Classical olfactory conditioning in the cockroach Periplaneta americana

We established a classical conditioning procedure for the cockroach, Periplaneta americana, by which odors were associated with reward or punishment. Cockroaches underwent differential conditioning trials in which peppermint odor was associated with sucrose solution and vanilla odor was associated with saline solution. Odor preference of cockroaches was tested by allowing them to choose between peppermint and vanilla sources. Cockroaches that had undergone one set of differential conditioning trials exhibited a significantly greater preference for peppermint odor than did untrained cockroaches. Memory formed by three sets of differential conditioning trials, with an inter-trial interval of 5 min, was retained at least 4 days after conditioning. This conditioning procedure was effective even for cockroaches that had been harnessed in plastic tubes. This study shows, for the first time in hemimetaborous insects, that both freely moving and harnessed insects are capable of forming olfactory memory by classical conditioning procedure. This procedure may be useful for future electrophysiological and pharmacological studies aimed at elucidation of neural mechanisms underlying olfactory learning and memory.     

Olfactory conditioning with single chemicals in the German Cockroach, Blattella germanica (Dictyoptera: Blattellidae)

Many insects find resources by means of the olfactory cues of general odors after learning. To evaluate behavioral responses to the odor of a particular chemical after learning with reward or punishment quantitatively, we developed a standardized odor-training method in the German cockroach, Blattella germanica (Linnaeus), an important urban pest species. A classical olfactory conditioning procedure for a preference test was modified to become applicable to a single odor, by which a (?)-menthol or vanillin odor was independently associated with sucrose (reward) or sodium chloride solution (punishment). The strength of the association with the odor was evaluated with the increase or decrease in visit frequencies to the odor source after olfactory conditioning. The frequency increased after (?)-menthol was presented with a reward, while it did not change with the rewarded vanillin odor. With both odors, the frequency decreased significantly after training with a punishment. These results indicate that cockroaches learn a single compound odor presented as a conditioned stimulus, although the association of the odor with a reward or punishment depends on the chemical. This olfactory conditioning method can not only facilitate the analysis of cockroach behavior elicited by a learned single chemical odor, but also quantify the potential attractiveness or repellency of the chemical after learning.     

桔小实蝇雄成虫联系性嗅觉学习

【目的】为了探究桔小实蝇 Bactrocera dorsalis (Hendel)雄成虫的嗅觉学习能力。【方法】本研究采用经典性嗅觉条件反射训练法（classical olfactory conditioning）在室内对固定的羽化后14-17日龄的桔小实蝇雄成虫进行气味与食物的联合学习训练, 即薄荷精油和10%蔗糖溶液联合的奖赏性训练（appetitive conditioning）以及甲基丁香酚(methyl eugenol, ME)和饱和盐溶液联合的惩罚性训练（aversive conditioning）,并以伸喙反射行为（proboscis extension reflex, PER）作为学习与否的判定标准。【结果】经过奖赏性训练后,桔小实蝇雄成虫对薄荷精油的伸喙反射率可从0%增加至68%;而经过惩罚性训练后,桔小实蝇对甲基丁香酚的伸喙反射率可从100%降低至36.54%,且这种伸喙反射率的变化是通过气味条件刺激（conditioned stimulus）和食物非条件刺激（unconditioned stimulus）的对称性联合而产生的。【结论】结果表明,桔小实蝇雄性成虫具有较强的联系性嗅觉学习能力,并且两种刺激的联合是形成学习记忆的必要条件。     

Odor preference in honeybees as a function of amount of reward: Tests of two explanations

How does a foraging honeybee come to prefer a color or odor paired with the large of two amounts of sucrose solution to a color or odor paired with the smaller amount? One hypothesis is that the attractiveness of a color or odor is based on the strength of its association with the taste of sucrose, which increases with the duration of concurrent color-taste or odor-taste stimulation. Another hypothesis is that the attractiveness of a color or odor is based on association with the afferent consequences of feeding, which are different for the two amounts. Both hypotheses were tested in experiments on proboscis-extension conditioning, a technique that provides better control of stimulation than is possible in work with free-flying foragers. In Experiments 1–3, which were designed to test the first hypothesis, an odor that accompanied the ingestion of sucrose on training trials was found to elicit extension of the proboscis when subsequently it was presented alone, but the duration of concurrent stimulation had no significant effect on the probability or persistence of the response. In Experiments 4 and 5, which were designed to test the second hypothesis, an odor that immediately followed the ingestion of sucrose on training trials failed to elicit extension of the proboscis when subsequently it was presented alone. The results support neither hypothesis.     

