Massed and spaced learning in honeybees: the role of CS, US, the intertrial interval, and the test interval

Conditioning the proboscis extension reflex of harnessed honeybees (Apis mellifera) is used to study the effect temporal spacing between successive conditioning trials has on memory. Retention is monitored at two long-term intervals corresponding to early (1 and 2 d after conditioning) and late long-term memory (3 and 4 d). The acquisition level is varied by using different conditioned stimuli (odors, mechanical stimulation, and temperature increase at the antenna), varying strengths of the unconditioned stimulus (sucrose), and various numbers of conditioning trials. How learning trials are spaced is the dominant factor both for acquisition and retention, and although longer intertrial intervals lead to better acquisition and higher retention, the level of acquisition per se does not determine the spacing effect on retention. Rather, spaced conditioning leads to higher memory consolidation both during acquisition and later, between the early and long-term memory phases. These consolidation processes can be selectively inhibited by blocking protein synthesis during acquisition.     

Food Avoidance Learning in Squirrel Monkeys and Common Marmosets

Using a conditioned food avoidance learning paradigm, six squirrel monkeys (Saimiri sciureus) and six common marmosets (Callithrix jacchus) were tested for their ability to (1) reliably form associations between visual or olfactory cues of a potential food and its palatability and (2) remember such associations over prolonged periods of time. We found (1) that at the group level both species showed one-trial learning with the visual cues color and shape, whereas only the marmosets were able to do so with the olfactory cue, (2) that all individuals from both species learned to reliably avoid the unpalatable food items within 10 trials, (3) a tendency in both species for quicker acquisition of the association with the visual cues compared with the olfactory cue, (4) a tendency for quicker acquisition and higher reliability of the aversion by the marmosets compared with the squirrel monkeys, and (5) that all individuals from both species were able to reliably remember the significance of the visual cues, color and shape, even after 4 months, whereas only the marmosets showed retention of the significance of the olfactory cues for up to 4 weeks. Furthermore, the results suggest that in both species tested, illness is not a necessary prerequisite for food avoidance learning but that the presumably innate rejection responses toward highly concentrated but nontoxic bitter and sour tastants are sufficient to induce robust learning and retention.     

Decision-making and associative color learning in harnessed bumblebees (Bombus impatiens)

In honeybees, the conditioning of the proboscis extension response (PER) has provided a powerful tool to explore the mechanisms underlying olfactory learning and memory. Unfortunately, PER conditioning does not work well for visual stimuli in intact honeybees, and performance is improved only after antennal amputation, thus limiting the analysis of visual learning and multimodal integration. Here, we study visual learning using the PER protocol in harnessed bumblebees, which exhibit high levels of odor learning under restrained conditions. We trained bumblebees in a differential task in which two colors differed in their rewarding values. We recorded learning performance as well as response latency and accuracy. Bumblebees rapidly learned the task and discriminated the colors within the first two trials. However, performance varied between combinations of colors and was higher when blue or violet was associated with a high reward. Overall, accuracy and speed were negatively associated, but both components increased during acquisition. We conclude that PER conditioning is a good tool to study visual learning, using Bombus impatiens as a model, opening new possibilities to analyze the proximate mechanisms of visual learning and memory, as well as the process of multimodal integration and decision-making.     

A cGMP-dependent protein kinase gene, foraging, modifies habituation-like response decrement of the giant fiber escape circuit in Drosophila

The Drosophila giant fiber jump-and-flight escape response is a model for genetic analysis of both the physiology and the plasticity of a sensorimotor behavioral pathway. We previously established the electrically induced giant fiber response in intact tethered flies as a model for habituation, a form of nonassociative learning. Here, we show that the rate of stimulus-dependent response decrement of this neural pathway in a habituation protocol is correlated with PKG (cGMP-Dependent Protein Kinase) activity and foraging behavior. We assayed response decrement for natural and mutant rover and sitter alleles of the foraging (for) gene that encodes a Drosophila PKG. Rover larvae and adults, which have higher PKG activities, travel significantly farther while foraging than sitters with lower PKG activities. Response decrement was most rapid in genotypes previously shown to have low PKG activities and sitter-like foraging behavior. We also found differences in spontaneous recovery (the reversal of response decrement during a rest from stimulation) and a dishabituation-like phenomenon (the reversal of response decrement evoked by a novel stimulus). This electrophysiological study in an intact animal preparation provides one of the first direct demonstrations that PKG can affect plasticity in a simple learning paradigm. It increases our understanding of the complex interplay of factors that can modulate the sensitivity of the giant fiber escape response, and it defines a new adult-stage phenotype of the foraging locus. Finally, these results show that behaviorally relevant neural plasticity in an identified circuit can be influenced by a single-locus genetic polymorphism existing in a natural population of Drosophila.     

