1
Introduction
Anxiety disorders, such as post-traumatic stress disorder, social phobia, specific
phobias, panic and obsessive-compulsive disorders, are among the most common mental
disorders in humans (Olesen et al., 2012). These disorders are associated with problems
to extinguish learned fear responses and also to consolidate extinction memories (Myers
and Davis, 2007; Milad et al., 2009). To date, the most effective strategies in the
treatment of anxiety disorders include use of pharmacological and cognitive strategies
(Nutt, 2005). However, significant drawbacks to current therapy exist including lack
of treatment response and also a lack of long-term beneficial effect of combining
available drugs with exposure-based therapy (Barlow et al., 2000; Davidson et al.,
2004; Foa et al., 2005; Yehuda and LeDoux, 2007; Norberg et al., 2008).
Clinical interventions which rescue deficits in fear extinction acquisition and consolidation
would constitute important treatment options in these disorders. Along these lines,
pharmacological and genetic silencing studies, predominantly in normally behaving
rodents and using massed extinction training paradigms, have identified a number of
neurobiological mechanisms that govern acquisition of fear extinction learning and
extinction consolidation. Acquisition of extinction learning requires N-methyl-d-aspartate
(NMDA) receptor 2B (Sotres-Bayon et al., 2007), α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic
acid (AMPA) (Zushida et al., 2007; Yamada et al., 2009) and metabotropic glutamate
7 (mGlu7) (Fendt et al., 2008) receptor activities in addition to GABAergic signalling
[for example, see (Harris and Westbrook, 1998; Chhatwal et al., 2005; Akirav et al.,
2006; Lin et al., 2009; Dalton et al., 2012)], amongst others. Following acquisition
of extinction, there is a consolidation phase which lasts up to several hours which
serves to stabilize plastic events into long-term memory (McGaugh, 2000). Activation
of NMDA receptors (Davis, 2011), brain-derived neurotrophic factor (BDNF) signalling
(Chhatwal et al., 2006; Soliman et al., 2010) and inhibition of histone deacetylases
(HDAC) (Guan et al., 2009; McQuown et al., 2011), amongst others, are important for
consolidation of extinction [for detailed reviews, see (Myers and Davis, 2007; Quirk
and Mueller, 2008; Herry et al., 2010; Pape and Pare, 2010; Steckler and Risbrough,
2011; Orsini and Maren, 2012; Tronson et al., 2012)].
The present study used isogenic 129S1/SvImJ (S1) mice which display deficient extinction
acquisition, poor recovery of fear-induced suppression of heart-rate variability,
enlarged dendritic arbours in basolateral amygdala neurons and functional abnormalities
in cortico-amygdala circuitry mediating fear extinction (Hefner et al., 2008; Camp
et al., 2009, 2012; Whittle et al., 2010). In the current study we reduced the intensity
of fear conditioning and observed that “weak” fear conditioning allowed for extinction
acquisition in S1 mice but revealed deficient extinction retrieval. Recent findings
show that impaired extinction acquisition and dysfunctional cortico-amygdala circuitry
can be rescued by a novel zinc restricted diet (Whittle et al., 2010), which interestingly
did not affect either fear learning or fear expression. Also, recent findings reveal
that the S1 fear extinction phenotype can be rescued by fluoxetine (a serotonin-selective
reuptake inhibitor) (Camp et al., 2012) which is of clinical relevance as serotonin-selective
reuptake inhibitors are first-line treatment for anxiety disorders (Nutt, 2005). Considering
the well-known disadvantages of long-term SSRI treatments, we screened for novel treatments
which rescue both deficient extinction acquisition and extinction consolidation/retrieval
in S1 mice.
Evidence suggests that deep brain stimulation (DBS) of the nucleus accumbens (AcbC)
can enhance extinction-like behaviour in intractable obsessive-compulsive disorder
patients, which is characterised by avoidance behaviours that fail to extinguish (Lipsman
et al., 2007; Burdick et al., 2009; Goodman et al., 2010; Greenberg et al., 2010;
Grant et al., 2011). Furthermore, a small clinical study involving patients suffering
from treatment-resistant depression has shown that DBS of the AcbC can enhance various
measures of cognition independent of mood status (Bewernick et al., 2012). However,
there are also reports of no effect of AcbC-DBS in mood disorder patients (Grubert
et al., 2011). Very recently, DBS of the AcbC during extinction training reduced fear
expression and strengthened extinction memory in normally behaving rats (Rodriguez-Romaguera
et al., 2012) potentially showing the utility of DBS of the AcbC as a novel fear reducing
adjunct during exposure-based therapy. Here, we went beyond using normally behaving
rodents and assessed whether DBS of the AcbC during extinction training can rescue
deficient extinction acquisition and extinction consolidation/retrieval in S1 mice.
