Trace fear conditioning is enhanced in mice lacking the subunit of the GABAA receptor

Article (PDF Available)inLearning & Memory 12(3):327-33 · May 2005with26 Reads
DOI: 10.1101/lm.89705 · Source: PubMed
Abstract
The delta subunit of the GABA(A) receptor (GABA(A)R) is highly expressed in the dentate gyrus of the hippocampus. Genetic deletion of this subunit reduces synaptic and extrasynaptic inhibition and decreases sensitivity to neurosteroids. This paper examines the effect of these changes on hippocampus-dependent trace fear conditioning. Compared to controls, delta knockout mice exhibited enhanced acquisition of tone and context fear. Hippocampus-independent delay conditioning was normal in these animals. These results suggest that reduced inhibition in the dentate gyrus facilitates the acquisition of trace fear conditioning. However, the enhancement in trace conditioning was only observed in female knockout mice. The sex-specificity of this effect may be a result of neuroactive steroids. These compounds vary during the estrus cycle, can increase GABAergic inhibition, and have been shown to impair hippocampus-dependent learning. We propose that activation of GABA(A)Rs by neuroactive steroids inhibits learning processes in the hippocampus. Knockouts are immune to this effect because of the reduced neurosteroid sensitivity that accompanies deletion of the delta subunit. Relationships between neurosteroids, hippocampal excitability, and memory are discussed.
Research
Trace fear conditioning is enhanced in mice lacking
the subunit of the GABA
A
receptor
Brian J. Wiltgen,
1,2
Matthew J. Sanders,
1,2
Carolyn Ferguson,
3
Gregg E. Homanics,
3,4
and Michael S. Fanselow
1,2,5
1
Psychology Department, and
2
The Brain Research Institute, UCLA, Los Angeles, California 90095, USA;
3
Department of Anesthesiology, and
4
Department of Pharmacology, University of Pittsburgh School of Medicine,
Pittsburgh, Pennsylvania 15261, USA
The subunit of the GABA
A
receptor (GABA
A
R) is highly expressed in the dentate gyrus of the hippocampus.
Genetic deletion of this subunit reduces synaptic and extrasynaptic inhibition and decreases sensitivity to
neurosteroids. This paper examines the effect of these changes on hippocampus-dependent trace fear conditioning.
Compared to controls, knockout mice exhibited enhanced acquisition of tone and context fear. Hippo-
campus-independent delay conditioning was normal in these animals. These results suggest that reduced inhibition in
the dentate gyrus facilitates the acquisition of trace fear conditioning. However, the enhancement in trace
conditioning was only observed in female knockout mice. The sex-specificity of this effect may be a result of
neuroactive steroids. These compounds vary during the estrus cycle, can increase GABAergic inhibition, and have
been shown to impair hippocampus-dependent learning. We propose that activation of GABA
A
Rs by neuroactive
steroids inhibits learning processes in the hippocampus. Knockouts are immune to this effect because of the reduced
neurosteroid sensitivity that accompanies deletion of the subunit. Relationships between neurosteroids, hippo-
campal excitability, and memory are discussed.
In the central nervous system, fast inhibitory transmission is
mediated primarily by -aminobutyric acid type A receptors
(GABA
A
Rs) (Macdonald and Olsen 1994; Olsen and Homanics
2000). These ligand-gated receptors are pentamers composed of
various subunit combinations (, , , , , , and ) that exhibit
differential expression in the brain (Barnard et al. 1998; Sieghart
and Sperk 2002). For example, the subunit is highly expressed
in the dentate gyrus, but not CA3 or CA1 regions of the hippo-
campus (Persohn et al. 1992; Peng et al. 2002). The pharmaco-
logic and kinetic properties of GABA
A
Rs are highly dependent on
the specific configuration of these subunits (Quirk et al. 1994;
Gunther et al. 1995; Smith and Olsen 1995; Khan et al. 1996;
Benke et al. 1997). Recent studies have shown that genetic dele-
tion of the subunit of the GABA
A
R produces a significant re-
duction in neurosteroid sensitivity (Mihalek et al. 1999; Vicini et
al. 2002; Porcello et al. 2003; Spigelman et al. 2003) and acceler-
ates the decay of spontaneous mIPSCs and evoked IPSPs in hip-
pocampal granule cells and thalamic relay neurons (Mihalek et
al. 1999; Porcello et al. 2003; Spigelman et al. 2003). These data
suggest that -containing GABA
A
Rs contribute to synaptic and
extrasynaptic inhibition and facilitate modulation by neuroac-
tive steroids.