Inhibitory neurotransmission and olfactory memory in honeybees

In insects, gamma-aminobutyric acid (GABA) and glutamate mediate fast inhibitory neurotransmission through ligand-gated chloride channel receptors. Both GABA and glutamate have been identified in the olfactory circuit of the honeybee. Here we investigated the role of inhibitory transmission mediated by GABA and glutamate-gated chloride channels (GluCls) in olfactory learning and memory in honeybees. We combined olfactory conditioning with injection of ivermectin, an agonist of GluCl receptors. We also injected a blocker of glutamate transporters (L-trans-PDC) or a GABA analog (TACA). We measured acquisition and retention 1, 24 and 48 h after the last acquisition trial. A low dose of ivermectin (0.01 ng/bee) impaired long-term olfactory memory (48 h) while a higher dose (0.05 ng/bee) had no effect. Double injections of ivermectin and L-trans-PDC or TACA had different effects on memory retention, depending on the doses and agents combined. When the low dose of ivermectin was injected after Ringer, long-term memory was again impaired (48 h). Such an effect was rescued by injection of both TACA and L-trans-PDC. A combination of the higher dose of ivermectin and TACA decreased retention at 48 h. We interpret these results as reflecting the involvement of both GluCl and GABA receptors in the impairment of olfactory long-term memory induced by ivermectin. These results illustrate the diversity of inhibitory transmission and its implication in long-term olfactory memory in honeybees.     

Trace classical conditioning in rainbow trout (Oncorhynchus mykiss): what do they learn?

There are two main memory systems: declarative and procedural memory. Knowledge of these two systems in fish is scarce, and controlled laboratory studies are needed. Trace classical conditioning is an experimentally tractable model of declarative memory. We tested whether rainbow trout (Oncorhynchus mykiss) can learn by trace conditioning and form stimulus–stimulus, as opposed to stimulus–response, associations. We predicted that rainbow trout trained by trace conditioning would show appetitive behaviour (conditioned response; CR) towards the conditioned stimulus (CS; light), and that the CR would be sensitive to devaluation of the unconditioned stimulus (US; food). The learning group (L, N = 14) was trained on a CS + US contingency schedule with a trace interval of 3.4 s. The control group (CtrL, N = 4) was kept on a completely random schedule. The fish that learnt were further trained as either an experimental (L, N = 6) or a memory control (CtrM, N = 3) group. The L group had the US devalued. The CtrM group received only food. No fish in the CtrL group, but nine fish from the L group conditioned to the light. When tested, five L fish changed their CRs after US devaluation, indicating learning by stimulus–stimulus association of the light with the food. CtrM fish retained their original CRs. To the best of our knowledge, this experiment is the first to show that rainbow trout can learn by trace classical conditioning. The results indicate that the fish learnt by ‘facts-learning’ rather than by reflex acquisition in this study.     

Genetic dissociation of acquisition and memory strength in the heat-box spatial learning paradigm in Drosophila

Memories can have different strengths, largely dependent on the intensity of reinforcers encountered. The relationship between reinforcement and memory strength is evident in asymptotic memory curves, with the level of the asymptote related to the intensity of the reinforcer. Although this is likely a fundamental property of memory formation, relatively little is known of how memory strength is determined. Memory performance at different levels in Drosophila can be measured in an operant heat-box conditioning paradigm. In this spatial learning paradigm, flies learn and remember to avoid one-half of a dark chamber associated with a temperature outside of the preferred range. The reinforcement temperature has a strong effect on the level of learning in wild-type flies, with higher temperatures inducing stronger memories. Additionally, two mutations alter memory-acquisition curves, either changing acquisition rate or asymptotic memory level. The rutabaga mutation, affecting a type-1 adenylyl cyclase, decreases the acquisition rate. In contrast, the white mutation, modifying an ABC transporter, limits asymptotic memory. The white mutation does not negatively affect classical olfactory conditioning but actually improves performance at low reinforcement levels. Thus, memory acquisition/memory strength and classical olfactory/operant spatial memories can be genetically dissociated. A conceptual model of operant conditioning and the levels at which rutabaga and white influence conditioning is proposed.     