Pharmacological compounds which enhance glutamatergic signalling, including DCS (NMDA
receptor partial agonist), PEPA (AMPA receptor potentiator) or AMN082 (mGlu7 receptor
agonist), can facilitate fear extinction in normally extinguishing animal models and
in clinical studies (Myers et al., 2011). We have previously shown that prior extinction
training administration of DCS is ineffective in rescuing deficient extinction learning
in S1 mice (Hefner et al., 2008). However, it remains unknown whether DCS can rescue
deficient extinction consolidation/retrieval once at least some extinction learning
is induced, which we revealed in “weak” fear conditioned S1 mice. Furthermore, it
is not known whether PEPA or AMN082 can reduce fear in an animal model of impaired
extinction.
Drugs inhibiting HDAC proteins to prevent the removal of acetyl groups on histone
tails is a promising approach in central nervous system disorders associated with
disturbed learning and memory (Kazantsev and Thompson, 2008). Interestingly, inhibiting
HDACs has been shown to enhance fear learning (Guan et al., 2009) and rescue deficits
in fear learning in mouse models of anxiety or neurodegeneration (Li et al., 2006;
Dash et al., 2009; Kilgore et al., 2010). Facilitation of fear extinction by HDAC
inhibition has been reported in normally extinguishing mice (Bredy et al., 2007; Lattal
et al., 2007; Bredy and Barad, 2008; Guan et al., 2009; Monsey et al., 2011). However,
it is not known whether inhibiting HDACs can improve extinction learning in animal
models of impaired extinction. Thus, we tested whether valproic acid (VPA), a dual
HDAC inhibitor and enhancer of GABAergic signalling or MS-275, a more specific HDAC
inhibitor (Khan et al., 2008; Bantscheff et al., 2011), can rescue deficient extinction
in S1 mice.
2
Materials and methods
2.1
Subjects
Subjects were male 3–5 month old 129S1/SvImJ (S1) mice. For experiments performed
at the University of Innsbruck, mice (obtained from Charles River, Germany) were housed
(4–5 per cage) in a temperature- (22 ± 2 °C) and humidity- (50–60%) controlled vivarium
under a 12 h light/dark cycle (lights on at 7:00 A.M.). For experiments conducted
at the National Institutes of Health, mice (obtained from The Jackson Laboratory,
USA) were housed (2 per cage) in a temperature (22 ± 1 °C)- and humidity (45 ± 15%)-controlled
vivarium under a 12 h light/dark cycle (lights on, 6:00 A.M.). All experimental procedures
were approved by the Austrian Animal Experimentation Ethics Board and by the National
Institute on Alcohol Abuse and Alcoholism Animal Care and Use and Austrian Ethical
Committees on Animal Care and Use (Bundesministerium für Wissenschaft und Verkehr,
Kommission für Tierversuchsangelegenheiten) and followed the National Institutes of
Health guidelines outlined in Using Animals in Intramural Research and the local animal
care and use committees.
2.2
General procedures for fear conditioning and extinction
For experiments performed at the University of Innsbruck, mice were conditioned in
a 25 × 25 × 35 cm chamber with transparent walls and a metal rod floor, cleaned with
water and illuminated to 300 lux (‘context A’). After a 120 s acclimation period,
there were 3× pairings of a 120 s, 80 dB white noise conditioned stimulus (CS) and
a 2 s scrambled foot shock unconditioned stimulus (US) (0.6 mA unless stated otherwise),
with a 120 s inter-pairing interval. There was a 120 s no-stimulus consolidation period
after the final pairing before mice were returned to the home cage. Extinction training
was performed the next day in a 25 × 25 × 35 cm cage with a solid grey floor and black
walls, cleaned with a 100% ethanol and illuminated to 10 lux (‘context B’). After
a 120 s acclimation period, there were CS-alone trials (16 unless stated otherwise),
with a 5-sec inter-CS interval. Stimulus presentation was controlled by a TSE operant
system (TSE, Bad Homburg, Germany). Extinction retrieval was conducted the following
day by repeating the extinction training procedure but with 2× CS trials. Freezing
was measured as an index of fear (Blanchard and Blanchard, 1969), manually scored
based on DVD recordings of the duration of the CS, defined as no visible movement
except that required for respiration, and converted to a percentage [(duration of
freezing within the CS/total time of the CS) × 100] by a trained observer blind to
the animals' group.
For experiments performed at NIAAA, mice were conditioned in a 27 × 27 × 11 cm chamber
with a metal-rod floor, cleaned with a 79.5% water/19.5% ethanol/1% vanilla-extract
solution (‘context A’). After a 180 s acclimation period, there were 3× pairings (60–120 s
inter-pairing interval) of the conditioned stimulus (CS; 30 s 80 dB, 3 kHz tone) and
the unconditioned stimulus (US; 2 s, 0.6 mA scrambled foot shock), in which the US
was presented during the last 2 s of the CS. The session ended 120 s after the final
CS–US pairing. Stimulus presentation was controlled by the Med Associates VideoFreeze
system (Med Associates, Burlington, VT, USA). 24 h after conditioning, extinction
training was conducted in a novel context (‘context B’) (cylinders with black/white-chequered
walls and a solid Plexiglas opaque floor cleaned with a 1% acetic acid/99% water solution)
housed in a different room from conditioning. After a 180 s acclimation period, there
were 16× CS presentations (5-sec inter-CS interval). Extinction retrieval was tested
in context B 48 h after extinction training. After a 180 s acclimation period, there
were 3× CS presentations (5-sec inter-CS interval). Freezing (no visible movement
except respiration) was scored every 5 s by an observer blind to condition/treatment
and converted to a percentage [(freezing observations/total observations) × 100].