Classical Olfactory Conditioning in the Oriental Fruit Fly,Bactrocera dorsalis

The oriental fruit fly, Bactrocera dorsalis, is a serious pest of fruits and vegetables. Methyl eugenol (ME), a male attractant, is used to against this fly by mass trapping. Control effect may be influenced by learning, which could modify the olfactory response of the fly to this attractant. To collect the behavioral evidence, studies on the capability of this fly for olfactory learning are necessary. We investigated olfactory learning in male flies with a classical olfactory conditioning procedure using restrained individuals under laboratory conditions. The acquisition of the proboscis extension reflex was used as the criterion for conditioning. A high conditioned response level was found in oriental fruit flies when an odor was presented in paired association with a sucrose reward but not when the odor and sucrose were presented unpaired. We also found that the conditioning performance was influenced by the odor concentration, intertrial interval, and starvation time. A slight sensitization elicited by imbibing sucrose was observed. These results indicate that oriental fruit flies have a high capacity to form an olfactory memory as a result of classical conditioning.     

Olfactory discrimination of structurally similar alcohols by cockroaches

The capability of the cockroach Periplaneta americana to discriminate odors of structurally similar aliphatic alcohols was studied by using an operant conditioning paradigm. Cockroaches were trained to discriminate three odors: one odor associated with sucrose solution (reward) and two odors associated with NaCl solution (non-reward). After training, their odor preferences were tested by counting the number of visits to each odor source. We tested the capability of cockroaches to discriminate (1) three normal aliphatic alcohols with different numbers of carbon (1-pentanol, 1-hexanol and 1-octanol), (2) three C6 aliphatic alcohols (1-hexanol, 2-hexanol and trans-2-hexen-1-ol), (3) binary mixtures of two of these three alcohols and their components, and (4) 1-hexanol solution of three different concentrations (1, 10 and 100 micro g micro l(-1)). Cockroaches exhibited higher preferences for the odors associated with reward in these tests, and we therefore conclude that cockroaches can discriminate these odors. However, discrimination of 1-hexanol and trans-2-hexen-1-ol and their binary mixture was imperfect, in that some statistical tests suggested significant level of discrimination but other tests did not. In addition, the cockroaches learned to associate a 1-hexanol solution of the highest or lowest concentration with sucrose reward but failed to learn to associate 1-hexanol of an intermediate concentration with reward.     

Altered representation of the spatial code for odors after olfactory classical conditioning; memory trace formation by synaptic recruitment

In the olfactory bulb of vertebrates or the homologous antennal lobe of insects, odor quality is represented by stereotyped patterns of neuronal activity that are reproducible within and between individuals. Using optical imaging to monitor synaptic activity in the Drosophila antennal lobe, we show here that classical conditioning rapidly alters the neural code representing the learned odor by recruiting new synapses into that code. Pairing of an odor-conditioned stimulus with an electric shock-unconditioned stimulus causes new projection neuron synapses to respond to the odor along with those normally activated prior to conditioning. Different odors recruit different groups of projection neurons into the spatial code. The change in odor representation after conditioning appears to be intrinsic to projection neurons. The rapid recruitment by conditioning of new synapses into the representation of sensory information may be a general mechanism underlying many forms of short-term memory.     

Nutrient-Specific Learning in an Omnivorous Insect: The American Cockroach Periplaneta americana L. Learns to Associate Dietary Protein with the Odors Citral and Carvone

Experiments were performed to test for nutrient-specific olfactory learning in the American cockroach, Periplaneta americana L. In a conditioning period, cockroaches were presented with two complementary foods, one of which contained protein and the other carbohydrate, this combination allowing them to select a nutritionally balanced diet. The foods were separated in space, and each was paired with one of two odors, citral or carvone. The cockroaches were then selectively deprived of one of the nutrients for 24 or 48 h. In the final (test) phase of the experiment the movement of the cockroaches toward the nutrient-associated odors was monitored. Associative learning was demonstrated with respect to protein, with protein-deprived cockroaches moving more frequently toward the protein-associated odor. No learned associations between carbohydrate and odor were demonstrated. These data are contrasted with similar experiments on an herbivorous insect, the locust Locusta migratoria.     

