Neuroscience Letters 343 (2003) 4952

www.elsevier.com/locate/neulet

Acupuncture increases neuropeptide Y expression in hippocampus of

maternally-separated rats
Sabina Lim, Yeun-Hee Ryu, Seung-Tae Kim, Mee-Suk Hong, Hi-Joon Park*
Department of Acupuncture and Meridian, East-West Medical Research Institute, Kyung Hee University, 1 Hoegidong, Dongdaemoongu,
Seoul, 130-701, South Korea
Received 2 January 2003; received in revised form 27 February 2003; accepted 7 March 2003

Abstract
Maternal separation (MS) is a risk factor in the development of mood-related disorders such as depression. Human and animal studies
support the involvement of neuropeptide Y (NPY) in the pathology of depression. To investigate the effect of acupuncture on depression-like
behavior and examine changes in NPY expression associated with MS, we observed body weight and locomotor activity, and performed NPY
immunohistochemistry in the hippocampus. MS for 7 days beginning on postnatal day 14 induced a significant decrease in body weight and
locomotion, while acupuncture treatment at acupoint Shenmen (HT7) resulted in a significant increase in both. NPY-immunoreactive cells in
area CA1 and the dentate gyrus were decreased in the MS group, but significantly increased in the acupuncture group. These findings suggest
that acupuncture has an effect on the depression-like disorder caused by MS, possibly by modulating NPY expression in the hippocampus.
q 2003 Elsevier Science Ireland Ltd. All rights reserved.
Keywords: Acupuncture; Maternal separation; Depression; Neuropeptide Y; Hippocampus

Maternal separation (MS) or a lack of care is a risk factor

in the development of mood-related disorders like
depression in adulthood [11,20]. Early-life stress may
cause changes in the central nervous system (CNS), such
as hypothalamic-pituitary adrenal dysregulation, and be
associated with an increased risk of depressive psychopathology in adult life [2]. MS has therefore been suggested
as a model for studying depression [1,6].
Acupuncture can be used to manipulate and balance
homeostasis when the bodys innate homeostatic potential is
overwhelmed by acute or chronic stress [17]. Acupuncture
has also been found to have therapeutic effects on
depression in human [4,5,19] and animal [18,23] studies.
Previously, we showed that acupuncture enhanced brainderived neurotrophic factor mRNA levels in the rat
hippocampus after stress induced by immobilization [23],
and augmented hippocampal neuronal cell proliferation
following MS [18].
Neuropeptide Y (NPY) is one of the most abundant
neuropeptides in the CNS. It has been implicated in the
regulation of many autonomic functions, including cardio* Corresponding author. Tel.: 82-2-961-9435; fax: 82-2-961-7831.
E-mail address: acufind@khu.ac.kr (H.J. Park).

vascular control and central stress responses [8,9]. NPY is

thought to play a role in the pathophysiology of certain
mood disorders and in the mechanism of action of
antidepressant drugs [7,8]. Therefore, it has been postulated
that altered NPY in the limbic regions might be a trait
marker of vulnerability to depressive disorders, as these
changes are common to several pre-clinical models of
disease [7,9,10,15]. Rats subjected to MS have lower
hippocampal NPY immunoreactive (ir) levels [7,9]. Interestingly, antidepressants [14], electroconvulsive stimulation
(ECS) [10], and lithium [8] increase hippocampal NPYergic
neurotransmission.
This study investigated the effects of acupuncture during
MS by (1) evaluating body weight and locomotion as
behavioral markers of depression and by (2) observing
changes in NPY expression in the hippocampus to elucidate
the mechanism of the therapeutic effect that we found.
Adult Sprague Dawley rats were housed in pairs at
standard temperature (22 ^ 3 8C) under a standard 12 h
light/dark cycle (lights on at 7:00 a.m.) with free access to
food and water. The experimental procedures were carried
out according to the animal care guidelines of the NIH and
the Korean Academy of Medical Sciences. The male in each
pair was removed once the female was clearly pregnant, and

0304-3940/03/$ - see front matter q 2003 Elsevier Science Ireland Ltd. All rights reserved.
doi:10.1016/S0304-3940(03)00317-3

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S. Lim et al. / Neuroscience Letters 343 (2003) 4952