2.3
Extinction in S1 mice after weak versus normal fear conditioning
Procedures and apparatuses for normal fear conditioning were as described above and
involved 3× pairings of the CS with a 0.6 mA US. The weak fear conditioning procedure
was the same with the exception that the US was 0.3 mA. Mice were randomly assigned
to either weak or normal fear conditioning and tested for extinction training and
retrieval as above (Fig. 1A).
2.4
Effects of deep brain stimulation of the nucleus accumbens
Mice were anaesthetized with a mixture of ketamine/xylazine (80/5 mg/kg) injected
intraperitoneally (i.p.). Using a stereotaxic frame, electrodes (MS303-3-B-SPC, bipolar,
twisted, 8 mm, Plastics ONE, USA) were implanted unilaterally, targeting the core
of the nucleus accumbens. The coordinates were +1.10 mm anterior to Bregma, +1.45 mm
lateral from midline, −4.65 mm ventral according to the atlas of (Franklin and Paxinos,
2008). Electrodes were fixed to the skull with 2 stainless steel screws and dental
cement (Havard Cement, Richter & Hoffmann Harvard Dental GmbH, Germany). After surgery
animals were individually caged and received buprenorphin (0.1 mg/kg injected intraperitoneally)
every 8 h for 3 days to minimise pain.
After 4–5 days of recovery, mice were conditioned via 5× pairings of the CS with a
0.7 mA US. Extinction training began with 2× CS-alone trials without DBS to measure
fear expression in the absence of any influence of DBS before presenting 16× CS-alone
extinction trials. DBS (130 Hz, 100 μA, 60 μs pulse width) was applied via an A310
Accupulser and A365 Stimulus Isolator (World Precision Instruments, Sarasota, USA)
throughout the CS period during each 16× CS extinction trials (Fig. 2A). Sham controls
were connected to the stimulator but received no current. At the completion of testing,
the localization of electrodes was confirmed from inspection of cresyl violet stained
coronal sections (Fig. 2C), and mice with misplaced electrodes were excluded.
2.5
Effects of VPA administration
Mice were conditioned via 3× pairings of the CS with a 0.6 mA US. 100 mg/kg VPA (Sigma,
St. Louis, USA) or saline vehicle was injected i.p. in a volume of 10 ml/kg body weight
2 h prior to extinction training (Fig. 3A). The dose, route and timing of administration
was based on previous studies showing extinction facilitating effects of VPA in mice
(Bredy et al., 2007; Bredy and Barad, 2008) and that 2 h is required before maximal
histone acetylation is observed (Tremolizzo et al., 2005).
2.6
Effects of AMN082 administration
Mice were conditioned via 3× pairings of the CS with a 0.6 mA US. 6 mg/kg AMN082 (Tocris
Bioscience, Bristol, United Kingdom) or saline + 0.5% methylcellulose vehicle was
administered per oral (p.o.) via gavage in a volume of 10 ml/kg body weight 2 h prior
to extinction training (Fig. 4A). The dose, route and timing of administration was
based on previous studies showing extinction facilitating effects of AMN082 in mice
(Fendt et al., 2008).
2.7
Effects of PEPA administration
Mice were conditioned via 3× pairings of the CS with a 0.6 mA US. 10 or 30 mg/kg PEPA
(Sigma, St. Louis, USA) or saline + 0.45% NaCl + 33% (2-hydroxypropyl)-β-cyclodextrin
vehicle was injected i.p. in a volume of 10 ml/kg body weight 15 min prior to extinction
training (Fig. 5A). The doses, route and timing of administration was based on previous
studies showing extinction facilitating effects of PEPA in mice (Zushida et al., 2007;
Yamada et al., 2009).
2.8
Effects of MS-275 administration
Mice were conditioned via 3× pairings of the CS with a 0.6 mA US. 5 or 10 mg/kg MS-275
(Sigma, St. Louis, USA) or saline + 25% DMSO vehicle was injected i.p. in a volume
of 10 ml/kg body weight 2 h prior to extinction training (Fig. 6A). The doses, route
and timing of administration was based on a previous study showing that maximal increases
in histone acetylation occurs 2 h following s.c. injection of MS-275 (Simonini et al.,
2006).
