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.Learning and memory formation in vertebrates and invertebrates have been studied on the basis of a large range of classical and operant conditioning paradigms. Typically, the interpretation of experimental results relies on performance measures that were derived by averaging over behavioral observations from identically treated animals. However, several studies have recognized the inadequacy of group-average measures to capture the characteristics of individual behavior and, consequently, the learninginduced changes in individual brains (Krechevsky 1932;Restle 1965;Hanson and Killeen 1981;Estes 2002;Brown and Heathcote 2003;Cousineau et al. 2003). Most notably, Gallistel et al. (2004) found that the gradually increasing learning curve observed in many vertebrate learning paradigms reflected an artifact of group averaging. The behavioral performance of individuals appeared to be characterized by an abrupt and often step-like increase in the level of response.To our knowledge and in contrast to the vertebrate literature (see Gallistel et al. 2004), surprisingly little is known of a possibly heterogeneous expression of behavior for the most frequently applied invertebrate conditioning paradigms. For the fruit fly (Drosophila melanogaster) it appears to be common sense that the group-average behavioral measures adequately represent the probabilistic expression of behavior in individuals-a notion that goes back to an early study by Quinn et al. (1974).In the following,...

Honeybees (Apis mellifera) are well known for their communication and orientation skills and for their impressive learning capability 1,2 . Because the survival of a honeybee colony depends on the exploitation of food sources, forager bees learn and memorize variable flower sites as well as their profitability. Forager bees can be easily trained in natural settings where they forage at a feeding site and learn the related signals such as odor or color. Appetitive associative learning can also be studied under controlled conditions in the laboratory by conditioning the proboscis extension response (PER) of individually harnessed honeybees 3,4 . This learning paradigm enables the study of the neuronal and molecular mechanisms that underlie learning and memory formation in a simple and highly reliable way [5][6][7][8][9][10][11][12] . A behavioral pharmacology approach is used to study molecular mechanisms. Drugs are injected systemically to interfere with the function of specific molecules during or after learning and memory formation [13][14][15][16] .Here we demonstrate how to train harnessed honeybees in PER conditioning and how to apply drugs systemically by injection into the bee flight muscle. Video LinkThe video component of this article can be found at https://www.jove.com/video/2282/ Protocol 1. Catching Bees from the Hive 1. One day before the experiment starts, between 2 and 4 p.m., bees leaving the hive are caught. To do so, a UV light-permeable plexiglass pyramid (height = 30 cm, apex 3,5 x 3, 5 cm, base 18 x 18 cm), which is closable at the apex and the base, is held at a 20-30 cm distance in front of the hive entrance with the base open and the apex closed so that bees leaving the hive enter the base of the pyramid. The base is then closed and the captured bees are brought into the lab for further handling. Transferring Bees from the Pyramid Into Glass Vials1. In the lab, the pyramid is placed on its base. The walls of the pyramid are darkened (e.g. with a towel) but the apex is left uncovered. Because of their positive phototaxis, bees will leave the pyramid through the apex when opened. One by one, bees are transferred from the pyramid into glass vials by holding the vials over the open apex. One vial is used per bee. Therefore, the apex is closed when one bee enters the vial. Harnessing Bees in Tubes1. Bees are immobilized by cooling them in the glass vials on ice for 2.5-3.5 min. It is advisable to watch the bee and remove it from the ice as soon as it stops moving. 2. A single immobilized bee is harnessed in a small plastic tube with sticky tape, such that it is able to move its proboscis freely but not its head, thorax or legs. It is important that the neck is not compressed. 3. Every bee fixed in a plastic tube is put into a numbered borehole on a rack for better handling and identification. After it has been removed for conditioning or memory retrieval the tube is always returned to the exact same borehole.

Hymenopteran eusociality has been proposed to be associated with the activity of the transcription factor CREB (cAMP-response element binding protein). The honeybee (Apis mellifera) is a eusocial insect displaying a pronounced age-dependent division of labor. In honeybee brains, CREB-dependent genes are regulated in an age-dependent manner, indicating that there might be a role for neuronal honeybee CREB (Apis mellifera CREB, or AmCREB) in the bee's division of labor. In this study, we further explore this hypothesis by asking where in the honeybee brain AmCREB-dependent processes might take place and whether they vary with age in these brain regions. CREB is activated following phosphorylation at a conserved serine residue. An increase of phosphorylated CREB is therefore regarded as an indicator of CREB-dependent transcriptional activation. Thus, we here examine the localization of phosphorylated AmCREB (pAmCREB) in the brain and its age-dependent variability. We report prominent pAmCREB staining in a subpopulation of intrinsic neurons of the mushroom bodies. In these neurons, the inner compact cells (IC), pAmCREB is located in the nuclei, axons, and dendrites. In the central bee brain, the IC somata and their dendritic region, we observed an age-dependent increase of pAmCREB. Our results demonstrate the IC to be candidate neurons involved in age-dependent division of labor. We hypothesize that the IC display a high level of CREB-dependent transcription that might be related to neuronal and behavioral plasticity underlying a bee's foraging behavior.

In honeybees, age-associated structural modifications can be observed in the mushroom bodies. Prominent examples are the synaptic complexes (microglomeruli, MG) in the mushroom body calyces, which were shown to alter their size and density with age. It is not known whether the amount of intracellular synaptic proteins in the MG is altered as well. The presynaptic protein Bruchpilot (BRP) is localized at active zones and is involved in regulating the probability of neurotransmitter release in the fruit fly, Drosophila melanogaster. Here, we explored the localization of the honeybee BRP (Apis mellifera BRP, AmBRP) in the bee brain and examined age-related changes in the AmBRP abundance in the central bee brain and in microglomeruli of the mushroom body calyces. We report predominant AmBRP localization near the membrane of presynaptic boutons within the mushroom body MG. The relative amount of AmBRP was increased in the central brain of two-week old bees whereas the amount of Synapsin, another presynaptic protein involved in the regulation of neurotransmitter release, shows an increase during the first two weeks followed by a decrease. In addition, we demonstrate an age-associated modulation of AmBRP located near the membrane of presynaptic boutons within MG located in mushroom body calyces where sensory input is conveyed to mushroom body intrinsic neurons.We discuss that the observed age-associated AmBRP modulation might be related to maturation processes or to homeostatic mechanisms that might help to maintain synaptic functionality in old animals.

The transcription factor cAMP-response element-binding protein (CREB) is involved in neuronal plasticity. Phosphorylation activates CREB and an increased level of phosphorylated CREB is regarded as an indicator of CREB-dependent transcriptional activation. In honeybees (Apis mellifera) we recently demonstrated a particular high abundance of the phosphorylated honeybee CREB homolog (pAmCREB) in the central brain and in a subpopulation of mushroom body neurons. We hypothesize that these high pAmCREB levels are related to learning and memory formation. Here, we tested this hypothesis by analyzing brain pAmCREB levels in classically conditioned bees and bees experiencing unpaired presentations of conditioned stimulus (CS) and unconditioned stimulus (US). We demonstrate that both behavioral protocols display differences in memory formation but do not alter the level of pAmCREB in bee brains directly after training. Nevertheless, we report that bees responding to the CS during unpaired stimulus presentations exhibit higher levels of pAmCREB than nonresponding bees. In addition, Trichostatin A, a histone deacetylase inhibitor that is thought to enhance histone acetylation by CREB-binding protein, increases the bees' CS responsiveness. We conclude that pAmCREB is involved in gating a bee's behavioral response driven by an external stimulus.