RETROGRADE AMNESIA AND RECONSOLIDATION OF A CONTEXT-NO US ASSOCIATION. A dissertation submitted. to Kent State University in partial

Transcription

1 RETROGRADE AMNESIA AND RECONSOLIDATION OF A CONTEXT-NO US ASSOCIATION A dissertation submitted to Kent State University in partial fulfillment of the requirements for the degree of Doctor of Philosophy by Gary W. Barnes August, 2011

2 Dissertation written by Gary W. Barnes B.A., Kent State University, 2000 M.A., Kent State University, 2005 Ph.D., Kent State University, 2011 Approved by, Chair, Doctoral Dissertation Committee David C. Riccio, Ph.D., Members, Doctoral Dissertation Committee Stephen B. Fountain, Ph.D., Benjamin H. Newberry, Ph.D., Richard Hirschman, Ph.D., Brent C. Bruot, Ph.D., Douglas W. Kline, Ph.D. Accepted by, Chair, Department of Psychology Maria S. Zaragoza, Ph.D., Dean, College of Arts and Sciences John R. D. Stalvey, Ph.D. ii

3 TABLE OF CONTENTS LIST OF FIGURES..... iv LIST OF TABLES v ACKNOWLEDGEMENTS. vi CHAPTER I INTRODUCTION II EXPERIMENT III EXPERIMENT 2 34 IV GENERAL DISCUSSION AND CONCLUSIONS REFERENCES. 48 iii

4 LIST OF FIGURES Figure Page 1 Mean total amount of time that subjects spent in the white side of the experimental device during stage 3 of Experiment Mean total amount of time that subjects spent in the white side of the experimental device during stage 3 of Experiment 2 40 iv

5 LIST OF TABLES Table Page 1 Experimental Design for Experiment Pairwise-Comparisons for Statistical Analysis of Experiment Experimental Design for Experiment v

6 Acknowledgements Credit is first due to both Ida Mae and the late Darrell Ray Barnes for giving me every opportunity to gain life experiences, and explore my world with enthusiasm. Thanks also to Dr. David C. Riccio for providing an enriched graduate experience with resources, guidance, and generous portions of his time. vi

7 CHAPTER I INTRODUCTION Amnesia is a pathological form of memory loss that results from brain trauma (see Kapur, 1999 for a review of clinical findings). Persons affected with amnesia cannot recollect experiences that occurred immediately prior to and/or soon following the traumatic event. [Neural damage is often assumed, but is not apparent in many cases (De Renzi, Lucchelli, Muggias, & Spinnler. 1997; see Danek, Uttner, & Straube, 2002 for an example)]. Information learned at times remote to trauma remain operationally intact, implying that only information confined to a small focus of time (during which it being processed by the brain) is vulnerable. It appears that memory systems are only momentarily affected by the amnesic agent, and that the memory becomes less vulnerable over time. The retroactive component of memory loss is referred to as retrograde amnesia (RA) and the proactive component is termed anterograde amnesia (AA). H.M. is a well documented case of AA (Scoville & Milner, 1957). In order to lessen debilitating epileptic seizures H.M. underwent a bilateral temporal-lobectomy. When assessed for memory of information acquired prior to surgery H.M. demonstrated a fully intact repertoire but when tested for memory of facts and events that transpired after surgery, H.M. exhibited profound amnesia. This behavior suggests that the temporal lobes are not the site of memory storage. Instead, this region is likely responsible for the retrieval of memories for experienced events. 1

8 2 Aside from demonstrating profound AA, H.M. maintained a healthy range of mental capacities; perceptual (Milner, 1970), sensory-response (Woodruff-Pak, 1993) and motor (Milner, 1965) forms of learning were unaffected by temporal lobectomy. Such resilience is common in clinical cases of amnesia in that other cognitive faculties remain fully intact (Spiers, Maguire & Burgess, 2001). Questions concerning the normally functioning memory systems can often be addressed by its response to induced pathology (see Spear & Riccio, 1994). As such, the fate of information lost to experimentally induced amnesia is of paramount importance among students of memory processing. The nature of AA has been largely ignored by the experimental framework due to an inherent confound (see Morgan, Burch-Vernon and Riccio, 1993 and Ahlers and Riccio, 1987 for exceptions). Namely the empirical investigations of AA require that the learning event follow soon after amnesic treatment. Residual perceptual and motor deficits can persist for some time after the amnesic agent is delivered. The experimenter cannot determine whether deficits are the result of true memory loss, or alternatively, an acquisition failure. RA is an ideal candidate for the study of memory loss because the amnesic agent is not delivered until training is complete. Because faculties of learning were intact at the time of training, the experimenter can rule-out perceptual and/or motor side effects, nullifying the possibility of acquisition failure. In the most elementary form, studies of RA are three-part. Subjects are first trained to perform a simple behavioral task. Soon after being trained an amnesia-inducing

9 3 trauma is delivered. [A plethora of amnesic agents are available, differing in route of administration and mechanism of action (see Glickman, 1961 for a review)]. Subjects are returned to the original training context at some later time and performance is measured. The severity of memory loss is calculated as a discrepancy in performance between animals that received amnesic trauma after training as opposed to those that did not. Electroconvulsive shock therapy emerged around 1937 and was soon followed by reports of RA among recipients. Zubin and Barrera (1941) were the first to investigate this phenomenon in a systematic fashion. Subjects were trained to recall separate lists of paired associates in a morning and an evening session. Electroconvulsive shock treatment was applied soon after the morning session and subjects were tested the next evening. Subjects performed accurately when tested for material that was learned on the prior evening, but memory for material from the morning session was impaired. Zubin and Barreras (1941) findings were soon replicated by a number of other research groups (Fleschner, 1941; Williams, 1950; Chronholm & Molander, 1958). Aside from the fact that the electroconvulsive shock treatment produced RA, a time-dependent quality was evident as well. Information learned during the morning session, immediately preceding electroconvulsive shock treatment, was disrupted while memory for information learned during the evening session, remote to electroconvulsive shock, remained operationally intact. The presence of memory for information from the evening session demonstrate that amnesic trauma affects information confined to only

