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Factors affecting long-term habituation in Caenorhabditis elegans Beck, Christine Daily O’Brien 1995

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FACTORS AFFECTING LONGTERM HABiTUATION IN CAENORHABDITIS ELEGANS by CHRISTINE DAILY O’BRIEN BECK  B.Sc., University of Alberta, 1987 M.A., University of British Columbia, 1991  A THESIS SUBMiTTED IN PARTIAL FUFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES Department of Psychology  We accept this thesis as conforming to the required standard  THE UNIVERSITY OF BRITISH COLUMBIA June 1995 © Christine Daily O’Brien Beck, 1995  In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission.  (Signature)  Department of  cJ ôLi(’ry’j  The University of BritisF( Columbia” Vancouver, Canada Date  DE-6 (2188)  LAAIJ2  II  Abstract The objective of these experiments was to explore long-term memory in Caenorhabditis elegans. This examination of memory in a simple organism with accessible genetics and a well understood biology may permit later work to defme the cellular processes that underlie longterm memory. Habituation training with a vibrational stimulus was administered on Day 1, and the retention test of a block of stimuli was given 24 h after the end of training on Day 2. Longterm retention of habituation was evident as a lower level of responding on Day 2 relative to the level of responding on Day 2 of untrained controls or the initial level of responding of worms on Day 1. In Experiments 1 and 2, a habituation training protocol that produced long-term retention of habituation was established, and the effects of stimulus number, interstimulus interval (1ST), and distribution of training on both short-term and long-term habituation were examined. In Experiment 1 (10-s 1ST), there appeared to be a floor effect which resulted in a low level of responding regardless of training on Day 1; thus no evidence for long-term habituation after training at a 10-s 1ST could be found. In Experiment 2 (60-s ISI), worms that received distributed and massed habituation training with 60 stimuli showed a significantly lower level of responding relative to untrained controls. The distributed habituation training appeared to be more effective at inducing long-term habituation and was used in the subsequent experiments. To characterize the effects of heat shock treatments used in the behavioral experiments that follow, the effects of heat shock on two assays, the induction of a heat shock protein gene, hspl6, and the rate of egg-laying were measured in Experiment 3. All heat shock treatments used caused the induction of hspl6. In addition, the number of eggs laid during a fixed interval after heat shock was sensitive to the heat shock treatments given in Experiments 4 through 8. In Experiments 4 through 8, the effects of heat shock on short- and long-term habituation were examined. Heat shock, which acts as a general cellular stressor, was  ifi  administered at different times before, during and after training. In Experiment 4, heat shock (45 mm, 3 2°C) was administered, ending 2 h before training on Day 1. Heat shock before training did not affect the initial level of responding on Day 1, habituation during training, short-term retention of habituation between blocks of training or long-term retention of habituation. In Experiment 5, heat shock (45 mm, 32°C) was administered during the rest periods of distributed training in the 1-h interval after each training block. While heat shock during training had no significant effect on responding on Day 1, long-term habituation was blocked. In Experiment 6, the possibility that heat shock before training would prevent the disruption of long-term habituation by heat shock during training by inducing thermal tolerance was examined. This was tested by administering heat shock (45 mm, 32°C) that ended 2 h before training and heat shock during training. It was found that heat shock before training did not prevent the disruption of long-term habituation by heat shock during training. In Experiment 7, the effect of heat shock that ended 2 h before the retention test on Day 2 on the retention of long-term habituation was examined. It was found that heat shock on Day 2 did not disrupt the retention of habituation. Finally, in Experiment 8, the effect of brief heat shock (15 mm, 32°C) at different intervals in the rest period following the training blocks was examined in an attempt to more narrowly defme a critical period for consolidation of long-term habituation. Although there was no significant effect of brief heat shock on retention of habituation, the pattern of the data suggests that there may be a period of greater vulnerability worth further investigation. In summary, heat shock given before training or before the retention test did not affect long-term habituation, while heat shock during training disrupted consolidation of long-term habituation. Taken together, these behavioral results provide the foundation for an investigation of the cellular processes underlying long-term memory in  .  elegans. By exploring the  dynamics of the formation of long-term habituation, intervals of time critical to the formation of long-term habituation were defmed. This in turn will help to focus attention on the cellular  iv processes whose activity during those intervals of time may be important to the consolidation of long-term memory.  V  Table of Contents ii  Abstract List of Tables  vii  List of Figures  viii  Acknowledgments  x  INTRODUCTION Habituation is the Product of Multiple Processes Long-term Habituation as a Form of Memory elegans as Simple Model System for Learning Long-term Habituation in elegans Interference as a Tool to Defme Memory Consolidation Overview of Experiments  1 1 9 15 22 23 28  General Methods Subjects and Materials Stimulation and Behavioral Observations Habituation Training Procedure  28 28 29 29  .  .  Chapter One  Heat Shock Scoring and Statistical Analysis  30 31  Experiment 1: Short- and Long-term Habituation at a Short ISI (10-s ISI) Methods Results  39 39 40  Chapter Two Experiment 2: Short-term and Long-term Habituation at a Long ISI (60-s ISI) Methods Results Chapter Three Experiment 3: The Effects of Heat Shock on hspl6 Induction and the Rate of Egg-laying Experiment 3A: The Effect of the Heat Shock Treatments on hspl6 Induction Methods Results Experiment 3B: The Effect of the Heat Shock Treatments on the Rate of Egg-laying Methods Results  47 48 48 56 56 57 57 60 60 61  Chapter Four Experiment 4: Short-term and Long-term Habituation with Pre-exposure to Heat Shock 64 Methods 64 Results 65 Chapter Five Experiment 5: Short-term and Long-term Habituation with Heat Shock During Training  71  vi  Chapter Six  Methods Results  71 72  Experiment 6: Short-term and Long-term Habituation with Pre-exposure to Heat Shock and Heat Shock During Training Methods Results  78 78 80  Chapter Seven Experiment 7: The Effects of Heat Shock Just Prior to Testing on the Retention of Long-term Habituation Methods Results  87 87 91  Chapter Eight Experiment 8: Titration of the Effects of Heat Shock During the Rest Period 91 92 Methods Results 93 Chapter Nine Synthesis of the Results: An Analysis Including Experiment 2, and Experiments 4 through 8 Chapter Ten  Discussion Interference as a Tool to Examine Memory Consolidation The Distributed Training Effect: A Psychological Perspective Differentiation of Types of Long-term Memory Through Interference Treatments The Use of Heat Shock as an Interference Treatment  96 106 109 114 117 122  List of Abbreviations  123  Bibliography  125  Appendix 1  The Distribution of Missing Values  136  vil List of Tables Table  1.  The post-hoc comparisons between the LTH and LTH-HS groups in Experiments 2, 4, 5, 6, 7 and 8 of the mean block magnitude of the first twenty stimuli on Day 1.  106  vm List of Figures Figure  1.  The nematode Caenorhabditis elegans with the apparatus used behavioral observations.  17  Figure  2.  The tap-withdrawal circuit.  20  Figure  3.  The countemull value and the calculation of effect size.  35  Figure  4.  Habituation curves at a 10-s 1ST.  42  Figure  5.  Habituation by block at a 10-s 1ST.  46  Figure  6.  Habituation curves at a 60-s 1ST.  50  Figure  7.  Habituation by block at a 60-s 1ST.  54  Figure  8.  The expression of JZ in transformed  Figure  9.  The number of eggs laid by five worms in a 30 mm test period, 15 to 45 mm after the end of the heat shock treatment.  63  Habituation curves: distributed habituation training with pre-exposure to heat shock.  67  Habituation by block: distributed habituation training with pre-exposure to heat shock.  70  Habituation curves: distributed habituation training with heat shock during training.  74  Habituation by block: distributed habituation training with heat shock during training.  77  Habituation curves: distributed habituation training with pre-exposure to heat shock and heat shock during training.  82  Habituation by block: distributed habituation training with pre-exposure to heat shock and heat shock during training.  86  Habituation by block: distributed habituation training with heat shock on Day 2, 2 hr before testing.  90  Habituation by block: distributed habituation training alone or with brief heat shock during training either early, mid or late in the 1-h rest period following each training block.  95  The mean Day 1 and Day 2 response levels across experiments: LTH groups.  98  The mean Day 1 and Day 2 response levels across experiments: LTH-HS groups.  100  Figure 10. Figure 11. Figure 12. Figure 13. Figure 14. Figure 15. Figure 16. Figure 17.  Figure 18. Figure 19.  .  elegans.  59  x 139  Figure 20.  The number of missing values in Experiments 1 and 2  Figure 21.  The number of missing values in the three blocks of training on Day 1 in the 142 massed and distributed habituation training group.  Figure 22  The number of missing values in the twenty stimuli control group, and the massed and distributed training groups.  Figure 23.  The distribution of missing values during distributed habituation training on 146 Day 1 and on Day 2.  Figure 24.  The distribution of missing values during massed habituation training on Day 1 and on Day 2.  148  Figure 25.  The number of missing values in Experiment 4: pre-heat shock.  152  Figure 26.  The distribution of missing values during training in the LTH and PRE HS I 155 LTH groups on Day 1 and on Day 2.  Figure 27.  LTH-HS (Exp. 5) and LTH (Exp. 2; distributed training) and the distribution of missing values during habituation training on Day 1.  Figure 28.  The distribution of missing values during training in the LTH (Experiment 160 2) and LTH-HS (Experiment 5) groups on Day 1 and on Day 2.  Figure 29.  The distribution of missing values during training in the LTH, LTH-HS and 164 the PRE HS / LTH-HS groups on Day 1 and on Day 2.  Figure 30.  The distribution of missing values during training in the LTH and LTH / D2HS groups on Day 1 and on Day 2.  166  Figure 31.  The distribution of missing values during training in the LTH and LTH early HS groups on Day 1 and on Day 2.  169  Figure 32.  The distribution of missing values during training in the LTH-mid HS and 171 LTH-late HS groups on Day 1 and on Day 2.  Figure 33.  The distribution of missing values during training in all the LTH groups (Exp. 2, 4, 6, 7 and 8) on Day 1 and on Day 2.  174  Figure 34.  The distribution of missing values during training in both of the LTH-HS groups (Exp. 5 and 6) on Day 1 and on Day 2.  178  144  158  x Acknowledgments First, I want to acknowledge the tremendous support and encouragement I received from my advisor, Cathy Rankin, whose patience with my struggles during this time seemed limitless. I also owe thanks to the other members of my thesis committee, Eric Eich and Tony Phiffips, who helped keep the standards of the work high. I wish to acknowledge the help of the others that were involved in the experiments I report here. Tracey Smillie, Richard Faber and Michael McCready, as undergraduate students, all ran projects which were part of this work. Peter Candido (Department of Biochemistry, UBC) kindly supplied me with the transgenic strain of. elegans used in Experiment 3A, and Don Jones offered his expertise in staining technique. Finally, I would like to thank Steve Wicks for staining and photographing the transgenic worms in Experiment 3A. Last but not least, I would like to gratefully acknowledge the support, encouragement and patience of my family, Sean, Caitlin and Rowan without whom, I cannot imagine having come this far. —  I would like to dedicate this thesis to my mother, Kathleen Beck, and my father, Charles Beck. Thank you both for passing your love of science on to me.  1 Introduction Long-term memory may be defined as the lasting changes in behavior that are brought about by experience and are mediated by cellular processes in the nervous system. Studying long-term memory in a simple model system provides an opportunity to defme both the behavioral patterns that indicate the operation of memory and the cellular mechanisms that must support memory. The experiments presented here using the nematode Caenorhabditis elegans begin an analysis of the behavioral changes produced by a form of long-term memory in this simple organism. Habituation is the product of multiple processes The focus of this work is the analysis of memory for a simple form of learning, habituation. Habituation is a ubiquitous form of learning, found in organisms ranging from protozoa to mammals (Harris, 1943; Thompson & Spencer, 1966; Wood, 1988) and has been described in a variety of behavioral responses, such as bristle-cleaning in the fruit fly Drosophila melanogaster (Corfas & Dudai, 1989), desynchronization of electroencephalography (BEG) to auditory stimuli in rats (Dworkin & Dworkin, 1990), the respiratory startle response to light in goldfish (Laming & McKinney, 1990), the galvanic skin response to visual stimuli in humans (Barry & Sokolov, 1993), and the escape response to electric shock in the crab (Rakitin, Tomsic & Maldonado, 1991). Habituation may be defined as a decrease in responding seen with repeated stimulation (Groves & Thompson, 1970). It is distinguished from simple fatigue by a number of features including the expression of dishabituation, which is the facilitation of the habituated (decremented) response by a novel or noxious stimulus, and a sensitivity of both habituation and spontaneous recovery from habituation to interstimulus interval (Staddon, 1993; Groves & Thompson, 1970). Retention of habituation may be observed after a repeated series of habituation training and spontaneous  2 recovery; this training leads to progressively greater habituation (Groves & Thompson, 1970; Petrinovich, 1984). Groves and Thompson (1970) proposed that the habituation observed during a single training session is composed of two antagonistic processes: habituation, which is stimulusspecific, and sensitization, which affects the state of the whole organism. Basing their dualprocess theory of habituation on work with the habituation of spinal reflexes in the cat, they argued that the connection between stimulus (S) and response (R) is affected by these two independent processes; habituation weakens the connection specifically between the stimulus used in habituation training and its response, while sensitization facilitates all S R connections -  by altering the state of the organism (Groves, DeMarco & Thompson, 1969; Groves, Glanzman, Patterson & Thompson, 1970; Groves, Lee & Thompson, 1969; Groves & Thompson, 1970). Generally, the habituation process starts small and builds gradually; the sensitization process starts relatively more strongly, and in some paradigms increases for a short time before fading gradually. They proposed that the dynamics of habituation are the product of the interaction between the dynamics of these two processes, habituation and sensitization (Groves & Thompson, 1970). Although there is some controversy about the role of stimulus perception in habituation (Hall, 1991), there appears to be a consensus that habituation is the product of multiple processes (Groves & Thompson, 1970; Hall, 1991; Peeke and Petrinovich, 1984; Staddon, 1993; Barry & Sokolov, 1993). The notion of multiple behavioral processes has implications for the cellular processes underlying the behavior. One question that must be answered is where in the nervous system habituation is mediated and whether different components are localized in different loci. The processes underlying habituation may be highly localized, for example, in the sensory neuron synapses (e.g. Castellucci, Pinsker, Kupfermann & Kandel, 1970) or may be distributed throughout the nervous system (e.g. Falk, Wu, Cohen & Tang, 1993). Studies correlating  3 electrophysiological changes in neuronal activity have focused on this question. The efforts to characterize the cellular processes underlying habituation have been confounded by the complex relationship between the decrement in response expressed in the sensory neurons transducing the stimuli and the behavioral habituation induced by the stimulation of the sensory neuron. In support of a simple and direct relationship between the activity of the sensory neurons and habituation, Hernández-Peón (1960) showed in the cat that the induction of habituation was accompanied by presynaptic inhibition of neurons in the periphery; he proposed that habituation results from inhibition of sensory input which may occur as early as the first sensory relay nucleus. However, Groves, Glanzman, Patterson and Thompson (1970) showed that habituation of spinal reflexes in the cat was not due to presynaptic inhibition of sensory fibers; there are no necessary changes in excitability of afferent terminals during the development of response habituation. This finding argues against a peripheral locus for habituation (Groves & Thompson, 1970). In addition, Weinberger, Goodman and Kitzes (1969) did not fmd a relationship between habituation of the eye-movement response to auditory stimuli and the ascending auditory evoked potentials; although they did find a significant relationship between reticular formation activity and eye-movement habituation, which may indicate the role of arousal state in habituation. It may be that the locus of habituation depends on the system that is mediating the plasticity. A traditional way of probing for the locus of processes that support learning is to lesion structures of the brain that may mediate those processes. There are few examples of disruption of habituation without the loss of sensory or response ability by lesions of specific neural structures; however, lesions of the telencephalic area blocks habituation in both the startle response to light stimulation of the goldfish and the attack response to visual stimuli in larval salamander (Laming & McKinney, 1990; Pietsch & Schneider, 1990). Pietsch and Schneider  4 (1990) noted that while the telencephalon was not formerly believed to play an important role in vision in the salamander, it may mediate an active-negative component of the visual behavior of the salamander. Laming and McKinney (1990) hypothesized that in the goldfish, the telencephalon acts as a holding center for short- and long-term memories, and may also play a role in processing temporal stimuli. In both organisms, the role of novelty in stimulusprocessing appears to have been disrupted by these lesions. Likewise, in the honeybee, there is evidence for inhibition of the proboscis extension reflex by central mechanisms (Braun & Bicker, 1992). The proboscis extension reflex is an appethive response to sugar solution applied to one of the bee’s antennae; Menzel, Hammer and Sugawa (1989) demonstrated that hunger improved the learning index in the classical conditioning of the proboscis extension reflex. Braun and Bicker (1992) examined whether hunger and satiation would influence habituation of the reflex. They found that both the initial response to the stimuli was higher and the rate of habituation was slower in hungry bees than in satiated bees, supporting the theory that the internal state of the organism (i.e. sensitization) affects habituation. Habituation of the reflex was limited to the antenna stimulated; the contralateral antenna did not show response decrement when tested. Dishabituation of the habituated reflex could be evoked by stimulating the contralateral antenna. Through pharmacological blockers of neural pathways it was determined that expression and habituation of the reflex depended upon the functioning of the antenna lobes, but not mushroom bodies, which mediate olfactory conditioning in the honeybee (Braun & Bicker, 1992). Although there appears to be no universal locus for habituation, it is possible that the cellular processes that support synaptic plasticity share characteristics across systems. A candidate for a cellular analogue of habituation is the phenomenon of synaptic depression, which is the decrease in post-synaptic activity with a consistent level of stimulation of the pre synaptic neuron (Zucker, 1989). Quantal analysis in the mollusk Aplysia californica,  5 demonstrated that synaptic depression of sensory neurons is accompanied by a decrease in the number of quanta of neurotransmitter released (Castellucci & Kandel, 1974). In addition, in the siphon sensory neuron motor neuron synapse, the development of synaptic depression is -  correlated with the kinetics of a long-lasting presynaptic calcium current inactivation (Klein, Shapiro & Kandel, 1980). The calcium current is responsible for presynaptic neurotransmitter release; with calcium current inactivation, the amount of neurotransmitter release would be diminished, resulting in synaptic depression (Klein, Shapiro & Kandel, 1980; Byrne, 1982). Further analysis demonstrated that a combination of transmitter depletion and inactivation of the presynaptic calcium current are likely responsible for gill sensory neuron synaptic depression (Gingrich & Byrne, 1985). Interestingly, it now appears that the gill-withdrawal reflex of Aplysia is not mediated solely by the monosynaptic pathway of the siphon sensory neuron to motor neuron connection (Lukowiak, 1978; Hawkins, Castellucci & Kandel, 1981; Frost, Clark & Kandel, 1988); it is clear that the circuit that supports the gill-withdrawal reflex involves polysynaptic pathways which may differentially modulate the response to siphon stimulation (Trudeau & Castellucci, 1992). Optical measurements of neuronal activity in the abdominal ganglion show that over 200 neurons are activated by the sensory stimulus that produces the gill-withdrawal reflex (Zecevic, Wu, Cohen, London, Hopp & Falk, 1989; Nakashima, Yamada, Shiono, Maeda & Satoh, 1992). In an additional study, the activity of neurons in the abdominal ganglion was characterized during habituation of the gill-withdrawal reflex with optical measurements (Fallc, Wu, Cohen & Tang, 1993). It was found that habituation was expressed nonuniformly by neurons in the abdominal ganglion; the neurons could be separated into classes based on their activity (e.g. remaining steady during habituation, decreasing with habituation, increasing with habituation) during the formation of habituation (Falk et al., 1993). While the synaptic depression exhibited by the sensory neuron-to-motor neuron synapse may still be important to  6 the formation of habituation of the withdrawal response in Aplvsia, it may not completely describe the cellular processes underlying habituation. An additional problem with homosynaptic depression as a cellular analogue for habituation in Aplysia is the finding that tail sensory neuron activity in the intact organism during habituation does not seem to decrease with stimulation (Stopfer & Carew, 1994). Stopfer and Carew (1994) defmed the stimulation necessary to produce behavioral habituation, decreased activity in the tail motor neurons, and homosynaptic depression in cell culture. When stimulation with these parameters was applied to tail sensory neurons while they simultaneously recorded from that sensory neuron and the motor neuron it was contacting, it actually facilitated rather than depressed the excitatory presynaptic potentials (EPSPs) of the tail sensory neuron while decreasing motor neuron activity (Stopfer & Carew, 1994). In addition, other tail sensory neurons also showed facilitation, demonstrating that the effect was heterosynaptic (Stopfer & Carew, 1994). These results indicate that homosynaptic depression is not an adequate description of the all of the cellular processes underlying this form of habituation (Stopfer & Carew, 1994). Stopfer and Carew (1994) proposed that either increased sensory neuron output to inhibitory intemeurons, or increased inhibition at intemeuronal sites may contribute to habituation in the tail withdrawal reflex. On the whole, work with Aplvsia has given us a tremendous amount of power to examine plasticity in the elements of the nervous system. However, it seems from the recent evolution of understanding of the nature of the gffl-withdrawal circuit (Fallc et al., 1993), and the fmding that homosynaptic depression may not be responsible for habituation of the tail withdrawal response (Stopfer & Carew, 1994), that information about cell-to-cell interactions must be examined in the context of the whole organism to test the validity of their participation in mediating a form of behavioral plasticity.  