Unimpaired trace classical eyeblink conditioning in Purkinje cell degeneration (pcd) mutant mice

Young adult Purkinje cell degeneration (pcd) mutant mice, with complete loss of cerebellar cortical Purkinje cells, are impaired in delay eyeblink classical conditioning. In the delay paradigm, the conditioned stimulus (CS) overlaps and coterminates with the unconditioned stimulus (US), and the cerebellar cortex supports normal acquisition. The ability of pcd mutant mice to acquire trace eyeblink conditioning in which the CS and US do not overlap has not been explored. Recent evidence suggests that cerebellar cortex may not be necessary for trace eyeblink classical conditioning. Using a 500 ms trace paradigm for which forebrain structures are essential in mice, we assessed the performance of homozygous male pcd mutant mice and their littermates in acquisition and extinction. In contrast to results with delay conditioning, acquisition of trace conditioning was unimpaired in pcd mutant mice. Extinction to the CS alone did not differ between pcd and littermate control mice, and timing of the conditioned response was not altered by the absence of Purkinje cells during acquisition or extinction. The ability of pcd mutant mice to acquire and extinguish trace eyeblink conditioning at levels comparable to controls suggests that the cerebellar cortex is not a critical component of the neural circuitry underlying trace conditioning. Results indicate that the essential neural circuitry for trace eyeblink conditioning involves connectivity that bypasses cerebellar cortex.     

Olfactory fear conditioning paradigm in rats: Effects of midazolam,propranolol or scopolamine

In rodents, fear conditioned responses are more pronounced toward olfactory stimulus, since olfaction is a dominant sense in these subjects. The present study was outlined to investigate if the association between coffee odor (CS1) and electrical footshock (US) would be an effective model for the study of fear-induced behavior and whether compounds used in humans for emotional-related disorders such as midazolam, propranolol, or scopolamine, applied during the different stages of fear conditioning (acquisition, consolidation and expression), affect the defensive responses to both, the olfactory CS1, and the context (CS2) where the CS1 had been presented (second order conditioning). The results revealed that five pairings between coffee odor (CS1) and electrical footshock (US) were able to elicit consistent defensive responses and a second order conditioning to the context (CS2). Midazolam (0.375–0.5 mg/kg; i.p.) treatment was able to interfere with the CS1–US association and with the consolidation of the aversive information. The propranolol (5–10 mg/kg; i.p.) treatment interfered with the CS1–US association, with the retention of fear memory and with the CS1–CS2 association. Propranolol also attenuated the expression of conditioned fear responses when applied before the CS1 test session. Scopolamine (0.6–1.2 mg/kg; i.p.) treatment impaired the acquisition of CS1–US and CS1–CS2 associations, and also disrupted the expression of conditioned fear responses when injected prior to the CS1 test session. These findings have pointed out the usefulness for the olfactory fear conditioning paradigm to investigate drug effects on the acquisition, consolidation and expression of fear conditioned responses.     

Linking Pavlovian Disgust Conditioning and Eating Disorder Symptoms: An Analogue Study

Although the experience of disgust is commonly endorsed among women with eating disorders, it remains unclear how to best model this emotion in relation to disordered eating. The present study sought to identify potential disgust conditioning abnormalities that may underlie the development of eating disorder symptoms. Individuals high and low in eating disorder symptoms (HED, n = 19; LED, n = 18) completed a Pavlovian conditioning procedure in which one neutral food item (conditioned stimulus; CS +) was followed by disgusting videos of individuals vomiting (unconditioned stimulus; US) and another neutral food item (CS–) was not reinforced with the disgusting video. Following this acquisition procedure, there was an extinction procedure in which both CSs were presented unreinforced. The results showed that ratings of disgust, fear, and willingness to eat the CS + and CS- did not significantly vary as a function of the conditioning phase for the LED group. However, the HED group rated the CS + as significantly more disgusting and fear inducing than the CS- after the acquisition phase and this pattern persisted after extinction. These conditioning effects were also observed to be significantly larger for disgust compared to fear. The HED group also reported being significantly less willing to eat the CS + compared to the CS- after the acquisition phase and this pattern also persisted after extinction. In the full sample, only discriminant disgust responding after acquisition was associated with the amount of calorie consumption over the 24-hour period after conditioning. These findings suggest that eating disorder symptoms may result from a heightened proneness to associate disgusting outcomes with otherwise neutral food items. This pattern of disgust learning may reinforce food avoidance in eating disorders and appears to be difficult to fully unlearn.     

