Neuroscience Letters 368 (2004) 7–10

Restraint stress affects hippocampal cell proliferation

differently in rats and mice
Megan J. Baina , Suzanne M. Dwyera , Benjamin Rusaka,b,∗
a Department of Psychology, Life Sciences Centre, Dalhousie University, Halifax, NS, Canada B3H 4J1
b Department of Psychiatry and Pharmacology, Lane Building, QEII HSC, Dalhousie University, Halifax, NS, Canada B3H 2E2

Received 8 January 2004; received in revised form 15 March 2004; accepted 27 April 2004

Abstract

Granule cell neurogenesis occurs in the dentate gyrus of the mammalian hippocampus throughout adult life, and incorporation of bromo-
deoxyuridine (BrdU) into DNA can serve as a marker of cell division associated with such neurogenesis. We examined the effects of a stressor
(3 h of restraint) on hippocampal cell proliferation in Sprague–Dawley rats and C57BL/6J mice. Animals were killed immediately following
restraint stress and their brains were prepared for immunohistochemical studies. Restraint stress caused similar significant increases in c-Fos
immunoreactivity among cells in the hypothalamic paraventricular nucleus of both species, indicating that the stress experienced was similar.
The restraint procedure also caused a significant decrease in BrdU labeling in the dentate gyrus of rats, as previously reported, but a significant
increase in the same region in mice. Hippocampal cell proliferation appears to respond differently to restraint stress in these species.
© 2004 Elsevier Ireland Ltd. All rights reserved.

Keywords: Bromodeoxyuridine; BrdU; Neurogenesis; Hippocampus; c-Fos; Paraventricular nucleus; Hypothalamus; Dentate gyrus; Acute stress.

Granule cell neurons are added throughout adulthood to
the dentate gyrus of the hippocampus (e.g., [1,7,8]). Stress-
induced activation of the hypothalamic–pituitary–adrenal
(HPA) axis has been reported to interfere with hippocampal
cell proliferation, giving rise to anatomical abnormali-
ties and pathological responses to stress [6,13]. In rats
and marmosets, a variety of stressful experiences reduce
cell proliferation in the dentate gyrus [5,8,18]. Blocking
corticosterone production prevents these effects [18],
while injections of corticosterone alone can inhibit cell
proliferation in the dentate gyrus [3].
Some acute stressors (predator odor or intruders) inhibited
granule cell proliferation in rats, tree shrews and marmosets
[7,8,18] while one study reported that acute restraint stress
did not affect cell proliferation in rats [17]. Studies of the
physiological consequences of stress in other species, such
as mice, have not assessed hippocampal cell proliferation
∗ Corresponding author. Tel.: +1 902 494 2159; fax: +1 902 494 6585.
E-mail address: rusak@dal.ca (B. Rusak).
[2,9,20]. Because of the interest in mice for genetic studies,
we compared the effects of an acute stressor (restraint) on
cell proliferation in the hippocampus of rats and mice.
To compare stress responses across species, it is valuable
to have an independent measure of the degree to which a
particular manipulation is stressful in these species. Stressful
events are known to increase levels of c-Fos immunoreactiv-
ity among cells in the paraventricular nucleus (PVN) of the
hypothalamus, presumably as part of the increased activity of
corticotrophin releasing factor (CRF) neurons that contribute
to the release of corticotrophin and ultimately of corticos-
terone [4,19,20]. We, therefore, assessed c-Fos levels in the
PVN after restraint stress in both species. In order to identify
currently dividing cells, we administered bromodeoxyuridine
(BrdU), a thymidine analogue that is incorporated into DNA
during the S phase of mitosis [7,15], and examined levels of
BrdU immunoreactivity in the dentate gyrus.
Male Sprague–Dawley rats and C57BL/6J mice (Charles
River, Montréal, Québec) were assigned to either Control
or Restraint treatment groups (11 rats and 18 mice in the

