SUPPLEMENT DOCUMENT
Predictable Chronic Mild Stress Improves Mood, Hippocampal Neurogenesis and Memory
V. K. Parihar1,2, B. Hattiangady1,2, R. Kuruba1,2, B. Shuai1,2, and A. K. Shetty1,2*
1
Medical Research & Surgery Services, Veterans Affairs Medical Center, Durham, North Carolina
27705. 2Department of Surgery (Division of Neurosurgery), Duke University Medical Center,
Durham NC 27710.
CONTENTS
A. Pertaining to Materials and Methods:
1. Supplemental figure 1
2. Analyses of depressive-like behavior using Forced swim test (FST).
3. Characterization of anxiety-like behavior using elevated plus maze (EPM) test
4. Quantification of the numbers of newly born BrdU+ cells and DCX+ neurons in the
hippocampus
5. Analyses of the neuronal fate-choice decision of newly born cells
6. Analysis of the dendritic growth of DCX+ newly born neurons
7. Characterization of learning and memory function using the water maze test (WMT)
8. Analyses of memory function using novel object recognition test (NORT)
B. Pertaining to Results:
1. Neurodegeneration or neuroinflammation after predictable chronic mild stress (PCMS)
2. Supplemental Figure 2
3. Supplemental Figure 3
4. Dendritic growth as analyzed by concentric circle analyses of Sholl
5. Supplemental Figure 4
C. References
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A. Pertaining to Materials and Methods:
1. Supplemental Figure 1
2
Legend: Schematic representation of the experimental design. Two groups of male Sprague-Dawley
rats were subjected to 5 minutes of restraint stress for 28 days (predictable chronic mild stress
[PCMS] groups). Two additional groups of age-matched Sprague-Dawley rats were handled
similarly but not exposed to restrain stress (control groups). In one set of PCMS and control rats
(n=6/group; Experiment #1), depressive and anxiety-like behaviors were analyzed on 29th day using
the forced swim test (FST) and elevated plus maze (EPM) test. Following this, these rats received
four i.p injections of 5’-bromodeoxyuridine (BrdU; 50mg/kg) over 18 hrs (one injection every 6
hrs) and were killed at 6 hours after the last BrdU injection. Tissues were processed for BrdU and
DCX single immunostaining, and BrdU-DCX dual immunofluorescence and confocal microscopic
analyses. The overall production of cells per day, percentages of new cells that differentiate into
neurons and net neurogenesis were quantified for subgranular zone and granule cell layer (SGZ &
GCL) using stereological counting of BrdU+ cells and extrapolation of the percentages of BrdU+
cells expressing DCX with numbers of BrdU+ cells in both groups. Additionally, the overall status
of hippocampal neurogenesis was ascertained through quantification of the total number of DCX+
neurons in the SGZ & GCL. In a second set of PCMS and control rats (n=6/group; Experiment #2),
learning and memory function were analyzed at 1.5 months after PCMS/handling regimen using the
water maze test (WMT) and novel object recognition test (NORT). To examine the persistence of
antidepressant and anxiolytic effects of PCMS at prolonged periods after PCMS, these rats used of
WMT and NORT were also examined at 2 months after PCMS for depressive and anxiety-like
behaviors using FST and EPM test.
2. Analyses of depressive-like behavior using Forced swim test (FST). One day after the
completion of 28-day PCMS or handling regimen, each rat was first placed in an acrylic glass
cylinder (having an inner diameter of 18.7 cm and depth of 42 cm) filled with tap water (~25°C) to
a depth of 30 cm. The depth of water used ensured that the animal could not touch the bottom of the
container with their hind paws or tail. The FST was conducted in a single session comprising 10
minutes and data were collected every minute for swimming, climbing (or struggling) and
immobility (or floating) during the procedure by two independent observers who are blind to animal
groups. Swimming in the FST is defined as the horizontal movement of the animal in the swim
chamber and climbing refers to the vertically directed movement with forepaws mostly above the
water along the wall of the swim chamber. On the other hand, immobility or floating is defined as
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the minimum movement necessary to keep the head above the water level. Rats were removed
from the water at the end of 10 minutes and gently dried and placed back in their home cages. From
the collected data, the total time spent in immobility for the trial duration was calculated for every
rat and utilized as an index of depressive-like behavior.