Pharmacologic or genetic manipulations that reduce
GABAergic inhibition often enhance synaptic plasticity and
learning (Introini-Collison et al. 1994; Crestani et al. 1999, 2002;
Staubli et al. 1999; Shumyatsky et al. 2002). For example, recent
experiments on GABA
A
R mutants (5 and 2) found enhanced
trace but normal delay conditioning (Crestani et al. 1999, 2002).
Trace conditioning is a form of hippocampus-dependent learn-
ing in which the conditional stimulus (CS) and unconditional
stimulus (US) are separated in time (Solomon et al. 1986; Moyer
Jr. et al. 1990; McEchron et al. 1998; Huerta et al. 2000; Quinn et
al. 2002). This is distinct from hippocampus-independent delay
conditioning in which the CS and US are presented contiguously.
Previous work has demonstrated that trace conditioning en-
gages the CA1 region of the hippocampus (Moyer Jr. et al. 1996,
2000; McEchron and Disterhoft 1999; McEchron et al. 2001,
2003; Leuner et al. 2003) and requires NMDAR-dependent plas-
ticity in this region (Huerta et al. 2000). Relatively little is known
about the contribution of the dentate gyrus to trace condition-
ing, although recent studies suggest this region also plays an
important role. For example, exposure to a trace-conditioned cue
produces significant increases in immediate early gene expres-
sion in the dentate gyrus (Weitemier and Ryabinin 2004). Trace
conditioning also increases neurogenesis in the dentate gyrus
(Gould et al. 1999) and is impaired by a reduction in the number
of newly generated neurons in this region (Shors et al. 2001). The
current experiments used knockout (KO) mice to examine the
effects of reduced inhibition and neurosteroid sensitivity in the
dentate gyrus on the acquisition of trace fear conditioning. We
also looked for nonspecific effects of subunit deletion by ex-
amining performance on open-field and RotaRod tests.
Results
Trace conditioning
Females
Data from female mice during the tone test are displayed in Fig-
ure 1A. The data are presented as percent time spent freezing
during four periods: Baseline (BL), Tone, Trace, and Intertrial
Interval (ITI). BL freezing levels were low and did not differ
among genotypes (F
(2,27)
= 2.34, p > 0.05). Mice showed a signifi-
cant increase in freezing when the trace-conditioned tone was
presented (F
(1,27)
= 47.5, p < 0.05), and this effect interacted with
genotype (F
(2,27)
= 3.8, p < 0.05). Post hoc tests (Fisher’s PLSD;
5
Corresponding author.
E-mail fanselow@psych.ucla.edu; fax (310) 206-5895.
Article published online ahead of print. Article and publication date are at
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Learning & Memory 327
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p < 0.05) revealed that homozygous (Homo) KOs exhibited a
larger increase in tone fear than either heterozygous (Het) KO or
wild-type (WT) mice. This enhancement was observed during the
entire test (F
(2,27)
= 3.52, p < 0.05) and did not interact with
stimulus period (F
(4,54)
= 1.22, p > 0.05). The day after the tone
test, mice were placed in the original training environment for a
context test. Figure 1B shows the freezing scores for females av-
eraged across the entire 8-min test. As observed during the tone
test, female Homo KOs froze significantly more than Het or wild-
type mice (F
(2,27)
= 4.66, p < 0.05).
Males
Male freezing during the tone test is illustrated in Figure 1C.
Freezing levels were low during the BL period and did not differ
among genotypes (F
(2,24)
= 1.1, p > 0.05). Mice exhibited an in-
crease in freezing during the tone presentations (F
(1,24)
= 30.2,
p < 0.05) that did not interact with genotype (F < 1). A repeated-
measures ANOVA on the Tone, Trace, and ITI periods found a
significant effect of stimulus period (F
(2,48)
= 19.27, p < 0.05) and
post hoc tests (Fishers PLSD; p < 0.05) revealed that most freez-
ing occurred during the tone. There were no overall freezing dif-
ferences among genotypes during these periods (F < 1). The day
after the tone test, male mice were placed back in the original
training environment for a context test. Figure 1D illustrates
freezing scores averaged across the entire 8-min test. Homo, Het,
and wild-type mice exhibited similar levels of context freezing
during this test (F < 1).
