Download View - OhioLINK ETD

INVESTIGATING THE DIFFERENTIAL AMNESTIC EFFECTS OF A MILD HYPOTHERMIC TREATMENT
ON THE MEMORY FOR EXTINCTION
A dissertation submitted
to Kent State University in partial
fulfillment of the requirements for the
degree of Doctor of Philosophy
by
Devin Alan Fava
AUGUST, 2012
Dissertation written by
Devin Alan Fava
B.S., Denison University, 2006
M.A., Kent State University, 2009
Ph.D., Kent State University, 2012
Approved by
David C. Riccio
, Chair, Doctoral Dissertation Committee
Stephen B. Fountain
, Members, Doctoral Dissertation Committee
Richard D. Hirschman
,
Christopher Was
,
Wilson Chung
,
Accepted by
Maria Zaragoza
, Chair, Department of Psychology
Raymond Craig
, Dean, College of Arts and Sciences
ii
TABLE OF CONTENTS
LIST OF FIGURES……………………………………………………………………………………………………….…..IV
LIST OF TABLES………………………………………………………………………………………………………….……V
INTRODUCTION.................................................................................................................1
EXPERIMENT 1…………………………………………………………..………………………………………...………16
METHODS………………………………………………………………………………………………..……….16
GENERAL RESULTS……………………………………………………………………………………..….….19
RESULTS…………………………………………………………………………………………………………….20
DISCUSSION………………………………………………………………………………………………………23
EXPERIMENT 2……………………………………………………………………………………………………………..24
METHODS………………………………………………………………………………………………………….25
RESULTS…………………………………………………………………………………………………………...25
DISCUSSION………………………………………………………………………………………………………30
EXPERIMENT 3……………………………………………………………………………………………………………..30
METHODS………………………………………………………………………………………………………….32
RESULTS……………………………………………………………………………………………………………33
EXPERIMENT 3-B………………………………………………………………………………………………………….33
RESULTS……………………………………………………………………………………………………………33
DISCUSSION……………………………………………………………………………………………………..34
GENERAL DISCUSSION………………………………………………………………………………………………….35
REFERENCES…………………………………………………………………………………………………………………49
APPENDICES…………………………………………………………………………………………………………………53
Appendix A: Experiment 1 Data………………………………………………………………………..53
Appendix B: Experiment 2 Data…………………………………………………………………………54
Appendix C: Experiment 3 Data…………………………………………………………………………55
Appendix D: Experiment 3B Data………………………………………………………………………56
iii
LIST OF FIGURES
FIGURE 1 – EXPERIMENT 1 TTS SCORES………………………………………………………………….......22
FIGURE 2A – EXPERIMENT 2 TTS SCORES……………………………………….…………………………... .28
.
FIGURE 2B – EXPERIMENT 2 LINEAR CORRELATION……………………………………………………..29
iv
LIST OF TABLES
TABLE 1 – EXPERIMENT 1 DESIGN…………………………………………………………………………………19
TABLE 2 – EXPERIMENT 2 DESIGN…………………………………………………………………………………26
TABLE 3 – EXPERIMENT 3 DESIGN…………………………………………………………………………………32
v
Investigating the Differential Amnestic Effects of a Mild Hypothermic Treatment
on the Memory for Extinction
The Nature of Extinction. As a neutral stimulus is repeatedly paired with an
Unconditional Stimulus (US), that neutral stimulus becomes associated with the US.
Eventually, these repeated associations elicit responding to the previously neutral
stimulus which suggests expectation of the US; such a neutral stimulus is thereafter
referred to as a Conditional Stimulus (CS). The association between these two stimuli
(CS and US) is the basis for Classical Conditioning as described first by Pavlov (1927).
From the earliest days of Classical Conditioning, it has been known that such
associations were not permanent fixtures in the brain. By way of example, the
associations could be disrupted by repeatedly presenting a CS without the associated US
in a process known as Extinction. By presenting the CS in absence of the US repeatedly,
the CS seems to lose its value as a predictor for the US: an animal no longer responds to
the CS in a way which suggests expectation of the US (Pavlov, 1927).
This process of presenting the CS in absence of the US (extinction) has a very
important role with regard to basic learning processes in both laboratory animals and
humans. During extinction, the association between the CS and US is altered. In humans
seeking therapy, the previously-neutral CS has generally been associated with an
anxiety-producing US; this association creates anxiety when the patient is exposed to
1
2
the CS. The goal in breaking this association is to create a situation wherein the CS no
longer elicits the anxiety response.
Pavlov (1927) suggested that extinction was a special type of inhibition to the
existing memory. He noted that CS, which was originally neutral, but elicited anxiety
after being paired with the US, might return to the original (neutral) state if repeatedly
presented in absence of the US. Clinical therapies involving extinction of conditioned
anxiety date back to Wolpe (1958). In Wolpe’s systematic desensitization, the patient is
instructed in relaxation techniques which are then paired with an “anxiety hierarchy”
which consists of various tiers of exposure to the stimulus from simply imagining the
stimulus to interacting with it. By creating a new association between the CS and
relaxation, the CS no longer elicits anxiety. More recently reported clinical techniques
may apply the principles of extinction directly: either by having the patient imagine the
stimulus for a prolonged period of time (implosive therapy) or by exposing the patient
to the stimulus directly (flooding; Stampfl & Levis, 1967). The basis of these therapies is
still applied in modern clinical interventions (Lovibond, Davis, & O’Flaherty, 2000).
Extinction, however, must be more complicated than merely breaking an
association: even the modern clinical interventions are not universally successful (e.g.,
Schiller et al., 2010). The field of learning and memory has not, however, always
accepted the complicated nature of extinction. For example, interpretations of the
Rescorla-Wagner Model (1972) predict that the process of extinction will reduce the
associative strength between the CS and the US over a series of trials, representing an
3
erasure or an “unlearning” of the association. This model of extinction was thought to
decrease, or otherwise reset, the associative value of the CS to zero; a finding which is
supported at face value by an attenuation of behavioral responding to a CS over
repeated, non-reinforced trials. The attenuation of the previous behavior led many to
assume that extinction represented an “unlearning” of the original association.
However, Pavlov (1927) identified extinction as a special type of inhibition to the
existing memory and noted that the original memory may spontaneously recover after a
retention interval. The failure of the Rescorla-Wagner Model (1972) to explain this
spontaneous recovery is cited as one of the weaknesses of that model (Miller, Barnet, &
Grahame, 1995).
There is substantial evidence which suggests that an unlearning interpretation of
extinction is not true: that extinction represents additional learning rather than any sort
of decrease in the original association. Were extinction sessions to actually reduce the
associative strength between the CS and the US, many of the learning phenomena
which have been observed could not be explained adequately. Perhaps the greatest
evidence that memory erasure is not occurring is the ability for an extinguished
association to return, as Pavlov (1927) observed.
Models of Relapse. Bouton (2002) reviewed four primary models of relapse in
which an extinction memory is disrupted and the memory of primary conditioning is
expressed on a test. The mechanism of determining which of two “ambiguous”
4
memories (the primary conditioning memory or the extinction memory) were retrieved
seems to be context.
Relapse Models: Renewal and Spontaneous Recovery. If primary conditioning and
extinction do not occur in the same environment, then memory retrieval will be
context-dependent. In this situation, only testing in the extinction context will cause the
memory of extinction to be retrieved; testing in the primary conditioning context, a
novel context, or even a neutral context will likely produce a failure to retrieve the
extinction memory. This laboratory model of relapse is known as Renewal.
This context-dependent model of determining the retrieval of extinction
memories has been expanded to include temporal cues as a context: if a period of time
passes between extinction training and the performance test, then the contextual cues
of the performance test may change over time, therefore preventing extinction memory
retrieval. The phenomenon of seeing relapse of primary conditioning after a retention
interval is often referred to, using Pavlov’s (1927) term, as Spontaneous Recovery.
Despite similarities with Renewal, and the evidence that the two phenomena operate
on the same mechanism of contextual cues, these are still recognized as separate
phenomena (Bouton, 2002).
The recovery of anxiety after extinction due to changes in context cues is not
limited to laboratory animals. Rachman (1979) has reviewed cases in the clinical
literature where human subjects regain a fear that they were previously believed to
have extinguished. The researchers note specifically that longer intervals between
5
desensitization therapy and test trial seem to produce a “return of fear”. This is seen as
a parallel to the Spontaneous Recovery seen in laboratory animals (e.g., Bouton, 2002).
The Renewal and Spontaneous Recovery models of relapse strongly suggest that
representations of memory for both acquisition of a conditioned association and the
extinction memory for the same association exist simultaneously. An example of the
simultaneous existence of both types of memory comes from the literature by Brooks
and Bouton (1993). Food-deprived animals were trained in a paradigm where food was
be delivered following a tone; these animals were measured with regard to a foodseeking behavior. Brooks and Bouton utilized a “lights-off” cue to attenuate
spontaneous recovery. Animals trained with the “lights-off” cue during extinction
(where tone was not followed by food) showed facilitated extinction if the cue was also
present during testing after a retention interval: the acquisition response did not
spontaneously recover. This was not due to a contextual change: animals which
received the “lights-off” cue during testing, but were not trained with the cue, showed
spontaneous recovery. As such, the memory of both acquisition and extinction can be
seen to exist simultaneously, and be selectively retrieved via cues. The same paradigm
has been used to attenuate the Renewal effect (Brooks & Bouton, 1994). If extinction
involved erasure, the acquisition memory would not spontaneously recover.
Furthermore, this result does not represent a failure of extinction: extinction is still
shown when cues are employed.
