Neuropeptides

Neuropeptides 37 (2003) 149–156

www.elsevier.com/locate/npep

Long-term effects of short and long periods of maternal separation

on brain opioid peptide levels in male Wistar rats q
Karolina Ploj, Erika Roman, Ingrid Nylander *

Department of Pharmaceutical Biosciences, Division of Pharmacology, Box 591, Uppsala University, SE-751 24 Uppsala, Sweden
Received 2 January 2003; accepted 19 April 2003

Abstract

Environmental manipulations early in life may induce persistent alterations in adult behaviour and physiology. The underlying
neural mechanisms of these responses are not yet clear. We have previously reported long-term changes in brain opioid peptide
levels in male and female Sprague–Dawley rats after short periods (15 min, known as neonatal handling) of maternal separation
(MS) until weaning. To study this further, we investigated behavioural and neurochemical effects of repeated MS in male Wistar
rats. The rat pups were separated from their dams in litters for either 360 min (MS360) or 15 min (MS15) daily from postnatal day 1
to 21 or exposed to normal animal facility rearing. Behavioural analysis showed that MS360 rats had increased ultrasonic calls on
postnatal day 5 compared to MS15 rats, but not on postnatal day 6. Moreover, the MS360 rats had more animals with higher
frequency of calls at day 5 than 6 than the MS15 rats. Analysis of the opioid peptides dynorphin B and Met-enkephalin-Arg6 Phe7
with radioimmunoassay 7 weeks after the MS procedure, revealed long-term neurochemical changes in several brain areas and in the
pituitary gland. Immunoreactive dynorphin B and Met-enkephalin-Arg6 Phe7 levels were affected in the hypothalamus and dy-
norphin B levels in the neurointermediate pituitary lobe, amygdala, substantia nigra and the periaqueductal gray. Together, these
findings show that repeated periods of MS early in life in male Wistar rats affect the development of the ultrasonic call response and
induce long-lasting and possibly permanent alterations in the opioid peptide systems.
Ó 2003 Elsevier Science Ltd. All rights reserved.

1. Introduction mechanisms underlying these long-lasting changes in

Adverse experiences early in life may imply effects with
consequences for the normal development of physiolog-
ical and mental functions (Nemeroff, 1998; Willner,
1990). It has been suggested that stressful events during
certain neonatal periods may permanently influence
stress-responsive neural networks (Anand et al., 1999;
Heim et al., 1997) and thereby increase the vulnerability
of an individual to develop psychopathology and/or drug
dependence later in life. Accordingly, there is evidence
that people with adverse early life experiences may have
an increased risk of developing alcohol dependence
(Cadoret et al., 1985; Kendler et al., 2000; Moncrieff
et al., 1996; Spak et al., 2001). The neurochemical
q
This study was supported by the Alcohol Research Council of the
Swedish Alcohol Retailing Monopoly (98/21) and the Swedish Medical
Research Council (K00-14X-12588).
*
Corresponding author. Tel.: +46-18-4714163; fax: +46-18-4714847.
E-mail address: ingrid.nylander@farmbio.uu.se (I. Nylander).
behaviour are not completely understood. Systematic
and controlled animal studies are important in order to
find out the neurobiological basis of how early adverse
stimuli may have developmental effects that may imply a
risk for personality disorders. This means manipulation
of the environment of the individual during its postnatal
development followed in adulthood by behavioural and
neurobiological screening. Several studies have described
the effects of maternal separation (MS) on adult physi-
ology and behaviour. The duration of separation is cru-
cial for the outcome. Thus, prolonged periods (>1 h) of
MS during the first weeks of life result in animals with
behavioural and neuroendocrine signs of elevated stress
reactivity as adults. However, contrary findings have
been reported also (Lehmann and Feldon, 2000). Rat
pups separated from their dam for a short period (3–20
min) during the neonatal period, known as neonatal
handling, has been found to decrease anxiety-like be-
haviours and biological stress responsivity compared to
non-separated rats (Anisman et al., 1998; Hall, 1998;