Encoding specific associative memory: evidence from behavioral and neural manipulations

Within-subjects procedures with rats assessed the associative structures acquired during conditioning trials in which the interval between the stimuli and food was either short or long (i.e., A-10 s→food and B-40 s→food). In Experiments 1 and 2, after these conditioning trials, A and B served as second-order reinforcers for 2 further stimuli (i.e., X→A and Y→B); whereas Experiment 3 used a sensory preconditioning procedure in which X→A and Y→B trials occurred before the conditioning trials, and rats were finally tested with X and Y. In each experiment, Y elicited greater responding at test than did X. This finding supports the contention that the long-lived trace of B (associated with food on B-40 s→food trials) is more similar to the memory of B that was associatively provoked by Y, than is the short-lived trace of A (associated with food on A-10 s→food trials) to the memory of A that was associatively provoked by X. These conclusions were reinforced by the effects of a neural manipulation that disrupted discrimination learning involving the short traces of stimuli but not the long traces of the same stimuli.     

Trace and contextual fear conditioning require neural activity and NMDA receptor-dependent transmission in the medial prefrontal cortex

The contribution of the medial prefrontal cortex (mPFC) to the formation of memory is a subject of considerable recent interest. Notably, the mechanisms supporting memory acquisition in this structure are poorly understood. The mPFC has been implicated in the acquisition of trace fear conditioning, a task that requires the association of a conditional stimulus (CS) and an aversive unconditional stimulus (UCS) across a temporal gap. In both rat and human subjects, frontal regions show increased activity during the trace interval separating the CS and UCS. We investigated the contribution of prefrontal neural activity in the rat to the acquisition of trace fear conditioning using microinfusions of the γ-aminobutyric acid type A (GABA_A) receptor agonist muscimol. We also investigated the role of prefrontal N-methyl-d-aspartate (NMDA) receptor-mediated signaling in trace fear conditioning using the NMDA receptor antagonist 2-amino-5-phosphonovaleric acid (APV). Temporary inactivation of prefrontal activity with muscimol or blockade of NMDA receptor-dependent transmission in mPFC impaired the acquisition of trace, but not delay, conditional fear responses. Simultaneously acquired contextual fear responses were also impaired in drug-treated rats exposed to trace or delay, but not unpaired, training protocols. Our results support the idea that synaptic plasticity within the mPFC is critical for the long-term storage of memory in trace fear conditioning.The prefrontal cortex participates in a wide range of complex cognitive functions including working memory, attention, and behavioral inhibition (Fuster 2001). In recent years, the known functions of the prefrontal cortex have been extended to include a role in long-term memory encoding and retrieval (Blumenfeld and Ranganath 2006; Jung et al. 2008). The prefrontal cortex may be involved in the acquisition, expression, extinction, and systems consolidation of memory (Frankland et al. 2004; Santini et al. 2004; Takehara-Nishiuchi et al. 2005; Corcoran and Quirk 2007; Jung et al. 2008). Of these processes, the mechanisms supporting the acquisition of memory may be the least understood. Recently, the medial prefrontal cortex (mPFC) has been shown to be important for trace fear conditioning (Runyan et al. 2004; Gilmartin and McEchron 2005), which provides a powerful model system for studying the neurobiological basis of prefrontal contributions to memory. Trace fear conditioning is a variant of standard “delay” fear conditioning in which a neutral conditional stimulus (CS) is paired with an aversive unconditional stimulus (UCS). Trace conditioning differs from delay conditioning by the addition of a stimulus-free “trace” interval of several seconds separating the CS and UCS. Learning the CS–UCS association across this interval requires forebrain structures such as the hippocampus and mPFC. Importantly, the mPFC and hippocampus are only necessary for learning when a trace interval separates the stimuli (Solomon et al. 1986; Kronforst-Collins and Disterhoft 1998; McEchron et al. 1998; Takehara-Nishiuchi et al. 2005). This