleus of the amygdala or Meynert basal
nucleus, and in the other seven cases neuronal damage
to the Ce was incomplete.
Illustrations of excitotoxin-induced lesion sites in 17 of
the rats that received bilateral infusion of NMDA into the
BST are shown in Fig. 1B. The excitotoxic lesions encompassed a significant amount of the dorsal (BSTLD), posterior (BSTLP), ventral (BSTLV) and juxtacapsular
(BSTLJ) parts of the lateral division, and the anterior
(BSTMA) and ventral (BSTMV) parts of the medial division
of the BST, including the anterior commissure and internal
capsule, although the neuronal cells in the surrounding
sites were not damaged. In some animals, there was neuronal damage in portions of the caudate putamen, lateral
globus pallidus, ventral pallidum, substantia innominata
and lateral septal nucleus. The data from 27 rats were
excluded from the final analysis, i.e. in 20 cases neuronal
damage substantially extended into these surrounding
sites, and in the other seven cases neuronal damage to the
BST was incomplete.
Effects of Ce- or BST-lesions on morphine
withdrawal-induced CPA
The effects of bilateral excitotoxic lesions of the Ce or
the BST on naloxone-precipitated morphine withdrawalinduced CPA are shown in Fig. 2.
As we previously reported (Watanabe et al., 2002b), Celesions significantly attenuated the morphine withdrawal-
induced CPA. In the present study, the time that Ce-shamoperated control rats spent in the morphine-withdrawal
paired compartment in the test session was 310⫾38.8 s,
which was significantly shorter than that in the preconditioning session (521⫾23.4 s, P⬍0.001). On the other
hand, the time that Ce-lesioned rats spent in the morphinewithdrawal paired compartment in the test session was
472⫾49.8 s, which was not significantly different compared with that in the preconditioning session (550⫾27.2
s). The CPA score of Ce-lesioned rats was significantly
smaller than that of Ce-sham-operated control rats (Fig.
2A).
Similarly, the time that BST-sham-operated control rats
spent in the morphine-withdrawal paired compartment in
the test session was 330⫾20.3 s, which was significantly
shorter than that in the preconditioning session (511⫾16.8 s,
P⬍0.001). The time that BST-lesioned rats spent in the
morphine-withdrawal paired compartment in the test session was 395⫾26.6 s, which was significantly shorter than
that in the preconditioning session. However, the CPA
score of BST-lesioned rats was significantly smaller than
that of BST-sham-operated control rats (Fig. 2B).
Effects of Ce- or BST-lesions on morphine
withdrawal-induced somatic signs
The effects of bilateral excitotoxic lesions of the Ce or BST on
the naloxone-precipitated morphine withdrawal-induced somatic signs are shown in Table 1. In the Ce- and BST-shamoperated control rats, i.p. injection of naloxone elicited char-
T. Nakagawa et al. / Neuroscience 134 (2005) 9 –19
13
Table 1. Effects of Ce- or BLA-lesions on the somatic signs induced by
naloxone-precipitated morphine withdrawal
Withdrawal signs Ce
Fig. 2. Effect of bilateral excitotoxic lesion of the Ce (A) or the BST (B)
on the naloxone-precipitated morphine withdrawal-induced CPA in
rats. Sham-operated (open bars) or lesioned (closed bars) rats were
given a morphine pellet for 3 days, and then were injected i.p. with
naloxone (0.3 mg/kg) under the place-conditioning paradigm. Each
column represents the CPA score expressed as mean⫾S.E.M.
* P⬍0.05, ** P⬍0.01 compared with respective sham-operated group
by Student’s t-test. Numerals in parentheses indicate the numbers of
rats used.
acteristic somatic signs such as weight loss, rearing, stretching, wet-dog shaking, teeth chattering, paw shaking, head
shaking, ejaculation, penile grooming, grooming, diarrhea,
lacrimation, rhinorrhea and ptosis, and rarely salivation and
jumping in the present paradigm. In the Ce- and BST-lesioned rats, naloxone elicited morphine withdrawal-induced
somatic signs similar to those in the sham-operated control
rats, although Ce- and BST-lesion significantly facilitated paw
shaking (P⬍0.01), and BST-lesion significantly attenuated
teeth chattering (P⬍0.05). There were no significant differences in any other signs between the respective sham-operated and lesioned groups.