0143-4179/03/$ - see front matter Ó 2003 Elsevier Science Ltd. All rights reserved.
doi:10.1016/S0143-4179(03)00043-X
150 K. Ploj et al. / Neuropeptides 37 (2003) 149–156
Heim and Nemeroff, 2001; Lehmann and Feldon, 2000;
Meaney et al., 1996; Newport et al., 2002).
The endogenous opioid peptides act at the l, d and j
receptors to mediate a variety of physiological effects,
such as pain modulation (Terenius, 1992), motivation,
reward (Nylander and Silberring, 1998) and neuroen-
docrine secretion (Pfeiffer and Herz, 1984) in the central
and peripheral nervous system. Numerous studies
support an important contribution of endogenous opi-
oid peptide systems in the mediation, modulation and
regulation of stress responses including endocrine, au-
tonomic nervous system and behavioural responses
(Calcagnetti and Holtzman, 1992; Dijkstra et al., 1992;
Drolet et al., 2001; Vanderah et al., 1992; Vander-
schuren et al., 1995; Watkins et al., 1992). We have
recently shown that daily MS for 15 min (MS15) or
360 min (MS360) from postnatal day (PND) 1 to 21
can affect voluntary ethanol intake in adult male Wistar
and ethanol preferring AA (Alko, Alcohol) rats (Ploj,
2002; Roman et al., 2003). In addition, we have pre-
viously reported long-term changes in opioid prody-
norphin- and proenkephalin-derived peptides in male
and female Sprague–Dawley rats after short periods of
MS until weaning (Ploj et al., 1999; Ploj et al., 2001).
The aim of this study was to determine if MS15 or
MS360 from PND 1 to 21 could induce long-term
behavioural and neurochemical effects on these opioid
peptide systems in the pituitary gland and the brain in
male Wistar rats. The opioid peptides dynorphin B
(DYNB) and Met-enkephalin-Arg6 Phe7 (MEAP),
markers of prodynorphin and proenkephalin, respec-
tively, were measured using specific radioimmunoassays
(RIAs).
2. Materials and methods
2.1. Animals
Time-mated pregnant Wistar rats were obtained from
Scanbur BK AB, Sweden for arrival at our animal de-
partment on gestation day 15–19. Upon arrival, the
dams were singly housed and maintained on food and
water ad libitum. All animals were housed in a tem-
perature (22
1 °C) and humidity (50
10%) controlled
animal room on a 12:12 light–dark cycle (light on at
6:00 h). Behavioural tests were run during the light pe-
riod. All animal experiments were performed under an
approved protocol in accordance with the Swedish
Animal Protection Legislation.
2.2. Maternal separation treatment
The litters were sexed, culled and cross-fostered from
45 time-mated Wistar rats to 11 pups per litter on day 0
(nine males, two females; day of birth ¼ day 0), although
in a small number of litters this was not possible. The
pups were randomly assigned to one of two treatments,
15 min (MS15) or 360 min (MS360) of MS from PND
1–21, or no treatment (animal facility reared ¼ AFR).
Seven litters were used for the MS15 treatment, six lit-
ters for MS360 and nine litters served as AFR rats.
Three of the AFR litters were assigned to establish the
weight measurements and ultrasonic vocalisation and
were not included in the neurochemical analysis. The
remaining AFR rats were not disturbed until weaning,
except for cage changes. The litters were weighed on
PND 6 (n ¼ 16), 14 (n ¼ 16) and 22 (n ¼ 22). All nine
AFR litters were weighed on PND 22. Cages were
changed, with old bedding material mixed with clean
bedding material, once a week during the separation
period for AFR litters as well as for MS15 and MS360
litters. The separation occurred once a day for the first
three postnatal weeks. First the dam and then the whole
litter were removed from the nest. Each litter was placed
in macrolon cages (26  20  14 cm) containing wood
chip bedding material and moved to an adjacent room
(25 °C). In the MS360 group the dams were kept in their
home cages during the separation period but taken out
prior to the return of the litters. After 15 or 360 min,
first the pups and then the dam were returned to their
home cage. Separation sessions were always performed
in the same room and by the same two persons who also
changed the cages and were the only people with per-
mission to enter the animal room during the experiment.
The separation treatment occurred every day between
09:00 and 09:15 h for the MS15 group and between
09:00 and 15:00 h for the MS360 group. On PND 22, the
animals were weaned and thereafter housed in same-
treatment groups of three to five. Only male rats were
used for the behavioural and neurochemical analysis.
From a total number of 234 rats, 30 rats (10 rats/group)
were randomly selected from the different treatment
groups 7 weeks after weaning, weighed and then sacri-
ficed by decapitation and used for analysis of immuno-
reactive (ir) peptide levels. Each rat was singly housed
30 min before decapitation in order to control for con-
ditions regarding the decapitation. To avoid the use of
related subjects, not more than two subjects derived
from the same dam were used. The remaining rats were
used in other studies on behavioural and neurochemi-
cal effects of MS. The treatment schedule is shown in
Fig. 1.
2.3. Ultrasonic call test
On PND 5 and 6, two to four pups/litter were ran-
domly selected from the MS15 (n ¼ 12), MS360 (n ¼ 12)
and the AFR (n ¼ 12) litters and were transferred to an