7 An alternative approach to the investigation of the cellular processes underlying learning is the isolation and cloning of mutants with impaired learning. By identifying the gene product coded by the gene affected by these mutations and defining the physiological role of the gene product in the nervous system, the cellular processes underlying learning may be probed (Heisenberg, 1989). This approach has been successfully used in the fruit fly Drosophila melanogaster (Dudai, 1988; Heisenberg, 1989; Tully, Preat, Boynton & Del Vecchio, 1994). The biochemical characteristics of the classic Drosophila learning mutants dunce and rutabaga have been defined (Byers, Davis & Kiger, 1981; Dudai & Zvi, 1984; Livingstone, Sziber & Quinn, 1984). Both mutants affect the level of cAMP, an intracellular second messenger believed to be involved in sensitization and classical conditioning in Aplysia (Byrne et al., 1993). dunce is deficient in cAMP phosphodiesterase; thus it has reduced cAMP hydrolysis (Byers, Davis & Kiger, 1981). rutabaga is deficient in the Ca 2 stimulation of adenylate cyclase, the enzyme that controls the production of cAMP; thus rutabaga has abnormally low levels of cAMP. These mutants were isolated through a screen for learning defective mutants in the olfactory classical conditioning paradigm; both show deficits in the retention of olfactory conditioning (Dudai, Jan, Byers, Quinn & Benzer, 1976; Aceves-Pina et al., 1983). Their performance in a variety of habituation paradigms has been examined; no consistent pattern of effects has emerged (Heisenberg, 1989; Corfas & Dudai, 1989, 1990; Wittekind & Spatz, 1988; Duerr & Quinn, 1982; O’Dell, 1994). Duerr and Quinn (1982) found that habituation of the proboscis extension reflex was slower and less profound in dunce and rutabaga flies than in wild-type (normal) flies. In contrast, the habituation of the landing response to visual stimuli in dunce and rutabaga flies was more rapid and deeper than that seen in wild-type flies. The habituation of the male courtship response was more rapid and deeper in dunce and inactive, a mutant strain deficient in octopamine, a neurotransmitter with a putative role in learning  8 (O’Dell, 1994). Clearly, there is no simple relationship between the deficits in olfactory conditioning exhibited by the classic learning mutants and abnormal expression of habituation. In the bristle-cleaning reflex of Drosophila, the relationship between the response decrement seen at the cellular level and the response decrement seen at the behavioral level was examined. The rate and depth of habituation of the bristle-cleaning reflex in response to a deflected bristle was unchanged in dunce and rutabaga when compared to wild-type ffies; however, memory for habituation was attenuated (Corfas & Dudai, 1988). Dishabituation of the habituated bristle-cleaning reflex was evoked by stimulation of the dorsalcentral microchaetae (Corfas & Dudai, 19989). The response decrement of the sensory neuron that transduces the bristle-bending stimulus, which might have been considered a cellular analogue of the habituation of the bristle-cleaning reflex, actually proved to be affected differently by the dunce and rutabaga mutants and was not facilitated by stimulation that produced dishabituation of the behavioral response (Corfas & Dudai, 1990). dunce showed a more rapid and deeper degree of fatigue to very brief stimulation at a short ISI (stimulus duration 400 ms; 5-s 1ST) than wild-type; rutabaga showed slower fatigue with longer stimuli at a longer 1ST (stimulus duration 6  5;  20-s 1ST) when compared to wild-type flies (Corfas & Dudai, 1990). In addition,  in dunce, the degree of fatigue was not 1ST-dependent when ISIs were varied between 1 and 2 s, while in both rutabaga and wild-type flies, fatigue was deeper at the shorter 1ST. In addition, the injection of cAMP phosphodiesterase inhibitors produced a dunce-like phenotype in fatigue exhibited by wild-type flies, suggesting that dunce has its effect on fatigue through the deficient cAMP phosphodiesterase (Corfas & Dudai, 1990). The phenotype of rutabaga is not affected by cAMP phosphodiesterase inhibitors, as would be expected since rutabaga has a low level of cAMP available. Because of the differential expression of habituation and fatigue in these mutant strains, Corfas and Dudai (1990) concluded that the cellular fatigue exhibited by the sensory neuron does not have a simple, direct relationship  9 with the behavioral habituation of the bristle-cleaning reflex, and that central mechanisms may be involved. In Drosophila, as with the other systems examined here, the relationships between learning processes and neural processes are rarely simple and direct. Even so, in most cases examined, a mutant strain that was isolated through a screen for abnormal olfactory conditioning exhibits some form of abnormal habituation as well. The continued analysis of the genetics of habituation would be enhanced by precise descriptions of the learning processes and the neural pathways that different learning paradigms share and ways in which they are unlike each other. For example, the disparate effects of dunce and rutabaga on various forms of habituation may be influenced by the state of the organism and how it affects that particular form of habituation. In summary, habituation is a highly significant and commonly observed form of learning. Despite its ubiquitous nature, the cellular processes underlying habituation are not yet well understood. It may be that investigations focused on the processes underlying long-term memory for habituation will help to elucidate the common elements of the processes that underlie habituation. Long-term habituation as a form of memory Long-term habituation has been demonstrated in a range of organisms and responses, such as the acoustic startle response and the response to spatial novelty in rats (Leaton & Supple, 1991; Cerbone & Sadile, 1994), the gill-withdrawal reflex in Aplysia (Carew, Pinsker & Kandel, 1972), the response of PC 12 cells (cultured rat pheochromocytoma cells) to ATP (Cheever & Koshland, 1992), and the proboscis extension reflex in the honeybee (Bicker & Hähnlein, 1994). Groves and Thompson (1970) define long-term habituation as the progressively greater habituation observed with repeated habituation training. In work with Aplysia and other  10 invertebrates, memory for training lasting at least 24 h is considered a characteristic of longterm memory for various types of learning including habituation (Carew, Pinsker & Kandel, 1972; Carew & Kandel, 1973; Tully, Preat, Boyton & Del Vecchio, 1994), although there are some examples of consolidated memory that last less than 12 h (Tully et aL, 1994; Tempel, Bonini, Dawson & Quinn, 1983; Ghirardi, Montarolo & Kandel, 1995). Long-term habituation has been observed as a decrease in the magnitude of the initial stimulus of a habituation run and as a more rapid rate of habituation during the retention test (e.g. long-term habituation was measured as a decrease in overall responsiveness, Carew, Pinsker & Kandel, 1972; increased rate of habituation, Cheever & Koshland, 1992; decrease in initial response and overall responsiveness, Leaton & Supple, 1991; increase in the rate of habituation and a decrease in response level, Lozado, Romano & Maldonado, 1990; decrease in the number of stimuli required for habituation, Bicker & Hahnlein, 1994; decrease in the overall responsiveness, Cerbone & Sadile, 1994). Long-term habituation may occur in the same cells or structures that are involved in short-term habituation or it may involve additional cells or structures. Likewise, it may involve the same cellular processes as short-term habituation, or it may include novel ones. Long-term habituation of the acoustic startle response in the rat appears to be mediated by the cortex and nuclei of the cerebellum, as lesions to these areas block the formation of long-term habituation of that response without disrupting short-term habituation, while lesions to other areas of the cerebellum did not disrupt long-term habituation of the acoustic startle response (Leaton & Supple, 1991). In addition, long-term habituation of the lick suppression response was not affected by the lesions to the medial cerebellum, so it appears that only processes associated with the formation of long-term memory for habituation of acoustic startle are localized there (Leaton & Supple, 1991).  11 Because short-term habituation of the acoustic startle reflex was unaffected by medial cerebellar lesions, Leaton and Supple (1991) suggested that the processes mediating long-term habituation of this response are separate from the processes that mediate the response itself. However, the finding that long-term habituation of the lick-suppression response was not affected by the lesions that disrupted long-term habituation of the acoustic startle response indicates that there is stimulus-specificity in the processes that mediate this form of long-term habituation (Leaton & Supple, 1991). It has also been found that lesions to the mesencephalic reticular formation block long-term habituation without disrupting short-term habituation, suggesting that this structure also plays a part in the circuit that mediates long-term memory for habituation of acoustic startle (Jordan, 1989). As with habituation during training, the internal state of the animal may contribute to long-term memory for habituation. Long-term habituation of the exploratory response to spatial novelty in the rat appears to be strongly influenced by factors that alter the internal state of the animal, such as wakefulness, stimulants, tranquilizers and strain differences in vasopressin levels (Cerbone & Sadile, 1994; Sadile, Cerbone & Cioffi, 1981). Stimulation of the medial forebrain bundle, a manipulation that enhances learning in some active and passive avoidance paradigms (Huston, Mueller & Mondadori, 1977; Mondadori, Huston, Ornstein & Waser, 1976), disrupts the formation of long-term habituation (Cerbone & Sadile, 1994). Cerbone and Sadile (1994) suggested that long-term habituation is the product of the interaction of subcortical structures which supply excitation and cortical structures which supply inhibition. The cellular processes underlying long-term memory may involve lasting changes in the state of a circuit or the activity of a structure (Dudai, 1988). There is evidence for responses at the level of the genome accompanying induction of long-term habituation of the response to spatial novelty (Cerbone & Sadile, 1994). Cycloheximide, a protein synthesis inhibitor,  12 blocked long-term habituation (Cerbone & Sadile, 1994). Arousal and habituation to novelty were accompanied by distributed changes in the expression of c-fos and c-jun immunoreactivity in the brain, possibly reflecting a change in gene expression induced by these altered internal states (Papa, Pellicano, Welzl & Sadile, 1993). Exposure to novelty altered the distribution of unscheduled DNA synthesis, which is thought to underlie DNA remodeling (Sadile et al., 1995). Taken together, the work on the habituation to spatial novelty in rats provides evidence that the internal state of the organism is capable of influencing molecular events, and thus may help mediate long-term habituation (Cerbone & Sadile, 1994). Long-term habituation may involve a number of neural structures in most systems, but it may be the case that the cellular processes underlying long-term habituation may be contained within individual cells as well. Cultured, neuronally differentiated PC12 cells from the rat respond with neurosecretory responses to the application of acetyicholine or ATP, and with repeated stimulation will exhibit response decrement with many of the characteristics of habituation (McFadden & Koshland, 1990a; McFadden & Koshland, 1990b). Cheever and Koshland (1992) found that with repeated training, the rate of response decrement increased. This increase in rate was dependent on the number of stimuli received, and was retained for at least 90 mm. It would be interesting to examine the interaction between the interstimulus interval, and retention of response decrement as evidenced by an increased rate of habituation with repeated training sessions. Although the plasticity expressed in this preparation cannot be viewed as a form of learning, as the sensitivity of spontaneous recovery from response decrement to ISI and dishabituation of the decremented response have not yet been described, it may be a useful tool in understanding what cellular events may accompany habituation in intact systems. The long-term memory for habituation training in Aplysia has been studied at both the behavioral and cellular levels (e.g.. Carew et al., 1972; Montarolo, Kandel & Schacher, 1987;  13 Bailey & Chen, 1983, 1988a, b). Carew et al. (1972) demonstrated that Aplysia is capable of long-term retention of habituation of two defensive withdrawal responses, the siphon withdrawal response and the gill withdrawal response, and demonstrated that the retention of long-term habituation lasts at least three weeks. Furthermore, they showed that long-term retention of habituation in the siphon withdrawal response was greater after distributed training than after massed training. Distributed training was given over a series of four days, while massed training was given on a single day. Distributed training contributed to better retention on the first test, one day later, and better retention after one week. In addition, Carew and Kandel (1973) showed that distributed training with blocks of training separated by only 1.5 hours rather than by entire days was also effective at producing long-term habituation. The study of long-term memory in Aplysia has led to an examination of the physiological changes in the cells and morphological changes at the synapse (Castellucci, Carew & Kandel, 1978; Bailey & Chen, 1983). Castellucci et al. (1978) showed that the incidence of excitatory postsynaptic potentials of the sensory neuron motor neuron synapse -  decreased significantly with long-term habituation. Bailey and Chen (1983) used electron microscopy to survey the ultrastructure of the siphon sensory neuron synapses in animals in which long-term memory for habituation and sensitization had been induced. They found that the number and size of presynaptic active zones and the complement of vesicles increased with long-term sensitization and decreased with long-term habituation. In view of the demonstrations that polysynaptic pathways play a role in the gillwithdrawal and the tail-withdrawal circuit, it is important to consider the possibility that longterm memory for plasticity expressed in the gill-withdrawal reflex may be mediated by cells other than the stimulated sensory neurons. In support of this possibility is the finding that longterm homosynaptic depression may not be demonstrable in cell culture, while heterosynaptic depression was successfully induced by the application of FMFRamide (Montarolo, Kandel &  14 Schacher, 1987). Work has continued to characterize long-term memory for sensitization in Aplysia. However, little further progress in the investigation of the processes underlying longterm habituation has occurred. In summary, a number of manipulations that affect the expression of long-term habituation have been explored in various systems; however, there is as yet no consensus on the loci or mechanisms of long-term habituation. In the rat, long-term habituation of the acoustic startle response can be abolished with a lesion to the medial cerebellum without disrupting short-term habituation of the acoustic startle response or long-term habituation of the lick-suppression response (Leaton & Supple, 1991). Also in the rat, long-term habituation of the exploratory response to spatial novelty was affected by agents affecting the arousal level of the organism and depressing activity in the forebrain (Cerbone & Sadile, 1994). In addition, there is evidence that events at the level of the genome may be triggered by changes in the internal state of arousal or habituation of the organism (Cerbone & Sadile, 1994). A simple model of habituation with cultured neuronally differentiated PC12 cells from the rat may help elucidate the activity of individual cells during habituation and long-term habituation (Cheever & Koshland, 1994). Finally, work with Aplysia on long-term habituation of the gillwithdrawal reflex has demonstrated that uhrastructural changes at the sensory neuron synapse accompany the induction of long-term memory for habituation (Bailey & Chen, 1983). Although a number of manipulations which block the consolidation of long-term habituation in the preparations have been discussed above, as yet little is known about the specific time during which memory consolidation for long-term habituation occurs. If critical periods during which memory consolidation was vulnerable were known, the roles of changes in gene expression or protein synthesis during those periods in the mediation of long-term habituation could be probed.  15 C. elegans as a simple model system of learning The purpose of the experiments proposed here is to examine the characteristics of a form of long-term habituation in the free-living (i.e. nonparasitic) nematode C. elegans. in order to investigate the processes proposed to underlie learning and memory in a new system and search for insights into these processes (see Figure 1 A). C. elegans has been successfully used as a model for the genetic control of development (Wood, 1988), and the extensive background of information on the organism’s biology make it an excellent candidate for the investigation of cellular and molecular mechanisms of learning and memory (Rankin, Beck & Chiba, 1990). Compared to the nervous systems of other organisms studied, the nervous system of C. elegans is extremely simple; all 302 neurons and their cell lineages have been identified, and the connectivity of all the neurons is known (White et al., 1986; Hall & Russell, 1991). Despite this simplicity, C. elegans has a rich behavioral repertoire and is capable of a variety of forms of behavioral plasticity (Bargmann, 1993; Rankin et al., 1990). Short-term habituation has been studied in the tap withdrawal reflex in C. elegans. The tap-withdrawal reflex is a set of behavioral responses that are evoked by vibrational stimuli (Ranicin, Beck & Chiba, 1990; see Figure 1 B and C for apparatus). This behavioral reflex is mediated by the tap-withdrawal circuit in the nervous system of C. elegans; this circuit has been functionally described through lesion studies (for a diagram of the tap-withdrawal circuit, see Figure 2; Wicks & Ranicin, 1995). The tap-withdrawal circuit is composed of seven mechanosensory neurons, nine interneurons, and a pooi of 60 motorneurons that control the locomotion of the worm (Wicks & Rankin, 1995). The tap-withdrawal circuit can be separated into two functional circuits that drive forward and backward swimming (Wicks & Rankin, 1995). A direct touch to the tail of the animal consistently evokes an acceleration in forward swimming; a direct touch to the head consistently evokes a reversal response, in which the  16  Figure 1. A. The nematode Caenorhabditis elegans. B. The behavioral apparatus. The worm rests on an agar-filed plate. Behavioral observations are made through the stereomicroscope. Behavioral responses are recorded on video-tape for future scoring. C. Vibrational stimuli are given by a mechanical tapper with an electromagnetic relay controlled by an S88 generator. (Adapted from Rankin, Beck & Chiba, 1990.)  17  A pharynx Intestine  tall  head eggs anus  0.1 mm  B  Grass S-88 stimulus generator  C tap  Pet r I plate holder  18 worm stops, swims tail-first or backward for a distance and then turns to resume forward swimming (Chalfie & Sulston, 1981). The tap is a vibrational stimulus that can evoke accelerations or reversals, and thus must activate both forward- and backward-swimming subcircuits. These two subcircuits of the tapwithdrawal circuit appear to functionally inhibit one another, so that the locomotory behavior emitted is the interaction of the activity of both subcircuits (Ranlcin, 1991; Wicks & Rankin, 1995). Making use of the worm’s transparent body structure and the knowledge of the somatic cell lineage, it is possible in  .  elegans to lesion individual neurons through laser microsurgery  early in development, and then test the behavior of these worms at maturity (Sulston & White, 1980). When the posterior mechanosensory neurons are ablated early in development, worms move normally at maturity but are insensitive to direct touches to the tail (Chalfie & Sulston, 1981). Worms with these neurons ablated responded to the vibrational stimulation of the tap with only backward swimming or reversal responses (Wicks & Rankin, 1995). The reversal responses from worms in which the posterior mechanosensory neurons were ablated were significantly larger in magnitude than those elicited from control worms by the same tap stimulus (Wicks & Rankin, 1995). The greater magnitude of the reversal responses in worms that have received lesions to the posterior mechanosensory cells indicates that with the loss of the acceleration response, a source of inhibition had been removed from the circuit driving the reversal response. Likewise, when the anterior mechanosensory neurons are ablated, worms are insensitive to touch to the area behind to phaiynx (Chalfie & Sulston, 1981). In worms with these neurons ablated, the tap stimulus evoked only accelerations (Wicks & Ranldn, 1995). A computational model based on the known anatomical structure of the tap-withdrawal circuit has been developed to confirm that the known configuration of neurons can indeed support this set of competing responses to tap (see Figure 2; Wicks, Roehrig & Rankin, unpublished observations).  19  Figure 2. The simplified tap-withdrawal circuit. The hypothesized circuit that mediates the nematode tap withdrawal reflex consists of seven sensory neurons (squares), nine interneurons (circles), and two motoneuron pools (not shown) that produce forward and backward locomotion (triangles). All cells represent bilateral classes of cells except AVM and DVA, which are single cells. Chemical connections are represented by arrows; the width of the arrows is proportional to the number of synaptic contacts as determined in electron microscopic studies of the  .  elegans nervous system (White et al., 1986). Gap  junctions are indicated by dashed lines. Putative excitatory (green) and inhibitory (red) chemical connections are indicated, based on anatomical work mentioned above and a computational model of the circuit (Wicks, Roehrig & Rankin, unpublished observations). This circuit has been simplified for ease of presentation in two ways. First, the bilateral symmetry of the circuit has been collapsed, and second, only connections with an average of greater than five synapses are shown. (Adapted from Wicks and Rankin, 1995.)  20  The Tap Withdrawal Circuit  —  --  —  —  --  Chcmica  lflhibitory  Electrical  Excitatory  21 The competitive relationship between the acceleration response and reversal response to tap brings one to the question of whether the analysis of just the reversal response, as in these experiments, is an adequate assay of habituation of the tap-withdrawal reflex. To answer this question, it is important to understand the relationship between the competing responses during habituation. In normal worms, accelerations do not occur at a high enough frequency during habituation to make an analysis of the habituation of the magnitude of accelerations useful (Rankin, Beck & Chiba, 1990). However, through habituation studies of worms that have been subject to laser ablations of mechanosensory neurons such that they emit only reversal responses or only acceleration responses, the habituation of the two subcircuits may be analyzed independently (Wicks & Rankin, unpublished observations). The two competing responses of the tap withdrawal circuit do, in fact, exhibit different dynamics during habituation. In worms with only posterior touch receptors, the acceleration response did exhibit habituation, but very slowly and with evidence of an early facilitation at short ISIs. In worms with only anterior touch receptors, the reversal response habituated more slowly than in unaltered controls. When summated, the dynamics of these two competitive responses approximate the dynamics of habituation exhibited in the reversal response to tap in normal worms (Wicks & Rankin, unpublished observations). Thus, an analysis of the habituation of the reversal response, the predominant response during habituation training in normal worms, actually reflects the habituation of both responses to the tap-withdrawal reflex. Habituation of the reversal response in C. elegans exhibits many of the characteristics of habituation described by Groves and Thompson (1970), including dishabituation, spontaneous recovery, and sensitivity to interstimulus interval (1ST). A major finding in our work is that interstimulus interval has important effects on the dynamics of a number of components of habituation, affecting the rate of habituation, depth of habituation, and the rate of spontaneous recovery from habituation (Broster & Rankin, 1994; Rankin & Broster, 1992).  22 Long ISIs (e.g. 60-s 1ST) lead to slower, shallower response decrement than short ISIs (e.g. 10-s ISI), but also lead to slower recovery from habituation (Rankin & Broster, 1992). The slower recovery exhibited after training with a long ISI suggests that short-term retention of habituation is better with longer ISIs. Long-term habituation in C. elegans In addition to short-term habituation, Rankin et al. (1990) showed that habituation training could be retained and affect behavior up to 24 hours after the training, a signfficant length of time in an organism whose reproductive cycle is three days. This fmding demonstrated that  .  elegans is capable of a form of long-term memory, long-term habituation.  To demonstrate long-term habituation, Rankin et al. (1990) used a distributed training procedure in which the experimental group received 100 stimuli in five blocks of 20 stimuli at a 10-s 151 separated by periods of rest on Day 1, while the control group received only one block of 20 stimuli. A test block of 20 stimuli was given on Day 2. The Day 2 performance of each group was compared to its Day 1 performance; the experimental group, receiving 100 stimuli, showed a significant decrease from Day 1 to Day 2 while the control group, receiving 20 stimuli, did not. This decrease on Day 2 exhibited by the experimental group was considered evidence of long-term habituation. Attempts to replicate these results using a variety of procedures have met with mixed success  —  in some experiments long-term habituation was  seen, and in others it was not (Rankin, unpublished observations). The objectives of Experiments 1 and 2 were to establish a procedure that reliably produces long-term habituation, and to examine the role of several stimulus parameters (stimulus number, stimulus grouping and interstimulus interval) in long-term habituation in C. elegans.  23 Interference as a tool to define memory consolidation Once a habituation training protocol that produces long-term retention of habituation has been established, the next step in the investigation of this form of LTM is to attempt to perturb the formation and retention of LTM so that the dynamics of these processes can be examined. Work with both vertebrates and invertebrates has suggested that protein synthesis is necessary for the cellular processes of LTM (Flood, Bennett, Orme & Jarvik, 1977; Mizumori, Rosenzweig, & Bennett, 1985; Frey, Krug, Reymann & Matthies, 1988; Davis & Squire, 1984; Montarolo, et al., 1986). Thus, protein synthesis inhibitors have been used to probe for critical periods in memory formation. The investigation of the time course of the cellular processes underlying formation of long-term memory for sensitization in Aplysia began with the demonstration that protein synthesis inhibitors administered just before training and lasting for 1 to 3 hr after training block both long-term sensitization of the gill-withdrawal response induced by repeated electric shocks to the tail in a reduced preparation and long-term facilitation in cultured cells by infusions of 5-HT (Castellucci, Blumenfeld, Goelet & Kandel, 1989; Montarolo et al., 1986). The structural changes at the synapse associated with sensitization, such as increases in the number of presynaptic active zones, the size of each zone and the vesicle complement of each zone (Bailey and Chen, 1983; 1988a, b), are also blocked by transcriptional and translational inhibitors in cell culture (Schacher, Montarolo, Kandel, Chen & Bailey, 1991), strengthening the argument that the protein synthesis inhibitors may be directly affecting memory formation. The results from experiments on memory and protein synthesis inhibitors with Aplysia have led investigators to examine the cellular processes that are altered during the period of time when protein synthesis inhibitors are effective at blocking the induction of long-term sensitization or its analogues (Castellucci, Kennedy, Kandel & Goelet, 1988; Barzilai, Kennedy, Sweatt & Kandel, 1989; Eskin, Garcia & Byrne, 1989). This work has led to the  24 identification and classification of a number of proteins, the synthesis of which alters during the induction of long-term sensitization or its analogues; these proteins have been classified by their time of induction as early, intermediate and late proteins (Barzilai et al., 1989; Castellucci et al., 1988; Kuhi, Kennedy, Barzilai & Kandel, 1992). Thus, the work with Aplvsia on the effect of protein synthesis blockers on LTM has led to the investigation of the roles of specific proteins in the formation of memory. Although in Aplysia there appears to be clear evidence for the blockage of long-term sensitization with protein synthesis blockers, not all areas of invertebrate research support the hypothesis that protein synthesis is always necessary for LTM. In the Drosophila olfactory classical conditioning paradigm, a number of treatments have been used to distinguish between different types of memory. Anesthetic in the form of cold shock (4 °C for 1 to 2 mm) within 30 mm of training disrupted memory, but did not disrupt memory if administered after 2 h (Tempel, Bonini, Dawson & Quinn, 1983; Tully et al., 1994). However, cycloheximide administered before training such that protein synthesis was inhibited by 90 to 95% in the brain during and immediately after training failed to block the formation of anesthesia-resistant memory (lasting about 4 days) for associative training, but blocked long-term memory (lasting over 7 days) from distributed training but not massed training (Tully et al., 1994). In addition, Wittstock, Kaatz and Menzel (1993) showed that when protein synthesis is inhibited by more than 99.5% in honeybees by cycloheximide, there was still no impairment of the one-trial longterm olfactory learning exhibited by bees  —  support for the notion that there may be multiple  mechanisms for LTM, some of which require protein synthesis, others of which do not. Olfactory learning resists disruption by protein synthesis inhibitors in vertebrate systems as well (Staubli, Faraday & Lynch, 1985). Clearly, the issue of the role of protein synthesis and gene activation in learning is far from resolved.  25 One problem that has interfered with the interpretation of the results from studies examining the blocking of memory formation with protein synthesis inhibitors is that many agents that affect protein management have systemic effects; the changes in memory formation observed after treatment with the agent may be a result of these effects, not a specific failure to form LTM (Davis, Rosenzweig, Bennett & Squire, 1980; Flexner, Flexner, & Roberts, 1966; Squire & Barondes, 1974; Squire, Geller & Jarvik, 1970; Gold, 1989). In work with vertebrates, attempts have been made to disassociate the systemic effects of the protein synthesis inhibitors such as depletion of constitutive proteins in the nervous system, sickness, lethargy, etc., from the specific ability of protein synthesis inhibitors to inhibit de novo protein synthesis at the time of training (Davis, Rosenzweig, Bennett & Squire, 1980; Squire, Emanuel, Davis & Deutch, 1975; Pilcher & Booth, 1975; Segal, Squire & Barondes, 1971; Squire & Barondes, 1974). In work with Aplysia, this problem is partly dealt with by using reduced preparations or cell culture to test long-term sensitization or its analogues (Byrne, Zwartjes, Homayouni, Critz & Eskin, 1993); but with these approaches, behavioral information from the whole organism is not accessible. In addition, the studies with Aplsia on the role of protein synthesis focus on sensitization. Sensitization involves the enhancement of a response (Groves & Thompson, 1970); thus, a treatment which blocks long-term sensitization decreases the responsiveness of the organism. Although protein synthesis blockers permit short-term sensitization while preventing long-term sensitization (Castellucci et al., 1989), it is possible to imagine that the history of the treatment may have a long-term effect of depressing the organism’s responsiveness, and thus its ability to express long-term sensitization, by impeding its overall functioning. One advantage of using habituation as a learning paradigm in this context is that the successful block of LTM for habituation by an agent would result in a higher Day 2 level of responding relative to trained, untreated controls, while systemic effects from the treatment impairing the ability of the organism to function  26 normally should result in a lower Day 2 level of responding. Thus, in a habituation paradigm, it is less likely that the systemic effects of the treatment will confound a positive effect on the retention of training. The agent used to disrupt LTM in the present experiments is heat shock (32°C). The cellular response to heat shock (body temperature elevated at least 2 to 5 °C above the optimal level) was first observed in Drosophila (Schlesinger, Tissieres & Ashbumer, 1982) and has since been described in every system examined, from bacteria to humans (Schlesinger, Tissieres & Ashburner, 1982; Lindquist, 1986; Nowak, 1993). The cellular response to heat shock is also evoked by other types of cellular stress such ischemia, glucose deprivation, toxins, cold, viral infections and other damaging agents (Lindquist, 1986). Heat and these other disruptive agents cause damage in the form of the denaturation and agglutination of proteins. In response, cells stop protein synthesis of all proteins except for a class of proteins called heat shock proteins (HSPs), the production of which are massively increased. These HSPs are the most widely conserved families of proteins described and appear to play a role in damage-control (Schlesinger, Tissieres & Ashburner, 1982; Lindquist, 1986). They are classified into three loosely grouped families by molecular weight (the HSP2Os or low molecular weight HSPs, the HSP7Os or intermediate molecular weight HSPs, and the HSP9Os or high molecular weight lISPs). Some HSPs are present constitutively and are not induced by cellular stress, others are present constitutively and are also induced by cellular stress, and still others are not present constitutively and are only induced by cellular stress (Feige & Polla, 1994; Schlesinger, Tissieres & Ashburner, 1982; Lindquist, 1986). In a cell, the presence of last kind of HSPs, the strictly inducible HSPs, is an indicator of cellular stress (Nowak, 1994; Sharp & Sagar, 1994; Koroshetz & Bonventre, 1994; Stringham, Dixon, Jones & Candido, 1992; Stringham & Candido, 1993).  