6
Relapse Model: Reinstatement. Extinction may also fail when unsignaled
presentations of the US are delivered after the extinction training. An animal that has
received extinction training to break the association of a basic tone-shock pairing shows
a relapse in fear behaviors when the US is administered in absence of the CS:
subsequent tests with the CS show elevated fear. This model of relapse is known as
Reinstatement (Rescorla & Heth, 1975). In the classic Reinstatement experiment,
animals are trained to press a bar for food reward, and at the same time, there is a tone
(CS) which is paired with a foot-shock (US). If animals pause in the lever-press behavior
with presentation of the tone, this “conditioned suppression” is measured as an
indicator for the memory of fear. Rescorla and Heth demonstrated strong conditioned
suppression after just a few tone-shock pairings, though they were able to extinguish
this fear on the following day through a tone-no shock extinction procedure. Animals
which received one additional, unsignaled shock after extinction, however, showed
significantly higher levels of conditioned suppression during tone presentation than
animals that did not receive that shock; these animals also showed higher levels of
conditioned suppression than animals that had no prior tone-shock pairings. This result
strongly suggests that the extinction procedure does not erase the fear memory: it
merely creates an alternative learning experience.
Reinstatement, like other models of relapse, is not limited to laboratory animals.
Hermans, Dirikx, Vansteenwegen, Baeyens, and Eelen (2005) demonstrated that a
conditioned fear of neutrally-rated images of faces paired with electroshock (applied to
7
the forearm) could be extinguished, and subsequently reinstated via unsignaled shock in
humans. In this experiment, fear was measured by subjective measures of USexpectancy and fear rating (as reported by the participant). Earlier efforts to
demonstrate reinstatement in humans seem to have been overlooked due to a focus on
heart-rate increase as a measure of fear (e.g., Rachman & Whittal, 1989). This earlier
study used electroshock in an attempt to reinstate a different type of fear: fear of
spiders or snakes. While it has been demonstrated that another type of aversive event
can create reinstatement for a previous fear association (Rescorla & Heth, 1975,
experiment 3), Rachman and Whittal were not able to show significant heart-rate
increases from electroshock. They did, however, demonstrate an increase in the
subjective fear experience or arousal reported by participants; a result which they
largely ignored, but which supports a model for reinstatement in human subjects.
Relapse Model: Rapid Reacquistion. Bouton (2002) also described the Rapid
Reacquisition of extinguished responses; a conditioned association which has been
extinguished, can be reacquired to full strength with fewer CS-US pairings than were
required to establish the association the first time. In other words, the original
conditioned association remains, even after substantial extinction. In a study by Ricker
and Bouton (1996), animals previously trained with a stimulus (tone) predicting food
were indistinguishable with regard to food-seeking behaviors from their baseline scores
after extinction. These animals, however, showed more rapid re-learning of the tonefood association than naïve counterparts, or even in rats which have equivalent
8
experience with the food US alone, or the context alone. This “savings” of association
suggests that the memory of the tone-food association is never actually erased: if
erasure occurred, acquisition and re-acquisition curves should be identical. Instead,
rapid reacquisition of the original response (as with these other models) suggests that
extinction has not destroyed the original association.
Erasure, in the conventional sense, would imply that the acquired conditioning is
lost and cannot be recovered; this is simply not the case. Numerous studies have found
that extinction is not a permanent process. Whether through recovery, savings, or the
disruption of an apparent extinction memory, the acquired conditioning persists, and
there is little evidence to support the view that simple behavioral extinction will ever
completely erase it. On the other hand, extinction memories are less resilient to failures
related to time, interference, and other forms of disruption. This would seem
particularly true when comparing the extinction memory to the long-lasting changes in
behavior brought about by the primary conditioning.
Retrograde Amnesia. Perhaps the most intriguing evidence, however, that
extinction is new learning comes from literature showing that the memories of
extinction are susceptible to treatments which disrupt the formation or consolidation of
other memories. In 1968, Riccio, Hodges and Randall discovered that inducing
hypothermia in animal subjects shortly after one-trial training in a passive avoidance
9
task creates retrograde amnesia for the events of that passive-avoidance training. The
prototypical passive-avoidance experiment involves an apparatus with two chambers:
one with black walls and one with white walls. Traditionally, the animal is placed in the
white chamber and allowed to cross into the black chamber; rats, being nocturnal, show
strong preference for the black chamber. Crossing into the black chamber is paired with
foot-shock (training), and memory is assessed on later tests by placing the animal in the
white chamber again and measuring its avoidance of the black chamber. When animals
are tested 24 hr. after training, they demonstrate extreme avoidance behaviors under
normal circumstances. When the training event is followed by administration of the
hypothermic agent, in which animal body temperatures were reduced to 68.5ºF
(20.3ºC), memory retrieval on the test was impaired: avoidance behaviors decreased.
Further experiments demonstrated that this effect could not be attributed to the
stressful effects of restraint, water submersion (Riccio, Hodges & Randall, 1968), or the
punishing effects of the cold water immersion (Riccio, Gaebelein, & Cohen, 1968).
Amnesia as Retrieval Failure. Early work by Spear (1978), highlighted that the
memory for a behavior might be more difficult to retrieve if the context in which a
memory was encoded was too different from the context in which retrieval was
attempted. This conclusion was based on previous papers, such as the Encoding
Specificity Hypothesis presented by Tulving and Thomson (1973). Tulving had found that
memory retrieval was improved if the cues in the environment when the target memory
10
was encoded matched the cues present during attempted retrieval. In the case of
hypothermia-induced retrograde amnesia, the apparent disappearance of a memory
can be attributed to the difference in context between the time when the memory was
encoded (low body temperature) and the time at which memory retrieval is attempted
(normal body temperature); contextual cues may be internal as well as external.
Even before the Encoding Specificity Hypothesis, McGeoch and Irion (1952)
noted that a previously observed conditioned behavior may appear to be forgotten in
the absence of the cues which are present during training. The effects of contextual
cues, they note, may be seen as contexts naturally change over time, though these
effects are even clearer when the context is manipulated by the researcher. Specifically,
McGeoch and Irion referenced human experiments in which recall of word lists was
dramatically improved in the presence of paired meaningful words or background visual
cues (e.g., a different classroom). They also noted that an alternate context may not
simply fail to elicit a response learned in a primary context, but may actually elicit
competing responses which block the original ones (McGeoch & Irion, 1952). These
findings suggest that a memory, blocked from retrieval by a lack of cues, may be
retrieved if those cues are returned.
In the area of hypothermia research, it was demonstrated that an animal, given
the hypothermic treatment shortly after training, would show an attenuation of the
expected retrograde amnesia if the animal’s body temperature was lowered prior to the
test (Hinderliter, Webster, & Riccio, 1975; Mactutus & Riccio, 1978). In other words,
11
adjusting the animal’s internal context prior to the test to be more similar to the posttraining interval (when the memory was presumably encoded) facilitated retrieval and
attenuated the hypothermia-induced retrograde amnesia. Additionally, if memory
consolidation in the “cold state” was interrupted by re-warming the animals in nonhypothermic water, the efficacy of the hypothermic treatment was diminished: animals
then consolidated the memory in a state similar to their normal body temperature state
(Mactutus, McCutcheon, & Riccio, 1980). This result demonstrates that memory
retrieval is largely dependent on similarity of cues between memory encoding and
memory retrieval: merely lowering the animal’s body temperature is insufficient to
produce retrograde amnesia. Taken together, this research led to a “retrieval failure”
model of laboratory-induced retrograde amnesia (e.g., Spear & Riccio, 1994).
Alternatives to Retrieval Failure. The retrieval failure model of amnesia is not
without critics. Recently, a study by Hardt, Wang, and Nader (2009) provided a
challenge to the retrieval failure model. This study took advantage of properties of the
NMDAr antagonist AP5, which creates amnesia for memories of first, but not second,
contexts. In other words, the AP5 injection will not have an effect on the memories of
animals which have been trained in a similar paradigm. Hardt established that, if the
first memory consolidation was disrupted by injection a different protein synthesis
inhibitor (anisomycin) following training, that the AP5 would still have an effect if used
to disrupt memory consolidation of the second training. The authors argued that, had
12
anisomycin only disrupted memory retrieval, the memory trace would have been
sufficient to prevent the amnestic effects of AP5. Therefore, the authors concluded that
anisomycin had completely blocked the memory storage, rather than affecting the
memory retrieval.
The Hardt, Wang, and Nader (2009) study does not, however, disprove a
retrieval failure model. The model assumes, for instance, that the dorsal hippocampus is
the only area which stores context-based memory, and this is not supported by the
literature (Matzel & Miller, 2009). More importantly, notable counterexamples to
Hardt’s study were not addressed by the authors. The first counterexample is an early
study by Bradley and Galal (1988) in which young chicks are trained to passively avoid
pecking at a chrome bead covered in an aversive liquid. Animals receiving protein
synthesis inhibitor injections prior to training showed amnesia for the training during
the test, but this amnesia was reversed by a pre-test injection of the same protein
synthesis inhibitor. In other words, if the drug was present in the animal’s system during
training, but not testing, amnesia was evident; if the drug was present in the animal’s
system during both training and testing, amnesia was attenuated.
The second counterexample to the Hardt, Wang, and Nader (2009) study is an
earlier Canal, Chang, and Gold (2007) study. In the Canal study, a protein synthesis
inhibitor produced retrograde amnesia, but it was also shown to increase levels of
endogenous neurotransmitters such as norepinephrine, serotonin, and dopamine by as
much as 17,000%. Co-administration of substances designed to decrease this surge of
13
neurotransmitter along with the protein synthesis inhibitor attenuated the amnesia
created by the protein synthesis inhibitor. Similarly, pre-test administration of the
previously-increased neurotransmitter itself also attenuated the retrograde amnesia.
