Neurobiology of Learning and Memory 87 (2007) 209–217
1074-7427/$ - see front matter © 2006 Elsevier Inc. All rights reserved.
Methylene blue facilitates the extinction of fear in an animal
model of susceptibility to learned helplessness
Kathryn M. Wrubel, Douglas Barrett, Jason Shumake,
S. Elizabeth Johnson, F. Gonzalez-Lima¤
Department of Psychology and Institute for Neuroscience, University of Texas at Austin, Austin, TX 78712, USA
Received 29 June 2006; revised 22 August 2006; accepted 23 August 2006
Available online 2 October 2006
The objectives were to (1) extend previous Wndings on fear extinction deWcits in male congenitally helpless rats (a model for susceptibil-
ity to learned helplessness) to female congenitally helpless rats, and (2) attempt a therapeutic intervention with methylene blue, a meta-
bolic enhancer that improves memory retention, to alleviate the predicted extinction deWcits. In the Wrst experiment, fear acquisition (four
tone-shock pairings in operant chamber) was followed by extinction training (60 tones in open Weld). Congenitally helpless rats showed
fear acquisition similar to controls but had dramatic extinction deWcits, and did not display the gradual extinction curves observed in con-
trols. Congenitally helpless rats demonstrated greater tone-evoked freezing as compared to controls in both the acquisition and extinction
contexts one week after extinction training, and also in the extinction probe conducted one month later. In the second experiment (which
began one month after the Wrst experiment) congenitally helpless subjects were further exposed to tones for 5 days, each followed by 4mg/kg
methylene blue or saline IP, and had a fear renewal test in the acquisition context. Methylene blue administration improved retention of
the extinction memory as demonstrated by signiWcant decreases in fear renewal as compared to saline-administered congenitally helpless
subjects. The impaired ability to extinguish fear to a traumatic memory in congenitally helpless rats supports the validity of this strain as
an animal model for vulnerability to post-traumatic stress disorder, and this study further suggests that methylene blue may facilitate fear
extinction as an adjunct to exposure therapy.
© 2006 Elsevier Inc. All rights reserved.
Keywords: Extinction; Post-traumatic stress disorder; Congenitally helpless rats; Methylene blue; Fear conditioning; Metabolic enhancer; Animal model;
Disease susceptibility; Memory retention; Exposure therapy; Cytochrome oxidase
Inescapable electric shock prevents animals from subse-
quently learning an escape response, a phenomenon termed
learned helplessness (Overmier & Seligman, 1967). Vulnera-
bility to learned helplessness is hereditable and can be
enhanced through selective breeding, as evidenced by the
creation of a strain of congenitally helpless rats (Henn &
Vollmayr, 2005). Studies show that up to 95% of the con-
genitally helpless oVspring typically show the learned
helpless phenotype, as opposed to the 5–20% of randomly
bred rats (Henn, Johnson, Edwards, & Anderson, 1985;
Lachman et al., 1992). Since epidemiological studies show
that 40–50% of the risk of susceptibility to depression and
30% of the risk of susceptibility to post-traumatic stress
disorder (PTSD) is genetic (Fava & Kendler, 2000; Sanders,
Detera-Wadleigh, & Gershon, 1999; True et al., 1993), an
appropriate animal model of congenital vulnerability to
these disorders could be very useful, especially in the devel-
opment of therapeutic treatments.
A recent paper reported behavioral characteristics of
male rats predisposed to learned helplessness (Shumake,
Barrett, & Gonzalez-Lima, 2005). Compared to normal
rats, congenitally helpless rats demonstrated reduced
*Corresponding author. Fax: +1 512 471 4728.
E-mail address: firstname.lastname@example.org (F. Gonzalez-Lima).
K.M. Wrubel et al. / Neurobiology of Learning and Memory 87 (2007) 209–217
reward sensitivity, high novelty seeking, an increase in con-
ditioned fear, and a deWcit in fear extinction. This behav-
ioral phenotype appears similar in many respects to that of
PTSD patients. For example, patients with PTSD show
stronger acquisition and reduced extinction of aversively
conditioned responses (Orr et al., 2000), and a personality
proWle of low reward dependence, high novelty seeking, and
high behavioral inhibition (Richman & Frueh, 1996; Wang
et al., 1997). Also, humans with PTSD show deWcits in
extinction of conditioned fears (Orr et al., 2000; Peri, Ben-
Shakhar, Orr, & Shalev, 2000) and congenitally helpless
rats also demonstrated similar deWcits when compared with
normal control rats (Shumake et al., 2005). Human studies
have shown that females are more vulnerable to PTSD
(with approximately twice the risk), and that their symp-
toms persist longer than those of males (NemeroV et al.,
2006). Based on the human studies, one would expect con-
genitally helpless females to show greater extinction deWcits
than males. The Wrst objective of the present study was to
extend our previous Wndings on fear extinction deWcits to
female congenitally helpless rats.
Methylene blue (MB), a metabolic enhancer, has been
shown to improve memory for extinction of fear condition-
ing in normal rats (Gonzalez-Lima & Bruchey, 2004). The
second objective of the present study was to determine if
MB could facilitate extinction in congenitally helpless sub-
jects. Congenitally helpless rats are postulated to be an ani-
mal model of PTSD, demonstrating extinction deWcits
observed in patients with the disorder. Since humans with
PTSD often go through extinction training to reduce their
fears, we administered MB during extinction training to
determine if it would facilitate retention of extinction in
congenitally helpless subjects. If so, MB may be a useful
therapeutic adjunct to exposure therapy for patients with
PTSD to aid in retention of extinction of conditioned fears
to traumatic memories.