Physiological properties and response modulations of mushroom body feedback neurons during olfactory learning in the honeybee, Apis mellifera

Mushroom bodies are central brain structures and essentially involved in insect olfactory learning. Within the mushroom bodies γ-aminobutyric acid (GABA)-immunoreactive feedback neurons are the most prominent neuron group. The plasticity of inhibitory neural activity within the mushroom body was investigated by analyzing modulations of odor responses of feedback neurons during olfactory learning in vivo. In the honeybee, Apis mellifera, feedback neurons were intracellularly recorded at their neurites. They produced complex patterns of action potentials without experimental stimulation. Summating postsynaptic potentials indicate that their synaptic input region lies within the lobes. Odor and antennal sucrose stimuli evoked excitatory phasic-tonic responses. Individual neurons responded to various odors; responses of different neurons to the same odor were highly variable. Response modulations were determined by comparing odor responses of feedback neurons before and after one-trial olfactory conditioning or sensitisation. Shortly after pairing an odor stimulus with a sucrose reward, odor-induced spike activity of feedback neurons decreased. Repeated odor stimulations alone, equally spaced as in the conditioning experiment, did not affect the odor-induced excitation. A single sensitisation trial also did not alter odor responses. These findings indicate that the level of odor-induced inhibition within the mushroom bodies is specifically modulated by experience. Accepted: 9 September 1999     

Light-induced activation of distinct modulatory neurons triggers appetitive or aversive learning in Drosophila larvae

During classical conditioning, a positive or negative value is assigned to a previously neutral stimulus, thereby changing its significance for behavior. If an odor is associated with a negative stimulus, it can become repulsive. Conversely, an odor associated with a reward can become attractive. By using Drosophila larvae as a model system with minimal brain complexity, we address the question of which neurons attribute these values to odor stimuli. In insects, dopaminergic neurons are required for aversive learning, whereas octopaminergic neurons are necessary and sufficient for appetitive learning. However, it remains unclear whether two independent neuronal populations are sufficient to mediate such antagonistic values. We report the use of transgenically expressed channelrhodopsin-2, a light-activated cation channel, as a tool for optophysiological stimulation of genetically defined neuronal populations in Drosophila larvae. We demonstrate that distinct neuronal populations can be activated simply by illuminating the animals with blue light. Light-induced activation of dopaminergic neurons paired with an odor stimulus induces aversive memory formation, whereas activation of octopaminergic/tyraminergic neurons induces appetitive memory formation. These findings demonstrate that antagonistic modulatory subsystems are sufficient to substitute for aversive and appetitive reinforcement during classical conditioning.     

Associative learning in ants: Conditioning of the maxilla-labium extension response in Camponotus aethiops

Associative learning has been studied in many vertebrates and invertebrates. In social insects, the proboscis extension response conditioning of honey bees has been widely used for several decades. However, a similar paradigm has not been developed for ants, which are advanced social insects showing different morphological castes and a plethora of life histories. Here we present a novel conditioning protocol using Camponotus aethiops. When the antennae of a harnessed ant are stimulated with sucrose solution, the ant extends its maxilla-labium to absorb the sucrose. We term this the “maxilla-labium extension response” (MaLER). MaLER could be conditioned by forward pairing an odour (conditioned stimulus) with sucrose (unconditioned stimulus) in the course of six conditioning trials (absolute conditioning). In non-rewarded tests following conditioning, ants gave significantly higher specific responses to the conditioned stimulus than to a novel odour. When trained for differential conditioning, ants discriminated between the odour forward-paired with sucrose and an odour forward-paired with quinine (a putative aversive stimulus). In both absolute and differential conditioning, memory lasted for at least 1 h. MaLER conditioning allows full control of the stimulation sequence, inter-stimulus and inter-trial intervals and satiety, which is crucial for any further study on associative learning in ants.     