The evidence, therefore, strongly suggests that protein synthesis inhibitors
create retrograde amnesia by creating a discrepancy of internal context between
memory consolidation and memory retrieval. Given that a retrieval failure model for
retrograde amnesia still seems the most tenable model for mature animals, as used in
the present study, it will remain the focus of this review.
“Mild” Treatments. Attention to a retrieval failure model is particularly
interesting with regard to the aforementioned difference between new memories and
memories reactivated using a reminder. A study by Mactutus, Riccio and Ferek (1979)
found that an amnestic agent which was too weak to disrupt a newly formed memory
(lowering animal body temperatures to 86ºF or 30ºC) was capable of disrupting an old
memory which had been reactivated using a reminder treatment; the temperature
animal bodies were reduced to for this amnestic treatment was higher (a milder
treatment) than treatments employed in the earlier work on hypothermia-induced
retrograde amnesia (e.g., Riccio, Hodges, & Randall, 1968).
The study by Mactutus, Ferek, and Riccio (1979) produces results similar to a
later study by Anokhin, Tiunova, and Rose (2003). In the Anokhin study, the necessary
dose of a protein synthesis inhibitor used to create retrograde amnesia was lower for a
14
reactivated memory than a newly formed memory. Both studies suggest that the
reactivated memory is more susceptible to the respective amnestic agent relative to the
memory as it is originally formed. Such findings raised several interesting questions. One
implication based on the results of these experiments is that not all types of memory
are equally resilient against disruption. Some types of memory may be “weaker” or
more susceptible to degradation. These weaker memories, therefore, may be affected
by a milder amnestic agent, such as a hypothermia exposure which is so mild that it has
no detectable effect on performance if applied immediately after training.
Amnesia for Extinction. Briggs and Riccio (2007) demonstrated that a
hypothermic agent, like the one used to induce retrograde amnesia in rats for a fearconditioning task, can also induce retrograde amnesia for extinction training associated
with that task. Briggs trained animals in a passive-avoidance paradigm and conducted an
extinction session on the following day; for some groups, extinction was followed by
administration of hypothermia (the amnestic agent). Animals receiving hypothermia
after extinction showed amnesia for the extinction training and a return of the original
fear memory. This suggests that extinction creates a memory rather than erases one: if
extinction represented memory erasure, then extinction itself could not be disrupted by
an amnestic treatment. Instead, the finding than an amnesia-inducing treatment has an
effect on extinction strongly suggests that extinction is a form of learning. Additionally,
15
it is a precedent which was fundamental for the goals of some of the following
experiments.
It is clear that the process of extinction creates a memory representation. It is
also reasonable, given the vulnerability of the extinction memory as evidenced by
several laboratory models of relapse, to consider that such an extinction memory may
be susceptible to forms of disruption (amnestic agents) which would not significantly
affect or disrupt the training memory. In other words, the memory for extinction may be
weaker, or more vulnerable to disruption, than the original associative memory it is
meant to eliminate. If true, a weaker form of existing amnestic agents may not affect a
memory for fear conditioning, but it might be enough to disrupt a memory of extinction
learning for that fear conditioning.
Present Study. To begin such an investigation, researchers used a mild
hypothermic agent similar to the treatment which disrupted a reactivated memory in
the Mactutus, Ferek, and Riccio (1979) study. By comparing the disruptive effects of a
mild hypothermic treatment on extinction to the effects of the same treatment on the
“training” or fear memory, the relative vulnerability of primary conditioning to
extinction can be examined.
16
Experiment 1
Experiment One was designed to test the hypothesis that an extinction memory
is more susceptible to disruption than the primary conditioning memory. It was
hypothesized that a mild hypothermia treatment, which should be too weak to disrupt
the primary conditioning memory (see Amnesia Control Group below), would be
capable of disrupting another learning event: the memory for Extinction.
Methods for Experiment 1
Subjects. Experiment One utilized 48 male and female Long-Evans Rats, bred at
the Kent State University Breed Colony. Animals were approximately 90 days old at the
beginning of experimental procedures, assigned to groups randomly, and
counterbalanced for sex. Animals were housed as same-sex with littermates in the Kent
State Animal Lab Main Colony for the duration of the experiment.
Handling. (Day 1 and 2, all subjects) On each of the two days prior to training,
subjects were handled for five minutes. This handling procedure took place in the
neutral environment of the housing colony to avoid unintended contact with any
contextual stimuli in the lab.
Training. (Day 3, all subjects) Subjects were trained in a standard passiveavoidance apparatus. The plexi-glass box has dimensions of approximately 45.5 x 17.5 x
23.5 cm, and is separated into a black compartment and a white compartment by a
partition. Subjects in the task were allowed to cross through the partition when a
17
guillotine door was opened by the researcher. The floor of the apparatus is constructed
from 2mm metal bars spaced 1cm apart which are connected to a Foringer Model SC901 Scrambler used to deliver foot-shock.
Upon being brought into room with the apparatus, subjects were handled for 30s
while they became acclimated to some of the contextual cues. After handling, the
subject was placed into the white “safe” side of the apparatus. The top of the apparatus
was closed, and subjects were allowed to explore for 20s. After this period, the
guillotine door was opened, and latency to cross into the black side of the apparatus
with all four paws was recorded. Once the subject had crossed, the guillotine door was
closed, confining the subject to the black side of the apparatus. After a 5s delay, the
subject was given inescapable foot-shock (.5mA, 1s duration, administered through the
metal flooring).
Extinction. (Day 4, Extinction Groups Only) Subjects were brought into room with
the apparatus and handled for 30s while they became acclimated to some of the
contextual cues. After handling, the subject was placed into the white “safe” side of the
apparatus. The top of the apparatus was closed, and subjects were allowed to explore
for 15s. The experimenter then removed the animal from the white side of the
apparatus, and placed the animal in the black “unsafe” side of the apparatus. The
subjects were allowed to explore for 12 minutes, during which time the experimenter
left the room. No shocks were delivered during the Extinction phase.
18
Animals not in Extinction Groups were brought into a neutral lab space and left
in their home cages for 13 minutes before being returned to the colony.
Body Cooling. (Day 3 or 4, Amnesia Groups Only) Subjects were restrained in
plastic tubes and lowered in 4.0ºC water up to the neck to induce body cooling or
hypothermia. Animals in the “Extinction + Amnesia” Group received hypothermia
immediately following Extinction. Animals in the “Amnesia Control” Group received
hypothermia immediately following training: this mild hypothermia treatment had not
been effective on original training memories in past experiments, but this control group
was necessary to demonstrate differential effects of hypothermia on training and
Extinction memories.
Subjects’ core temperatures (normally 37ºC or 98.6ºF) were lowered to 26.7ºC
(80ºF) in experimental groups. Subjects’ core temperatures were recorded via rectal
probe after removing them from the water.
Water was cooled and maintained at a stable temperature by a 2095 Forma
Temp Jr. water bath and circulator device. Animals from these Experimental Groups
were dried and wrapped with a towel and allowed to re-warm naturally in the Recovery
Room of the Animal Colony before being returned to their home cages.
Test. (Day 5, all subjects) After 24 hours, the subjects were returned to the lab.
Upon being brought into room with the apparatus, subjects were handled for 30s while
they became acclimated to some of the contextual cues.
19
After handling, the subjects were placed into the white “safe” side of the
apparatus. The top of the apparatus was closed, and subjects were allowed to explore
for 20s. After this period, the guillotine door was opened, and the testing “exploration”
period began. During this period, No shocks were administered, and the guillotine door
was never closed.
Researchers recorded the latency to cross into the black side of the apparatus
with all four paws. In addition, the total time spent in the white “safe” side of the
apparatus was also recorded. The testing period lasted for 10 minutes. At the end of
testing, subjects were returned to the lab colony.
Group
Training
Retention
Extinction + Amnesia
Extinction Control
Amnesia Control
shock
shock
shock
shock
Post-Training
Hypo
no
no
no
yes
Extinction
no
yes
yes
no
Post-Ext
Hypo
no
yes
no
no
Test
10min.
10min.
10min.
10min.
Table 1 – Experiment 1 Design
General Results
In all experiments, data were screened for rats that were outliers within their
own groups; animal Latency and Total Time in the Safe Area (TTS) scores were
standardized, and scores greater than two standard deviations from the group mean
were discarded. In groups as small as the ones used in this study, this was a rare
occurrence.
20
A ten-minute latency ceiling was imposed on all subjects during the tests. Rats
which failed to cross over to the black side of the shuttle-box were assigned a latency
score of 600 seconds for the test portion. There were no missing data in the
experiments: some subjects were removed due to mechanical failures of the apparatus,
or environmental disruptions (outside interruptions of the experiment). One subject was
removed due to injury. Replacement subjects were added where needed to fill groups.
Results for Experiment 1
Data for Experiment 1 are presented in Appendix A. A One-Way ANOVA
indicated that there was no effect of any Group on Training Latencies, F (3, 44) = 1.91, p
> .05. There were also no effects of animal sex on Testing Latencies or TTS scores (all Fs
< 1). Groups were, therefore, collapsed across sex.
A One-Way ANOVA revealed an effect of Group on Test Latency scores, F (3, 44)
= 2.879, p = .047). A Bonferroni post-hoc test showed a significant effect of the
Extinction treatment when compared to Retention Control animals (p = .035), but no
other effects on Test Latency Scores.
A One-Way ANOVA revealed an effect of Group on Test TTS scores, F (3, 44) =
5.71, p = .002. Bonferroni post-hoc tests showed that the Extinction Group TTS scores
were significantly lower than the scores in the Retention Group (showing significant
Extinction, p = .007), the Amnesia Control Group (showing no effect of Hypothermia
following training, p = .001), and the Extinction + Amnesia Group (suggesting that the
21
mild hypothermia significantly attenuated the Extinction Memory, p = .007). As
predicted, there were no other differences between groups. These scores are shown in
Figure 1.
