1. Sex Differences in Hippocampus-dependent Tasks: Female Dominance in
Fear Conditioning, Novel Object Recognition, and Morris Water Maze
Mahal A1, Nguyen J1, Tiacharoen M1, Cushman JD1,2,3
1Department of Psychology and Brain Research Institute UCLA, 2Integrative Center for Learning and Memory UCLA, 3Behavioral Testing Core Facility UCLA
Abstract Results
Methods
Groups
•36 C57Bl/6j mice (18 male and 18 female) were divided into three groups among ten students taking a
UCLA Psychology 111 Learning Laboratory course. The students first attended a Mouse User Wet Lab
conducted by the UCLA Deparment of Laboratory Animal Medicine. They then received specific training
in proper mouse handling for three days prior to the experiments. Each task was conducted over three
consecutive days per week for three weeks. Each group received task training in a different order. All
procedures were conducted in accordance with NIH guidelines and were approved by the UCLA Office for
the Protection of Research Subjects and the Chancellor’s Animal Research Committee.
Auditory Fear Conditioning: Tone/Noise Generalization
DAY 1: Acquisition
• Foot shock was associated with Context A and a discrete tone
• The training context consisted of a standard grid floor, a fan, and 50% Simple Green scent
• The discrete tone was a 2800 Hz, 75 dB, rise/fall 50, and played for 20 seconds per presentation.
• At the end of each tone, mice were shocked at 0.5 mA for 2 seconds, followed by an inter-trial interval
of 3 minutes.
• 20 mice (10 male, 10 female) received two tone-shock and 16 mice (8 males, 8 females) received 5
tone-shock pairings.
DAY 2: Context Test
• The animals were returned to the original training context for an 8 minute freezing test.
DAY 3: Pure Tone Test
• The chambers were modified with a plastic white floor, a plastic black A-frame, and Windex scent.
Tones were presented in an identical fashion to training, with shock omitted.
Measures
• Percent time freezing was determined using Video Freeze computer software (Med-Associates, Inc.).
Novel Object Recognition
DAY 1: Habituation
•Mice were placed in an a square open field (28 cm x 28 cm) in a dimly lit (5 lux) room for 20 minutes.
DAY 2: Familiarization
• Two 50 ml Erlenmeyer flasks were placed in the top-left and bottom-right corners of each open field
cage, 6 cm from each side. The flasks were filled with green tissue paper for half of the animals and were
empty for the other half.
DAY 3: Novelty Preference Test
• One of the objects was replaced with a novel object (either a filled or empty 50 ml Erlenmeyer flask
depending on the familiar objects from Day 2) with the position of the replaced counterbalanced across
groups
• Objects were carefully cleaned with 70% ethanol between animals.
Measures
• Time spent sniffing the objects were determined using TopScan software (CleverSys Inc.) and was
validated using hand scoring. Preference ratios were calculated as: (Novel – Familiar)/(Novel + Familiar).
Morris Water Maze
DAYS 1-2: Acquisition
• Set-up consisted of a 1.2 m diameter tank filled with water to 1 cm above a hidden platform that was
placed in one corner quadrant of the pool (designated as the target quadrant).
• The water was made opaque with white tempura paint and the thermostat was set to 24 C.
• Each mouse was placed in the pool at one of three starting quadrants (excluding the target quadrant).
The stopwatch was started soon as the mice entered the pool. Once the mouse found the platform, they
remained there for 15 seconds before being removed and placed into the drying cage. If 60 seconds
passed and the mice still had not found the platform, they were placed on the platform for 15 seconds
before being placed into the drying cage.
• Each mouse had three trials per day, with each trial starting from the middle of a non-target quadrant.
• One group of 12 mice (6 male, 6 female) were trained with only distal room cues. Another group was
trained with distal room cues as well as a yellow curtain hanging on one side of the pool (between two
starting locations opposite to the target quadrant) to serve as a landmark cue.
DAY 3 Acquisition + “Probe” Trial
• Mice were tested as they had been on Days 1 and 2.
• For the Probe Trial, the platform was removed and the mice were placed the mice into the pool for 60
seconds.
Measures
• Escape latency was measured with a stopwatch as researchers viewed live video feed of mice behavior
during training and testing sessions. Quadrant preference during the “probe” trial was measured using
TopScan (CleverSys Inc.) video tracking software.