An ANOVA on the tone test data revealed a main effect of
sex (F
(1,51)
= 10.64, p < 0.05) as males froze significantly less than
females. This effect did not interact with genotype (F < 1), sug-
gesting a general increase in freezing for females across all peri-
ods. In contrast, an ANOVA conducted
on the context freezing scores from male
and female mice did not find an effect of
sex (F
(1,51)
= 2.29, p > 0.05).
Delay conditioning
Females
Female data during the tone test are il-
lustrated in Figure 2A. BL freezing levels
were low and did not differ among geno-
types (F < 1). All groups showed a signifi-
cant increase in freezing when the de-
lay-conditioned tone was presented
(F
(1,24)
= 477.39, p < 0.05), and this ef-
fect did not interact with genotype
(F
(2,24)
= 1.09, p > 0.05). Freezing to
the tone following delay conditioning
was significantly greater than that ob-
served following trace conditioning
(F
(1,55)
= 37.49, p < 0.05). There was a
significant effect of stimulus period
during the test (F
(2,48)
= 103.78, p < 0.05)
as most freezing occurred during the
tone (Fishers PLSD; p < 0.05). There
were no freezing differences among
genotypes during any of the stimulus
periods (F
(2,24)
= 1.76, p > 0.05). The
day after the tone test, female mice
were placed in the original training en-
vironment for a context test. Figure 2B
shows the freezing scores for females av-
eraged across the entire 8-min test.
Homo, Het, and wild-type mice exhib-
ited similar levels of context freezing during this test (F
(2,24)
= 1.3,
p > 0.05).
Males
Male data during the tone test are illustrated in Figure 2C. Freez-
ing levels were low during the BL period and did not differ
among genotypes (F
(2,24)
= 1.03, p > 0.05). All groups exhibited a
subsequent increase in freezing during the tone presentation
(F
(1,24)
= 218.45, p < 0.05), which did not interact with genotype
(F
(2,24)
= 1.33, p > 0.05). The tone acquired more fear in the delay
procedure compared to the trace procedure used in the previous
experiment (F
(1,52)
= 51.45, p < 0.05). Freezing levels during the
tone were the same across genotypes (F
(2,24)
= 1.82, p > 0.05).
Mice also exhibited elevated freezing during the Trace and ITI
periods. A repeated-measures ANOVA on the Tone, Trace, and ITI
periods found a significant effect of stimulus period
(F
(2,48)
= 142.02, p < 0.05), and post hoc tests (Fishers PLSD;
p < 0.05) revealed that most freezing occurred during the tone.
There were no freezing differences among genotypes during any
of these periods (Fs < 1). The day after the tone test, the same
mice were placed back in the original training environment for a
context test. Figure 2D illustrates freezing scores averaged across
the entire 8-min test. All genotypes exhibited similar levels of
context freezing during this test (F
(2,24)
= 1.06, p > 0.05).
An ANOVA comparing the tone test data from male and
female mice revealed a main effect of sex (F
(1,48)
= 7.47, p < 0.05)
as females froze significantly more than males. This effect did not
interact with stimulus period (F < 1), suggesting a general in-
crease in freezing across all periods. In contrast to the tone data,
an ANOVA on context freezing scores from male and female mice
did not reveal an effect of sex (F < 1).
Figure 1. Data from the tone and context tests following trace fear conditioning. (A) Mean (SEM)
percent freezing for female mice during each period of the tone test. (B) Mean (SEM) percent
freezing for female mice during the context test. (C) Mean (SEM) percent freezing for male mice
during each period of the tone test. (D) Mean (SEM) percent freezing for male mice during the
context test. An asterisk (*) indicates a significant group difference (p < 0.05).
Wiltgen et al.
328 Learning & Memory
www.learnmem.org
Openfield and RotaRod performance
The subunit is expressed in granule cells in the cerebellum
(Peng et al. 2002), an area intricately involved in motor function.
Therefore, we determined if KOs had any changes in motor
activity or coordination. Motor impairments could produce per-
formance deficits on learning tasks and confound evaluations of
memory function.