22
600
**
**
**
Total Time in Safe (seconds)
500
400
300
200
100
0
Retention
Extinction
Extinction + Amnesia
Amnesia Control
Group
Figure 1 – Mean TTS Scores by Group (sexes combined) +95% C.I. for Experiment 1.
Animals in the Extinction Group spent significantly less time in the white (safe) side of
the apparatus than retention animals, demonstrating extinction. Additionally, animals
given the mild hypothermia following extinction performed as though they did not
retrieve extinction memories (significantly higher scores). This hypothermia-induced
retrograde amnesia did not, however, affect animals given the treatment immediately
following training, suggesting that the training memory was more resilient to the
hypothermia treatment than the extinction memory. ** indicates differences from
Extinction, p < .01.
23
Discussion
It was hypothesized that the memory of extinction would be disrupted by the
mild hypothermia treatment administered shortly after Extinction training in the
Extinction + Amnesia Group. This effect was detected; animals in the Extinction +
Amnesia Group showed TTS scores similar to Retention Animals, and significantly
different from animals in the Extinction Group. The disruptive properties of the mild
hypothermia treatment seem to be specific to the Extinction Memory: animals in the
Amnesia Control Group did not differ from the Retention Group nor from the Extinction
+ Amnesia Group.
These effects were more difficult to detect using the Latency scores. While used
for several decades as the primary measure of animal fear in passive-avoidance tasks,
the Latency score is susceptible to inaccuracies when studying very impulsive or highlystressed animals: animals which are highly stressed may cross from the safe side of the
apparatus quickly as an escape behavior, only to recall the passive-avoidance training in
the presence of contextual cues, and quickly return to the safe side. When this occurs,
Latency scores may be low, even while TTS scores are very high. Researchers generally
focused on the TTS scores because the observed power of this measure was higher than
the power of the Latency score. The observed power of the ANOVA conducted on
Latency scores in Experiment One (.648) was substantially lower than the observed
power of the ANOVA conducted on TTS scores (.929). It is worth noting, however, that
24
these values represent retrospective power analyses and should therefore be
interpreted with some caution (Thomas, 1997).
The findings of Experiment One suggest that the mild hypothermic agent is
insufficient to disrupt the original memory of fear conditioning, but that it is sufficient to
disrupt the memory for extinction. Specifically, the less severe hypothermic treatment
was effective at disrupting the extinction memory as evidenced by significant
attenuation of extinction which resulted in avoidance behavior (indicative of a return of
the training or fear memory). This mild treatment, however, was insufficient to disrupt
the fear memory itself when administered with the same intensity following the initial
training session. This finding suggests that the memory for Extinction is more vulnerable
to contextual differences between memory encoding and memory retrieval than a
memory for fear conditioning.
Experiment 2
Successful attenuation of an extinction memory separate from the disruption of
the primary conditioning suggests greater vulnerability of the extinction memory.
Experiment Two was designed to be a parametric study testing several degrees of
hypothermia in order to determine the “cut-off” for extinction memories to be
disrupted. Previous studies have suggested that stressors, such as water immersion or
restraint, were insufficient to disrupt a memory for stimulus attributes (Fava, Barnes,
and Riccio, in prep.) but the question of vulnerability remained for an extinction
25
memory: if a very mild treatment were sufficient to disrupt an extinction memory, one
must consider that perhaps the results of Experiment One were disruptions of memory
resulting from stress or interference.
Experiment Two, therefore, attempted to demonstrate a functional relationship
between contextual change and extinction memory. The hypothesis for this study was
that a significant negative correlation would exist between post-extinction body
temperature and measures of memory (Latency and TTS). Such a finding would replicate
the results from Experiment One. Additionally, such a result would support the
hypothesis that it is the contextual difference between the time extinction memories
are encoded and test memories are retrieved (rather than a stressor like restraint)
which is primarily responsible for the disruption of the extinction memory.
Methods for Experiment 2
Fifty-one animal subjects were used as described in Experiment 1. Handling,
Training, and Extinction procedures occurred as described in Experiment 1. This
experiment also utilized a Retention Control and an Extinction Control Group which
were run as described in Experiment 1.
Body Cooling. (Day 3 or 4, Amnesia Groups Only) Body Cooling Procedures
occurred as described in Experiment 1, except where noted below.
Subjects’ core temperatures (normally 37ºC or 98.6ºF) were lowered to either
26.7ºC (80ºF, to replicate the results of Experiment 1), 29.4ºC (85ºF), or 32.2ºC (90ºF)
26
through immersion in water which is 4ºC. Differences of as little as 5ºF have been
shown to have different disruptive properties in regards to different types of memories
(Mactutus, Ferek, and Riccio, 1979; Fava, Barnes, and Riccio, in prep.).
Test. (Day 5, all subjects) After 24 hours, the subjects were returned to the lab,
and testing proceeded as described in Experiment 1.
Group
Training
Extinction
Retention
Extinction Control
Hypothermia 80º
Hypothermia 85º
Hypothermia 90º
shock
shock
shock
shock
shock
no
yes
yes
yes
yes
Post-Ext
Hypo
no
no
to 80º
to 85º
to 90º
Test
10min.
10min.
10min.
10min.
10min.
Table 2 – Experiment 2 Design
Results for Experiment 2
Data for Experiment 2 are presented in Appendix B. The Results for Experiment
Two are found in Figure 2.
A One-Way ANOVA indicated that there was no effect of any Group on Training
Latencies, F (4, 46) = 1.71, p = .163. There were also no effects of animal sex on Testing
Latencies, F < 1; or on TTS Scores, F (1, 46) = 1.14, p = .291. Groups were, therefore,
collapsed across sex.
A One-Way ANOVA revealed a significant effect of Group on Test Latency Scores,
F (4, 46) = 4.583, p = .004. A Bonferroni post-hoc test demonstrated that the animals in
the Hypothermia 80º Group had significantly higher Latency scores when compared to
27
Extinction Control animals (p = .034) and Hypothermia 90º animals (p = .023). There
were no other significant differences between Groups.
A One-Way ANOVA revealed a significant effect of Group on TTS, F (4, 46) = 6.36,
p < .001. A Bonferroni post-hoc test showed a significant effect of the Extinction
treatment when compared to Retention Control animals (p = .03), replicating a
significant extinction effect. Furthermore, the animals receiving the 80º Hypothermic
Treatment had significantly higher TTS scores than animals in the Extinction Group (p =
.001). Animals receiving 85º and 90º Hypothermic Treatments did not significantly differ
from either Retention or Extinction Controls. These data are presented in Figure 2A.
A Pearson Correlation was also conducted using actual post-treatment colonic
temperatures (opposed to using categorical groups) in the animals given the
hypothermia treatments. This test suggested that the relationship between body
temperature and memory retrieval was a linear relationship, rather than merely a
stepwise effect. The relationship between post-treatment colonic temperatures and TTS
scores approached significance; r = -.378, p = .052. These data are presented in Figure
2B.
28
Total Time in Safe (seconds)
600
**
**
500
400
300
200
100
0
Retention
Extinction
Hypo 80
Hypo 85
Hypo 90
Group
Figure 2A – Mean TTS Scores by Group (sexes combined) +95% C.I. for Experiment 2.
Animals in the Extinction Group spent significantly less time in the white (safe) side of
the apparatus than animals in the Retention Group, demonstrating extinction.
Additionally, animals in the Hypo 80 Group performed as though they did not retrieve
extinction memories (significantly higher scores), though animals in Hypo 85 and Hypo
90 Groups did not significantly differ from animals in the Extinction Group. This result
suggests that a minimum amount of body-cooling is necessary to create disruption of
the extinction memory. Asterisks indicate differences from Extinction, ** p < .01.
29
600
Total Time in Safe (seconds)
500
400
300
200
100
0
75
77
79
81
83
85
87
89
91
93
95
Post-Extinction Body Temperature (ºF)
Figure 2B – Individual TTS scores are displayed as a function of Body Temperature; line
indicates best fit. A negative trend, approaching significance (R2 = .143, p = .052),
demonstrates that lower Post-Extinction Body Temperatures result in greater disruption
of extinction as indicated by higher TTS scores. Conversely, the closer an animal’s body
temperature is to normal following extinction, the better extinction memories are
retrieved on the retrieval test 24 hr. later (when body temperature is also normal).
30
Discussion
This result replicates the findings of Experiment One: lowering an animal’s
temperature to 80ºF after extinction disrupts the memory for extinction. In Experiment
One, it was shown that an 80ºF treatment was ineffective at attenuating performance
for the fear memory when administered following training, which suggests a difference
in strength between the memory of training and the memory for extinction.
More importantly, this experiment helps to further investigate the relationship
between post-extinction contextual cues (temperature) and later memory retrieval.
Specifically, as the body temperature of the animal in the post-extinction period (when
the memory is presumably encoded) becomes more similar to the temperature of the
animal during the test (via weaker treatments) the retrieval of the memory for
extinction is better. This is a relationship demonstrated by a linear trend which
approaches significance: Test TTS scores increase across treatment groups from least- to
most-severe hypothermic treatment, suggesting that extinction memories are more
disrupted by more severe hypothermia treatments; treatments which are still too mild
to disrupt the original fear memory.
Experiment 3
In Experiment 2, more severe post-extinction treatments seemed to increase the
effectiveness of hypothermia as an agent for inducing retrograde amnesia of the
extinction memory. This is likely due to differences in the internal context of the animal
31
between the post-extinction interval when extinction memories were encoded, and the
test: specifically, as the treatments were weakened, animal body temperature during
the post-extinction interval was higher and therefore more similar to the test interval,
facilitating retrieval of the extinction memory. The Briggs and Riccio (2007) study
supported this finding. Early experiments in the Briggs and Riccio study utilized
hypothermia to create retrograde amnesia for extinction memories. In their later
experiments, Briggs adjusted animal body temperatures prior to the test to facilitate
retrieval of the extinction memory by making the internal context more similar to the
post-extinction period. This pre-test recooling facilitated retrieval of the extinction
memory in animals which had been treated with the hypothermic treatment after
extinction. As animals re-warmed, extinction memories were no longer retrieved.