adjacent room with the same conditions as the animal
room. The rat pups were placed singly in a circular re-
cording chamber, made of aluminium with a diameter of
 K. Ploj et al. / Neuropeptides 37 (2003) 149–156
Fig. 1. Treatment schedule. Abbreviations: PND, postnatal day; MS15, maternal separation for 15 min during postnatal day 1–21; MS360, maternal
separation for 360 min during postnatal day 1–21; AFR, animal facility reared; and ir, immunoreactive.
17 cm, kept at room temperature (22
1 °C). After 1
min of adaptation, the number of ultrasonic calls emit-
ted during 1 min was recorded. A bat detector (Petter-
son Elektronik AB, Uppsala, Sweden) linked to an
electronic counting device (developed by Department of
Medical Pharmacology, Uppsala University, Uppsala,
Sweden) was used. The recording chamber was wiped
clean with water after each test. The test was performed
before the separation treatment in the morning, to avoid
the acute effect of the separation treatment. The ultra-
sonic calls were measured two times in order to inves-
tigate if there would be any changes in ultrasonic calls
between PND 5 and 6.
2.4. Serum corticosterone assays
Trunk blood was collected after decapitation for se-
rum corticosterone assay. The blood samples were
stored at room temperature for 20–30 min and then
centrifuged at 1500g for 10 min. The serum was then
collected and stored at )20 °C until analysis. The cor-
ticosterone analysis was performed using the commer-
cial ImmunChemTM Double Antibody Corticosterone
125
I RIA kit for rats and mice (ICN Biomedicals, Costa
Mesa, CA, USA), which uses a highly specific cortico-
sterone antiserum. The samples were analysed in ac-
cordance with the protocol in the RIA kit. All samples
were assayed in duplicates and in the same assay in
order to avoid inter-assay variations.
2.5. Radioimmunosassays
Rats from different litters from the MS15 (n ¼ 10),
MS360 (n ¼ 10) and AFR (n ¼ 10) groups were used in
the analysis that occurred 7 weeks after the separation
procedure. All dissected tissues were immediately fro-
zen on dry ice and stored at )80 °C until peptide ex-
traction. Tissue extraction was performed with 1 M
acetic acid. The samples were heated at 95 °C for 5 min
and, after cooling on ice, homogenised using sonication.
The samples were reheated at 95 °C for 5 min, cooled on
ice and then centrifuged for 15 min at 12,000g. The tissue
extracts were then subjected to a purification step using
151
an ion exchange procedure (Christensson-Nylander et al.,
1985) before RIA. The peptides were analysed using
specific RIAs for DYNB and MEAP described in detail
elsewhere (Ploj et al., 2000). The DYNB and MEAP
tracer peptides were labelled using chloramine T and
purified by high-performance liquid chromatography.
Samples subjected to MEAP assay were oxidised prior to
the RIA procedure. The samples, dissolved in methanol:
0.1 M hydrochloric acid (1:1), were incubated with an-
tiserum and 125 I-labelled peptide for 24 h at 4 °C. The
MEAP (90:3D II) and DYNB (113+) antiserum was di-
luted 1:180,000 and 1:562,500, respectively, in gelatine
buffer. Following the incubation period, the DYNB
samples were incubated for 1 h with a sheep–antirabbit
antiserum (Pharmacia Decanting Suspension; Pharmacia
Diagnostics, Uppsala, Sweden). After centrifugation
(10 min) the pellet was counted in a gamma counter. To
separate free and antibody-bound peptide in the MEAP
assays, a charcoal suspension was added and the samples
were incubated for 10 min. The samples were then cen-
trifuged (2 min) and an aliquot (300 ll) of the superna-
tant was counted in the gamma counter. The DYNB
antiserum did not show cross-reactivity with either
DYNA (1–17) or DYNA (1–8). Cross reactivity with
DYNB 29 was 1% and with big dynorphin (DYN 32)
100%. Other opioid peptides did not cross-react with the
DYNB antiserum. Cross-reactivity with MEAP antise-
rum and Met-enkephalin, Met-enkephalin-Arg6 , Met-
enkephalin-Arg6 Gly7 Leu8 , Leu-enkephalin or DYNA
(1–6) was less than 0.1%.
2.6. Statistical analysis
For comparisons of mean ir peptide levels and body
weight the parametric one-way ANOVA combined, if
significant, with FisherÕs post hoc test was used, whereas
repeated measures ANOVA was used for comparisons
of the mean body weight gain during the MS treatment.
Behavioural data and values not within normal distri-
bution, that is, general treatment effect on numbers of
ultrasonic calls on PND 5 and 6 and the serum corti-
costerone levels, were also analysed with the non-para-
metric Kruskal–Wallis test. When a significant difference
was found, the data were analysed further with pairwise
152 K. Ploj et al. / Neuropeptides 37 (2003) 149–156
comparisons by using the Mann–Whitney U test. The v2
test was used to determine whether there was a signifi-
cant effect within and between the groups on the change
of ultrasonic calls from PND 5 to 6. Statistical analyses
were made using the Statview 4.5 computer program.
Differences were considered statistically significant at
p < 0:05.
3. Results
3.1. Ultrasonic call test
Figs. 2A–B illustrate the mean
SEM numbers of
ultrasonic calls during 1 min on PND 5 and 6 (n ¼ 12/
group). The corresponding median
MAD (Median
Absolute Deviation) values were: PND 5: AFR 121