27 The optimal temperature range for growth and development in C. elegans is 15° to 25° C (Wood, 1988). C. elegans reacts with the production of HSPs to temperatures over 29° C (Snutch & Baillie, 1983). In C. elegans, the production of HSPs occurs in all cell types including neurons, demonstrating that all tissues are affected by the stress of the heat shock (Stringham et a!., 1992). Heat shock proteins belonging to the HSP2O, HSP70 and HSP9O families are expressed in C. elegans (Snutch & Baihie, 1983; Heschl & Baillie, 1990; Russnak & Candido, 1985; Dailey & Golomb, 1992). As little as 15 mm of exposure to 33°C results in the production of HSPs (Stringham et al., 1992). Yet after strong heat shock (33°C, for two shifts of 2 hr each), the worms are still viable; after heat shock they continue to reproduce and respond normally to tactile stimuli (Beck, unpublished observations). When observed within 2 mm after heat shock (45 mm, 32°C), worms are eating and moving in a coordinated fashion; they respond normally to tactile and vibrational stimuli, but their spontaneous behavior remains slightly lethargic for 3 to 5 mm (Beck, unpublished observations). For this reason, in the experiments presented here, an interval of 10 mm at room temperature was allowed after heat shock before habituation training resumed. The key characteristic of heat shock as an agent to disrupt LTM formation is its nonspecificity, as it both causes damage directly to cellular proteins through denaturation; and it evokes an active response from the affected cells, in the form of a reduction of general protein synthesis accompanied by the stress-induced production of HSPs (Lindquist, 1986; Morimoto, Tissieres & Georgopoulos, 1990). The number of cellular processes that might be affected by one or more of these effects of heat shock is great. This breadth of effect limits the narrowness of the assertions that may be made about the cellular processes underlying LTM based on the results of this study, but greatly increases the chance of disrupting some essential process. Importantly, heat shock can be delivered for short, defined periods of time, which permits an examination of the effects of a disrupting stimulus lasting as little as 15 min. Thus, using heat  28 shock, it is possible to examine the effects of a potentially disrupting treatment on LTM not only before, during and after training, but also to examine critical periods of memory formation more closely by shifting short duration treatments through them systematically. An additional advantage of heat shock is that its effect may be measured in more than its possible behavioral consequences. Heat shock causes the induction of heat shock proteins; this induction may be demonstrated in transgenic worms with a reporter gene after a heat shock promoter so that induction of heat shock proteins results in the induction of that reporter gene (Stringham et al., 1992). Heat shock affects the rate of egg-laying; this is a sensitive assay to the severity of the heat shock. Thus, a profile of the consequences of heat shock on the organism and on the consolidation of long-term memory may be developed. Overview of Experiments In Experiments 1 and 2, a protocol that produces long-term habituation was defined by examining factors affecting LTM for habituation in . elegans such as stimulus number, interstimulus interval, and distribution of training. In Experiments 3, the effect of heat shock on the induction of the heat shock gene hspl6 and the rate of egg-laying was examined. In Experiments 4 through 8, the effects of heat shock before, during and after habituation training on immediate habituation, short-term retention of habituation and long-term retention of habituation were investigated with the objective of defining a critical period for the consolidation of LTM for habituation. General Methods Subjects and Materials  £. elegans Bristol (N2) were maintained in 4 cm diameter Petri plates filled with 10 ml Nematode Growth Medium agar. The data from a total of 399 worms were included in the analyses of these experiments. The nematodes fed on E i (0P50), as described in Brenner (1974). At four days post-hatching (the peak of egg-laying), subjects were individually placed  29 on labeled agar-filled plates at least two hours before training and were maintained on the same plates throughout the procedure. Worms were transferred individually from the breeding plate to the labeled plates using a bent wire pick; simultaneously, small amounts of bacteria sufficient to feed each subject during the study were seeded on each plate. Stimulation and Behavioral Observations Observations were made through a stereomicroscope with attached videorecording equipment (see Figure 1B; Wild M3Z, Wild Zeiss Canada; Panasonic Digital 5100 camera;  Panasonic AG 1960 VCR; NEC monitor). A time-date generator was used to superimpose the experimental time, time of day and date on the video record (Panasonic WJ-810). The vibrational stimulus used in this work was a 6 Hz train of six taps delivered to the side of the Petri plate holding the subject (see Figure 1C). Each tap exerted approximately 1 2 N of force -  on the plate. The stimuli were produced by a mechanical tapper with an electromagnetic relay triggered by a Grass S88 stimulus generator controlled by the experimenter. The dependent measure used in this work was a measure of the magnitude of the reversal response to tap, in which the worm, either lying still or swimming forward, responds to a vibrational stimulus by initiating tail-first or backward swimming for a distance. The distance traveled during the reversal response can be quantified using stop-frame video analysis and computer-driven digitizing equipment (Macintosh computer; Bit Pad Plus digitizing tablet; Macmeasure software). Habituation Training Procedure In Experiments 1 and 2, worms were trained using one of four different training procedures on Day 1 (4 days post-hatching) and tested on Day 2 (5 days post-hatching). The four Day 1 training groups were: (a) a distributed training group that received 60 stimuli in three blocks of twenty stimuli with one hour rest periods between blocks of training, (b) a massed training group that received 60 stimuli together, (c) a twenty stimuli control group that  30 received 20 stimuli together, and (d) a single stimulus control group that received only one stimulus. The twenty stimuli control group was included to examine the effects of a small number of stimuli during training on long-term habituation. The single stimulus control group, that received no habituation training on Day 1, was included to control for the effects of age and handling. On Day 2, at least 24 hours (24 to 28 hours) after the end of training on Day 1, all subjects received 20 stimuli. The performance on Day 2 of the single stimulus control group was compared with the performance on Day 1 and on Day 2 of any group that showed a lower level of responding on Day 2. Long-term habituation was evident when there was a decrease in the level of responding from Day 1 to Day 2 in a trained group and at the same time the Day 2 performance of the untrained single stimulus group was comparable to the Day 1 performance of the trained groups. The results from Experiments 1 and 2 indicated that habituation at a 60-s 1ST with 60 stimuli was the most effective at producing LTM for habituation, and that with an inspection of Figure 4, distributed habituation training with 60 stimuli appeared slightly more effective than massed habituation training. Therefore, in Experiments 4 through 8, which involved heat shock, the 60-s 1ST distributed habituation protocol and the single stimulus control protocol were used. Heat shock Heat shock (32°+I- 1°C), used in Experiments 4 through 8, was delivered by immersing the plate sealed with Parafilm in which the worm rested in a temperature controlled water bath. When measured with a probe thermometer, the temperature of the agar when a plate was submerged in the water bath reached 32 ° C in about 2 mm. When removed from the bath and returned to a room-temperature environment, the temperture of the agar returned to room temperature in about 30 s. Tn different experiments, heat shock was given before training,  31 during training or after training on Day 2. When heat shock was delivered on Day 1 during training, a minimum of 10 mm was allowed after the end of heat shock exposure before training was resumed. In Experiment 3, the effects of the heat shock treatments used in the subsequent behavioral experiments were examined in two contexts. In Experiment 3A the induction of hspl6, the gene of a low molecular-weight heat shock protein HSP16, by the heat shock treatments used in the subsequent experiments was tested. In Experiment 3B, the effect of these heat shock treatments on the rate of egg-laying, a sensitive and easily quantifiable assay, was tested. Scoring and statistical analyses The statistical package, SPSS (Version 4.0.5 Macintosh), was used for these analyses. Planned comparisons were done by hand. General analyses. In general, ANOVAs were employed to test for differences between the means of groups and different stages of habituation (see below for a description of the application of ANOVAs to the analysis of short- and long-term habituation). The alpha level was set at .05. The error rate of each analysis was controlled by dividing the alpha level by the number of comparisons within the analysis; for example, in the examination of long-term habituation in Experiments 1 and 2, the asymmetry of the design and experimental hypotheses made the application of two ANOVAs, one at each level of day of training, appropriate in the analysis of long-term habituation. The alpha level of these ANOVAs was reduced: .05 /2 = .025. Between-subject, within-subject and mixed-design ANOVAs were employed as appropriate. When a repeated-measures design was used, Mauchly’s test for sphericity, which examines the validity of the assumption of sphericity in the repeated-measures data, was done (Lindman, 1991). When a significant result from this test indicated that the assumption of the  32 homogeneity of variance and covariance was violated, the degrees of freedom of the corresponding E-test were adjusted downward using the Huynh-Feldt epsilon generated by sPss. Planned comparisons focusing on contrasts that have significance in the context of the study were employed when the overall ANOVA reached significance. When multiple planned comparisons were used in one analysis, the alpha level was reduced by dividing by the number of comparisons (Lindman, 1991). As planned comparisons are not robust to violations of the homogeneity of variance, Bartlett’s test of homogeneity of variance was applied across between-subjects factors (Lindman, 1991). When the test indicated that the assumption of homogeneity was violated, the error term of the E ratio for that planned comparison, which would nonnally be the error term representing the variance from the entire analysis, was replaced with the error term reflecting only the groups being compared (Lindman, 1991). if, in a repeated measures design, Mauchly’s test of sphericity indicated that the assumption of the homogeneity of variance and covariance was violated, planned comparisons were not employed, as planned comparisons are not robust to violations of this assumption. The results of Bartlett’s test of the homogeneity of variance and Mauchly’s test for sphericity are only reported here when they are significant. The measurement of effect size. An estimate of effect size (ES) which is independent of the units of measurement used in the dependent measure may be calculated from the information in an ANOVA table with the following formula: effect size (ES)  ((MSeffect I  fl)  / MS error)  1/2  As can be seen, the effect size has a direct relationship with the value of the f test. F  =  2 (MSeffect / (MS error) = ES  (fl)  33 The countemull value of the observed mean and the interpretation of statistical significance. When the contrast of two means is not statistically significant (for example, if alpha is set at .05, and p> .05), the common practice is to infer that the experimental manipulation had no effect at all on the group receiving treatment. Likewise, when the contrast between two means is significantly different, the common practice is to assume that the statistical significance automatically signals scientific importance (Rosenthal & Rubin, 1994). Both of these assumptions may be invalid in some situations. A consideration of the counternull value of the observed mean, a statistic which corresponds to the value of the mean for which there is as much evidence as the mean of the null hypothesis, but on the opposite side of the observed mean, may help clarify these situations (Rosenthal & Rubin, 1994). In Figure 3A, the distribution of the mean under the null hypothesis is shown. The value of the obtained mean corresponds to the probability of finding that mean on the distribution of the expected mean, which is determined by the null hypothesis. However, there is just as much evidence (the same probability) for a mean with twice the effect size of the observed mean, assuming the expected effect size of the mean of the null hypothesis is zero. As can be seen in Figure 3B, the observed mean corresponds to the same probability regarding a distribution the mean of which is symmetrical and in the opposite direction from the mean of the null hypothesis (Rosenthal & Rubin, 1994). The mean of this symmetric but opposite distribution is the counternull value of the observed mean (Rosenthal & Rubin, 1994). A consideration of the countemull value of the observed mean may be helpful in two situations. First, when the observed mean is not found to be statistically significant, the counternull value, for which there is as much evidence as the expected mean of the null hypothesis, may be considered; if the effect size of the countemull value would be significant there is no justification for saying conclusively that the experimental manipulation had nj effect on dependent measure. In other words, considering the countemull value of the observed  34  Figure 3. The counternull value of the obtained effect size. In Fig. 3A, the distribution corresponding to the null hypothesis with the expected mean is shown; the probability of the obtained mean is the area under the curve away from the center of the distribution (shaded area). In Fig. 3B, the obtained mean has the same probability regarding the distribution of the counternull mean; again the probability of the obtained mean is the area under the curve away from the center of the distribution.  35  X obtained  X expected  X expected  X obtained  X counternull  For example: ES expected  =  0  ES obtained  =  .68  ES counternull  =  2 (ES obtained  =  2 (.68)  =  1.36  -  0  -  ES expected)  36 mean may prevent the misinterpretation of a failure to observe significance as incontrovertible evidence for a lack of a relationship between the experimental manipulation and the result (Rosenthal & Rubin, 1994). A second situation in which the counternull value of the observed mean may be helpful in the interpretation of statistical results, is in judging the scientific significance of a difference  between the expected mean and the observed mean. The counternull mean corresponds to the value of mean when the effect size is doubled (see Figure 3B). If the difference between the observed mean and the counternull mean is not big enough to be of scientific importance in the context of the work, then a statistically significant difference between the expected mean and the observed mean probably does not correspond to a scientifically important difference either (Rosenthal & Rubin, 1994). The countemull value is not a test of significance, but rather an aid to the interpretation of statistical results and their importance in the context of the work (Rosenthal & Rubin, 1994). The counternull value of an obtained effect size will be considered here when the interpretation of a fmding is in doubt or is of special theoretical significance. Scoring of responses. Reversal responses were quantified by measuring the distance traveled backward in response to the tap stimulus. A pause in forward swimming was scored as a reversal response of zero, as was no response to the tap stimulus. Increases in forward swimming immediately following the tap (accelerations), less than 30% of the responses, were considered missing data since the magnitude of accelerations cannot be directly compared with the magnitude of reversal responses. In Appendix 1, the significance of the distribution of missing values to the interpretation of the habituation data is explored in Experiments 1 and 2 and 4 through 8. Treatment of data. Differences in performance during habituation training were examined through analyses of the response magnitudes averaged over blocks of either five or  37 twenty stimuli. Obtaining these means decreased some the variability exhibited in these data, and helped deal with the missing data points. The means of responses were calculated for every subject and then used in the analyses. The comparisons of the mean block magnitudes were used in the analyses of habituation, short-term retention of habituation and long-term retention of habituation. Analysis of short-term habituation. Short-term habituation was examined during habituation training on Day 1. The mean of five responses is more sensitive to short-term changes in the levels of responding than the mean of twenty responses, so it was used as the dependent measure. The effects of different types of training on habituation during training on Day 1 were compared across the trained groups in Experiments 1 and 2 with a mixed-design ANOVA; the effect of training was measured by comparing the mean of the first five responses to the mean of the last five responses. A significant decrease from the initial response level to the final response level on Day 1 was considered evidence of short-term habituation. The nature of the distributed habituation training protocols used in Experiments 1 and 2 made it appropriate to expand the analysis of short-term habituation of the distributed training groups. As this approach was used in the analyses of Experiments 4 through 6 as well as Experiments 1 and 2, it will be described here. In distributed training, stimuli are delivered in three blocks of twenty stimuli with rest intervals between them. A two-way repeated-measures ANOVA with Block (3 levels: blocks 1, 2 and 3) and Training (2 levels: the initial mean of five responses, INIT, and the final mean of five responses, HAB, in each block) was employed. Thus, habituation within blocks (a significant main effect of Training, in which the mean of the initial five responses is higher than the mean of the fmal five responses), the short-term retention of habituation between blocks (a significant main effect of Block, in which the levels of responding decreased across blocks), and differences in the habituation expressed in each block (a significant Training x Block interaction) were examined through this analysis.  38 ant in the context Because the expression of spontaneous recovery from habituation was import of last five responses of of the work, a planned comparison was employed to contrast the mean se with Blocks 2 and 3. Block 1 with the mean of the first five responses of Block 2 and likewi in more than In Experiments 4, 6,7 and 8, the distributed training procedure was used was of particular one group; in Experiments 4 and 6, the expression of short-term habituation groups with a interest. In these experiments, short-term habituation was compared across ry from mixed-design ANOVA (Block x Training x Group); again spontaneous recove risons. habituation between blocks of training was examined with planned compa was defmed as a Analysis of long-term habituation. Long-term retention of habituation In the analysis of longsignificant decrease in the level of responding from Day 1 to Day 2. measure as it is less term memory, the mean of twenty responses was used as the dependent erm habituation. For variable than the mean of five responses used in the analysis of short-t 1 and the twenty each worm, the mean of the responses to the first twenty stimuli on Day l groups that received stimuli on Day 2 were calculated. In Experiments 1, 2, 5 and 7, contro ng effects. Worms in one stimulus on Day 1 were included to control age, treatment, or handli twenty responses these groups received habituation training only on Day 2, thus the mean of two ANOVAs were on Day 2 was calculated for these subjects. In Experiments 1, 2, 5 and 7, employed at the different levels of day (Day 1 and Day 2). experimental In Experiments 4, 6 and 8, no untrained control group was included in the groups with a designs. In these experiments, long-term habituation was analyzed across 1 and Day 2 were mixed-design ANOVA (Day x Group). The level of responding on Day contrasted using planned comparisons. the data from Experimental design affected the particular planned comparisons done on in the methods and results Day 2 in all experiments. These planned comparisons are discussed sections of each experiment.  39 Experiment 1 Short- and long-term habituation at a short ISI (10-s 151) To clarify some of the inconsistent findings from a variety of 10-s 1ST long-term habituation experiments with different handling and training procedures (Rankin, unpublished observations), both short and long-term habituation after training at a 10-s 1ST with massed and distributed procedures was examined. Methods Subjects and Materials. Worms were maintained as described in the general methods. Twenty-one subjects were run in each of four conditions for a total of 84 subjects, and the data from 83 subjects were used in the analyses (one subject was dropped because of a variety of technical errors such as failure to record trials or equipment difficulties). The use of the apparatus, behavioral observations and scoring of behavioral responses were performed as described in the general methods section. Procedure. The Day 1 training procedures for the four groups are described in the general methods (distributed training, ),  21; massed training, j], = 21; twenty stimuli control  training, j, = 21; and single stimulus control, n = 20. Testing was 24 28 hr later on Day 1. -  Statistics. The analysis of short-term habituation and long-term habituation was performed in the same manner for Experiment 1 (10-s ISI) and Experiment 2 (60-s ISI). The analysis of short-term habituation, using means of the first five responses and last five responses as the measure, was performed as described in the general methods. A two-way, mixed design ANOVA (Training x Group; alpha = .025) compared the initial response level and final response levels of the trained groups. A two-way, repeated-measures ANOVA (Block x Training; alpha = .025) compared the initial and final level of responding across blocks of training in the distributed training procedure. Mauchly’s test of sphericity indicated that the assumption of homogeneity of variances and covariances was violated (Mauchly’s sphericity  40 test, chi-square approximation = 7.17,  =  .03), so planned comparisons examining recovery  from habituation were not performed, and the degrees of freedom of the relevant E tests were adjusted with the Huynh-Feldt epsilon. Long-term habituation, using means of the first twenty stimuli on Day 1 and the twenty stimuli on Day 2, was analyzed with pair of ANOVAs (each with an alpha = .025), one on each level of Day, and were followed with planned comparisons designed to test specific hypotheses. A planned comparison was used to compare the Day 2 response level of single stimulus control subjects to the average response level of the trained subjects on Day 1 (alpha = .025). This contrast was done to examine whether the untrained subjects of the single stimulus control group were responding on Day 2 at the same level as the other groups at the beginning of training on Day 1 as would be expected if age and handling did not affect the responding of the subjects of the single stimulus group. To examine the effects of training, the average of the Day 2 level of responding of the distributed and massed training groups was compared to the Day 2 level of responding of the single stimulus controls (alpha  =  .0 1). Long-term habituation may be expressed as a  significantly lower response level on Day 2 in the distributed and massed group than the single stimulus control group. To examine the effects of the different training procedures, the Day 2 response of the distributed and massed training groups were contrasted with each other (alpha =  .01).  Results Habituation 10-s 1ST. Short-term habituation was examined across groups comparing the mean of the initial five responses with the mean of the final five responses on Day 1. Distributed training (Fig. 4A), massed training (Fig. 4B) and twenty stimuli control training (Fig. 4C) all produced significant short-term habituation on Day 1 (mixed-design Group x Training ANOVA (alpha  =  .05 I 2  =  .025): Training: E(1, 55)  =  173.85,  <  .01). The  41  Figure 4. Habituation curves at a 10-s 1ST. The mean response magnitude (mm) to each stimulus given during training on Day 1 and testing on Day 2 is shown for subjects receiving distributed training (Fig. 4A; j = 21; 60 stimuli in three blocks of 20 stimuli with one hour rests between blocks), massed training (Fig. 4B;  n = 21; 60 stimuli  consecutively), and twenty stimuli control training (Fig. 4C; consecutively).  n = 21; 20 stimuli  42  DAY2  DAY1 A 3u-I-—  cnE  2.5-  ZE  b  i.::  1• 0.5  0  20  40  21  41  60  0  20  STIMULI B 3LLJ  ZE  25-  1•  STIMULI C 3Ui—  øE  2.5  ZE 21.:1W  0.50-  ST I MU LI  L  43 groups did not differ from each other in this measure of the expression of short-term habituation (Group: E(2, 55)  =  .02,  n& Group x Training: E(2,  .41,  55)  a).  However, it is worth noting that the lack of difference observed between the three procedures may have been produced by a floor effect, as for all groups the mean block magnitude was close to zero by the end of the training runs (mean block magnitude (in mm) +1SE to the last five stimuli in distributed training: 0.20 +1- .07; massed training: .09 +1- .03, twenty stimulus control: 0.20 +I .05). To examine habituation duing distributed training on Day 1, the means of the initial five responses and final five responses of each block were compared in a two-way repeatedmeasure Block x Training ANOVA (alpha blocks overall (Training: (1, 12)  =  9.39,  = =  .025). There was significant habituation within .01), and this habituation did not vary  significantly between blocks (Training x Block: E( 2, 24)  =  .64, n). Interestingly there was  evidence for short-term retention of habituation; the level of responding within blocks exhibited a significant decrease during training on Day 1 (Block: E(1.46, 17.5)  =  5.16,  =  .02; degrees  of freedom adjusted with the Huynh-Feldt epsilon). As the Mauchly test for sphericity (Training x Block: chi-square approximation = 7.17,  =  .03; Huynh-Feldt epsilon = .73)  indicated that the assumption of the homogeneity of variances and covariances had been violated, planned comparisons examining recovery from habituation between blocks of training were not carried out. However, an inspection of Figure 4A indicates that there appeared to be recovery from habituation between Blocks 1 and 2 and Blocks 2 and 3. Long-term retention of habituation 10-s 1ST. Retention of habituation training should be evident as a lower level of responding in trained groups than the untrained control group on Day 2, while the level of responding on Day 1 of the trained groups should be the same as the level of responding on Day 2 of the untrained control group. Two factorial ANOVAs across groups on the level of response as measured by the mean of the first twenty responses on Day  44 1 and the twenty responses on Day 2, including the single stimulus control group were calculated, one at each level of Day (Day I and Day 2; alpha  .05 / 2 = .025).  