Antennal sucrose perception in the honey bee (Apis mellifera L.): behaviour and electrophysiology

Physiological mechanisms of antennal sucrose perception in the honey bee were analysed using behavioural and electrophysiological methods. Following sucrose stimulation of the tip of a freely moving antenna, the latency of proboscis extension was 320–340 ms, 80–100 ms after the first activity in muscle M17 controlling this response. When bees were allowed to actively touch a sucrose droplet with one antenna, contacts with the solution were frequent with durations of 10–20 ms and average intervals between contacts of approximately 40 ms. High sucrose concentrations led to short and frequent contacts. The proboscis response and M17 activity were largely independent of stimulus duration and temporal pattern. Taste hairs of the antennal tip displayed spike responses to sucrose concentrations down to at least 0.1%. The first 25 ms of the response were suitable for discrimination of sucrose concentrations. This time interval corresponds to the duration of naturally occurring gustatory stimuli. Sucrose responses between different hairs on the same antenna showed a high degree of variability, ranging from less than five to over 40 spikes per 0.5 s for a stimulus of 0.1% sucrose. This variability of receptor responses extends the dynamic range of sucrose perception over a large range of concentrations.     

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.     

Critical Period of Memory Enhancement during Taste Avoidance Conditioning in Lymnaea stagnalis

The present study investigated the optimal training procedure leading to long-lasting taste avoidance behavior in Lymnaea. A training procedure comprising 5 repeated pairings of a conditional stimulus (CS, sucrose), with an unconditional stimulus (US, a tactile stimulation to the animal’s head), over a 4-day period resulted in an enhanced memory formation than 10 CS-US repeated pairings over a 2-day period or 20 CS-US repeated pairings on a single day. Backward conditioning (US-CS) pairings did not result in conditioning. Thus, this taste avoidance conditioning was CS-US pairing specific. Food avoidance behavior was not observed following training, however, if snails were immediately subjected to a cold-block (4°C for 10 min). It was critical that the cold-block be applied within 10 min to block long-term memory (LTM) formation. Further, exposure to the cold-block 180 min after training also blocked both STM and LTM formation. The effects of the cold-block on subsequent learning and memory formation were also examined. We found no long lasting effects of the cold-block on subsequent memory formation. If protein kinase C was activated before the conditioning paradigm, snails could still acquire STM despite exposure to the cold-block.     

From antenna to antenna: lateral shift of olfactory memory recall by honeybees

Honeybees, Apis mellifera, readily learn to associate odours with sugar rewards and we show here that recall of the olfactory memory, as demonstrated by the bee extending its proboscis when presented with the trained odour, involves first the right and then the left antenna. At 1-2 hour after training using both antennae, recall is possible mainly when the bee uses its right antenna but by 6 hours after training a lateral shift has occurred and the memory can now be recalled mainly when the left antenna is in use. Long-term memory one day after training is also accessed mainly via the left antenna. This time-dependent shift from right to left antenna is also seen as side biases in responding to odour presented to the bee's left or right side. Hence, not only are the cellular events of memory formation similar in bees and vertebrate species but also the lateralized networks involved may be similar. These findings therefore seem to call for remarkable parallel evolution and suggest that the proper functioning of memory formation in a bilateral animal, either vertebrate or invertebrate, requires lateralization of processing.     

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。     

Stable and unstable phase of memory in classically conditioned fly, Phormia regina: Effects of nitrogen gas anaesthesia and cycloheximide injection

This study examined the effects of nitrogen anaesthesia and cycloheximide injection on memory of the classically-conditioned fly, Phormia. 1 M NaCl solution was given to each fly as a conditioning stimulus and 0.5 M sucrose solution was the unconditioned stimulus that induced the proboscis extension response. The training period was as short as 2 min and testing was usually carried out 2 hr later. At varying times (0–60 min) between training and testing, flies were anaesthetized with nitrogen gas for 25 sec. When flies were anaesthetized immediately after training the effect of nitrogen gas was the greatest and few flies showed any conditioned response, but the sensitivity of memory to nitrogen gas declined as the interval between training and nitrogen treatment became longer, and such treatment had no effect on memory when the interval was longer than 30 min. The effect on memory of cycloheximide, an inhibitor of peptide bond synthesis, was also investigated. The injection of cycloheximide (0.37 μg) immediately after training diminished the memory, but when given 1 hr after training it had no effect on memory. These results show that the memory in Phormia has two phases, stable and an unstable phase, like long-term and short-term memory in vertebrates.     