39, MS15 89
34, MS360 150
37 and PND 6: AFR
108
41, MS15 102
38, MS360 122
26. The num-
bers of ultrasonic calls in all animals were between eight
and 242 calls/min. On PND 5, MS360 rats had an in-
creased number of ultrasonic calls compared to MS15
rats, whereas there were no significant differences in
response between MS360 and AFR rats or MS15 and
AFR rats. No statistically significant treatment effects
were seen on PND 6. A detailed analyses of the change
of ultrasonic calls from PND 5 to 6 revealed no statis-
tically significant difference within groups over days but
an obvious difference between groups as to number of
animals with higher frequency of calls PND 5 than PND
Fig. 2. (A)–(B) The mean
SEM number of ultrasonic calls during 1
min on postnatal day 5 (A) and 6 (B) in the MS360, MS15, and AFR
rats (n ¼ 12/group). #p < 0:05 compared to MS360 rats (one-way
Kurskal–Wallis and Mann–Whitney U test).
6 (p < 0:05). Seventy-five % of MS360 rats, 25% of
MS15 rats and 50% of AFR rats had higher frequency
of calls PND 5 than PND 6.
3.2. Body weight
The mean
SEM rat body weight in the MS15, MS360
and AFR litters on PND 6 (n ¼ 16), 14 (n ¼ 16) and 22
(n ¼ 22) is shown in Fig. 3. No significant treatment ef-
fects were seen on body weight or body weight gain during
the separation period. Each rat (n ¼ 10/group) that was
used for evaluation of the neurochemical effects of MS
was weighed before decapitation, at 10 weeks of age (266–
354 g). No statistically significant differences on body
weight were seen at the time of decapitation.
3.3. Serum corticosterone levels
The median
MAD serum corticosterone levels
(n ¼ 30) 7 weeks after MS were 181.1
100.7 (MS15),
111.1
47.1 (MS360) and 151.5
89.6 (AFR) ng/ml. No
statistically significant differences in serum levels of
corticosterone were seen between the groups at the time
of decapitation.
3.4. Ir opioid peptide levels
The mean
SEM ir opioid peptide levels in various
brain areas 7 weeks after MS are shown in Table 1.
MS360 and MS15 rats had higher ir DYNB levels in the
neurointermediate pituitary lobe (NIL) and MS15 rats
had higher ir DYNB levels in the hypothalamus com-
pared to those of the AFR rats. In the hypothalamus,
higher ir MEAP levels were found in the MS15 rats
Fig. 3. The mean
SEM rat body weight (g) in the MS15, MS360, and
AFR litters on postnatal day (PND) 6 (n ¼ 16), 14 (n ¼ 16) and 22
(n ¼ 22).
 K. Ploj et al. / Neuropeptides 37 (2003) 149–156 153