Effects of cold immobilization and recovery period on honeybee learning, memory, and responsiveness to sucrose

In addition to human error and variation in laboratory conditions, there are numerous factors that can complicate comparisons among studies. Furthermore, differences in how experimental methods are executed can make it difficult to distinguish between effects of focal versus extraneous variables. Insect neural function is commonly evaluated using Pavlovian conditioning techniques; learning and memory in many species can be assessed using the proboscis extension reflex (PER). However, there are significant inconsistencies in methods used to immobilize insects prior to PER tests. We compared responses of honeybees immobilized in a refrigerator, on ice, and in a freezer, and evaluated influence of recovery interval before testing. Ice-chilling weakly decreased learning (response to an originally neutral odor) more so than refrigeration or freezing, but not 24-h recall of odor. We found no significant differences in responsiveness to sucrose relative to cooling method, but responsiveness was significantly lower among honeybees left to recover for only 0.75 h versus 1.5 or 3 h. Finally, we observed increased responsiveness to sucrose and geraniol between June and August. Our results suggest that inconsistencies in cold immobilization methods could confound interpretation and comparison of results from a large body of work on honeybee learning and memory.     

Maternal attenuation of hypothalamic paraventricular nucleus norepinephrine switches avoidance learning to preference learning in preweanling rat pups

Infant rats learn to prefer stimuli paired with pain, presumably due to the importance of learning to prefer the caregiver to receive protection and food. With maturity, a more 'adult-like' learning system emerges that includes the amygdala and avoidance/fear learning. The attachment and 'adult-like' systems appear to co-exist in older pups with maternal presence engaging the attachment system by lowering corticosterone (CORT). Specifically, odor-shock conditioning (11 odor-0.5 mA shock trials) in 12-day-old pups results in an odor aversion, although an odor preference is learned if the mother is present during conditioning. Here, we propose a mechanism to explain pups ability to 'switch' between the dual learning systems by exploring the effect of maternal presence on hypothalamic paraventricular nucleus (PVN) neural activity, norepinephrine (NE) levels and learning. Maternal presence attenuates both PVN neural activity and PVN NE levels during odor-shock conditioning. Intra-PVN NE receptor antagonist infusion blocked the odor aversion learning with maternal absence, while intra-PVN NE receptor agonist infusion permitted odor aversion learning with maternal presence. These data suggest maternal control over pup learning acts through attenuation of PVN NE to reduce the CORT required for pup odor aversion learning. Moreover, these data also represent pups' continued maternal dependence for nursing, while enabling aversion learning outside the nest to prepare for pups future independent living.     

Can Honey Bees Learn the Removal of a Stimulus as a Conditioning Cue?

Experiments investigated a Pavlovian conditioning situation where the presence and absence of the stimulus are reversed temporally with respect to the presentation of a reward. Instead of a conditioned stimulus (e.g. odor) signaling the presence of a reward, the stimulus (e.g. odor) is present in the environment except just prior to the presence of the reward. Thus, the absence of the stimulus, or offset of the stimulus (e.g. absence of odor), serves as a conditioned stimulus and is the reward cue. Honey bees (Apis mellifera) were used as a model invertebrate system, and the proboscis‐conditioning paradigm was used as the test procedure. Using both simple Pavlovian conditioning and discrimination‐learning protocols, animals learned to associate the onset of an odor as conditioned stimuli when paired with a sucrose reward. They could also learn to associate the onset of a puff of air with a sucrose reward. However, bees could not associate the offset of an order stimulus with the presentation of a sucrose reward in either a simple conditioning or a discrimination‐learning situation. These results support the model that a very different cognitive architecture is used by invertebrates to deal with certain environmental situations, including signaled avoidance.     

Appetitive odor learning does not change olfactory coding in a subpopulation of honeybee antennal lobe neurons

Odors elicit spatio-temporal patterns of activity in the olfactory bulb of vertebrates and the antennal lobe of insects. There have been several reports of changes in these patterns following olfactory learning. These studies pose a conundrum: how can an animal learn to efficiently respond to a particular odor with an adequate response, if its primary representation already changes during this process? In this study, we offer a possible solution for this problem. We measured odor-evoked calcium responses in a subpopulation of uniglomerular AL output neurons in honeybees. We show that their responses to odors are remarkably resistant to plasticity following a variety of appetitive olfactory learning paradigms. There was no significant difference in the changes of odor-evoked activity between single and multiple trial forward or backward conditioning, differential conditioning, or unrewarded successive odor stimulation. In a behavioral learning experiment we show that these neurons are necessary for conditioned odor responses. We conclude that these uniglomerular projection neurons are necessary for reliable odor coding and are not modified by learning in this paradigm. The role that other projection neurons play in olfactory learning remains to be investigated.     