In order to confirm that results in Experiments 1 and 2 are due to internal
context change, Experiment 3 was designed to replicate the Briggs and Riccio (2007)
result. It was hypothesized that returning animals to a hypothermic state prior to the
test would facilitate memory retrieval for events (extinction) which were encoded
during a similar state (the post-extinction interval).
Animal subjects were used as described in Experiment 1. Handling, Training, and
Extinction procedures were conducted as described in Experiment 1. This experiment
also utilized a Retention Control and an Extinction Control Group which were run as
described in Experiment 1. Body Cooling procedures occurred as described in
Experiment 2, except where noted below.
32
Methods for Experiment 3
Body Cooling. (Day 3 or 4, Amnesia Groups Only) Based on the results of
Experiment 2, subjects’ core temperatures were lowered to 26.7ºC (80ºF) by immersion
in water which was 4ºC.
Test. (Day 5, all subjects). After 24 hours, the subjects were returned to the lab.
Testing in Retention Control and Extinction Control Groups proceeded as described in
Experiment 1. A third group (Extinction-Hypo Group) was employed to replicate the
results of the Hypothermia 80º Group from Experiment Two.
The Experimental Group (Pre-Test Re-exposure Group) was given a second brief
hypothermia treatment prior to the test. Subject core temperatures were lowered to
approximately 85ºF, mirroring the treatment used in the Briggs and Riccio (2007) study
which successfully allowed the extinction memory to be retrieved after a more severe
post-extinction hypothermia treatment.
Group
Training Extinction
Retention
Extinction Control
Extinction-Hypo
Pre-Test Re-exposure
shock
shock
shock
shock
Table 3 – Experiment 3 Design
no
yes
yes
yes
Post-Ext
Hypo
no
no
yes
yes
Pre-Test
Hypo
no
no
no
yes
Test
10min.
10min.
10min.
10min.
33
Results for Experiment 3
Data for Experiment 3 are presented in Appendix C. A One-Way ANOVA
conducted on the TTS scores of subjects in Experiment Three showed no effect of Group
F (3, 28) = 1.401, p = .263. This result is interpreted as anomalous because it failed to
replicate any of the results of previous experiments, including an extinction effect which
is well-documented in the literature. Possible causes for this anomaly are discussed
below.
Experiment 3, Part B
In order to further investigate the anomaly of Experiment Three, the ExtinctionHypo Group and the Pre-Test Re-exposure Group were re-run using 14 naïve animals.
Results for Experiment 3, Part B
Data for Experiment 3B are presented in Appendix D. A t-test conducted on the
TTS scores of subjects in Experiment Three showed no effect of t(11) = -1.90, p = .084.
This result approaches significance, but animals in the 80º Hypothermia Group show
much lower TTS scores than animals in the animals in the Pre-Test Re-Exposure Group,
which runs counter to our hypothesis. The scores of the 80º Hypothermia Group are far
lower than seen in the previous experiments, and they are thought to be a product of
disruptions in the lab environment, which are discussed below.
34
Discussion
Anomalous findings in Experiment Three are likely attributable to changing lab
conditions. Ongoing construction and preparations for a National Inspection by the
Association for Assessment and Accreditation of Laboratory Animal Care International
(AAALAC) brought many new individuals and contextual cues into the laboratory, which
may have been disruptive to extinction memories. Both Construction and AAALAC
Inspection are necessary for the continued function of the animal lab, and the situation
was unavoidable.
As noted earlier, extinction memories are particularly sensitive to changes in
context. If a context of testing does not match the context in which extinction occurred,
a Renewal Effect is often observed. This Renewal of the original conditioned response
occurs whether the testing context closely resembles the original conditioning context
(ABA Renewal) or whether it is a novel context (ABC Renewal; Bouton, 2002). Although
researchers and technicians did not observe any specific changes to the lab
environment, it is likely that what resembles an important or obvious change to a
human researcher is not the same as the same representation to an animal subject.
Alternatively, ongoing operations may have changed the internal context of the animal
by elevating stress levels; such a context shift would not be observable to a researcher
who was not actively measuring it.
35
General Discussion
Contributions. To the best of my knowledge, a test for the differential
susceptibilities of extinction versus primary conditioning memories for disruption has
never been attempted. Examples can be found wherein neurotransmitter injections
facilitate or disrupt extinction processes (e.g., Koh & Bernstein, 2003), but such findings
(especially disruption of extinction) could be explained by memory models of relapse:
specifically, the chemical shifts may represent contextual change indicative of a renewal
effect.
This research also provides further insight into extinction memories. As
previously stated, it has long been known that extinction represents new learning rather
than an erasure of a previous association. Additionally, it is well-established that
extinction memories are susceptible to disruption by the various models of relapse (e.g.,
Bouton, 2002). It has also been demonstrated that extinction memories can be
disrupted by an amnestic agent like the cholinesterase inhibitor physostigmine (Deutsch
& Wiener, 1969) or hypothermia (Briggs & Riccio, 2007).
The findings of Experiments One and Two provide another example that
extinction memories can be disrupted by an amnestic agent. Moreover, our results
support the hypothesis that an extinction memory is actually more susceptible to
disruption than the fear memory which it extinguishes. Specifically, the results of
Experiments One and Two demonstrate that a milder body cooling treatment is able to
disrupt a newly formed memory for extinction, but not a newly formed fear memory.
36
Contributions: Effects on “Weaker” Memory. The results of Experiment One and
Two are similar to previous findings, including those by Fava, Barnes, and Riccio (in
prep.) which showed that a memory for stimulus attributes (contextual memory) seems
to be susceptible to a mild hypothermia treatment. The Fava, Barnes, and Riccio study
relied heavily on the so-called “context shift effect” (attributed to Spear, 1978). The
context shift effect is a phenomenon in which memory retrieval fails because the
contextual cues present at the time of a retrieval test do not match the cues present
when that memory was encoded; in other words, when the context is “shifted”, the
performance of the animal suggests that it does not remember the original training. In
the Fava, Barnes, and Riccio study, untreated (control) animals displayed a context shift
effect in a passive-avoidance task identical to the one used in the present study, but
animals treated with mild hypothermia following training showed no context shift
effect. The results of that study suggest that the memory for stimulus attributes (that is,
the memory of the contextual cues themselves) is more susceptible to disruption by an
amnestic agent than the memory of fear itself: post-training treatment with the mild
hypothermia actually causes a generalization of fear across multiple contexts rather
than amnesia for the fear memory.
We might compare the results for the memory for stimulus attributes with the
memory of extinction: Bouton (2002) has suggested that extinction relies heavily on
contextual cues. Specifically, extinction is most successful when the contextual cues of a
performance test match the contextual cues present during extinction; when cues are
37
ambiguous, animals seem to revert to the training memory. Based on the results of the
Fava, Barnes, and Riccio study, the results of the present study may reflect the reliance
of extinction on contextual cues: if a mild hypothermic treatment changes those
contextual cues, extinction memories may not be retrieved.
Contributions: Reactivation vs. Extinction. Previous research (e.g., Mactutus,
Ferek, & Riccio, 1979; Anokhin, Tiunova, & Rose, 2002) has demonstrated that milder
treatments are capable of disrupting memories reactivated by a reminder treatment but
not newly formed memories. It is, however, important to note the distinction that exists
between reactivating a memory and extinguishing a memory. Although both the
reactivation process and the reconsolidation process involve re-exposure to the fearinducing apparatus, an extinction treatment is generally longer in duration. Pedreira and
Maldonado (2003) demonstrated that longer exposures (> 1 hr) to a fear-inducing
stimulus facilitate extinction to a visual danger stimulus in the Chasmagnathus crab: fear
to the stimulus decreases, but injections of a protein synthesis inhibitor (cycloheximide)
shortly after this extinction session disrupts that extinction. On the other hand, brief
exposures to the stimulus (< 1 hr) may act as a reminder to reactivate the memory: no
decrease of fear is observed, and cycloheximide injections serve to disrupt fear rather
than disrupt extinction.
Although the threshold between extinction and reactivation is not 1 hr. in the
rat, it seems clear that the present study disrupted the extinction memory rather than
the training memory. Specifically, if the extinction procedures in the present study acted
38
to reactivate, rather than extinguish, the fear memory, one would expect a result more
similar to the results of the Mactutus, Ferek, and Riccio (1979) study: minimal fear
resulting from disruption of the reactivated fear memory. It is interesting to note that
there is an emerging pattern wherein a memory that is not the primary memory, such as
a reactivated memory (Mactutus, Ferek, & Riccio, 1979), a contextual memory (Fava,
Barnes, & Riccio, in prep.) or an extinction memory (present study) is susceptible to
disruption via a less-severe treatment. Future research may be able to demonstrate that
extinction is especially susceptible to disruption specifically because it is a “secondary”
memory.
Alternative Models of Explanation. There are a few other models, however,
which may be used to describe these findings in a different way: retrograde amnesia is
not the only mechanism by which a memory can be disrupted. Innumerable other
memory phenomena can cause the disruption of memory. The limitations of memory
are seen most clearly in its failures.
Alternative Model: Interference. One limitation to our memory is our “memory
load,” which was described as early as 1958 by Broadbent. Broadbent suggested that
environmental stimuli could be responsible for filling this memory load, and otherwise
causing interference which disrupts the formation of new memory. One might attempt
to explain these findings using an interference model and suggesting that the added
stimuli of hypothermia retroactively interfered with the memory of extinction.