Table 1
Dynorphin B (DYNB) and Met-enkephalin-Arg6 Phe7 (MEAP) immunoreactive levels in various brain areas and in the pituitary gland 7 weeks after
15 min (MS15) or 360 min (MS360) of maternal separation daily during postnatal day 1–21 and in animal facility reared (AFR) rats (n ¼ 7–10/group)
Region DYNB MEAP
MS15 MS360 AFR MS15 MS360 AFR
Anterior pituitary lobe 9.43
1.05 10.41
1.56 7.95
1.22 1.10
0.24 1.02
0.19 1.31
0.29
Neurointermediate pituitary 155.71
16.82 135.03
11.10 96.10
10.33 n.d. n.d. n.d.
lobe
Hypothalamus 22.09
2.06 18.60
1.04 14.42
0.99 64.20
4.67### 43.39
1.78 44.07
2.65
Frontal cortex 2.00
0.27 1.77
0.26 1.43
0.32 4.27
0.44 4.14
0.39 4.29
0.50
Medial prefrontal cortex 1.47
0.17 1.54
0.26 1.06
0.08 3.31
0.27 4.15
0.48 3.37
0.34
Nucleus accumbens 38.12
4.20 35.45
2.39 30.88
2.78 43.99
5.72 49.39
6.58 35.61
3.70
Striatum 12.54
0.49 12.32
0.67 11.05
0.73 37.68
3.29 33.60
2.46 28.83
2.52
Hippocampus 13.44
0.75 13.14
0.81 13.17
0.68 11.71
0.36 10.93
0.73 10.03
0.66
Amygdala 2.82
0.34## 4.41
0.35 3.88
0.49 10.54
1.17 11.84
2.18 10.00
1.28
Substantia nigra 88.97
7.72### 57.01
4.34 55.79
5.11 5.86
0.59 3.74
0.44 5.65
0.90
Ventral tegmental area 1.67
0.18 2.07
0.36 2.04
0.30 8.74
1.15 6.17
0.69 8.86
1.67
Periaqueductal gray 7.52
1.17## 11.84
0.89 5.81
1.03 27.53
3.89 29.35
3.85 27.78
3.54
The values represent means
SEM and are expressed as fmol/mg tissue. ## p < 0:01, ### p < 0:001 in comparison with MS360 rats,  p < 0:05,