Openfield
Females
To assess general activity levels, mice were placed on an openfield
for 20 min. Overall, Het and Homo KOs exhibited normal activ-
ity levels relative to controls (Fig. 3A) (F
(2,16)
= 1.31, p > 0.05).
Across the session, there was a significant decrease in activity
(F
(19,304)
= 9.21, p < 0.05) that did not differ among genotypes
(F < 1).
Males
Both Het and Homo KOs exhibited activity levels that were simi-
lar to wild-type controls (Fig. 3C) (F < 1). Across the session, there
was a general decrease in activity (F
(19,437)
= 18.49, p < 0.05) that
was observed in all genotypes. The magnitude of this decrease
did not differ among genotypes (F < 1). Overall, there was no
effect of sex on motor activity in the openfield (F < 1). Males and
females also displayed similar decreases in activity across the 20-
min session (F < 1).
RotaRod
Females
After the openfield, motor coordination was analyzed on the
RotaRod. Female mice displayed an increase in latency to fall
across days (Fig. 3B) (F
(6,102)
= 9.71,
p < 0.05) that did not differ among geno-
types (F < 1). Overall, there was no effect
of genotype (F < 1).
Males
There was an increase in latency to fall
across days (Fig. 3D) (F
(6,144)
= 20.79,
p < 0.05) that did not differ among geno-
types (F
(12,144)
= 1.6, p > 0.05). Overall,
there was no effect of genotype
(F
(2,24)
= 1.3, p > 0.05). There was also no
difference between the performance of
males and females overall (F <1) or
across days (F <1)
Discussion
Our results demonstrate that deletion of
the subunit of the GABA
A
R enhances
the acquisition of hippocampus-depen-
dent trace fear conditioning in female
mice. Delay fear conditioning was nor-
mal in KOs, suggesting the augmenta-
tion was not due to a general increase in
motivation or performance. Normal mo-
tor activity and coordination were also
observed on the RotaRod and openfield
tests. Genetic deletion of the subunit
decreases neurosteroid modulation and
reduces inhibition in the dentate gyrus
(Spigelman et al. 2003). Therefore, our
results suggest that GABAergic inhibi-
tion in this region plays an important
and selective role in the acquisition of
trace fear conditioning. It should be noted that the subunit is
conspicuously absent from several brain regions known to be
essential for fear conditioning (e.g., amygdala, periaqueductal
gray) (Fendt and Fanselow 1999; Peng et al. 2002). However, this
subunit is highly expressed in the cerebellum, a structure re-
cently implicated in the consolidation of fear learning (Peng et
al. 2002; Sacchetti et al. 2002, 2004). Despite this fact, there are
several reasons that make it unlikely that the enhanced condi-
tioning we observed resulted from -subunit deletion in this re-
gion. First, motor learning was completely normal in KO mice.
Second, manipulations of the cerebellum do not selectively affect
hippocampus-dependent learning. In fact, hippocampus-
independent delay conditioning is most sensitive to changes in
cerebellar activity or placticity (Sacchetti et al. 2002, 2004). This
contrasts with the selective enhancement of hippocampus-
dependent learning that we observed in the KO mice.
The current results replicate our previous finding that tone
and context fear are normal in KOs following delay condition-
ing (Mihalek et al. 1999). We also found that females acquired
significantly more tone fear than males following both delay and
trace conditioning. Increased tone fear in females is consistent
with the rat eyeblink literature, where females acquire tone con-
ditioning at a faster rate and to a higher asymptote than males
(Shors et al. 2000). In contrast, previous experiments have found
that females tend toward slower acquisition of context fear
(Maren et al. 1994). In the current study, context conditioning
was not different for male and female mice. This could be ex-
plained by the amount of exposure our animals had to the con-
text. A recent study showed that sex differences in the acquisi-
tion of context fear depend on the amount of exposure to the
training environment (Wiltgen et al. 2001). With longer
amounts of context exposure, females acquire the same amount
Figure 2. Data from the tone and context tests following delay fear conditioning. (A) Mean (SEM)
percent freezing for female mice during each period of the tone test. (B) Mean (SEM) percent
freezing for female mice during the context test. (C) Mean (SEM) percent freezing for male mice
during each period of the tone test. (D) Mean (SEM) percent freezing for male mice during the
context test.
