The nature of forgetting: the case for storage impairment or
retrieval impairment in experimental amnesia
Jie Jane Zhang
Department of Psychology
McGill University, Montreal
November 2013
A thesis submitted to McGill University in partial fulfillment
of the requirements of the degree of Master of Science
© Jie Jane Zhang 2013
Table of Contents
Abstract........................................................................................................................................... 1
Résumé............................................................................................................................................ 2
Acknowledgements......................................................................................................................... 3
Introduction
Forgetting............................................................................................................................ 4
Interference-based forgetting.............................................................................................. 5
Decay-based forgetting....................................................................................................... 7
Storage impairment versus retrieval impairment in experimental amnesia........................ 9
Second learning and the role of NMDAR......................................................................... 10
Rationale............................................................................................................................13
Second training with object location................................................................................. 13
Hypotheses........................................................................................................................ 14
Methods
Open fields........................................................................................................................ 15
Procedure.......................................................................................................................... 16
Infusions.............................................................................................................................17
Surgery.............................................................................................................................. 17
Histology........................................................................................................................... 17
Analyses
Behavioural measurements of object location recognition............................................... 18
Statistics............................................................................................................................ 18
Results
First learning..................................................................................................................... 19
Second learning following unimpaired first learning....................................................... 19
Second learning following impaired first learning........................................................... 19
Validity of presence of first learning................................................................................ 21
Discussion..................................................................................................................................... 22
Figures........................................................................................................................................... 29
References..................................................................................................................................... 35
Abstract
Although much work has been done to unravel the mechanisms by which memory forms,
is retained, and is brought back up again, there remains many unanswered questions of the
opposing side of phenomenon: the issue of forgetting. Forgetting is often debated as being
caused by either an impairment of retrieval mechanisms or a failure of storing the memory trace.
Recently, a novel approach to differentiate between these two theories has been devised by using
a second learning protocol, which takes advantage of the unique properties of second learning
acquisition. We show that unlike first, naive learning, any subsequent learning of the dorsal
hippocampus-dependent object location task does not require NMDAR (N-methyl-Daspartate receptors) for acquisition. By experimentally impairing first learning memory with
infusions of PKMζ-inhibitor ZIP, we show that second learning memory of the task is blocked
by infusions of NMDAR-antagonist AP5 ((2R)-amino-5-phosphonovaleric acid), and therefore
does require NMDAR for acquisition. This implies that first learning memory has been lost in
such a way that second learning has reverted to first learning mechanisms. Overall, our results
offer support for the storage failure theory of experimental amnesia. Although this study is
limited to the effects of experimental amnesia only and further research is needed to provide
more conclusive evidence, these theories are nevertheless useful as a guide to infer
neurobiological mechanisms of typical, non-pathological forgetting.
1
Résumé
Bien que beaucoup de recherches aient été faites pour démystifier les mécanismes par
lesquels les souvenirs se forment, se retiennent et sont rappelés, il reste plusieurs questions sans
réponse en ce qui concerne le phénomène inverse : l’oubli. Il y a un débat à savoir si l’oubli est
causé par une déficience des mécanismes de rappel ou par une incapacité d’emmagasiner le
souvenir. Récemment, une nouvelle approche expérimentale pour différencier entre ces deux
théories a été élaborée en utilisant un protocole de second apprentissage, lequel tire avantage de
ces propriétés uniques. Nous montrons que, contrairement au premier apprentissage, tous les
apprentissages subséquents de la tâche de reconnaissance de l’emplacement d’objets, qui dépend
de l’hippocampe dorsal, ne requièrent pas les récepteurs NMDA (N-méthyl-D-aspartique). En
réduisant expérimentalement le souvenir du premier apprentissage avec l’infusion de ZIP, un
antagoniste de PKMζ, nous avons montré que le souvenir du second apprentissage est bloqué par
l’infusion de AP5 (acide (2R)-amino-5-phosphonovalerique), un antagoniste des récepteurs
NMDA et que, par conséquent, il ne requière pas ces récepteurs pour apprendre la tâche. Ceci
implique que le souvenir du premier apprentissage a été perdu de telle façon que le second
apprentissage retourne à l’utilisation de mécanismes de premier apprentissage. Dans l’ensemble,
nos résultats supportent la théorie de l’oubli basée sur l’incapacité d’emmagasiner le souvenir.
Bien que cette étude est limitée aux effets amnésiques produits expérimentalement, et que
d’autres recherches sont nécessaires pour fournir des évidences plus concluantes, ces théories
sont néanmoins utiles pour déduire les mécanismes typiques et normaux de l’oubli.
2
Acknowledgements
My heartfelt gratitude goes out to my supervisor, Karim Nader, for his patience and
guidance; my project supervisor, Oliver Hardt, who taught me more than I knew I had to know;
Yun and Carmelo who stuck out early mornings and late afternoons of infusions with me; Karine
for offering help with everything I could possibly need; the entire Nader Lab, who encouraged
me and suffered through my data talks and were there to complain with me when I needed
commiseration; Judy, for good advice, support, food, and company; Julian, for sharing in on the
journey for two years; my committee, Jeff Mogil, Andy Baker, and Wayne Sossin, for not
judging when I presented disappointing results; and of course our graduate school coordinators,
Gio and Bärbel, who amazingly did not go crazy while waiting for me to finish up this paper.
And lastly, my parents, who were always there.
Thank you very much everyone.
3
Introduction
I. Forgetting
Throughout the history of psychology and neuroscience, the study of how to learn, store,
and recall memories has always been needed for our understanding of the world. For many
species of organisms, memory is a necessary process to help make sense of their environment, so
they may utilize information gained from the past to adapt to new experiences. Memory
acquisition, maintenance, and retrieval are therefore of great research interest for academic,
clinical, and philosophical purposes. By breaking down the neurobiological processes of
memory, we shed light on how memories can keep or deteriorate, which in turn allows research
into both pathological and non-pathological, natural memory loss. It is the aim of this thesis to
distinguish between two main theories of experimental amnesia: storage impairment and
retrieval impairment, and offer new evidence for the role of decay processes in non-pathological
forgetting.
Much research already exists on the multiple phases of memory. Long-term potentiation
(LTP), the long-lasting enhancement of synaptic transmission strength caused by synaptic
stimulation (Bliss & Collingridge, 1993), is thought to be the cellular basis of a memory. Once
the memory is acquired, it stabilizes under a process called consolidation, where a memory shifts
from a stable to unstable state (Dudai, 2004). The memory is retained in either short-term or
long-term memory storage (Cowan, 1988; Squire, Knowlton, & Musen, 1993). Retrieving a
memory through reactivation initiates a process called reconsolidation, in which the memory
returns to a labile state for up to several hours before it once again restabilizes (Debiec, LeDoux,
& Nader, 2002; Nader, Schafe, & Le Doux, 2000). Overall, memory has been discussed in depth
within three phases: acquisition, consolidation, and retrieval (Abel & Lattal, 2001; Davis &
Squire, 1984; McGaugh, 2000).