Methylene blue is a redox dye commonly administered
as an antidote for methemoglobinemia, a condition in
which (usually due to metabolic poisoning) the body is
unable to convert methemoglobin to hemoglobin to allow
oxygen transport (Bodansky & Gutmann, 1947; Bradberry,
2003; Clifton & Leiken, 2003; Etteldorf, 1951). MB is able
to reverse this process, and has been used safely in humans
for over a century. The memory retention enhancing eVects
of MB were Wrst reported by Martinez, Jr. and colleagues
(1978), who discovered that low dose post-training admin-
istration of MB improved memory retention in an inhibi-
tory avoidance task. MB has also been shown to enhance
memory retention in a spatial memory task, an object rec-
ognition task, and to aid in between-days habituation to a
familiar environment (Callaway, Riha, Bruchey, Munshi, &
Gonzalez-Lima, 2004; Riha, Bruchey, Echevarria, & Gonz-
alez-Lima, 2005). More relevant to the present study is that
Gonzalez-Lima and Bruchey (2004) reported that memory
retention of extinction of Pavlovian fear conditioning could
be enhanced with post-extinction administration of MB in
normal rats. Total post-extinction freezing scores were
lower in subjects receiving MB than saline, and MB-treated
subjects also had a longer lasting eVect of extinction. There-
fore, the current study was undertaken to determine if con-
genitally helpless rats, which are resistant to extinction, can
likewise have their extinction learning improved by methy-
2. Materials and methods
For the Wrst experiment, subjects were 23 female congenitally helpless
rats (bred in our laboratory from breeding pairs obtained from the Central
Institute for Mental Health in Mannheim, Germany, courtesy of Dr. Fritz
Henn), and 12 female Sprague–Dawley control rats obtained from Harlan
(Houston, TX), all weighing approximately 300g at the beginning of the
experiment. The congenitally helpless subjects used in this study were bred
in our laboratory and were not tested for susceptibility to learned helpless-
ness, as this would involve administration of foot shocks prior to acquisi-
tion training. Subjects were housed 2–3 per cage under standard
laboratory conditions with a 12 h light/dark cycle and free access to food
and water. Animal experimentation was approved by the University of
Texas Institutional Animal Care and Use Committee. Male rats would
also have been used, but, because of the need for male subjects for another
experiment and high infertility in our colony, we were unable to breed a
suYcient number of male subjects for this purpose. Rats were given daily
vaginal smears and began training on the Wrst day of their estrus cycle in
both experiments. For the second experiment, female congenitally helpless
rats were subdivided into 2 groups: 12 were treated with MB and 11 were
DiVerent apparati were utilized during extinction training and probes,
in order to parse out any eVects of contextual fear on freezing behavior.
The acquisition session was conducted in standard operant chambers and
extinction sessions were conducted in open-Weld activity chambers in a
2.2.1. Acquisition context (Context A)
Pavlovian tone foot-shock acquisition training was conducted in four
operant conditioning chambers, each measuring 22£ 25£32cm (MED
Associates, St. Albans, VT) and enclosed in sound-attenuated boxes illu-
minated by red lights. The two sides of each chamber were aluminum, and
the front, back, and top were made of clear plexiglas. Tones were gener-
ated by a Wavetek Sweep/Modulation Generator (Wavetek, San Diego,
CA) and presented through speakers mounted at the top of each chamber.
The acoustic conditioned stimulus (CS) was a frequency-modulated tone
of 1–2kHz, 2 sweeps/s, 15s in duration, with an intensity of 68dB, mea-
sured at the center of the Xoor of the chamber with a decibel meter. The
unconditioned stimulus (US) was a mild foot shock of 0.5 mA, 0.75s in
duration, delivered through metal bars (separated by 1.2cm) which
formed the Xoor of the chamber, and were wired to shock generators
(MED Associates). Stimulus presentations were controlled by computer
programs, written by the experimenters using the MED-PC for Windows
programming language (MED Associates). A Bioclean solution (Stanbio
Laboratory, Boerne, TX) was placed in the tray beneath the chamber to
provide a distinct olfactory cue for the acquisition context.
2.2.2. Extinction context (Context B)
Extinction training occurred in a diVerent context. Two open-Weld
activity boxes measuring 31£ 45£ 45cm (MED Associates) with Wber-
glass bottoms, clear plexiglas sides, and an open top were utilized for
extinction training. Horizontal activity was detected by arrays of infrared
motion detectors (16£ 16, 2.54 cm apart), with two arrays located 1cm
above the Xoor of the chamber. Rearings were detected with a vertical-axis
K.M. Wrubel et al. / Neurobiology of Learning and Memory 87 (2007) 209–217
array positioned 13cm above the surface of the Xoor to ensure that only
those rearing movements in which the subject’s forepaws left the ground
would register as rearing counts. Open-Weld boxes were controlled by the
MED Associates Activity Monitor program (Version 5.10), which
recorded various parameters related to a subject’s ambulation (sequential
horizontal beam breaks), immobility (no beam breaks), and rearing behav-
iors (vertical beam breaks). Stereotypic movement is deWned by the com-
puter-controlled infrared beam detectors as movement without
displacement, i.e., short movements that break a beam without the subject
entering into ambulation. The tone CS which was 1–2kHz, 2 sweeps/s, and
15 s in duration, was digitally recorded from the tone generator used in the
acquisition context and presented through a computer speaker above the
open Welds (measured at 68dB in the center of the Xoor of each open Weld
with a decibel meter).
2.3. Freezing scores
Freezing scores were utilized as an index of tone-evoked fear. Experi-
menters trained to score freezing with high inter-rater reliability recorded
a freezing score during pre-CS and CS periods through direct observation.
Freezing was operationally deWned as the subject having all four feet on
the Xoor, with minimal head movements, and shallow, rapid breathing. A
score of 1 was recorded if the subject froze for the majority of a 3s bin,
with a maximum freezing score of 5 for complete freezing during the 15-s
pre-CS and CS periods. Immobility time was scored automatically as time
spent with no new infrared beam breaks (no horizontal or vertical move-
ment). Immobility time overlapped with freezing scores, but whereas freez-
ing was scored in Wve 3-s bins by an experimenter, immobility time was
continuously scored by the computer. The two independent measures were
highly correlated (rD 0.81).
2.4. Statistical analysis
The SPSS 11.5 for Windows statistical software package was used for
statistical analysis. All data were analyzed with analyses of variance
(ANOVAs), and p-values were reported as Huynh–Feldt corrected values,
with an ?-value of .05 regarded as signiWcant. When warranted, simple-
eVects tests of signiWcant interactions were performed and adjusted by
3. Experiment 1: Extinction of Pavlovian fear conditioning in
the congenitally helpless female rat
3.1. Behavioral training
Subjects were handled every day for 7 days prior to the
start of training. During this time, each subject was habitu-
ated to the acquisition context (Context A) in the absence
of tones or shocks for 1h a day. Vaginal smears were used
to determine the estrus cycle phase for each individual sub-
ject before training began. An estrus cycle of 4 days was
conWrmed and training was staggered between subjects
according to estrus cycle, such that the Wrst day of acquisi-
tion training occurred while each subject was in estrus.