No effect of group was evident on Day 1 or Day 2 (Day 1: E(3, 79) 2: E(3, 79)  =  10.2, p  =  <  2.39, n; Day  2.55, n), although the counternull values of the effect sizes were significantly  different from the null effect size (Day 1, Group: obtained ES  E(3, 79)  =  9.59,  <  .01; Day 2, Group: obtained ES  =  =  .34, counternull ES  .35, counternull ES  =  .68,  .70, (3, 79)  =  .01) indicating that it should not be concluded that the training has no effect on  response levels on Day 1 and Day 2. While the lack of a significant difference between groups was expected on Day 1, the failure to see differences between the groups on Day 2 was not, as long-term habituation might be apparent as a difference between the Day 2 level of responding among groups. These results supply no evidence of long-term retention of habituation after training at a 10-s 1ST. A particular concern in the ANOVA on the Day 1 response levels of the trained groups and the Day 2 response level of the single stimulus control group was whether the Day 2 response level of the single stimulus control group was the same as the average response level of the trained groups on Day 1. A planned comparison contrasted the average Day 1 response level of the trained groups (distributed and massed training groups and the twenty stimuli control group) with the Day 2 response level of the single stimulus control group (alpha = .025). It was found that the difference between the trained groups and the single stimulus control was not significant (E(1, 79)  =  4.34,  i&).  although the counternull value of the effect  size was significant (obtained ES: .46; counternull ES: .91, F(1, 79)  =  17.18,  <  .01). An  inspection of Figure 5 suggests that the Day 2 response level of the single stimulus group may these be depressed when compared to the average of the trained groups on Day 1. Based on results, it cannot be concluded that age and handling did depress response levels on Day 2  45  Figure 5. Habituation by block at a 10-s ISI. Fig. 5 shows the mean block magnitude (mm) for Day 1 and Day 2 of each of the three groups that received training on Day 1: the distributed training group  (a = 21), massed training group ( = 21), and the twenty stimuli  control group  (a = 21), and the Day 2 mean block magnitude (mm) of the single stimulus  control group  ( = 20; error bars show +I SE).  46  3  2.5 JDAY1 U.’  (o C’1  B  2  DAY 2  1.5  1  C.)  0 -J  0.5  0  DISTRIB  MASSED TWENTY  SINGLE.  47 independent of training, but on the other hand, it cannot be concluded that age and handling did  nQi affect Day 2 response levels of the single stimulus group. From the results, it might be concluded that habituation training at a 10-s 1ST did not induce long-term habituation. However, an inspection of Figure 5, in which the Day 1 and Day 2 mean block magnitudes for the trained groups are compared with the Day 2 mean block magnitude of the single stimulus control group, suggests two possible confounding factors: a) an age and handling effect, or b) a floor effect. Age and handling effects may have depressed response levels on Day 2 in all groups, preventing the expression of long-term habituation. This would explain the failure to find a significant difference between the trained and untrained Day 2 levels of responding. Alternatively, the 10-s 151 used in these procedures may produce such strong response decrement that it is not possible to exhibit a further decrease. Both of these factors may contribute to produce the pattern of results seen here. Therefore, at a 10-s 151, there was no conclusive evidence that habituation training with 60 stimuli produced significant long-term habituation even though short-term habituation in all trained groups and retention of habituation between blocks of training on Day 1 in the distributed training group was evident. Experiment 2 Short- and long-term habituation at a long 151 (60-s 151) As discussed earlier, Rankin and Broster (1992) showed that habituation training at a long 1ST (60 s) resulted in slower spontaneous recovery from habituation than training at a short ISI (10 s). This observation led to the hypothesis that habituation training at a longer 1ST such as 60 s might also produce better long-term retention of the training than habituation at a 10-s 1ST. In addition, the shallower habituation resulting from training at a 60-s ISI may help prevent a floor effect. Here, this hypothesis is tested by examining short and long-term habituation at a 60-s 1ST.  48 Methods Subjects and Materials. Worms were maintained as described in the general methods. Twenty-one worms were run in each of four conditions for a total of 84 worms (4 worms were dropped because of a variety of technical errors so that the final total was 80 worms). The use of the apparatus, behavioral observations and scoring of behavioral responses were performed as described in the general methods. Procedure. The Day 1 training procedures for the four groups described in Experiment 1 were employed in Experiment 2 as well (distributed training, j = 21; massed training,  =  20;  twenty stimuli control training, n = 20; and single stimulus control, n = 19). Testing was at least 24 hours later on Day 2. The only procedural difference between Experiments 1 and 2 was that in Experiment 2 habituation training and testing were given at a 60-s ISI instead of a 10-s 1ST. Statistics. The statistical analysis was performed as described in the general methods and the statistics section of the methods of Experiment 1. In the analysis of short-term habituation, two planned comparisons (alpha = .01) were employed to examine recovery from habituation during the two intervals of the distributed procedure between Blocks 1 and 2 and Blocks 2 and 3 by contrasting the mean of the last five responses of one block with the mean of the first five responses of the next block. Results Habituation 60-s 151. At the 60-s 1ST, short-term habituation was again produced by all three training procedures. Comparisons of the response magnitudes of the first and last stimuli  on Day 1 in the trained groups show that the distributed (Fig. 6A), massed (Fig. 6B) and twenty stimuli control (Fig. 6C) procedures produced a significant decrease in mean response magnitude during training on Day 1 (mixed-design Group x Training ANOVA; alpha = .025; Training: F(l, 56)  =  81.64,  <.01). While the group did not affect the overall level of  49  Figure 6. Habituation curves at a 60 s 1ST. The mean response magnitude (mm) to each stimulus given during training on Day 1 and testing on Day 2 is shown for subjects receiving distributed training (Fig. 6A; n = 21; 60 stimuli in three blocks of 20 stimuli with one hour rests between blocks), massed training (Fig. 6B; n = 20; 60 stimuli consecutively), and twenty stimuli control training (Fig. 6C;n = 20; 20 stimuli consecutively).  50  —DAY2—  DAY1 A 3.  cn 2 O  2.5’ 2’  J 1’ LU<  0.5’ 0’  I  0  20  40  21  60  41  0  20  0  20  0  20  STIMULI B 3.  cnE Z 2  o  2.5  0.5 0•  0  I  I  20  40  60  STIMULI C 3.  cn 2  2.5  ZE  1’  <0  11.1<  0.5’  0’  I  0  20  STIMULI  51 responding (Group: (2, 56)  =  1.96, n), the groups did differ in the way they exhibited  habituation (Group x Training: E(2, 56)  =  5.46,  <  .01). In Figure 6, it seems clear that  habituation with fewer stimuli (the twenty stimuli control group Fig. 3C) led to less profound habituation than massed or distributed training with 60 stimuli (X +1- SE in mm; distributed training HAB: .68 +1- .11; massed training HAB: .599 +1- .09; twenty stimuli control HAB: 1.25 +1- .0 1). The habituation during distributed training was considered separately. Overall, training produced habituation during the blocks of training (two repeated-measures Block x Training ANOVA, alpha = .025; Training: E(1, 19)  =  40.79,  <  .01). There were no  significant differences in the way habituation was expressed within blocks (Block x Training: E(2, 38)  =  .7, j. However, there was evidence of short-term retention of habituation  between the blocks (Block: E(2, 38)  =  9.99,  <.01).  Spontaneous recoveiy between the blocks of training, a significant increase in response magnitude from the end of one block to the beginning of the next, was examined by planned comparisons of Block 1 HAB with Block 2 NIT, and Block 2 HAB with Block 3 INIT (alpha =  .025 I 2  =  .01). The recovery between Block 1 and Block 2 was not significant (E(1, 38)  =  3.39, n) although the value of the counternull effect size suggests that some recovery may have occurred (obtained ES  =  .40, counternull ES  =  .80, E(1, 38)  =  13.57,  <  .01).  However, there was evidence of recovery from habituation between Blocks 2 and 3 (E( 1, 38) =  14.89,  <  .01).  Comparisons of habituation on Day 1: lOs- and 60-s 151. The analyses of short-term habituation during training at 10-s and 60-s ISIs showed many similarities, though differences were also apparent. At both ISIs, there was habituation on Day 1 in all trained groups; however, at a 60-s ISI, the depth of habituation was affected by the type of training, while at a 10-s 151, it was not. This difference between ISIs is likely to be related to the rapid and profound habituation exhibited during habituation training at a 10-s 151.  52 As noted in Rankin and Broster (1992), the depth of habituation after training at a short 1ST is greater that after a long 1ST. In the present experiments, it is clear from a comparison of Figures 4 (short-term habituation at a 10-s 1ST) and 6 (short-term habituation at a 60-s 1ST) that habituation training at a short 1ST produced greater response decrement than did habituation training at a long 1ST with the same number of stimuli and the same procedures. This rapid decrement seen during training at a 10-s 1ST may make training at a 10-s ISI insensitive to particular training procedures. However, at both the 10-s and 60-s ISIs, there was evidence of retention of habituation between blocks training in the distributed, which would not be expected if the habituation produced by training at a 10-s 151 were completely insensitive to training. Long-term habituation 60-s 151. As can be seen in Figure 7, the single stimulus control group had a level of responding on Day 2 comparable to the levels of responding on Day 1 of the trained subjects (alpha  =  .025; E(3, 76)  =  1.46, j. In addition, when the levels of  responding on Day 2 were compared, the training received on Day 1 significantly affected responding (alpha  =  .025; E(3, 76)  =  3.45,  =  .02). From an examination of Figure 7, it is  apparent that distributed or massed training groups may have expressed long-term habituation, while training with twenty stimuli did not affect response levels on Day 2. In a planned comparison, the average of the Day 2 response levels of the two groups that received 60 stimuli during training, the distributed and massed training groups, was contrasted with the Day 2 response level of the single stimulus group. Subjects receiving distributed and massed training showed evidence of long-term habituation (alpha = .025 /2  =  .01; (1, 76)  =  9,195,  .005). An inspection of Figure 7 indicates that distributed training may be slightly more effective than massed training at inducing long-term habituation. However, as seen in a second planned comparison, the response level on Day 2 after distributed training was not significantly  53  Figure 7. Habituation by block at a 60-s ISI. The mean block magnitude on Day 1 and Day 2 of the three trained groups, distributed  (= 21; 60 stimuli in three blocks of 20 stimuli  with one hour rests between blocks), massed  (n = 20; 60 stimuli consecutively) and twenty  stimuli (11=20) training and on Day 2 of the single stimulus control group (fl= 19; 20 stimuli consecutively) are shown (error bars show +1- SE).  z G) r m  C,)  -I  m z  -I  m  Cl) C,)  Cl) -I  CII  p j43  Cfl  1%) C)  I43L  20 STIMULI BLOCK MAGNITUDE (mm)  L1  55 different from the Day 2 response level on Day 2 after massed training (alpha = .01; E(1,76) .72,  n). Age and handling effects did not appear to contribute to the decrease in responding seen  in the distributed training group on Day 2. Therefore, the decrease in responding in the 60-s 1ST seen in the distributed and massed training groups can be attributed to the long-term retention of habituation training received on Day 1. Comparisons of lone-term habituation 10 s- and 60-s ISIs. At a 10-s 1ST there appeared to be age and handling effects that depressed Day 2 response levels regardless of training; in addition with habituation at a 10-s 1ST, the depth of the habituation may result in a floor effect thus preventing the expression of long-term habituation (Figure 5). These effects were not evident at a 60-s 1ST (Figure 7). Long-term habituation was expressed after training at a 60-s 1ST; no evidence for long-term habituation was seen after training at a 10-s 1ST. Although this failure to express long-term habituation after training at a 10-s 151 cannot be interpreted as a failure to remember training, it is clear that habituation training at 60-s 151 would be a better procedure to use in the examination the processes underlying long-term habituation. In conclusion, these experiments have shown that number of stimuli, 1ST, and distribution of training all influence the expression of long-term memory in  .  elegans. The  distributed and massed habituation training procedures at a 60-s 1ST, taken together, were effective at producing LTM for habituation. As the selection of one procedure was required for the subsequent experiments, the distributed training procedure, which appeared from an inspection of Figure 7 to be slightly more effective than the massed training procedure at inducing long-term habituation, was chosen to be used in the experiments on the effects of heat shock on LTM that follow.  56 Experiment 3 The effects of heat shock on hspl6 induction and rate of egg-laying In the series of experiments that follow (Experiments 4 through 8), the behavioral consequences of heat shock treatments at different times in relation to training will be examined. Here, in Experiment 3, the effects of the heat-shock treatments used in those behavioral experiments on the induction of a heat-shock protein gene, hspl6, and on the rate of egg-laying, a sensitive measure of organism-wide stress, will be characterized. Experiment 3A: The effect of the heat shock treatments on hsp 16 induction It is possible to insert a segment of DNA into an organism’s genome such that when a specific gene is induced, the inserted strand is also transcribed and the resulting protein is synthesized. This type of transformation was performed to mark the transcription of a gene for a low molecular weight HSP (HSPI6) in  .  elegans (Stringham, Dixon, Jones & Candido,  1992). The reporter gene in this strain is iZ, the induction of which leads to the expression of B-galactosidase. The induction of hspl6 is tightly controlled by heat shock; induction does not occur constitutively, making this gene an excellent marker for the effects of heat shock. After heat shock, the worms may be fixed and stained for B-galactosidase expression. The expression of hsp 16 as reported by the expression of 8-galactosidase may then be examined in situ. While the expression of 8-galactosidase after induction of hspl6 cannot be used as a  strict quantitative assay of heat shock protein production, there are still relative differences in staining that relate to treatment intensity (Stringham, Dixon, Jones & Candido, 1992). This strain was used to examine the relative effectiveness of the different heat shock treatments used in the behavioral experiments that follow.  57 Methods Subjects. This transgenic strain, hsp16-1Z (48.1C), was obtained from the Candido laboratory (Stringham et al., 1992). At the time of treatment, worms were 3 to 4 days old (at the peak of egg-laying). Twenty to thirty worms were placed on plates together. Five plates were prepared. Apparatus. Heat shock was delivered by immersing the plates (sealed with Paraflim) in a temperature controlled water bath at 32 ° C. Fixing and staining of the worms was done as described by Stringham et al. (1992) with a histochemical stain containing X-gal (Fire, WhiteHarrison & Dixon, 1990). Procedure. The effects of four heat shock treatments on the expression of B galactosidase were examined as well as a no-heat shock control. The four heat shock treatments were: a) single heat shock (45 mm, 32°C), b) three heat shocks (45 mm, 32 °C) given at the same intervals as are used in Experiments 5 and 6, starting 1 h, 20 mm apart, c) single heat shock (15 mm, 32°C), d) three heat shocks (15 mm, 32° C) given at the same intervals as the 45 mm heat shocks. After the end of heat shock treatment, the worms were allowed to rest for 30 mm to permit the 8-galactosidase to develop (Stringham, et al., 1992). The animals were then fixed and stained as described by Stringham et al. (1992). Analysis. The analysis was a qualitative judgment of staining intensity. Photographs of representative staining after each of the treatments are presented here. A positive heat shock control (same strain, heat shock at 33°C, for 30 min) performed by the Candido laboratory is also presented for comparison. Results. As can be seen in Figure 8, there was staining after all heat shock treatments, but none in the no-heat shock control group. Overall, it appears there was a dose-effect in staining intensity expressed between the 15 mm and 45 mm heat shock. Interestingly, it seems  58  Figure 8. The expression of JZ in transformed C. elegans. In this strain, JZ has been inserted behind the promotor of hsp 16. The heat shock treatments were the ones used in the behavioral experiments (Experiments 4 through 8). No-heat shock controls. Positive heat shock controls, 33°C, 30 mm (Candido laboratory). Single heat shock (15 mm, 32°C). Three heat shocks (15 mm, 32°C) at 1 h 20 mm intervals. Single heat shock (45 mm, 32°C). Three heat shocks (45 mm, 32°C) at I h 20 mm intervals.  59  7..  /  NEGATIV  SINGLE HS (15 MIN, 32°C)  SINGLE HS (45 MIN, 32°C)  (30 MIN, 33°C)  r HS (15 MIN, 32°C)  I THREE HS (45 MIN, 32°C) 4r  .  60 that staining after three heat shocks (45 mm, 32°C) was no more intense than the staining after a single heat shock (45 mm, 32°C; see Fig. 8E and F). These results clearly indicate that the heat shock treatments used in Experiments 4 through 8 caused cellular stress marked by the induction of hspl6, a gene for HSP16, a low molecular weight heat shock protein. The effects of these heat shock treatments on the rate of egg-laying was examined next. Experiment 3B. The effects of the heat shock treatments on the rate of egg-laying The rate of egg-laying in  .  elegans is under optimal conditions, fairly regular: one  worm at the peak of egg-laying, 4 days of age, lays about 8 to 12 eggs per hour. This rate is sensitive to conditions in the environment; particularly to the absence of food, but also to overcrowding, tactile stimulation and the presence of contaminants. The sensitivity of egglaying to heat shock was examined by heat shocking worms then counting the number of eggs five worms laid in a 1/2 h interval, 15 to 45 min after the end of the heat shock. Methods Subjects. The strain used in the behavioral experiments, N2 (Bristol) was maintained as described in the general methods. The age of the worms was synchronized by laying gravid adults on a plate with a lawn of. iii for 2.5 to 3 h, then removing the adults, but leaving the eggs laid during that time. The worms were handled in the same way regardless of the heat shock treatment received; all worms received the same number of transfers from plate to plate at the same times relative to the time of the egg test. Apparatus. Heat shock was delivered as described above in the general methods section. The apparatus described in the general methods used for behavioral observations was used to count the eggs laid during the test interval, after the adults had been removed. The assay for the number of eggs was done on plates streaked with 0.1 ml of an previously and incubated at 20°C.  .  jj culture 24 h  61 Procedure. Three heat shock treatments and a no-heat shock control were tested. The heat shock treatments were: a) single heat shock (45 mm, 32°C), b) three heat shocks (45 mm, 32°C) at the intervals described above, and c) three heat shocks (15 mm, 32°C) at the intervals described above. Egg-laying was assayed by placing five worms on a plate 15 to 45 mm after the end of the heat shock treatment. The worms were left on the plate for 30 mm and then removed. The number of eggs laid was then counted. The egg-laying after each treatment was assessed with five plates  ( (plates) = 20).  Analysis. The mean number of eggs laid by five worms in 30 mm was compared across groups using nonparametric statistics (Kruskal-Wallis one-way ANOVA for independent samples; Siegel, 1956). Results. As can be seen in Figure 9, there was a significant difference in the number of eggs laid between groups receiving different heat shock treatments. It appears as though the single 45 mm heat shock produced an intermediate depression in egg-laying, while the triple heat shock (both the 15 mm and the 45 mm) caused a more severe decrease in egg-laying. This is interesting in light of the results from the staining which failed to show much change in intensity between single and triple heat shocks of either 45 mm or 15 mm duration. It is not surprising that heat shock affects different aspects of the physiology of the worm differently. However, these results do reinforce the necessity of examining a number of aspects of habituation in the experiments that follow; based on what was seen here, it is certainly possible to imagine that one aspect of learning or memory would be affected by one parameter of heat shock while another aspect of learning might be more sensitive to a different parameter of heat shock. Taken together, the results of Experiments 3A and B indicate that the heat shock treatments used in these experiments have consequences for the worm; while these treatments  62  Figure 9. The number of eggs laid by five worms in a 30 mm test period, 15 to 45 mm after the end of the heat shock treatment (5 worms per plate, 5 plates per group). The control group received no heat shock.  63  50-  z 40C  cø 0 >0  w  30-  20-  10-  0CONTROL  SINGLE 45M, 32°C HEAT SHOCK  TRIPLE 45M, 32°C HEAT SHOCK  TRIPLE 15M, 32°C HEAT SHOCK  64 do not affect its ability to move or respond to tactile stimuli (Beck, unpublished observations), they do cause the induction of hspl6 and they do alter the rate of egg-laying. Experiment 4 Short- and long-term habituation with pre-exposure to heat shock In this experiment, the effects the pre-exposure to heat shock on the expression of habituation and long-term habituation are examined. The presence of the HSPs themselves may affect the later expression of short- or long-term habituation. In addition, a history of cellular stress may have consequences for learning and memory processes. To test these possibilities, heat shock was administered before training, and the habituation expressed during training and testing was compared with that of similarly trained controls that did not receive heat shock. Methods Subjects and Materials. Worms were maintained as described in the general methods. Twenty subjects were run in each of two groups for a total of 40 subjects. The administration of heat shock (45 mm, 32°C), the use of apparatus, behavioral observations and scoring of behavioral responses were performed as described in the general methods. Procedure. The first group, LTH (n = 20), received habituation training on Day 1 and was tested on Day 2 as described in the general methods. The second group, PRE HS I LTH (n  =  20), received a heat shock (45 mm, 32°C) that ended 2 hr before the beginning of the  behavioral training, and then received the same training and testing as the LTH group. Statistics. Short- and long-term habituation was analyzed as outlined in the general methods. In the analysis of short-term habituation, habituation during distributed training was compared across groups with a three-way, mixed design ANOVA (Block x Training x Group; alpha = .05). Mauchly’s test of sphericity indicated that the assumption of homogeneity of variances and covariances was violated (Mauchly’s sphericity test, chi-square approximation =  65 7.54, p = .02), so planned comparisons examining recovery from habituation were not performed, and the degrees of freedom of the relevant E tests were adjusted with the Huynh Feldt epsilon. Long-term habituation was assessed with a mixed-design ANOVA (Day x Group; alpha =  .05). Expression of long-term habituation may be seen as a significantly lower level of  responding on Day 2 than on Day 1 across both groups, or as a significant interaction between Day and Group. To examine whether any interaction was the product of a difference the Day 2 level of responding, a planned comparison contrasting the Day 1 level of responding of the two groups was performed (alpha = .05). Results Habituation. Overall, the LTH (Fig. 1OA) and PRE HS / LTH (Fig. lOB) groups showed significant habituation during blocks of training on Day 1 (mixed-design Training x Block x Group ANOVA; alpha = .05; Training: F(0.9, 34.2)  =  125.56, p  test of sphericity for the effect of Block: chi-square approximation Feldt epsilon  =  =  <  .01; Mauchly’s  7.54, p = .02; Huynh  .90).  There were no overall differences in the level of responding on Day 1 between the group that received heat shock (45 mm, 32°C) before training (PRE HS I LTH) and the group that did not (LTH) (Group: f(1, 38)  =  .64, n&). Taken together, subjects in both groups  showed short-term retention of habituation between the blocks of training (Block: E(1.8, 68.4) =  48.5, p < .01) and there was no evidence that pre-exposure to heat shock affected the  expression of short-term habituation (Group x Block: (1.8, 68.4)  =  .06, j, In addition,  the pre-exposure to heat shock did not appear to affect the expression of habituation within blocks of training (Group x Training: E(1, 38)  =  .01,  n..&).  Interestingly, when the groups  were taken together, subjects habituated differently within the three blocks of training on Day 1 (Block x Training: E(2, 76)  =  4.89, p = .01). The pre-exposure to heat shock did not appear  66  Figure 10. Habituation curves: distributed habituation training with pre-exposure to heat shock (45 mm, 32°C). The mean response magnitude (mm) to each stimulus given during training on Day 1 and testing on Day 2, 24 hr after the end of training, is shown for subjects receiving distributed habituation training only (LTH; Fig. 1OA;  n  20; 60 stimuli  in three blocks of 20 stimuli at a 60 s ISI with one hour rests between blocks), and subjects receiving distributed habituation training 2 hr after the end of a 45 mm heat shock (PRE HS /LTH; Fig. 10B;=20).  67  —DAY2—  DAY1 A  3—  3,  2.5I  2.5fç  —.  ZE 0— uJ  ZZ  40  uJ4  1  20  21  40  41  B  nI LI ,g.  1  ZZ  20  1  20  3-,  w  40  1  60  IW L91  2.54Q 2  05  -I 1  20  21  40  STIMULI  41  60  68 to affect this interaction either (Group x Block x Training: (2, 76)  =  1.26,  j&)  although the  countemull effect size was significantly different from zero, so it cannot be assumed that pre exposure to heat shock had no effect on the Block x Traiiiing interaction (obtained ES counternull ES  =  .50, (2, 76)  =  =  .25;  5.05, p < .0 1). An inspection of Figure 10 suggests that the  amount of habituation may be diminishing with each successive block of training. This may be a result of a floor effect of response decrement or it may reflect a limitation of the learning procedure. Recovery from habituation could not be evaluated with planned comparisons as Mauchly’s test of sphericity indicated that the assumption of the homogeneity of variances and covariances was violated). However, an inspection of Figure 10, comparing the amounts of response increment during the 1-h rest period following habituation, suggests that recovery might be observed between blocks 2 and 3 but not between blocks 1 and 2. Long-term retention of habituation after pre-exposure to heat shock. The Day 1 response levels of the two groups were contrasted with a planned comparison to determine whether the pre-exposure to heat-shock had affected initial response levels; there was no significant difference between the groups (see Fig. 10; alpha = .05; F(l, 38)  =  2.49,  jj.)  although the significance of the counternull effect size indicates that there may be an effect of pre-exposure to heat shock on initial response levels (obtained ES: .35, counternull ES: .70, E(1, 38)  =  9.96, p < .0 1). Retention of habituation training should be evident as a lower level  of responding on Day 2 than on Day 1. Taken together, the worms in the LTH and PRE HS I LTH groups showed significant long-term habituation (see Fig. 11; mixed design Day x Group ANOVA; alpha = .05; Day: E(1, 38)  =  37.65, p < .0 1). Pre-exposure to heat shock did not  affect the expression of long-term habituation (Day x Group: F(1, 38)  =  .16,  t).  