On the opposing side of the spectrum, research is less established on the neurobiological
basis of forgetting. Instead a cognitive perspective is emphasized. After acquisition has taken
place, impairments in both memory storage and recall can lead to inability to express a specific
memory. The mechanisms that lead to non-pathological memory loss are commonly debated
from an interference and memory instability (i.e., new learning blocking old learning, or old
4
learning inhibiting new learning) or decay (i.e., old learning fading over time, caused by a loss of
the memory substrate) framework (Dewar, Cowan, & Sala, 2007; Oliver Hardt, Nader, & Nadel,
2013). Research from the days of Ebbinghaus' nonsense syllables have provided support for
interference-based forgetting, and past trends in cognitive psychology show greater focus on
interference-based than decay-based forgetting (Ebbinghaus, 1913; Richardson, 2007). However,
recent research on the neurobiology of decay-based forgetting have shed new light on how longterm memory is lost, leading to novel perspectives of the mechanisms of forgetting in the brain.
II. Interference-based forgetting
In 1885, Hermann Ebbinghaus studied the pattern of long-term memory loss (LTM).
LTM is memory that persists after initial learning at lengths of several hours to the indefinite
future (Richardson, 2007). Ebbinghaus trained himself on lists of nonsense syllables, then tested
his recall of these syllables at predetermined breaks of time, without rehearsal. His results,
visualized in his well-published forgetting curve, show a sharp decline of accurately recalled
syllables after the first twenty minutes of training (58% retention of original list), a smaller
decline an hour later (44%), an even smaller decline by the end of the first day (33%), and so on.
Over time, his performance levelled (21% by 31 days), which he attributed to the stabilization of
LTM. He concluded that retention decreases at an exponential rate over time barring use of
techniques to strengthen memory (for example, mnemonics) (Ebbinghaus, 1913).
Interference is thought to mediate the effects of this pattern. From a neurobiological
standpoint, interference is thought to arise from either disturbing the cellular consolidation
processes following initial learning, or from disturbances occurring while retrieving a
consolidated memory (Bouton, 1993). Retrieval, once thought to be a passive reactivation of the
memory trace (J. R. Anderson, Fincham, & Douglass, 1999), has in recent years been suggested
to have an effect on the memory itself by temporarily returning the memory trace to a labile state
after reactivation (Abel & Lattal, 2001; Nader et al., 2000). During this plastic stage, new
information may be added to the memory trace to increase or diminish the original memory
strength (M. C. Anderson & Neely, 1996). In experiments such as Ebbinghaus', the delay interval
between recall trials gives opportunity for new learning to usurp resources needed for perfect
retention, which leads to increased forgetting as time passes.
5
According to Underwood (1957), proactive interference is responsible for the bulk of day
to day forgetting. This type of interference describes what occurs when previously learned
information results in increased forgetting of newly learned information due to larger salience of
one set of stimuli over the other. Much of this effect can be attributed to cognitive load limits of
working memory, the ability to train attention on the stimulus and away from distractions from
the environment (i.e. interference) (Bouton, 1993). Underwood proposed that the large decrease
in performance exhibited from Ebbinghaus' study was caused by interference from previous
learning in the laboratory. When the source of interference was removed, he showed that percent
recall the first 24 hours after training dropped from a 75% loss to only 25% (Underwood, 1957).
In general, previously learned information that conflicts with new information reduces response
accuracy in short-term memory tasks (Kuhl, Bainbridge, & Chun, 2012). Similar effects can be
found in animal models, such as pigeons, monkeys, and rats (Dunnett & Martel, 1990; Edhouse
& White, 1988; White, 1985). For example, White (1985) showed the effects of proactive
interference with a standard delayed matching-to-sample task. In this task, a brief stimulus is
presented, then extinguished after a set time duration. Memory is assessed by the subject
choosing the previously presented stimulus from a set of similar stimuli (White, 1985). The
experiment showed that the trial immediately preceding the response trial (N - 1) can interfere
with the accuracy of the response trial (N). This effect is minimized when the interval between
trials is raised from five seconds to 15 (Dunnett & Martel, 1990). Similar patterns of behaviour
are shown when rats are taught radial and three-choice maze tasks (Roitblat & Harley, 1988).
In the opposite direction, retroactive interference occurs when newly learned information
increases forgetting of older memories. Retroactive interference mechanisms differ from those of
proactive interference. For instance, in contrast to their detrimental performances on delayed
matching-to-sample tasks, rats have been shown to exhibit some measure of invulnerability to
retroactive interference even when performance of a spatial task is interrupted with delays. Using
an eight-arm radial maze task, Maki et al. (1979) trained rats to choose each of the eight arms
one by one without repetition. They found that interrupting performance at test with a delay
varying from 15 seconds to two minutes did not disrupt rats' memories of their prior choices.
However, most studies have shown that retroactive interference has large effects on recall.
Certain stimuli remain in memory better than others. When human participants are tested for
recognition memory of faces and landscapes directly after studying images, they experience
6
interference for all images, but less interference for concrete nouns and pictures of common
objects (Deffenbacher, Carr, & Leu, 1981). As the delay after study increases, susceptibility to
interference of all stimuli increases, just as in Ebbinghaus' forgetting curve.
Interference studies such as these have led to two-factor theories of response competition
and unlearning (Postman & Underwood, 1973). These theories state that stimuli compete for
limited cognitive resources. As a new set of stimuli is learned, it can weaken memories of a
previously acquired set. Participants taught two different responses to the same cue (e.g. the A-B
list is different from the A-C list) show that correct recall of the A-B list declined in proportion
to the amount of training the A-C list received. Thus the previously learned A-B association is
progressively unlearned as the secondary A-C list is acquired. Interference caused by this
competition is theorized to arise from associating novel memory traces to the first learning
retrieval cue, or by adding traces to strengthen an already existing competitor cue, which forms a
new associative structure (Anderson, 2003; McGeoch, 1942).
From these studies we know that interference theory can account for many forgetting
phenomenon. On the other hand, these A-B, A-C paradigm involved in cue-overload interference
procedures are noted for their validity in laboratory settings, but lack applicability in real, day to
day life (Wixted, 2004b). Real life interference occurs because memory traces that have not fully
consolidated are susceptible to intervening forces, such as other mental activity, pharmaceutical
influence, and sleep (Wixted, 2004b). Some other types of recall performance are not easily
explained by the dominant proactive interference theory. For example, Jenkins' and Dallenbach's
1924 study showed how sleep could benefit recall. After learning lists of nonsense syllables,
participants who slept through the retention interval was shown to lead to better recall than those
who stayed awake, suggesting a beneficial role of sleep in consolidation. These results were not
attributable to proactive interference since new learning accumulated during awake stages
weakened old learning, and were best attributed to a reduction of retroactive interference. As
forgetting theory developed, Underwood's original influential theory of proactive interference
was regarded as less and less comprehensive.
Still, because interference theories reliably explained a host of data from forgetting
experiments, they have long taken the spotlight in forgetting research. Less research has been
done with decay theories of forgetting. But as theories of interference became increasing
complicated and difficult to apply to real life, more inclusive theories were needed. Research on
7
the temporal gradient of amnesia, for example, showed that memories do need time to
consolidate and newer memories are more easily impaired than older ones. This threw attention
back on how forgetting could be characterized by decay (Gold, 2006).
III. Decay-based forgetting
The role of decay in LTM is more difficult to assess than interference. Research on decay
lagged heavily behind research on interference (J. Brown, 1958; Lewis, 1979) even though decay
as a concept spans back to Jost's 1897 law of forgetting (Wixted, 2004a). Jost stated that given
two memory traces of the same strength, over time, the younger trace will decay more rapidly
than the older. In other words, the amount of forgetting was a function of the age of the memory.