the females were monitored until delivery. The day of

delivery was designated P0. On P14 [13], the pups were
randomly assigned to one of six groups (n 50): (1) a
control group subjected to MS (MS, n 10); (2) an
acupuncture group subjected to MS, using acupoint Shenmen (HT7), which is used to treat mental disorders in
Oriental medicine [2] (MS HT7, n 10); (3) an
acupuncture group subjected to MS, using acupoint Zusanli
(MS ST36, n 8); (4) a non-acupoint group subjected to
MS (MS NA, n 8) to observe the specificity of the
effect of acupoint HT7; (5) a normal group with maternal
care (NOR, n 8); and (6) a normal group given
acupuncture at HT7 (NOR HT7, n 6). Pups in the
normal groups were housed with their mothers under
standard conditions; those in the remaining groups were
maintained individually. For the HT7 groups (MS HT7
and NOR HT7), the rat pups were lightly immobilized by
hand to minimize stress and acupuncture needles were
inserted to a depth of 3 mm on either side of acupoint HT7,
at the end of the transverse crease of the ulnar wrist of the
forepaw. The needles were twisted twice a second for 30 s
and then removed. For the MS ST36 group, the same
manipulation was applied to acupoint ST36, which is near
the knee joint of the hind limb, 2 mm lateral to the anterior
tubercle of the tibia [17]. For the MS NA group, the same
manipulation was applied to a non-acupoint located on the
hip. The NOR and MS groups were also lightly immobilized
using the same methods for 30 s, and then returned to their
cages. All treatments were repeated on 7 consecutive days.
During treatment, the animals were weighed and observed
in their home cages daily [13,18,22]. On P21, locomotor
activity was measured in a stabilimeter (60 60 30 cm).
The animals were placed on the stabilimeter and its
sensitivity was adjusted so that it recorded gross body
movements. The walls and floor were made of clear
Plexiglas painted black. Data were collected for 1 h [22].
On day P22, the rats were deeply anesthetized and
perfused transcardially with 0.05 M phosphate-buffered
saline (PBS, pH 7.4), followed by chilled 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4). Following
postfixation, frontal sections (40 mm thick) were cut using a
freezing microtome (Shandon, UK). Free-floating tissue
sections were washed twice in 50 mM PBS and then
permeabilized in 0.2% Triton X-100 for 30 min. After
washing twice with PBS, the sections were incubated
overnight with rabbit anti-NPY antibody (DiaSorin, Stillwater, MN) at a dilution of 1:4000, then washed twice in
PBS again and incubated for 1 h with biotinylated antirabbit antibody (1:200). Bound secondary antibody was
then amplified using a Vector Elite ABC kit (Vector
Laboratories, Burlingame, CA). The antibody-biotin-avidin-peroxidase complexes were visualized using 0.05%
diaminobenzidine. The sections were mounted on gelatinized glass slides and air-dried, and cover slips were applied
with Permountw [21].
An average of eight sections within the dorsal hippo-

campal region, spanning from bregma 2 3.30 to 2 4.16 mm

in the atlas of Paxinos and Watson, were selected from each
brain. Stained neurons were counted separately for the
stratum oriens, stratum pyramidale and stratum radiatum of
the CA1, CA2 and CA3 regions, and for the granule cell
layer and hilus of the dentate gyrus (DG) region after
staining with methyl green (DAKO, Carpinteria, CA). The
NPY-ir cells were counted from each section using a brightfield microscope (Olympus, Japan) and analyzed using an
image analyzer (Optimas version 6.5, Media Cybernetics,
MD). Cell counts were expressed as the mean number of
NPY-positive cells per unit area (mm2). All of these
quantification procedures were performed blind; an independent investigator (Dr Y.H. Ryu) implemented a code that
concealed the identities, and the code was not broken until
the correlations were complete. All data were presented as
the mean ^ SEM and were analyzed by ANOVA with the
Newman Keuls post-hoc test.
Within 24 h of separation, the pups in the MS groups
moved to the dark side of their containers or burrowed under
the wood-chip bedding, and became virtually quiescent
[13]. After the second day, the MS HT7 group no longer
avoided brightness and moved more than the MS group, as
we reported previously [17]. The average body weights of
the NOR and NOR HT7 groups increased normally to
61.3 and 58.8% of the basal weight over 7 days. That of the
MS group, however, decreased for the first 2 days after MS,
and then gradually increased to 12.0% of the basal weight.
The MS HT7 group showed a similar pattern to that of the
MS group, but the rates of increase 6 and 7 days after MS
were greater (19.5 and 28.6%, each P , 0:01 vs. the MS
group, respectively). The MS ST36 and MS NA
groups did not differ statistically from the MS group (Fig.
1).
Fig. 2 shows that MS for 7 consecutive days beginning

Fig. 1. Changes in body weight. Pups kept with their mothers (NOR). Pups
kept with their mothers, with acupuncture at HT7 (NOR HT7). Pups
subjected to MS (MS). HT7-acupuncture treated pups subjected to MS
(MS HT7). ST36-acupuncture treated pups subjected to MS
(MS ST36). Non-acupoint-acupuncture treated pups subjected to MS
(MS NS). Values are given as the mean ^ SEM (bar). *P , 0:05,
**P , 0:01, and ***P , 0:001 vs. the MS group at each time point.