p < 0:01 and  p < 0:001 in comparison with AFR rats (one-way ANOVA, FisherÕs post hoc test). n.d., not detectable.

compared to both MS360 and AFR rats. In the sub-
stantia nigra, MS15 rats had higher ir DYNB levels
compared to those of the MS360 and AFR rats. Lower
ir DYNB levels in the amygdala were detected in the
MS15 rats compared to MS360 rats. MS360 rats had
higher ir DYNB levels in the periaqueductal gray (PAG)
compared to those of the MS15 and AFR rats. No
statistically significant differences in ir opioid peptide
levels were seen in other regions analysed.
4. Discussion
A significant environmental factor during the early
development of mammals is the interaction between the
infant and its mother. Ultrasonic calls play an important
communicative role in mother-offspring interaction
since they elicit in the dam a prompt response con-
cerning caregiving behaviours (Smotherman et al.,
1974). Infant rats typically respond to separation from
their dams and littermates with ultrasonic calls, as a
reaction to decreased body temperature, novel and
stressful environment (Allin and Banks, 1971; Hofer and
Shair, 1978; Noirot, 1972). Rates of ultrasonic calls are
an established measure of neonatal anxiety (Carden
et al., 1991; Hofer, 1996; Hofer and Shair, 1978; Kehoe
and Blass, 1986; Takahashi, 1994). The development of
rat pup ultrasonic calls displays a characteristic time-
course that may reflect stages of mental development.
The calls start around PND 2–3, reach a plateau level
during PND 4–6 and then gradually decrease (Data
from a thesis summary by Johansson-Wallsten 1993,
published in Acta Universitatis Upsaliensis compre-
hensive summaries of Uppsala dissertations from the
Faculty of Medicine). We expected the frequency of calls
to be the same at PND 5 and PND 6, that is the time for
the mentioned plateau level. This was true for the AFR
rats. However, the MS360 rats had more animals with
higher frequency of calls at PND 5 than 6 than the
MS15 rats indicating a difference in anxiety-like behav-
iour and also in the time course of the development of
the ultrasonic call response.
Information regarding neural mediators of MS re-
sponses is still rudimentary. Interpretation of results
from previous studies is complicated by a large variation
in the experimental protocols used. In the present study,
we have used a protocol established to examine long-
term effects of MS on neurochemistry and behaviour.
The animals are, for instance, subjected to MS the entire
period prior to weaning and the pups are separated in
litters to avoid variability due to time period of sepa-
ration and isolation stress. The pregnant females were
shipped during gestation, a factor that may affect the
outcome of later postnatal separation. However, the
results presented herein are comparable with other
studies using the same protocol. The hypothalamic–
pituitary–adrenal (HPA) axis is clearly affected by short
periods of MS until weaning (neonatal handling), as
evidenced by a reduced response to stressors later in life,
whereas in animals exposed to longer periods of MS
both hyper- and hypo-responsiveness have been re-
ported (Lehmann and Feldon, 2000). Except from the
glucocorticoid system, MS produces alterations in other
neurotransmitter systems, such as the dopamine, c-am-
inobutyric acid, serotonin, norepinepherine, neuropep-
tide Y and the nociceptin/orphanin FQ system in
rodents (Meaney et al., 2002; Newport et al., 2002; Ploj
et al., 2002). The involvement of the endogenous opioid
system in the MS responses has been implicated by a
number of studies (Bartolome et al., 1991; DÕAmore
et al., 1993; Irazusta et al., 1999; Kehoe and Blass, 1986;
Kalinichev et al., 2001; Wang et al., 1996). Opioid
154 K. Ploj et al. / Neuropeptides 37 (2003) 149–156
peptides have been suggested to play a role in develop-
ment, and therefore postnatal manipulations may
disturb normal behavioural and physiological develop-
ment. Peptides from the opioid peptide family are
present in the fetus and generally reach peak levels at the
third postnatal week (McDowell and Kitchen, 1987).
Although both peptides and receptors are present at
PND 1–21, MS during this period will most probably
interfere with continued development like proliferation
of terminals and synaptic contacts. We have shown that
MS induces long-term changes in the opioid system (Ploj