Effect of Ce-lesions on morphine withdrawal-induced
c-Fos expression in the BST
The effects of bilateral excitotoxic lesions of the Ce on the
naloxone-precipitated morphine withdrawal-induced c-Fos
expression in the BST are shown in Fig. 3 and Table 2. In
the non-operated and placebo pellet-implanted naive rats
injected i.p. with naloxone, only a few c-Fos-immunoreactive neurons were observed in any subdivision of the BST
(data not shown). On the other hand, in the Ce-shamoperated and morphine-dependent rats, i.p. injection of
naloxone induced c-Fos-immunoreactive neurons in the
BST, including the BSTLD, BSTLP, BSTLV, BSTLJ,
BSTMA and BSTMV (Fig. 3A, C, D). In the Ce-lesioned
and morphine-dependent rats, the induction of c-Fosimmunoreactive neurons was decreased in the lateral and
medial BST, such as the BSTLD, BSTLP, BSTLV, BSTLJ
and BSTMA, but not in the BSTMV (Fig. 3B, E, F). Quantitative analysis showed that bilateral excitotoxic lesion of
the Ce significantly reduced the number of morphine with-
Weight loss (%)
Stretching
Wet-dog shaking
Teeth chattering
Jumping
Paw shaking
Head shaking
Ejaculation
Penile grooming
Grooming
Diarrhea
Salivation
Lacrimation
Rhinorrhea
Ptosis
BST
Sham
(12)
Lesion
(10)
Sham
(12)
Lesion
(9)
2.9⫾0.3
4.9⫾1.5
9.8⫾1.5
70.7⫾6.5
0.3⫾0.3
4.0⫾1.2
11.5⫾2.1
1.6⫾0.4
3.8⫾0.8
7.3⫾1.8
11/12
2/12
6/12
10/12
12/12
2.8⫾0.4
3.1⫾0.4
3.0⫾0.5
2.4⫾1.0
5.3⫾1.3
6.0⫾2.6
16.0⫾3.0
11.0⫾1.9 15.6⫾4.3
57.7⫾7.4
106.7⫾13.6 63.7⫾9.0*
0.2⫾0.2
0
1.6⫾1.0
10.7⫾2.1**
6.1⫾1.3 14.0⫾2.0**
13.8⫾3.5
10.6⫾1.9 12.2⫾3.9
1.0⫾0.3
0.9⫾0.3
1.2⫾0.4
2.7⫾1.0
4.3⫾0.8
4.7⫾1.2
7.2⫾1.9
11.3⫾2.4 11.8⫾1.9
9/10
11/12
9/9
2/10
2/12
4/9
4/10
3/12
2/9
6/10
8/12
6/9
8/10
11/12
8/9
Weight loss is presented as the mean⫾S.E.M of percentage body
weight loss during 1 h. Signs are shown as the means⫾S.E.M of total
numbers for 1 h. The occurrence of diarrhea, salivation, lacrimation,
rhinorrhea and ptosis is expressed as the number of rats showing
positive signs divided by the total number of rats tested. * P⬍0.05,
** P⬍0.01 compared with respective sham operated group by Student’s t-test. Numerals in parentheses indicate the numbers of rats
used.
drawal-induced c-Fos immunoreactive neurons in the
BSTLP (P⬍0.05), BSTLV (P⬍0.05), BSTLJ (P⬍0.01) and
BSTMA (P⬍0.01) compared with those in the Ce-shamoperated group (Table 2).
Effect of BST-lesions on morphine
withdrawal-induced c-Fos expression in the Ce
The effects of bilateral excitotoxic lesion of the BST on the
naloxone-precipitated morphine withdrawal-induced c-Fos
expression in the Ce are shown in Fig. 4 and Table 3. In the
non-operated and placebo pellet-implanted naive rats injected i.p. with naloxone, only a few c-Fos-immunoreactive
neurons were observed in any subdivision of the Ce (data not
shown). On the other hand, in the BST-sham-operated and
morphine-dependent rats, i.p. injection of naloxone dramatically induced c-Fos-immunoreactive neurons in the CeC,
while it induced only a few c-Fos-immunoreactive neurons in
the CeL and CeM (Fig. 4A, C). In the BST-lesioned and
morphine-dependent rats, the induction of c-Fos-immunoreactive neurons in the CeC was similar to that of BST-shamoperated and morphine-dependent rats (Fig. 4B, D). Quantitative analysis showed that there was no significant difference in the number of morphine withdrawal-induced c-Fos
immunoreactive neurons in any subdivision of the Ce between the BST-sham-operated and BST-lesioned groups
(Table 3).