10 4 small periods of time surrounding the event; memory systems were only momentarily affected by the amnesic agent. The first paradigm introduced for the study of amnesia in animals was activeavoidance (Duncan, 1949). These tasks require that the animal make a volitional or active response in order to avoid occurrence of an aversive event. When returned to this same situation at a later time animals that have experienced amnesic trauma behave as they did prior to training. However, those with intact memory for the training event perform the active-avoidance response. Two confounds within the active-avoidance paradigm were noted early on (Duncan, 1949; Madsen & McGaugh, 1961). First, when animals are placed in the apparatus to be tested, the experimenter assumes that absence of the trained behavior is the result of induced memory loss. Opponents argue however that absence of the trained response could be attributed to amnesia or punishment with equal parsimony. When amnesic treatment follows soon after the training session its aversive qualities may be perceived and remembered, lessening the probability of the behavior s occurrence in the future. The active-avoidance paradigm produces the same response to punishment as that resulting from loss of memory for the training event. If animals do not display the trained behavior it can be interpreted as a result of memory loss or, on the other hand, the behavior is withheld as a consequence of its previously being followed by an unpleasant event (i.e., amnesic treatment).

11 5 The second problem with the active-avoidance paradigm is that multiple training sessions are required before the animal will display active-avoidance behavior to a reasonable criterion. Because multiple training trials are standard for active-avoidance tasks the experimenter is not able to define a specific time-point where the memory was acquired; this is a weakness of the task in light of the RA gradient. When studying RA, an experimenter must be able to pinpoint exactly the interval of time that passes between the animal learning the task and the point where the amnesic agent was applied. Passive-avoidance (PA) tasks have been specifically designed to circumvent these complications that accompany the active-avoidance paradigm. The prototypical PA paradigm is as follows (see Madsen et al, 1961 for an example). A naïve animal is initially placed within the safe region of a training apparatus, and can wander about freely. Foot-shock is applied when the animal enters a pre-designated unsafe region of the apparatus (i.e., stepping off of a raised platform or entering into black regions of a white box). Soon after performing the punished response an amnesic agent is administered. Animals are returned to the training environment at some later time so that memory for the original training event can be assessed. There are two major advantages to PA tasks (Madson & McGaugh, 1961). First, a sufficient degree of learning occurs within a single training session. Because the effectiveness of amnesic agents is time-dependent this is of utmost importance, allowing an accurate definition of the time-point were learning of the aversive association originally occurred.

12 6 Second, as mentioned above another fault of the active-avoidance paradigm is that it does not allow a definitive differentiation between behaviors associated with aversive aspects of the training/treatment episode and those that arise from amnesic memory loss. The PA paradigm however produces a different response if the animal s behavior is motivated by fear of the training/treatment episode as opposed to loss of memory for the training event. On the one hand fear generated by memory of the train/treatment episode is manifest as avoidance of the black region (i.e., unsafe side ) of the apparatus. On the other hand loss of memory for the aversive training/treatment event is manifest as a failure to avoid the black region (i.e., unsafe side ). Memory for PA training is often measured as the animal s latency to cross from safe to unsafe regions of the experimental apparatus (see Mactutus & Riccio, 1978 for an example). On average animals with intact memory for the training event demonstrate a greater amount of time elapsing before crossing from safe to unsafe regions of the device while those having less recollection show lesser durations. Total time in white (TTW) is another standard measure of memory for PA training (see Mactutus et al, 1978 for an example). This takes advantage of the animals preferences for location within the chamber. If animals have experienced a foot-shock upon crossing into the black side of the box at training, they will avoid this region when tested. As such animals that have intact memory for the training experience will spend a greater amount of time in white than black regions.

13 7 A well documented method of inducing amnesia is through body-cooling treatments (Mactutus et al, 1978). Following the standard protocol (see Hinderliter, Webster, & Riccio, 1975 as an example), animals are inserted into a plastic restraining tube and placed upright inside of a circulating bath of cold water. Upon being removed the experimenter takes a measure of core BT and subjects are returned to their homecages. The hypothermic bout is comprised of four components (see Barnes, Bogart, & Riccio, 2007b): 1) the interval separating completion of the training session from the initiation of treatment, 2) the absolute depth of hypothermia that is achieved, 3) the length of time that is spent at a target temperature, and 4) the rate of recovery from the hypothermic state. This technique results in profound memory loss when applied soon after the training session. There is a direct relationship between the depth of hypothermia and severity of memory loss (Riccio, Hodges, & Randall, 1968). Rats were immersed in a bath of cold water for 14, 11, 6, 4 or 2 ½ minutes after PA training, resulting in BTs of 15, 18, and 29 degrees Celsius respectively. Animals experiencing BTs of 15, 18, and 21 degrees Celsius demonstrated profound amnesia while those experiencing higher BTs did not. So, the threshold for hypothermia-induced memory loss is approximately 23 degrees C. A lesser drop in body temperature has no effect on memory for a previously trained behavior. The biological substrates of hypothermia-induced memory loss are unknown. Hypothermic temperatures appear to affect cellular activity and the formation and later