2.9
Effects of MS-275 and DCS administration after weak conditioning
Mice were conditioned via 3× pairings of the CS with a 0.3 mA US. 10 mg/kg MS-275
(Eubio, Vienna, Austria), 15 mg/kg DCS (Sigma, St. Louis, USA) or saline + 25% DMSO
vehicle was injected i.p. in a volume of 10 ml/kg body weight immediately (<2 min)
after extinction training (Fig. 7A). The doses, route and timing of administration
of MS-275 was based on our earlier experiment and the doses, route and timing of administration
of DCS was based on previous studies showing extinction facilitating effects in rats
and humans (Walker and Davis, 2002; Ressler et al., 2004; Richardson et al., 2004).
2.10
Statistical analysis
All data were examined for equal variances using Levene's test before performing parametric
tests. Data were analyzed using analysis of variance (ANOVA), with repeated measures
for trial/trial-block, or by Student's t-test. Significant ANOVA results were followed
by Fisher's LSD post hoc tests.
3
Results
3.1
Weak fear conditioning in S1 mice permits fear reduction during extinction training,
but not retrieval
We have previously found that the level of fear expression in S1 mice can be modulated
by the intensity (shock amplitude, shock-tone trial number) of fear conditioning (Camp
et al., 2009). Therefore, we asked whether extinction in S1 was graded by the intensity
of conditioning. There was a significant and similar increase in freezing across 3
conditioning trials in mice given 0.3 mA and 0.6 mA shocks [ANOVA effect of trial:
F
(2, 20) = 80. 15, p < 0.001; effect of group: p > 0.05, interaction: p > 0.05, n = 6/group]
(Fig. 1B). During extinction training, there was a significant group × trial–block
interaction for freezing (F
(7, 70) = 8.71, p < 0.001). Post hoc tests revealed that the weak shock group displayed
lower freezing than the 0.6 mA group on blocks 3–8, but not blocks 1 or 2 (Fig. 1B).
However, during extinction retrieval, freezing was not different between groups (t-test:
p > 0.05) (Fig. 1B). These results show that while this weak conditioning procedure
did not reduce fear learning or expression compared to normal conditioning, it did
allow for significant fear reduction during the extinction learning trial in S1 mice.
However, extinction memory was not successfully consolidated/retrieved on the next
day.
3.2
Deep brain stimulation of the nucleus accumbens rescues impaired extinction retrieval
We first examined the effects of DBS of the AcbC, applied during extinction training,
on impaired extinction in S1 mice. During conditioning, freezing increased across
trials and did not differ between groups [ANOVA effect of trial: F
(4, 64
) = 109.80, p < 0.001, effect of group: p > 0.05, interaction: p > 0.05, n = 9/group]
(Fig. 2B). DBS during extinction training did not affect freezing, as compared to
sham-stimulated controls, as both groups showed no change in freezing across trial-blocks
[ANOVA effect of group: p > 0.05, effect of trial-block: p > 0.05, group × trial-block
interaction: p > 0.05] (Fig. 2B). During extinction retrieval, freezing was significantly
lower in DBS than sham controls during extinction retrieval (t
16 = 2.96, p < 0.01) (Fig. 2B). These results show that DBS of the nucleus accumbens
rescued extinction retrieval, but not extinction acquisition, in S1 mice.
3.3
VPA rescues impaired extinction learning and retrieval
We next examined the effects of 3 diverse pharmacological compounds, applied acutely
prior to extinction training, on impaired S1 extinction. Our first experiment tested
the effects of VPA. During conditioning, freezing increased across trials and did
not differ between groups [ANOVA effect of trial: F
(2, 32
) = 347.05, p < 0.001, effect of group: p > 0.05, interaction: p > 0.05, n = 8–9/group]
(Fig. 3B). There was a significant group × trial block interaction for freezing during
training (F
(7, 98) = 3.24, p < 0.01). Post hoc tests showed freezing was significantly lower
in the VPA group relative to vehicle, on trial-blocks 2–8 (Fig. 3B). During extinction
retrieval, freezing was again significantly lower in the VPA than the vehicle group
(t
15 = 4.75, p < 0.001) (Fig. 3B). These data show that VPA rescues impaired extinction
learning and retrieval in S1 mice.
3.4
AMN082 rescues impaired extinction learning and retrieval
Our next experiment examined the effects of pre-extinction-administered AMN082. During
conditioning, freezing increased across trials and did not differ between groups [ANOVA
effect of trial: F
(2, 28
) = 90.25, p < 0.001, effect of group: p > 0.05, interaction: p > 0.05, n = 8/group]
(Fig. 4B). There was a significant treatment × trial-block interaction for freezing
during extinction training (F
(7, 98) = 4.82, p < 0.001). Post hoc tests showed significantly lower freezing in
the AMN082 treated group as compared to vehicle on trial-blocks 3–8 (Fig. 4B). Freezing
was also significantly lower in the AMN082 group than the vehicle group during extinction
retrieval (t
14 = 4.24, p < 0.001) (Fig. 4B). The results of this experiment indicate rescue of
impaired S1 extinction learning and retrieval by AMN082.