However, studies like that of Jenkins and Dallenbach (1924) lent strong support for retroactive
interference as the cause of forgetting, and interference theory dominated the literature for LTM
loss. Additionally, decay processes were not well operationally defined, making them difficult to
assess empirically. Wickelgren proposed a mathematical model of LTM storage and decay using
a power law. His law included properties of resistance and strength of the LTM trace, where
resistance is defined as the capacity to endure against the force of decay (Wickelgren, 1972), but
did not specify just how the decay process was taking place. Decay seemed to be taken for
granted as a conceptual phenomenon that that involved some gradual loss of memory over time
via unknown mechanisms.
Many studies on the decay of memory were limited to immediate memory and short-term
memory, with retention intervals in the range of seconds. Brown (1958) argued that decay theory
could help explain the mechanisms of a memory span, which he defined as the capacity for
reliable short-term memory in the interval between stimulus perception and its subsequent recall.
He assumed that the memory trace decayed rapidly after acquisition. If the trace decayed over a
critical point, accurate recall would not be possible. Memory span was theorized to be a function
of the amount of stimulus given (in his study's case, the length of the sequence of items). If the
interval between sequence presentation and recall exceeded a certain time, then decay would
reach its critical point, resulting in incorrect recall. It should be noted that length of memory span
can also be partially attributed to interference, where increased retention time allows opportunity
for new learning to build on top of and perhaps destabilize old learning (returning us to the
theory of retroactive interference).
8
Reitman (1971) showed that interference theory could not explain all recall phenomenon.
She studied short-term memory of simple English nouns given while participants were presented
with one of three signal-detection tasks (tonal detection, silent syllabic detection, vocal syllabic
detection) during the 15-second interval between word presentation and the free recall test. The
signal-detection tasks were added to prevent participants from rehearsing the word list. Results
showed that despite lack of rehearsal, mean retention rate of the three-word list was high (93%),
initially showing no support for memory decay. A follow-up study in 1974, however, concluded
that the high level of performance was influenced by ceiling effects and undetected covert
rehearsal. When given more list items to remember, participants showed significant forgetting
within 15 seconds, an effect exacerbated by performing simple detection tasks before recall test.
Additionally, it was found that when given a string of stimuli to memorize, the training itself can
act as a type of inherent rehearsal mechanism. Participants who could generate a rule to follow in
order to remember a set of items showed better recall than participants who had to memorize
unrelated items (Reitman, 1974). Therefore without interference or rehearsal, memory could still
be lost during retention.
Despite the empirical evidence for decay theory, interference theory was still heavily
favoured as the cause for forgetting. The main contention for decay-based theory seemed to be
that the process was originally thought to be rather passive (Altmann & Gray, 2002; J. R.
Anderson et al., 1999). Common issues in memory research included the distortions in memory
that occurred after recall, and how memory interference relied on some level of similarity
between first- and second-learned stimuli. These issues highlighted a dynamic aspect of
forgetting that could not be explained with decay theory. Instead, because decay did not negate
other forgetting theories, it was often seen as a complementary process to interference-based
forgetting. Additionally, experiments that showed how partially decayed memories may still be
able to produce accurate recall (such as Gold et al., 1973) added further nuances to the complex
mechanisms of memory decay.
IV. Storage impairment versus retrieval impairment in experimental amnesia
The unbalanced amount of research between interference and decay shows that forgetting
is a more dynamic process than previously thought, and requires further research. In recent
decades, another perspective developed which examined forgetting in an unique manner. This
9
perspective asked if forgetting is caused by a storage failure (i.e. enough of the memory trace is
physically lost from the brain that the memory cannot be expressed) or a retrieval failure (i.e. the
memory trace is present, but is blocked from being recalled) (Gold & King, 1974; O. Hardt,
Wang, & Nader, 2009; Spear, 1973). Evidence for memory loss as caused by storage failure
would provide support for the decay theory of forgetting. Evidence for retrieval failure would
support impaired recall as the basis of forgetting. However, the difficulty in finding evidence lies
implicitly in the behaviour of forgetting: if the memory is lost, how is it possible to verify what
caused that loss? Previous research has focused heavily on spontaneous recovery from amnesia
experiments, for example, recall tasks (Miller & Springer, 1973). In these, the return of memory
could be attributed to spontaneous recovery from partial memory loss (Nader & Wang, 2006;
Zinkin & Miller, 1967) or new learning that formed connections on the residual traces of
memory (Cherkin, 1972; Nader & Wang, 2006). In the latter's case, a reminder-like cue, such as
giving a weak foot shock to a fear-conditioned animal, has been show to result in test
performance that resembles the behaviour of a full recovery of memory (Miller & Springer,
1972). Gold et al.'s study about reminder effects (1973) explicitly showed this result. Animals
given shocks immediately after training showed decreased performance on subsequent retention
tests, but when given non-contingent footshocks (NCFS) in a different context, performance on
the second retention test improved. Gold hypothesized that the NCFS itself was acting as a weak
training experience. Giving a NCSF to an animal that had weak retention of training caused later
test performance to match the performance of animals that had initially received strong training.
Animals with partial retrograde amnesia given the NCSF also showed identical high
performance. However, despite these findings, new learning building on a residual memory trace
does not explain all types of spontaneous recovery (Hardt et al., 2009). Nevertheless, this
experiment vitally showed how spontaneous recovery could have multiple origins, and
highlighted the ambiguity of conclusions from experiments that tested recovery from amnesia
(Nader & Wang, 2006; Squire, 1980).
The ambiguity is twofold. If recovery from amnesia does not occur, this can be caused by
loss of the memory trace (storage failure), as well as the inability to retrieve the memory
(retrieval failure). If recovery from amnesia does occur, only then it is possible to conclude that
there was not a physical loss of the memory, and therefore the experimental amnesia was more
likely to have been caused by retrieval failure. Notably, whether there is recovery from amnesia
10
or not, the retrieval failure model cannot be disproven. The storage failure theory is also at a
stalemate as it relies on negative findings. Experimentally, reaching conclusions from negative
findings is not ideal. In this case, negative findings do not provide undisputable evidence of the
physical absence of memory—the memory trace may still be present, merely inexpressible.
Therefore a paradigm must be made for positive predictions of amnesic recovery. Needed
is a double-dissociation paradigm that can differentiate whether experimental amnesia was
caused by either storage or retrieval failure. Under this scheme, behaviour that supports the
storage failure theory cannot support the retrieval failure theory, and vice versa.
V. Second learning and the role of NMDAR
Such a protocol can be done with the unique role of NMDAR second learning. N-methylD-aspartate receptors (NMDAR) are a key player in synaptic learning and plasticity. They are a
necessity in the coding and immediate storage of memory traces in new learning (Lamprecht &
LeDoux, 2004) and spatial memory (Nakazawa, McHugh, Wilson, & Tonegawa, 2004; Tsien,
Huerta, & Tonegawa, 1996). However, NMDAR have a different relationship in regards to
subsequent, or second, learning.