Trace conditioning and the subunit
Learning & Memory 329
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of context fear as males. In the current study, all animals were in
the training context for an extended period of time, which likely
alleviated any sex difference in context fear.
Using a trace conditioning procedure, we found enhanced
tone and context fear in female KOs. A performance ceiling did
not preclude an effect in male KOs as they showed less trace
conditioning than females. Males were also capable of freezing at
higher levels, for example, following delay conditioning (Fig.
2C). The sex-specificity of this effect suggests the involvement of
neuroactive steroids. There are sex differences in neurosteroid
levels and in the response of GABA
A
Rs to these compounds (Ro-
bel and Baulieu 1995; Wilson and Biscardi 1997; Reddy and
Kulkarni 1999). Neurosteroid levels and their efficacy also vary
throughout the estrus cycle (Finn and Gee 1993; Palumbo et al.
1995; Reddy and Kulkarni 1999). The general action of these
compounds is to increase GABAergic inhibition and impair hip-
pocampus-dependent learning (Finn and Gee 1993; Palumbo et
al. 1995). Therefore, the enhanced conditioning observed in fe-
male KOs could be a consequence of their reduced responsive-
ness to neurosteroids. The fact that the enhancement was only
observed following trace conditioning, a task that depends on
the hippocampus, suggests the dentate gyrus is the site of action.
There is evidence that fluctuations in GABAergic inhibition
during the estrus cycle produce changes in learning. In eyeblink
conditioning, learning is enhanced in females during proestrus
and reduced during estrus and diestrus (Shors et al. 2000). The
enhanced learning observed during proestrus results from a rise
in estrogen, which increases the formation of dendritic spines.
This proliferation is a direct result of reduced GABAergic inhibi-
tion (Murphy et al. 1998; Segal and Murphy 2001). In the hip-
pocampus, estrogen receptors are located on glutamic acid decar-
boxylase (GAD) positive interneurons necessary for the synthesis
of GABA. The presence of estradiol significantly reduces the
amount of GAD found in these neurons (Murphy et al. 1998).
Conversely, GABA agonists increase inhibition and block estro-
gen-induced increases in dendritic spines (Segal and Murphy
2001). Therefore, enhanced learning
during proestrus likely results from a re-
duction in GABAergic inhibition and the
subsequent increase in dendritic spines.
Progesterone, which peaks during
estrus, acts to oppose the effects of estro-
gen. Progesterone increases GABAergic
inhibition and reduces the number of
dendritic spines in the hippocampus
(Woolley and McEwen 1993; Murphy
and Segal 2000; Segal and Murphy
2001). It is likely that these changes are
mediated by the synthesis of endog-
enous neurosteroids, natural metabo-
lites of progesterone (Murphy and Segal
2000). Neurosteroid levels and their effi-
cacy vary in the hippocampus during
the female cycle (Finn and Gee 1993; Pa-
lumbo et al. 1995), and direct adminis-
tration of these compounds increases in-
hibition and impairs hippocampus-
dependent learning (Paul and Purdy
1992; Johansson et al. 2002). Therefore,
it is possible that neurosteroid-mediated
inhibition acts to reduce the number of
dendritic spines in the hippocampus
during estrus and diestrus and impair
subsequent learning. KOs would be im-
mune to these effects because deletion of
this subunit substantially reduces the
sensitivity of GABA
A
Rs to neurosteroids. A closer analysis of our
trace conditioning data supports this idea. Histograms of the
trace conditioning scores (averaged across all test periods) are
shown in Figure 4. Female wild type (WT) (Fig. 4A) and Hets (Fig.
4B) showed varying amounts of conditioning with most animals
freezing at low to moderate levels (0%40%) and very few mice
freezing at high levels (>40%). This distribution was completely
reversed in Homo KOs (Fig. 4C). The majority of these animals
froze at high levels, and very few exhibited low to moderate
conditioning. This suggests that deletion of the subunit in-
creased freezing in animals that normally would show low levels
of conditioning. The fact that the distribution was completely
reversed in KOs (and not simply shifted to the right) suggests that
deletion of the subunit did not generally enhance learning.
Instead, there was a selective benefit for those animals exhibiting
the lowest levels of conditioning. These results are exactly what
one would predict if neurosteroids impaired hippocampus-
dependent learning during the estrus cycle. Male mice did not
show a similar profile (Fig. 4DF). Mice from each genotype, in-
cluding Homo KOs, showed the same distribution of freezing
scores.