69  Figure 11. Habituation by block: distributed habituation training with pre-exposure to heat shock (45 mm, 32°C). The mean block magnitudes on Day 1 and Day 2 of the two groups (LTH, distributed training only, exposure to heat shock,  j =  =  20, and PRE HS I LTH, distributed training with pre  20) are shown (error bars show +1- SE).  -D r 3 -rn cj)  =  -I  TC1  P I  I  .(1l  -L  r4)  -‘  •EJ  M  20 STIMULI BLOCK MAGNITUDE (mm)  71 Thus, it seems that neither short-term nor long-term habituation were affected by the pre-exposure to heat shock (45 mm, 32°C) that ended 2 h before training. It may be that heat shock given during training would be more effective at blocking the consolidation of memory. Experiment 5 Short- and long-term habituation with heat shock during training Previous work with agents that disrupt LTM has showed that memory formation was vulnerable to disruption during and immediately after training (Castdllucci et aL, 1989; Frey et al., 1988). Here, the effects of heat shock delivered during each of the 1-h rest periods immediately after each training block on the expression of habituation, and the retention of short- and long-term habituation are examined. Methods Subjects and Materials. Worms were maintained as described in the general methods. Twenty subjects were run in each of two groups. The data from 4 subjects were not included because of technical errors in stimulus administration; the fmal total was N = 36 subjects. The administration of heat shock (45 min, 32°C), the use of apparatus, behavioral observations and scoring of behavioral responses were performed as described in the general methods. Procedure. The first group, LTH-HS (ii = 18), received habituation training as described in the general methods. Immediately after each of the three training blocks during the 1-hr rest periods, worms were exposed to a heat shock. A minimum of 10 mm passed after the heat shock exposure was finished before training began again. Testing was given on Day 2 as described in the general methods. The second group, HS ONLY  (jj =  18), received only a  single stimulus on Day 1, but received the heat-shock exposures at the same intervals as the LTH-HS group. Testing was given on Day 2 as described in the general methods. This control group was included to examine the effects of the heat-shock treatment on performance on Day 2.  72 Results Habituation. As can be seen in Figure 12A by comparing the response magnitudes to the initial and fmal stimuli of training, the LTH-HS group exhibited habituation during the blocks of training (two-way repeated-measures Training x Block ANOVA; alpha = .05; Training: F(.64, 9.6) =  13.84,  <  49.41,  <  .01; Mauchly’s test of sphericity chi-square approximation  .01; Huynh-Feldt epsilon  =  .64).  The worms receiving heat shock during training showed a significant decrease in the level of responding between blocks (Blocks: E(1.28, 19.2)  =  18.23,  <  .0 1). However, there  were no differences between blocks in the habituation exhibited within blocks (Training x Blocks: E(2, 30)  =  .38,  n&).  Because Maucffly’s test of sphericity indicated that the assumption of the homogeneity of variances and covariances was violated, the planned comparisons to describe recovery from habituation between blocks of training cannot be performed. However, an inspection of Figure 12A suggests that spontaneous recovery might be evident between Blocks 2 and 3 but not between Blocks 1 and 2. The effects of heat shock (45 mm, 32°C) during training on responding on Day 1 are not yet clear. It may be, for example, that heat shock has caused an accumulating depression in the level of responding which has been masked by the expected development of habituation. Thus we cannot be sure that heat shock is not having an effect on responding; however, because the dynamics of habituation on Day 1 are similar to that seen on Day 1 in the distributed training group of Experiment 2 (see Figure 6A), it is clear that short-term habituation has not been blocked by heat shock. Long-term retention of habituation. In this experimental design, retention of habituation is seen as a significantly lower Day 2 level of responding of a trained group relative to an untrained control. Two contrasts using two-tailed, unpaired I tests were made: the first  73  Figure 12. Habituation curves: distributed habituation training with heat shock (45 mm, 32°C) during training. The mean response magnitude (mm) to each stimulus given during training on Day 1 and testing on Day 2, 24 hr after the end of training, is shown for subjects receiving distributed habituation training (60 stimuli in three blocks of 20 stimuli at a 60 s 1ST with one hour rests between blocks) with heat shock (45 mm, 32°C) after each of the three training blocks (LTH-HS; Fig. 12A;  n = 18), and subjects receiving heat shock  (45 mm, 32°C) at the same intervals as the LTH-HS subjects, but without habituation training (HS ONLY; Fig. 12B; = 18).  74  —DAY2  DAY1 A 3. 2.5  zE O o.uJ  2 1.5  ZZ 40  w4  1 05• =  0• 21  20  F-  41  40  1  60  B 2.5  E z E  1I1  uJ  t4CI W’Ll  0  o  <0  uJ4  I-  2  bI N 0  DI°L  piovI  I-.  I’iD  1.5  1  STIMULI  20  75 was between Day 2 of the heat shock alone group and Day 1 of the trained, heat shocked group to examine the effects of heat shock on responding on Day 2. The second contrast was between Day 2 of the heat shock alone group and the Day 2 of the trained group to examine the effect of heat shock during training on long-term habituation. As there were two contrasts, alpha was reduced: .05/2  =  .025.  The effects of heat shock on habituation on Day 2 were tested by the heat-shock alone group. The comparison of the heat-shock alone group Day 2 level of responding with the Day 1 responding of the trained group (before heat shock) will demonstrate if heat shock may facilitate or depress responding by itself. An unpaired, two-tailed test was used to contrast the Day 2 level of responding of the heat shock alone group with the Day 1 level of responding of the LTh-HS group. It was found that the response level on Day 2 of the HS ONLY group was not significantly different from the initial response level on Day 1 in the LTH-HS group (i(34) =  1.44, n) although the significance of the counternull effect size indicates that it cannot be  concluded that heat shock alone has no effect on responding on Day 2 (counternull effect size: I (34)  =  2.88,  <  .01). Heat shock during training appeared to block the expression of  habituation training (LTH-HS Day 2 vs. HS ONLY Day 2; unpaired t test, t(34)  =  .96,  An inspection of Figure 13 indicates that heat shock during training did not affect responses levels on Day 2 when given alone on Day 1, but that the induction of long-term habituation was blocked by heat shock given during training on Day 1. While the expression of habituation during training and the short-term retention of habituation was not blocked by heat shock during training, long-term retention for habituation was lost. One possible explanation for the failure to exhibit long-term habituation after heat shock during training is that the LTH-HS happened to have an unusually low initial level of responding on Day 1, making it unlikely that significant long-term habituation would be expressed. As can be seen by comparing Figures 7 and 13, in Experiment 2 (habituation at a  76  Figure 13. Habituation by block: distributed habituation training with heat shock (45 mm, 32°C) during training. The mean block magnitudes on Day 1 and Day 2 of the two groups are shown (LTH-HS, heat shock during distributed training, j = 18, and HS ONLY, heat shock without training on Day 1,  =  18, error bars show +1- SE).  z r  0  Cl)  Cl)  -I  r  0 I Cli I  p I  ..L I  -‘  %)  h)  ;.n  -‘  20 STIMULI BLOCK MAGNITUDE (mm) C*)  78 60-s ISI) the Day 1 response level of the distributed training group was comparable to the Day 1 response level of the LTh-HS in this experiment (Exp. 2, distributed training group, Day 1 (X +1- SE; in mm): 1.422 +1- .132; Exp. 5, LTH-HS, Day 1: 1.5437 +1- .149). Thus, this does not seem like a plausible explanation. Experiment 6 Short- and long-term habituation with pre-exposure to heat shock and heat shock during training One of the earliest and best documented phenomena related to the heat shock response is the thermal tolerance effect (Lindquist, 1986). When given a severe heat shock, cells in culture died. However, if the cell culture was given a pre-treatment of a mild heat shock, then given the severe heat shock, the cells survived. In some systems, the accumulation of HSPs from the early mild heat shock is responsible for the thermal tolerance of the severe heat shock (Lindquist, 1986; Parsell, Taulien & Lindquist, 1993). In this context, the accumulation of HSPs from pre-exposure to heat shock may protect the formation of LTM for habituation training from disruption of heat shock during training. In this experiment, the effects of pre exposure to heat shock on the disruption of LTM formation by heat shock during training were examined. Methods Subjects and Materials. Worms were maintained as described in the general methods. Twenty subjects were run in each of two groups for a total of 40 subjects. The administration of heat shock (45 min, 32°C), the use of apparatus, behavioral observations and scoring of behavioral responses were performed as described in the general methods. Procedure. The first group, PRE HS I LTH-HS  (n = 20), received a heat shock that  ended 2 h before the beginning of habituation training on Day 1. During habituation training, heat shock was presented after each of the three training blocks during the 1-h rest periods.  79 Testing on Day 2 was as described in the general methods. The second group, LTH-HS  (jj =  20), was a replication of the disrupting effects of heat shock during treatment observed in Experiment 5. As in Experiment 5, the LTH-HS group received habituation training with heat shock (45 mm, 32°C) after each of the three training blocks during the 1-hr rest periods. Statistics. The analysis of short- and long-term habituation was performed as outlined in the general methods. Habituation on Day 1 was assessed with a three-way, mixed design ANOVA (Block x Training x Group; alpha = .05). Recovery from habituation was examined by comparing the mean of the last five responses at the end of one block with the mean of the first five responses of the next block. Three planned comparisons were used to assess recovery from habituation between blocks, and to examine the effects of pre-exposure to heat shock and heat shock during training on the degree of recovery expressed (alpha = .05 / 3  .0 1). In the first planned comparison,  the average of the habituated response levels (Blocks 1 and 2) across groups and the two intervals was contrasted with the average of the initial response levels (Blocks 2 and 3) to confirm that recovery from habituation did, overall, occur. The second planned comparison was used to examine the effects of pre-exposure to heat shock on the degree of recovery expressed by contrasting the average recovery (Block 1 to 2 and Block 2 to 3) of the groups that did not receive pre-exposure to heat shock (LTH and LTH-HS) with the average recovery expressed by the PRE HS / LTH-HS group. The third planned comparison was used to examine the effects of heat shock during training on the degree of recovery expressed. Here, the average recovery (Block 1 to 2 and Block 2 to 3) of the LTH group was compared to the average recovery exhibited by the two groups that received heat shock during training (LTH HS and PRE HS I LTH-HS). Long-term habituation was assessed with a two-way, mixed-design ANOVA (Day x Group; alpha = .05). The expression of long-term habituation in all groups taken together  80 would be seen as a significantly lower response level on Day 2 than on Day 1. A significant interaction between Day and Group might indicate a difference in the expression of long-term habituation. Three planned comparisons were used to examine these data. To examine the effects of pre-exposure to heat shock on initial response levels on Day 1, the average of the Day 1 response levels of the two groups that did not receive pre-exposure to heat shock (LTH and LTH-HS) were contrasted with the Day 1 response level of the PRE HS / LTH-HS group. The capacity of pre-exposure to heat shock to induce thermal tolerance for the disruptive effects of heat shock during training on long-term habituation was tested by contrasting the change from Day 1 to Day 2 of the LTH-HS group and the PRE HS / LTH-HS group. To examine whether the PRE HS I LTH-HS group exhibited the disruption of long-term habituation previously seen in Experiment 5 and whether the LTH group exhibited long-term habituation, the average of the change from Day 1 to Day 2 of the LTH group was contrasted with the average of the change from Day 1 to Day 2 of LTH-HS group and the PRE HS / LTH-HS group. Results Habituation with pre-exposure to heat shock and heat during training. Short-term habituation occurred in all three groups (see Figures 14A, B and C; mixed design ANOVA; alpha  =  .05; Training: E(1, 51)  =  155.81,  <  .01). There was significant retention of  habituation between blocks of training W(2, 102)  =  15.61,  <  .01). The heat shock treatments  did not significantly affect the overall level of responding (Group: F(2, 51)  =  1.92,  n.&)  although the significance of the countemull effect size indicates that it cannot be assumed that the heat shock treatments had no effect on response levels (obtained ES: .31, countemull ES: 61, F(2, 51)  =  7.68, p  <  .01). Furthermore, habituation within blocks did not vary between  blocks (Block x Training: E(2, 102) =  =  .27,  n&) or across groups (Training x Group: F(2, 51)  .05, n.s.). However, there was a significant interaction between Block, Training and Group  81  Figure 14. Habituation curves: distributed habituation training with pre-exposure to heat shock (45 mm, 32°C) and heat shock during training (45 mm, 32°C). The mean response magnitude (mm) to each stimulus given during training on Day 1 and testing on Day 2,24 hr after the end of training, is shown for subjects receiving distributed habituation training (60 stimuli in three blocks of 20 stimuli at a 60 s 1ST with one hour rests between blocks) with pre-exposure to heat shock and heat shock after each of the three training blocks (PRE HS / LTH-HS; Fig. 14A; =20), subjects receiving distributed training with heat shock (45 min, 32°C) after each of the three training blocks (LTH-HS; Fig. 14B; fl = 20), and subjects receiving only training (LTH, Fig. 14C, n= 19).  82  —DAY2—  DAY1 A  =  z 2.5 ZE .  LU  (n  w  u  in  q  2  C)  1.5:  ujx  0.5  0’.  ç  I  20  3  ZE  I  I  I  41  60  2  05  I  C  20  r  1  21  40  L  20  3 2.5 in e  C) i.!  w  Ui  Ui  1  O’j  z  in  .0%  ox  40  x  2  1.5’  I  1.5’  ZZ <0  2.5 in  21  z  2.5  Cl)  c  z  x  1  B  3.  I  I  41  60  3  IJt3J”  0.5 0’, 1  20  3.  2.5’  2.5  ZE  0’  LU  (n UJ  1%kT  ZZ  <0 UJ<  0.5 0, 1  0.5’ I  20  21  40  STIMULI  I  I  41  60  0-, 1  20  83 (E(4, 102)  =  .04). An inspection of Figure 14 does not suggest any obvious pattern.  2.66, p  It may be that there is a subtle difference that wifi be evident in an analysis of recovery from habituation. The presence of recovery from habituation was assayed by contrasting the average of the habituated response levels of one block with the initial response levels of the following block (alpha = .0 1). Overall, there was recovery from habituation between blocks of training (E(1, 51)  =  87.45,  <.0 1). The effect of pre-exposure to heat shock on the expression of  recovery was tested by contrasting the average difference between the habituation response levels of one block with the initial response level of the next of the LTH and LTH-HS groups with the average difference of the PRE HS I LTH-HS group (alpha  =  .0 1). Pre-exposure to  heat shock did not appear to affect recovery from habituation ((1, 51)  =  .97, ni). The  effects of heat shock during training on the recovery from habituation expressed was examined by contrasting the averaged mean of the differences between habituated and initial response levels of the LTH group with the averaged mean of the differences between habituated and initial response levels of the LTH-HS group and the PRE HS / LTH-HS group. No effect of heat shock on recovery from habituation was observed (E(1, 51)  =  .10,  n.&).  The analysis of short-term habituation has determined that the habituation exhibited by the three groups, LTH, LTH-HS and PRE HS I LTH-HS has much in common, such as retention of habituation between blocks of training, the degree of habituation during training and the degree of recovery from habituation. The interaction between Block, Training and Group has not yet been accounted for. Without a context for this effect or any discernible pattern in the data, it may be that this interaction, while statistically significant, is not of any theoretical importance. Long-term retention of habituation training.  Response levels on Day 1 and 2 in the  three groups were compared with an overall mixed design ANOVA (see Fig. 15; Day x Group;  84 alpha = .05). While the overall response level did not vary with either group or day (Group: E(2, 56)  =  .14,  n; Day: E(1, 56) = .04, n). the change from Day  across the groups (Day x Groups: F(2, 56)  =  3.7,  =  1 to Day 2 did vary  .03).  One effect that may have contributed to such an interaction is the effect of the pre exposure to heat shock on the initial response levels on Day 1. The effect of pre-exposure of heat shock on the initial response levels was considered in a planned comparison that contrasted the average Day 1 response level of the LTH and LTh-HS groups with the Day 1 response level of the PRE HS I LTH-HS group (alpha heat shock was observed (see Figure 10; (1, 56)  .0 1). No effect of pre-exposure to  .024,  n.&).  Although it cannot be explained by an effect of the pre-exposure to heat shock on the initial level of responding, the significant interaction between Day and Group from the overall ANOVA is not surprising, as the LTH group should exhibit long-habituation (Experiments 2 and 4) while the LTH-HS group should exhibit a block in long-term habituation (Experiment  5). From an inspection of Figure 14, it is evident that in the LTH group, the level of responding decreased from Day 1 to Day 2, while in the LTH-HS group, the level of responding increased slightly from Day 1 to Day 2. Two planned comparisons were used to define the effects of pre-exposure to heat shock on the disruption of long-term habituation by heat shock during training and to contrast the changes from Day 1 to Day 2 in response levels between the LTH-HS and LTH groups. In the first comparison, the difference between Day 1 and Day 2 response levels of the PRE HS I LTH-HS group was contrasted with the differences in Day 1 and 2 response levels. If pre exposure to heat shock prevented the disruption by heat shock during training, the difference between Day 1 and Day 2 would significantly greater in the PRE HS I LTH-HS group than the LTH-HS group (alpha = .01). There was no effect of pre-exposure to heat shock on the  85  Figure 15. Habituation by block: distributed habituation training with pre-exposure to heat shock (45 mm, 32°C) and heat shock during training (45 mm, 32°C). The mean block magnitudes on Day 1 and Day 2 of the three groups that received training on Day 1: the PRE HS I LTH-HS group the LTH-HS group group  (ij, =  (jj, =  20) which received heat shock before and during training,  20) that received heat shock during training only, and the LTH  (n = 19) that received training only are shown (error bars show -i-I- SE).  86  J  DAY1  •  _LU 1.  co c1  0 0 -J  LTH  PRE HS I LTH-HS  LTH-HS  87 change from Day 1 to Day 2 expressed by the PRE HS / LTH-HS; the two groups changed the same amount between Day 1 and Day 2 (F(l, 38)  =  .275, n).  A fmal planned comparison was used to compare the change in response level from Day 1 to Day 2 exhibited by the group that received only training on Day 1 with the averaged change in response level from Day 1 to Day 2 in the groups that received heat shock during training (alpha  .01). It was found that the change exhibited by the LTH group was  significantly different from the change exhibited by the LTH-HS and PRE HS / LTH-HS groups ((1, 56)  =  7.09, p <.0 1). One interpretation of these results is that the LTH group  exhibited long-term habituation, the LTH-HS group did not, and the pre-exposure to heat shock failed to prevent the block of LTH by heat shock during training. However, this is not the only possible interpretation. As can be seen in Figure 15, the Day 2 response level of the LTH group is similar to the Day 2 response level of the LTH-HS group, and that the Day 1 response levels of these two groups are not as similar. It may be that the significant contrast between the differences in Day 1 to Day 2 response levels have been affected by differing initial response levels. This possibility is explored in the synthesis of results after Experiment 8. Experiment 7 The effects of heat shock just prior to testing on the retention of long-term habituation In Experiment 4, pre-exposure to heat shock did not prevent the formation of long-term memory; in Experiment 5, heat shock during training blocked the formation of LTM. In this experiment, the effect of heat shock on the retention of memory, is tested. Heat shock before the Day 2 retention test long after training may affect the processes necessary for the retention of long-term habituation. Methods Subjects and Materials. Worms were maintained as described in the general methods. Twenty worms were run in each of two groups for a total of 40 worms. The administration of  88 heat shock (45 mm, 32°C), the use of apparatus, behavioral observations and scoring of behavioral responses were performed as described in the general methods. Procedure. The first group, LTH / DAY 2 HS  (n = 20), received habituation training  on Day 1 as described in the general methods and received a heat shock ending 2 hr before testing on Day 2. The second group, DAY 2 HS ONLY  (n = 20), received only a single  stimulus on Day 1, but like the LTH I DAY 2 HS group, received a heat shock ending 2 hr before testing on Day 2. The DAY 2 HS ONLY group was included to control for the direct effects of Day 2 heat shock on the level of responding during testing. Only the long-term retention of habituation will be analyzed in the results as the heat shock was presented after training on Day 1 was complete. Statistics. The analysis of long-term habituation was performed as outlined in the general methods. Two factorial ANOVAs (alpha = .05 / 2  =  .025) were performed, one on  each level of Day (Day 1 and Day 2), comparing the performance of the different groups. In a planned comparison on the response levels of Day 1, the average of the Day 1 response levels of the trained groups (LTH and LTH I D2HS) was contrasted with the Day 2 level of responding of the untrained Day 2 heat shock only group to examine the effects of training (alpha  =  .025).  Two planned comparisons were performed on the data from Day 2 (alpha = .025 I 2  =  .0 1). The Day 2 response levels of the trained groups, LTH and LTH / DAY 2 HS, were contrasted to examine the effects of Day 2 heat shock on Day 2 responding (alpha = .01). To determine whether both trained groups, overall, exhibited long-term habituation, the average of Day 2 response levels of these two groups was contrasted with the Day 2 response level of the Day 2 response level of the DAY 2 HS group (alpha = .01).  89  Figure 16. Habituation by block: distributed habituation training with heat shock (45 mm, 32°C) on Day 2, 2 hr before testing. The mean block magnitudes on Day 1 and Day 2 of the group which received only training, LTH  (n = 19), the group that received training on  Day 1 and heat shock before the retention test on Day 2, LTH I D2 HS (ii = 20), and the DAY 2 HS ONLY (11=20) group are shown (error bars show +1- SE). The DAY 2 HS ONLY group received only a single stimulus on Day 1, no habituation training, and a heat shock on Day 2 that ended 2 hr before the retention test.  90  0  •  LU  —z  DAY 1  1  IT  0<  c1 C.) 0 -J  LTH  LTH I D2 HS  D2 HS ONLY  91 Results Long-term retention of habituation after Day 2 heat shock. The effects of training and heat shock on Day 2 on the levels of responding on Day 1 and Day 2 were compared with two factorial ANOVAs, one on each level of Day (alpha = .025). The ANOVA on Day 1 included the Day 2 response levels of the D2 HS control group. There was no difference between the groups (see Fig. 16; Day 1, Group: (2, 56)  =  .400, n). A planned comparison that  contrasted the average of the Day 1 response levels of the trained groups with the Day 2 response level of the DAY 2 HS control group confirmed that heat shock on Day 2 did not depress or elevate responding from baseline (E(1, 56)  =  .01, nj. On Day 2, there was a  significant difference between the response levels of three groups (Day 2, Group: E(2, 56)  =  .93, n). The effects of heat shock on Day 2 on the retention of long-term habituation was examined with a planned comparison contrasting the Day 2 response levels of the LTh and LTH / DAY 2 HS groups. There was no effect of heat shock on Day 2 on the response levels after training on Day 2 (E(1, 56)  =  3.44,  The average of the Day 2 response levels of the  trained groups was contrasted with the Day 2 response levels of the D2 HS control group. Taken together, the LTH and LTH I DAY 2 HS groups express long-term habituation, as seen by a significantly lower response level than the controls (E(1, 56)  9.54,  <  .01). Thus, it  appears that heat shock on Day 2 does not affect responding on Day 2 or the retention of longterm habituation. Experiment 8 Titration of the effects of heat shock during the rest period Work with Aplysia and other systems has emphasized the dynamics of the cellular processes that support LTM (Byrne et al., 1993). In Experiments 5 and 6, we have seen that in  .  elegans, heat shock delivered during the rest periods of distributed training disrupts the  retention of LTM for habituation training. Unlike the protein synthesis inhibitors used in work  92 with Aplysia, heat shock can be delivered in brief, discrete pulses. Thus, it may be possible to titrate the timing of heat shock to determine whether there is a shorter interval within the 1-h rest period during which LTM formation is particularly vulnerable to disruption. In this experiment, the effects of brief heat shock (15 mm, 32°C) given either early, mid-way or late in the 1-h rest periods on the retention of habituation training were examined. Methods Subjects and Materials. Worms were maintained as described in the general methods. Twenty subjects were run in each of four groups for a total of 80 subjects. The administration of heat shock (15 mm, 32°C), the use of apparatus, behavioral observations and scoring of behavioral responses were performed as described in the general methods. Procedure. All groups received habituation training on Day 1 and were tested on Day 2 as described in the general methods. The first group, LTH  (n = 20), received no heat shock in  the 1-hr rest periods during training. The second group, LTH-EARLY HS  (j =  20), received  15 mm heat shock from 0 to 15 min in the 1-hr rest periods during training. The third group, LTH-MID HS (n = 20), received 15 mm heat shock from 15 to 30 mm in the 1-hr rest periods during training. The fourth group, LTH-LATE HS  ( = 20), received 15 mm heat shock from  30 to 45 min in the 1-hr rest periods during training. Only long-term habituation was considered in the analysis because the longer duration 45 mm heat shock used in Experiments 3, 4 and 5 did not affect the expression of habituation during training on Day 1 nor the shortterm retention of habituation. Statistics. The levels of responding on Day 1 and Day 2 were compared across groups with a mixed-design ANOVA (Day x Group; alpha = .05). Although there was no significant interaction between Day and Group, five comparisons were made to examine the data for patterns that may help direct future efforts to narrow the critical period of memory consolidation during long-term habituation. To compare the initial response levels, a factorial  93 ANOVA was performed across groups on the Day 1 response levels. With a significant interaction between Day and Group in the overall ANOVA, the long-term habituation expressed in each group would be assessed with a planned comparison of that group’s Day 1 and Day 2 response levels. The alpha level for these comparisons was reduced: alpha = .05 / 5  =  .01.  Results Long-term retention of habituation training. The overall ANOVA (Day x Group) compared levels of responding on Day 1 and Day 2 across the groups tested. Overall, the level of responding on Day 1 was significantly higher than the level of responding on Day 2 (see Fig. 17; (1, 76)  =  30.10, p <.01). The level of responding overall did not vary significantly  with the heat shock treatment (Group: F(3, 76)  =  nor was there a significant  2.13,  difference in the change in response levels between Day 1 and Day 2 across groups (Day x Group: E(3, 76)  =  1.29,  n). However, the countemull values of the effect sizes of Group  and the interaction of Day and Group were significant, so it cannot be assumed that Group had no effect on the overall level of responding or the change in response levels from Day 1 to Day 2 (Group: obtained ES: .32, counternull ES: .65, f(3, 76) obtained ES: .25, counternull ES: .50, E(3, 76)  =  =  8.52, p  5.16, p < .01).  <  .01; Day x Group:  The initial response levels  on Day 1 were compared across groups with a factorial ANOVA; there was no significant variation between groups in the initial response level (alpha  =  .01; Day 1, Group: E(3, 76)  =  .449, nj. Therefore any apparent differences between groups in the change from Day 1 to Day 2 in response levels may be attributed to a difference in the Day 2 level of responding. Because no significant interaction between Day and Group was found, the planned comparisons between Day 1 and Day 2 within each group were not carried out. However, as can be seen in Figure 17, it is possible that there was a weakening of the long-term habituation expressed by the LTH-early HS and LTH-mid HS groups perhaps representing a time dependent vulnerability to disruption in memory consolidation. However, these effects are far  94  Figure 17. Habituation by block: distributed habituation training alone or with brief heat shock (15 mm, 32°C) during training either early, mid or late in the 1-h rest period following each training block. The mean block magnitudes on Day 1 and Day 2 of all four of the groups: the LTH group  (n = 20; no heat shock during 1-h rest periods), the LTH  EARLY HS group (11=20; heat shock from 0 to 15 min in the 1-h rest periods), the LTH MID HS  (ja = 20: heat shock from 15 to 30 mm in the 1-h rest periods, and the LTH-LATE  HS group  (= 20; heat shock from 30 to 45 min in the 1-h rest periods), are shown (error  bars show +1- SE).  -I -<I  m  rr  I x-I  3:  0 01 I  I  -L I  20 STIMULI BLOCK MAGNITUDE  DO . > -< -<  I  a’  1%)  (mm) C’,  Lit  96 too small to interpret these results as an indication of defined critical period. In future studies, an effort should be made to sharpen the possible disruption of long-term habituation by brief heat shock by altering the training procedure, the intensity of heat shock, or both. Synthesis of results: An analysis including Experiment 2 and Experiments 4 through 8 There are several issues in this work which may be addressed by comparing the results of the experiments with each other, as has been done throughout the results, and by the performance of direct comparisons across experiments. Differences between experiments in the overall level of responding may be examined. The variability in initial response levels observed between groups receiving the same treatment but in different experiments, or different treatments in the same experiments may be examined, and the impact on the interpretation of the results of individual experiments discussed. The effect size for those phenomena over all of the experiments may be calculated. The objective here is to synthesize the results from the different experiments, examine the data for systematic differences by experiment and treatment, and discuss the pattern of the results and the appropriate interpretation of this pattern. This discussion will be limited to the analyses of long-term habituation, as this is where the most striking and theoretically important differences with training and treatment were observed. One possible problem with the interpretation of these experiments may be differences between experiments in overall response levels; such differences may affect the outcome of individual experiments, making it difficult to interpret the results of each experiment in the context of the others presented here. To examine this possibility, a comparison of the overall level of responding across experiments was done with the groups that received only distributed habituation training (60 stimuli at a 60-s ISI) on Day 1 and the retention test of 20 stimuli on Day 2 with a mixed-design ANOVA (Day x Experiment; alpha from Experiments 2  (j =  21), 4  =  .05). The five LTh groups  (n= 20), 6 (ii = 19), 7 (a = 19) and 8 (a = 20) were included  97  Figure 18. The mean Day 1 and Day 2 response levels across experiments: LTH groups. A. The effects of training by Day and Group. B The effects of training by Day.  98  A  3-  D  2.5-  EXP. 2 EXP. 4  —Ui -Jo  2-  I-z  1.5-  EXP. 6 T  U  EXP. 7  Co  e  EXP. 8  1—  C.,  0  0.50-  B E _LU  I-z  C1  DAY1  DAY 2  DAY1  DAY2  3— 2.5-  2 1 .5 1  C)  0 -J  0.5 0  99  Figure 19. A. The effects of training with heat shock by Day and Group. B. The effects of training with heat shock by Day.  100  A  Q  EXP. 5  •  EXP. 6  Ui  z  0  0 -J  DAYI  DAY2  DAYI  DAY2  B  Ui  z  0 0 -J  101 (see Figures 18A and B). This analysis showed that the level of responding on Day 2 was lower than on Day 1 across experiments (Day: (1, 94)  = 33.875,  <  .0 1). There was no  overall significant difference in the level of responding between experiments (Experiment: F(4, 94)  = 1.374,  Furthermore, the change between Day 1 and Day 2 did not vary  significantly between experiments (Day x Experiment: E(4, 94)  = .538,  This last result  is particularly reassuring, as it suggests that the expression of long-term habituation did not vary between the experiments. The countemull value of the effect size of Experiment was significant, while the countemull value of the effect size of the interaction between Experiment and Day was not (Experiment: obtained ES: .25; counternull ES: .50, E(4, 94) .01; Day x Experiment: obtained ES: .16; countemull ES: .32, E(4, 94)  = 5.51,  = 2.15, . While it  is possible that there are differences in the overall response level between experiments, it can be said with some confidence that differences between the experiments does not affect the change in the level of responding from Day 1 to Day 2 (see Figures 18A and B).  A mixed-design  ANOVA (Day x Experiment) comparing the two groups that received heat shock during training was also performed (alpha = .05). The two LTH-HS groups from Experiment 5 18) and 6  (j,  (=  = 20) were included (see Figures 19A and B). There was no significant change  with Day overall (Day: E(1, 36)  3.243, ii.&). In addition, response levels did not vary  overall with the Experiment (Experiment: E(1, 36)  = 3.395, j). Finally, the response levels  of Day 1 and Day 2 did not interact significant with experiment (Day x Experiment: f(1, 36) .452,  =  It is worth noting that the effects of Day and Experiments came close to  significance; the counternull values of the effect size (ES) of each are statistically significant: Day: observed ES  = .41; counternull ES = 2(.41) 0 = .82, E(1, 36) = 12.97,  Experiment: observed ES  -  = .42; counternull ES = .84, E(1, 36) = 13.58,  <  <  .01;  .01). Thus  while Day and Experiment did not have a significant effect on the level of responding in these  102 LTH-HS experiments, it cannot be concluded that there is no effect of Day and Experiment in this type of training. Overall, while there was no significant effect of Experiment or interaction between Day and Experiment on the response levels observed on Day 1 and Day 2 in the LTH and LTH-HS groups, the possibility that differences between experiments may contribute to overall levels of responding in both LTH and LTH-HS groups and to the changes in the level of responding from Day 1 to Day 2 in the LTH-HS groups cannot be discounted. However, as can be seen from Figures 18 and 19, the effect of training and heat shock during training is apparent when the results from different groups and experiments are taken together. To quantify the effects of training and heat shock during training, the effect size for day of training across all LTH groups and across the two LTH-HS groups may be calculated. The effect size associated with the Day main effect in the ANOVA comparing the LTH groups was 1.30, while the effect size associated with the Day main effect in the ANOVA comparing the LTH-HS groups was 0.40. As can be seen in Figures 18 and 19, these effects go in the opposite direction. In studies from other laboratories examining long-term habituation, expression of LTH was sometimes observed as a decrease in the initial response magnitude from training to test (Leaton & Supple, 1991). Here, the change in the initial response magnitude from training to test was examined across the LTH groups (Experiments 2, 4, 6, 7 and 8) and the LTH-HS groups (Experiments 5 and 6) using a factorial design ANOVAs (Day x Experiment; a factorial ANOVA was used because the number of missing values made a repeated-measures analysis of Day of training inappropriate). Across the LTH groups, there was a significant decrease from Day 1 to Day 2 in the mean magnitude of the initial response (E(1, 150)  =  8.42 1,  <  .0 1),  while there was no difference between experiments in the overall initial response magnitude or the difference between Day 1 and Day 2 in initial response magnitude (Experiment: f(4, 150)  =  103 .72,  n; Experiment x Day: E(4, 150)  =  .61,  Across the LTH-HS groups, there was  no significant difference between Day 1 and Day 2 in the initial response level ((2, 59) 1.326, n); in addition there was no significant difference between experiments in the overall initial response magnitude or the initial response level on Day 1 and Day 2 (Experiment: E(1, 59)  =  .16, ; Experiment x Day: F(1, 59)  =  .40, ii.aJ. Overall, these results indicate that  the change in initial response magnitude from Day 1 to Day 2 reflects both the retention of habituation in the LTH groups and the disruption of habituation after heat shock during training. One issue that is of concern in the interpretation of the results from the LTH-HS groups of Experiments 5 and 6 is the possibility that the failure to observe long-term habituation on Day 2 in these groups was not caused by the heat shock treatment during training, but rather, to an unusually low initial level of responding on Day 1. If so, the mean initial response level of the LTH-HS groups should be consistently lower than the Day 1 response levels and comparable to Day 2 response levels of the LTH groups. While the LTH-HS group from Experiment 6 did exhibit a lower level of responding on Day 1 than any of the LTH groups (Day 1 mean +1- SE (in mm): 1.245 +1- .075), the LTH-HS group of Experiment 5 had a level of responding on Day 1 higher than two of the five LTH groups (LTH-HS, Exp. 5 Day 1: 1.544 +1- .146; LTH, Exp. 2, 1.422 ÷1.132; LTH, Exp. 7: 1.429 +1- .113). However, such comparisons only go so far in answering such concerns. The factors that resulted in a low level of responding in the LTH-HS group of Experiment 6 are not known (note that during the first block of training, the worms have not yet received the first heat shock treatment). An overall ANOVA comparing the Day 1 response levels across all groups that received distributed training and did not receive pre-exposure to heat shock showed a significant effect of group despite the fact that all  104 groups received the same treatment up to that point (eleven groups included; E( 10, 206) =  2.463,  <  .01). Fisher’s PLSD post-hoc comparisons showed significant  differences between a number of groups (see Table 1). The variability in initial response levels is consistent with the findings in other studies of habituation of the reversal response to tap from this laboratory; in these studies, as with this one, the characteristics of habituation are consistent despite the variability of the initial response level (Rankin & Broster, 1992; Broster & Rankin, 1994). Therefore, the failure to see long-term habituation in the groups of the present experiments that received heat shock during training need not be attributed to a depressed initial level of responding. In Experiment 4, the effects of pre-exposure to heat shock on levels of responding on Day 1 and Day 2 were explored. To examine the effects of pre-exposure to heat shock on Day 1 response levels more closely, the data from Experiments 4 and 6 may be considered together. Both the PRE HS I LTH (Exp. 4) and the PRE HS I LTH-HS (Exp. 6) groups received heat shock 2 h before the first 20 stimuli block on Day 1, while the LTH (Exp. 4) and the LTH-HS (Exp. 6) groups did not. A two-way factorial ANOVA (PRE HS x Experiment) was used to compare the effects of pre exposure to heat shock on response levels to the first twenty stimuli on Day 1 across experiments (alpha = .05). The results showed a significant effect of Experiment, with subjects in Experiment 4 responding at a significantly higher level than subjects in Experiment 6 (Experiment: E(1, 95)  =  18.07,  <  .0 1). There was no significant effect  of the pre-exposure to heat shock, nor any interaction between the effect of experiment and the presence of the pre-exposure to heat shock (PRE HS: F(1, 95) Experiment x PRE HS: E(1, 95)  =  =  .76, n.&;  1.07, n). Clearly, while there were experimental  factors that affected the initial response levels, pre-exposure to heat shock did not have  105 an impact on initial response levels measured during the first twenty stimuli of training on Day 1. In summary, the pattern of results of the experiments using the distributed training procedure at a 60-s ISI indicate that while there were uncontrolled factors that affected response levels, the effects of training on the change in responding from Day 1 to Day 2 and the disruption of that change by heat shock during training were robust. Factors which may have contributed to the differences observed between experiments using the same treatments include differences in experimenter, temperature of testing environment (19° 24°C), variations in the worm strain, scorer differences and so on. -  It is important to consider the results of each experiment in the context of the others. In this manner, by comparing the performance over all of the groups that received training only, that received heat shock during training, or pre-exposure to heat shock before training, the factors which affect the development of retention for habituation may become apparent. Discussion The experiments in this dissertation examine factors that affect the development of long-term habituation in  .  elegans. The objective was to determine training  parameters that affect the development of long-term habituation, establish a procedure that produces long-term habituation, and then use interference with memory consolidation as a tool to explore the dynamics of long-term habituation. In Experiment 1 (10-s 1ST), neither distributed nor massed habituation training at a 10-s 1ST produced unambiguous LTH. The possibility that a longer interstimulus interval would lead to the expression of long-term memory was tested in Experiment 2 (60-s 1ST). At a 60-s 1ST, distributed and massed habituation training taken together led to long-term memory for habituation.  106  Fishers PLSD for dl Effect: Day 1 Groups SIgnIflcarn Level: 5 %  LTH (E2), LTH(E4) LW (E2), LTH-HS (ES) LTH (E2). LTH (E6) LTH (E2). LTH (E7) LW (E2), LTH (ES) LTH (E2). LTH-HS (E6) LTH (E2), LTH / 1)2 MS (El) LW (E2), LTH-eHS (ES) LTH (E2). LTH-mHS (ES) LW (E2), LTH-LHS (ES) LTH(E4). LTH-HS (E5) LTH(E4), LTH (E6) LTH(E4). LTH (El) LTH(E4), LTH (ES) LTI-1(E4), LTH-HS (E6) LTH(E4), LTH / 1)2 MS (E7) LTh(E4), LTH-eHS (E8) LTH(E4), LTH-mHS (E8) LTH(E4), LTH-IHS (ES) LTM-HS (ES). LTH (E6) LTH-HS (E5). LTH (El) LTH-HS (E5). LTH (E8) LW-MS (E5). LTH-HS (E6) LTH-HS (E5). LTH / 1)2 MS (E7) LW-MS (E5), LTH-eHS (ES) LTH-HS (ES), LTH-znHS (ES) LTh-HS (ES), LTH-IHS (ES) LTH (E6), LTH (El) LW (E6). LTH (ES) LTH (E6). LTH-HS (E6) LW (E6), LW I D2 MS (E7) LW (E6), LW-eHS (ES) LW (E6). LTH-mHS (E8) LW (E6), LTH-114S (ES) LW (El), LW (E8) LW (El), LW-MS (E6) LW (El), LW /1)2 MS (El) LW (E7), LW-eMS (ES) LW (E7), LW-mHS (ES) LW (E7), LW-hiS (E8) LW (ES), LW-HS (E6) LW (ES), LW / 1)2 HS (E7) LW (ES), LW-eNS (ES) LW (ES), LW-mHS (ES) LW (ES), LTh-IHS (ES) LW-MS (E6), LW /1)2 HS (E7) LW-HS (E6). LW-eHS (ES) LW-MS (E6). LW-mHS (ES) LW-MS (E6), LW-INS (ES) LW / 1)2 MS (El). LW-eMS (E8) LW / 1)2 HS (E7), LTh-miIS (ES) LW / 1)2 MS (E7), .LTH-IHS (ES) LW-eMS (ES), LW-inNS (ES) LTH-eHS (Es). LW-INS (ES) LW-mNS (ES), LW-iNS (ES)  Mean Diff. -.320 -.122 -.106 • 007 -.258 .176 -216 -.386 -.443 -.304 .198 .214 .313 .062 .496 .104 -.066 -.123 .016 .016 .115 -.136 .298 -.094 -.264 -.321 -.182 .098 -.152 .282 -.110 -.280 -.33 8 -.198 -.250 .184 -.209 -.378 -.436 -.297 .434 .042 -.128 -.186 -.046 -.392 -.562 -.620 -.480 -.170 -.227 -.088 -.058 .082 .139  Crit. Diff .330 .339 .334 .334 .330 .330 .330 .334 .326 .330 .343 .338 .33S .334 .334 .334 .338 .330 .334 .341 .347 .343 .343 .343 .347 .339 .343 .342 .338 .338 .338 .342 .334 .338 .338 .338 .338 .342 .334 .338 .334 .334 .338 .330 .334 .334 .338 .330 .334 .338 .330 .334 .334 .338 .330  —  P-Va... 0571 .4787 .5333 .9655 .1249 .2929 .1980 0239 .0079 .0706 2562 .2129 0697 .7130 .0037 .5394 .7021 .4620 .9244 .9265 .5155 .4362 .0877 .5896 .1351 .0631 .2968 .57 15 .3761 .1015 .5212 .1085 .0418 .2492 .1458 .2853 .2251 .0305 .0108 .0852 .0111 .8056 .4562 .2684 .7849 .0214 .0012 .0003 .0050 .3234 .1756 .6038 .7344 .6340 .4057  S S  S  S  S S S  S S S S  107 It is interesting that the age and handling dependent effect was seen with testing at a 10-sec ISI but not at a 60-s ISI. This pattern was observed in an additional experiment in two untrained groups (a single stimulus control group and a zero stimulus control group) at a 10-s 1ST (Marion, Beck & Rankin, 1992). However, in their work on the effects of age on habituation, Beck and Rankin (1993) did not find any change in the depth of response decrement after habituation training at a 10-s 1ST when four day old worms were compared to seven day old worms using handling procedures similar to those used in the present study. The present experiments compare the habituation of four day old worms with five day old worms; it is possible that the difference in habituation observed here over 24 h may have been missed in Beck and Rankin’s (1993) earlier comparison of four and seven day old worms. More work on the nature of the effects of age and prior stimulation on habituation and longterm retention of habituation is needed to clarify this issue. Although the short 1ST (10-s) produced a greater response decrement during training on Day 1 than the long TSI (60-s), LTH was evident after training with the long 151. In addition, on Day 1, the expression of habituation was affected by the training procedure (distributed, massed or twenty stimuli control training) when training was given at a 60-s 151, but not at a 10-s 1ST. Whether habituation training at a 10-s 1ST is capable of producing long-term habituation in  .  elegans is not known. Retention of  habituation across blocks of training, or short-term retention of habituation, was evident with training at a 60-s ISI and at a 10-s ISI. It should be noted that the failure to find evidence for long-term habituation at a 10-s 1ST may be due to a floor effect resulting from the very rapid and deep habituation to training at a 10-s 1ST. It seems paradoxical that greater response decrement on Day 1 should lead to less retention of habituation; however, this same pattern is apparent in the rate of spontaneous recovery  108 from short-term habituation. As discussed above, the rate of recovery from habituation is faster after habituation at a 10-s ISI than after habituation at a 60-s 1ST, despite the greater depth of habituation during training at a 10-s ISI. These findings suggest that the rate of spontaneous recovery from habituation may be a better predictor of retention for habituation training than the amount of response decrement expressed during training. Broster and Rankin (1994) hypothesized that 10-s and 60-s ISIs may recruit different cellular processes during habituation training. If this is the case, the cellular processes recruited by habituation training at a 60-s 1ST may be necessary for the formation of memory for habituation. Testing the effects on habituation of treatments that alter specific cellular processes, such as ablations of subclasses of neurons in the touch circuit (Wicks and Rankin, unpublished observations), may help characterize the contributions of these processes to habituation at different ISIs. The effects of the distribution of training at a 60-s 1ST on LTH are similar to the results seen in experiments on long-term habituation with Aplvsia. and support the idea that memory formation benefits from training that is distributed over time rather than training that is condensed (Carew et al., 1972; Carew and Kandel, 1973). There are two explanations that could account for the effect of distributed training on retention. The first is that memory for training is formed during training itself. During distributed training, a subject habituates three times. Breaking the habituation training into three distinct blocks permits recovery from short-term habituation. Each further block of training leads to rehabituation and thus greater accumulation of memory. According to this hypothesis, available time for an appropriate amount of recovery from habituation before the next block of training would be critical, so that the rehabituation would have a maximal impact on the long-term memory for habituation training.  109 The second possible explanation of the benefit of distributed training over massed training is that the long-term memory for training forms after training has finished. In the distributed procedure, the 1-h rest periods would permit the active encoding of the habituation into long-term memory. A number of training blocks with rest periods after each permit greater accumulation of memory for the training. According to this hypothesis, short-term habituation and recovery processes would not govern the formation of long-term memory for habituation. Rather, other independent processes (e.g. up- or down-regulation of specific proteins) that control memory formation would determine the optimal rest period between blocks of training in distributed procedure. Interference as a tool to examine memory consolidation To test these possibilities, the role of the rest period of distributed training in memory formation was examined. In this approach, conditions were introduced during the rest period that would perturb the cellular processes which may be necessary for memory formation without interrupting spontaneous recovery from habituation in an attempt to determine whether there is an interval of the rest period critical for memory formation. Heat shock (45 min, 32°C) induces heat shock protein production as evidenced by the B-galactosidase staining in transgenic animals after this heat-shock treatment (see Figure 8; Stringham et al., 1992). The presence of heat shock proteins is a marker of cellular stress (Stringham et al., 1992; Lindquist, 1986); therefore worms that have received heat shock (45 mm, 32°C) show evidence of a history of cellular stress. The damage resulting from the stress or the active cellular response to stress, which includes a reduction in protein synthesis and the production of heat shock proteins, may alter the processes which mediate learning or consolidation of memory. Thus, heat  110 shock may be used to probe the dynamics of long-term habituation for an interval or intervals critical to memory formation. In Experiment 4, heat shock (45 mm, 32°C) was delivered so that it ended 2 h before the beginning of training. Heat shock before training might affect responding on Day 1 or it might affect the change in the level of responding from Day 1 to Day 2. This could occur due to residual cell damage, altered cellular processes or the accumulation of heat shock proteins. However, the findings indicated that a history of cellular stress produced by this heat shock treatment did not affect either the responding on Day 1 or the expression of long-term habituation as seen by the change of responding from Day 1 to Day 2 (see Figures 10 and 11). The failure of a single heat shock (45 mm, 32°C) to produce a change in behavior is interesting, in light of the demonstration that a single heat shock (45 min, 32°C) caused the production of heat shock proteins (from Experiment 3A; see Figure 8), and depressed egg-laying measured 15 mm after the end of the heat shock treatment (Experiment 3B; see Fig. 9). Overall, these findings suggest that this heat shock treatment was indeed a significant cellular stressor, and that its lack of effect on behavior is a reflection of the time it was given: two hours before training. In Experiment 5, the effects of heat shock (45 mm, 32°C) given during training on habituation, short-term retention of habituation, and long-term retention of habituation were examined. Heat shock given without training had no effect on responding on Day 2. In addition, heat shock during training did not affect the expression of habituation or short-term retention of habituation training; however, it did prevent the expression of long-term habituation (see Figures 12 and 13). Heat shock given in this procedure was certainly sufficient to cause the induction of heat shock proteins (Experiment 3A; see Figure 8); however, the induction of 8-galactosidase did  111 not appear to be any more intense after three heat shocks for 45 mm each than after a single heat shock of 45 mm. On the other hand, the triple heat shock (45 mm, 32°C) definitely appeared to produce a greater depression in egg-laying than a single heat shock (45 mm, 32°C). Thus, while the results of Experiment 3A on the induction of hsp 16 support an interpretation of the effect of heat shock on training being a result of the time the heat shock treatment was given, the possibility of a dose-effect of the single vs. triple heat shock cannot be set aside. In many systems, the effects of severe heat shock can be alleviated by an earlier milder heat shock; this phenomenon is called induced thermal tolerance (Lindquist, 1986; Parsell, Taulien & Lindquist, 1993). In Experiment 6, to examine whether thermal tolerance could be induced to the effects of heat shock during training on longterm habituation, a pre-treatment of a heat shock (45 mm, 32°) was given as in Experiment 4 ending 2 h before training, followed by training with heat shock as in Experiment 5. If thermal tolerance for the effects of heat shock (45 min, 32°C) during training were induced by the exposure to heat shock before training, long-term habituation should not be blocked. However, the findings indicated that thermal tolerance for the effects of heat shock during training on long-term habituation was not induced; in the group that received heat shock before training as well as during training, long-term habituation was blocked as it was in the group that received only heat shock during training (see Figures 14 and 15). In Experiment 7, retention of long-term habituation was examined by introducing a heat shock treatment (45 mm, 32°C) that ended 2 h before the retention test on Day 2. Studies examining the effects of interference on retention of memory have generally found that interference is not effective at disrupting long-term memory long after training has finished (Squire & Davis, 1985). The results of Experiment 7  112 showed that heat shock (45 mm, 32°C) on Day 2 without training on Day 1 did not affect responding on Day 2. In addition, heat shock on Day 2 had no effect on the expression of long-term habituation. The caveats that were noted in the discussion of Experiment 4 and 5 apply here as well; the single heat shock treatment (45 mm, 32°C) may not be of a comparable magnitude to the triple heat shock during training (45 mm, 32°C) that disrupted long-term habituation. While this possibility must be considered, these findings still suggest that memory is not vulnerable to disruption long after training has been completed. In Experiment 8, the 1-h periods following blocks of training in the distributed procedure during which long-term habituation was vulnerable to disruption were examined further for short intervals within the 1 h periods during which memory consolidation was particularly vulnerable to disruption by heat shock. Three brief heat shocks were used (15 mm, 32°C), which in Experiment 3A, caused the expression of 8-galactosidase, and in Experiment 3B, depressed the rate of egg-laying as much as the triple 45 min heat shocks. Heat shock (15 mm, 32°C) was given either early (0 15 -  mm), mid-way (15 30 mm) or late (30 45 mm) in the 1-h periods following training -  -  blocks. Although the overall ANOVA showed no significant interaction between Day and Group, an inspection of Figure 17 suggests that there may be a weakening of longterm habituation when brief heat shock is given early or mid-way in the 1 h rest period. Little weight can be placed on these results as the overall ANOVA failed to detect any differences between groups in the change in the level of responding from Day 1 to Day 2. However, these data do suggest that the first 30 mm following training blocks may be particularly important to memory consolidation. In future studies, the definition of a critical period for the consolidation of long-term memory may be resolved by using a  113 brief heat shock at a higher temperature (e.g. 34°C) to emphasize and clarify the effect of brief heat shock at different intervals on long-term habituation. In addition, it would fascinating to examine whether the 1-h intervals after each block were equally important to memory consolidation. This could be done by treating groups with heat shock after different combinations of two blocks, or after only one block. If, for example, there was a differential effect of heat shock after the first block compared with the last block, that would have implications for the dynamics of longterm habituation and the processes that support the distributed training effect. The behavioral experiments, considered separately, each describe some aspect of the factors affecting the development of long-term habituation and the effects of various heat shock treatments on learning and memory in  .  