Acute ethanol ingestion impairs appetitive olfactory learning and odor discrimination in the honey bee

Invertebrates are valuable models for increasing our understanding of the effects of ethanol on the nervous system, but most studies on invertebrates and ethanol have focused on the effects of ethanol on locomotor behavior. In this work we investigate the influence of an acute dose of ethanol on appetitive olfactory learning in the honey bee (Apis mellifera), a model system for learning and memory. Adult worker honey bees were fed a range of doses (2.5%, 5%, 10%, or 25%) of ethanol and then conditioned to associate an odor with a sucrose reward using either a simple or differential conditioning paradigm. Consumption of ethanol before conditioning significantly reduced both the rate of acquisition and the asymptotic strength of the association. Honey bees also exhibited a dose dependent reduction in arousal/attention during conditioning. Consumption of ethanol after conditioning did not affect recall 24h later. The observed deficits in acquisition were not due to the affect of ethanol on gustatory sensitivity or motor function. However, honey bees given higher doses of ethanol had difficulty discriminating amongst different odors suggesting that ethanol consumption influences olfactory processing. Taken together, these results demonstrate that an acute dose of ethanol affects appetitive learning and olfactory perception in the honey bee.     

Appetitive and aversive olfactory learning induce similar generalization rates in the honey bee

Appetitive and aversive learning drive an animal toward or away from stimuli predicting reinforcement, respectively. The specificity of these memories may vary due to differences in cost–benefit relationships associated with appetitive and aversive contexts. As a consequence, generalization performances may differ after appetitive and aversive training. Here, we determined whether honey bees show different rates of olfactory generalization following appetitive olfactory conditioning of the proboscis extension response, or aversive olfactory conditioning of the sting extension response. In both cases, we performed differential conditioning, which improves discrimination learning between a reinforced odor (CS+) and a non-reinforced odor (CS?) and evaluated generalization to two novel odors whose similarity to the CS+ and the CS? was different. We show, given the same level of discriminatory performance, that rates of generalization are similar between the two conditioning protocols and discuss the possible causes for this phenomenon.     

Side-specificity of olfactory learning in the honeybee: generalization between odors and sides

Honeybees (Apis mellifera) can be trained to associate an odor stimulus with a sucrose reward. The neural structures involved in the detection and integration of olfactory stimuli are represented bilaterally in the brain. Little is known about the respective roles of the two sides of the brain in olfactory learning. Does each side learn independently of the other, or do they communicate, and if so, to what extent and at what level of neural integration? We addressed these questions using the proboscis extension response (PER) conditioning paradigm applied in a preparation that allows the separation of the two input sides during olfactory stimulations. Bees conditioned to two odorants A and B, one being learned on each side (A+/B+ training), showed in extinction tests rather unspecific responses: They responded to both odorants on both sides. This could be attributable to either a transfer of the learned information between sides, or to a generalization between odorants on each side. By subjecting bees to conditioning on one side only (A+/0 training), we found that the learned information is indeed transferred between sides. However, when bees were trained explicitly to give opposite values to the two odorants on the two sides (A+B-/B+A- training), they showed clear side-specific response patterns to these odorants. These results are used in the elaboration of a functional model of laterality of olfactory learning and memory processing in the honeybee brain.     

Nitric oxide is involved in appetitive but not aversive olfactory learning in the land mollusk Limax valentianus

The land slug Limax performs both aversive and appetitive olfactory learning, and we investigated neurotransmitters involved in each type of learning. Slugs were conditioned by presenting a vegetable juice (appetitive conditioning) or a mixture of vegetable juice and quinidine (aversive conditioning), and the latency to reach the juice became shorter (appetitive conditioning) or longer (aversive conditioning) after conditioning. L-NAME injected either before conditioning or testing blocked the reduction in latency in appetitive conditioning but had no significant effects in aversive conditioning. 5,7-dihydroxytryptamine had no significant effects in appetitive conditioning. These results suggest different mechanisms for appetitive and aversive learning.     