Diestrus females received up to three additional days of
habituation and handling.
The training procedure is presented in Table 1 under
“Experiment 1”. For acquisition training, all subjects were
placed in the conditioning chambers (Context A) and
received four tone-shock pairings over 15min, with pseudo-
random inter-trial intervals averaging 3min. Each 15-s tone
CS co-terminated with the foot shock US.
Extinction training occurred over two-consecutive days
while female subjects were in the diestrus phase, to consis-
tently synchronize the phase of the cycle with the context,
such that animals would be tested in the same context at
the same phase of the estrus cycle (Birke & Archer, 1975)
and avoid interference with conditioned freezing mea-
sures in the open Weld (Context B, used for extinction
training). Subjects were placed in the open Weld, and 30
tones were presented over 60min, with 2min between
each tone onset. The Wrst minute in the open-Weld context
served as a baseline measure of activity (prior to the Wrst
tone CS presentation).
Post-extinction probe trials occurred one week later in
the acquisition context (when subjects were in estrus), in
order to measure context-dependent renewal of fear. Each
subject was returned to the operant chamber (Context A)
and presented with four 15-s tones (in the absence of foot
shock) over 10min, with 2min intervals between each tone
onset. Post-extinction probe trials (consisting of four tone
CS presentations, as described above) occurred in the
extinction context on the following day, when subjects were
in diestrus, and served to measure retention of extinction
learning. Experimenters recorded freezing scores during
pre-CS and CS periods in both contexts.
Helpless and control subjects showed similar baseline open-
Weld activity prior to extinction training. In contrast, they
showed dramatic group diVerences during extinction, with
congenitally helpless rats exhibiting a large extinction deWcit.
3.2.1. Baseline motor activity and excitatory conditioned
behavior in the open Weld
There were no diVerences in baseline motor activity mea-
sures between congenitally helpless and control subjects
during the Wrst minute of Extinction Session 1 (prior to the
onset of the Wrst tone CS) in any of the parameters ana-
lyzed, which included ambulatory time, stereotypic move-
ment time, immobility time, and rearing time (Table 2).
Design for Experiments 1 and 2
T, 1–2 kHz 68dB 15s tone CS; S, 0.5mA 0.75s foot shock US; MB, methylene blue.
Experiment 1 Experiment 2
Day 1Days 2–3Day 9Day 10Day 1Days 2–6Day 7
4 T! S
4 T/Day + MB or Saline
K.M. Wrubel et al. / Neurobiology of Learning and Memory 87 (2007) 209–217
There were no signiWcant diVerences between groups in
time spent in the center of the open Weld during the Wrst min-
ute of Extinction Session 1 (Table 2), which is considered a
measure of fearfulness since timid subjects have been found
to spend more time along the walls of the apparati (Treit &
Fundytus, 1988). This indicates that the diVerences observed
in extinction behavior between congenitally helpless rats and
control subjects cannot be attributed to baseline strain diVer-
ences in general motor activity or fearfulness.
To evaluate possible group diVerences in the strength of
the original excitatory conditioning, tone-evoked freezing
and motor activity measures during the Wrst tone after the
acquisition session were evaluated in the open Weld. There
were no signiWcant group diVerences in freezing time, immo-
bility time, ambulatory time, rearing time or stereotypic
movement time (Fig. 1). This indicates that both groups
showed a similar strength of fear conditioning after the origi-
nal acquisition session. The lack of a group eVect on the Wrst
extinction trial is consistent with the lack of an acquisition
eVect, but this may not entirely rule out an acquisition eVect.
3.2.2. Short-term extinction
Short-term extinction was deWned as the extinction behav-
ior seen in the Wrst two 1-h sessions of training (Days 2–3).
Data were evaluated with repeated measures ANOVA, with
session and trial serving as within-subject factors. Behavioral
measures from each 15-s tone CS presentation were averaged
into six bins representing Wve tone presentations every 10min.
There was a signiWcant group eVect on freezing behavior
during extinction training, F(1,29)D139.67, p<0.001, with
congenitally helpless subjects freezing much more than con-
trols. The degrees of freedom are 29 instead of 33 for the
extinction sessions because of missing values from 4 out of
the 23 congenital helpless subjects. Control rats showed grad-
ual decreases in freezing, whereas congenitally helpless rats
show a paradoxical enhancement of freezing during the Wrst
session of extinction trials. There was a signiWcant
group£trial£session interaction, F(5,145)D4.13, p<0.01.
There was also a signiWcant group£trial interaction in ses-
sion 1, F(5,145)D10.92, p<0.001, but not in session 2,
F(5,145)D0.12. In session 1, there was a simple main eVect of
trial for the control group, which decreased freezing,
F(5,55)D9.56, p<0.001; however, the helpless group showed
increased freezing, F(5,100)D2.75, p<0.05. While control
rats showed similar rates of extinction in both sessions with
smooth learning curves, congenitally helpless subjects dis-
played no evidence of extinction in Extinction Session 1, and
slowly began to extinguish freezing behavior in Extinction
Session 2 (Fig. 2A).
3.2.3. Context-dependent renewal
In order to assess the eVects of context-dependent fear
renewal after extinction, the data from the four tone probes
delivered on Day 9 in the acquisition context, Context A,
were analyzed (Probe-Acq). Congenitally helpless subjects
showed signiWcantly more freezing relative to controls,
F(1,33)D230.94, p<0.0001 in this session (Fig. 2B). The
main reason for the large F value is that the group eVects of
helpless vs. control are extremely large.