DISCUSSION
The present studies using the place-conditioning paradigm
and immunohistochemistry for c-Fos protein show the in-
14
T. Nakagawa et al. / Neuroscience 134 (2005) 9 –19
Fig. 3. Brightfield light micrographs of the BST showing the distribution of naloxone-precipitated morphine withdrawal-induced c-Fos expression in the
Ce-sham-operated (A, C, D) and Ce-lesioned (B, E, F) morphine-dependent rats. The upper left panel represents the approximate area shown in light
micrographs A and B. (C–F) Light micrographs at higher magnification in the BSTLJ (C, E) and BSTMA (D, F) of the Ce-sham-operated (C, D) and
Ce-lesioned (E, F) morphine-dependent rats. ac, anterior commissure; ic, internal capsule; st, stria terminalis. Scale bar⫽400 ␮m for A and B; 100 ␮m
for C–F.
volvement of activation of the BST mediated by the Ce in
the morphine withdrawal-induced CPA. This finding further
supports the recent notion that the extended amygdala,
including the Ce and the BST, plays a role in the negative
affective component of morphine withdrawal (Delfs et al.,
2000; Koob, 1999; Koob and Le Moal, 2001).
There is a body of evidence for the involvement of the
Ce in the negative affective component of morphine withdrawal. Stinus et al. (1990) showed that microinjection of
methylnaloxonium, a hydrophilic opioid antagonist, into the
Ce elicited CPA in morphine-dependent rats. We previously showed that bilateral excitotoxic lesions of the Ce,
but not the basolateral nucleus of the amygdala, attenuated the naloxone-precipitated morphine withdrawal-induced CPA (Watanabe et al., 2002b), a finding consistent
with those of the present study. In addition, we have re-
ported that intra-Ce injection of glutamate receptor antagonists or ␤-adrenoceptor antagonists attenuated acquisition of morphine withdrawal-induced CPA, while they had
little effect on the expression of somatic signs (Watanabe
et al., 2002a, 2003). On the other hand, limited studies
have directly shown the role of the BST in the negative
affective component of morphine withdrawal (Delfs et al.,
2000), although there are numerous findings regarding
positive and negative affective components of other drugs
of abuse, such as cocaine (Epping-Jordan et al., 1998; Erb
et al., 2001; McFarland et al., 2004), heroin (Walker et al.,
2000) and ethanol (Eiler et al., 2003). Nevertheless, it was
reported that morphine withdrawal precipitated by low
doses of naloxone (7.5–30 ␮g/kg) induced CPA and c-Fos
or c-fos gene expression in the BST and the Ce, while it did
not induce somatic signs (Frenois et al., 2002; Gracy et al.,
T. Nakagawa et al. / Neuroscience 134 (2005) 9 –19
Table 2. Effect of Ce-lesions on the number of c-Fos immunoreactive
cells in the subdivisions of the BST induced by naloxone-precipitated
morphine withdrawal
Subdivision of BST
BSTLD
BSTLP
BSTLV
BSTLJ
BSTMA
BSTMV
Mean number of c-Fos
immunoreactive cells
Ce-sham
Ce-lesion
154.0⫾18.5
29.3⫾7.3
8.7⫾0.7
49.6⫾5.7
155.7⫾23.8
72.4⫾1.9
103.7⫾15.5
9.75⫾2.0*
6.1⫾0.4*
19.8⫾2.3**
49.4⫾6.3**
69.0⫾2.5
The mean total number of c-Fos immunoreactive cells was counted
in each subdivision of the BST from four Ce-sham operated or Celesioned morphine-dependent rats 90 min after an injection of naloxone (0.3 mg/kg). Of note, the numbers of c-Fos immunoreactive cells
in any subdivisions of the BST from placebo pellet-implanted naive
rats after naloxone challenge are 0 – 4. * P⬍0.05, ** P⬍0.01 compared
with Ce-sham operated group by Student’s t-test.