elegans. However, it is  not so much in the results of any one experiment that the emphasis should be placed but on the overall pattern of the results. With this in mind, an analysis of the effects of factors varying between experiments on the overall response levels, the initial response levels and the expression of the main effects observed: the effects of training, and the block of the effects of training with heat shock during training. The results indicate that there are factors which vary between experiments which must affect at least the initial level of responding. Knowing this, it is interesting to note that the effects of training and of heat shock during training on response level were consistent across experiments (see Figures 18 and 19). The expression of these effects was robust across experiments in which differences in the worms strains, ambient temperature of the testing environment, experimenters and scorers may have all contributed to produce different levels of responding. In addition, when the groups were considered together, long term habituation in the LTH groups was evident in a second measure, the change in the  114 magnitude of the response to the initial stimulus from Day 1 to Day 2; the disruption of LTH was evident in this measure of initial response magnitude in the groups that  received heat shock during training. The distributed training effect: a psychological perspective Up to this point in this work, the implications of the effect of distributed training have been discussed from a biological perspective. However, the effect of distributed training is frequently discussed in psychological literature, and is considered a robust effect, particularly in verbal learning tasks (Melton, 1970; Hintzman, Block & Summers, 1973; Shaughnessy, 1976; Dellarosa & Bourne, 1986). While most of these experimental paradigms have focused on tasks which involve a conscious effort to retrieve information (either free recall or recognition tasks), a small effect of distributed training on implicit memory has also been demonstrated (Pemichet, 1994). There is no consensus as yet on the psychological processes that produce the advantage for memory formation associated with distributed training (Perruchet, 1994; Dellarosa & Bourne, 1986; Toppino, Kasserman & Mracek, 1991; Ross & Lindauer, 1978; Cornell, 1980, Hintzman, 1974). In the psychological literature, there are at least three classes of theories explaining the effect of distributed training. The first theory explaining the distributed training effect is the encoding variability hypothesis, which attributes greater recall of repeated items to the variety of subjective contexts in which the target is encountered during training, thus increasing the number of retrieval routes (Dellarosa & Bourne, 1986). There has been relatively little empirical support for this theory; in one study there was a demonstration that variable contexts of encoding actually impeded memory rather than improving it (Postman & Knecht, 1983).  115 The second theory is the processing-inhibition hypothesis, which suggests that massed training inhibits processing of stimuli through habituation, while distributed training permits encoding of each item before the next is presented (Cornell, 1980; Hintzman, 1974). However, Dellarosa and Boume (1986) suggest that the failure to demonstrate the distributed training effect in infants (Toppino & DiGeorge, 1984) indicates that habituation is unlikely to be the mechanism behind the distributed training effect, as infants are capable of habituation. The third is the previous encoding-accessibility hypothesis, which argues that the likelihood of encoding processes being engaged upon presentation of a stimulus depends on how accessible the last encoding of that stimulus is. Thus the likelihood of engaging the encoding processes should vary directly with the length of the interval between stimuli (Rose, 1980; Rose & Rowe, 1976). It has been found that the effect of distributed training on recall is lost when other manipulations that increase the likelihood of encoding were performed (Cuddy & Jacob, 1982; Dellarosa & Bourne, 1986). Although in this literature, habituation is viewed as a hard-wired process (Dellarosa & Bourne, 1986), the encoding-accessibility hypothesis is in some senses closer to our present understanding of retention for habituation training. As seen in the earlier discussion of habituation and long-term retention of habituation, habituation is not a unitary, hard-wired process, but rather a form of plasticity which integrates information about stimulus familiarity and the organism’s internal state in sophisticated ways. The encoding-accessibility hypothesis permits, through the flexibility of the processes that control the engagement of encoding, the influence of organism-wide states on the process of memory consolidation.  116 This hypothesis predicts that anything that increases the likelihood of engaging memory processes would tend to mask the effect of distributed training on memory, while manipulations that decrease the likelihood of engaging memory processes should emphasize the effect of distributed training (Dellarosa & Bourne, 1986). If so, it may be possible to manipulate the expression of the distributed training effect by increasing or decreasing the likelihood of encoding. One possible way to increase the likelihood of encoding during habituation in C. elegans may be to alter the interstimulus interval. Habituation in  .  elegans is  highly sensitive to interstimulus interval; in recent work, there is evidence that interstimulus interval is encoded and that anticipatory responding may be observed in the worm’s behavior (Rankin & Broster, 1992, Broster & Rankin, 1994; Wicks & Rankin, unpublished observations). If so, changing the interstimulus interval for short periods during massed training and distributed training may stimulate encoding and mask the distributed training effect on retention of habituation. Encoding of the stimuli may be depressed by an agent such as magnesium chloride that causes reversible paralysis by affecting motor neuron synapses in invertebrates. Touch neurons in the head are stimulated as the worm moves forward through its environment (Kaplan & Horvitz, 1993). It is possible that blocking the movement of the worm would prevent such stimulation from triggering encoding. Such a treatment maintained during training with monotonous stimulation should exaggerate the distributed training effect on memory, although it may have an overall effect on memory as well. No attempts have been made yet to habituate  .  elegans while it is  unable to respond. This psychological perspective would provide a theoretical context for doing so.  117 While the long-term habituation expressed is not a large effect in the present experiments, the procedure does appear to consistently produce significant retention of habituation (see Experiments 2,4, 6,7, and 8). Building an understanding of the factors affecting the processing of stimuli in  .  elegans by testing predictions based on  theories of memory may help us conceptualize the behavioral plasticity observed in  .  elegans as learning and memory. This theoretical approach makes the work on the cellular processes underlying these forms of behavioral plasticity in C. elegans more relevant to our understanding of learning and memory phenomena. Differentiation of types of long-term memory through interference treatments Studies on the effects of protein synthesis inhibition and other interference treatments on memory consolidation have been used to differentiate between different forms of memory in Drosophila. Tully et al. (1994) found that mid-term memory (lasting 7 h) for olfactory conditioning was vulnerable to anesthesia produced by cold shock (flies are immobilized by cold temperatures), while the longer lasting anesthesiaresistant memory (lasting 4 days) was attenuated in the radish memory mutant. Longterm memory (lasting over 7 days) induced by distributed training was disrupted by cycloheximide while long-term memory induced by massed training was not (Yin et al., 1994). The finding that LTM resulting from distributed training was blocked by cycloheximide while LTM resulting from massed training was not, suggests an interesting experiment in C. elegans: the effects of the application of a protein synthesis inhibitor on memory resulting from distributed and massed training could be studied. If LTM for distributed training were blocked while LTM for massed training were spared, the contention that distributed and massed habituation training produce qualitatively different types of memory would be substantiated. Contrasting the effects  118 of heat shock on the retention of massed and distributed training with the effects cycloheximide on the retention of the same might suggest whether heat shock has its effect on long-term habituation through protein synthesis or through interference with some other cellular process. If protein synthesis is induced during memory consolidation, one likely pathway for this induction is the cAMP second-messenger pathway, which has been implicated in learning in Aplysia (Schacher et aL, 1988; Dash et aL, 1990). Through reverse genetics, the role of cAMP-responsive transcription in memory was examined. There is evidence that the transcriptional factor CREB may play a role in mediating this cAMP-induced transcription during long-term facilitation in Aplysia, as an antagonist of CREB prevented development of long-term facilitation (Dash et aL, 1990). In addition, work with rats has demonstrated that long-term potentiation (LTP), a form of longlasting synaptic plasticity induced by tetanic stimulation, may be mediated by a cAMPresponsive transcriptional factor (Frey et al., 1993; Huang & Kandel, 1994; Bourtchuladze et al., 1994). The role of CREB in Drosophila was tested using the gene called dCREB2 that exists in several naturally-occurring Drosophila CREB isoforms (Yin et al., 1994). One isoform, dCREB2-a, is a cAMP-dependent protein kinase A (PKA)-responsive transcriptional activator, while another isoform, dCREB2-b, blocks PKA-responsive transcription (Yin et al., 1994). Transcription is the process by which the DNA of the genome is read and the corresponding RNA is generated, while translation is the process by which a protein molecule is constructed from the template of the RNA strand (Watson, Hopkins, Roberts, Steitz & Weiner, 1987). The antagonistic isoform dCREB2-b was placed under the control of a heat-shock promoter, so that the expression of the antagonistic dCREB2-b isoform could be induced by heat shock.  119 before training, When heat shock was given to these transgenic flies three hours blocked, while it was the long-term memory for distributed olfactory conditioning was heat shock in the unimpaired in the transgenic strain without heat shock, and after (Yin et al., 1994). matched control strain without the antagonistic dCREB2-b isoform esponsive This experiment is an elegant demonstration of the role of the PKA-r the block of PKA transcription pathway in memory. Significantly, the effects of animals. This fact responsive transcription were measured in the behavior of intact lends validity to their results. training in It is interesting to note that the heat shock treatment given 3 h before induce isoforms of the the work on the role of CREB in memory consolidation to al., 1994). This finding fits protein did not affect learning and memory by itself (Yin et given 2 h before in with the present results, where it was observed that heat shock training did not affect learning and memory in C. elegans. particularly well The approach of reverse genetics is one to which C. elegans is the expression of hspl6 suited. The transgenic strain used in Experiment 3a to examine this case, iZ) inserted after various heat shock treatments is an example of a gene (in product, 8-galactosidase, after a heat shock promoter (the hspl6 promoter), so the jZ et al., 1992). An is produced whenever hspl6 is induced by heat shock (Stringham colleagues (Yin et al., interesting twist on the experiment performed by Tully and his shock individual 1994) described above could be done employing the laser to heat this technique, the neurons in adult worms (Stringham & Candido, 1993). With in an individual neuron antagonistic isoform of the CREB gene could be induced only s responsible for longor subset of neurons thought to be the locus for structural change blocked long-term memory term memory. If induction of the antagonistic CREB gene  120 while heat shock of the same cells in genetically matched controls did not, this would be strong evidence for the locus of structural changes due to learning in those neurons. Work with Aplysia has also revealed more than one type of retention for training based on interference studies using transcriptional and translational inhibitors  (Ghirardi, Montarolo & Kandel, 1995). Long-term facilitation, a cellular analogue for long-term sensitization, can be separated into two components; the weaker intermediate form (lasting 3 to 6 h) is blocked by anisomycin, a translational inhibitor, but not actinomycin, a transcriptional inhibitor. This memory process must require the synthesis of proteins from RNA stored in the cell but not new manufacture of RNA from DNA. On the other hand, the long-term form (lasting over 24 h) is blocked by either translational or transcriptional inhibitors, and thus requires both the synthesis of proteins from RNA and the synthesis of RNA from DNA to form LTM (Ghirardi et al., 1995). This result opens the interesting possibility that other systems may also have forms of memory that rely differentially on translation and transcription. This possibility could be tested in  .  elegans by comparing the effects of exposure to a  translational inhibitor with the effects of exposure to a transcriptional inhibitor during and after training on the development of long-term memory for habituation. One of the common themes in the survey of literature in the introduction of the present work was the importance of examining factors affecting long-term memory in whole intact animals, in the behavior as well as cellular analogues of the behavior, and in more than one form of learning. From the Drosophila work, it is clear that the characterization of the impact of the learning mutants on learning and memory processes will require a synthetic approach to the results from a great variety of learning paradigms. This principle also applies to the investigation of the cellular processes  121 underlying LTM in  .  elegans. If we wish to characterize long-term memory and the  cellular processes that mediate it in this organism, we must examine memory in as great a range of behavioral paradigms as possible. This is true because it is difficult to predict what value can be gained by studying a specific form of learning; an investigation that begins simply as a description of a specific form of learning may produce lines of research which lead to interesting insights or the development of new methodologies that may be applied to many systems. But even more importantly, just as the study of learning and memory in every organism has its unique advantages, every form of learning offers the opportunity to examine a unique aspect of the animal’s behavior. To achieve an understanding of the principles that guide learning and memory  and the cellular processes that mediate learning and memory, the results of studies focusing on a great range of behaviors must be intelligently synthesized. What other forms of behavioral plasticity does  .  elegans offer as choices for  future research on the cellular processes underlying memory? There are many possibilities, but a logical choice would be an examination of sensitization and the retention of sensitization in the tap withdrawal reflex. Sensitization to tap has been demonstrated in C. elegans (Rankin et al., 1990). Generally speaking, sensitization is thought to play a critical role in habituation (Groves & Thompson, 1970). The cellular processes underlying sensitization have been implicated in classical conditioning in Aplysia (Byrne et al., 1993). The results from an exploration of factors affecting sensitization and memory for sensitization in the tap withdrawal circuit may help to characterize the role of sensitization in the habituation and retention of habituation observed in the same circuit. Interestingly, work examining habituation to tap in worms in which the anterior touch cells were ablated through laser microsurgery so that the animals consistently  122 accelerate forward to tap has indicated that the acceleration response appears to be more sensitive to sensitization of the tap withdrawal reflex than the reversal response (Wicks, unpublished observations); while, as discussed earlier, the reversal response is more sensitive to the habituation of the tap withdrawal reflex. Already the comparison of these two forms of learning, habituation and sensitization, has shed some light on the nature of the relationship of the two competitive responses of the tap withdrawal reflex and perhaps on the circuit that supports it. The use of heat shock as an interference treatment As a tool to investigate the dynamics of memory consolidation, heat shock has some critical advantages. First, it can be administered easily, without disturbing the organism, or introducing any exogenous substances. Second, unlike cold shock and other anesthetics, it does not cause paralysis. C. elegans continues to move and forage during moderate heat shock, and is able to respond to tactile stimuli normally immediately after treatment. Third, unlike chemical agents that affect protein synthesis, heat shock can be administered for a defined, brief period of time. Fourth, heat shock evokes an active cellular response, which is interesting in and of itself; but more importantly, the interaction of the cellular response to heat shock and the plasticity expressed in the tap withdrawal reflex may be considered a model system for the examination of the interaction between organism-wide responses to changes in the environment and plasticity specifically by the nervous system. Finally, the fmding that heat shock disrupts the formation of long-term memory means that we now have a fme-grained tool for the investigation of the temporal parameters of memory consolidation and for the expansion of our understanding of the processes underlying memory.  123 List of Abbreviations ANOVA  analysis of variance  ATP  adenylyl triphosphate  °C  degrees centigrade  cAMP  cyclic adenylyl monophosphate  C. elegans  Caenorhabditis elegans  CREB  cAMP response element binding protein  Da  Dalton  E. jj  Escherichia ij  ES  effect size ratio, a statistical test in the analysis of variance  h  hour  HAB  mean of the responses to the last five stimuli  HSPs  heat shock proteins  HS  group receiving heat shock treatment  INJT  mean of the responses to the first five stimuli  ISI  interstimulus interval  LTH  group receiving long-term habituation training  LTH-HS  group receiving heat shock during training  LTM  long-term memory  LW  long-term potentiation  mm  minute  MS  mean sum of squares number of subjects in group  N  number of subjects in experiment  124 N  Newton not statistically significant  p  probability  PKA  protein kinase A  PRE-HS  group receiving heat shock that ended 2 h before training  R  response  s  second  S  stimulus  SE  standard error of the mean  SPS S  Statistical Package for the Social Sciences test, a statistical test comparing two means  x  mean  5-HT  serotonin  125 Bibliography Aceves-Pifla, E. 0., Booker, R., Duerr, J.S., Livingstone, M. 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Biological Sciences. 314. 1-340. Wicks, S. R., & Rankin, C. H. (1995). Integration of mechanosensory stimuli in Caenorhabditis elegans. Journal of Neuroscience. 15. 2434 2444. -  Wittekind, W. C., & Spatz, H.-C. (1988). Habituation of the landing response of Drosophila. In G. Hertting & H.-C. Spatz (Eds.), Modulation of sYnaptic transmission and plasticity in the nervous system (pp. 351 368). New York: Springer Verlag. -  Wittstock, S., Kaatz, H. H., Menzel, R. (1993). Inhibition of brain synthesis by cycloheximide does not affect formation of long-term memory in honeybees after olfactory conditioning. Journal of Neuroscience. 13, 1379 1386. -  Wood, D. C. (1988). Habituation in Stentor: a response-dependent process. Journal of Neuroscience. 8, 2248 2253. -  Wood, W. B. (Ed.). (1988). The nematode Caenorhabditis elegans. Cold Spring Harbor: Cold Spring Harbor Laboratory. Yin., 3. C. P., Wallach, J. S., Del Vecchio, E. L., Wilder, E. L., Zhou, H., Quinn, W. G., & Tully, T. (1995). Induction of a dominant-negative CREB transgene specifically blocks long-term memory in Drosophila. Cell. 79, 49 58. -  Zecevic, D., Wu, J.-Y., Cohen, L. B., London, J. A., Hopp, H.-P., & Fallc, C. X. (1989). Hundreds of neurons in the Aplysia abdominal ganglion are active during the gillwithdrawal reflex. Journal of Neuroscience. 9, 3681 3689. -  Zucker, R. S. (1989). Short-term synaptic plasticity. Annual Review of Neuroscience. U, 13 31. -  136 Appendix 1 Analysis of the Distribution of Missing Values in the Habituation Data Because there are a sizable number of missing values due to the presence of the acceleration response to tap in the habituation data, it is appropriate to consider how the distribution of missing values affects the interpretation of reversal response data. The relationship between the habituation of acceleration responses to tap and the habituation of the reversal responses to tap, which was discussed in the introduction of the thesis, bears on this issue. There, it was argued that because of the relationship between the two responses, reversals and accelerations, the habituation of the magnitude of the reversal response reflects the habituation of both responses (Wicks and Rankin, unpublished observations). In this analysis, the distribution of missing values will be examined for characteristics that may help to interpret this relationship between the habituation of the acceleration and reversal responses. In the present experiments, the overall percentage of missing data values was 26.5%. Fewer than 1% of the responses to stimuli were not scored due to technical errors in stimulus administration or video recording, so the percentage of missing data values represents the proportion of acceleration responses to stimuli. The proportion of accelerations expressed may vary across training and between groups and experiments. Wicks and Rankin (unpublished observations) found that the proportion of accelerations varied significantly during habituation training in normal worms and that the proportion of accelerations was significantly higher overall during habituation training at a 10-s ISI than at a 60-s 1ST; these findings support the possibility that the distribution of missing values may vary significantly over habituation training in the present experiments. Therefore, the consequences of uneven distributions of accelerations must be explored. One concern is that the distribution of accelerations within blocks will vary during training on Day 1 or between training and testing days, affecting the expression of reversal  137 responses. For example, if in the first block, accelerations occurred late in the habituation run, displacing relatively short reversal responses; but on Day 2, accelerations occurred early in the habituation run, displacing relatively long reversal responses, this may affect the mean block magnitudes of Day 1 and Day 2. Another possibility is that the number of accelerations will differ significantly between groups or experiments, thus affecting the data. The analyses that follow address these concerns. This analysis is intended to investigate overall patterns in the distribution of missing values. Consequently, only a little emphasis will be placed on individual significant results. In addition, the alpha level will not be adjusted when multiple comparisons are done; multiple comparisons are limited as no strong hypotheses as to what patterns may be evident were formed a priori. Experiment 1: Short- and lone-term habituation with a short interstimulus interval (10-s 1SF) The distribution of missing values was uneven between the training groups— overall, the massed training group had a greater proportion of missing values than the other groups, while the single stimulus control group had fewer (see Fig. 20A; distributed vs. massed training groups, number of missing values across all 80 stimuli, unpaired, two-tailed test: (40)  =  2.911,  <  .01; Fig. 20B; distributed, massed training groups, twenty stimuli control,  number of missing values across the first 20 stimuli on Day 1 and the 20 stimuli on Day 2, factorial ANOVA: f(2, 60)  =  7.043,  <  .01; Fig. 20C; distributed, massed training groups,  twenty stimuli and single stimulus controls, number of missing values from the twenty Day 2 stimuli, factorial ANOVA: E(3, 79)  =  11.998, p < .01). The unusually high proportion of  accelerations in the first twenty stimuli of the massed training group is difficult to interpret, as there were no differences in the treatment that worms in the distributed and massed training groups, as well as the twenty stimuli control group received in the first twenty stimuli.  138  Figure 20. The number of missing values in Experiments I (10-s ISI) and 2 (60-s ISI). A. The number of missing values across all training and testing in the massed (60 stimuli together on Day 1) and distributed (60 stimuli, in 3 blocks of 20 stimuli on Day 1) habituation training. B. The number of missing values totaled over the first 20 stimuli given on Day 1 and the 20 stimuli on Day 2 in the twenty stimuli control group (20 stimuli on Day 1), massed and distributed habituation training groups. C. The number of missing values in the 20 stimuli given on Day 2 in the single stimulus control group (one stimulus on Day 1), the twenty stimuli control group, and the massed and distributed habituation training groups.  139  MISSING VALUES DAY 1 AND DAY 2 EXP. 1 AND EXP. 2  A Cl) 0 LU .J  8060-  K  >cJ  10-s  ISI  60-s  ISI  0 c 40200DIST  MASSED B  40  Cr, Ui  .  >  30 20-  LJ  1 0-s  ‘SI  K  60-s  151  10-  41:  TWENTY  DIST  MASSED  C 20Cl) LU D  -J 0  >..— a..,  10-s  ‘SI  60-s  (SI  1510  CI)  41:  0 MASSED  DIST  TWENTY  SINGLE  140 When the proportion of missing values were considered across three blocks of 20 stimuli on Day 1 in the distributed and massed training groups, there was no significant change in the proportion of missing values across the blocks; however, there was a difference between the distributed and massed training groups, and a significant interaction between block and group (see Fig. 21A; mixed design ANOVA, Block: f(2, 80) 8.993, p  <  .01; Block x Group: E(2, 80)  =  5.63, p  <  =  .49,  n&; group: E(1, 40) =  .01). Interestingly, none of the three  trained groups, distributed, massed or twenty stimuli control, showed a change in the number of accelerations when the number of missing values in the data from the first 20 stimuli on Day 1 were compared to those from the 20 stimuli on Day 2 (Fig. 22A; paired, two-tailed tests; distributed: 1(20) .681,  =  massed: (20)  1.671,  =  .677, n.&; twenty stimuli control: 1(20)  =  u.). Within blocks of training, the distribution of missing values during habituation was  considered by separating the responses to the 20 stimuli into four sets of five stimuli (stimuli 1 -  5, 6 10, 11 -  -  15, 16 20). The number of missing values to the first 20 stimuli on Day 1 -  and the 20 stimuli on Day 2 were analyzed in the distributed and massed training groups using two-way repeated measures ANOVAs. In the massed training group, the number of missing values changed significantly during habituation, while in the distributed training group, the number of missing values did not vary significantly during training (Fig. 23A and 24A; massed: (3, 60)  =  7.65, p  <  .01; distributed: E(3, 60)  =  2.08, i&). The day of training did  not significantly affect the number of missing values in either group (massed: E(1, 20)  =  .46,  n&; distributed: f(l, 20) = 2.79, nj, nor was there an interaction between habituation stimuli and day of training (massed: E(3, 60)  =  .57,  n..&; distributed: E(3, 60) = 1.01, nJ.  In summary, there were significant differences between groups in the total number of missing values, but no differences in the number of missing values between the two days of training. The differences between the groups may affect the interpretation of the results from  141  i each) Figure 21. The number of missing values in the three blocks of training (20 stimul Day 1) and on Day 1 in the massed habituation training group (60 stimuli given together on stimuli the distributed habituation training group (60 stimuli given in three blocks of 20 e ÷1each). A. Experiment 1: 10-s ISI. B. Experiment 2: 60-s ISI. Error bars indicat SEMs.  142  MISSING VALUES ON DAY 1 MASSED AND DISTRIBUTED TRAINING A  EXP. 1 10-s ISI  20-  0  csJ  U  MASSED  I DIST  15Cl) Ui  D -J  >  10-  0 z Cl) Cl)  5-  4$:  0-  BLOCK 1  B  BLOCK 2  BLOCK 3  EXP. 2 60-s ISI  20—  0  MASSED 1 5-  I DIST  Cl) Ui D -J  >  10  0 z Cl) Cl)  5  4$:  0  BLOCK 1  BLOCK 2  BLOCK 3  143  Figure 22. The number of missing values on Day 1 (first 20 stimuli of training) and on Day 2 of the massed (60 stimuli given together on Day 1), distributed (60 stimuli given in 3 blocks of 20 stimuli on Day 1) and the twenty stimuli control group (20 stimuli on Day 1). A. Experiment 1: 10-s 1ST. B. Experiment 2: 60-s 1ST. Error bars indicate +1- SEMs.  