3. Characterization of anxiety-like behavior using elevated plus maze (EPM) test. The EPM
comprised of a fiber glass cross with four elevated arms (50 cm from the floor, 50 cm long and 10
cm wide) with two opposing arms closed with 40 cm high walls (i.e. closed arms). This facilitated
the exploration of rats in 5 different zones: two open arm zones, two closed arm zones, and a central
zone (measuring 10 X 10 cm area) where the arms cross over. Two days after the 28-day PCMS or
handling regimen (and one day after the FST), each rat was first placed in the central zone of the
EPM with the head facing towards the closed arm. The rat was allowed to freely explore the maze
for 5 minutes and the entire test was done under dim light conditions. Data were collected by two
independent observers who are blind to animal groups and sat quietly at a distance of 2.5 meters
from the maze. The frequency of entries and the amount of time spent in each arm was recorded.
An entry to each zone was considered when the rat had all four paws in the zone. At the end of 5
minutes, the rat was removed from EPM and placed back in its home cage. The EPM apparatus was
thoroughly cleaned with 70% alcohol before each trial to avoid any influence of odor related clues
from the previous session. The EPM test is typically used to determine the rodent’s unconditioned
response to a potentially dangerous environment. Anxiety-related behavior is measured by the
degree to which the rodent avoids the unenclosed (or the open) arms of the maze. It is well known
that rats exhibiting increased anxiety-like behavior spend more time in the closed arms and less
time in the open arms. Therefore, the number of open arm entries and duration of time spent in open
arms for the duration of the test were used for calculation of the extent of anxiety-like behavior in
each rat.
4. Quantification of the numbers of newly born BrdU+ cells and DCX+ neurons in the
hippocampus. The counting of cells utilized the StereoInvestigator system from Microbrightfield,
which comprised a color digital video camera (Optronics Inc.,) interfaced with a Nikon E600
microscope (Nikon Instruments). The detailed optical fractionator counting procedure used in this
study is described in our earlier reports.1-3 In each animal (n=6 per group), BrdU+/DCX+ cells were
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counted from 37 (in sections through the anterior most parts of the hippocampus) to 563 (in sections
through posterior parts of the hippocampus) randomly and systematically selected frames (each
measuring 40 × 40μm, 0.0016 mm2 area) in every 15th section through the entire hippocampus using
the 100X oil immersion lens. The Gundersen coefficient error (CE) was in the range of 0.05-0.10
for all counts in this study.
5. Analyses of the neuronal fate-choice decision of newly born cells. The sections were first
processed for DCX staining using a goat polyclonal primary antibody (1:250; Santa Cruz
Biotechnology). After an overnight incubation, the sections were treated with biotinylated anti-goat
IgG (1:200; Vector labs) and subsequently visualized using streptavidin Alexa Fluor 594 (1: 200)
which gave red color to the cytoplasm in both soma and dendrites of DCX+ neurons under the
fluorescence microscope. Following this, the sections were processed for BrdU
immunofluorescence using a rat anti-BrdU (1: 300; SeroTech) and visualized using goat anti-rat
Alexa Flour 488 (1:200; InVitrogen) which gave green color to BrdU-positive nuclei under the
fluorescence microscope. The sections were mounted using slowfade-antifade mounting medium
and subsequently analyzed using laser scanning confocal microscope (LSM-510, Carl Zeiss). The
Z-stacks taken at 1µm intervals of double labeled cells (i.e. cells positive for both BrdU and DCX)
were analyzed using LSM-510 image explorer program and the percentages of BrdU+ cells
expressing DCX were calculated for both groups. This quantification involved analyses of ~100
BrdU+ cells per animal (n=4/ group) in both groups.