3.5
PEPA does not rescue either impaired extinction learning or retrieval
We next studied the effects PEPA applied before extinction. During conditioning, freezing
increased across trials and did not differ between groups [ANOVA effect of trial:
F
(2, 42
) = 140.25, p < 0.001, effect of group: p > 0.05, interaction: p > 0.05, n = 8/group]
(Fig. 5B). There was no effect of treatment on freezing during extinction training
and neither group showed a reduction in freezing across trial-blocks (Fig. 5B). Freezing
was also no different between groups on extinction retrieval (Fig. 5B). These data
show that, unlike VPA and AMN082, PEPA failed to rescue impaired S1 extinction.
3.6
MS-275 does not rescue either impaired extinction learning or retrieval
In our next experiment, we examined the ability of MS-275, administered prior to extinction
training, to rescue impaired S1 extinction retrieval. Freezing increased across conditioning
trials, and treatment groups did not differ from one another [ANOVA effect of trial:
F
(2, 42
) = 90.82, p < 0.001, effect of group: p > 0.05, interaction: p > 0.05, n = 7–10/group]
(Fig. 6B). There was no effect of treatment on freezing during extinction training
and neither group showed a reduction in freezing across trial-blocks (Fig. 6B). Freezing
was also no different between groups on extinction retrieval (Fig. 6B). This experiment
shows that, like PEPA, application of MS-275 prior to extinction training failed to
rescue impaired S1 extinction.
3.7
After weak conditioning, MS-275 and DCS rescues impaired extinction retrieval
Our observation that S1 mice can exhibit extinction learning after weak conditioning,
but still have a deficit in extinction retrieval, led us to test whether two drug
treatments shown here (MCS-275) and previously (DCS) (Hefner et al., 2008) to be ineffective
in rescuing extinction after normal conditioning would be effective after weaker conditioning.
Freezing increased across conditioning trials, and treatment groups did not differ
from one another [ANOVA effect of trial: F
(2, 50
) = 163.80, p < 0.001, effect of group: p > 0.05, interaction: p > 0.05, n = 9–10/group]
(Fig. 7B). During extinction training, there was a significant reduction in freezing
across trials that was similar between groups [ANOVA effect of trial: F
(7, 175) = 88.18, p < 0.001, effect of group: p > 0.05, interaction: p > 0.05] (Fig. 7B).
Mice were treated immediately after training. There was a significant effect of treatment
on freezing during extinction retrieval (F
(2, 25) = 7.21, p < 0.01). Post hoc tests showed that freezing was significantly lower
in the DCS (p < 0.05) and MS-275 (p < 0.01) treated groups as compared to vehicle
(Fig. 7B). The results of this experiment show that MS-275 and DCS can rescue impaired
extinction retrieval in S1 mice given weak conditioning.
4
Discussion
The present study revealed a number of novel findings. Behaviourally, we show that
using a “weak” fear conditioning paradigm S1 mice were able to reduce fear during
extinction training, but were unable to consolidate/retrieve this memory during an
extinction retrieval test. This result, in addition to the well described profound
extinction acquisition deficit observed following “normal” conditioning (Hefner et al.,
2008; Camp et al., 2009; Whittle et al., 2010), reveal that S1 mice, by using graded
conditioning paradigms, can serve as an ideal model to dissociate whether extinction-promoting
interventions act via inducing extinction acquisition and/or by enhancing extinction
consolidation/retrieval. We next assessed a variety of treatments in their ability
to rescue the profound extinction acquisition deficit seen after normal fear conditioning
in S1 mice. Here we show that DBS of the AcbC during extinction training lowers fear
in extinction retrieval and that pharmacological compounds which enhance GABAergic
signalling in tandem with HDAC inhibition, or drugs that activate mGlu7 receptors,
rescue extinction acquisition deficits and lower fear during extinction retrieval
in S1 mice. AMPA receptor potentiation on the other hand was ineffective. Lastly,
we revealed that compounds which enhance NMDA receptor activity or inhibit HDACs rescue
the impaired extinction consolidation/retrieval deficit in S1 mice.
4.1
Weak fear conditioning in S1 mice permits fear reduction during extinction training,
revealing specific deficiency in extinction consolidation/retrieval
Present data reveal a novel behavioural finding that “weak” fear conditioned S1 mice
display impaired extinction consolidation/retrieval despite successful reduction of
fear during extinction training. No significant alterations in either fear learning
or fear expression were observed between “weak” and “normal” fear conditioned S1 mice
demonstrating the selectivity of the extinction consolidation/retrieval deficit. Importantly,
it is unlikely that sensitisation to the CS contributed to the observed extinction
retrieval deficit as we have previously shown that S1 mice do not sensitise to the
CS (Camp et al., 2009). Collectively, our current data and that of previously published
behavioural findings showing S1 mice display deficient extinction acquisition following
“normal” fear conditioning (Hefner et al., 2008; Camp et al., 2009; Whittle et al.,
2010), reveal that S1 mice represent a unique model to determine whether extinction
promoting treatments act via inducing extinction acquisition and/or enhancing extinction
consolidation by using “normal” and “weak” fear conditioning paradigms.