Second learning, such as learning of a second language, builds upon the neural networks
previously laid down by first learning. Animals can acquire second learning if they are trained in
a distinguishably different context from the first, but the type of task is kept the same (i.e.
forming and retaining both memories is dependent on the same region of the brain). Studies have
shown how initial learning in the hippocampus affects subsequent learning such that only
modifications to the entire hippocampus is enough to impair second learning (Wang, Finnie,
Hardt, & Nader, 2012). Bannerman et al. (1995) crucially showed the unique properties of
experimental second learning when he trained animals in two different Morris water maze
contexts. Rats were pre-trained in a second "downstairs" water maze and infused with NMDAR
antagonist, AP5 ((2R)-amino-5-phosphonovaleric acid), immediately prior to training of the
"upstairs" water maze. They were shown to have comparable learning to controls. Rats that were
not given the pre-training did not exhibit a decrease in escape latency over consecutive test trials
and therefore showed the predicted impairment to water maze learning. This novel result caused
by giving pre-training provided evidence that NMDAR were not required in acquisition of
second learning memory. Because NMDAR is a glutamate receptor critical for acquisition of
11
memory (Lamprecht & LeDoux, 2004), it is a key element in producing long-term potentiation
(LTP), a crucial step in learning and memory. When NMDAR binds to magnesium ions at the
same time as depolarization of the synapse caused by α-Amino-3-hydroxy-5-methyl-4isoxazolepropionic acid receptor (AMPAR) activation, calcium ions flow into the neuron,
triggering an intracellular mechanism that is the basis of neuronal structural growth and change.
The influence of AMPAR and NMDAR on learning and memory is not the focus of this paper,
but it is crucial to note the role of NMDAR in plasticity and increasing synaptic strength,
specifically, for our purposes, with respect to spatial memories (Tsien et al., 1996). Blocking
NMDAR-dependent LTP with AP5 infusions should result in impaired training memory. As
Bannerman has shown, AP5 infusions into the dorsal hippocampus (dHC) prior to training does
in fact disrupt first learning acquisition of spatial tasks, but not second learning acquisition.
These results, while inherently interesting, also provide a unique method of functionally
separating the performance of a two-level experimental amnesia protocol. Taking advantage of
the unique properties of second learning and NMDAR, Hardt et al. (2009) devised a novel
paradigm to test for storage or retrieval deficit within a single experiment, therefore bypassing
the need to interpret changes in performances through negative or non-falsifiable results. By
impairing first learning, we can investigate if subsequent learning requires NMDAR for
acquisition. If yes, this indicates that first learning memory was erased in such a way that
acquisition of subsequent (second) learning has reverted to first learning mechanisms. If second
learning does not revert to first learning mechanisms, it can be assumed that the memory trace of
first learning is still intact enough for the memory system to acquire subsequent learning under
normal second learning mechanisms.
In Hardt and colleagues' study with contextual fear memory, one group of animals was
infused with protein synthesis inhibitor anisomycin (ANI) following the first contextual fear
training, and then infused with AP5 prior to second training in a different context (ANI-AP5
group). Animals showed significantly low levels of conditioned fear response during test of first
learning and second learning. In contrast, animals who were infused with vehicle (VEH) during
first training and AP5 prior to second training (VEH-AP5 group) showed high levels of fear at
second learning test, identical to the control VEH-VEH group and experimental ANI-VEH
group. Of note is the lack of impairment caused by AP5 in the VEH-AP5 group and the evidence
of impairment in the ANI-AP5 group. Hardt et al. concluded that AP5 infused prior to second
12
training only impeded acquisition when first training protein synthesis mechanisms were blocked
by ANI infusion, which impaired first learning memory. Their data were thus consistent with the
storage failure theory.
VI. Rationale
We can now see a method that makes positive predictions for the recovery of memory
from amnesia, or lack thereof. Our goal of this thesis is to distinguish between storage and
retrieval theories of experimental amnesia. This can be done with the second learning protocol. If
amnesia of first learning is caused by a storage failure, then it assumes the memory trace itself is
physically lost. Therefore any second training must follow the mechanisms of naive, first
learning. Following this logic, infusing AP5 before second learning training should impair the
acquisition of the task. The retrieval failure theory maintains that even if first learning memory is
blocked, the memory trace is still present in some capacity. Second training will therefore still be
acquired under normal second learning mechanisms, and thus will not be impaired by AP5
infusion prior to training. By evaluating performance on the second learning, we can clearly
provide evidence for either storage failure or retrieval failure theory.
VII. Second training with object location
For this thesis, we were interested in testing the second learning paradigm with a
recognition of object location task. The object location task used in this study is a spatial task
that is heavily dependent on the dHC (Ennaceur, Neave, & Aggleton, 1997; Mumby, Gaskin,
Glenn, Schramek, & Lehmann, 2002). Rats are trained over several sessions to become familiar
with two or more objects located at fixed spots within the open field. After a period of retention,
one of the objects is moved to a different location within the field, and rats are then reintroduced
to the context. Typically, rats will spend significantly more time exploring the object in the novel
location than the object(s) in the old location (Dix & Aggleton, 1999). This novelty preference
pattern in exploratory behaviour implies that the rat remembered the object's old location and is
less interested in it. Rats that show no memory of the previous object location will spend equal
amounts of time exploring old and new object locations.
To carry out the second learning paradigm, two separate open field contexts were created.
All characteristics of the contexts were kept notably different (see Methods), but both were
13
object location tasks. The aim was to create enough visual disparity between the contexts that the
animal will recognize second training as a separate event from first training, but still acquire first
and second learning training on the same neuronal network (the dHC).
VIII. Hypotheses
Following the example of Hardt et al. (2009), rats will be trained in two stages of first
learning and second learning. As per the literature, we intend to establish that first learning of
object location memory does require NMDAR for acquisition, and that second learning does not.
If rats infused with AP5 prior to second training show no preference for the novel object location
during second learning probe test, we may assume that their memory of first learning has been
effectively "wiped out," leaving second learning to revert to first learning mechanisms. This
supports storage failure theory. On the other hand, if rats infused with AP5 prior to second
training do show preference for the novel object location, second learning mechanisms have
necessarily occurred, which implies the successful retention of first learning memory.
We also intend to impair first learning and then examine the sensitivity of second
learning to AP5. Similar to Hardt and colleagues impairing memory with ANI infusions, our
study will assess memory loss in cases when memory is functionally erased. Blocking
mechanisms of PKMζ, a protein needed for maintenance of long-term memories (Migues et al.,
2010; Pastalkova et al., 2006; Sacktor, 2008), has been shown to cause experimental amnesia in
rats. Infusing PKMζ-inhibiting peptide ZIP into the dHC after learning will impair first learning
memory and allow examination of second learning memory properties. If ZIP-infusions causes
impairment similar to ANI-infusions, results can provide an alternate avenue of support for
whether absence of experimental first learning memory is caused by storage or retrieval failure.
Experiments with ZIP can provide the pivotal next step in unravelling the issues of
forgetting. But it must be emphasized that experimental amnesia is analogous, not identical, to
natural, non-pathological forgetting. The goal of this thesis on experimental amnesia is to
provide a valid mechanism to which non-pathological forgetting can be similarly compared. It is
therefore necessary to create protocols which imitate the duration and environment associated
with natural forgetting. However, much of that extends past the breadth of this paper. Overall,
the intent of my thesis is to show support for the storage failure theory of forgetting, and provide
evidence that decay does indeed cause loss of memories over time.