2001; Higgins and Sellers, 1994; Le Guen et al., 2001).
These indirect studies suggested the possibility that the
neural activation of the BST and the Ce correlated with the
negative affective, rather than somatic, component of morphine withdrawal. In this study, we found that excitotoxic
lesions of the BST, as well as the Ce, attenuated morphine
withdrawal-induced CPA. The present results give direct
evidence that the BST, as well as Ce, plays a critical role
in the negative affective component of morphine withdrawal, consistent with previous findings (Delfs et al.,
2000). However, because the Ce- and BST-lesions were
performed before the induction of morphine dependence, it
is difficult to determine whether the lesions affected the
development of morphine dependence related to negative
affective component, acquisition or expression of morphine withdrawal-induced CPA. Our and other group studies conducting intra-Ce or intra-BST injection of receptor
antagonists prior to the conditioning suggest that the Ce
and the BST are, at least, involved in acquisition of morphine withdrawal-induced CPA (Watanabe et al., 2002a,
2003; Delfs et al., 2001), while, to our knowledge, there are
no studies investigating the involvement of these regions in
development of negative affective dependence and/or expression of morphine withdrawal-induced CPA. Further
investigations are needed to elucidate it
In some animals that were finally analyzed, neuronal
damage by BST-lesion slightly extended to surrounding
areas, including the lateral septal nucleus. It has been
reported that the lateral septal nucleus was involved in
contextual fear conditioning (Desmedt et al., 1999) and
that morphine withdrawal precipitated by low doses of
naloxone induced c-fos gene in this area (Frenois et al.,
2002). Therefore, we cannot fully exclude the possibility for
a contribution of the lateral septal nucleus.
On the other hand, the roles of the Ce and the BST in
the morphine withdrawal-induced somatic signs are controversial. Electrolytic lesions of the amygdala failed to
alter somatic withdrawal signs (Kelsey and Arnold, 1994)
except for jumping behavior (Calvino et al., 1979). Micro-
15
injections of some types of glutamate receptor antagonists,
␤-adrenoceptor antagonists or ␣-adrenoceptor agonists
into the Ce (Taylor et al., 1998; Watanabe et al., 2002a,
2003) or the BST (Delfs et al., 2000) significantly but
partially attenuated the expression of somatic withdrawal
signs, while other types of antagonists had no effects
(Watanabe et al., 2002a, 2003). On the other hand, in the
present study, excitotoxic lesions of the Ce or BST unexpectedly facilitated a somatic sign, paw shaking. Although
we cannot fully explain this discrepancy, the Ce and the
BST do not seem to play essential roles in the mediation of
somatic withdrawal signs. At least, it is considered that the
inhibitory effects of Ce- and BST-lesion on the morphine
withdrawal-induced CPA and c-Fos expression are unlikely to be due to the reduction of the development of
morphine physical dependence and expression of morphine withdrawal somatic signs.
The pattern of c-Fos expression induced by naloxoneprecipitated morphine withdrawal in the present immunohistochemical study is in good agreement with the findings
of previous studies (Frenois et al., 2002; Gracy et al., 2001;
Hamlin et al., 2004; Le Guen et al., 2001; Veinante et al.,
2003), although there are some discrepancies. In the
present study, naloxone at a relatively low dose of
0.3 mg/kg was intraperitoneally injected in morphine-dependent rats, which were s.c. implanted with a 75-mg
morphine pellet for 3 days. This procedure induced significant CPA and moderate somatic withdrawal signs. Under
these conditions, we found that c-Fos immunoreactive
neurons were observed in the CeC and throughout the
lateral and medial BST, but there were few such neurons in
the CeL and CeM. On the other hand, some other studies
reported the induction of c-Fos immunoreactive neurons
even in the CeL as well as in the CeC (Hamlin et al., 2004;
Le Guen et al., 2001; Veinante et al., 2003). However,
those previous studies used higher doses of naloxone
(⬎2 mg/kg), which could precipitate strong morphine withdrawal-induced somatic signs (Higgins and Sellers, 1994;
Le Guen et al., 2001), and induce c-Fos immunoreactive
neurons in the some regions, including the Ce and BST
even of naive rats. Thus, the discrepancies about the
c-Fos induction in the CeL might have been due to differences in the degree of morphine withdrawal. The CeC
appears to receive inputs directly from the several thalamic
areas (Turner and Herkenham, 1991), the pontine parabrachial area (Bernard and Besson, 1990; Bernard et al.,
1993), and the cortical areas (Mcdonald and Mascagni,
1996). Indeed, we and other groups have reported that a
variety of aversive and/or stressful stimuli induced the
expression of c-Fos or c-fos gene (Frenois et al., 2002;
Nakagawa et al., 2003; Swank, 1999), and activated neurons, especially in the CeC (Bernard et al., 1992). These
findings suggest that the CeC may be a key gate associating aversive information, including morphine withdrawal,
with aversive emotion.