14 8 use of memory traces. Lipp (1964) noted an increase in seizure-related activity, when hypothermia was induced among un-anaesthetized rabbits and cats. Also, cortical and sub-cortical areas of the brain exhibit a decrement in electrical activity when BT is reduced (Horsten, 1949). Sub-optimum body temperatures are known to diminish cellular metabolism as well (Blair, 1965). Changes that are initiated by, and persist beyond a learning episode, (e.g., production of proteins, Schafe & LeDoux, 2002) require metabolic activity. As presented above, Zubin, et al (1941) showed that memory for recent events is more easily distorted by amnesic trauma than those for remote events, a time-dependent quality referred to as the gradient of RA (for a discussion see Spear & Riccio, 1994, p. 217). From direct clinical observation Ribot (1882) proposed a law of regression in order to frame observations that brain trauma at any given time will affect consolidation of memory for the most recent events, and then proceed in a decremented pattern to those for the earliest events. Hebb (1949) is credited with the first theory of memory formation at the neuronal level. Upon registering a given stimulus, a population of neural cells becomes active and remains so for an extended period of time. A constellation of strengthened anatomical connections emerges as a result of this activity, representing associations of the initial event in the animal s environment. If the same stimuli are experienced at a later time the cellular network and associations represented therein will become active once again - the

15 9 event will be remembered. The gradient of RA that is observed behaviorally corresponds to a time-dependent fixation of the representation within the brain. With a more modern approach Dudai (2004) posits a dual process underlying the observations of varied degrees of loss as a function of train-insult duration. The first process is termed synaptic-consolidation and is thought to create a rudimentary engram that is formed and initiates creation of a fully functional memory for an event. If this synaptic process runs its course unperturbed the brain is altered on a larger scale, termed systems-consolidation. At the systems level larger brain regions dedicated to memory processing and storage are reorganized to accommodate the information in a functional and permanent form. Consolidation theorists (for a review see McGaugh, 2000) believe that younger memories exist in a transient and labile state for a short (ill-defined) period of time during which they are susceptible to the detriments associated with amnesic treatment. After a lengthy duration of processing they become immune to this effect; the trace has been transformed into a more resilient form. Retrieval theories of memory loss are a response to recent evidence suggesting that traumatic injury does not completely abolish representations within the brain, but merely renders them inaccessible to the animal (see Spear, 1978). Though the information is present its location cannot be ascertained by some, as of yet, vaguely described retrieval mechanism. Retrieval theorists take residual traces as evidence that

16 10 much of the information is encoded within a span of milliseconds or seconds of the trauma. Retrieval oriented theorists refer to an entire body of evidence that to support a brief cellular process followed by a lengthier systems process. Some of the most basic characteristics of retrieval theory are presented below. Lynch and Yarnell (1973) accumulated data of the onset of RA in a field study of football players. Athletes were assessed for memory of recent plays proceeding experience of a concussive injury during the game. Players were questioned repeatedly about these strategic plays at various points after being removed from the game. Retention was accurate immediately after being removed from the field, but the same report of plays was deteriorated at later points of recall. The concussion subjects were compared to a control group of players who were removed from the game for nonconcussive injuries, such as fractures or dislocations. Players questioned after nonconcussive injuries exhibited no decline in memory for events that occurred on the field at any point of testing. Results of the Lynch, et al (1973) study demonstrate that the memory for a prior event is represented in the brain for some duration beyond the point of amnesic trauma. The memory exists in some functional form prior to becoming undetectable. This phenomenon is frequently observed and leaves the suspicion that the memory is fixed within the CNS prior to the moment of trauma.

17 11 Lewis, Miller, and Misanin (1968; 1969) found that the gradient of RA could be nullified if animals were allowed to explore the training apparatus before aversive conditioning occurs. Allowing animals to freely explore a training apparatus prior to aversive conditioning reduced the amount of memory loss produced by subsequent ECS treatment. If ECS treatment was applied as early as.5 seconds after the training episode, memory for the event remained intact. This suggests that once a representation has been formed it can facilitate processing of memories for events that occur there at a later time - termed familiarization. Spear (1978) explains that a representation of the experimental context is formed during pre-exposure. So, when the animal is returned there to be conditioned it must simply elaborate on the representation, adding the memory for PA training. Because the representation of the apparatus need only be tweaked slightly, in order to add information about the conditioning episode before amnesia is induced, the amnesic treatment is less effective. Also, if a reminder treatment is applied prior to the retrieval episode it can successfully reinstate previously inaccessible memories (Miller & Springer, 1972; 1973). The animal is able to remember events at test that would have been otherwise rendered the memory irretrievable due to the amnesic trauma. The success of reminder treatments can be quantified and compared between amnesic animals that do receive reminder treatments prior to test and performance of amnesic-treated animals that do not.

18 12 Retrieval theory assumes that the learning event is always embedded within a context. Successful retrieval is a function of the similarity between both the physiological (Mactutus, McCutcheon, & Riccio, 1980) and environmental (Gordon & Mowrer, 1980) mileu present during the initial learning event and those of the retrieval context (Tulving & Thompson, 1973). Animals that are both trained and tested under the same conditions perform more accurately than those placed in differing ones. To further characterize amnesic memories Morgan & Riccio (1994) asked whether an inaccessible memory could be altered by current, ongoing events. On the first day animals experienced amnesic trauma, followed by training on an aversion task. This procedure induced AA for the training episode as was intended. On Day 2 some animals were returned to the experimental apparatus and allowed to explore for a 0-, 5-, or 10- minute extinction session; the aversive stimulus was not presented. On Day 3 animals were given a reminder of their experience on Day 1, and returned to the apparatus shortly thereafter to assess behavior. This reminder was intended to reinstate memory for the training event of Day 1. A decreasing level of aversion-related behavior was measured across groups extinguished for 0-, 5-, and 10-minutes respectively. This pattern indicated that reexposure to the training context on Day 2 had generated extinction of the aversive memory in a stepwise function. These authors thereby demonstrated that memory for the initial training event was influenced by events occurring at a later time. AA treatment had