6. Analysis of the dendritic growth of DCX+ newly born neurons. Only the DCX+ neurons that
exhibited the following features were chosen in both groups: (i) vertically oriented dendrites
reaching the molecular layer; (ii) no truncated branches near the soma; (iii) lack of overlap with
dendrites of neighboring neurons; and (iv) soma located in or near the middle of the section
thickness. Twelve such neurons were selected in each animal belonging to the two groups (n=6
animals/group; 72 neurons per group) and were traced at 40X magnification using the Neurolucida
neuron tracing system (Microbrightfield), as described in our earlier reports.2-4 The data for various
morphological measurements such as the total dendritic length, numbers of dendritic nodes and
endings were measured and compared. In addition, the numbers of dendritic intersections at
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different distances from the soma were measured using Sholl’s analyses, as described in our earlier
report.3
7. Characterization of learning and memory function using the water maze test (WMT). Rats
underwent learning and memory testing during the day-light period. The water maze tank (a circular
fiberglass pool measuring 170 cm in diameter and 75 cm in height) was filled with room
temperature water to 35 cm height and extra-maze cues were placed on the walls of the room. Rats
were first trained to find the circular platform (15 cm in diameter) submerged in water within one of
the 4 quadrants using spatial cues. The movement of rat in the water maze tank was continuously
video-tracked and recorded using the computerized ANY-Maze video-tracking system. The training
comprised 7 sessions over 7 days with 4 acquisition trials per session. Each trial lasted 90 seconds
and the inter-trial interval was 120 seconds. During different trials, the rat was placed in the water
facing the wall of the pool in a pseudo-random manner so that each trial commenced from a
different start location. Once the rat reached the platform, it was allowed to stay there for 30
seconds. When a rat failed to find the platform within the ceiling period of 90 seconds, it was
guided into the platform where it stayed for 30 seconds. The location of platform remained constant
across all days and trials for an individual animal. After each trial, the rat was wiped thoroughly
with dry towels, air dried and placed in the home cage. During the 7 days acquisition period, the
latency to reach the platform was measured as an indicator of learning ability. The latency to find
the platform was recorded for every trial. From these, the mean latency to reach the platform in
every session was calculated using the Anymaze software and compared between the two groups.
Learning curves in both groups were assessed using the mean latency to reach the platform in all 7
sessions and regression analyses. The overall improvement in learning over the training sessions
was also measured by comparing the latencies to reach the platform between the first and last
sessions. One-day after the above 7-day learning paradigm, rats were subjected to 45 second
retention (probe) test. For this, the platform was removed and rats were released from the quadrant
opposite to the original position of the platform. Latency to reach the platform area, time spent in
platform area, platform area crossings and dwell time in the platform quadrant were measured.
Typically, rats that are capable of easily retrieving the learned memory head straight to the platform
area after release, spend most of the trial (45 sec) searching within the quadrant (or area) where the
platform was originally placed and exhibit many platform area crossings. Thus, rats exhibiting
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shortest latency to reach the platform area, greater dwell time in the platform area and increased
numbers of platform area crossings are considered to have superior memory than rats exhibiting
greater latency to reach the platform area, reduced dwell time in the platform area and fewer
platform area crossings.
8. Analyses of memory function using the novel object recognition test (NORT). This involved
one session of testing for each rat with 3 successive trials, which involves the following. The rat
was first placed in the center of the empty open field box (60 x 60 cm) having no objects and was
allowed to freely explore for 5 minutes (1st trial; habituation phase). Following this, the rat was
removed, held in its home cage for 3 minutes (inter-trial interval), placed again in the center of the
open field box with two identical plastic spheres placed in opposite corners of the box and allowed
to freely explore the objects for 5 minutes (2nd trial; sample phase). The rat was then removed and
held in its home cage for 3 minutes (inter-trial interval), placed in the center of the box with a
plastic sphere used in trial #2 in one corner and a new cylindrical wooden object in another corner
and allowed to freely explore for 5 minutes to assess novel object recognition ability (3rd trial;
memory testing phase). We considered that the rat is exploring the novel object in trial #3 when its
nose is within 2 cm of the novel object. The entire trial #3 was video-recorded and videos were
analyzed later by two independent investigators who are blind to animal groups. Time spent with
familiar object, time spent with novel object, and time spent with familiar object + novel object (i.e.