The hippocampus contributes to important memory functions such as fear learning, latent
learning, and spatial learning. Because sex differences are often ignored in experimental analyses
(Clayton and Collins, 2014), it is important to investigate these effects on hippocampal dependent
learning to study how males and females react differently to stressors and other stimuli. This will
provide the scientific community with baseline measures of sex differences that may affect future
experimental designs. Furthermore, this will influence translational research in a clinical setting by
allowing physicians to better understand disease susceptibility and development, and formulate
specific treatment options for each sex. Using equal numbers of male and female C57Bl6 mice, the
present study investigated sex differences in learning and memory utilizing three highly defined
experimental paradigms: auditory fear conditioning, novel object recognition, and Morris water
maze training. Previous studies have shown sex differences in context fear conditioning (Wiltgen et
al., 2001), novel object recognition (Bettis and Jacobs, 2012), and Morris water maze training (Roof
and Stein, 1999). Using a simplified three-day experimental paradigm for each respective
investigation, we found that females acquire auditory fear faster than males, females explore a
similar novel object more than males, with prior stress influencing this observation, and females
outperform males in a spatial navigation task when a prominent landmark cue is available. The
implications of these results may affect future experiments, as experimenters and clinicians should
be aware of these significant findings in their research.
Conclusions
Fear Conditioning
Sex differences were only observed in the stronger 5 tone-shock pairings protocol. Selection pressures may mediate this effect since females transmit important information to other animals such as predatory attacks (Jones et al., 2014) and are encouraged to react quickly in order to return to their nest to care for their young.
Freezing was maximal after the second tone-shock pairing—specifically, tones three and five are driving the sex difference seen in acquisition.
Freezing was lower during the context test for the 2 tone-shock pairings protocol than for the 5 tone-shock pairings protocol. This indicates that an animal’s fear in certain contexts is a function of the number of threats it has faced in that environment.
Novel Object Recognition
Females preferentially investigate the novel object more than males, consistent with previous findings of enhanced novel object recognition for similar object pairs in female mice (Bettis and Jacobs, 2012).
Prior fear conditioning and water maze training training reduced exploration of the novel object. This may be a result of the prior stressful experiences encountered by the mouse because the novel object recognition task relies on spontaneous exploratory performance behavior. Both fear conditioning and Morris water maze training
are stressful experiences in which the animals receive aversive foot shocks and must escape from water, respectively. Thus, after experiencing these stressful situations, the typical preference for exploring novel objects may be reduced.
Morris Water Maze
When a prominent landmark cue was available (a large yellow curtain hanging from the ceiling) acquisition occurred at a slightly faster rate; however, no sex differences were observed.
Without the landmark cue neither sex was able to acquire a spatial bias for the target quadrant. When a landmark cue was available, however, females showed a target quadrant preference whereas males did not. This is consistent with interpretations in the literature that females rely on landmark cues and males rely on geometric
cues in spatial navigation (Roof and Stein, 1999) .
Implications of Results
The results examined in this study will provide baseline measures of sex differences in three commonly used hippocampal dependent learning and memory experimental paradigms. Uncovering sex differences will influence bench-to-bedside research in a clinical setting by allowing physicians to better understand disease susceptibility
and development, and formulate specific treatment options for each sex.
Fear Conditioning More rapid acquisition of tone fear in females
(A) Mean percent freezing (±SEM) for each of the five
tone auditory presentations during training. Both male
and female mice showed normal auditory fear acquisition
(RM ANOVA p<0.001). However, freezing rates differed
between the two groups. Females acquired fear faster
than males (RM ANOVA intxn: p = 0.002) and showed
significantly higher percent freezing during the third
(*p=0.021 ANOVA) and fifth (*p=0.031 ANOVA) tone
presentation. (B) Mean percent freezing (±SEM) during
training with two tone-shock pairings. Both male and
female mice showed normal auditory fear acquisition (RM
ANOVA p<0.001). No sex differences in acquisition were
observed (ANOVA p = 0.322). (C, D) Mean percent freezing
(±SEM) during the context and tone tests. No sex
differences were observed in the context (ANOVA p =
0.769) or tone test (ANOVA p = 0.851). However, the 2
tone-shock pairing protocol resulted in lower freezing
during the context test compared to the 5 tone-shock
pairing protocol (ANOVA p = 0.028). Both male and female
mice were able to discriminate between the trained
context and test context, evident by the lower freezing
rates observed in the baseline measurements at test
(ANOVA p < 0.001).