3.2.4. Long-term extinction
Long-term extinction was deWned as extinction behavior
after 1 week, and was assessed by comparing freezing behav-
ior during the Wrst four tones at the beginning of extinction
session 1 with freezing during the four probe trials on Day
10, which were conducted in the same context, the extinction
context B (Probe-Ext). Both control and congenitally help-
less rats showed long-term reductions in freezing as a conse-
quence of extinction training: main eVect of session for
control group, F(1,11)D214.53, p<0.001, and for helpless
group, F(1,22)D47.51, p<0.001. However, congenitally help-
less subjects showed signiWcantly less long-term extinction
relative to controls: main eVect of group, F(1,33)D23.91,
p<0.001 (Fig. 2B).
4. Experiment 2: The eVects of methylene blue on memory
retention of extinction of Pavlovian fear conditioning in
congenitally helpless rats
4.1. Behavioral training
Subjects were the same 23 female congenitally helpless
rats used in the Wrst experiment. The training procedure is
Motor activity (s) during baseline open Weld (1min) in helpless and con-
No signiWcant group diVerences.
Helpless nD 23
Fig. 1. Conditioned freezing and motor activity during the Wrst tone CS in
extinction session 1 in the open Weld was assessed in the post-acquisition
probe, to investigate possible group diVerences in strength of the original
excitatory conditioning. Freezing scores (0–5) were transformed to freez-
ing time (0–15s) to match the 15-s timeframe of the open-Weld parameters.
There were no signiWcant group diVerences in freezing or ambulatory
K.M. Wrubel et al. / Neurobiology of Learning and Memory 87 (2007) 209–217
presented in Table 1 under “Experiment 2”. This experiment
began one month after the context renewal probe trial con-
ducted in Experiment 1. Congenitally helpless females were
given daily vaginal smears to assess their estrus cycle. On
their Wrst day of estrus, they were returned to the acquisition
context (Context A) for probe trials consisting of four 15-s
tones (in the absence of foot-shock) over 10min, with 2min
intervals between each tone onset. These probe trials veriWed
that all congenitally helpless subjects continued to show sig-
niWcant freezing one month after the end of Experiment 1.
Subjects were matched based on these freezing scores and
assigned to either the MB treatment or saline control group.
For each of the next 5 days, subjects were presented with
four 15-s tones (in the absence of foot shock) over 10min,
with 2min intervals between each tone onset in the extinc-
tion context (Context B, Probe-Ext Days 2–6). Extinction
trials were conducted in a context diVerent from the origi-
nal acquisition context in order to reduce confounds result-
ing from contextual conditioning. The rationale was to
match the extinction protocol that the laboratory previ-
ously developed for MB testing and also to avoid a Xoor
eVect that more intensive extinction training would have
induced. Thirty minutes following each daily probe session
in the extinction context, subjects were injected with either
MB (4mg/kg) or saline I.P. Freezing scores recorded during
pre-CS and CS presentations were recorded and analyzed
as previously described.
On Day 7, subjects were returned to the acquisition con-
text (Context A) for a post-treatment probe session consist-
ing of four 15-s tones (in the absence of foot shock) over
10min, with 2min intervals between each tone onset
(Probe-Acq Day 7). Freezing scores were recorded by an
experimenter blind to group assignment. Freezing scores to
the tone CS in pre-treatment (Day 1) and post-treatment
(Day 7) probe sessions recorded in the acquisition context
were compared between groups to determine if MB aided in
the facilitation of memory retention of extinction.
Methylene blue treatment following extinction training
signiWcantly improved memory retention of extinction in
the acquisition context (Context A) in congenitally helpless
subjects. While MB did not improve extinction learning in
extinction context (Context B) sessions, it did signiWcantly
improve the generalization of that learning to the acquisi-
tion context. The relatively low levels of freezing in both
groups at the start made it very diYcult to see an initial
facilitation of extinction by MB. However, the MB-treated
rats showed suYcient freezing to the Wrst tone in the
renewal context, and the improvement is shown by an
acceleration of extinction in this context, when the tone is
not followed by shock (Fig. 3).
4.2.1. EVects of methylene blue treatment in the extinction
context (Context B) probe sessions
Freezing to the tone CS in the extinction context was
analyzed with repeated measures ANOVA, with the aver-
age of the four trials from each extinction session serving as
the repeated measure. No signiWcant eVects related to treat-
ment were obtained (Fs<1), but a signiWcant main eVect of
session indicated that both MB and saline-administered
subjects froze less with each session, F(4,84)D9.50, p<.001.
In addition to freezing, general activity measures recorded
by the infrared arrays in the extinction context were analyzed
using the same repeated measures design. There were no sig-
niWcant treatment-related diVerences in ambulation, rearing,
immobility, or stereotypic movement times, demonstrating
that MB did not alter general motor activity (Table 3).
4.2.2. EVects of methylene blue treatment in the acquisition
context (Context A) probe sessions
There were no group diVerences in pre-CS freezing in the
post-injection renewal test (Fig. 3B). DiVerences in freezing
to the tone CS in the acquisition context were analyzed
with repeated measures ANOVA, with pre-treatment and
Fig. 2. (A) Extinction curves (means §SE). The conditioned response dur-
ing Extinction Sessions 1 and 2 was measured in terms of freezing behav-
ior. Each 1h extinction session consisted of one tone CS every 2min,
averaged by Wve tones for each 10-min bin for the repeated measures
ANOVA. SigniWcant diVerences (p<.01) between congenitally helpless
and control groups were found at every time point in both sessions.
¤p<.001. (B) Freezing scores from post-extinction probe trials
(means §SE) in both the acquisition (Probe-Acq, Context A) and extinc-
tion (Probe-Ext, Context B) contexts. Each probe test consisted of four
tones in 10min. Congenitally helpless subjects showed signiWcantly more
freezing relative to controls. ¤p<.0001.
K.M. Wrubel et al. / Neurobiology of Learning and Memory 87 (2007) 209–217
post-treatment probes serving as repeated measures. A sig-
niWcant two-way (treatment£probe) interaction was
obtained, F(1,21)D21.59, p<.0001, indicating that congen-
itally helpless rats treated with MB showed signiWcantly
reduced freezing in the acquisition context. Therefore, MB
facilitated fear extinction in these subjects (Fig. 3C).