In the present study, we found that Ce-lesion significantly reduced not only morphine withdrawal-induced
CPA, but also the number of c-Fos-immunoreactive neurons in the lateral and medial BST, including the BSTLJ,
16
T. Nakagawa et al. / Neuroscience 134 (2005) 9 –19
Fig. 4. Brightfield light micrographs of the Ce showing the distribution of naloxone-precipitated morphine withdrawal-induced c-Fos expression in the
BST-sham-operated (A, C) and BST-lesioned (B, D) morphine-dependent rats. The lower left panel represents the approximate area shown in the light
micrographs A and B. (C, D) Light micrographs at higher magnification in the CeC of the BST-sham-operated (C) and BST-lesioned (D) morphinedependent rats. BLA, basolateral nucleus of the amygdala; Scale bar⫽400 ␮m for A and B; 100 ␮m for C–F.
BSTLP, BSTLV, and BSTMA, and tended to reduce the
number in the BSTLD. These results suggest that the Ce,
at least in part, contributes to activation of the BST neurons
during morphine withdrawal. Previous retrograde and anterograde tracing studies indicated that that all subdivisions of the Ce have extensive projections to specific
areas of the BST in rats, and that the neurons arising from
the CeC project to the lateral BST, such as the BSTLP and
BSTLV, and to some extent the BSTLD (Bourgeais et al.,
2001; Dong and Swanson, 2004; Krettek and Price, 1978;
Petrovich and Swanson, 1997; Sun et al., 1991), although
they primarily and extensively project to the substantia
innominata dorsalis (Bourgeais et al., 2001). Those findings taken together with the above finding that morphine
withdrawal induced c-Fos immunoreactive neurons especially in the CeC among of the subdivisions of the Ce imply
that it is likely that the neural pathway from the CeC could
activate the BST neurons. Indeed, half of the morphine
withdrawal-induced c-Fos immunoreactive neurons in the
CeC were retrogradely labeled following injection of a retrograde tract tracer into the BST (our unpublished data).
However, the number of morphine withdrawal-induced
c-Fos immunoreactive neurons in the BSTMA, which receives little or no input from the CeC, was also decreased
significantly by Ce-lesion. It has been reported that the
neurons arising from the CeL extensively project to the
BSTLP and BSTLV, and to some extent the BSTLD and
BSTMA, and that the neurons arising from the CeM project
mainly to the lateral BST, similarly to those from the CeC
and CeL, and clearly to the BSTMA (Bourgeais et al.,
2001; Dong and Swanson, 2004; Krettek and Price, 1978;
Petrovich and Swanson, 1997; Sun et al., 1991). Taken
together, these observations imply that it is conceivable
that the decrease in the BSTMA and other BST subdivisions might be due to the loss of projections from the CeL
and/or CeM due to the excitotoxic lesions, although these
Ce subdivisions had few morphine withdrawal-induced
c-Fos immunoreactive neurons. It was reported that some
neurons projecting from the CeL and CeM to the medial
BST, including the BSTMA, are GABAergic (Sun and Cassell, 1993). It seems to be of interest to examine the
possibility of the disinhibition of the GABAergic neurons
during morphine withdrawal. Another potent candidate is
corticotropin-releasing factor (CRF), which is known to be
T. Nakagawa et al. / Neuroscience 134 (2005) 9 –19
Table 3. Effect of BST-lesions on the number of c-Fos immunoreactive
cells in the subdivisions of the Ce induced by naloxone-precipitated
morphine withdrawal
Subdivision of Ce
CeC
CeL
CeM
Mean number of c-Fos
immunoreactive cells
BST-sham
BST-lesion
220.5⫾6.1
9.9⫾3.8
7.5⫾3.1
215.9⫾4.9
7.3⫾5.3
7.3⫾2.2
The mean total number of c-Fos immunoreactive cells was counted
in each subdivision of the Ce from four BST-sham operated or BSTlesioned morphine-dependent rats 90 min after an injection of naloxone (0.3 mg/kg). Of note, the numbers of c-Fos immunoreactive cells
in any subdivisions of the Ce from placebo pellet-implanted naive rats
after naloxone challenge are 0 – 6.