14
Methods
Animals were co-housed in pairs in their plastic home cage when not participating
actively in the experiment. Colonies followed a 7am-7pm light:dark cycle. Each cage was fed 10
pellets per meal, twice a day. Water was provided ad libitum. Rats were handled for two to three
days prior to training or surgeries.
I. Open fields
Two stages of object location training were needed in this experiment. The two training
contexts were kept as different as possible to reduce contextual generalization, while still
keeping the base training protocol identical.
a. Open field 1: First learning training was given in a dim room in a darkened corridor lit
only by red light. The open field was made of sixteen transparent Plexiglas panels held together
by hinges, arranged in a circular shape (total area 160 cm2). Flooring was a wooden pegboard
and bedding was a 1:1 mixture of sawdust to mouse corn bedding. Three objects (two identical,
one dissimilar) were arranged at one end of the open field (see Figure 2), which could be
screwed and unscrewed to other positions on the pegboard. Objects were two green incense
candle burners and one silver plastic Buddha head statuette. Objects were all around 15 cm in
height and eight cm in width.
Naive animals were placed into open field 1 with no habituation. This was done to ensure
that the learning acquired would be first learning within that context.
b. Open field 2: Second learning was given in an alternate room, well-lit, with distinct
different poster decorations, in a lit corridor. A cubic open field (40 cm width × 40 cm length ×
60 cm height, no top covering) with the same area as open field 1 was set up. Walls were made
of thin, white-laminated wood-based panelling. Sawdust bedding was used to carpet the bottom
of the box. Two identical objects were screwed into position (see Figure 1), which could be
removed and anchored again at other regularly-spaced holes within the base board.
Positions of objects were always switched across groups for counterbalance. Objects
were mounted upon glass mason jars. Both objects were matte black sanitary-tee tubes, also
mounted on glass mason jars. Objects were around 10 cm in height.
Training and tests were recorded by overhead security cameras that had an aerial view of
the entire open field.
15
II. Procedure
a. Experiment 1: Rats were infused with AP5 or VEH immediately prior to first training
in open field 2. To save time, training took place at an accelerated schedule of twice daily
sessions of ten minutes (Figure 1). Training sessions occurred at least five hours apart, once in
the morning and once in the afternoon. For each session, rats were allowed to roam freely within
the open field. When the session ended, rats were returned to their home cage. After two days of
rest, one object was moved to the other side of the open field, and rats were brought back to test
for first learning of object location memory in a three minute probe test. Only the first minute of
probe was measured for the novelty ratio; the following two minutes were used as a control to
confirm no presence of irregular behaviour.
b. Experiment 2: Rats were given first learning training in open field 1 at a more sedate
pace across seven consecutive days, in daily sessions of five minutes each (Figure 2). No
infusions were made at this time. After two days of rest, protocol for second learning continued
identical to Experiment 1.
As outlined above, contextual conditions of open field 1 and 2 were kept notably
different so rats would be able to reliably differentiate between first and second learning training
sessions.
c. Experiment 3: Rats were trained in open field 1 with seven daily sessions of ten
minutes each. Training sessions were extended to ensure adequate learning prior to drug
infusion. One day after first training, rats were infused with ZIP or an inactive form of the
peptide, scrambled-ZIP (SCR) in the dHC. Rats were then given one day of rest before given
second training in open field 2. Second training proceeded as in Experiment 1. (See Figure 4A.)
There were four conditions total: ZIP-AP5, ZIP-VEH, SCR-AP5, and SCR-VEH.
d. Experiment 4: Rats were trained in open field 1 with seven daily sessions of ten
minutes each. Retention time before probe test varied with group (see Figure 5A). No surgeries
or infusions were made as our primary interest was confirming that rats were learning normally
in the novel open field 1 context, as it had never been previously used in our lab. We also
partially explored how long first learning memory remained intact without pharmacologic
intervention.
16
III. Infusions
In first learning experiments, AP5 or vehicle was infused immediately prior to placing the
animal within their first training context. Infusions of 1μl AP5 (concentration 5μg/μl) or vehicle
PBS (ph 7.0) were administered at 0.25μL/min bilaterally in the dHC. In second learning
experiments with ZIP, ZIP (Myr-SIYRRGARRWRKL-OH), or scrambled-ZIP (MyrRLYRKRIWRSAGR-OH) (10 nmol/µl), was dissolved in TRIS-saline (pH 7.0) and infused
0.25μL/min for a total of 1μL/hemisphere. Injector tips were left within the cannula for a minute
after infusions to allow full diffusion of the drug into the brain.
Infusions of ZIP and SCR were performed in a room adjacent to open field 1 under dim
light to remain consistent with open field 1's context. Infusions of AP5 and VEH were performed
in a room adjacent to open field 2 under normal light to remain consistent with open field 2's
context.
III. Surgery
Stereotaxic surgeries were performed on 300g-350g male Long Evans rats from the
Charles River, Quebec, animal facility. Rats were first anesthetized with an intraperitoneal (IP)
injection (anesthesia made of Dexdomitor, 0.27mg/mL, xylazine, 3.33mg/mL, and ketamine,
55.55mg/mL) matched at 1mL/kg of body weight. Two 22-gauge cannula were bilaterally
inserted into the dorsal hippocampus (measurements: anteroposterior, -3.6mm; medial-lateral,
3.1mm; dorsoventral, -2.4mm, 10º away from midline) and then glued in place with dental
cement. Following surgery, an intramuscular injection of analgesic (Carprofen, 5mg/mL) was
given. An IP injection of recovery drug (Antisedan, 7.5mg/kg) was given to awaken the animal
from anesthesia.
V. Histology
Brains were removed after decapitation and sliced at 50μm sections with a Micron
cryostat. Slices were stained purple with formal thionin and cannula placement was checked
under a light microscope for correct infusion into the CA1 area of the dHC (Figure 6).
17
Analyses
I. Behavioural measurements of object location recognition
The measure of object recognition memory in rats is dependent on rats' preference for
novel stimuli. In the case of object location, they will spend more time exploring areas or objects
that are new to them (Dix & Aggleton, 1999). By quantifying the amount of time that rats spend
with each object in the open field, we can use the ratio tnovel /(tnovel + tsample) where tnovel is the
time spent with the new location and tsample is the total exploratory time during the probe test
(Mumby et al., 2002). A naive animal will show a novelty ratio equal to chance, 0.5. Contrarily,
animals who remember the previous arrangement of objects will spend more time exploring the
changed object location and thereby have a significantly higher novelty ratio, >0.5.
Novelty ratios for open field 1, which had a set-up with three objects (two unchanged
locations and one changed), required a different equation than the one above. However, the same
principle was used of time spend with the novel location divided by total time spent exploring.
The equation devised was tnovel /(tnovel + [tsample1+ tsample2 ]/2), where tsample1 is the time spent
exploring one of the old object locations, and tsample2 is the time spent exploring the other.
All ratios were calculated from the first minute of exploratory information in the three
minute probe test. Data was gathered by visual inspection and manual timing with a stopwatch.
Specific exploration of an object location was counted when the animal made any sort of overt
gesture or turning attention towards the object, such as sniffing, touching, circling, or
characteristically pointing the nose at the object. Climbing onto the object itself and sniffing at
the context walls were not counted towards exploratory time.