Congenitally helpless female rats exhibited abnormally
high freezing in response to a tone formerly predictive of
electric shock, even after 60 presentations of the tone in the
absence of shock. This extinction deWcit could not be attrib-
uted to diVerences in the strength of the original excitatory
conditioning. While congenitally helpless females showed a
trend for higher freezing during the Wrst tone after extinc-
tion, they showed no signiWcant group diVerences in freez-
ing, immobility or any of the behaviors measured in the
open Weld. They failed to show the gradual decrement in
freezing characteristic of the normal extinction curves seen
in the control subjects during the extinction sessions, and
continued to show signiWcantly greater tone-evoked fear
one week after extinction training, when tested in both the
acquisition and extinction contexts as we hypothesized.
These Wndings are consistent with those previously
reported in the congenitally helpless male rat (Shumake
et al., 2005).
The behavioral deWcit observed in congenitally helpless
rats cannot be explained by a general suppression of motor
activity or by an overall increase in non-speciWc fearfulness,
which can be indicated by thigmotaxis, or time spent in the
periphery versus the center of the open Weld (Treit &
Fundytus, 1988). There were no baseline group diVerences
in open-Weld behavior prior to the onset of the fear-associ-
ated tones, in terms of motor activity or thigmotaxis.
This experiment demonstrated that congenitally helpless
rats show deWcits in both extinction performance and reten-
tion: an inherently neutral tone CS is capable of evoking
maladaptive fearful responses in the helpless subjects, even
outside of the context in which they experienced the aver-
sive shock US. Another animal model of post-traumatic
stress disorder is likewise characterized by the formation
and persistence of memories that evoke fear across multiple
contexts (Rau, DeCola, & Fanselow, 2005). Post-traumatic
stress disorder patients show increases in autonomic
arousal (heart rate, skin conductance) in response to aver-
sive Pavlovian conditioning, and these conditioned
responses are resistant to extinction (Orr et al., 2000; Peri
et al., 2000). This phenomenon is known as paradoxical
enhancement (an increase in conditioned responding as a
consequence of extinction training), and was demonstrated
by congenitally helpless subjects in the Wrst extinction ses-
sion, making an even stronger argument for their role as an
animal model of vulnerability to PTSD. In our previous
study (Shumake et al., 2005), helpless males showed
approximately twice as much freezing as controls after
long-term extinction; whereas in the present study, helpless
Fig. 3. (A) Conditioned freezing (mean §SE) to the tone in Context B
(extinction/open Weld) averaged over 5 days (Probe-Ext Days 2–6) of MB
and saline injections (4mg/kg I.P.) in congenitally helpless rats. No signiW-
cant eVects related to treatment were obtained (Fs<1). (B) No signiWcant
diVerences were found in freezing scores (mean §SE) during the pre-CS
period in the acquisition context (Context A) between MB and saline-
treated groups of congenitally helpless rats (Fs<1). (C) EVect of methy-
lene blue on extinction of freezing to the tone in the acquisition context
(Context A). Pre-treatment (Probe-Acq Day 1) and post-treatment
(Probe-Acq Day 7) freezing scores can be seen in the congenitally helpless
subjects. Conditioned freezing (mean §SE) to the tone before and after
daily post-training injections of 4mg/kg I.P. of MB or saline is shown.
Subjects were matched into groups with respect to pre-injection freezing
scores, then tested again after 5 days of tone presentations in the
extinction context. Conditioned freezing was signiWcantly reduced in con-
genitally helpless subjects receiving MB injections as compared to the
saline-injected group. ¤p<.0001.
Post-injection average motor activity (s) in open Weld (600s) in helpless
No signiWcant group diVerences.
Saline nD 11
Stereotypic movement time
K.M. Wrubel et al. / Neurobiology of Learning and Memory 87 (2007) 209–217
females showed approximately three times more freezing
than controls in the long-term extinction probe. This con-
curs with data from human studies showing that females
are more vulnerable to PTSD than males.
The brains of congenitally helpless rats have been com-
prehensively mapped using metabolic brain imaging tech-
niques, and these subjects show extensive alterations in
regional brain metabolism as compared to non-helpless
subjects (Shumake, Poremba, Edwards, & Gonzalez-Lima,
2000; Shumake, Edwards, & Gonzalez-Lima, 2001, 2002,
2003; Conejo-Jimenez, Gonzalez-Pardo, & Gonzalez-Lima,
2004; Shumake & Gonzalez-Lima, 2003). Congenitally
helpless rats have reduced metabolism in prefrontal cortical
regions (Shumake et al., 2000), which are highly engaged in
behavioral extinction (Barrett, Shumake, Jones, & Gonz-
alez-Lima, 2003; Gonzalez-Lima & Bruchey, 2004; Milad &
Quirk, 2002; Nair, Berndt, Barrett, & Gonzalez-Lima,
2001a, Nair, Berndt, Barrett, & Gonzalez-Lima, 2001b).
This could lead to persistent fear-related conditioned
responses to a Pavlovian conditioned tone, which may be
more resistant to extinction than in control rats. Subjects
with higher prefrontal cortical activity are also more suc-
cessful at inhibiting a conditioned emotional response (Bar-
rett et al., 2003).
Medial prefrontal cortex may be crucial for the inhibi-
tion of the conditioned response during extinction (Barrett
et al., 2003; Milad & Quirk, 2002; Quirk, Garcia, & Gonz-
alez-Lima, 2006; Sotres-Bayon, Cain, & LeDoux, 2006;
Weible, McEchron, & Disterhoft, 2000), and PTSD patients
show decreased metabolic activity in this region during
extinction (Bremner et al., 1999). In newborn congenitally
helpless rats, brainstem regions are metabolically uncou-
pled from networks of frontal and limbic regions (Shumake
et al., 2004). This functional decoupling between brainstem
and forebrain regions may be indicative of a developmental
disorder in which brainstem regions are removed from the
inhibition provided by the frontal cortex during successful
Pavlovian extinction. A disruption of prefrontal–amygdala
interactions could also selectively impair Pavlovian extinc-
tion while leaving acquisition relatively intact (Phelps, Del-
gado, Nearing, & LeDoux, 2004; Sotres-Bayon, Bush, &
Successful fear extinction training in PTSD patients may
be facilitated by pharmacotherapy (Davis, Myers, Chhat-
wal, & Ressler, 2006). Methylene blue is an FDA-approved
drug which can be safely used in humans. It acts as an elec-
tron carrier in mitochondria, improving cellular respiration
(Visarius, Stucki, & Lauterburg, 1997), and enhancing neu-
ronal metabolism. Administration of MB to congenitally
helpless rats signiWcantly ameliorated their extinction deW-
cit in the fear-evoking acquisition context, as predicted in
our hypothesis. It did not enhance memory retention of
extinction during extinction sessions in Context B, possibly
due to a Xoor eVect on conditioned freezing in the extinc-
Gonzalez-Lima and Bruchey (2004) demonstrated the
eYcacy of MB in Pavlovian fear extinction and on regional
brain metabolism in normal rats. Methylene blue success-
fully improved Pavlovian fear extinction memory, and
resulted in higher metabolic activity in the frontal cortical
regions previously implicated in extinction (Barrett et al.,
2003). In addition, this study found that the regional meta-
bolic increases were correlated with the degree of extinction
retention. The eVects of MB on the congenitally helpless
subjects seen here, together with our prior Wndings, suggest
that MB may be therapeutically useful in PTSD patients.