39
“Interference”, as it is generally discussed in the literature, refers to associative
interference (Spear & Riccio, 1994). Specifically, as two representations compete for
associative strength with a particular memory element or item, the competition of each
of those representations weakens the associative strength of each. In hypothermiainduced retrograde amnesia, there is little evidence of an association between the
apparatus and the hypothermic treatment: Hinderliter (1978) demonstrated that the
treatment could be used as a punisher, but only after many trials.
There have been isolated arguments for a general physiological trauma which
some have referred to as a type of interference, but these generally lack support and
are thought to be of little use theoretically (Spear & Riccio, 1994). Given that there is
little evidence for any association between the events of the apparatus (training and
extinction) and the hypothermia, it would be incorrect to interpret the results of the
present study from the standpoint of associative interference.
On the other hand, Wixted (2004) reviews over a century of interference
literature with specific emphasis on retroactive interference. Retroactive interference
occurs when the memory formation of a second event or association disrupts the
formation of the first. Many of the studies in Wixted’s review demonstrate that the
interference does not have to be strictly associative. In other words, the memories
which are “competing” for consolidation do not need to be related to the same task or
stimulus: a taxing, but otherwise unrelated, mental exercise following a learning event is
sufficient to create retroactive interference for that learning event. Therefore, a model
40
of retroactive interference might describe the memory disruption created by
hypothermia as being the product of mentally taxing properties of the cold water
treatment which serve to block the prior training event (or, in this study, the prior
extinction event).
There is, however, sufficient evidence to suggest that retroactive interference is
not the mechanism of disruption in the hypothermia literature. A model for retroactive
interference would not, for example, predict that re-warming the animals after coldwater immersion would reverse the effects of the retrograde amnesia: the cold water
treatment should be interfering on the prior training regardless of what follows.
Contrary to this prediction, Mactutus, McCutcheon, and Riccio (1980) demonstrated
that a re-warming treatment immediately following body-cooling reduced the amnestic
effects of the cold-water treatment. Additionally, a model for retroactive interference
would not predict that additional re-cooling would help the memory recover. On the
contrary, re-cooling the animal prior to the test would likely be viewed as a second
event that would enhance, rather than diminish, the effects of retroactive interference.
Instead, the opposite effect is seen: re-cooling the animal prior to the test enhances
memory retrieval, presumably by making the internal context of the animal during the
test similar to the internal context of the animal when the memory was encoded
(Hinderliter, Webster, & Riccio, 1975).
Without the anomalies of Experiment Three, an even stronger case could have
been made for the specificity of hypothermia as the mechanism for memory disruption.
41
Specifically, using body cooling as a reminder treatment (in Experiment Three) would
have provided an excellent counter-example to the interference model. Such examples,
however, are not absent from the literature. Briggs and Riccio (2007) demonstrated that
a body-cooling treatment prior to a test apparently aids in the retrieval of a memory of
extinction which was disrupted by hypothermia. For one to argue that the results in the
present study are the result of interference, we would need evidence that the results of
this study are due to a fundamentally different mechanism than the results of the Briggs
and Riccio (2007) study. In other words, acceptance of the interference model suggests
that the milder treatment seen in this study creates the retrograde amnesia observed in
Experiments One and Two by way of a different mechanism than the amnesia resulting
from a more severe treatment in the Briggs study.
Alternative Model: Punishment. One of the most obvious alternatives to a
contextual-change model for these results is a model of punishment. In this model,
prolonged exposure to the black (unsafe) side of the apparatus during the extinction
phase represents a stimulus which is then paired with a punishing cold water treatment.
Later avoidance of the black side of the apparatus, in this case, is not indicative of a
failure to retrieve the extinction memory, but an intact association between the black
chamber and a punishment (hypothermia).
If retrograde amnesia is to be attributed to a discrepancy between contextual
cues between encoding and retrieval, a punishment model must be ruled out. Past
research indicates that animals are frequently amnestic for the stimulus which causes
42
their amnesia, and only associate a CS with the amnestic agent after repeated pairings.
In 1965, Chorover and Schiller conducted a study which utilized an electroconvulsive
shock (ECS; the most popular amnestic treatment at the time) following a step-down
task. Animals in this study did not show fear of stepping off a platform until the pairing
had occurred at least three times. Furthermore, the ECS stimulus had its greatest
“punishing” effect if there was a short delay (10 sec.) between CS and ECS suggesting
that the effect was not truly a punishment effect at all: our basic understanding of
classical conditioning suggests there should be as small a delay as possible between CS
and US. The authors suggest that the “punishing” properties of ECS may be due to
another phenomenon as the ECS does not follow the expectations of classical
conditioning.
Early research utilizing hypothermia as an agent to create retrograde amnesia
ruled also out possibility that the cold water treatment decreases behavior due to a
punishment effect. Riccio, Gaebelein, and Cohen (1968) used a passive-avoidance task
where entry into the black chamber was paired with a hypothermic treatment, but no
shock. Animals receiving hypothermia following entry into the black chamber did not
show fear of the black chamber on a test 24 hr. later: the hypothermia, therefore,
should not be thought of as a punisher. This effect was replicated in the aforementioned
study by Hinderliter (1978), who demonstrated that hypothermia could only be used as
a punisher after several exposures. In the Hinderliter study, animals did not show
hesitation to enter the chamber paired with hypothermia until the seventh such pairing;
43
therefore, it seems that the single pairing of the test apparatus and hypothermia in the
present study should not be interpreted as a punishing event.
Furthermore, the Briggs (2007) study also contests a punishment model: pre-test
re-exposure to the hypothermic treatment resulted in better retrieval of the extinction
memory (Briggs & Riccio, 2007). The present study was unable to replicate this result,
but for apparently unrelated reasons. It could therefore be argued that the 80º
hypothermia treatment, which was milder than the treatment used in the Briggs study,
was not strong enough to create amnesia, but instead acted as a punisher. Such an
argument, however, is less tenable because of the failure to obtain extinction in the 90º
Hypothermia Group, as shown in Experiment 2. It could be argued that a very precise
“zone of punishment” exists between 70º (the temperature used to induce retrograde
amnesia for extinction; Briggs & Riccio, 2007) and 90º (used in this study to no effect)
where the cold water treatment is remembered and interpreted as punishment. This
does not rule out the possibility that a more severe hypothermic treatment could be
considered more punishing within that particular range, but there is no evidence to
support this claim. While a punishment explanation has been ruled out for
temperatures around 75ºF (23.9ºC; Riccio, Gaebelein, & Cohen, 1968; Hinderliter, 1978)
the punishment alternative persists within the range tested in this study. The
researchers would like to replicate this study and expand on the Briggs and Riccio (2007)
which found that a pre-test re-exposure does, in fact, facilitate retrieval of the
44
extinction memory. A demonstration that pre-test re-exposure facilitates retrieval
would eliminate a punishment explanation more thoroughly.
Alternative Model: Disinhibition. One last alternative explanation for the results
of this study could be a disinhibition model. Disinhibition, described by Pavlov (1927), is
a phenomenon in which the presentation of a novel, usually excitatory, stimulus breaks
previous inhibitory associations. One could argue that the cold water treatment is such
a stimulus. Disinhibitory stimuli, however, are reported to work best when they occur
shortly before or during a performance test. The cold water stimulus, which occurs
immediately after extinction and 24 hours prior to the test, is therefore not a good
example of a disinhibitory stimulus.
Furthermore, a graded disinhibition effect has, to the best of my knowledge,
never been observed. One should expect a disinhibition model to predict that the 90º
Hypothermia Group in Experiment 2 would show as much loss of extinction as the 80º
Hypothermia Group if hypothermia was capable of serving as a disinhibitory stimulus. In
other words, while a distinct gradient was not observed, the significance of the
correlational tests suggests that the relationship between post-extinction temperature
and memory retrieval is linear whereas a disinhibition model would predict an “all or
nothing” effect.
Applications. Conditioned fears and other aspects of panic disorders are thought
to originate from the sorts of associative learning phenomena found in the classical
conditioning literature (e.g., Bouton, Mineka, & Barlow, 2001). Extinction of conditioned
45
associations is thought to be the mechanism by which many clinical therapies operate
(Rachman, 1979; Reiss, 1991; Lovibond, Davis, & O’Flaherty, 2000). Exposure to a feared
CS in the absence of the US, particularly in close temporal proximity to the incident that
creates the initial CS-US association, may help to weaken or break that association,
preventing the development or continuance of a conditioned fear (Rachman, 1979).
Although such therapies are not always successful, and the association may return at a
later time, exposure therapy in some form is the basis for many behavioral
interventions.
Additionally, models for drug addiction are often thought of with regard to
classical and operant models of conditioning (Littleton, 2000). Specifically, drug-taking
behaviors can be thought of as an operant conditioning phenomenon: a response which
becomes associated with a particular outcome like the rewarding effects of that drug.
Many of the addictive properties of the drug can be thought of as classically conditioned
tolerances and processes learned by the organism (e.g., Siegel, 1975). While the
laboratory animal model does not always parallel the human model precisely, extinction
is still the basis of many of the behavior therapies used to treat drug addiction (Littleton,
2000). If extinction, however, is thought of as another training event designed to
attenuate responding, such as fear responding, then the primary learning is never
completely erased, and may return. Indeed, several laboratory models of relapse have
been used to describe why extinction, in both humans and laboratory animals, may fail.
46
The relative strengths and vulnerabilities of extinction and primary conditioning
memories, however, have not been directly tested in the past. Utilizing mild
hypothermia as a measure of the sensitivity to disruption of an extinction memory, it
may be possible to find behavioral or pharmacological means to strengthen the
extinction memory. Specifically, if a behavioral or pharmacological intervention can
strengthen the extinction memory, it may be possible to prevent relapse of the primary
conditioning. To this end, I have several goals with regard to future research, discussed
below.