II. Statistics
Experiments comparing a AP5-infused group with a vehicle-infused group were analyzed
with one-sample t-tests. For experiments involving two different conditions, ZIP- or SCRinfused after first training, and either AP5- or vehicle-infusion prior to second training, a oneway ANOVA was used with an ɑ=0.05.
18
Results
All groups of rats in all experiments did not show any significant difference in
exploratory time within the first minute of the probe test.
I. First learning
To test for the requirement of NMDAR in first learning of object location memory, rats
were infused with either AP5 or vehicle (VEH) in the dHC immediately prior to first training in
open field 2 (Figure 2). AP5-infused rats had a novelty preference ratio of chance (n=8, t=-1.25,
p>0.05), while VEH-infused rats showed significantly more preference to the novel object
location (n=8, t=1.99, p<0.05) (Figure 3). We conclude that NMDAR is needed for acquisition of
first learning of object location in the dHC.
II. Second learning following unimpaired first learning
To test for the requirement of NMDAR in second learning of object location memory,
rats were given first training in open field 1 without infusions, then infused with either AP5 or
VEH in the dHC prior to second training in open field 2 (Figure 2). At second learning test, both
groups displayed significant preference for the new object location in open field 2 (VEH-infused
rats: n=8, t=3.22, p<0.05; AP5-infused rats: n=8, t=2.49, p<0.05), showing that NMDAR are not
required in the dHC for acquisition of second learning of object location memory (Figure 3).
Incidentally, these results also confirm that object location training procedures in open
field 1 followed by open field 2 do function in a first learning, second learning pattern. If they
did not, second learning would not be affected by AP5 infusions.
III. Second learning following impaired first learning
To test for the requirement of NMDAR in second learning after first learning was
impaired, rats were infused with ZIP or SCR immediately following first training in open field 1,
and infused with AP5 or vehicle immediately prior to second training in open field 2 (Figure
4A). We hypothesized both SCR-AP5 and SCR-VEH groups would show unimpaired first
learning and unimpaired second learning, showing significant preference for the new object
location during second learning probe test. SCR-AP5 would have intact first memory so there
would be no effect of AP5-infusions on second memory. SCR-VEH would show no effect of
19
treatment on either first or second memory. On the other hand, ZIP-AP5 and ZIP-VEH groups
would have impaired first learning memory due to ZIP infusion after first training. With first
learning memory effectively erased, ZIP-AP5 group animals will acquire second training as if it
were first training, and therefore be susceptible to AP5 impairment. This group would display atchance object location preference during second training probe test. ZIP-VEH would show
significant object location preference at second training probe test, however; even if second
learning will be acquired as first learning, VEH infusions will not impair acquisition of its object
location memory.
During probe test of second learning, rats infused with SCR-AP5 showed a novelty
preference ratio of 0.71, SCR-VEH 0.65, ZIP-AP5 0.55, and ZIP-VEH 0.46, of which groups
SCR-AP5 showed significant preference for the new object location over chance (n=8, t=3.35,
p<0.01), and SCR-VEH showed near significance for the new location (n=7, t=1.42, p<0.1)
(Figure 4B). The two ZIP-infused groups showed novelty preference behaviour close to chance.
This indicated that the SCR-groups did successfully acquire second learning of open field 2,
whereas the ZIP-groups did not.
Within these results, no significant difference was found between the four groups
(F3,27=1.56; Fcrit=2.96, p=0.22), but when split up, animals infused with SCR after first training
showed near significant preference for the novel object location compared to animals infused
with ZIP (F1,29=4.08, p=0.053). This difference is greatly reduced if comparisons are split up by
drug of second infusion, i.e. AP5 or VEH (F1,29=0.02, p=0.87).
These results show that there is indeed an effect of ZIP-infusions that impedes first
learning memory such that second learning memory is affected by AP5-infusions. Animals
infused with SCR after first learning memory do not perform in the same way in open field 2 as
animals infused with ZIP. Specifically, because of the erasure of first learning memory by ZIPinfusions, behaviour at the second training probe test by the ZIP-AP5 group shows no significant
preference for the new object location, showing that second learning memory was not reliably
acquired. We conclude the second learning acquisition has reverted to a first learning-like
mechanism. These results were not shared by the two groups infused with SCR after first
learning, both of which showed either significant or a trend of significance towards the new
object location during second training probe test, displaying normal second learning memory.
20
However, group ZIP-VEH, which was hypothesized to show unimpaired second learning
memory and normal object location novelty preference, showed a novelty ratio not significantly
different from 0.5 at second training probe test. Possible reasons for this unexpected result
include residual effects of ZIP-infusion and damage caused by experimental procedures. These
are more fully explored in the discussion.
IV. Validity of presence of first learning memory
Being that open field 1 was a new training set-up built for this study, it was important to
verify that animals were reliably acquiring the training as they would with the standard, cubic
open field 2. To this end, a series of side experiments were carried out to test first learning
memory over different retention times. No infusions were given. The purpose of the tests were to
verify if learning could be acquired in the novel circular context, as well as to explore the limits
of how long this first learning memory lasts. Results are presented in Figure 5. All results show a
significant preference for the new object location at first learning probe test, proving that there
were no difficulties acquiring memory in open field 1.
21
Discussion
This thesis had two objectives. Firstly, we wanted to shed light on the two theories used
to conceptualize forgetting. Secondly, we wanted to provide a method of differentiating between
these theories. Both storage failure and retrieval failure mechanisms have been proposed as ways
to explain forgetting. However, it has been difficult to provide positive, unambiguous evidence
for either one theory. To this end we employed the second learning protocol, which hinges on the
necessity of NMDAR activation for initial but not subsequent learning acquisition. From it we
formed a double-dissociation paradigm which allows for attribution of behavioural results to
either storage failure and decay mechanisms, or retrieval failure and interference mechanisms. If
experimentally induced amnesia of first learning memory causes second learning memory to
require NMDAR-dependent acquisition, we can infer that the first learning memory has been
physically lost, causing second learning to revert to first learning mechanisms.
To address our hypothesis of second learning mechanisms differing from those of first
learning, our experiments used the spatial task of recognition of object location. Learning the
object location paradigm has been shown to be dependent on the functioning of the intact dHC
(Ennaceur et al., 1997; Mumby et al., 2002). In our first experiment, we infused NMDAR
antagonist AP5 or vehicle (VEH) into the rodent dHC immediately prior to training them in open
field 2. After two days of retention, animals were tested on object location preference. Those
with intact memory are expected to spend significantly more time exploring the object moved to
the new location than the object which remained at the old location. During the testing session,
the group infused with AP5 showed no significant preference for the new object location over
the old one, showing that they had not sufficiently acquired the training. In comparison, the
control/VEH group showed significant preference for the new object location, displaying normal
novelty preference behaviour.
To explore the characteristics of second learning, in our second experiment we gave first
learning training in open field 1 without infusions, then infused AP5 or VEH into the dHC prior
to second training in open field 2. In contrast to Experiment 1, AP5-infused animals performed
on par with controls, showing significant preference for the new object location. These results
replicate the findings from previous second learning dHC-dependent experiments (Bannerman,
22
Good, Butcher, Ramsay, & Morris, 1995; O. Hardt et al., 2009) which show the requirement of
NMDAR in first learning acquisition, but not in second learning acquisition.
