Olfactory Interference during Inhibitory Backward Pairing in Honey Bees

Citation: Dacher M, Smith BH ( Olfactory Interference during Inhibitory Backward Pairing in Honey Bees Matthieu Dacher 0 Brian H. Smith 0 Bjo rn Brembs, Freie Universitaet Berlin, Germany 0 School of Life Sciences, Arizona State University , Tempe, Arizona , United States of America 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. - Insects have proven to be invaluable for studying not only the basic forms of learning but also for understanding how higher-order cognitive processes might be supported by their smaller, more accessible nervous systems [13]. Use of insects such as fruit flies and honey bees has clearly provided insight into the cellular and molecular events that underlie sophisticated behavioral plasticity in higher vertebrates [4,5]. Likewise, well-conceived mechanistic studies of insect behavior can reveal components of the more complex cognitive phenomena found in mammals [611]. In particular, there is a rich history of using the honey bee for studying both non-associative and associative learning processes [13,1217] as well as cognitive processes such as choice behavior [18,19], non-elemental learning [20,21], the use of conceptual rules [911], spatial orientation [22,23], visual categorization [24], delayed alternation [25], lateralization [26] and inter-individual communication about location of food via the dance language [23,27]. Non-associative and associative learning have been extensively studied in the laboratory with restrained bees using a well-defined behavioral response called the proboscis extension response (PER, the proboscis is the main mouthpart of the bee). PER is an appetitive response triggered when sucrose solution -the unconditioned stimulus (US)- is applied to the antennae and/or the proboscis. The animal then extends its proboscis to consume the sucrose solution. If an odor the conditioned stimulus (CS)precedes and slightly overlaps sucrose presentation (forward pairing), the bee will form an excitatory association between this odor and the sucrose (and/or the PER) by way of classical (Pavlovian) conditioning [28]. On the other hand, presentation of an odor 15 s after sucrose delivery (backward pairing) will produce inhibitory learning about this odor, i.e. bees will display poorer performance during subsequent training [29]. The PER olfactory conditioning protocol allows for the precise control of stimulation parameters in behavioral studies as well as for simultaneous linkage to neurophysiological and imaging analyses of brain activity [3033]. Furthermore PER conditioning allows for pharmacological and molecular manipulation of identified modulatory pathways involved in processing stimuli, learning and reinforcement [3448]. Recent studies have begun to extend PER conditioning to cognitive processes such as choice behavior [18,19] and non-elemental learning [20,21] using restrained bees (rather than free-flying bees) in the controlled conditions of a laboratory. These studies provide the opportunity for associating more complex kinds of learning with neurophysiological measurements and molecular manipulations. are significantly lower than 1 during the 1519 s time period (the odor presentation period), which means that the odor-treated groups have a significantly lower survival rate than the air-treated or the control group during the odor presentation; in other words, they retract their proboscis more often. Note that the scale is logarithmic. This is because Cox regression uses an exponential equation that produces very large (and asymmetric) error bars. Furthermore, the error bars increase in size when the number of animals still displaying a PER decreases at later time points, because the sample size is decreasing and the estimation of the ratio loses precision. Hence, the error bars are larger during the last time period (1934 s) because few bees are still displaying a PER. doi:10.1371/journal.pone.0003513.g001 Backward pairing of sucrose and odor has received relatively less attention [29]. Here we report on the observation of a new PER related phenomenon, which we call olfactory interference. During the olfactory interference protocol, a hungry bee consumes a small droplet of sucrose solution, which elicits PER that continues for several seconds after the sucrose has been consumed. After the end of the feeding, the proportion of bees displaying the ongoing PER decays smoothly. This ongoing PER can be abruptly interrupted by presentation of an odor stimulus (backward pairing), but only within a specific time frame. Olfactory interference elicits inhibitory conditioning as with other backward pairing stimuli [29]. We argue that studying olfactory interference will provide insight into an important process that gates how excitatory and inhibitory memories for odor-PER associations are formed in the brain. It is well-established that inhibitory neurotransmitters (such as histamine) are involved in olfactory processing in well defined areas of the honey bee brain [33,37,48,49]. We show that olfactory interference can be disrupted by blockade of cimetidine-sensitive pathways specifically in the deuterocerebrum. Olfactory interference In this first experiment, sucrose was presented to the (...truncated)