19 13 not rendered the amnesic memory impervious to processing or integration with later events. A more recently developed hypothesis on memory processing suggests that it is not the memory s age that determines whether it is likely to succumb to amnesia, but its degree of activity when the amnesic agent is delivered (Land, Bunsey, & Riccio, 2000; Misanin, Miller, & Lewis, 1968; Nader, Schafe, & LeDoux, 2000). As an operational definition, memories are re-activated when the animal is presented some attribute of the learning episode (i.e., the animal remembers the original event). It appears as though reactivation of a memory transfers it from the state of systems-consolidation, which is immune to amnesic trauma, to the synaptic state where it is once again vulnerable to disruption. In other words the information must go through a reiteration of the consolidation process each time it is retrieved - reconsolidation (see Przybyslawksin & Sara, 1997 for an example). As of yet no clear consensus has emerged as to whether the anatomical and physiological processes are identical to, or recapitulate the initial consolidation (see Lee, Everitt, & Thomas, 2004). [Reminder-treatment that is intended to reactivate an older memory, also serves as an extinction trial. This means that when animals are returned to attributes of the experimental situation in order to initiate reconsolidation, the environment that was initially associated with aversion is now associated or paired with a non-event. Any drop in performance at test may be due to an extinction of fear within that context rather than amnesia for the initially conditioned aversion. This problem is circumvented by

20 14 including a group that experiences the reactivation treatment in absence of hypothermia. This group can then be compared to one that only experienced the training manipulation. If extinction does occur it is observed as less conditioned performance among the former at testing]. Mactutus, Ferek, George, & Riccio (1982) explored the similarities and differences between a newly acquired memory and an older memory that had been recently reactivated. First, the onset of memory loss occurred more quickly for old reactivated memories than for more recently acquired memories. Second, lost memories could be recovered with the same reminder treatment. Regardless of whether amnesia was induced after reactivation or initial training they were both recovered with equal ease. Finally, the reactivated memory was lost with a less intense level of amnesic trauma than the newly acquired memory, suggesting that it is more fragile than newly learned material. So, the old reactivated memory is augmented more rapidly, and with a lesser trauma than the young memory, and reconsolidation appears to be more rapid than the process of initial consolidation. The majority of experimental literature devoted to amnesia is concerned with loss of memory for either CS-US or context-us associations. These associations involve learned contingencies whereby the presence of a CS or context predicts the occurrence of a biologically relevant stimulus, the US. For instance, in PA training, as described above, the PA chamber itself comes to predict occurrence of foot-shock.

21 15 Two types of learning that differ from this usual stimulus predicting contingency are extinction and latent inhibition (LI). Rather than predict occurrence of a biologically significant event, these two phenomena involve stimulus-alone exposures whereby the animal learns that no biologically significant event occurs in their presence. The stimuli surrounding the experimental situation actually predict a non-event. Westbrook and Bouton (2010) argue that both extinction and latent inhibition are similar in both content and conditions of learning. Both involve a CS- or context-no US, but differ according to a number of signature characteristics. These distinctions will be outlined separately below and then their susceptibility to amnesic loss will be addressed. Extinction is said to occur when the value of a conditioned stimulus as a predictor becomes weakened following its presentation in absence of a previously associated US. Methodologically, pairing of the PA chamber with foot-shock creates the context-us association thereby forming a predictive contingency between the two. If the context is later presented over a period in absence of the foot-shock, the contextual association becomes extinguished. Following this treatment the animal will display less of the conditioned fear or avoidance behavior. On the other hand animals that did not experience the extinction trial will continue to display PA behavior when returned to the context on a subsequent occasion. There are four general characteristics that can be used to define extinction behavior. These attributes demonstrate the context-dependent nature of the extinction learning, while also revealing a competition for control between the initially conditioned

22 16 association and that acquired during extinction procedure. The first is renewal (Bouton & Bolles, 1979a). If an association is conditioned in experimental context A, but the extinction trials occur in context B, the initially conditioned behavior will be observed when returned to context A. This renewal phenomenon is a demonstration of the importance that contextual components to the extinguished association. If extinction trials occur outside of the environmental constraints surrounding conditioning, the conditioned CS- or context-no US association will not be observed. A second feature of the extinction association is reinstatement (Bouton & Bolles, 1979b). This is said to occur when the previously extinguished behavior returns following a treatment whereby the animal is presented the US alone, outside of the experimental context, and then tested in the experimental context. For instance, the initially trained aversion related behavior will return if the animal is returned to the apparatus after receiving a foot-shock in a context different from that of initial training. Third, placing a simple lapse in time between the extinction trials and testing will result in a spontaneous recovery of conditioned responding (Rescorla, 2004) when the animal is returned to the experimental context. This transience of the extinction association is a demonstration of the persistence of the conditioned response. The learning that occurs during extinction does not erase the previously learned behavior, but rather a separate association is formed that vies with the earlier one for control over the animal s behavior.

23 17 Another example of the presence of the extinguished association is the ease with which it can be re-conditioned. Once extinguished, a conditioned association will be reacquired more quickly if the CS or context is again paired with the US (Napier, Macrae, & Kehoe, 1992). Bouton (1993) developed a memory-based account of extinction whereby the coexisting and contradictory associations compete for retrieval at the time of testing. Memory for the initially conditioned stimulus is not framed with contextual components of the training situation. The conditioned association between a CS and US will be displayed across contexts regardless of whether or not the environmental situation of training is present. On the other hand the memory for an extinction episode exists within a contextual framework. Contextual features present at the time of extinction, such as physical, emotional, and temporal attributes must be present at the time of testing. The animal that has been conditioned and then extinguished thereby balances two contrasting memories associated with the experimental situation (Bouton, 1993). Responding during the testing episode is guided by the association that is retrieved at the time of testing. If contextual attributes present at the time of extinction are not present at the time of testing the conditioned behavior will return, as illustrated by its remergence through spontaneous recovery and renewal. However, as demonstrated by the reinstatement phenomenon, presentation of the US prior to conditioning will prime, or enhance the salience of the initial CS-US association and thereby interfere with retrieval of the CS- or context-no US association.