A new method to assess the influence of odor on food selection in dogs

Previous research provides evidence that odor is a key driver in food selection in dogs. Dogs' flavor preferences are generally assessed through paired comparison tests based on food intake. Methods for evaluating odor preference in canines are lacking. In this study, the paired comparison test was modified by replacing standard bowls with false‐bottom bowls (FBBs). Made of two compartments separated by a drilled, stainless‐steel plate, FBBs enable odorant compounds to be placed under the food that is presented to the dogs. Several paired comparison trials were conducted on a trained canine panel with FBBs containing various odorant substances under the kibbles. Results showed that dogs were able to perceive the hidden substances and to distinguish between the bowls accordingly. These results demonstrate that the false‐bottom bowl paired comparison method could be helpful in evaluating the role of odor in dogs' food preferences, thus, also as a way of assessing food odor performance.

Practical applications

The false‐bottom bowl method is an adaptation of the paired comparison test that enables the influence of odor on dog behavior to be isolated from that stimulated by vision, taste or textural parameters. The odor impact of a hidden substance is tested under pet meal conditions. This new method could be useful in pet food industry to measure the odor potential of a new ingredient, or to understand the key food selection drivers for dogs and cats. In addition, as the olfactory stimulus is not eaten by the animal, the influence of odor in non‐food products for dogs, such as pet care and pet medicines, could also be evaluated using this method.     

The Effect of Sucrose Concentration on Olfactory-Based Associative Learning in Culex quinquefasciatus Say (Diptera: Culicidae)

A classical conditioning approach was used to examine the ability of Culex quinquefasciatus Say (Diptera: Culicidae) to associate odor with a sugar-meal of varying quality. The objectives of this study were to investigate the impact of different sucrose concentrations (5 %, 10 %, or 50 %) on positive response to conditioning and to examine sucrose concentration preference following exposure to a 10 % solution. Mosquitoes conditioned in conjunction with all three sucrose concentrations showed evidence of learning; including the concentration of the conditioning stimulus, and the sex of the mosquito. Using colored solutions to determine feeding patterns of experienced mosquitoes indicated male mosquitoes showed no preference but females showed a preference for 10 % over 5 % sucrose but not between 10 % and 50 % sucrose solutions.     

Conditioning odor-shock associations in the black blowfly,Phormia regina

Discriminative classical conditioning of an olfactory avoidance response was demonstrated in the blowfly, Phormia regina.Learning indices were calculated as the fraction of flies avoiding the CS+ (conditioned stimulus paired with electric shock reinforcement) minus the fraction of flies avoiding the CS-(conditioned stimulus not paired with electric shock), averaged over two different groups of flies, in which reciprocal odors were used as the CS+. Avoidance responses to both odors presented simultaneously at a T-maze choice point yielded mean learning indices of zero for naive flies or pseudoconditioned (shock alone) or sensitized (odors alone) controls. In contrast, pairing an odor with electric shock produced a mean learning index significantly greater than zero. These results are similar to those reported for Drosophila melanogaster.Blow flies, however, showed much lower levels of associative learning than fruit flies.     

Analysis of associative learning in the terrestrial mollusc Limax maximus. II. Appetitive learning

Summary The odor and taste processing systems of the terrestrial mollusc Limax maximus have been shown capable of a number of complex computations. Most of the complex higher-order features of Limax learning have been demonstrated using differential aversive conditioning. The present experiments probe the appetitive learning ability of Limax. In the first experiment a differential appetitive classical conditioning procedure was used. An aversive CS⁺ odor was paired with an attractive taste while a CS^– odor was explicitly unpaired with the attractive taste. This appetitive conditioning procedure dramatically increased the preference for the CS⁺ odor. Further experiments determined the time course of acquisition, the effect of an extinction procedure and long-term retention of the appetitive conditioning. Now that Limax has been shown capable of appetitive conditioning, the neural network simulation of Limax learning, called LIMAX, can be examined for its ability to display appetitive conditioning.Abbreviations AA amyl acetate - CS conditioned stimulus - ISI interstimulus interval - ITI intertrial interval - MCH methylcyclohexanol - US unconditioned stimulus     

The evolutionary aspects of the dynamic acquisition of simple conditioned reflexes

The dynamics of elaboration of simple classical and instrumental conditioned reflexes in snails, rats, cars, dogs and humans was investigated. The dependence between the number of correct responses (in percentages) and the number of reinforcements was significantly approximated with exponential equation including the coefficient describing the initial level of conditioning. Regression analysis gave the opportunity to compare the dynamics of conditioning in different animal species.