Characteristics of the infant arousal response

Arousal is considered to be an importantresponse to a life-threatening stimulus. Recently, it has been shownthat the infant arousal response to an elevated inspiredCO₂ level occurs as a sequence ofevents involving presumptive brain stem responses before awakening (A. Lijowska, N. Reed, B. Chiodini, and B. T. Thach. Am.J. Respir. Crit. Care Med. 151: A151, 1995; A. S. Lijowska, N. W. Reed, B. A. Mertins Chiodini, and B. T. Thach.J. Appl. Physiol. 83: 219-228,1997). We wanted to further evaluate the relationship ofsubcortical reflexes to cortical arousal in infants. We used anonrespiratory (tactile) stimulus to elicit arousal in infants duringnon-rapid-eye-movement (NREM) and rapid-eye-movement (REM) sleep. Wefound that a tactile stimulus elicited an arousal sequence thatcommenced with a spinal withdrawal reflex, was followed by brain stemresponses (respiratory and startle responses), and ended in a corticalarousal. The entire pathway or part of it in the order of spinal tocortical responses could be elicited. REM and NREMresponses were similar except for significant differences in thelatencies of spinal and subcortical reflexes. These observations suggest that the infant arousal response to a tactile stimulus involvesa progression of central nervous system activation from the spinal tocortical levels. The different components of the arousal pathway may beimportant for an infant to respond appropriately to stimuli duringsleep without necessarily disturbing sleep.

     

Volatile exposure within the honeybee hive and its effect on olfactory discrimination

Honeybees of different ages and reproductive castes cohabit in the hive where they are exposed to many odors that might affect associative learning. Our aim was to analyze the role of odors pre-exposed as volatiles on appetitive learning in honeybees of different ages and search for their long-term effect both under natural and laboratory conditions. By evaluating memory acquisition and retention through a differential proboscis extension response conditioning, we found that hive-exposed odors offered as a reinforced conditioned stimulus during training promoted a learning-reduced effect [latent inhibition (LI)]. On the other hand, no effect was found when the non-reinforced conditioned stimulus was pre-exposed. The LI effect varied with the odor identity. However, only slight differences were found with the age of the bees. Exposure-conditioning intervals longer than 24 h did not show an LI effect unless the odor concentration was increased or exposure was prolonged. Our results show that pre-exposed volatiles could either reduce learning performance, if this odor is later associated with food, or be irrelevant in the case that alternative scented resources circulate within the colony. The differential effects found according to the olfactory exposure characteristics could strongly influence the propagation of chemosensory information within the hive.     

Classical reward conditioning in Drosophila melanogaster

Negatively reinforced olfactory conditioning has been widely employed to identify learning and memory genes, signal transduction pathways and neural circuitry in Drosophila. To delineate the molecular and cellular processes underlying reward-mediated learning and memory, we developed a novel assay system for positively reinforced olfactory conditioning. In this assay, flies were involuntarily exposed to the appetitive unconditioned stimulus sucrose along with a conditioned stimulus odour during training and their preference for the odour previously associated with sucrose was measured to assess learning and memory capacities. After one training session, wild-type Canton S flies displayed reliable performance, which was enhanced after two training cycles with 1-min or 15-min inter-training intervals. Higher performance scores were also obtained with increasing sucrose concentration. Memory in Canton S flies decayed slowly when measured at 30 min, 1 h and 3 h after training; whereas, it had declined significantly at 6 h and 12 h post-training. When learning mutant t beta h flies, which are deficient in octopamine, were challenged, they exhibited poor performance, validating the utility of this assay. As the Drosophila model offers vast genetic and transgenic resources, the new appetitive conditioning described here provides a useful tool with which to elucidate the molecular and cellular underpinnings of reward learning and memory. Similar to negatively reinforced conditioning, this reward conditioning represents classical olfactory conditioning. Thus, comparative analyses of learning and memory mutants in two assays may help identify the molecular and cellular components that are specific to the unconditioned stimulus information used in conditioning.     