24 18 Literature concerning amnesia for an extinction experience is sparse (see Berman & Dudai, 2001 for an exception). Briggs & Riccio (2007) however documented loss of memory for an extinguished context-us association following hypothermia treatment. Avoidance behavior was extinguished 24 hours after animals had been trained on a PA task. Immediately, 30 minutes, or 60 minutes after extinction animals experienced a duration of deep body-cooling. Varying this delay between extinction and hypothermia treatment resulted in a time-dependent reduction in the severity of loss of memory for the extinction episode. Animals that had experienced the 0- and 30-minute delays demonstrated a normal amount of avoidance at testing, as though the extinction event had not occurred. On the other hand, amnesic treatment did not reduce the amount of extinction observed among animals that had experienced the 60-minute delay. The lack of avoidance-behavior within the 60-minute treatment group shows that the memory for extinction had been completely consolidated within that span of time. In a second experiment Briggs, et al (2007) demonstrated a state-dependence in the memory for extinction treatment by varying the depth of body temperature at the time of testing in a between groups fashion. Animals were recooled to 25 degrees Celsius and then tested after they had warmed to a BT of 30, 33, or 37 degrees. Groups that had been tested at the 30 or 33 degrees Celsius demonstrated extinction equal to animals that had not been rendered amnesic. Animals that were allowed to warm to 37 degrees Celsius demonstrated a lack of extinction at test. This state-dependent return of memory for

25 19 extinction treatment demonstrates transience in memory for extinction that fluctuates with the animal s physiological state. Similar in many ways to the extinction phenomenon outlined above, LI is a deficit in the acquisition or expression of an association between two stimuli as the result of the animal having been previously exposed to one in absence of the other. As an example of LI: During stage 1 the animal is placed in the experimental context for some duration and no significant event occurs. A context-no US association is created. During stage 2 the same experimental context is paired with an aversive foot-shock (US), thereby creating a context-us association. This event contradicts the earlier context-no US association. Finally, during stage 3 these same animals are again placed in the experimental environment and the amount of conditioned behavior is measured. When performance in stage 3 is compared between a group of animals that were not exposed to the context prior to conditioning and others that were exposed, it appears that the latter did not establish a functional context-us association during stage 2. This apparently weakened association is demonstrated during a later testing phase (stage 3), during which the context is presented in absence of the shock, and fearfulness is measured in some operational fashion. LI can be reasonably explained by either acquisition- or performance-deficit theories. More simply, the deficit in avoidance-behavior within the context can be reasonably interpreted as either a failure to learn the association created in stage 2, or as a failure to exhibit the response that was otherwise learned. As an example of an

26 20 acquisition failure, attention-models explain LI as a loss of informative value during preexposure, resulting in laxation in conditioning during the phase 2. The association between the context and shock is not acquired in stage 2 because the subject has less reason to devote attention to the context (see Lubow, Weiner, & Schnur, 1981). Because the animal first learns that the context is irrelevant, serving no predictive value, it attends less to this environment when it is later presented in combination with the US. It is as though the animal has learned to ignore the context. Performance-deficit models of LI assume that the conditioned association has been acquired in stage 2, but that the subject is not able to translate this memory into behavior when observed during stage 3. This detriment is interpreted as an inability to either retrieve (Bouton, 1993; 1997) or express (Miller & Matzel, 1988) the conditioned association. The retrieval account of LI suggests that the memory created during conditioning must compete with that created during pre-exposure when placed in the situation for stage 3. In stage 3 the subject behaves according to the association that is of the greatest relative strength. Either the context-no US is exhibited as a lack of avoidance behavior, or context-us association is observed as an escalation in this same behavior. The second species of expression-deficit models propose that memories do not compete for retrieval during stage 3, but rather that they compete for control of the animal s ensuing behavior. Following this logic, if multiple associations are linked to the experimental context a balance must be achieved. The response that is observed during

27 21 stage 3 is the result of relative associative strength. Does the animal behave as though context predicts that nothing will occur, or that a shock is about to occur? Traverso, Ruiz, & De la Casa (2003) demonstrated an RA-induced reduction in the LI of a CS-no US association using a conditioned taste-aversion paradigm. Animals have a natural tendency to consume biologically reinforcing foods. However animals that experience malaise after eating a satisfying food will avoid or consume less of it at future times; the taste of the food becomes a predictor of illness. In Traverso et al (2003) rodents were preexposed to a saccharine flavored drink in stage 1 of an LI paradigm, thereby experiencing its reinforcing quality. Some animals were injected with the amnesia-inducing agent MK-801 (an NMDA antagonist) immediately following preexposure while others were injected with a benign saline solution. Animals freely consumed the same saccharine solution a second time during stage 2 of the study but some animals were given a nausea-inducing dose of LiCl immediately thereafter, and controls were injected with saline. In this way, animals that had been injected with LiCl learned to associate the previously experienced solution (the CS) with malaise (the US). During stage 3 subjects were again presented the flavored drink and experimenters measured the amount that animals consumed. Animals that had been preexposed to the taste in stage 1, prior to conditioning, drank as though the solution had not been paired with illness, thereby demonstrating complete LI. On the other hand, it was apparent that animals injected with MK-801 immediately after stage 1 did not

28 22 remember the preexposure session. These animals demonstrated a profound tasteaversion in the form of reduced consumption of the solution at test.