Using pavlovian higher-order conditioning paradigms to investigate the neural substrates of emotional learning and memory

In first-order Pavlovian conditioning, learning is acquired by pairing a conditioned stimulus (CS) with an intrinsically motivating unconditioned stimulus (US; e.g., food or shock). In higher-order Pavlovian conditioning (sensory preconditioning and second-order conditioning), the CS is paired with a stimulus that has motivational value that is acquired rather than intrinsic. This review describes some of the ways higher-order conditioning paradigms can be used to elucidate substrates of learning and memory, primarily focusing on fear conditioning. First-order conditioning, second-order conditioning, and sensory preconditioning allow for the controlled demonstration of three distinct forms of memory, the neural substrates of which can thus be analyzed. Higher-order conditioning phenomena allow one to distinguish more precisely between processes involved in transmission of sensory or motor information and processes involved in the plasticity underlying learning. Finally, higher-order conditioning paradigms may also allow one to distinguish between processes involved in behavioral expression of memory retrieval versus processes involved in memory retrieval itself.     

Learning in honey bees with brain lesions: how partial mushroom-body ablations affect sucrose responsiveness and tactile antennal learning

Honey bees are ideal organisms for studying associative learning, because they can rapidly learn different sensory cues, even under laboratory conditions. Classical olfactory learning experiments have shown that the mushroom bodies (MBs), a prominent neuropil of the central nervous system of the bee, are involved in olfactory learning and memory formation. We tested whether the MBs are also involved in tactile antennal learning. As in olfactory learning, bees use the antennae during tactile learning to sense tactile cues. We produced specific MB ablations by applying the mitotic blocker hydroxyurea (HU). In Drosophila, HU-induced brain lesions of the MBs strongly impaired olfactory memory formation. As treatment with HU might also interfere with the processing of the reward stimulus, sucrose, we measured the responsiveness to sucrose stimuli in these bees. Treatment with HU led to partial ablations of the median MB sub-units on one or both sides of the brain. We analysed side-specific effects in double-blind tests, testing sucrose responsiveness separately for each antenna, and conditioning first one antenna and then the other in a reversal learning assay. HU-treated bees without detectable ablations were less responsive to sucrose and had a poorer learning performance than untreated controls. Partial MB ablation did not additionally affect responsiveness to sucrose or tactile antennal learning. Interestingly, bees with bilateral MB ablations did not differ from untreated controls in their learning performance during the first learning phase. During reversal learning, acquisition in these bees was significantly lower than that in untreated controls. It is concluded that HU treatment affects sucrose responsiveness and tactile learning even without detectable ablation of neuropils. The effects of MB ablations on tactile learning are not side-specific and not correlated with the volume of the ablated neuropil. Accepted after revision: 15 January 2001 ❚ Electronic Publication     

Feeding behavior of Aplysia: a model system for comparing cellular mechanisms of classical and operant conditioning

Feeding behavior of Aplysia provides an excellent model system for analyzing and comparing mechanisms underlying appetitive classical conditioning and reward operant conditioning. Behavioral protocols have been developed for both forms of associative learning, both of which increase the occurrence of biting following training. Because the neural circuitry that mediates the behavior is well characterized and amenable to detailed cellular analyses, substantial progress has been made toward a comparative analysis of the cellular mechanisms underlying these two forms of associative learning. Both forms of associative learning use the same reinforcement pathway (the esophageal nerve, En) and the same reinforcement transmitter (dopamine, DA). In addition, at least one cellular locus of plasticity (cell B51) is modified by both forms of associative learning. However, the two forms of associative learning have opposite effects on B51. Classical conditioning decreases the excitability of B51, whereas operant conditioning increases the excitability of B51. Thus, the approach of using two forms of associative learning to modify a single behavior, which is mediated by an analytically tractable neural circuit, is revealing similarities and differences in the mechanisms that underlie classical and operant conditioning.     

A systems approach to the cellular analysis of associative learning in the pond snail Lymnaea

We show that appetitive and aversive conditioning can be analyzed at the cellular level in the well-described neural circuitries underlying rhythmic feeding and respiration in the pond snail, Lymnaea stagnalis. To relate electrical changes directly to behavior, the snails were first trained and the neural changes recorded at multiple sites in reduced preparations made from the same animals. Changes in neural activity following conditioning could be recorded at the level of motoneurons, central pattern generator interneurons and modulatory neurons. Of significant interest was recent work showing that neural correlates of long-term memory could be recorded in the feeding network following single-trial appetitive chemical conditioning. Available information on the synaptic connectivity and transmitter content of identified neurons within the Lymnaea circuits will allow further work on the synaptic and molecular mechanisms of learning and memory.     