the total exploration time) were scored using the videos. From these data, the discrimination index
for the novel object was calculated by using the formula, (novel object recognition time/total
exploration time)-(familiar object recognition time/total exploration time) × 100. Memory analysis
with NORT is based on the natural tendency of rodents to investigate a novel object instead of a
familiar one. The choice to explore the novel object reflects the use of learning and (recognition)
memory processes.5
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B. Pertaining to Results:
1. Neurodegeneration or neuroinflammation after predictable chronic mild stress (PCMS)
exposure. Analyses of serial (every 15th) sections through the hippocampus with Fluoro-Jade B
staining revealed no neurodegeneration in rats belonging to both PCMS and control groups while
positive control sections from animals treated with kainic acid revealed the validity of Fluoro-Jade
B staining for identifying degenerated neurons.6 Characterization of additional serial (every 15th)
sections with NeuN immunostaining demonstrated that PCMS does not alter the structural
organization of the hippocampus, as the overall cytoarchitecture was found to be similar between
the PCMS treated and control rats (see Fig. 2 in this supplement).
Furthermore, analyses of serial
sections with markers of reactive astrocytes (nestin)7-8 and activated microglial cells (ED-1)1,9
revealed no signs of inflammation with PCMS. Nestin positive reactive astrocytes (see Fig. 3 in this
supplement) and ED-1+ activated microglial cells were rarely observed in both control and PCMS
treated rats.
2. Supplemental Figure 2:
Legend: Neuronal cytoarchitecture of the hippocampus from an age-matched control rat (A1) and a
rat that underwent PCMS for 28 days (B1), visualized by NeuN immunostaining. A2 and B2 are
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magnified views of dentate gyrus regions from A1 and B1. Note that the overall neuronal
cytoarchitecture of the hippocampus is comparable between the age-matched control rat and the rat
that underwent PCMS for 28 days. Scale bar, A1, B1 = 400 µm; A2, B2 = 200 µm.
3. Supplemental Figure 3:
Legend: Nestin immunostaining of the dentate gyrus from an age-matched control rat (A1) and a rat
that underwent PCMS for 28 days (B1). Note that, nestin immunostaining mostly identified the
vasculature (capillaries) in both groups. Nestin+ reactive astrocytes (indicated by arrows in A1, B1)
are rare in both control and PCMS groups, suggesting that PCMS does not induce the generation of
reactive astrocytes. Scale bar, A1, B1 = 200 µm.
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4. Dendritic growth as analyzed by the concentric circle analyses of Sholl. The concentric circle analyses
of Sholl10 revealed that most of the apical dendrite branches of relatively mature DCX+ neurons in both
groups extend beyond the 100-µm distance from the soma and end between 100 and 150 µm distance from
the soma (see Fig. 4 in this supplement). However, neurons in the PCMS group showed greater numbers of
dendritic nodes at 50–100 µm and 100–150µm distances from the soma than their counterparts in the control
group (Fig. 4 [A]). Furthermore, neurons from the PCMS group exhibited greater numbers of dendritic
intersections and dendritic endings between 100 and 150 µm distance from the soma than neurons from the
control group (p<0.05 to p<0.001; supplemental Fig. 4 [B-C]). Additionally, neurons in the PCMS group
exhibited increased dendritic length at 50-100 µm, 100-150 µm, and 150-200 µm distances from the soma, in
comparison to their counterparts in the control group (p<0.01 to p<0.001; supplemental Fig. 4 [D]). Thus,
during the DCX expression phase, newly born neurons in the PCMS group exhibit considerably enhanced
dendritic growth than newly born neurons in the control group.
5. Supplemental Figure 4:
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