29 CHAPTER II EXPERIMENT 1 The current experiment was designed to demonstrate experimentally-induced RA for memory of a context-no US association using a PA paradigm. Amnesia for the context-no US association created during stage 1 has not yet been reported in the literature. The comparisons made between groups described below are defined in more detail in the Methods section of this manuscript. Two conditioned control groups, designated [Pa] and [Pa7], were run to ensure that the context-us had been created using the protocol described below. These animals had experienced only PA training and were tested 1 or 7 days later. It was intended that these two groups display a similar and robust amount of avoidance behavior during the testing session. The basic LI effect of the context-no US was to be revealed in a comparison of Group [PrePa] which was exposed to the device prior to conditioning, and groups [Pa] and [Pa7]. Group [PrePa] was expected to exhibit the basic context-no US association as a lessening of avoidance behavior when compared to the conditioned controls. In order to reveal amnesia for the context-no US association a comparison was made between Group [PreHypPa], which experienced preexposure followed by hypothermia, and Group [PrePa]. The former would exhibit amnesia for the context-no 23

30 24 US association as a greater amount of avoidance-behavior when tested, or conversely, a dampening of the LI effect. A failure to demonstrate amnesia for memory of the context-no US association in Group [PreHypPa] could be reasoned to have been due to an insufficient depth of hypothermia. In order to address this possibility, animals of Group [PaHyp] were subjected to the same intensity of hypothermia immediately after PA training. This group was compared to Groups [Pa1] and [Pa7]. If memory for PA training was dampened by hypothermia in Group [PaHyp] the treatment would be deemed sufficient and replicating the intensity frequently reported in literature. Subjects in Group [Pre2hrHypPa] experienced a 2-hr delay between preexposure and hypothermia treatment in order to verify that lessening in the LI effect within in Group [PreHypPa] resulted from a disruption in memory processing, and not some extraneous psychological factor. Accordingly memory for the context-no US association should be immune to amnesic loss because consolidation of memory for the context-no US association would otherwise be completed by the time that hypothermia was administered (see discussion of the RA gradient above). Does memory for the context-no US associations undergo reconsolidation as do memories for context-us associations? In order to answer this question Group [PreRemHypPa] experienced a reactivation treatment just prior to hypothermia treatment. Reactivation served to initiate reconsolidation of memory for the context-no US association just prior to amnesic trauma. If reactivation returned the initial memory for

31 25 preexposure to a vulnerable state its comparison to Group [PrePa] would be significantly different, and a process of reconsolidation will be assumed to have occurred. Methods Table 1 presents the design of Experiment 1. Subjects. Eighty-four male, Long-Evans rats, between the ages of 90 and 120 days, weighing between 350 and 500 grams were used. Animals were housed individually, provided food and water freely, and maintained on a 12-hour light/dark cycle. This study is represented as protocol 252-GB-DR It was reviewed and approved by the ACUC of Kent State University. Apparatus. The PA chamber had Plexi-glass walls (dimensions: 45.5 X 17.5 X 23.5 cm) and was partitioned at the center by a removable guillotine-style door that could be raised and lowered from the ceiling. Half of the box was white and the other half was black. The lid of the entire chamber consisted of two glass window panes that could be opened and shut by the experimenter. The PA apparatus was equipped with metal grid flooring (2.0-mm grids spaced 1.0 cm apart). Grids on the black side of the box were connected to an electrical source and those of the white side were not. The fixedimpedance shock source delivered an electrical pulse through grids of the black side under the experimenter s control.

32 26 Hypothermia was induced with a model 2095 Forma Temp Jr. water bath and circulator. Animals were restrained in a plastic tube throughout immersion. A rectal probe was employed in order to ascertain each rat s body temperature following treatment. Table 1. Treatment Conditions for Experiment 1. Group Preexpose Hypo Delay Pa Delay Test Pa1* N N 1 Y 1 Y Pa7* N N 1 Y 7 Y PaHyp N N 1 Y-hypo-1 PrePa Y N 1 Y 1 PreHypPa Y Y 1 Y 1 Pre2hrHypPa Y - 2hr - Y 1 Y 1 PreRemHypPa Y -rem- Y 1 Y 1 * Groups [Pa1] and [Pa7] were collapsed into Group [CollPa] during statistical analysis. A digital video camera was positioned over the passive-avoidance chamber so that the entire field of the box was in view for recording throughout testing. A Logitech Quickcam Express web camera was positioned three feet above the ceiling of the PA chamber at all times. The camera was interfaced with a Compax Presario computer for video storage and analysis.

33 27 A white noise generator was functioning in the laboratory room throughout all phases of this study. All time intervals throughout this study were measured with a stopwatch. Procedure. On each of the two days, prior to being randomly assigned to groups and introduced into the study. All animals were handled and acclimated to the experimenter in the animal colony for four minutes. The handling experience ensured that subjects were accustomed to the experimenter prior to initiation into the study. After the handling procedures all animals were randomly assigned to treatment groups, they were not matched on body weight. During stage 1 Groups [PrePa], [PreHypPa], [PreRemHypPa], and [Pre2hrHypPa] were pre-exposed to the PA chamber for a duration of 60 minutes. [Pilot data has shown that the lengthy stage 1 preexposure of 60 minutes is necessary in order to generate an effective context-no US association with the device]. The partition was in the raised position throughout preexposure treatment. From the moment that animals were placed in the chamber they could freely explore both sides of the PA apparatus. Animals in groups [Pa], [Pa7], and [PaHyp] were not preexposed to the device. Immediately after the stage 1 preexposure a body-cooling treatment was administered to animals in Group [PreHypPa]. This treatment consisted of an 8- to 9- minute period of immersion up to the neck in water having a temperature of 4 degrees Celsius (this time interval was measured with a stopwatch). Animals were restrained in a