Color modulates olfactory learning in honeybees by an occasion-setting mechanism

A sophisticated form of nonelemental learning is provided by occasion setting. In this paradigm, animals learn to disambiguate an uncertain conditioned stimulus using alternative stimuli that do not enter into direct association with the unconditioned stimulus. For instance, animals may learn to discriminate odor rewarded from odor nonrewarded trials if these two situations are indicated by different colors that do not themselves become associated with the reward. Despite a growing interest in nonelemental learning in insects, no study has so far attempted to study occasion setting in restrained honeybees, although this would allow direct access to the neural basis of nonelemental learning. Here we asked whether colors can modulate olfactory conditioning of the proboscis extension reflex (PER) via an occasion-setting mechanism. We show that intact, harnessed bees are not capable of learning a direct association between color and sucrose. Despite this incapacity, bees solved an occasion-setting discrimination in which colors set the occasion for appropriate responding to an odor that was rewarded or nonrewarded depending on the color. We therefore provide the first controlled demonstration of bimodal (color-odor) occasion setting in harnessed honeybees, which opens the door for studying the neural basis of such bimodal, nonelemental discriminations in insects.     

Successive olfactory reversal learning in honeybees

Honeybees Apis mellifera can associate an originally neutral odor with a reinforcement of sucrose solution. Forward pairings of odor and reinforcement enable the odor to release the proboscis extension reflex in consecutive tests. Bees can also be conditioned differentially: They learn to respond to a reinforced odor and not to a nonreinforced one. They can also learn to reverse their choice. Here we ask whether honeybees can learn successive olfactory differential conditioning tasks involving different overlapping pairs of odors. The conditioning schedules were established in order to train the animals with 3, 2, 1, or 0 reversals previous to a last differential conditioning phase in which two additional reversals were present. We studied whether or not successive reversal learning is possible and whether or not learning olfactory discrimination reversals affects the solving of subsequent discrimination reversals. Therefore we compared the responses of bees that had experienced reversals with those of bees that had not experienced such reversals when both are confronted with a new reversal situation. In experiment 1 we showed that bees that had experienced three previous reversals were better in solving the final reversal task than bees with no previous reversal experience. In experiment 2, we showed that one reversal learning is enough for bees to perform better in the final reversal task. The successive different reversals trained in our experiments resemble the natural foraging situation in which a honeybee forager has to switch successively from an initial floral species to different ones. The fact that experiencing such changes seems to improve a bee's performance in dealing with further new exploited food sources has therefore an adaptive impact for the individual and for the colony as a whole.     

Salivary conditioning with antennal gustatory unconditioned stimulus in an insect

Classical conditioning of olfactory conditioning stimulus (CS) with gustatory unconditioned stimulus (US) in insects has been used as a pertinent model for elucidation of neural mechanisms underlying learning and memory. However, a conditioning system in which stable intracellular recordings from brain neurons are feasibly obtained while monitoring the conditioning effect has remained to be established. Recently, we found classical conditioning of salivation in cockroaches Periplaneta americana, in which an odor was associated with sucrose solution applied to the mouth, and this conditioning could be monitored by activities of salivary neurons. Application of gustatory US to the mouth, however, leads to feeding movement accompanying a movement of the brain that prevents stable recordings from brain neurons. Here we investigated whether a gustatory stimulus presented to an antenna could serve as an effective US for producing salivary conditioning. Presentation of sucrose or sodium chloride solution to an antenna induced salivation and also increased activities of salivary neurons. A single pairing trial of an odor with antennal presentation of sucrose or sodium chloride solution produced conditioning of salivation or of activities of salivary neurons. Five pairing trials led to a conditioning effect that lasted for one day. Water or tactile stimulus presented to an antenna was not effective for producing conditioning. The results demonstrate that gustatory US presented to an antenna is as effective as that presented to the mouth for producing salivary conditioning. This conditioning system provides a useful model for studying the neural basis of learning at the level of singly identifiable neurons.     