Taken together, these two experiments lay the preliminary groundwork to untangle the
mechanisms involved in experimental amnesia. To dissociate the hypothesized results of storage
failure and retrieval failure, first learning memory must be experimentally impaired and second
learning subsequently tested for its dependency on NMDAR. Hardt et al. (2009) infused proteinsynthesis inhibitor ANI or VEH into the dHC following first learning of contextual fear
conditioning. This impaired LTM of first learning. To find out how this impairment affected
mechanisms of second learning, animals were then infused with either AP5 or VEH prior to
second training of fear conditioning in a different context. Out of the four experimental groups,
ANI-VEH, ANI-AP5, VEH-VEH, and VEH-AP5, only the ANI-AP5 rats displayed significant
lack of fear behaviour to the second training context. Hardt and colleagues concluded that the
ANI-induced amnesia of first learning rendered second learning sensitive to AP5 impairment.
When first learning memory was unimpaired, second learning acquisition was unimpaired by
either AP5 infusions or VEH infusion. These results are better explained with storage failure
theory than retrieval failure theory. The latter theory assumes that first learning memory
impairment would be caused by inability to properly retrieve the memory, but the memory trace
itself remains intact within the neural network. Thus with first memory still retained in some
capacity, second learning would not be AP5-sensitive. Storage failure theory attributes first
learning memory impairment to a physical loss of the memory trace. Without initial learning
present, second learning will be treated as naive learning and therefore require NMDAR for
acquisition. Hardt et al.'s results were consistent with storage failure theory, which led to the
conclusion that ANI infusions disrupted first learning memory storage.
The experiments for this thesis followed Hardt and colleague's general protocol.
However, instead of protein synthesis inhibitor anisomycin, ZIP was infused, a peptide which
selectively inhibits PKMζ (Migues et al., 2010; Shema, Sacktor, & Dudai, 2007). ZIP has
previously been shown to cause memory erasure and disruption of LTP in many types of
memory processes (including object location memory, see Migues et al., 2010). The question
then arises if this ZIP-impairment affects memory the same way as ANI did in Hardt et al.'s
study. Subsequently, we can make inferences about the similarities between experimental
amnesia and natural, non-pathological forgetting. However, the data gathered from Experiment 3
23
in this thesis did not specifically disambiguate the mechanisms of ZIP-induced amnesia. We
created four conditions based on what was infused one day after first learning training and what
was infused immediately prior to second learning training: ZIP-AP5, ZIP-VEH, SCR-AP5, and
SCR-VEH. Our original hypothesis stated that if ZIP caused impairments similar to those from
ANI infusions, then only the ZIP-AP5 test group would show impaired object location memory
during the second learning probe test. Thus, groups infused with an inactive, scrambled version
of ZIP (SCR) after first learning would not be impaired by either AP5 or VEH infusions during
second learning. ZIP-infused rats infused with VEH during second training should be able to
acquire second learning effectively despite first learning memory being impaired because VEH
leads to no impairment on learning. However, our data showed that the ZIP-VEH group was
impaired during the second learning test.
The unexpected behaviour of the ZIP-VEH group cannot be explained by ZIP treatment
being ineffective. If all ZIP-infusions were ineffective and did not impair first learning memory,
second learning acquisition in the ZIP-AP5 group would not have been affected by AP5
infusions at second training. The study's ZIP-AP5 infused rats did show impairment of second
learning memory. The discrepant effects of ZIP-VEH alternatively may be explained by
experimental restrictions (for example, small sample size). Although eight animals is sufficient
to show reliable statistical significance for a single condition, variability increases with multiple
manipulations due to individual differences. Minor damage incurred during the surgical
placement of cannula in the dHC may potentially be a factor in this variability, but histological
analysis did not reveal any notable damage. Nevertheless, uncontrolled effects could have been
exacerbated by a variety of nonspecific factors.
Successful impairment of second learning can be attributed to infusions of AP5 alone, not
ZIP. ZIP has been shown to wash fully out of brain tissue by 24 hours after initial infusion, but is
present at comparable levels between five and 22 hours after infusion (Kwapis, Jarome,
Gilmartin, & Helmstetter, 2012). However, while inactivation of PKMζ with ZIP has been
shown to impair long term memory of first learning up to seven days after training, it does not
impair acquisition of new tasks taught afterwards (Gámiz & Gallo, 2011). Studies have
consistently shown that PKMζ inactivation impairs retention, but not new acquisition of spatial
tasks and fear conditioning tasks (Pastalkova et al., 2006; Serrano, Friedman, Kenney,
Taubenfeld, Zimmerman, Hanna, Alberini, Kelley, Maren, Rudy, Yin, Sacktor, & Fenton, 2008;
24
Migues at al., 2010). In this project, second training is begun within 24 hours after infusion, and
therefore first learning memory is impaired by ZIP infusions, but any second learning acquisition
impairment must be caused by AP5-sensitivity.
Both ZIP-VEH and ZIP-AP5 groups displaying impaired second learning may be caused
by an inability to learn second training. We considered the possibility of nonspecific effects from
ZIP infusions impairing second learning acquisition. Many studies use low concentrations of ZIP
to block PKMζ mechanisms (Pastalkova et al., 2006; Sacktor, 2008; Shema et al., 2007) so there
is little research to clarify what these nonspecific effects may be. Theories of the mechanisms of
ZIP focus instead on its effect of impairing memory. Past studies (Pastalkova et al., 2006)
investigated whether this impairment is caused by storage or memory retrieval. If ZIP were
impairing memory retrieval only, removing ZIP should reveal intact memory. Alternatively, if
ZIP impaired memory storage, removal of ZIP will not be enough to rescue the memory.
Therefore to differentiate between the two possibilities, Pastalkova et al. injected ZIP after
conditioned rats with long-term place-avoidance, waited for ZIP to be naturally eliminated from
the animals, then tested to see if the memory returned. They found that memory was indeed
impaired: ZIP-infused rats did not avoid the context associated with shock, whereas the VEHinfused rats did. Additional tests were performed to determine if ZIP infusions were causing
other types of lasting memory impairment, but it was shown that retrained animals had no
trouble acquiring new memory or maintaining it as LTM. Overall these results indicate that ZIP
erases storage of previously acquired long-term memories, but does not significantly impede
memory function in any other way. More research will therefore be needed to discover why the
ZIP-VEH group in Experiment 3 did not show normal object location learning in open field 2.
The results of our study nevertheless suggest that PKMζ-based inactivation erasure of
first learning memory by ZIP will return second learning to first learning mechanisms, that is, in
an AP5-sensitive, NMDAR-dependent state. From this we infer that the first learning memory
trace has been lost, so the animal acquires second learning under the mechanisms of first
learning. Coupled with previous research showing memory retrieval not being affected by ZIP
infusions, our results strongly support the storage failure theory of forgetting.
