SUPPLEMENTAL MATERIALS
Supplemental Methods
Acoustic startle
Startle chambers (San Diego Instruments, San Diego, CA, USA) consisted of
nonrestrictive Plexiglas cylinders 5 cm in diameter resting on a Plexiglas platform in a ventilated
chamber. High-frequency speakers mounted 33 cm above the cylinders produced all acoustic
stimuli. Piezoelectric accelerometers mounted under the cylinders transduced movements of the
animal, which were digitized and stored by an interface and computer assembly. Beginning at
startling stimulus onset, 65 consecutive 1 ms readings were recorded to obtain the amplitude of
the animal's startle response. For all the acoustic startle sessions, the inter-trial intervals between
stimulus presentations averaged 15 sec (range of 7-23 sec). Startle pulses were 40 ms in
duration, prepulses were 20 ms in duration, and prepulses preceded the pulse by 100 ms (onsetonset). All sessions began with 5-min acclimation period.
One week prior to stress exposure, baseline startle was assessed over 3 consecutive days
(Session 1). Session 1 consisted delivery of 10 each of 105 dB startle pulses (40 ms) over 50 dB
background in dark chambers. One week after stress exposure, startle reactivity was re-assessed
in two consecutive sessions. ‘Session 1’ consisted the same parameters as before in baseline
assessment except that 10 additional pulses (in a pseudorandom order ) were presented with
houselights coming on for 2.95 s prior to the startle stimulus to replicate the acoustic startle
session previously described for the mouse predator stress model of PTSD (1). To further assess
startle habituation and inhibition as measured by prepulse inhibition (PPI) a second session
(Session 2) was presented immediately after the first. In Session 2, 65 dB background was
presented continuously throughout the session in chambers lights on during the whole session
and it included five blocks beginning with the delivery of five each of 120 dB startle pulses
(Block1) allowing startle to reach a stable level before specific testing. The second block tested
response threshold and included four each of five different acoustic stimulus intensities: 80, 90,
100, 110 and 120 dB in a pseudorandom order. The third block consisted of 42 total trials
consisting of twelve 120dB startle pulses and ten each of three different prepulse intensity trials
(69, 73 and 81 dB 20 ms prepulse preceding a 120 dB pulse with 100 ms from prepulse to pulse
onset) to assess PPI. The fourth block totaled 28 trials consisting of eight 120dB startle pulses
and four each of five different prepulse-pulse onset trials (i.e. interstimlus interval (ITI): 73 dB
prepulses preceding 120 dB pulses by 25, 50, 100, 200, or 500 ms) to assess PPI with different
ITI intervals. The session ended with five pulses of 120 dB (Block 5) to assess habituation of the
startle response. Prepulse inhibition (PPI) was calculated as percent change compared to 120 dB
pulses without prepulse using 120 dB alone trials in Block3 using the following formula:
%PPI=(100-(average startle magnitude in prepulse trial/average startle magnitude to pulse alone
trial)*100). Habituation was measured by comparing the response to 120dB pulses across 5
blocks in the session. In all experiments, the average startle magnitude over the record window
(i.e., 65 msec) was used for all data analysis.
Composite avoidance (z-)score analysis
We calculated and analyzed composite avoidance scores, which is a common method to
create an overall index of different symptoms in clinical research when multiple measures
(subdimensions) of the same construct are conducted. First, we calculated z-scores of time spent
in the aversive arenas (i.e. center of open field; light compartment of light-dark box; 3-cm radius
zone near the tube filled with cat litter) using the following formula:
(individual score – average of the whole experimental cohort) / standard deviation of the whole experimental cohort
Z-scores were calculated for each test separately for males and females because of
significant effects of sex. To create composite z-scores, we used two methods: (1) we averaged
z-scores of the above avoidance tests, or (2) calculated weighted composite z-scores based on
their factor loadings in a preceding factor analysis of the three individual tests. Accordingly,
composite z-scores represent overall avoidance (of three tests) by indicating group values
normalized for the whole same-sex cohort (z-score=0). We added these variables in order to
determine overall effect size of stress x CRH, and reduce family-wise error, and hence, reduce
the probability of false positive outcomes generated by repeated testing.
qRT-PCR
Crf1 (Mm00432670_m1), Crf2 (Mm00438303_m1), and Fkbp51 (Mm00487401_m1)
were assessed using commercially available primers (Life Technologies, Carlsbad, CA). First,
RNA extraction was performed using the RNeasy Lipid Kit (Qiagen, Valencia, CA) according to
manufacturer’s instructions. On-column DNase digestion was performed using the RNase-Free
DNase Kit (Qiagen, Valencia, CA). RNA quantity and quality was measured using a Nanodrop
2000 (Thermo Scientific, Wilmington, DE) and total RNA across each sample was standardized
and used for first-strand cDNA synthesis using the Superscript III first-strand kit (Life
Technologies, Carlsbad, CA). TaqMan quantitative PCR (Applied Biosystems, Carlsbad, CA)
was performed at the UCSD core facility (http://cfar.ucsd.edu/).