involved in drug addiction (Koob, 1999). It has been reported that some neurons projecting from the CeL to the
lateral and medial BST contain CRF (Sakanaka et al.,
1986). Heinrichs et al. (1995) reported that suppression of
CRF receptor by a CRF receptor antagonist ␣-helical CRF
(9 – 41) or impairment of CRFergic neurons by immunotargeted toxins in the Ce reduced morphine withdrawalinduced CPA. Furthermore, it was shown that morphine
dependence and withdrawal increased the expression of
CRF mRNA in the Ce, but not the BST (McNally and Akil,
2002). Taken together, it is considered that the CRFergic
neurons projecting from the CeL might contribute to the
activation of the neurons in the medial and lateral BST,
including the BSTMA (Erb et al., 2001). Indeed, our preliminary data showed that microinjection of ␣-helical CRF
(9 – 41) into the BST reduced morphine withdrawal-induced CPA.
On the other hand, it is certain that the Ce receives
inputs from the BST, and that the activity of the Ce is
regulated by GABAergic neurons originating from the BST
(Sun and Cassell, 1993; Veinante and Freund-Mercier,
1998). In the present study, we found that the BST-lesion
had no effect on the number of c-Fos-immunoreactive
neurons induced by morphine withdrawal in the CeC, nor
in the CeL and CeM, although it significantly reduced the
morphine withdrawal-induced CPA. These results suggest
that the activation of the Ce neurons during morphine
withdrawal is not affected through the BST.
Both the Ce and the BST are innervated by noradrenergic afferents mainly through the ventral noradrenergic
bundle from the A1/A2 cell groups. It was shown that
morphine withdrawal-induced CPA was reduced by impairment of the ventral noradrenergic bundle or microinjection
of ␤-adrenoceptor antagonists into the Ce or the BST
(Delfs et al., 2000; Watanabe et al., 2003). Furthermore,
both the Ce and the BST are reciprocally connected with
and regulated by the mesocorticolimbic dopaminergic neurons arising from the ventral tegmental area (Georges and
Aston-Jones, 2002; Hasue and Shammah-Lagnado, 2002;
Phillips et al., 2003), and the dopaminergic terminals have
shown to contact CRFergic neurons in the Ce and the BST
(Asan, 1998; Phelix et al., 1994). The dopaminergic sys-
17
tem is known to be critical for not only drug addiction and
reward, but also aversive emotional responses (Borowski
and Kokkinidis, 1996; Shippenberg and Bals-Kubik, 1995).
Taken together, it is likely that the neural pathway from the
Ce to the BST might compose part of the neural circuit with
noradrenergic and dopaminergic system, and the neural
circuit might play an important role in the negative affective
component of morphine withdrawal (Koob, 1999).
CONCLUSION
In summary, we found that neural activation in some subdivisions of the BST during morphine withdrawal is mediated, at least in part, through the neural pathway from the
Ce, while the neural activation in the Ce is not affected by
BST-lesions. These results suggest that activation of the
neural pathway from the Ce to the BST during morphine
withdrawal is crucial for stimulating neurons within the
BST, which leads to the initiation of aversive behavior
induced by morphine withdrawal.
Acknowledgments—This work was supported by grants from the
Ministry of Education, Culture, Sports, Science and Technology of
Japan, and the Special Coordination Funds for Promoting Science
and Technology Target-oriented Brain Science Research
Program.
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(Accepted 25 March 2005)