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.

A detailed account is given by the octopaminergic innervation of the antennal heart in Schistocerca gregaria using various immunohistochemical methods. Anterograde axonal filling illustrates the unilateral innervation on the medial ventral surface of the pumping muscle of the antennal heart via the paired corpora cardiaca nerve III. In addition, antibody staining revealed that ascending axons of this nerve terminate at the ampullae of the antennal heart forming synaptoid structures and extensive neurohaemal release sites. Due to the innervation by two dorsal unpaired median neurons, the presence of the biogenic amines octopamine and tyramine could be visualized by immunocytochemistry in an insect antennal heart for the first time. The data suggest that tyramine acts as a precursor and not purely as an independent transmitter. While the octopaminergic fibers innervating the pumping muscle of the antennal heart indicate a cardioregulatory role, we conclude that octopamine released from the neurohaemal area is pumped into the antennae and an involvement in the modulation of the antennal sensory sensitivity is discussed.

No abstract