Supplemental Results
Table S1. Locomotor and exploratory activity over 30 min in the mouse BPM. Data are
presented as mean ± SEM. *p<0.05 (main effect of stress or CRFOEdev in three-way ANOVA
test); CRFOEdev: transient CRF over-expression before puberty.
CRF
Stress
Handled
Male Control
Stressed
Handled
Male CRFOEdev
Stressed
Handled
Female Control
Stressed
Handled
Female CRFOEdev
Stressed
Main effect of stress:
Main effect of CRFOEdev:
Distance travelled (cm)
4494 ± 93
4169 ± 100
5217 ± 130*
4449 ± 132*
4951 ± 195
4916 ± 150
5897 ± 233*
6136 ± 233*
F(1,66)<1, ns
F(1,66)=5.50, p=0.022
Number of rears
100.0 ± 25.1
81.7 ± 24.3
87.1 ± 30.8
95.5 ± 37.0
60.4 ± 24.6
73.0 ± 34.0
67.0 ± 30.1
66.3 ± 26.0
F(1,66)<1, ns
F(1,66)<1, ns
Number of hole-pokes
56.2 ± 25.2
43.4 ± 18.8*
43.6 ± 22.6
37.4 ± 18.7*
43.4 ± 23.3
28.6 ± 13.1*
49.9 ± 22.3
33.8 ± 18.9*
F(1,66)=8.20, p=0.006
F(1,66)<1, ns
Table S2. The intensity of predators stress during a 10-minutes-long interaction. UpperData are
presented as mean ± SEM. CRFOEdev: transient CRF over-expression before puberty. There is
no singificant difference between sexes, or controls and CRHOE groups.
Time duration (s)
of:
Near the mouse
(<1 ft)
Sniffing
Pawing
Mouthing
Male Control
383.3 ± 28.2
2.7 ± 2.1
157.0 ± 9.8
51.9 ± 20.8
Male CRFOEdev
360.0 ± 52.7
5.0 ± 2.0
149.5 ± 21.5
57.7 ± 18.5
Female Control
389.8 ± 29.3
1.2 ± 0.7
176.9 ± 14.1
52.0 ± 14.1
Female CRFOEdev
388.5 ± 24.1
2.2 ± 1.0
174.2 ± 11.8
41.3 ± 7.9
Frequency of:
Near the mouse
(<1 ft)
Sniffing
Pawing
Mouthing
Male Control
14.7 ± 1.5
3.3 ± 2.1
63.8 ± 6.4
28.0 ± 8.4
Male CRFOEdev
13.4 ± 2.7
3.8 ± 1.6
59.2 ± 10.6
27.5 ± 7.2
Female Control
15.8 ± 2.1
1.6 ± 1.0
64.7 ± 6.6
29.0 ± 6.1
Female CRFOEdev
17.8 ± 1.5
1.7 ± 0.6
68.8 ± 5.3
27.3 ± 3.8
Males
Females
80
Average Startle Magnitude
Average Startle Magnitude
100
80
60
40
20
60
40
20
0
0
Pre-stress
Pre-stress
Post-stress
Post-stress
Control
CRFOE
*
Fig.S1. Average startle magnitude exhibited in Session 1 in dark chambers (105 dB) before and
after predator stress. Data are collapsed across handled and predator stressed groups since stress
did not affect startle magnitude (Fstress(1,66)<1, p>0.674). In contrast, CRFOE increased the
magnitude of the reaction in both sexes indicated by three-way ANOVA test (*p<0.01 main
effect of CRFOEdev). Data are presented as mean ± SEM. CRFOEdev: transient CRF overexpression before puberty.
Supplemental References
1.
Adamec R, Fougere D, Risbrough V (2010): CRF receptor blockade prevents initiation
and consolidation of stress effects on affect in the predator stress model of PTSD. The
international journal of neuropsychopharmacology / official scientific journal of the Collegium
Internationale Neuropsychopharmacologicum. 13:747-757.