0304-3940/$ – see front matter © 2004 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.neulet.2004.04.096
8 M.J. Bain et al. / Neuroscience Letters 368 (2004) 7–10
Control groups; 12 rats and 19 mice in the Restraint groups).
Animals were housed individually in a 12 h:12 h light:dark
cycle with food and water available ad libitum (except during
restraint). BrdU was dissolved in 0.9% NaCl (15 mg/ml for
rats and 10 mg/ml for mice) and delivered via intraperitoneal
injections to rats (100 mg/kg [5]) and mice (75 mg/kg [14])
2 h after lights-on.
Following injection, animals in the Restraint group were
placed in 50 ml plastic tubes (mice) or in standard Plexi-
glass injection restrainers (17 cm × 4.5 cm × 6.5 cm; rats)
for 3 h. Both restraint devices had multiple air holes and al-
lowed animals to stretch their legs, but not to move within
the tubes. Control animals were left unhandled in their home
cages. Immediately after the restraint period, animals in both
the Restraint and Control groups were killed with an anes-
thetic overdose using sodium pentobarbital. Animals were
perfused transcardially with 0.9% NaCl, followed by cold 4%
paraformaldehyde in 0.1 M phosphate buffer, and brains were
prepared for immunohistochemical localization of BrdU and
c-Fos.
For BrdU immunohistochemistry [11,12], 40 ␮m brain
sections were cut through the hippocampus and were placed
in formamide solution (2 h at 65 ◦ C), rinsed in sodium cit-
rate, followed by 2N HCl (30 min at 37 ◦ C) and 0.1 M bo-
rate buffer. Sections were then rinsed in Tris-buffered saline
(TBS) and blocked in horse serum. Sections were incubated
in mouse anti-BrdU (1:10,000, Roche) and then processed us-
ing standard avidin–biotin processing kits (Vectastain) using
diaminobenzidine (DAB) as chromogen. For c-Fos labeling
[19], avidin–biotin/DAB procedures were similar to those
described above, but using goat blocking serum and rabbit
anti-Fos primary (1:20,000 Oncogene Science).
For each brain, BrdU-positive cells were counted through
ten sections of the dentate gyrus of the hippocampus (ev-
ery fourth section for mice, every eighth section for rats),
starting with the most rostral section in which the dentate
gyrus could be identified. The cells were identified by their
brown stain and were counted visually using an Olympus
BH-2 microscope at 20× objective magnification. Images of
c-Fos-labeled cells in the PVN were captured using a COHU
CCD camera on an Olympus BH-2 microscope at 20×. c-
Fos-labeled cells were counted using Scion Image (NIH) soft-
ware (Version 1.62C) and using templates based on the PVN
boundaries according to Paxinos and Watson [16]. Seven sec-
tions at the same levels of the PVN were counted from each
animal (every fourth section for mice, every eighth section
for rats). All brain sections were coded and counted by an ex-
perimenter blinded with respect to treatment condition. For
statistical analyses, group comparisons were made for each
species using parametric tests (t-tests) where possible or non-
parametric tests (Mann–Whitney U tests) where parametric
assumptions were not fulfilled.
Restraint stress affected BrdU labeling differently in rats
and mice (Fig. 1). In rats, restraint stress resulted in a sig-
nificant decrease in the number of BrdU-labeled cells in the
dentate gyrus compared to Control rats [t(21) = 3.41, P < 0.01
Fig. 1. (A and B) Acute restraint stress significantly reduced the number of
BrdU-labeled cells in the dentate gyrus of rats and increased BrdU-labeled
cells in the dentate gyrus of mice. (C and D) The same treatment significantly
increased numbers of c-Fos immunoreactive cells in the PVN of both rats
and mice. Values are means ± S.E.M. (∗ P < 0.01; ∗∗ P < 0.001).
means (±S.E.M.): 174.0 ± 19.4 cells for Control rats and
71.0 ± 23.2 cells for Restraint rats; see Fig. 2]. In contrast,
restraint stress in mice resulted in a significant increase in the
number of BrdU-labeled cells in the dentate gyrus [U = 267,
P < 0.01; 31.2 ± 12.9 cells in Control mice and 63.3 ± 10.3
cells in Restraint mice; see Fig. 2].
As a measure of whether mice and rats both experienced
the restraint period as stressful, we examined the number
of c-Fos immunoreactive cells in the PVN in each condi-
tion for both species (Fig. 1). The number of c-Fos-labeled
cells in the PVN of rats was increased significantly follow-
ing stress [t(21) = 6.3, P < 0.001; Control: 361.6 ± 45.8
cells and Restraint: 970.1 ± 84.2 cells; see Fig. 3]. Simi-
larly, restraint stress increased c-Fos immunoreactivity sig-
nificantly in the PVN of mice [t(35) = 7.7, P < 0.001; Control:
266.0 ± 62.8 cells and Restraint: 944.5 ± 62.0 cells; see
Fig. 3].
To address the possibility that changes in BrdU labeling
in the hippocampus might reflect non-specific effects of re-
straint stress on BrdU availability, the caudal subventricu-
lar zone of the lateral ventricles was also examined [12,18].
There were no significant changes in BrdU labeling in this
region in response to restraint stress in either species [rats: P
< 0.48; mice: P < 0.18].
Rats showed decreased hippocampal cell proliferation in
response to stress, as has been reported previously for some
stressors [5,8,18]. A recent study, however, did not find a
similar decrease in BrdU labeling in rat hippocampus after a
single restraint experience [17]. Among other methodolog-
ical differences, BrdU was administered in that study after,
 M.J. Bain et al. / Neuroscience Letters 368 (2004) 7–10 9