Activation of MAPK is necessary for long-term memory consolidation following food-reward conditioning

Although an important role for the mitogen-activated protein kinase (MAPK) has been established for memory consolidation in a variety of learning paradigms, it is not known if this pathway is also involved in appetitive classical conditioning. We address this question by using a single-trial food-reward conditioning paradigm in the freshwater snail Lymnaea stagnalis. This learning paradigm induces protein synthesis-dependent long-term memory formation. Inhibition of MAPK phosphorylation blocked long-term memory consolidation without affecting the sensory and motor abilities of the snails. Thirty minutes after conditioning, levels of MAPK phosphorylation were increased in extracts from the buccal and cerebral ganglia. These ganglia are involved in the generation, modulation, and plasticity of the feeding behavior. We also detected an increase in levels of MAPK phosphorylation in the peripheral tissue around the mouth of the snails where chemoreceptors are located. Although an increase in MAPK phosphorylation was shown to be essential for food-reward conditioning, it was also detected in snails that were exposed to the conditioned stimulus (CS) or the unconditioned stimulus (US) alone, suggesting that phosphorylation of MAPK is necessary but not sufficient for learning to occur.     

Differential involvement of the central amygdala in appetitive versus aversive learning

Understanding the function of the distinct amygdaloid nuclei in learning comprises a major challenge. In the two studies described herein, we used c-Fos immunolabeling to compare the engagement of various nuclei of the amygdala in appetitive and aversive instrumental training procedures. In the first experiment, rats that had already acquired a bar-pressing response to a partial food reinforcement were further trained to learn that an acoustic stimulus signaled either continuous food reinforcement (appetitive training) or a footshock (aversive training). The first training session of the presentation of the acoustic stimulus resulted in significant increases of c-Fos immunolabeling throughout the amygdala; however, the pattern of activation of the nuclei of the amygdala differed according to the valence of motivation. The medial part of the central amygdala (CE) responded, surprisingly, to the appetitive conditioning selectively. The second experiment was designed to extend the aversive versus appetitive conditioning to mice, trained either for place preference or place avoidance in an automated learning system (INTELLICAGE). Again, much more intense c-Fos expression was observed in the medial part of the CE after the appetitive training as compared to the aversive training. These data, obtained in two species and by means of novel experimental approaches balancing appetitive versus aversive conditioning, support the hypothesis that the central nucleus of the amygdala is particularly involved in appetitively motivated learning processes.     

Deficits in acquisition and extinction of conditioned responses in mGluR7 knockout mice

Metabotropic glutamate receptor 7 (mGluR7) is expressed in brain regions implicated in emotional learning and working memory, and previous behavioral experiments indicated contributions of mGluR7 to various complex behaviors. In the present study, we investigated the specific effects of mGluR7 deletion on a variety of conditioning paradigms that model crucial neurocognitive and psychopathological behavioral phenomena. Null-mutant mGluR7^−/− mice displayed defects during scheduled appetitive conditioning, acquisition and extinction of appetitive odor conditioning, extinction of response suppression-based conditioned emotional responding (CER), acquisition of discriminative CER, and contextual fear conditioning. mGluR7^−/− animals were slower to acquire the association between a conditioned stimulus and a positive or negative reinforcer, but eventually reached similar performance levels to their wildtype littermates. Notably, extinction learning of conditioned responses was slower in mGluR7^−/− compared to wildtype animals. The observed delays in the acquisition of complicated stimulus associations across conditioning procedures may suggest a critical role for mGluR7 in neurocognitive functions and psychopathology.     

The effects of lesions in the central nucleus of the amygdala on appetitive classical conditioning in rats

Abstract: Lesions in the central nucleus of the amygdala (cAMY) have been known to interfere with the acquisition of fear classical conditioning when footshock is used as an unconditioned stimulus (US). The present study examined whether or not a similar interference would occur with an appetitive US. Five rats with lesions in the cAMY (the cAMY group), and eight unoperated control rats were trained in an appetitive classical conditioning paradigm, which did not include elements of operant learning, using a visual conditioned stimulus (CS) (5 W of light for 10 s duration) paired with a food pellet US (45 mg, cheese flavor). The behavioral index of appetitive conditioning was an increase in rearing approach behavior to the CS after CS and US pairings. During CS and US pairings, the movement of the rat was limited so that this approach behavior could not occur. As a result, all control rats showed an increase in rearing, but the cAMY group did not. These results suggest that the cAMY is critical for appetitive as well as fear classical conditioning.     