forebrain dependence has led to the hypothesis that neural activity in these structures is necessary to bridge the CS–UCS temporal gap. In support of this hypothesis, single neurons recorded from the prelimbic area of the rat mPFC exhibit sustained increases in firing during the CS and trace interval in trace fear conditioning (Baeg et al. 2001; Gilmartin and McEchron 2005). Similar sustained responses are not observed following the CS in delay conditioned animals or unpaired control animals. This pattern of activity is consistent with a working memory or “bridging” role for mPFC in trace fear conditioning, but it is not clear whether this activity is actually necessary for learning. We address this issue here using the γ-aminobutyric acid type A (GABA_A) receptor agonist muscimol to temporarily inactivate cellular activity in the prelimbic mPFC during the acquisition of trace fear conditioning.The contribution of mPFC to the long-term storage (i.e., 24 h or more) of trace fear conditioning, as opposed to a strictly working memory role (i.e., seconds to minutes), is a matter of some debate. Recent reports suggest that intact prefrontal activity at the time of testing is required for the recall of trace fear conditioning 2 d after training (Blum et al. 2006a), while mPFC lesions performed 1 d after training fail to disrupt the memory (Quinn et al. 2008). The findings from the former study may reflect a role for prelimbic mPFC in the expression of conditional fear rather than memory storage per se (Corcoran and Quirk 2007). However, blockade of the intracellular mitogen-activated protein kinase (MAPK) cascade during training impairs the subsequent retention of trace fear conditioning 48 h later (Runyan et al. 2004). Activation of the MAPK signaling cascade can result in the synthesis of proteins necessary for synaptic strengthening, providing a potential mechanism by which mPFC may participate in memory storage. To better understand the nature of the prefrontal contribution to long-term memory, more information is needed about fundamental plasticity mechanisms in this structure. Dependence on N-methyl-d-aspartate receptors (NMDAR) is a key feature of many forms of long-term memory, both in vitro and in vivo. The induction of long-term potentiation (LTP) in the hippocampus, a cellular model of long-term plasticity and information storage, requires NMDAR activation (Reymann et al. 1989). Genetic knockdown or pharmacological blockade of NMDAR-mediated neurotransmission in the hippocampus impairs several forms of hippocampus-dependent memory, including trace fear conditioning (Tonegawa et al. 1996; Huerta et al. 2000; Quinn et al. 2005), but it is unknown if activation of these receptors is necessary in the mPFC for the acquisition of trace fear conditioning. Data from in vivo electrophysiology studies have shown that stimulation of ventral hippocampal inputs to prelimbic neurons in mPFC produces LTP, and the induction of prefrontal LTP depends upon functional NMDARs (Laroche et al. 1990; Jay et al. 1995). If the role of mPFC in trace fear conditioning goes beyond simply maintaining CS information in working memory, then activation of NMDAR may be critical to memory formation. We test this hypothesis by reversibly blocking NMDAR neurotransmission with 2-amino-5-phosphonovaleric acid (APV) during training to examine the role of prefrontal NMDAR to the acquisition of trace fear conditioning.Another important question is whether mPFC contributes to the formation of contextual fear memories. Fear to the training context is acquired simultaneously with fear to the auditory CS in both trace and delay fear conditioning. Conflicting reports in the literature suggest the role of mPFC in contextual fear conditioning is unclear. Damage to ventral areas of mPFC prior to delay fear conditioning has failed to impair context fear acquisition (Morgan et al. 1993). Prefrontal lesions incorporating dorsal mPFC have in some cases been reported to augment fear responses to the context (Morgan and LeDoux 1995), while blockade of NMDAR transmission has impaired contextual fear conditioning (Zhao et al. 2005). Post-training lesions of mPFC impair context fear retention (Quinn et al. 2008) in trace and delay conditioning. Contextual fear responses were assessed in this study to determine the contribution of neuronal activity and NMDAR-mediated signaling in mPFC to the acquisition of contextual fear conditioning.     