It may also be of interest that extinction seems to be disrupted by a relatively
mild internal contextual change. Retrieval failure due to a change in contextual cues is,
as previously noted, understood as the primary mechanism for most forms of
retrograde amnesia (Spear, 1978; Spear & Riccio, 1994) and several models of relapse
(Bouton, 2002). With this in mind, therapies which utilize both behavioral and
pharmacological approaches might be considered in a different light.
It has been demonstrated in the animal model that chronic administration of the
selective serotonin reuptake inhibitor Fluoxetine (Prozac) combined with extinction
training produces faster, and more robust, extinction of fear on a basic Pavlovian
conditioning task than either treatment alone (Karpova, et al., 2011). Karpova explains
that Fluoxetine increases synaptic plasticity, allowing extinction training to have a
greater physiological impact on the brain. This conclusion is supported with regard to
understanding contextual change as well: animals were already receiving Fluoxetine
47
prior to the fear conditioning, so the facilitation of extinction cannot be explained by
any change of internal context by the drug alone. It would, however, be of interest to
see the effect of cessation of drug treatment. Based on the results of the present study,
I would predict that stopping the drug therapy would produce a relapse from the
behavioral extinction more readily than it would produce renewal for the fear memory;
the memory of extinction seems more susceptible to disruption than the memory for
training. While warning labels of many anti-depressant drugs indicate that sudden
cessation of an anti-depressant can be detrimental to the patient, it could be that this
detriment is due, in part, to a disruption of extinction memory caused by contextual
shift.
Future Research. The first follow-up experiment planned for this research is a
further investigation into the anomaly of Experiment Three. Replicating the findings of
the Briggs and Riccio (2007) study, wherein a re-exposure to the cold water prior to the
test facilitated retrieval of the extinction memory, would help to eliminate certain
alternative explanations (punishment, disinhibition) and provide substantial support to a
retrieval failure explanation of the results of Experiments One and Two.
Second, I would like to replicate these findings using an alternative amnestic
agent such as the beta-adrenergic receptor antagonist propranolol. Propranolol has
been used as a pharmacological treatment to create retrograde amnesia for a number
of decades (Gold & Van Buskirk, 1978). Since it has been demonstrated that lower doses
48
of pharmacological agents work to attenuate a reactivated memory (Anokhin, Tiunova,
& Rose, 2003), we might consider the similarities drawn between reactivated memories
and extinction memories in the present study. It would be of interest to determine if
milder internal context changes (such as low doses of administered pharmaceuticals)
could be decreasing the efficacy of extinction. It is reasonable that, in clinical settings
which often employ a combination of pharmaceutical interventions and
psychotherapies, such a finding would have relevance. This is especially true in light of
more recent research using reactivation and pharmaceuticals like propranolol to treat
conditioned fear responses (Kindt, Soeter, & Vervliet, 2009).
Lastly, I would like to begin studies to investigate behavioral interventions which
may “protect” these weaker memories against these seemingly minor changes in
context. For instance, determining whether a relationship exists between the amount of
extinction which occurs and the minimum threshold of contextual change necessary to
disrupt that extinction memory would be of significant interest. A major contribution of
this study is its ability to disrupt extinction by quantifiable means, establishing such a
threshold. Further investigations of the contextual changes necessary to disrupt
extinction memories could provide insights into the best methods (intensities,
frequencies, et cetera) to utilize when employing behavioral extinction.
References
Anokhin, K.V., Tiunova, A.A., Rose, S.P.R. (2003). Reminder effects – reconsolidation or
retrieval deficit? Pharmacological dissection with protein synthesis inhibits
following reminder for a passive-avoidance task in young chicks. European
Journal of Neuroscience, 15, 1759-1765.
Bouton, M.E. (2002). Context, Ambiguity, and Unlearning: Sources of Relapse after
Behavioral Extinction. Biological Psychiatry, 52(10), 976-986.
Bouton, M.E., Mineka, S., Barlow, D.H. (2001). A modern learning theory perspective on
the etiology of panic disorder. Psychological Review, 108(1), 4-32.
Bradley, P.M., Galal, K.M. (1988). State-dependent recall can be induced by protein
synthesis inhibition: behavioural and morphological observations.
Developmental Brain Research, 40, 243-251.
Briggs, J. F., Riccio, D.C. (2007). Retrograde amnesia for extinction: Similarities with
amnesia for original acquisition memories. Learning and Behavior, 35, 131-140.
Broadbent, D.E. (1958). Perception and communication. New York: Oxford University
Press. 338 pp.
Brooks, D.C., Bouton, M.E. (1993). A Retrieval Cue for Extinction Attenuates
Spontaneous Recovery. Journal of Experimental Psychology: Animal Behavior
Processes, 19(1) 77-89.
Brooks, D.C., Bouton, M.E. (1994). A Retrieval Cue for Extinction Attenuates Response
Recovery (Renewal) Caused by a Return to the Conditioning Context. Journal of
Experimental Psychology: Animal Behavior Processes, 20(4), 366-379.
Canal, C.E., Chang, Q., Gold, P.E. (2007). Amnesia produced by altered release
of neurotransmitters after intraamygdala injections of a protein synthesis
inhibitor. Proceedings of the National Academy of Sciences, 104(30), 1250012505.
Chorover, S.L., Schiller, P.H. (1965). Short-Term Retrograde Amnesia in Rats. Journal of
Comparative and Physiological Psychology, 59(1), 73-78.
49
50
Deutsch, J.A., Wiener, N.I. (1969). Analysis of Extinction Through Amnesia. Journal of
Comparative and Physiological Psychology, 59(1), 179-184.
Fava, D.A., Barnes, G.W., Riccio, D.C. (in prep.) Evaluating the Sensitivity for the Memory
of Stimulus Attributes. Unpublished Manuscript.
Gold, P.E., Van Buskirk, R. (1978). Effects of !a- and !b-adrenergic antagonists on posttrial epinephrine modulation of memory: Relationship to post-training brain
norepinephrine concentrations. Behavioral & Neural Biology, 24(2), 168-184.
Hardt, O., Wang, S., Nader, K. (2009). Storage or retrieval deficit: the yin and yang of
amnesia. Learning and Memory. 16, 224-230.
Hermans, D., Dirikx, T., Vansteenwegen, D., Baeyens, F., Van den Bergh, O., Eelen, P.
(2005). Reinstatement of fear responses in human aversive conditioning.
Behavior Research and Therapy, 43, 533-551.
Hinderliter, C.F. (1978). Hypothermia: Amnesic agent, punisher, and conditions
sufficient to attenuate amnesia. Physiological Psychology, 6(1), 23-28.
Hinderliter C.F., Webster, T., Riccio, D.C. (1975). Amnesia induced by hypothermia
as a function of treatment test interval and recooling in rats. Animal Learning
and Behavior. 3, 257-263.
Karpova, N.N., Pickenhagen, A., Lindholm, J., Tiraboschi, E., Kulesskaya, N., Ágústsdóttir,
A., Antila, H., Popova, D., Akamine, Y., Sullivan, R., Hen, R., Drew, L.J., Castrén, E.
(2011). Fear Erasure in Mice Requires Synergy between Antidepressant Drugs
and Extinction Training. Science, 334, 1731-1734.
Kindt, M., Soeter, M., Vervliet, B. (2009). Beyond extinction: erasing human fear
responses and preventing the return of fear. Nature Neuroscience, 12(3), 256258.
Koh, M., Bernstein, l. L. (2003). Inhibition of protein kinase A activity during conditioned
taste aversion retrieval: interference with extinction or reconsolidation of a
memory? NeuroReport: For Rapid Communication of Neuroscience Research,
14(3), 405-407.
Littleton, J. (2000). Can craving be modeled in animals? The relapse prevention
perspective. Addiction, 95(Suppl2), S83-S90.
51
Lovibond, P.F., Davis, N.R., O’Flaherty, A.S. (2000). Protection from extinction in human
fear conditioning. Behaviour Research and Therapy, 38(10), 967-983.
Mactutus, C.F., Riccio, D.C. (1978). Hypothermia-induced retrograde amnesia: role of
body temperature in memory retrieval. Physiological Psychology, 6(1), 18-22.
Mactutus, C.F., Riccio, D.C.., Ferek, J.M. (1979). Retrograde amnesia for old
(reactivated) memory: some anomalous characeristics. Science, 204(4399), 13191320.
Mactutus, C.F., McCutcheon, K., Riccio, D.C. (1980). Body Temperature Cues as
Contextual Stimuli: Modulation of Hypothermia-Induced Retrograde Amnesia.
Physiology and Behavior, 25, 875-883.
Matzel, L.D., Miller, R.R. (2009). Parsing storage from retrieval in experimentally induced
amnesia. Learning and Memory, 16, 670-671.
McGeoch, J.A., Irion, A.L. (1952). The Psychology of Human Learning. Longmans,
Green and Co., New York. 595pp.
Miller, R.R., Barnet, R.C., Grahame, N.J. (1995). Assessment of the Rescorla-Wagner
model. Psychological Bulletin. 117(3), 363-386.
Pavlov, I.P. (1927). Conditioned Reflexes. New York: Dover Publications, pp. 430.
Pedreira, M.E., Maldonado, H. (2003). Protein Synthesis Subserves Reconsolidation or
Extinction Depending on Reminder Duration. Neuron, 38, 863-869.
Rachman, S. (1979). The return of fear. Behaviour Research and Therapy, 17,164-165.
Rachman, S., Whittal, M. (1989) The Effect of an Aversive Event on the Return of Fear.
Behavioural Research Therapy, 27(5), 513-520.