34 28 plastic tube throughout body-cooling. Using these cooling parameters the experimenter recorded minimum a mean colonic temperature of 23 degrees Celsius immediately after each animal s removal from the water-bath. Group [Pre2hrHypPa] remained in a shoebox within the laboratory area for 2 hours between the preexposure and hypothermia treatment. Subjects in Group [PreRemHypPa] did not experience hypothermia treatment until twenty-four hours after the preexposure session. Immediately prior to body-cooling treatment these animals were returned to the white side of the PA device for 30 seconds with the partition lowered as a reminder treatment. All animals remained housed in the animal colony during the 24 hour duration of the stage 1-2 delay. During PA training animals were placed in the white side of the box with the center partition lowered, restricting them to this area. After a 20-second delay the partition was raised, allowing the animal to explore the black side of the box. Upon crossing into the black side of the box (all four feet) the partition was lowered, restricting the animal to this side of the box. Five seconds after the partition was lowered a 0.5 ma foot shock of one-second duration was administered through the grid flooring and occurred again, a second time, five seconds after the first. Subjects could not escape or avoid these shocks. Animals were removed from the chamber 10 seconds after the final shock. All time intervals were measured with a stopwatch.

35 29 Hypothermia treatment was induced among Group [PaHyp] immediately after experiencing the aversive conditioning of stage 2. Animals remained housed in the animal colony during the 24-hour stage 2-3 delay. Group [Pa7] experienced a 7 day delay between stages 2 and 3. Avoidance-behavior was measured in the experimental context during the stage 3 testing session. This observation period consisted placing each animal in the white side of the PA chamber with the center partition raised over the course of 20 minutes. Animals could explore the apparatus freely throughout the observation session. Digital video recording of each animal s movement began immediately upon placing the animal inside of the PA chamber. Results and Discussion A one-way ANOVA was conducted in order to assess whether PA behavior was affected by the manipulations under study. The dependent measure of memory for PA training was TTW scores. This variable served as the operational reflection of the LI effect. The LSD served as post-hoc analysis. The critical value of significance was held at.05, the power of effect was recorded, and eta squared was selected as the measure of effect size. See Table 2 for an illustration of the comparisons that were made in Experiment 1.

36 30 Figure 1 presents memory performance in the form of TTW scores during the observation period of stage 3. These scores represent the amount of time that an animal spends in the white compartment of the chamber. TTW scores did differ significantly between groups, F (5, 78) = 8.42, p=.00, eta square =.35, power = Groups [Pa] and [Pa7] demonstrated the effect that PA training has on behavior after a retention interval of 24 hours or 7 days respectively. These groups did not differ significantly from each other, suggesting that the conditioned aversion to the experimental context persisted throughout the entire course of this study. The data from these groups was collapsed into a single Group [CollPa] for further statistical analysis. Groups [PrePa] and [CollPa] were compared in order to assess whether the context-no US association was of sufficient strength to disrupt memory for PA training. These groups did differ significantly (p<.04), suggesting that the preexposure manipulation generated LI effectively. Groups [PaHyp] and [CollPa] were compared to ensure that the amnesic treatment was of sufficient intensity, comparable to that in outside literature. These groups did differ significantly (p<.00), suggesting that the amount of hypothermia applied produced results comparable to that commonly reported. Groups [PreHypPa] and [PrePa] were compared in order to assess whether hypothermia had disrupted memory for preexposure. These groups did not differ significantly suggesting that the hypothermic trauma had failed to nullify memory for the preexposure event.

37 31 The comparison between groups [Pre2hrHypPa] and [PrePa] was significant (p<.05). This data suggests that the trace of the context-no US was somehow strengthened by the 2 hour delay in combination with hypothermic trauma. The comparison between groups [PreRemHypPa] and [PrePa] shows a significant enhancement in performance of PA behavior after reactivation treatment (p<.02), suggesting that memory for the context-no US association was somehow affected by this treatment. Presence of the context-no US association was successfully detected through the measure of LI. Though trauma was sufficient to disrupt memory for PA training (i.e., a context-us association) there was no evidence of the context-no US association being nullified. Results of the reconsolidation group suggest that information underwent some form modification following the reactivation-hypothermia sequence. These subjects performed avoidance behavior to a lesser extent than the experimental group that had only been preexposed and then given PA training, thereby exhibiting an actual enhancement of the LI effect. The reactivation treatment may have served as reminder which strengthened the context-no US association in spite of its being followed by hypothermic trauma.

38 32 Table 2. Pair-wise comparisons in analysis of Experiment 1. Groups Comparison Degree of Significance (p values*)_ CollPa X PrePa**. P<.04 CollPa X PaHyp**.. p<.00 PrePa X PreHypPa. p<.32 PrePa X Pre2hrHypPa.. p<.05 PrePa X PreRemHypPa...p<.02_ *Only p-values at or below.05 were deemed significant in the ANOVA and post-hoc analysis. ** [CollPa] is composed of groups [Pa] and [Pa7]. The latter two groups were collapsed because they did not differ significantly.

39 33 Figure 1. Mean Total Time in White in seconds (+/- SEM) for Experiment 1 as a function of group treatment conditions.