Fig. 2. BrdU-labeled cells shown in representative coronal sections through the dentate gyrus of the hippocampus of rats (top row) and mice (bottom row) in
the Control (A and C) and Restraint stress (B and D) groups (scale bar = 0.1 mm).

Fig. 3. c-Fos immunoreactive cells shown in representative coronal sections through the paraventricular nucleus of rats (top row) and mice (bottom row) from
Control groups (A and C) and Restraint stress groups (B and D) (scale bar = 0.1 mm).
10 M.J. Bain et al. / Neuroscience Letters 368 (2004) 7–10
rather than before, the restraint procedure, which might have
contributed to the different results.
Unlike rats, mice showed a significant increase in cell pro-
liferation following the restraint treatment, even though the
treatment increased PVN c-Fos levels similarly in the two
species, as would be predicted by increased CRF and corti-
costerone in both species after restraint [2,9]. Despite the sim-
ilarities in responses of the PVN to restraint stress in rats and
mice, there are also documented differences in the HPA axis
response to stress. Thus, CRF receptor-1 mRNA was induced
in the PVN of rats, but not mice, following restraint stress ex-
posure, although c-fos mRNA levels were increased in both
species [9]. There may also be more general species differ-
ences in hippocampal plasticity, as suggested by evidence of
shrinkage of the molecular layer of the dentate gyrus in re-
sponse to entorhinal cortex damage in rats that was absent in
mice [10].
An apparent difference between species in baseline lev-
els of BrdU incorporation (see Fig. 1, Controls) will need to
be assessed further using a range of BrdU doses. Neverthe-
less, differences in BrdU availability cannot account for the
opposite effects of the restraint procedure in rats and mice.
This study demonstrated species differences in apparent cell
proliferation in response to stress; future studies will eval-
uate the longer term fate of the BrdU-labeled cells in both
species.
Acknowledgments
We are grateful to Donna Goguen, Debbie Fice, Karthika
Devarajan and Marc Goguen for their excellent technical
assistance. Supported by a grant from NSERC of Canada
(A0305) and fellowships from the Nova Scotia Health Re-
search Foundation and the Canadian Institutes of Health Re-
search/Wyeth.
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