Olfactory interference during inhibitory backward pairing in honey bees

Background

Restrained worker honey bees are a valuable model for studying the behavioral and neural bases of olfactory plasticity. The proboscis extension response (PER; the proboscis is the mouthpart of honey bees) is released in response to sucrose stimulation. If sucrose stimulation is preceded one or a few times by an odor (forward pairing), the bee will form a memory for this association, and subsequent presentations of the odor alone are sufficient to elicit the PER. However, backward pairing between the two stimuli (sucrose, then odor) has not been studied to any great extent in bees, although the vertebrate literature indicates that it elicits a form of inhibitory plasticity.

Methodology/Principal Findings

If hungry bees are fed with sucrose, they will release a long lasting PER; however, this PER can be interrupted if an odor is presented 15 seconds (but not 7 or 30 seconds) after the sucrose (backward pairing). We refer to this previously unreported process as olfactory interference. Bees receiving this 15 second backward pairing show reduced performance after a subsequent single forward pairing (excitatory conditioning) trial. Analysis of the results supported a relationship between olfactory interference and a form of backward pairing-induced inhibitory learning/memory. Injecting the drug cimetidine into the deutocerebrum impaired olfactory interference.

Conclusions/Significance

Olfactory interference depends on the associative link between odor and PER, rather than between odor and sucrose. Furthermore, pairing an odor with sucrose can lead either to association of this odor to PER or to the inhibition of PER by this odor. Olfactory interference may provide insight into processes that gate how excitatory and inhibitory memories for odor-PER associations are formed.     

Olfactory conditioning of proboscis activity in Drosophila melanogaster

Olfactory learning and memory processes in Drosophila have been well investigated with aversive conditioning, but appetitive conditioning has rarely been documented. Here, we report for the first time individual olfactory conditioning of proboscis activity in restrained Drosophila melanogaster. The protocol was adapted from those developed for proboscis extension conditioning in the honeybee Apis mellifera. After establishing a scale of small proboscis movements necessary to characterize responses to olfactory stimulation, we applied Pavlovian conditioning, with five trials consisting of paired presentation of a banana odour and a sucrose reward. Drosophila showed conditioned proboscis activity to the odour, with a twofold increase of percentage of responses after the first trial. No change occurred in flies experiencing unpaired presentations of the stimuli, confirming an associative basis for this form of olfactory learning. The adenylyl cyclase mutant rutabaga did not exhibit learning in this paradigm. This protocol generated at least a short-term memory of 15 min, but no significant associative memory was detected at 1 h. We also showed that learning performance was dependent on food motivation, by comparing flies subjected to different starvation regimes.     

Responsiveness to sucrose and habituation of the proboscis extension response in honey bees

In honey bees, complex behaviours such as associative learning correlate with responsiveness to sucrose. In these behaviours, the subjective evaluation of a sucrose stimulus influences the behavioural performance. Habituation is a well-known form of non-associative learning. In bees, the proboscis extension response can be habituated by repeatedly stimulating the antennae with a low sucrose concentration. A high sucrose concentration can dishabituate the response. This study tests whether habituation correlates with responsiveness to sucrose in bees of different behavioural states and in bees which are habituated with different sucrose concentrations. Habituation and dishabituation in newly emerged bees, 5-day-old bees and foragers strongly correlated with responsiveness to sucrose. Bees with high responsiveness to sucrose displayed a lower degree of habituation and showed greater dishabituation than bees with low responsiveness. The degree of habituation and dishabituation also depended on the concentration of the habituation stimulus. These experiments demonstrate for the first time in a non-associative learning paradigm that the subjective strength of a sucrose stimulus determines the behavioural performance. Non-associative learning shares this property with associative learning, which suggests that the two processes might rely on similar neural mechanisms.Abbreviations: GRS Gustatory response score - PER Proboscis extension response     

Glutamatergic and GABAergic effects of fipronil on olfactory learning and memory in the honeybee

We investigated here the role of transmissions mediated by GABA and glutamate-gated chloride channels (GluCls) in olfactory learning and memory in honeybees, both of these channels being a target for fipronil. To do so, we combined olfactory conditioning with injections of either the GABA- and glutamate-interfering fipronil alone, or in combination with the blocker of glutamate transporter l-trans-Pyrrolidine-2,4-Dicarboxylicacid (l -trans-PDC), or the GABA analog Trans-4-Aminocrotonic Acid (TACA). Our results show that a low dose of fipronil (0.1 ng/bee) impaired olfactory memory, while a higher dose (0.5 ng/bee) had no effect. The detrimental effect induced by the low dose of fipronil was rescued by the coinjection of l-trans-PDC but was rather increased by the coinjection of TACA. Moreover, using whole-cell patch-clamp recordings, we observed that l-trans-PDC reduced glutamate-induced chloride currents in antennal lobe cells. We interpret these results as reflecting the involvement of both GluCl and GABA receptors in the impairment of olfactory memory induced by fipronil.     