40 CHAPTER III EXPERIMENT 2 Experiment 1 failed to extend the findings of Traverso et al (2003) to memory for a context-no US association. Memory for preexposure was not affected by hypothermia treatment, suggesting that this form of memory is more deeply entrenched than the context-us association, which was greatly affected in the comparison between Groups [PaHyp] and [CollPa]. In Experiment 2 the age of the inaccessible memory for LI will be addressed as potential factor in the creation of newer memories. Outside literature has demonstrated that after memory for the context-no US has undergone an extended retention interval its influence over the CS-US association is lessened. In other words, inserting a lengthy interval between the preexposure and conditioning stages, stages 1 and 2, reduces the LI effect among intact subjects (see Hall & Schachtman, 1987). Does the effect of an inaccessible memory become even less profound when the age of the context-no US association is increased in conjunction with amnesic insult? A comparison was first made between a group of animals that experienced a 7-day delay between stages 1 and 2, designated Group [Pre7Pa], and Group [PrePa] of Experiment 1 which had only experienced the 1-day delay. Following the literature cited above, it was predicted that memory for the preexposure treatment would be weakened over the course of this 7-day delay, and so a greater amount of avoidance behavior would be exhibited by the former group, or conversely there would be a more robust LI effect among the latter. 34

41 35 To address the effect that hypothermia treatment has on memory for a context-no US association one group (i.e., [PreHyp7Pa]) experienced a combination of amnesic treatment and a 7-day delay between stages 1 and 2. If the detrimental effect of these two factors is additive, this group would demonstrate a greater amount of avoidance behavior than Group [PrePa] of Experiment 1. The current study also asked whether the amount of time that the amnesic and accessible memory coexisted within the animal s memory store is a factor in the relation between them. Studies show that latent inhibition of a stimulus wanes over the passage of time. Aguado, Symonds, & Hall (1994) measured a recovery of memory for the conditioned response, observed in stage 3, when stages 2 and 3 were separated by an extended interval. It may be that the duration of concurrent processing of the contradictory associations is a factor in the interaction between the accessible and inaccessible memories. Does the effect of an inaccessible memory become even less profound when the duration that the context-no US and context-us coexist (i.e., the interval between its stages 2 and 3) is extended, in conjunction with amnesic treatment? A comparison was first made between a group of animals that experienced a 7-day delay between stages 2 and 3, designated Group [PrePa7], and Group [PrePa] of Experiment 1 which had only experienced the 1-day delay. Following the literature cited above, it was predicted that memory for the preexposure treatment would be weakened over the course of the 7-day

42 36 delay and so a greater amount of avoidance behavior would be exhibited by the former group, or conversely there would be a more robust LI effect among the latter. Experiment 2 addressed the effect that amnesic trauma has on memory for a context-no US association in conjunction with a delay between stages 2 and 3. Group [PreHypPa7] experienced a combination of hypothermia treatment and a 7-day delay between stages 2 and 3. If the detrimental effects of the delay are additive between these two factors, this group would demonstrate a greater amount of avoidance behavior than [PrePa] of Experiment 1. Though the depth of hypothermia used in Experiment 1 did not affect memory for the context-no US association, Experiment 2 applied the same depth of hypothermia treatment. It was expected that animals would display the desired amnesic loss when this treatment was combined with the aforementioned delays; that the cumulative effect of the two manipulations would be sufficient. Methods Table 3 depicts the design of Experiment 2. Subjects. Ninety-six male, Long-Evans rats, between the ages of 90 and 120 days, weighing between 350 and 500 grams were used. Animals were housed individually, provided food and water freely, and maintained on a 12-hour light/dark cycle. This study

43 37 is represented as protocol 252-GB-DR It has been reviewed and approved by the ACUC of Kent State University. Table 3. Treatment Conditions for Experiment 2. Group Preexpose Hypo Delay Pa Delay Test PrePa* Y N 1 Y 1 PreHypPa* Y Y 1 Y 1 Pre7Pa Y N 7 Y 1 PreHyp7Pa Y Y 7 Y 1 PrePa7 Y N 1 Y 7 PreHypPa7 Y Y 1 Y 7 * Groups [PrePa] and [PreHypPa] are originally from Experiment 1. They were included in the statistical analysis of Experiment 2. Apparatus. Apparatus and the experimental situation used in Experiment 2 were identical to those of Experiment 1. Procedure. All animals experienced a handling procedure identical to that of Experiment 1, prior to being randomly assigned to a group. During stage 1 all animals experienced a preexposure session identical to that of Experiment 1. Immediately after

44 38 being preexposed to the PA device a body-cooling treatment was administered and body temperatures were assessed following a protocol identical to that of Experiment 1. All animals remained housed in the animal colony during the stage 1-2 delay. This delay lasted 24 hours for groups [PrePa7] and [PreHypPa7] and 7 days for groups [Pre7Pa] and [PreHyp7Pa]. All animals experienced an aversive-conditioning session in the PA chamber during stage 2 that was identical to that of Experiment 1. All animals remained housed in the animal colony during the stage 2-3 delay. This delay lasted 24 hours for groups [Pre7Pa] and [PreHyp7Pa] and 7 days for groups [PrePa7] and [PreHypPa7]. Avoidance-behavior was measured in the experimental context for all groups during the stage 3 observation period following the protocol outlined for Experiment 1. Results and Discussion Figure 2 presents memory performance in the form of TTW scores during the observation period of stage 3 of Experiment 2. These scores represent the amount of time that an animal spends in the white (i.e., safe) compartment of the chamber during this time. Groups [PrePa] and [PreHypPa] from Experiment 1 were included in the analysis. These groups were not run separately for Experiment 2. TTW scores did not differ significantly between groups, F (5, 66) = 1.00, p=.42, eta square =.07, power =.34.

45 39 Because the omnibus ANOVA did not detect significant differences between groups the analysis was concluded. Increasing the duration between stages did not decrement the intensity of LI to any extent, suggesting that the context-no US association was maintained regardless of these delays. Also, the LI effect was not weakened to a further degree when hypothermia was applied in combination with the 7-day delays.

46 40 Figure 2. Mean Total Time in White in seconds (+/- SEM) for Experiment 2 as a function of group treatment. Solid black bars represent groups that were initially run in Experiment 1, and were included in data analysis of Experiment 2. Hatched bars represent groups that experienced a 7-day delay between stages 1 and 2. White bars represent groups that experienced a 7-day delay between stages 2 and 3.