Enhanced novel object recognition in females.
(A) Mean exploration times (±SEM) show that males did
not explore the novel object more, regardless of testing
order. (B) Mean exploration times (±SEM) show that
when female mice did not receive any prior training
(group 1), they demonstrated a strong preference for the
novel object at test. When female mice had prior fear
conditioning (group 2), a reduction in exploration was
observed. Moreover, when female mice had prior
exposure to fear conditioning and Morris water maze
training, the effect was eliminated. (C) Mean preference
ratios (±SEM) show that novel object recognition
performance differed as a function of testing order
(ANOVA p = 0.017). (D) Mean exploration times (±SEM)
show that female mice explored a similar novel object
significantly more than male mice (*p=0.008 ANOVA
effect of object in females; ANOVA effect of object in
males p=0.795).
Enhanced landmark-based spatial learning in
females
(A) Mice reduced their latency to find the hidden
platform over three training days. (ANOVA p < 0.001). (B)
Mean latency (±SEM) shows that acquisition of Morris
water maze training occurred amongst all mice whether a
landmark cue was present or not (ANOVA p = .125).
When the landmark was present, acquisition occurred at
a slightly faster rate. (C) Mean percent occupancy
(±SEM) shows that when no landmark was present
during training and the probe trial, neither sex showed a
target quadrant preference. (D) Mean percent occupancy
(±SEM) shows that when a prominent landmark cue was
present during training and the probe trial females
demonstrated a significant preference for the target
quadrant whereas males did not (*p = 0.008 ANOVA).
Novel Object Recognition
Morris Water Maze
0
10
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BL Tone1 ITI1 Tone2 ITI2 Tone3 ITI3 Tone4 ITI4 Tone5 ITI5
PercentFreezing
Acquisition Day – 5 Tone Shock Pairings
Male
Female
0
5
10
15
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25
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35
40
45
1 2 3
ExplorationTime
Testing Order
Effect of Testing Order on NOR - Males
Novel
Familiar
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10
20
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90
100
BL Tone1 ITI1 Tone2 ITI2
PercentFreezing
Acquisition Day - 2 Tone Shock Pairings
Male
Female
0
10
20
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50
60
70
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90
100
Context Baseline Tone
Percentfreezing
Fear Conditioning - 5 Tone Shock
Male
Female
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60
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100
Context Baseline Tone
Percentfreezing
Fear Conditioning - 2 Tone Shock
Male
Female
0
5
10
15
20
25
30
35
1 2 3
ExplorationTime
Testing Order
Effect of Testing Order on NOR - Females
Novel
Familiar
-0.4
-0.3
-0.2
-0.1
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
1 2 3
PreferenceRatio
Testing Order
Effect of Testing Order on NOR
Females
Males
0
5
10
15
20
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30
35
Female Male
ExploratiojnTime
Effect of Sex on NOR
Novel
Familiar
0
10
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60
Day 1 Day 2 Day 3
Latency(s)
MWM Acquisition - Accross All Animals
Male
Female
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60
Day 1 Day 2 Day 3
Latency(s)
MWM Acquisition - Effect of Landmark
No Curtain
Curtain
0
5
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35
Females Males
PercentOccupancy
Probe Trial - No Landmark Group
Target Quadrant
Other Quadrants
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5
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45
Females Males
PercentOccupancy
Probe Trial – Landmark Group
Target Quadrant
Other Quadrants
Acknowledgements
We would like to thank the UCLA Psychology Department and the Behavioral Testing Core for providing the
funding, equipment and space for these experiments. We would also like to thank the UCLA Department of
Laboratory Animal Medicine for developing a specialized Mouse User Wet lab for this course. We would also
like to thank the other students in the class that helped to generate this data: Veronica Duarte, Neri Gallegos,
Ola Smith, Heidi Tan, Angelica Chaghouri, Yasmin Tontat, Sean Colaco and Guanchen Liu.
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A B C D
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A B C D
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