Because animals can be retrained after ZIP has been eliminated from their system, the
next step for our research will be to confirm this with first learning of object location memory
and see how second training is affected. From the results of Experiment 4, which was performed
25
to test for the valid presence of first learning memory in the open field 1, we can see that object
location memory in that context persists even after ten days of retention. The rats in these
experiments were not given ZIP infusions in order to establish a time frame for how long object
location memory in open field 1 lasts. As long retention time of unimpaired memory is longer
than the duration ZIP remains active in the brain, we can conclude that any impairments caused
by ZIP are not due to the memory naturally being forgotten by the rat. In future experiments, ZIP
can be infused after first learning memory has been established, and as per the protocol in
Pastalkova and colleagues' experiments, object location memory tested once ZIP has reliably
passed through the system. Based on previous results, we would expect to see no return of first
learning memory, as well as no learning impairment when animals are retrained in the task. This
will provide another line of support for the boundaries of ZIP impairment, as well as the storage
failure theory of memory.
Establishing the timeline of first learning memory retention also provides necessary
information for when LTM, unimpaired by experimental means, will be fully or partially lost
only over time. This process can be thought of as a parallel to memory naturally decaying as time
passes. As long as LTP is maintained and PKMζ mechanisms remain active, LTM should persist.
But LTP does decay after induction (Abraham, 2003; Villarreal, Do, Haddad, & Derrick, 2001).
Hippocampal LTP has been shown to decrease over time, with early-LTP returning to baseline
levels within three hours (Abraham, Dragunow, & Tate, 1991; Racine, Milgram, & Hafner,
1983), and late-LTP extending possibly to 25 days (Abraham, 2003). Under ideal conditions,
stable LTP has been shown to extend to a year (Abraham, 2003). These persistent LTM
conditions can be experimentally induced with pharmacological interventions such as regular
infusions of synthetic peptide GluR23Y. GluR23Y has been shown to prevent ZIP-induced
memory impairment by blocking GluR2-dependent AMPAR synaptic removal (Migues et al.,
2010). Our study can also extend LTM with such a protocol. If LTP and LTM of first learning is
experimentally maintained, that is, by artificial means, we can test if second learning is affected
by AP5 infusions. If second learning is found to be AP5-sensitive, this offers a new perspective
on long-lasting first learning memories: that they can be maintained by experimental means
despite loss of typical first learning mechanisms. The implications of this would open up many
new avenues of research into both memory maintenance and forgetting.
26
Previous research has shown that hippocampal-dependent spatial memory can last
upwards of weeks to months (Vnek & Rothblat, 1996), but a firm characterization of LTM
persistence in object location memory is still unclear. This trend may be due partially to the
emphasis of experimenters on impairing or sustaining memory at specific points in time to
clearly show the effects of the experimental intervention, rather than investigating a longitudinal
profile of non-experimental and non-pathological forgetting itself. However, the limits of
experimental amnesia must be investigated in order to compare and contrast results to the
progress of natural, non-pathological forgetting. Another future direction for this project is to
investigate the full timeline of LTM of first training in open field 1. Once object location
memory is no longer observed behaviourally (0.5 novelty ratio), we infer that the memory has
been naturally forgotten. Infusions of AP5 or VEH can then be administered prior to second
training (as was previously done in Experiments 2 and 3) to test for presence of first learning
memory. The aim of these studies would be to eventually find a mechanism of experimental
amnesia that can be reliably applied to natural forgetting.
Overall, the data described in this thesis have shown support for the storage failure theory
of LTM loss. We have shown similar findings with that of previous studies examining
experimental amnesia induced with the second learning paradigm. Specifically, we have shown
that impairing first learning memory with ZIP causes second learning to be acquired through first
learning mechanisms. We have therefore inferred that first learning memory is erased by ZIPinfusions. In order to apply these mechanisms to non-pathological forgetting, future studies can
test the boundaries of experimental amnesia with other pharmacological interventions such as
infusing GluR23Y to maintain memory, and allowing first learning memory to decay on its own
before carrying out the second learning paradigm. Although much of day-to-day forgetting is
thought to be due to interference processes (G. D. Brown & Lewandowsky, 2010), it is the
forgetting and retention of LTM that poses a significant issue for clinical populations, for
example those afflicted with Alzheimer's disease. There is still much to study in the forgetting
field. Recently, new perspectives have come forth supporting the theory that forgetting might not
be a passive memory trace loss, but rather an active neural mechanism which is used to pick and
choose necessary memories based on their relevance to our lives (Hardt et al., 2013). As such,
using terms such as storage "failure" is inaccurate and even misleading. Destabilizing memory
by the process of forgetting might have a larger purpose of improving efficiency in our memory
27
systems. It would therefore be better suited to think of forgetting as a storage impairment, caused
by decay mechanisms.
These theories of forgetting, in addition to research on experimental amnesia, can form a
more representative, comprehensive theory of forgetting that can lead to future manipulations to
improve memory ability. The data from this thesis has shown support for the storage impairment
theory of forgetting, which not only contributes to the current literature on experimental amnesia,
but also to the global perspective with which the phenomenon of forgetting is conceptualized.
28
Figures
Figure 1. Protocol and layout to test first learning requirement of NMDAR in open field 2.
Animals were trained infused with VEH or AP5 prior to ten min training sessions across two
days. After two days of rest, animals were given a three min probe test, the first one of which
was scored for behaviour.
29
Figure 2. Protocol and open field 1 (octagon) and 2 (square) layout to test second learning
requirement of NMDAR. Animals were trained for seven days (first training), given two days of
rest, then followed the same protocol of Experiment 1 (above) for second training.
30
Figure 3. Results for Experiments 1 and 2. A novelty ratio significantly above chance (0.5)
indicates that animals showed preference for the new object location and thus retained memory.
Test of first learning (Experiment 1) is shown to be at chance when animals were infused with
AP5, compared with controls, who did show significant object preference. Second learning
(Experiment 2) is shown to be insensitive to AP5-infusions. Both VEH and AP5 groups showed
significant novel object location preference at probe test. N of each group are indicated on their
respective bar. Vertical lines represent standard error. Numbers above are novelty scores; stars
indicate significance from 0.5.
31
A.
B.
Figure 4. A. Protocol for testing second learning requirement of NMDAR after first learning
memory is blocked with ZIP. Animals are given first learning training in open field 1, then
infused with ZIP or SCR 24 hours later. Animals are given one day of rest then given second
learning training in open field 2. B. Results show that SCR-AP5 and SCR-VEH groups showed
significant preference for the new object location. Infusions of SCR after first training did not
affect memory so that second learning acquisition became NMDAR-dependent. ZIP-AP5 and
ZIP-VEH groups performed close to chance, showing no significant preference for the new
location. N of each group are indicated on their respective bar. Vertical lines represent standard
error. Numbers above are novelty scores; stars indicate significance from 0.5.
32
A.
B.
Figure 5. A. Protocol to validate object location recognition task learning in novel context, open
field 1. Unsurgerized animals were trained for ten min across seven days, given two days of rest,
then a probe test for novel location preference. No drugs were infused. B. Compilation data for
varying retention times given after first training in open field 2. Results indicated consistent
object location learning even as retention time lengthened to ten days between training and test.
N of each group are indicated on their respective bar. Vertical lines represent standard error.
Numbers above are novelty scores; stars indicate significance from 0.5.
33
Figure 6. Histology. Images are of typical bilateral cannula placement in the CA1 region of the
dorsal hippocampus. Slices are 50μm and stained with formal thionin. Images at coordinates of
AP -3.6mm; ML 3.1mm; DV -2.4mm.
34
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