et al., 1999; Ploj et al., 2001) in areas that regulate the
activity of the HPA-axis (Calogero et al., 1996; Iyengar
et al., 1986; Plotsky, 1986). Male Sprague–Dawley
MS15 rats have higher ir DYN levels in the pituitary
gland and the hypothalamus compared to AFR rats
(Ploj et al., 1999), whereas these structures are un-
affected by MS15 in female Sprague–Dawley rats (Ploj
et al., 2001). This suggests a gender specific involvement
of opioids in the HPA-axis response to stress in Spra-
gue–Dawley rats. The present study shows that MS15
induced similar effects in male Wistar rats in the pitui-
tary gland and the hypothalamus as in male Sprague–
Dawley rats. In addition to MS15 rats, MS360 rats had
elevated DYNB ir levels in the NIL compared to AFR
rats. Given the known endocrine and behavioural profile
of short and long periods of MS, although as mentioned
above contrary findings have been reported after pro-
longed periods of MS, the increased DYNB ir levels in
both MS15 and MS360 rats was unexpected. Long-term
effects of prolonged periods of MS on DYN peptides
have to our knowledge not previously been reported and
further evaluation of the effects of MS on the pituitary
DYNB system are required prior to further speculation.
In addition to DYNB, elevated MEAP levels were found
in the hypothalamus in MS15 rats compared to MS360
and AFR rats. Altogether, these results provide further
evidence that the opioid system may play a role in the
altered HPA-axis responsiveness after MS.
There is evidence that MS induces changes not only
in areas directly related to the HPA-axis, but also in
other areas involved in the co-ordination and integra-
tion of sensory information. The amygdala has been
implicated in a variety of emotional/cognitive functions
ranging from fear and orienting responses, defensive and
aversive reactions and associative conditioning (Davis,
1992). Alterations in the opioid (Ploj et al., 2001),
corticotropin-releasing factor (Hunt et al., 2000) and
benzodiazepine (Caldji et al., 2000) systems in the
amygdala, in response to short (15 min) and/or long
periods (180 min) of MS, have been reported. Here we
report that male MS15 Wistar rats, like female Sprague–
Dawley rats (Ploj et al., 2001), had lower ir DYNB levels
in the amygdala, compared to MS360 and AFR rats.
Taken together these results show that several neuro-
transmission systems within the amygdala are involved
in the processes affected by environmental manipula-
tions early in life, such as MS. Opioid peptide levels were
also affected in the PAG, which receives projections
from the amygdala and, in turn, has reciprocal con-
nections with the amygdala (Davis, 1992). The MS360
rats had higher ir DYNB levels compared to MS15 and
AFR rats whereas ir DYNB levels in the MS15 rats were
unaffected compared to AFR rats, in contrast to previ-
ous findings in female rats (Ploj et al., 2001). Compared
to MS360 and AFR rats, MS15 rats also had higher ir
DYNB levels in the substantia nigra, a brain region
involved in locomotor behaviour and neuronal integra-
tion of motivational processes into behavioural output
(Angulo and McEwen, 1994). Dynorphins are present in
a striato-nigral pathway and we have previously re-
ported elevated ir DYNB levels in the striatum, where
dynorphinergic cells are abundant, in MS15 Sprague–
Dawley male rats, (Ploj et al., 1999) a finding not rep-
licated in this study using Wistar rats.
Taken together, these results demonstrate that MS15
and MS360 induce long-term changes in ir tissue levels
of opioid peptides, especially DYNB in the brain and
the pituitary gland. Discrepancies were found in certain
brain areas when comparing with previous results from
studies on MS15-induced effects on opioid peptides.
This gives further evidence that the outcome of MS is
dependent on the experimental set-up, gender and rat
strain. Of particular interest in this study was the
changes in the DYNB system. A dysregulation in this
system may contribute to differences in emotionality
between rats after short and long periods of MS and
may also affect voluntary drug intake, as described in
previous studies (Nylander et al., 1994; Nylander et al.,
1995; Ploj, 2002; Roman et al., 2003).
Acknowledgements
The authors thank Professor Bengt J. Meyerson for
valuable discussions and guidance in the behavioural
studies.
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