Methylene blue’s metabolic eVect may facilitate the adap-
tive inhibition of the conditioned fear response provided by
frontal cortical regions during successful Pavlovian extinc-
tion. Moreover, this facilitatory eVect is seen when subjects
are returned to the threatening (acquisition) context,
although subjects never received MB after exposure to this
The memory retention eVects of low dose MB cannot be
attributed to alterations in locomotor activity, motivation,
reward value, or fearfulness (Gonzalez-Lima & Bruchey,
2004; Riha et al., 2005). Despite these reports, in order to
control for any non-speciWc or state-dependent learning
eVects that may occur by giving pre-training MB, it has
generally been administered following training in behav-
ioral studies. Also, because MB enhances memory retention
of the events preceding its administration, the time of its
injection follows the target memory task.
It is worth noting that MB was not given during the
original extinction training, but after spontaneous recovery
and re-extinction. It seems unlikely that the eVect of MB
might be speciWc to this phase of training, but diVerential
eVects of drugs given in conjunction with initial extinction
exposure vs. subsequent exposures have been reported with
passive avoidance (Bevilaqua, da Silva, Medina, Izquierdo,
& Cammarota, 2005). Therefore, future experiments could
test the eVects of MB when given during other phases of
Since MB has a half-life of 5–6.5h (Peter, Hongwan,
Kupfer, & Lauterburg, 2000), it is unlikely that the facilita-
tion of the extinction memory observed 24h after the last
injection of MB reXects a continued direct action of the
drug. Rather, it is probably due to enhanced oxidative
energy metabolism (Riha et al., 2005) occurring at a critical
time in memory consolidation because memory retention in
rats is not aVected by MB given 15min before training, 6h
after training, or 15min before testing (Martinez, Jr. et al.,
1978). MB increases cytochrome oxidase enzymatic activity
in a use-dependent manner, with brain regions with the
highest metabolic demand during memory consolidation
after extinction showing the largest increases in cytochrome
oxidase activity (Gonzalez-Lima & Bruchey, 2004).
Studies with multiple learning paradigms including
appetitive and aversive tasks, object recognition memory
and long-term habituation show that MB works as a meta-
bolic memory enhancer by increasing cytochrome oxidase
activity (Callaway, Riha, Wrubel, McCollum, & Gonzalez-
Lima, 2002; Callaway et al., 2004; Gonzalez-Lima & Bru-
chey, 2004; Riha et al., 2005). Cytochrome oxidase is the
K.M. Wrubel et al. / Neurobiology of Learning and Memory 87 (2007) 209–217
terminal enzyme of the electron transport chain which is
tightly coupled to neuronal metabolism and ATP produc-
tion. It is the rate-limiting enzyme in mitochondrial respira-
tion (Wong-Riley, 1989), and cytochrome oxidase increases
are correlated with increased energy demand (Gonzalez-
Lima & Cada, 1998). Electrons can be donated from
reduced MB to enter the electron transport chain between
Complexes II and III (Visarius et al., 1997), resulting in
enzyme induction of cytochrome oxidase (Callaway et al.,
2004; Gonzalez-Lima & Bruchey, 2004) and elevated cellu-
lar oxygen consumption (Riha et al., 2005; Visarius et al.,
1997). Therefore, by increasing mitochondrial respiration
through cytochrome oxidase activity, MB can enhance the
amount of ATP available within a neuron in order to
improve memory retention and recall in certain cognitive
tasks. If administered post-training (during the time of
memory consolidation), MB has been shown to increase
cytochrome oxidase activity within brain regions utilized
during a memory task (Gonzalez-Lima & Bruchey, 2004).
An important consideration to make about the mech-
anism of action of MB is that unlike other pharmacolog-
ical treatments proposed for use in PTSD, MB does not
appear to be working as an anxiolytic compound or on a
speciWc neurotransmitter system (Berlau & McGaugh,
2006; Chhatwal, Davis, Maguschak, & Ressler, 2005;
Davis et al., 2006). Drugs working on pharmacologically
speciWc synapses have actions distributed throughout the
brain, in relevant regions, as well as other regions that
could lead to unwanted side eVects. Methylene blue selec-
tively enhances metabolism in the brain during memory
formation, in regions related to the task, without the side
eVects of drugs acting on synaptic transmission else-
where in the brain. Methylene blue can be used to facili-
tate extinction memory formation for a speciWc fear-
evoking stimulus, and can facilitate extinction training
through repeated administrations without losing its
Congenitally helpless rats showed somewhat enhanced
acquisition and dramatically impaired extinction of fear
evoked by an aversive memory—a crucial part of the
behavioral phenotype associated with PTSD (Charney,
Deutch, Krystal, Southwick, & Davis, 1993). The congeni-
tally helpless rat also shows diVerences in regional brain
metabolism which resemble biological abnormalities
detected in PTSD patients (Shumake & Gonzalez-Lima,
2003). Thus, congenitally helpless rats may be a useful
model for studying those biological factors which render a
subset of individuals vulnerable to PTSD, and for testing
novel treatments such as methylene blue, which can facili-
tate the extinction of conditioned fear.