“Clean up your plate”: Effects of child feeding practices on the conditioning of meal size

To obtain information on the development of the learned control of food intake, preschool children (N = 22) participated in conditioning and extinction trials to determine whether meal size could be conditioned. Each trial consisted of a two-part snack in which consumption of a fixed amount of a distinctively flavored high (145 kcal) or low (60 kcal) caloric density preload was followed by ad lib consumption of snacks. Following the pairs of conditioning trials, extinction test trials were given in which the flavors previously paired with either high or low caloric density were presented in intermediate, isocaloric preloads. Conditioning occurred in one of two child feeding contexts: one that focused children on internal cues of hunger and satiety, or one that focused the children on external cues, including the amount of food remaining on the plate. The external group also could receive rewards for eating. Only the children in the internal context showed evidence of responsiveness to caloric density cues and associative conditioning, eating more ad lib following the low-density preload than following the high density preload during conditioning, and eating more during extinction following the flavor previously paired with low caloric density. Children in the external context showed no evidence of responsiveness to caloric density cues, but increased in ad lib consumption across the conditioning trials.     

Average group behavior does not represent individual behavior in classical conditioning of the honeybee

Conditioned behavior as observed during classical conditioning in a group of identically treated animals provides insights into the physiological process of learning and memory formation. However, several studies in vertebrates found a remarkable difference between the group-average behavioral performance and the behavioral characteristics of individual animals. Here, we analyzed a large number of data (1640 animals) on olfactory conditioning in the honeybee (Apis mellifera). The data acquired during absolute and differential classical conditioning differed with respect to the number of conditioning trials, the conditioned odors, the intertrial intervals, and the time of retention tests. We further investigated data in which animals were tested for spontaneous recovery from extinction. In all data sets we found that the gradually increasing group-average learning curve did not adequately represent the behavior of individual animals. Individual behavior was characterized by a rapid and stable acquisition of the conditioned response (CR), as well as by a rapid and stable cessation of the CR following unrewarded stimuli. In addition, we present and evaluate different model hypotheses on how honeybees form associations during classical conditioning by implementing a gradual learning process on the one hand and an all-or-none learning process on the other hand. In summary, our findings advise that individual behavior should be recognized as a meaningful predictor for the internal state of a honeybee--irrespective of the group-average behavioral performance.     

Differential effects of muscarinic receptor blockade in prelimbic cortex on acquisition and memory formation of an odor-reward task

The present experiments determined the consequences of blocking muscarinic cholinergic receptors of the prelimbic (PL) cortex in the acquisition and retention of an odor-reward associative task. Rats underwent a training test (five trials) and a 24-h retention test (two retention trials and two relearning trials). In the first experiment, rats were bilaterally infused with scopolamine (20 or 5 microg/site) prior to training. Although scopolamine rats showed acquisition equivalent to PBS-injected controls, they exhibited weakened performance in the 24-h retention test measured by number of errors. In the second experiment, rats were injected with scopolamine (20 microg/site) immediately or 1 h after training and tested 24 h later. Scopolamine rats injected immediately showed severe amnesia detected in two performance measures (errors and latencies), demonstrating deficits in retention and relearning, whereas those injected 1 h later showed good 24-h test performance, similar to controls. These results suggest that muscarinic transmission in the PL cortex is essential for early memory formation, but not for acquisition, of a rapidly learned odor discrimination task. Findings corroborate the role of acetylcholine in consolidation processes and the participation of muscarinic receptors in olfactory associative tasks.     

Contextual modulation of memory consolidation

We investigate olfactory memory consolidation in honeybees. Three experiments are reported that include 1024 animals in 28 experimental groups. After one pairing of odorant and sucrose reward, retention is typically nonmonotonic with a minimum 3 min after conditioning. This corresponds to the “Kamin effect” in vertebrates; the postminimum rise in retention is usually interpreted as reflecting memory consolidation. First, we test for the generality of this effect across four different odorants. The postminimum rise in retention was reproducibly observed for 1-hexanol but not for 1-octanol, limonene, or geraniol. Second, we investigate whether previous learning about the training context modulates subsequent memory consolidation. On the day before training, a reward was applied either upon placement into the future training context for 1 min, halfway during exposure or just before removal from the context. In the latter group, the 3-min minimum in retention was eliminated; thus, in that group, forward pairings of context and reward (i.e., context exposure begins before reward is applied) lead to an associative context memory that can modulate subsequent olfactory memory consolidation. Third, we found no evidence for a modulation of olfactory memory consolidation by pre-exposure to the odorant.     