4.2
DBS of the AcbC during extinction training reduces fear in a retrieval test session
DBS of the AcbC during extinction training reduced freezing in the extinction retrieval
test indicating a novel treatment to overcome impaired extinction and thus to reduce
learned fear. The AcbC was chosen based on evidence that stimulation of the AcbC enhances
extinction-like cognitive performance in intractable obsessive-compulsive disorder
patients (Lipsman et al., 2007; Burdick et al., 2009; Goodman et al., 2010; Greenberg
et al., 2010; Grant et al., 2011) and is associated with long-term enhancement in
cognitive performance on tests including attention, learning and memory in remitted
depressed patients (Bewernick et al., 2012). Interestingly, the extinction promoting
effect of AcbC stimulation was independent of any observable reduction of fear during
extinction training. Reasons for this may be inferred from clinical studies showing
that DBS of the AcbC can induce/enhance symptoms of fear and panic (palpitations and
increased heart rate, flushing, fearful and panicky feelings) in patients (Shapira
et al., 2006; Okun et al., 2007). Thus, assessment of potential reductions in freezing
behaviour during the extinction session may be overlaid by enhancing fearful responses
by DBS of the AcbC per se.
The exact mechanisms via which DBS of the AcbC promotes extinction are not fully understood
and different hypotheses exist. DBS has been proposed to cause a functional lesion
by suppressing neural activity at the stimulated brain area, or that DBS elicits stimulation-induced
activation of axons resulting in a wide range of effects on local cells and at a circuit
level (McIntyre et al., 2004). However, existing data indicate that suppression of
AcbC activity may not mediate fear reductions directly since lesions of the AcbC (Riedel
et al., 1997; Levita et al., 2002; Josselyn et al., 2005), or temporary inactivation
(Haralambous and Westbrook, 1999; Rodriguez-Romaguera et al., 2012) do not disrupt
fear responses regardless of how fear is measured (fear-potentiated startle or conditioned
freezing). In addition, DBS of the AcbC does not evoke any change in local neurotransmitter
release within this brain region (van Dijk et al., 2011) further negating a direct
role of the AcbC mediating fear reduction. It is possible that DBS of the AcbC exerts
its fear reducing effect at a circuit level to modulate pathological network activity,
as our experiment used 130 Hz, 100 μA and 60 μs pulse width, conditions which can
influence fibres of passage (Nowak and Bullier, 1998a,b) and induce changes in excitability
and plasticity in brain regions critically involved in fear extinction. Specifically,
DBS of the AcbC selectively increases slow oscillatory activity (McCracken and Grace,
2009) and supresses pyramidal cell firing (McCracken and Grace, 2007) in the orbitofrontal
cortex, and increases spontaneous and stimulus-induced coherent activity between regions
in the thalamocortical system, which are implicated in extinction circuitry [e.g.
medial prefrontal cortex and mediodorsal thalamus (Pape and Pare, 2010; Lee et al.,
2011)]. Furthermore, increased expression of phosphorylated extra cellular-regulated
kinase (pERK), a marker of cell plasticity (Sweatt, 2004), is observed in brain regions
critical for extinction learning including the medial prefrontal cortex, orbitofrontal
cortex, and a small subregion of the lateral part of the central amygdala following
DBS in the AcbC (Rodriguez-Romaguera et al., 2012), indicating that many of exactly
those areas are targeted by AcbC DBS that have been shown to be aberrantly activated
in extinction-impaired S1 mice (Whittle et al., 2010). Collectively, our results reveal
for the first time that DBS of the AcbC rescued deficient extinction retrieval in
an animal model of impaired extinction, most likely by positively interfering with
the documented cortico-limbic extinction circuitry failure accompanying the impaired
extinction.
4.3
Enhancing mGlu7 receptor activity during extinction training reduces fear in S1 mice
We have previously revealed that enhancing the activity of ionotropic NMDA receptors,
via administration of DCS, is ineffective in inducing extinction acquisition in S1
mice (Hefner et al., 2008). Here, we investigated whether enhancing the activity of
ionotropic AMPA receptors by administration of PEPA, or metabotropic mGlu7 receptors,
by administration of AMN082, could rescue the extinction acquisition deficit in S1.
Administration of AMN082, but not PEPA, prior to an extinction training session induced
fear extinction and also reduced fear during a retrieval test in S1 mice. The lack
of extinction promoting effect of PEPA is in contrast with published studies in normally
behaving mice (Zushida et al., 2007). It is likely that PEPA, like DCS, facilitates
the consolidation of extinction learning rather than inducing extinction learning
per se (Davis et al., 2006). Evidence supporting this hypothesis is that long-term
potentiation elicited by the AMPA receptor glutamate receptor 1 is necessary for the
formation of emotional memories (Humeau et al., 2007).