The ability of neurosteroids to modulate neuronal plasticity
has generated substantial interest in their role in learning and
memory processes and their potential use as treatments for age-
related and cognitive diseases (Schumacher et al. 1997). Recent
evidence has also shown that these compounds play a critical
role in the mediation of anxiety, particularly in females (Engel
and Grant 2001; Toufexis et al. 2004). Consistent with these
ideas, we suggest that neurosteroids affect fear learning in fe-
males by acting on -containing GABA
A
Rs in the hippocampus.
Materials and Methods
Subjects
All mice were of a mixed C57Bl/6J 129Sv/SvJ genetic back-
ground. The mice were generated and genotyped as described
Figure 3. Data from the openfield and RotaRod tests. (A) Mean (SEM) activity scores for female
mice across the 20-min openfield test. (B) Mean (SEM) latency to fall from the RotaRod for female
mice across 5 d. (C) Mean (SEM) activity scores for male mice across the 20-min openfield test. (D)
Mean (SEM) latency to fall from the RotaRod for male mice across 7 d.
Wiltgen et al.
330 Learning & Memory
www.learnmem.org
previously (Mihalek et al. 1999). All experiments were conducted
with homozygous (Homo) and heterozygous (Het) KOs and their
wild-type littermates. The mice, which ranged from 4 to 6
months of age, were maintained on a 12 h light/12 h dark cycle
in the Psychology Department vivarium at UCLA. Animals were
group housed with free access to food and tap water. All proce-
dures were performed during the light phase of the light:dark
cycle.
Fear conditioning
Fear conditioning took place in four identical observation cham-
bers (28 21 21 cm; Lafayette Instrument Co.) located in a
well-lit room. A video camera was positioned in front of the
chambers, which allowed behavior to be observed and recorded
by an experimenter in an adjacent room. The floor of each cham-
ber consisted of 33 stainless steel rods (2 mm diameter) spaced 6
mm apart (center to center). The rods were wired to a shock
generator and scrambler (Med-Associates Inc.) for the delivery of
footshock. A speaker was mounted on the top of each chamber
and wired to an audio generator (Med-Associates Inc.) for deliv-
ery of the tone CS. Background noise (60 dB) was supplied by a
fan positioned underneath the video camera. Prior to condition-
ing, the chambers were cleaned with a 1% acetic acid solution
and pans containing a thin film of the same solution were placed
underneath the grid floors. The tone test was conducted in novel
chambers (B context) that were structurally different than the
conditioning chambers (A context). These chambers had a Plexi-
glas floor (28 21 cm) and two white plastic side walls (24 21
cm) placed at 60° to the floor, forming a triangular enclosure. The
room was dimly lit by a red lightbulb. Background noise (60 dB)
was supplied by a white noise generator. Prior to the tone test,
the chambers were cleaned with a 5% sodium hydroxide solu-
tion, and pans containing a thin film of the same solution were
placed underneath the Plexiglas floors.
On the conditioning day, mice were brought from the vi-
varium into a holding room and allowed to sit undisturbed in
their homecages for 10 min. Mice then underwent either trace or
delay fear conditioning. In each proce-
dure, the mice were placed in the condi-
tioning context and allowed to explore
for 3 min before the onset of the tone
(20 sec, 80 dB, 2800 Hz). In the trace
conditioning groups, tone termination
and shock onset were separated by a 20-
sec interval (trace interval). In the delay
conditioning groups, tone termination
was contiguous with shock (2 sec, 0.5
mA) onset. Both the delay and trace con-
ditioning groups received five condi-
tioning trials, each separated by a 200-
sec intertrial interval (ITI). The mice
were removed from the conditioning
chambers 2 min after the last shock
presentation and returned to their
homecages. Then, 24 h later, the mice
were placed in the B context for a tone
test. The tone test consisted of a 2-min
baseline period (BL) followed by three
20-sec tone presentations. Each tone
presentation was separated by a 220-sec
ITI. The freezing response (a defensive
posture defined as the absence of mo-
tion except that necessitated by breath-
ing) was used as a measure of condi-
tional fear (Bolles and Collier 1976). It
was measured using a time sampling
procedure in which an observer scored
the presence or absence of the freezing
response for each mouse every 2 sec.