Pharmacological dissociation of memory: anisomycin, a protein synthesis inhibitor, and leupeptin, a protease inhibitor, block different learning tasks

Inhibition of protein synthesis by anisomycin for a short duration impairs memory of a one-trial inhibitory avoidance task in rats. Memory of escape conditioning involving eight trials is disrupted only if the duration of protein synthesis is prolonged by repeated injections. In marked contrast, olfactory memory of rats trained on two odor discriminations is not affected by anisomycin even if the duration of inhibition is prolonged and the number of trials is reduced to a minimum. In previous work, leupeptin, a thiol proteinase inhibitor, was shown to impair olfactory discrimination learning, but left inhibitory and avoidance conditioning intact. Together, these results provide a pharmacological double dissociation of memory, and suggest that the same chemistries, or mixtures of chemistries, may not be involved in all types of memory.     

Cardiac arrhythmias in the monkey during classically conditioned fear and excitement

Rhesus monkeys(Macaca mulatto) were tested in classic aversive and appetitive conditioning paradigms following complete coronary artery occlusion (CO) to test the hypothesis that “emotional stress” induces ventricular arrhythmias. Findings were based upon conditioning trials conducted for one or more weeks after occlusion in 13 animals. When all data from each animal for the week following CO were considered, there was no demonstrable tendency for arrhythmias to increase during “fear” conditioned to unavoidable electric shock or during “excitement” in anticipation of food. However, selected trials from six monkeys did reveal instances when changes in the frequency of occurrence of arrhythmias were coupled with behavioral conditioning. While analysis of these trials did not reveal any simple relationship between emotional stress and the development of ventricular arrhythmias after myocardial infarction, certain of the behavioral situations may be more potentially arrhythmogenic than others. For these selected trials, with respect to control, the number of arrhythmias may have increased or decreased upon presentation of the conditional stimulus; the exact response appears to depend upon the immediate physiologic status of the animal as well as on the behavioral condition. “More stressful” situations, such as aversive conditioning, are not necessarily associated with greater numbers of arrhythmias than were “less stressful” situations, such as appetitive conditioning. Arrhythmias appear to occur more frequently when an animal’s heart rate is within a given range; this may reflect underlying cardiac sympathetic and parasympathetic nerve activity.     

The procerebrum is necessary for odor-aversion learning in the terrestrial slug Limax valentianus

The terrestrial slug Limax has a highly developed ability to associate the odor of some foods (e.g., carrot juice) with aversive stimuli such as the bitter taste of quinidine solution. The procerebrum (PC) is a part of the slug's brain thought to be involved in odor-aversion learning, but direct evidence is still lacking. Here we present evidence showing that the PC is essential for odor-aversion learning. Unlike sham-operated slugs, PC ablation 7 d prior to conditioning showed that most slugs did not avoid carrot juice in the memory retention test conducted 24 h after the conditioning. Slugs with the PC ablated 3 h, 1 d, 3 d, or 7 d after conditioning and examined by the memory retention test at 3 d after the PC ablation were also less likely to avoid carrot juice than sham-operated slugs. The PC ablation did not damage the ability of the slugs to sense attractive odor (everyday food) or innately aversive odor (onion or garlic). These results demonstrate that the PC is a necessary component in the retention and/or retrieval of odor-aversion memory.     

Learning in cod (Gadus morhua): long trace interval retention

Basic knowledge about learning capacities and awareness in fish is lacking. In this study we investigated which temporal gaps Atlantic cod could tolerate between two associated events, using an appetitive trace-conditioning paradigm with blinking light as conditioned stimulus (CS) and dry fish food as unconditioned stimulus (US). CS–US presentations were either temporally overlapping (delay conditioning, CS duration 24 s, interstimulus interval 12 s) or separated by 20, 60, or 120 s (trace conditioning, CS duration 12 s) or 2 h (control, CS duration 12 s). The percentage of fish in the feeding area increased strongly during CS presentation in all delay, 20 s, and 60 s trace groups and in one out of two 120 s trace groups, but not in the control groups. In the 20 and 60 s trace procedures, the fish crowded together in the small feeding area during the trace interval, showing strong anticipatory behaviour. In all the conditioned groups, the fish responded to the CS within eight trials, demonstrating rapid learning. At 88 and 70 days after the end of the conditioning experiments, the delay and 20 s trace groups, respectively, were presented the CS six times at 2-h intervals without reward. All groups responded to the light signal, demonstrating memory retention after at least 3 months. This study demonstrates that Atlantic cod has an impressively good ability to associate two time-separated events and long time retention of learnt associations.