S. Lim et al. / Neuroscience Letters 343 (2003) 4952

Fig. 2. Locomotion for 1 h after 7 days of MS. Pups kept with their mothers
(NOR). Pups kept with their mothers with acupuncture at HT7
(NOR HT7). Pups subjected to MS (MS). HT7-acupuncture treated
pups subjected to MS (MS HT7). ST36-acupuncture treated pups
subjected to MS (MS ST36). Non-acupoint-acupuncture treated pups
subjected to MS (MS NS). Values are given as the mean ^ SEM (bar).
*P , 0:05 vs. the MS group.

on P14 decreased total locomotion as compared to the NOR

group (NOR: 1874.6 ^ 47.3 cm/h; MS: 1617.4 ^ 44.1 cm/
h). The MS HT7 group recovered to the level of the NOR
group (1916.7 ^ 68.6 cm/h), while the MS ST36 and
MS NA groups did not differ statistically from the MS
group (1624.7 ^ 75.0 and 1509.1 ^ 110.1 cm/h, respectively).
The numbers of NPY-ir cells in area CA1 of the NOR
and NOR HT7 groups were 117.3 ^ 5.4 and
94.1 ^ 7.9/mm2, respectively, but did not differ from each
other statistically (P , 0:01 and 0.05 vs. the MS group,
respectively, Fig. 3). The number decreased to 58.3 ^ 10.0/
mm2 in the MS group, but became significantly elevated to

Fig. 3. Quantitative analysis of the number of NPY-positive cells per mm2

in areas CA1, CA2, and CA3, and the DG of the hippocampus. Pups kept
with their mothers (NOR). Pups kept with their mothers with acupuncture at
HT7 (NOR HT7). Pups subjected to MS (MS). HT7-acupuncture treated
pups subjected to MS (MS HT7). ST36-acupuncture treated pups
subjected to MS (MS ST36). Non-acupoint-acupuncture treated pups
subjected to MS (MS NS). Values are given as the mean ^ SEM (bar).
*P , 0:05, **P , 0:01 vs. the MS group for each region.

51

102.2 ^ 9.0/mm2 in the MS HT7 group (P , 0:05 vs.

MS group). The values for the MS ST36 and MS NA
groups did not differ from the MS group (71.5 ^ 4.5 and
49.9 ^ 4.5/mm2, respectively).
A similar pattern was observed in the DG. The numbers
of NPY-ir cells in the NOR and NOR HT7 groups were
199.89 ^ 13.8 and 185.3 ^ 30.7/mm2, respectively. The
number was reduced to 97.3 ^ 9.4/mm2 in the MS group,
and was significantly elevated to 172.6 ^ 16.1/mm2 in the
MS HT7 group. The values for the MS ST36 and
MS NA groups did not differ statistically from the MS
group (126.2 ^ 14.9 and 126.2 ^ 15.5/mm2, respectively)
(Fig. 3). The numbers of NPY-ir cells in areas CA2 and CA3
did not differ among groups (Fig. 3).
Depression is primarily characterized by a lowering of
mood and an inhibition of both mental and physical activity
[10]. In our study, rat pups subjected to MS showed
depression-like behavior: (1) they moved to the dark side of
their cages and became quiescent; (2) they refused to eat for
the first 2 days and then ate some food, causing their body
weights to decrease for 2 days and then increase very slowly
(20% of the body weight of the NOR group at P21); and (3)
their locomotion was decreased to 86% of the NOR group at
P21. However, these behavior patterns differed in the MS
group that received acupuncture at acupoint HT7: (1) they
did not avoid brightness and moved more than those in the
MS group; (2) their average weight increased more than that
of the MS group (47% of the body weight of the NOR group
at P21); and (3) their locomotion did not decrease (102% of
the NOR group).
NPY appears to play a role in the pathophysiology of
depression, and represents a potential novel target for the
treatment of this illness [20]. Repeated ECS increased NPY
expression in the hippocampus of rats [14]. Chronic
antidepressant treatment also increased NPY and NPY
Y1-type receptor mRNA levels in certain brain regions [3].
The Fawn Hooded (FH) rat, a genetic animal model of
depression, showed low baseline hippocampal NPY immunoreactivity concentrations, and ECS increased NPY levels
in this animal [15]. NPY had antidepressant-like activity in a
forced-swimming test of rats and mice [15]. In a clinical
study, NPY levels were found to be decreased in
cerebrospinal fluid and platelet-poor plasma from depressed
patients [16]. Recent experiments using a MS paradigm in
rats also support a role for NPY in depression [7,8,9].
Jimene z-Vasquez et al. [9] reported that MS caused lower
hippocampal NPY levels in rats. Our study also showed that
MS for 7 days beginning on P14 reduced NPY expression in
area CA1 and the DG of the hippocampus. However,
acupuncture at acupoint HT7 increased NPY levels in the
same regions, demonstrating the antidepressant effect of
acupuncture in this model. Animals in the MS ST36 and
MS NA groups, however, showed no significant changes
as compared to the MS group, indicating that the changes
seen in the MS HT7 group were not related to the stress
induced by needling. Acupoint HT7 has been used to treat

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S. Lim et al. / Neuroscience Letters 343 (2003) 4952

mental disorders, while acupoint ST36 is often used to

regulate gastrointestinal function and relieve pain [17]. Our
results support the clinical use of acupoints in Oriental
medicine.
In this study, acupuncture treatment at acupoint HT7
increased body weight, locomotion, and NPY-positive cells
in area CA1 and the DG of rat pups subjected to MS. These
results suggest that acupuncture improves mood-related
disorders, such as depression induced by MS.

[11]

[12]
[13]

Acknowledgements

[14]

This research was supported by the Korean Research

Foundation (KRF-2002-003-E00189).

[15]

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