MISSING VALUES DAY 1 AND 2 MASSED, DISTRIBUTED TRAINING, TWENTY STIMULI CONTROL A  EXP. 1 10-s ISI  0 CJ  EJ  Cl)  Ui D  DAY1  DAY2  -J  4  >  C’, z Cl) Co #4:  MASSED  B  01ST  TWENTY  EXP. 2 60-s ISI  0  c..J  1 Co  LU D -j  J  DAY1  I  DAY2  4 >  C!, z Co Cl)  5  44:  0  MASSED  DIST  TWENTY  144  145  Figure 23. The distribution of missing values during distributed habituation training (60 stimuli in three blocks of 20 stimuli) on Day 1 (first 20 stimuli of training) and on Day 2 (20 stimuli retention test). The number of missing values in five stimuli bins  (S 1-5,  s6-10,  si 1-15, and s16-20) was considered. A. Experiment 1: 10-s 1ST. B. Experiment 2: 60-s 1ST. Error bars indicate +1- SEMs.  146  DISTRIBUTION OF MISSING VALUES DURING DISTRIBUTED TRAINING ON DAY 1 AND DAY 2 A  EXP. 1 10-s ISI  5U)  DAY1 4-  Cl) Ui D -J  DAY2  3.  >  z Cl) Cl)  2  1•  0•  F Si  -  5  S6-1O  EXP. 2 60-s ISI  B 5. U)  IJ  4. Cl) Ui D -J  S16-20  S11-15  DAY I  •DAY2 3.  >  0 2  z  Cl) Cl)  1-  0 Si  -  5  S6  -  10  Sli  -  15  S16  -  20  147  Figure 24. The distribution of missing values during massed habituation training (60 stimuli given together) on Day 1 (first 20 stimuli of training) and on Day 2(20 stimuli retention test). The number of missing values in five stimuli bins (sl-5, s6-10, sl 1-15, and s16-20) was considered. A. Experiment 1: 10-s 1ST. B. Experiment 2: 60-s 1ST. Error bars indicate +1- SEMs.  148  DISTRIBUTION OF MISSING VALUES DURING MASSED TRAINING ON DAY 1 AND DAY 2 A  EXP. 1 10-s ISI  5—  LI)  Cl) Ui D -J  DAY1  4-  DAY2 3-  > C.,  z  2-  Cl) Cl) •1—  0S1-5  S6-10  B  S11-15  S16-20  EXP. 2 60-s ISI  LI)  LI DAY 1  Cl) Ui  DAY2  D -J  >  C., z Cl) Cl)  Si  -  5  S6  -  10  Sil  -  15  S16  -  20  149 direct comparisons between the groups. The findings that the day of training did not affect the total number of missing values or the distribution of missing values during habituation within each block, taken together, indicate that whatever differences are apparent in the levels of responding on Day 1 and Day 2 in this experiment cannot be attributed to changes in the frequency of acceleration responses. Thus, the presence of accelerations in the data does not limit the interpretation of differences within groups in the reversal response magnitude data. Experiment 2: Short- and long-teim habituation with a long interstimulus interval (60 s The distribution of missing values was found to be even across groups (Fig. 20B; distributed vs. massed, number of missing values in all 80 stimuli: unpaired, two-tailed i test, (39)  =  1.75 1, j; Fig. 20B; distributed, massed and twenty stimuli control, number of  missing values in the first 20 stimuli on Day 1 and the 20 Day 2 stimuli: factorial ANOVA, E(2, 58)  =  1.941,  n.&; Fig. 20C; distributed, massed, twenty stimuli control, and single  stimulus control, number of missing values in the 20 Day 2 stimuli: factorial ANOVA, E(3, 76) =  2.544, When the distributions of missing values were considered across three blocks of 20  stimuli on Day 1 in the distributed and massed training groups, a significant change across the blocks was observed, but no difference between the groups and no interactions were evident (see Fig. 21B; mixed design ANOVA, Blocks 1 to 3: E(2, 78) [distributed vs. massed]: E(1, 39)  =  1.026,  =  5.374,  <  .01; groups  The massed training group and twenty  stimuli control group both showed a significantly higher number of missing values on Day 2 than in the first 20 stimuli on Day 1 (Fig. 22B; paired, two-tailed tests; massed: (19) 3.337,  <  .01; twenty stimuli control: 1(19)  not (distributed: (20)  =  .81,  =  3.15 1,  <  =  .01), while the distributed group did  n.&).  Within blocks of training, the distribution of missing values during habituation was considered by separating the 20 stimuli in a training block into four sets of five  150 stimuli (stimuli 1 5, 6 10, 11 -  -  -  15, and 16 20). The first 20 stimuli on Day 1 and -  the 20 stimuli on Day 2 were analyzed in the distributed and massed training groups using mixed design ANOVAs. In the distributed group, there was no significant effect of training during habituation while in the massed training group, the number of missing values changed significantly during habituation (Fig. 23B and 24B; distributed: E(3, 60)  =  2.68,  j;  massed: (3, 57)  =  6.06,  <  .01). In the distributed group,  there was no significant change in the number of missing values from Day 1 to Day 2, while in the massed group there was a significant increase in the number of missing values on Day 2 (distributed: E(1, 20)  =  .66,  n; massed: E(1, 19)  =  11.14,  <  .01).  Most importantly, however, in neither group was there a significant interaction between habituation stimuli and day of training (distributed: f(3, 120) 114)  =  .18,  jj).  =  .40, n; massed: E(3,  As can be seen in Figures 22 and 23, the distribution of missing  values during habituation follows the same pattern on Day 1 and 2 in both the distributed and massed training groups. In summary, there were no differences between groups in the proportion of missing values overall; however, there were differences between Day 1 and Day 2 in two groups, and there was a significant pattern in the distribution of missing values during habituation within a block in the massed training group. To counter this finding, the absence of a significant interaction between day of training and habituation stimuli indicates that the pattern of missing value distribution during habituation within blocks is the same on Day 1 and 2 in both groups. This is a critical point, as it demonstrates that any difference in the response levels between Day 1 and 2 exhibited by these groups cannot be attributed to a change in the distribution of acceleration responses during habituation within blocks. In addition, as all trained groups exhibited an increase in the proportion of accelerations on Day 2 over Day 1, so any difference  151 between the groups in the change in level of responding from Day 1 to Day 2 cannot be attributed to the change in the proportion of accelerations. Thus, although the proportion of accelerations did vary during training, the presence of missing values in the data does not limit the interpretation of the reversal response magnitude data in Exp. 2. The effects of three types of heat-shock treatments on the distribution of missing values during habituation on Day 1 and Day 2 require examination: heat shock before training on Day 1, as used in Experiments 4 and 6; heat shock during training, as used in Experiments 4, 6 and 8; and heat shock before testing on Day 2, as used in Experiment 7. Experiment 4: the effects of heat shock (45 mm. 32°C) before training on the distribution of missing values An examination of the distribution of missing values between groups LTH and PRE HS / LTH, the days of training within those groups, the blocks of training on Day 1, and within the first block on Day 1 and the test block on Day 2 showed that heat shock (45 mm, 32°C) before training had no effect on the propensity to accelerate to tap. The distribution of missing values was not affected by the day of training in either group (see Fig. 25A; mixed design ANOVA; Group: E(1, 38) 38)  =  .996,  n;  Group x Day: E(1, 38)  =  =  .120, n&; Day: E(1,  .008, n.&). The mean number of missing  values did not change across the three blocks of training on Day 1 for either group (see Fig. 25B; mixed design ANOVA; Group: E(1, 76) .252,  n;  Group x Block: E(2, 76)  =  .658,  =  Block: (2, 38)  1.599,  =  n).  Finally, the distribution of missing values within blocks of training was considered by separating the responses to the 20 stimuli within blocks of training into four sets of five stimuli (stimuli 1 5, 6 -  -  10, 11  -  15, and 16 20). When the first -  152  Figure 25. The number of missing values in Experiment 4: pre-exposure to heat shock. A. the number of missing values on Day 1 (first 20 stimuli of training) and on Day 2 (the 20 stimuli of the retention test) in the LTH group (distributed training at a 60-s 1ST) and the PRE HS I LTH group (distributed training with pre-exposure to heat shock (45 mm, 32°C) ending 2 h before training begins). B. The number of missing values for the three blocks of training on Day 1 (each 20 stimuli) in the LTH and PRE HS I LTH groups. Error bars indicate +1- SEMs.  153  EXPERIMENT 3: PRE-EXPOSURE TO HEAT SHOCK A  DAY 1 AND DAY 2  0 C4  DAY2  Co Lii D -j  >  CD  z  CO Co  PREHS/LTH  LTH  B  20  BLOCKS ON DAY 1  0  csJ  D  15 Co Lii D  BLK 1 BLK 2  -j  >  10  BLK 3  CD  z Co Cl)  S  4*:  0  LTH  PREHS/LTH  154  Figure 26. The distribution of missing values during training in the LTH and PRE HS / LTH groups on Day 1 (first 20 stimuli of training) and on Day 2 (20 stimuli retention test).  The number of missing values in five stimuli bins (sl-5, s6-l0, si 1-15, and s16-20) was considered. A. LTH group (distributed training at a 60-s ISI). B. PRE HS / LTH group (distributed training at a 60-s ISI preceded by a heat shock (45 mm, 32°C). Error bars indicate +1- SEMs.  155  MISSING VALUES AFTER PRE-EXPOSURE TO HEAT SHOCK EXPERIMENT 4 A  LTH  U)  E:r  Cl)  uJ  D -J  DAY2  > C.,  z  C,, Cl)  S1-5  S6-iO  Sii-15  S16-20  PRE HS I LTH 4— .  3-  DAY1 DAY2  2-  1—  0-  1  Si-5  S6-i0  Sii-15  Si 6-20  156 block of training on Day 1 and the test block on Day 2 were examined, the distribution of missing values was found to be even within each block; furthermore, no significant differences were seen between days in either group (see Fig. 26A & B; mixed design ANOVA; Day: E(1,38) approximation =  .945,  =  11.85,  =  1.00, n.&; Mauchly’s sphericity test, chi-square <.05, Huynh-Feldt epsilon  n.; Day x Training: E(3, 114)  x Day: E(1,38)  =  =  .18,  =  .94; Training: E(2.82, 107.16)  Group: E(1,38)  .01, ; Group x Day x Training: E(3, 114)  =  .12,  =  Group The  2.00,  comparisons of the two groups of Experiment 4, LTH and PRE HS I LTH provide evidence that heat shock before training does not affect the propensity to accelerate to tap. ExperimentS: the effects of heat shock (45 mm. 32°C) during training on the distribution of missing values It is possible that the distribution of missing values during habituation changes with the presentation of heat shock during training and that this change affects the expression of habituation on Day 1 or on Day 2. This possibility was examined by an analysis of the distribution of missing values on Day 1 across blocks of training and between Day 1 (Block 1) and Day 2 of the LTh-HS group in Experiment 5 and the LTH of Experiment 2 (distributed training group, 60-s ISI) with mixed design ANOVAs (see Fig. 27A & B). In the analysis of the distribution of missing values on Day 1, there was no significant overall difference in the number of missing values between the LTH-HS and the LTH groups (Group: E(1, 37)  =  .59,  Across the  groups, the number of missing values between blocks changed significantly (Block: E(2, 74)  =  3.96,  =  .02); however, the distribution of missing values across blocks  was not significantly different between the groups (Block x Group: E(2, 74) jj.  =  1.35,  The distribution of missing values was uneven with the blocks of training  157  Figure 27. LTH-HS (Exp. 5) and LTH (Exp. 2; distributed training) and the distribution of missing values during habituation training on Day 1. The number of missing values in five stimuli bins (sl-5, s6-10, sll-15, and s16-20) was considered. A. LTH group (Experiment 2, distributed training at a 60-s 1ST) across three blocks of training on Day 1. B. LTH-HS group (Experiment 5, distributed training at a 60-s 1ST, with heat shock (45 mm, 32°C) between blocks of training) across three blocks of training on Day 1. Error bars indicate +1- SEMs.  158  DISTRIBUTION OF MISSING VALUES DURING TRAINING ON DAY 1 •A  LTH (EXP. 2)  5  JBLK1 b-..  ØBLK2 D  •BLK3  3-  z  2-  Cl) Cl)  1-  0-  Si  -  5  B  S6-i0  Sii-i5  S16-20  LTH-HS (EXP. 5) DBLK1  .  4-  t3BLK2  LU ••J  C_I,  z  •BLK3  3  -  iTr.  1 I  HL1 0 Si  -  5  S6  -  10  Sii  -  15  Si6  -  20  159  Figure 28. The distribution of missing values during training in the LTH (Experiment 2) and LTH-HS (ExperimentS) groups on Day 1 (first 20 stimuli of training) and on Day 2 (20 stimuli retention test). The number of missing values in five stimuli bins (s 1-5, s6- 10, si 1-15, and s16-20) was considered. A. LTH group (distributed training at a 60-s 1ST). B. LTH-HS group (distributed training at a 60-s ISI with heat shock (45 mm, 32°C) between blocks of training). Error bars indicate +1- SEMs.  160  DISTRIBUTION OF MISSING VALUES ON DAY 1 AND DAY 2 A 5U) Co LU  LTH (EXP. 2)  4.  EJ  DAY1  •  DAY2  3.  -J ‘I  2  z Cl) Cl)  1•  0-  Si  B  -  5  -I  S6-iO  Sli-15  LTH-HS (EXP. 5)  5-  U)  EJ  4Cl) LU D -J  S16-20  DAY1  • 3-  > 1%  z  2-  Cl) U)  1•  0•  1 Si  -  5  S6-i0  Sli -15  S16  -  20  161 (Training: E(3, 111)  =  10.71, p  <  .01); again, however, there was no significant  difference between the groups in this distribution (Training x Group: E(3, 111)  n&).  =  2.21,  Finally, there was no significant difference in the distribution of missing values  within blocks of training across blocks (Training x Block: E(6, 222)  =  .36,  n). or in  the expression of this between the groups (Training x Block x Group: E(6, 222)  =  .71,  The introduction of heat shock treatments (45 mm, 32°C) during training did not seem to affect the number or the distribution of missing values during training on Day 1. The distribution of missing values on Day 1 and Day 2 was considered in the LTH-HS group of Experiment 5 and the LTH group of Experiment 2 (distributed training, 60-s ISI; see Fig. 28A & B). There was no significant overall difference in the number of missing values between these groups (Group: (1, 37)  =  .52, n.s.).  The number of missing values did not differ significantly between Day 1 and Day 2 (Day: E(1, 37)  2.60, j, nor was it different between groups (Group: E(1, 37)  =  .12, jj. The distribution of missing values was uneven within blocks of training (Training: E(3, 111)  5.07, p < .01). However, this distribution of missing values  did not change significantly between Day 1 and Day 2 (Training x Day: E(3, 111)  =  .92, nj; nor did this pattern alter between the groups (Training x Group: f(3, 111)  =  1.59, Overall, these analyses suggest that heat shock (45 min, 32°C) did not affect the number of missing values. Therefore any difference observed in habituation on Day 1 or long-term habituation on Day 2 between these groups cannot be attributed to a change in the number or distribution of missing values seen here.  162 Experiment 6: the effects of heat shock (45 mm. 32°C) before training and heat shock (45 mm. 32°C) during training on the distribution of missing values between Day 1 and Day 2. In Experiment 6, the number of missing values seen in the LTH, LTH-HS and PRE HS / LTH-HS groups may be compared across days of training. The overall number of missing values expressed by the groups did not differ significantly (see Fig. 29A, B, & C; Group: E(2, 56)  =  .64,  n&).  There was no significant difference  between days of training in the number of missing values (Day: E(1, 56)  =  3.73,  nor was this distribution altered by heat shock treatments (Day x Group: F(2, 56) 1.68,  =  The distribution of missing values was uneven across training within  blocks (Training: (3, 168)  =  16.47, p  <  .0 1); however, this distribution was not  different across groups (Training x Group: E(6, 168) days of training (Training x Day: E(3, 168)  =  .84,  =  n).  1.64, n), or between the Finally, the distribution of  missing values within blocks of training across days of training was not significantly altered by the heat shock treatments (Training x Day x Group: E(6, 168)  =  1.35,  Overall, it appears that these heat shock treatments do not alter the distribution of missing values seen in these data. Therefore, any differences between the groups or days of training in the reversal magnitudes observed cannot be attributed to the number or distribution of missing values. Experiment 7: the effects of heat shock (45 mm. 32°C) on Day 2 on the number and distribution of missing values The possibility that the heat shock on Day 2 affected the distribution or number of missing values on Day 2 was examined with a mixed design ANOVA (Group x Day x Training). There was no significant difference overall in the number of missing values between groups (see Fig. 30A & B; LTH vs. LTH / D2 HS; Group: E(1, 37)  =  163  Figure 29. The distribution of missing values during training in the LTH, LTH-HS and the PRE HS I LTH-HS groups on Day 1 (first 20 stimuli of training) and on Day 2 (20 stimuli retention test). The number of missing values in five stimuli bins (sl-5, s6-10, sl 1-15, and s16-20) was considered. A. LTH group (distributed training at a 60-s 1ST). B. LTH HS group (distributed training at a 60-s 1ST with heat shock (45 mm, 32°C) between blocks of training). C. PRE HS I LTH-HS group (distributed training at a 60-s 1ST preceded by heat shock (45 mm, 32°C) and with heat shock (45 mm, 32°C) during training. Error bars indicate +1- SEMs.  MISSING VALUES IN EXPERIMENT 6 A U)  z  LTH  5.43-  —  164  E1  DAY1  •  DAY2  2I— 0S1-5  B  5-  U)  4-  z w  S6-lO  S11-15  S16-20  LTH-HS  D  DAY1  •  321— 0SI-5  5 C  z  ‘4)  4-  C1)  3-  S6-10  SI1-15  S16-20  PRE HS I LTH-HS  D  DAY1 DAY2  —  21— 0-  SI -5  S6-10  S1I-15  S16-20  165  Figure 30. The distribution of missing values during training in the LTH and LTH I D2HS groups on Day 1 (first 20 stimuli of training) and on Day 2 (20 stimuli retention test). The number of missing values in five stimuli bins (s 1-5, s6- 10, si 1-15, and s 16-20) was considered. A. LTH group (distributed training at a 60-s 1ST). B. LTH I D2HS group (distributed training at a 60-s 1ST with heat shock (45 mm, 32°C) on Day 2 ending 2 h before the retention test). Error bars indicate +1- SEMs.  166  MISSING VALUES IN EXPERIMENT 7 LTH  A U)  DAY1  C,) LU  DAY2  D  -I  > C!,  z  Cl) Cl)  S1-5  B  S6-1O  S1i-15  S16-20  LTH I D2HS  E:J  U)  DAY1 DAY2  Cl) Lii D -J  > C!,  z  Cl) Cl) *  Si -5  S6-1O  Si1-15  Si6-20  167 .64, n). The number of missing values did not differ overall with the day of training (Day: E(1, 37)  =  1.88,  n), nor was there a significant effect of the Day 2 heat shock  on this distribution of missing values across days of training (Group x Day: E(1, 37)  =  1.88, The distribution of missing values within blocks of training varied significantly (Training: E(3, 111)  =  3.60, p = .02). However, this distribution was not affected by  the heat shock on Day 2 (Training x Day: f(3, 111)  =  .57, n). In addition the  distribution of missing values within blocks of training did not differ significantly between days of training (Training x Day: F(3, 111)  =  nor was this affected  .89,  by the heat shock on Day 2 (Training x Group x Day: f(3, 111)  =  .32,  n.&).  These results indicate that the number and distribution of missing values was not affected by heat shock on Day 2. This implies that any differences in performance observed in this experiment cannot be attributed to the number or distribution of missing values. Experiment 8: the effects of brief heat shock on the number and distribution of missing values The effects of brief heat shock (15 mm, 32°C) during training may be different than that of the longer heat shock (45 min, 32°C) given during training in Experiments 5 and 6. To examine this, a mixed design ANOVA (Group x Day x Training) was performed. It was found that the overall number of missing values differed significantly between the groups (see Figures 31 and 32; Group: E(3, 76)  =  3.00, p =  .04). In addition, the overall number of missing values differed significantly between the days of training (Days: E(1, 76)  =  9.95, p < .01), and the difference between Day 1  and Day 2 was affected by the group (Group x Day: E(3, 76)  =  .3.34, p =.02).  168  Figure 31. The distribution of missing values during training in the LTH and LTH-early HS groups on Day 1 (first 20 stimuli of training) and on Day 2 (20 stimuli retention test). The number of missing values in five stimuli bins (sl-5, s6-10, sll-15, and s16-20) was considered. A. LTH group (distributed training at a 60-s ISI). B. LTH-early HS group (distributed training at a 60-s 1ST with heat shock (15 mm, 32°C) during training from 0 to 15 min in the 1-h interval following each training block). Error bars indicate ÷1- SEMs.  169  MISSING VALUES IN EXPERIMENT 8 LTH  A U,  .  b.-.  4  Cl)  DAY1 DAY2  Ui D .-J  < >  (  z  2-  Cl) Cl) 1*  0Si-S  B  5-  S6-10  S11-15  LTH-early  HS  S16-20  U,  E:  4Co Ui  DAY2  D -J  DAY1  3-  >  z  2-  Co Co  1•  0• Si-5  S6-i0  S11-15  Si 6-20  170  Figure 32. The distribution of missing values during training in the LTH-mid HS and LTH-late HS groups on Day 1 (first 20 stimuli of training) and on Day 2 (20 stimuli retention test). The number of missing values in five stimuli bins (sl-5, s6-10, sl 1-15, and s16-20) was considered. A. LTH-mid HS group (distributed training at a 60-s 1ST with heat shock (15 mm, 32°C) during training from 15 to 30 mm in the 1-h interval following each training block). B. LTH-late HS group (distributed training at a 60-s 1ST with heat shock (15 min, 32°C) during training from 30 to 45 mm in the 1-h interval following each training block). Error bars indicate +1- SEMs.  171  MISSING VALUES IN EXPERIMENT 8 A  LTH-mid HS  .  It)  DAY1  4. U)  DAY2  Lii  -J  3.  > C.,  2  z  Cl) Cl)  1•  0• Si -5  .. 5 B  S6-iO  LTH-Iate  S11-15  L  Si6-20  HS  L()  4Cl) UI D 1  J  DAY1 DAY2  3-  >  C.,  z  2-  U) U)  1-  0Si-5  S6-iO  S11-15  S16-20  172 The distribution of missing values was uneven during habituation (Training: E(3, 228)  =  24.86, p  <  .01); however, this distribution was not affected by the group  or by the day of training (Day x Training: E(3, 228) E(9, 228)  =  1.77,  =  1.43, n; Group x Training:  Day x Training x Group: E(9, 228)  =  1.40,  n.&).  An inspection of Figures 31 and 32 suggests that while there was variation in the number of missing values in the data between groups, that, overall, this difference between groups does not affect the distribution of missing values within training blocks. The significant interaction between group and day of training in the number of missing values found does not seem to follow any distinct pattern (for example, it does not appear to be different between the groups that received heat shock and the group that did not). Analysis of the number and distribution of missing values in the LTH and LTH-HS groups As was done for the habituation data (Chapter Nine, synthesis of results), it is possible to examine the LTH groups together and the LTH-HS groups together, in this case, to look for effects of experiment on the number and distribution of missing values. A mixed design ANOVA (Experiment x Day x Training) was performed on the five LTH groups (Exp. 2, 4, 6, 7, and 8) and the two LTH-HS groups (Exp. 5 and 6). In the analysis of the number and distribution of missing values in the data of the LTH groups, it was found that there was a significant difference in the number of missing values between experiments (see Figure 33, Experiment: E(4, 94)  =  3.03, p =  .02). The difference in the number of missing values between Day 1 and Day 2 was very nearly significant (E(l, 94)  =  3.76,  The observed difference in the number  of missing values between experiments did not interact with the distribution of missing values between days of training (Day x Experiment: F(4, 94)  =  1.01,  The  173  Figure 33. The distribution of missing values during training in all the LTH groups (Exp. 2, 4, 6, 7 and 8) on Day 1 (first 20 stimuli of training) and on Day 2 (20 stimuli retention test). The number of missing values in five stimuli bins (sl-5, s6-10, si 1-15, and s16-20) was considered. A. Day 1. B. Day 2. Error bars indicate +1- SEMs.  174  MISSING VALUES IN ALL LTH GROUPS A  DAY1  5  U) 3  0  EXP.2  I  EXP.4  0  EXP. 6  ISI  EXP. 7  EXP.6  >  0  z  S1-5  S6-1O  B  LC  S11-15  DAY2  4  0  XP. 2  L  EXP. 4  0  EXP. 6  D  EXP. 7  EXP.8  Cl) LU -J  S16-20  3  S1-5  S6-1O  S11-15  S16-20  175 distribution of missing values across habituation training was uneven (Mauchly’s test of sphericity, chi-square approximation Training: E(2.85, 267.9)  =  12.86,  <  =  16.46,  <  .01, Hunyh-Feldt epsilon = .95;  .01) and this distribution differed between  experiments (Training x Experiment: E(11.4, 267.9)  =  2.32,  <  .01).  However, the distribution of missing values during habituation training was not different between days of training (Day x Training: F(3, 282)  =  1.19, n.s.), which  suggests that any difference between Day 1 and Day 2 in the level of responding cannot be attributed to a change in the distribution of missing values during habituation. In addition, the distribution of missing values during habituation on Day 1 and on Day 2 is not affected by Experiment (Experiment x Training x Day: F(12, 282)  .65, n.s.).  Interestingly, these two experiments (Exp. 4 and 8) were run during the same 4-month period, though by different experimenters; worms for both experiments were drawn from the same worm colonies. Although the strain of worms used throughout these experiments remained constant and genetic variation should be slight, apparent differences in the propensity to accelerate to tap have been observed in the past in this laboratory (Rankin, Beck and Chiba, unpublished observations). It is possible that some factor affecting behavior such as slight changes in the temperature of the training environment (20° 24 °C) or colony population conditions alters the propensity to -  accelerate to tap, resulting in a lower mean number of accelerations overall in these experiments. Overall, an increase in the number of accelerations observed from Day 1 to Day 2 in the LTH groups is not surprising because it has been observed that greater habituation is accompanied by a greater proportion of accelerations (Wicks and Rankin, unpublished observations). The interaction between Day and Experiment may be produced by the loss of this pattern in the experiments such as Exp. 4 and 8 when the  176 number of accelerations becomes too low to observe this relationship between the  habituation process and acceleration distribution. The number and distribution of missing values was examined in the LTH-HS groups with a mixed design ANOVA (Day x Training x Experiment). It was found that the number of missing values did not vary significantly between experiments (see Figure 34; Experiment: F(1, 36)  =  1.03, n.s.); however, there was a significant  increase overall in the number of missing values between Day 1 and Day 2 (Day: F( 1, 36)  =  7.97, p  <  .01). This difference between Day 1 and Day 2 was not affected by  experimental factors (Experiment x Day: F( 1, 36)  =  .08, n.s.). The significant effect of  Day is interesting as it may have been expected that the number of accelerations would remain constant between days of training in the LTH-HS, which showed no long-term habituation between Day 1 and Day 2. It is clear from these results that the difference between the LTH and LTH-HS groups in the expression of long-term habituation is probably not produced by a difference between the conditions in the distribution of missing values between Day 1 and Day 2. There was a significantly uneven distribution of missing values across habituation training (Training:  E(3, 108) 7.73, p < .01). This pattern did not differ  significantly across experiments (Training x Experiment: E(3, 108) across days of training (Day x Training: E(3, 108) Training: E(3, 108)  =  2.43,  =  .32,  j;  =  .15, ) or  Day x Experiment x  n).  Overall, the analysis of missing values across the LTH and LTH-HS groups indicates that there are variations between experiments in the number and distribution of missing values observed that are not related to the training condition. These differences in the number and distribution of missing values may bear some relationship to the qualities of the expression of habituation and the unknown factors which affect  177  Figure 34. The distribution of missing values during training in both of the LTH-HS groups (Exp. 5 and 6) on Day 1 (first 20 stimuli of training) and on Day 2 (20 stimuli retention test). The number of missing values in five stimuli bins (s 1-5, s6- 10, sil- 15, and s16-20) was considered. A. Day 1. B. Day 2. Error bars indicate +1- SEMs.  178  MISSING VALUES IN ALL LTH-HS GROUPS A  DAY1  5.  Lf)  Cl) LU -J  4.  D  EXP.5  •  EXP. 6  3.  >  z  2-  T  T  Tr  ILi  Cl) U)  I  1-  0-  iI Si -5  B  S 6-10  DAY2  5.  4. Cl) Ui  D -J  S 16-20  S11-15  D  EXP. 5  •  EXP. 6  3.  >  z  2  T  U) Cl)  1—  T J  T I  I1T  41:  0Si-5  S6-1 0  S11-15  S 16-20  179  performance between experiments. However, as seen in the overall analysis of the habituation data (Chapter Nine, synthesis of results), the expression of long-term habituation and the effect of heat shock during training on LTH seems to be robust to these variations.  

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