This research was supported by National Institutes of
Health Grant R01 NS37755 to F. Gonzalez-Lima and
Texas Consortium in Behavioral Neuroscience Fellowships
T32 MH65728 to Kathryn M. Wrubel, Douglas Barrett,
and Jason Shumake. The original breeding pairs, which
produced the oVspring used for this study, were obtained
from the Central Institute for Mental Health (Mannheim,
Germany), courtesy of Dr. Fritz Henn and Dr. Barbara
Vollmayr. We thank Abby Schindler for her assistance with
data collection. Dr. Wrubel conducted this study in partial
fulWllment of the requirements for the Ph.D. degree at the
University of Texas at Austin.
Barrett, D., Shumake, J., Jones, D., & Gonzalez-Lima, F. (2003). Metabolic
mapping of mouse brain activity after extinction of a conditioned emo-
tional response. Journal of Neuroscience, 23, 5740–5749.
Berlau, D. J., & McGaugh, J. L. (2006). Enhancement of extinction mem-
ory consolidation: the role of the noradrenergic and GABAergic sys-
tems within the basolateral amygdala. Neurobiology of Learning and
Memory, 86, 123–132.
Bevilaqua, L. R. M., da Silva, W. N., Medina, J. H., Izquierdo, I., & Cam-
marota, M. (2005). Extinction and reacquisition of a fear-motivated
memory require activity of the Src family of tyrosine kinases in the
CA1 region of the hippocampus. Pharmacology, Biochemistry and
Behavior, 81, 139–145.
Birke, L. I., & Archer, J. (1975). Open-Weld behaviour of oestrous and
dioestrus rats: evidence against an ‘emotionality’ interpretation. Ani-
mal Behavior, 23, 509–512.
Bodansky, O., & Gutmann, H. (1947). Treatment of methemoglobinemia.
Journal of Pharmacology, 90, 46–56.
Bradberry, S. M. (2003). Occupational methemoglobinemia: mechanisms
of production, features, diagnosis and management including the use
of methylene blue. Toxicology Reviews, 22, 13–27.
Bremner, J. D., Staib, L. H., Kaloupek, D., Southwick, S. M., Soufer, R., &
Charney, D. S. (1999). Neural correlates of exposure to traumatic pic-
tures and sound in Vietnam combat veterans with and without post-
traumatic stress disorder: a positron emission tomography study.
Biological Psychiatry, 45, 806–816.
Callaway, N. L., Riha, P. D., Bruchey, A. K., Munshi, Z., & Gonzalez-
Lima, F. (2004). Methylene blue improves brain oxidative metabolism
and memory retention in rats. Pharmacology, Biochemistry and Behav-
ior, 77, 175–181.
Callaway, N. L., Riha, P. D., Wrubel, K. M., McCollum, D., & Gonzalez-
Lima, F. (2002). Methylene blue restores spatial memory retention
impaired by an inhibitor of cytochrome oxidase in rats. Neuroscience
Letters, 332, 83–86.
Charney, D. S., Deutch, A. Y., Krystal, J. H., Southwick, S. M., & Davis,
M. (1993). Psychobiologic mechanisms of posttraumatic stress disor-
der. Archives of General Psychiatry, 50, 294–305.
Chhatwal, J. P., Davis, M., Maguschak, K. A., & Ressler, K. J. (2005).
Enhancing cannabinoid neurotransmission augments the extinction of
conditioned fear. Neuropsychopharmacology, 30, 516–524.
Clifton, J., & Leiken, J. B. (2003). Methylene blue. American Journal of
Therapeutics, 10, 289–291.
Davis, M., Myers, K. M., Chhatwal, J., & Ressler, K. J. (2006). Pharmaco-
logical treatments that facilitate extinction of fear: relevance to psycho-
therapy. NeuroRx, 3, 82–96.
Etteldorf, J. N. (1951). Methylene blue in the treatment of methemoglobi-
nemia in premature infants caused by marking ink: a report of eight
cases. The Journal of Pediatrics, 38, 24–27.
Fava, M., & Kendler, K. S. (2000). Major depressive disorder. Neuron, 28,
Gonzalez-Lima, F., & Bruchey, A. K. (2004). Extinction memory improve-
ment by the metabolic enhancer methylene blue. Learning and Mem-
ory, 11, 633–640.
Gonzalez-Lima, F., & Cada, A. (1998). Quantitative histochemistry of
cytochrome oxidase activity: theory, methods, and regional brain vul-
nerability. In F. Gonzalez-Lima (Ed.), Cytochrome oxidase in neuronal
K.M. Wrubel et al. / Neurobiology of Learning and Memory 87 (2007) 209–217 Download full-text
metabolism and Alzheimer’s disease (pp. 55–91). New York and Lon-
don: Plenum Press.
Henn, F. A., Johnson, J., Edwards, E., & Anderson, D. (1985). Melancholia
in rodents: neurobiology and pharmacology. Psychopharmacology Bul-
letin, 21, 443–446.
Henn, F. A., & Vollmayr, B. (2005). Stress models of depression: forming geneti-
cally vulnerable strains. Neuroscience and Biobehavioral Reviews, 29, 799–804.
Lachman, H. M., Papolos, D. F., Weiner, E. D., Ramazankhana, R., Hart-
nick, C., Edwards, E., et al. (1992). Hippocampal neuropeptide Y
mRNA is reduced in a strain of learned helpless resistant rats. Brain
Research: Molecular Brain Research, 14, 94–100.
Martinez, J. L., Jr., Jensen, R. A., Vasquez, B. J., McGuinness, T., &
McGaugh, J. L. (1978). Methylene blue alters retention of inhibitory
avoidance responses. Physiological Psychology, 6, 387–390.
Milad, M. R., & Quirk, G. J. (2002). Neurons in medial prefrontal cortex
signal memory for fear extinction. Nature, 420, 70–74.
Nair, H. P., Berndt, J. D., Barrett, D., & Gonzalez-Lima, F. (2001a). Meta-
bolic mapping of brain regions associated with behavioral extinction in
preweanling rats. Brain Research, 903, 141–153.