Octopamine mediates rapid stimulation of protein kinase A in the antennal lobe of honeybees

In the honeybee octopamine mediates mechanisms of arousal that interfere with the appetitive proboscis extension response to food-indicating chemosensory stimuli. This study demonstrates that injections of octopamine or cyclic adenosine monophosphate (cAMP) into the primary chemosensory neuropil of the honeybee, the antennal lobe, evokes a rapid and transient activation of cAMP-dependent protein kinase (PKA). Other monoamines detectable in the antennal lobe, dopamine and serotonin, do not affect the level of PKA activity. Stimulation of the bees' antenna with the appetitive stimulus water or sucrose solution in vivo also causes a short-term activation of PKA in the antennal lobe. The increased PKA activity can be detected immediately (0.5 s) after stimulation but reverts to the basal level within 3 s. This effect can be abolished by monoamine depletion with reserpine. Since octopamine is the only monoamine that stimulates PKA, it appears to mediate the PKA activation after sucrose stimulus and may contribute to the processing of this chemosensory input. © 1995 John Wiley & Sons, Inc.     

Effects of Lanthanum Chloride Administration in Prenatal Stage on One-Trial Passive Avoidance Learning in Chicks

Lanthanum cations (La 3+) are well known for their inhibitory actions on calcium channels. Prenatal lanthanum exposure may affect the development of embryo and alter the capacity for learning and memory in adults, and the one-trial passive avoidance learning paradigm with day-old chicks is an excellent model to study several mechanisms of memory formation. In the present study, we examined the effects of prenatal lanthanum chloride exposure on memory consolidation using one-trial passive avoidance learning task in day-old chicks. The data suggest that chicks injected with lanthanum chloride (2 mg/kg) daily from E9 to E16 had significantly impaired long-term memory at 120 min after training (p < 0.05) but not the chicks injected with lanthanum chloride (0.1 mg/kg) daily from E9 to E16.     

Tactile learning and the individual evaluation of the reward in honey bees (Apis mellifera L.)

Using the proboscis extension response we conditioned pollen and nectar foragers of the honey bee (Apis mellifera L.) to tactile patterns under laboratory conditions. Pollen foragers demonstrated better acquisition, extinction, and reversal learning than nectar foragers. We tested whether the known differences in response thresholds to sucrose between pollen and nectar foragers could explain the observed differences in learning and found that nectar foragers with low response thresholds performed better during acquisition and extinction than ones with higher thresholds. Conditioning pollen and nectar foragers with similar response thresholds did not yield differences in their learning performance. These results suggest that differences in the learning performance of pollen and nectar foragers are a consequence of differences in their perception of sucrose. Furthermore, we analysed the effect which the perception of sucrose reward has on associative learning. Nectar foragers with uniform low response thresholds were conditioned using varying concentrations of sucrose. We found significant positive correlations between the concentrations of the sucrose rewards and the performance during acquisition and extinction. The results are summarised in a model which describes the relationships between learning performance, response threshold to sucrose, concentration of sucrose and the number of rewards. Accepted: 14 April 1999     

Visual associative learning in restrained honey bees with intact antennae

A restrained honey bee can be trained to extend its proboscis in response to the pairing of an odor with a sucrose reward, a form of olfactory associative learning referred to as the proboscis extension response (PER). Although the ability of flying honey bees to respond to visual cues is well-established, associative visual learning in restrained honey bees has been challenging to demonstrate. Those few groups that have documented vision-based PER have reported that removing the antennae prior to training is a prerequisite for learning. Here we report, for a simple visual learning task, the first successful performance by restrained honey bees with intact antennae. Honey bee foragers were trained on a differential visual association task by pairing the presentation of a blue light with a sucrose reward and leaving the presentation of a green light unrewarded. A negative correlation was found between age of foragers and their performance in the visual PER task. Using the adaptations to the traditional PER task outlined here, future studies can exploit pharmacological and physiological techniques to explore the neural circuit basis of visual learning in the honey bee.     

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.