The present finding that AMN082 rescued deficient extinction acquisition in S1 mice
extends recent studies showing that AMN082, a specific allosteric modulator of mGlu7
receptors (Mitsukawa et al., 2005), impairs the acquisition of conditioned fear as
well as facilitates between-session extinction in normally behaving rodents (Fendt
et al., 2008; Siegl et al., 2008). Thus, AMN082, in addition to dietary zinc-restriction
(see ‘Introduction’), constitute rare examples of treatments which enhance extinction
learning, but not fear learning. Moreover, these results show that AMN082 can rescue
extinction deficits in a psychopathological animal model, suggesting that AMN082 be
used during extinction-based therapy to induce extinction learning in individuals
with pathological fear.
At present, it is difficult to understand the extinction promoting effect of AMN082
(O'Connor et al., 2010). mGlu7 receptors, located entirely pre-synaptically, are widely
distributed throughout the central nervous system (Ohishi et al., 1995). Activation
of this receptor generally inhibits glutamatergic and GABAergic synaptic transmission
(Schoepp, 2001), although facilitation of glutamatergic transmission has also been
reported in specific brain regions (Millan et al., 2002; Li et al., 2008; Martin et al.,
2010). Given the wide-spread distribution of mGlu7 receptors and their location on
both glutamatergic and GABAergic axon terminals, the cumulative excitatory and inhibitory
effects of systemic application of AMN082 is difficult to predict at present as precise
knowledge of the location of the receptor in the microcircuits underlying fear extinction
acquisition is lacking. Further studies on localised effects of AMN082 are warranted.
Furthermore, recent findings reveal that a major metabolite of AMN082 inhibits serotonin-,
dopamine- and noradrenaline-transporters (Sukoff Rizzo et al., 2011). As chronic treatment
with the selective serotonin re-uptake inhibitor fluoxetine reduces fear in S1 mice
(Camp et al., 2012) and protects against the return of fear in normally extinguishing
mice (Karpova et al., 2011), we cannot exclude the contribution of enhanced monoaminergic
neurotransmission in AMN082-induced reduction of fear.
4.4
Enhancing NMDA receptor activity after successful extinction learning reduces fear
in S1 mice
A meta-analysis of published data shows that DCS is effective in reducing fear when
combined with fear extinction or exposure therapy (Norberg et al., 2008). Results
from normally extinguishing rodents show that DCS, administered immediately post extinction
training, can lower fear by enhancing extinction memory consolidation (Ledgerwood
et al., 2003). Clinically, DCS, combined with exposure-based therapy, is effective
in reducing fear in patients with so-called simple fears including acrophobia, social
anxiety, obsessive compulsive and panic disorders (Graham et al., 2010). However,
in most cases beneficial effects of DCS were only apparent after a few exposure sessions
further suggesting that the primary mechanism of DCS is to consolidate fear extinction
memories. Along these lines, we have previously published that prior extinction training
administration of DCS is ineffective in rescuing deficient extinction learning in
S1 mice (Hefner et al., 2008). Here, using a novel behavioural finding that S1 mice
display impaired extinction consolidation/retrieval we tested whether DCS is effective
in lowering fear when administered following successful fear reduction in extinction
learning. Results revealed that DCS rescued deficient extinction consolidation/retrieval
in S1 mice. Thus, present data and that of Hefner et al. (2008) conclusively demonstrate
that DCS can rescue deficient extinction learning only when administered during the
extinction consolidation phase and that the efficacy of DCS is gated by the ability
of S1 mice to acquire (at least to some extent) extinction acquisition. These results
using a psychopathological animal model have clinical implications as they strengthen
the suggestion that DCS be used as an add-on drug to facilitate reductions in fear
by exposure therapy in specific anxiety disorders (Norberg et al., 2008; Davis, 2011).
4.5
HDAC inhibition rescues deficient extinction consolidation/retrieval
Recent studies have demonstrated that histone acetylation is dynamically regulated
following successful extinction learning (Levenson et al., 2004; Fontan-Lozano et al.,
2008; Bousiges et al., 2010; Peleg et al., 2010; Monsey et al., 2011; Stafford et al.,
2012), and furthermore, that pharmacological or genetic silencing of HDAC enzyme isoforms
can enhance fear extinction (Bredy et al., 2007; Lattal et al., 2007; Bredy and Barad,
2008; Guan et al., 2009; Monsey et al., 2011). Here, we first assessed whether MS-275,
which is, using a chemoproteomic approach a HDAC1- 2- and 3-isoform inhibitor (Bantscheff
et al., 2011), or using purified rhHDACs primarily a HDAC1- and 9-isoform inhibitor
(Khan et al., 2008), could rescue deficient extinction consolidation/retrieval in
“weak” fear conditioned S1 mice. We revealed that post extinction training administration
of MS-275, at a dose with demonstrated HDAC inhibitory properties (Simonini et al.,
2006), rescued deficient extinction consolidation/retrieval in S1 mice. It is likely
that combined inhibition of HDAC1-, HADAC2- and HDAC3-isoforms contributes to the
observed rescue of deficient extinction consolidation/retrieval in S1 mice. Pharmacological
and/or genetic silencing of HDAC1- or HDAC2-isoforms have been shown to enhance the
consolidation of fear and/or extinction memories (Guan et al., 2009; Bahari-Javan
et al., 2012) and that genetic silencing of the HDAC3-isoform enhances long term memory
in the object recognition test (McQuown et al., 2011). Notwithstanding, our data show
for the first time that preferentially selective HDAC inhibitors such as MS-275 can
rescue profound extinction consolidation/retrieval deficits in a psychopathological
animal model and thus suggest that such compounds be used as an add-on drugs to facilitate
reductions in fear in exposure-based therapy.