The structure of individual differences in batteries of rapid acquisition tasks in mice

Two experiments examined the structure of individual differences in mice by means of tasks that produced significant acquisition within 1 session. In Experiment 1, 5 cognitive tasks-detour, winshift, olfactory discrimination, fear conditioning, and operant acquisition-were used in conjunction with two control procedures: an open field and a light- dark test. In Experiment 2, some modifications were made to the tasks used in the 1st experiment, and 3 new tasks were used in conjunction with the same control procedures. The battery consisted of 5 learning tasks: detour, Hebb-Williams, radial maze, olfactory foraging, and fear conditioning. Results of both experiments indicate that when cognitive tasks and control procedures were included in principal-components analyses most of the variance attached principally to individual tasks rather than to a general component as is found typically in human cognitive batteries. When control procedures were eliminated, there was better evidence for the presence of a general cognitive factor, particularly in Experiment 2.     

Associative mechanosensory conditioning of the proboscis extension reflex in honeybees

The present work introduces a form of associative mechanosensory conditioning of the proboscis extension reflex (PER) in honeybees. In our paradigm, harnessed honeybees learn the elemental association between mechanosensory, antennal stimulation and a reward of sucrose solution delivered to the proboscis. Thereafter, bees extend their proboscis to the antennal mechanosensory stimulation alone. We show that bees can learn such an association in a side-specific manner, that is, they learn the association on the antennal side that was rewarded and not on the side that was not rewarded. Responding produced by the paired training does likely contain a substantial Pavlovian component. Responding is only elicited by mechanosensory stimulation and not by spurious cues such as olfactory, visual, and contextual ones. The interstimulus interval (ISI) affects one-trial mechanosensory learning: a bell-shaped curve with a maximum of responding approximately 4 sec ISI was obtained. Mechanosensory memory is still operative 24 h after conditioning. Apart from absolute conditioning in which mechanosensory stimulation of one antenna is paired with sucrose, differential, side-specific, mechanosensory conditioning using two mechanosensory stimulations, one rewarded and the other not, is also possible. This paradigm constitutes, therefore, a new standard procedure for further learning studies in honeybees.     

Appetitive learning using visual conditioned stimuli in the pond snail, lymnaea

Feeding can be conditioned in the pond snail Lymnaea stagnalis to two different visual stimuli (a black panel or a 5-mm black and white check surround) by pairing the potential conditioned stimulus (CS) with sucrose. Exclusion of chemical cues (associated with differences between the water in home tank and that in training apparatus) that could serve as CS is important for successful visual conditioning. A featureless gray surround, used as an alternative to the check (to which it was matched in luminance) in counterbalanced training designs, was discriminated from the check, showing that resolution (for which the eyes would be necessary) was occurring. The gray surround was largely ineffective as a CS. Single-trial learning was possible with the black panel, but not with the check; it is argued that this may be due to lack of prior experience of stimuli like the check. Conditioning of feeding has now been obtained in Lymnaea to chemical, tactile, and visual cues, opening the way to comparative studies of the neural circuitry underlying appetitive conditioning in different senses, so far explored in Lymnaea only for tactile CS. Such comparative studies are as yet largely lacking in invertebrates.     

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.     

Evidence for associative learning in newly emerged honey bees (Apis mellifera)

The honey bee is a model organism for studies on the neural substrates of learning and memory. Associative olfactory learning using sucrose rewards is fast and reliable in foragers and older hive bees. However, researchers have so far failed to show any significant learning in newly emerged bees. It is generally argued that in these bees only part of the brain structures important for learning are fully developed. Here we show for the first time that newly emerged honey bees are capable of associative learning, if they are sufficiently responsive to sucrose. Responsiveness to sucrose, which can be measured using the proboscis extension response (PER), increases with age. Newly emerged bees are on average very unresponsive to sucrose. We show that if newly emerged bees displaying a PER to 10% sucrose or lower sucrose concentrations are conditioned to an odour, they show significant associative learning and early long-term memory. Nevertheless, the level of acquisition is still lower than in foragers. The general assumption that newly emerged honey bees are incapable of associative learning must therefore be reconsidered. Further, our study suggests that an age-dependent increase in responsiveness to rewarding stimuli is directly related to the development of early learning abilities. The decisive influence of responsiveness to rewarding stimuli in associative learning of newly emerged bees has far reaching consequences for studies on the development of associative learning capabilities in insects and vertebrates.