Reiss, S. (1991). Expectancy Model of Fear, Anxiety, and Panic. Clinical Psychology
Review, 11, 141-153.
Rescorla R.A, Wagner, A.R. (1972). A theory of Pavlovian conditioning: Variations in the
effectiveness of reinforcement and nonreinforcement. Classical Conditioning II:
Current Research and Theory (Eds Black AH, Prokasy WF) New York: Appleton
Century Crofts, pp. 64-99.
52
Rescorla, R.A., Heth, C.D. (1975). Reinstatement of Fear to an Extinguished Conditioned
Stimulus. Journal of Experimental Psychology: Animal Behavior Processes, 104(1),
88-96.
Riccio, D.C., Gaebelein, C., Cohen, P. (1968). Some behavioral aspects of retrograde
amnesia produced by hypothermia. Physiology & Behavior. 3(6), 973-976.
Riccio, D.C., Hodges, L.A., Randall, P.K. (1968). Retrograde amnesia produced by
hypothermia in rats. Journal of Comparative and Physiological Psychology. 66(3),
618-622.
Ricker, S.T., Bouton, M.E. (1996). Reacquisition following extinction in appetitive
conditioning. Animal Learning and Behavior, 24(4), 423-436.
Schiller, D., Monfils, M.-H., Raio, C.M., Johnson, D.C., LeDoux, J.E. Phelps, E.A. (2010).
Preventing the return of fear in humans using reconsolidation update
mechanisms. Nature, 463(7), 49-54.
Siegel, S. (1975). Evidence from Rats that Morphine Tolerance is a Learned Response.
Journal of Comparative and Physiological Psychology, 89(5), 498-506.
Spear, N.E. (1978) The processing of memories: forgetting and retention. Lawrence
Erlbaum, Oxford, England xiv, 553pp.
Spear, N.E., Riccio, D.C. (1994). Memory: Phenomena and Principles. Allyn and Bacon,
Boston, US, 402pp.
Stampfl, T.G., Levis, D.J. (1967). Essentials of Implosive Therapy: a Learning-TheoryBased Psychodynamic Behavioral Therapy. Journal of Abnormal Psychology.
72(6), 496-503.
Thomas, L. (1997). Retrospective Power Analysis. Conservation Biology, 11(1), 276-280.
Tulving, E., Thomson, D.M. (1973). Encoding specificity and retrieval processes in
episodic memory. Psychological Review, 80(5), 352-373.
Wixted, J.T. (2004). The Psychology and Neuroscience of Forgetting. Annual Review of
Psychology, 55, 235-269.
Wolpe, J. (1958). Psychotherapy by Reciprocal Inhibition. Stanford University Press,
Stanford, US, 239pp.
Appendix A: Experiment 1 Data
ANIMAL ID
GROUP
SEX
TRAIN LAT
1
Retention
Female
13
BODY TEMP
518
571
2
Retention
Female
13
9
134
3
Retention
Male
8
600
600
4
Retention
Female
10
18
510
11
Retention
Female
8
341
501
12
Retention
Female
12
489
489
13
Retention
Male
8
37
37
14
Retention
Male
9
600
600
15
Retention
Female
5
600
600
90
Retention
Female
13
535
578
92
Retention
Female
12
31
250
96
Retention
Female
6
37
300
97
Retention
Female
3
600
600
98
Retention
Male
5
381
572
5
Exti nction
Male
26
120
6
Exti nction
Male
600
600
7
Exti nction
Female
6
85
8
Exti nction
Female
85
95
9
Exti nction
Male
17
18
170
10
Exti nction
Male
7
20
173
16
Exti nction
Female
13
37
301
91
Exti nction
Female
24
108
274
93
Exti nction
Female
7
56
307
94
Exti nction
Female
6
46
335
95
Exti nction
Female
5
18
165
99
Exti nction
Female
7
35
128
100
Exti nction
Female
6
21
46
101
Ext Mild Hyp
Female
4
490
591
102
Ext Mild Hyp
Female
4
78.3
2
376
103
Ext Mild Hyp
Male
3
77.5
600
600
104
Ext Mild Hyp
Male
4
81
25
561
105
Ext Mild Hyp
7
79.5
365
471
106
Ext Mild Hyp
Female
5
80
20
199
107
Ext Mild Hyp
Female
5
78.5
37
596
108
Ext Mild Hyp
Female
3
77.5
109
Ext Mild Hyp
Female
5
110
Ext Mild Hyp
Female
12
215
Ret Mild Hyp
Female
4
216
Ret Mild Hyp
Female
213
Ret Mild Hyp
79
TEST LAT
TEST TTS
40
68
186
499
81.5
215
449
77.5
600
600
10
77.5
75
175
Male
8
79
9
138
590
214
Ret Mild Hyp
Male
25
80.5
4
2132
Ret Mild Hyp
Female
8
80
198
464
2142
Ret Mild Hyp
Female
10
79
20
596
223
Ret Mild Hyp
Female
2
79.5
22
569
224
Ret Mild Hyp
Female
7
79.5
600
600
2232
Ret Mild Hyp
Female
4
78.5
600
600
225
Ret Mild Hyp
Male
3
78
600
600
226
Ret Mild Hyp
Female
7
78.5
6
220
53
Appendix B: Experiment 2 Data
ANIMAL ID
GROUP
SEX
TRAIN LAT
15
Retention
Female
15
BODY TEMP
TEST LAT
600
TEST TTS
600
16
Retention
Female
6
600
600
27
Retention
Female
7
11
281
28
Retention
Female
6
129
198
43
Retention
Female
11
600
600
44
Retention
Female
11
600
600
113
Retention
Female
41
163
531
114
Retention
Female
39
186
478
7
Extinction
Male
16
19
81
8
Extinction
Male
9
2
93
11
Extinction
Female
2
13
26
12
Extinction
Female
12
600
600
23
Extinction
Female
6
210
210
24
Extinction
Female
2
13
322
25
Extinction
Female
6
28
143
26
Extinction
Female
15
600
600
31
Extinction
Male
5
104
252
32
Extinction
Male
2
258
301
33
Extinction
Female
6
13
264
34
Extinction
Female
5
25
182
103
Extinction
Female
44
151
223
104
Extinction
Female
37
122
180
107
Extinction
Female
33
112
482
108
Extinction
Female
39
107
410
6
Hypo80
Male
7
79
600
600
13
Hypo80
Female
6
82
106
545
14
Hypo80
Female
12
82
600
600
29
Hypo80
Female
7
80
600
600
35
Hypo80
Female
7
81
600
600
36
Hypo80
Female
3
80
160
352
38
Hypo80
Female
10
77
344
565
41
Hypo80
Female
3
81
177
541
42
Hypo80
Female
5
81
600
600
5
Hypo90
Male
6
89
28
205
17
Hypo90
Female
5
87.5
244
332
18
Hypo90
Female
9
87.5
5
112
19
Hypo90
Female
6
93
46
306
20
Hypo90
Female
11
89.5
11
587
30
Hypo90
Female
5
92
28
412
21
Hypo85
Female
15
85
218
465
22
Hypo85
Female
22
86.5
170
290
47
Hypo85
Male
6
84.5
58
176
48
Hypo85
Male
5
87
600
600
59
Hypo85
Male
7
82.6
600
600
61
Hypo85
Male
27
84
600
600
75
Hypo85
Male
15
83.5
199
266
54
Appendix C: Experiment 3 Data
ANIMAL ID
GROUP
SEX
57
Male
Retention
58
Male
Retention
67
Male
68
TRAIN LAT PST-TR TMP PRE-TS TMP
TEST LAT
TEST TTS
16
22
338
25
463
463
Retention
21
461
579
Male
Retention
23
600
600
69
Male
Retention
25
170
225
70
Male
Retention
8
600
600
39
Male
Retention
14
600
600
40
Male
Retention
15
59
154
49
Female
Extinction
7
125
382
50
Female
Extinction
6
56
241
152
Male
Extinction
22
146
459
53
Female
Extinction
11
20
87
54
Female
Extinction
12
261
495
55
Male
Extinction
20
205
347
56
Male
Extinction
15
39
251
60
Male
Extinction
6
52
259
51
Male
Hypo80
5
80.5
61
551
52
Male
Hypo80
13
80
43
325
62
Male
Hypo80
9
80
600
600
63
Male
Hypo80
5
80.3
56
156
64
Male
Hypo80
25
79.5
83
174
65
Male
Hypo80
7
80.6
600
600
66
Male
Hypo80
5
81.5
125
284
79
Male
Hypo80
4
79.8
600
600
71
Male
Recool
17
79.75
83.1
83
83
72
Male
Recool
4
80.5
85.1
600
600
73
Male
Recool
13
81.3
83.4
600
600
74
Male
Recool
13
81.5
83.8
600
600
76
Male
Recool
12
82.1
87.25
78
272
77
Male
Recool
9
78.5
83.1
600
600
78
Male
Recool
4
81
83.8
600
600
80
Female
Recool
25
79.7
86
600
600
55
Appendix D: Experiment 3B Data
ANIMAL ID
GROUP
SEX
TEST LAT
TEST TTS
86
Male
Hypo80
81.3
600
600
87
Male
Hypo80
79.3
18
156
103
Female
Hypo80
80.7
52
208
94
Male
Hypo80
82.3
26
74
96
Male
Hypo80
82.3
16
90
108
Female
Hypo80
81.3
600
600
89
Male
Recool
81.3
83.2
67
67
104
Female
106
PST-TR TMP PRE-TS TMP
Recool
79
85.4
600
600
Recool
79.7
83
600
600
95
Male
Recool
81.2
82.5
600
600
90
Male
Recool
78.8
78
600
600
91
Male
Recool
81.6
83.8
600
600
107
Female
Recool
81.9
84.3
600
600
56