Nair, H. P., Berndt, J. D., Barrett, D., & Gonzalez-Lima, F. (2001b). Matu-
ration of extinction behavior in infant rats: large-scale regional interac-
tions with medial prefrontal cortex, orbitofrontal cortex, and anterior
cingulate cortex. Journal of Neuroscience, 21, 4400–4407.
NemeroV, C. B., Bremner, J. D., Foa, E. B., Mayberg, H. S., North, C. S., &
Stein, M. B. (2006). Posttraumatic stress disorder: a state-of-the-science
review. Journal of Psychiatric Research, 40, 1–21.
Orr, S. P., Metzger, L. J., Lasko, N. B., Macklin, M. L., Peri, T., & Pitman,
R. K. (2000). De novo conditioning in trauma-exposed individuals with
and without post-traumatic stress disorder: a functional MRI study.
Journal of Abnormal Psychology, 109, 290–298.
Overmier, J. B., & Seligman, M. E. (1967). EVects of inescapable shock
upon subsequent escape and avoidance responding. Journal of Com-
parative and Physiological Psychology, 63, 28–33.
Peri, T., Ben-Shakhar, G., Orr, S. P., & Shalev, A. Y. (2000). Psychophysio-
logic assessment of aversive conditioning in posttraumatic stress disor-
der. Biological Psychiatry, 47, 512–519.
Peter, C., Hongwan, D., Kupfer, A., & Lauterburg, B. H. (2000). Pharma-
cokinetics and organ distribution of intravenous and oral methylene
blue. European Journal of Clinical Pharmacology, 56, 247–250.
Phelps, E. A., Delgado, M. R., Nearing, K. I., & LeDoux, J. E. (2004).
Extinction learning in humans: role of the amygdala and vmPFC. Neu-
ron, 43, 897–905.
Quirk, G. J., Garcia, R., & Gonzalez-Lima, F. (2006). Prefrontal mechanisms
in extinction of conditioned fear. Biological Psychiatry, 60, 337–343.
Rau, V., DeCola, J. P., & Fanselow, M. S. (2005). Stress-induced enhance-
ment of fear learning: an animal model of posttraumatic stress disor-
der. Neuroscience and Biobehavioral Reviews, 29, 1207–1223.
Richman, H., & Frueh, B. C. (1996). Personality disorder symptomatology
among Vietnam veterans with combat-related PTSD. Anxiety, 2, 286–295.
Riha, P. D., Bruchey, A. K., Echevarria, D. J., & Gonzalez-Lima, F. (2005).
Memory facilitation by methylene blue: dose-dependent eVect on
behavior and brain oxygen consumption. European Journal of Pharma-
cology, 511, 151–158.
Sanders, A. R., Detera-Wadleigh, S. D., & Gershon, E. S. (1999). Molecular
genetics of mood disorders. In D. S. Charney, E. J. Nestler, & B. S. Bun-
ney (Eds.), Neurobiology of mental illness. New York: Oxford.
Shumake, J., Barrett, D., & Gonzalez-Lima, F. (2005). Behavioral charac-
teristics of rats predisposed to learned helplessness: reduced reward
sensitivity, increased novelty seeking, and persistent fear memories.
Behavioural Brain Research, 164, 222–230.
Shumake, J., Conejo-Jimenez, N., Gonzalez-Pardo, H., & Gonzalez-Lima,
F. (2004). Brain diVerences in newborn rats predisposed to helpless and
depressive behavior. Brain Research, 1030, 267–276.
Shumake, J., Edwards, E., & Gonzalez-Lima, F. (2001). Hypermetabolism
of paraventricular hypothalamus in the congenitally helpless rat. Neu-
roscience Letters, 311, 45–48.
Shumake, J., Edwards, E., & Gonzalez-Lima, F. (2002). Dissociation of
septo-hippocampal metabolism in the congenitally helpless rat. Neuro-
science, 114, 373–377.
Shumake, J., Edwards, E., & Gonzalez-Lima, F. (2003). Opposite meta-
bolic changes in the habenula and ventral tegmental area of a genetic
model of helpless behavior. Brain Research, 963, 274–281.
Shumake, J., & Gonzalez-Lima, F. (2003). Brain systems underlying sus-
ceptibility to helplessness and depression. Behavioral and Cognitive
Neuroscience Reviews, 2, 198–221.
Shumake, J., Poremba, A., Edwards, E., & Gonzalez-Lima, F. (2000). Con-
genital helpless rats as a genetic model for cortex metabolism in
depression. Neuroreport, 11, 3793–3798.
Sotres-Bayon, F., Bush, D. E., & LeDoux, J. E. (2004). Emotional persever-
ation: an update on prefrontal–amygdala interactions in fear extinc-
tion. Learning and Memory, 11, 525–535.
Sotres-Bayon, F., Cain, C. K., & LeDoux, J. E. (2006). Brain mechanisms
of fear extinction: historical perspectives on the contribution of pre-
frontal cortex. Biological Psychiatry, 60, 329–336.
Treit, D., & Fundytus, M. (1988). Thigmotaxis as a test for anxiolytic
activity in rats. Pharmacology, Biochemistry and Behavior, 31, 959–962.
True, W. R., Rice, J., Eisen, S. A., Heath, A. C., Goldberg, J., Lyons, M. J.,
et al. (1993). A twin study of genetic and environmental contributions
to liability for posttraumatic stress symptoms. Archives of General Psy-
chiatry, 50, 257–264.
Visarius, T. M., Stucki, J. W., & Lauterburg, B. H. (1997). Stimulation of
respiration by methylene blue in rat liver mitochondria. Federation of
European Biochemical Societies Letters, 412, 157–160.
Wang, S., Mason, J., Charney, D., Yehuda, R., Riney, S., & Southwick, S.
(1997). Relationships between hormonal proWle and novelty seeking in
combat-related posttraumatic stress disorder. Biological Psychiatry, 41,
Weible, A. P., McEchron, M. D., & Disterhoft, J. F. (2000). Cortical
involvement in acquisition and extinction of trace eyeblink condition-
ing. Behavioral Neuroscience, 114, 1058–1067.
Wong-Riley, M. T. (1989). Cytochrome oxidase: an endogenous metabolic
marker for neuronal activity. Trends in Neurosciences, 12, 94–101.