Data were transformed into a percent
freezing score by dividing the number of
freezing observations by the total num-
ber of observations and multiplying by
100. For statistical analyses, freezing scores were averaged across
the entire test and grouped into four bins: BL, Tone, Trace, and
ITI. The Trace bin refers to the 20 sec immediately following tone
termination, and the ITI bin is the subsequent 200 sec. This pe-
riod corresponds to the trace interval used in the trace condition-
ing experiment. Although a trace interval was not used for the
delay conditioning groups during training, freezing was analyzed
during this period for comparative purposes. The day after the
tone test, the mice were placed back in the original conditioning
box (A context) for an 8-min context test. During this test, freez-
ing was scored for each mouse every 8 sec. For trace conditioning,
nine wild-type, nine Het, and nine Homo male mice and nine
wild-type, 10 Het, and 11 Homo female mice were used. For delay
conditioning, nine wild-type, nine Het, and nine Homo male
mice and nine wild-type, nine Het, and 10 Homo female mice
were used. All mice were naive prior to training.
Openfield
The activity monitors (Med-Associates) were made of polycar-
bonate plastic (27 27 20.3 cm.). Activity was tracked by 16
photobeams spaced evenly apart on both x- and y-axes. Mice
were placed in the chambers (located in a dimly lit room), and
ambulatory counts were calculated over a 20-min period. The
chambers were washed thoroughly with Windex between mice.
In this experiment, there were eight wild-type, nine Het, and 11
Homo male mice and five wild-type, 10 Het, and five Homo
female mice. Data from two males (Hets) and one female (WT)
were excluded from statistical analyses because of equipment
malfunction.
RotaRod
The RotaRod consisted of a drum made of ribbed plastic and
panels that separated the apparatus into five lanes (Technical and
Scientific Equipment). Mice were placed on the RotaRod while it
was rotating at 5 rpm. As soon as all mice were placed on the
apparatus, the acceleration and timer were activated and rota-
tional speed steadily increased from 5 to 60 rpm over the course
Figure 4. Histograms from the trace fear conditioning test. The data were averaged across all test
periods and are expressed as percent animals exhibiting low (0%–20%), moderate (20%–40%), and
high (40%–60%) freezing scores.
Trace conditioning and the subunit
Learning & Memory 331
www.learnmem.org
of 3 min. On each day, every mouse was tested three times se-
quentially. The average of the three trials was the latency value
used for that day. The same mice from the openfield experiment
were used on the Rotarod. One male (Het) was excluded from the
experiment because of injuries obtained in the homecage.
Acknowledgments
This work was supported by an NIH grant (P01NS35985) to M.S.F.
and a UCLA Research Mentorship Fellowship to B.J.W.
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Trace conditioning and the subunit
Learning & Memory 333
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    • "In addition, genetic deletion of the delta subunit of the GABA receptors reduces the synaptic inhibition in the hippocampus. Compared to controls, delta knockout mice exhibited an enhanced acquisition of tone and context fear [64]. In our experimental model, we asked whether in addition to the LTP found in the parallel fibres to Purkinje cell synapses, there was any long-term change in the inhibitory synapses. "
    [Show abstract] [Hide abstract] ABSTRACT: Great attention has been given so far to cerebellar control of posture and of skilled movements despite the well-demonstrated interconnections between the cerebellum and the autonomic nervous system. Here is a review of the link between these two structures and a report on the recently ac-quired evidence for its involvement in the world of emotions. In rodents, the reversible inactivation of the vermis during the consolidation or the reconsolidation period hampers the reten-tion of the fear memory trace. In this region, there is a long-term potentiation of both the excitatory synapses between the parallel fibres and the Purkinje cells and of the feed-forward inhibition mediated by molecular layer interneurons. This concomitant potentiation ensures the temporal fidelity of the system. Additional contacts between mossy fibre terminals and Golgi cells provide morphological evidence of the poten-tiation of another feed-forward inhibition in the granular layer. Imaging experiments show that also in humans the cerebellum is activated during mental recall of emotional personal epi-sodes and during learning of a conditioned or unconditioned association involving emotions. The vermis participates in fear learning and memory mechanisms related to the expres-sion of autonomic and motor responses of emotions. In humans, the cerebellar hemispheres are also involved at a higher emotional level. The importance of these findings is evident when considering the cerebellar malfunctioning in psychiatric diseases like autism and schizophrenia which are characterized behaviourally by emotion processing impairments. http://link.springer.com/article/10.1007%2Fs12311-015-0649-9