The extinction consolidation/retrieval-promoting effect of MS-275 was specific as
results revealed that following “normal” fear conditioning, in which S1 mice display
deficient extinction acquisition, prior extinction training administration of MS-275
did not induce extinction acquisition or reduce fear during a retrieval test. This
indicates that rescue of deficient extinction acquisition requires mechanisms beyond
HDAC inhibition. As enhancing GABAergic activity has been shown to significantly contribute
to the molecular mechanisms underlying fear extinction [for example, see (Harris and
Westbrook, 1998; Chhatwal et al., 2005; Akirav et al., 2006; Lin et al., 2009; Dalton
et al., 2012)], we tested whether VPA, a pharmacological compound which enhances GABAergic
signalling (Mimaki et al., 1984; Miller et al., 1988; Ko et al., 1997; Simonini et al.,
2006) in addition to exhibiting robust HDAC inhibition (Kramer et al., 2003; Khan
et al., 2008), can rescue impaired extinction acquisition and deficient extinction
consolidation/retrieval. Indeed, our results revealed that prior extinction training
administration of VPA rescued both deficient extinction acquisition and deficient
extinction consolidation/retrieval in S1 mice. This result in a psychopathological
animal model further extends findings showing prior extinction training administration
of VPA potentiates reductions in fear extinction in normally extinguishing B6 mice
(Bredy et al., 2007; Bredy and Barad, 2008) and, when administered prior to cognitive
behavioural therapy, facilitates extinction in healthy humans (Kuriyama et al., 2011).
Our current result using a psychopathological animal model reveals that prior exposure-based
extinction training administration of VPA, acutely applied, can rescue impaired extinction
acquisition and deficient extinction consolidation/retrieval. This result has clear
clinical applications. Specifically, to date, in preclinical animal models of impaired
fear learning and neurodegeneration, cognitive enhancing effects of VPA have been
shown only with chronic prior fear conditioning administration (Li et al., 2006; Dash
et al., 2009; Kilgore et al., 2010). Our present result in a psychopathological animal
model demonstrates the utility of a single VPA treatment in combination with exposure-based
therapy to rescue impaired extinction acquisition and deficient extinction consolidation/retrieval.
Furthermore, a number of small clinical studies demonstrate that chronic VPA monotherapy,
for a minimum of eight weeks, reduces fear in social anxiety- (Kinrys et al., 2003),
panic- (Primeau et al., 1990; Keck et al., 1993; Woodman and Noyes, 1994; Baetz and
Bowen, 1998) and obsessive-compulsive- (Cora-Locatelli et al., 1998) disorder patients.
Beneficial effects of chronic VPA monotherapy have also been reported in combat veterans
with PTSD (Fesler, 1991; Otte et al., 2004), however, lacking effects are also reported
(Davis et al., 2008; Hamner et al., 2009). A major drawback to chronic VPA monotherapy
is that up to 25% of patients within the afore-mentioned studies are reported to stop
medication due to adverse effects (including nausea, dizziness, fatigue, sedation
and gastrointestinal complaints). Our current data suggests that acute VPA treatment,
combined with exposure-based therapy may be an effective and novel clinical strategy
to reduce fear in anxiety patients with minimal adverse effects.
5
Conclusions
In summary, we show that S1 mice are a unique psychopathological animal model as they
display deficient extinction acquisition and deficient extinction retrieval, which
can be experimentally dissociated by choosing different experimental conditioning
setups. Using this mouse model the present data identify novel therapeutic targets
that induce fear reductions when combined with extinction, resembling exposure-based
therapy. Different pharmacological and non-pharmacological treatments that target
cellular and molecular mechanisms with a documented or putative role in extinction
memory formation within relevant brain areas and circuitries were chosen. This study
reveals that DBS of the AcbC during exposure-based therapy may be a novel non-pharmacological
treatment strategy to overcome impairments in reducing learned fear. Furthermore,
that pharmacological enhancement of NMDA receptor activity or inhibition of HDAC1-,
HDAC2- and HDAC3-isoforms, may rescue deficient extinction retrieval only following
successful reductions in fear during extinction training, which can have important
implications for the use of such agents in human exposure therapy. On the other hand,
enhancing mGlu7 activity or enhancement in GABAergic activity in tandem with broad
inhibition of HDAC isoforms could be a novel pharmacological treatment strategy to
rescue both, impairments in extinction acquisition and extinction retrieval.