    Full-text · Article · Jan 2015
    • "To investigate if the sensitivity of the GABAergic system was altered by life-long depletion of brain 5-HT we challenged male mice systemically with diazepam, a GABA-A receptor allosteric enhancer, and analyzed anxiety-like behavior in two behavioral paradigms (open field with low illumination and elevated plus maze with high illumination). Since diazepam has multiple effects depending on the particular receptor subunit (SU) com- position [64,65], allosteric modulation of the heteropentameric GABA-A receptor can induce different effects, including sedative myo-relaxation (1 SU is necessary), anxiolysis (2/3 SU), and cognitive-mnestic impairments (5 SU)66676869 . Therefore, benzodiazepines can affect serotonin-related dysfunctions such as anxiety disorders, reward-craving, hyperalgesia, and depressive disorders [70]. "
    [Show abstract] [Hide abstract] ABSTRACT: Tryptophan hydroxylase (TPH) is a rate limiting enzyme in the synthesis of serotonin (5-HT), a monoamine which works as an autacoid in the periphery and as a neurotransmitter in the central nervous system. In 2003 we have discovered the existence of a second Tph gene, which is expressed exclusively in the brain, and, therefore, is responsible for the 5-HT synthesis in the central nervous system. In the following years several research groups have independently generated Tph2-deficient mice. In this review we will summarize the data gained from the existing mouse models with constitutive or conditional deletion of the Tph2 gene, focusing on biochemical, developmental, and behavioral consequences of Tph2-deficiency.
    Full-text · Article · Jun 2014
    • "Thus, it will be of interest to examine the influence of ALLO on in vivo hippocampal synaptic plasticity and neuronal activity that represents the encoding of spatial and nonspatial memory, to more completely establish the mechanisms by which this neurosteroid affects learning and memory. In contrast to the ability of exogenous ALLO to disrupt memory, genetic deletion of the δ subunit of the GABA-A receptor enhanced hippocampal-dependent trace fear conditioning (Wiltgen et al., 2005). The δ subunit mRNA of the GABA-A receptor are preferentially expressed in the cerebellum, thalamus, and dentate gyrus (Pirker et al., 2000; Wisden et al., 1992 ). Given its effect on hippocampal memory , it will be of interest to examine whether the memory-impairing effects of systemic and intra-hippocampal ALLO can be recapitulated by direct infusion into the dentate gyrus, as well as to determine whether administration of ALLO into the dorsal vs. ventral hippocampus exerts differential effects on memory, since the ventral hippocampus may hold a more significant role in emotional memory. "
    [Show abstract] [Hide abstract] ABSTRACT: Allopregnanolone (ALLO, or 3α-hydroxy-5α-pregnan-20-one) is a steroid metabolite of progesterone and a potent endogenous positive allosteric modulator of GABA-A receptors. Systemic ALLO has been reported to impair spatial, but not nonspatial learning in the Morris water maze (MWM) and contextual memory in rodents. These cognitive effects suggest an influence of ALLO on hippocampal-dependent memory, although the specific nature of the neurosteroid's effects on learning, memory or performance is unclear. The present studies aimed to determine: i) the memory process(es) affected by systemic ALLO using a nonspatial object memory task; and (ii) whether ALLO affects object memory via an influence within the dorsal hippocampus. Male C57BL/6J mice received systemic ALLO either before or immediately after the sample session of a novel object recognition (NOR) task. Results demonstrated that systemic ALLO impaired the encoding and consolidation of object memory. A subsequent study revealed that bilateral microinfusion of ALLO into the CA1 region of dorsal hippocampus immediately following the NOR sample session also impaired object memory consolidation. In light of debate over the hippocampal-dependence of object recognition memory, we also tested systemic ALLO-treated mice on a contextual and cued fear-conditioning task. Systemic ALLO impaired the encoding of contextual memory when administered prior to the context pre-exposure session. Together, these results indicate that ALLO exhibits primary effects on memory encoding and consolidation, and extend previous findings by demonstrating a sensitivity of nonspatial memory to ALLO, likely by disrupting dorsal hippocampal function.
    Full-text · Article · May 2014
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