Paul Szyszka

Multiple memory traces after associative learning in the honey bee antennal lobe

We investigated the effect of associative learning on early sensory processing, by combining classical conditioning with in vivo calcium‐imaging of secondary olfactory neurons, the projection neurons (PNs) in the honey bee antennal lobe (AL). We trained bees in a differential conditioning paradigm in which one odour (A+) was paired with a reward, while another odour (B−) was presented without a reward. Two to five hours after differential conditioning, the two odour–response patterns became more different in bees that learned to discriminate between A and B, but not in bees that did not discriminate. This learning‐related change in neural odour representations can be traced back to glomerulus‐specific neural plasticity, which depended on the response profile of the glomerulus before training. (i) Glomeruli responding to A but not to B generally increased in response strength. (ii) Glomeruli responding to B but not to A did not change in response strength. (iii) Glomeruli responding to A and B decreased in response strength. (iv) Glomeruli not responding to A or B increased in response strength. The data are consistent with a neural network model of the AL, which we based on two plastic synapse types and two well‐known learning rules: associative, reinforcer‐dependent Hebbian plasticity at synapses between olfactory receptor neurons (ORNs) and PNs; and reinforcer‐independent Hebbian plasticity at synapses between local interneurons and ORNs. The observed changes strengthen the idea that odour learning optimizes odour representations, and facilitates the detection and discrimination of learned odours.

Differential odor processing in two olfactory pathways in the honeybee.

An important component in understanding central olfactory processing and coding in the insect brain relates to the characterization of the functional divisions between morphologically distinct types of projection neurons (PN). Using calcium imaging, we investigated how the identity, concentration and mixtures of odors are represented in axon terminals (boutons) of two types of PNs – lPN and mPN. In lPN boutons we found less concentration dependence, narrow tuning profiles at a high concentration, which may be optimized for fine, concentration-invariant odor discrimination. In mPN boutons, however, we found clear rising concentration dependence, broader tuning profiles at a high concentration, which may be optimized for concentration coding. In addition, we found more mixture suppression in lPNs than in mPNs, indicating lPNs better adaptation for synthetic mixture processing. These results suggest a functional division of odor processing in both PN types.

DNA Methylation Mediates the Discriminatory Power of Associative Long-Term Memory in Honeybees

Memory is created by several interlinked processes in the brain, some of which require long-term gene regulation. Epigenetic mechanisms are likely candidates for regulating memory-related genes. Among these, DNA methylation is known to be a long lasting genomic mark and may be involved in the establishment of long-term memory. Here we demonstrate that DNA methyltransferases, which induce and maintain DNA methylation, are involved in a particular aspect of associative long-term memory formation in honeybees, but are not required for short-term memory formation. While long-term memory strength itself was not affected by blocking DNA methyltransferases, odor specificity of the memory (memory discriminatory power) was. Conversely, perceptual discriminatory power was normal. These results suggest that different genetic pathways are involved in mediating the strength and discriminatory power of associative odor memories and provide, to our knowledge, the first indication that DNA methyltransferases are involved in stimulus-specific associative long-term memory formation.

Mind the Gap: Olfactory Trace Conditioning in Honeybees

Trace conditioning is a form of classical conditioning, where a neutral stimulus (conditioned stimulus, CS) is associated with a following appetitive or aversive stimulus (unconditioned stimulus, US). Unlike classical delay conditioning, in trace conditioning there is a stimulus-free gap between CS and US, and thus a poststimulus neural representation (trace) of the CS is required to bridge the gap until its association with the US. The properties of such stimulus traces are not well understood, nor are their underlying physiological mechanisms. Using behavioral and physiological approaches, we studied appetitive olfactory trace conditioning in honeybees. We found that single-odor presentation created a trace containing information about odor identity. This trace conveyed odor information about the initial stimulus and was robust against interference by other odors. Memory acquisition decreased with increasing CS–US gap length. The maximum learnable CS–US gap length could be extended by previous trace-conditioning experience. Furthermore, acquisition improved when an additional odor was presented during the CS–US gap. Using calcium imaging, we tested whether projection neurons in the primary olfactory brain area, the antennal lobe, contain a CS trace. We found odor-specific persistent responses after stimulus offset. These post-odor responses, however, did not encode the CS trace, and perceived odor quality could be predicted by the initial but not by the post-odor response. Our data suggest that olfactory trace conditioning is a less reflexive form of learning than classical delay conditioning, indicating that odor traces might involve higher-level cognitive processes.

Olfactory coding in the insect brain: molecular receptive ranges, spatial and temporal coding

Odor information is coded in the insect brain in a sequence of steps, ranging from the receptor cells, via the neural network in the antennal lobe, to higher order brain centers, among which the mushroom bodies and the lateral horn are the most prominent. Across all of these processing steps, coding logic is combinatorial, in the sense that information is represented as patterns of activity across a population of neurons, rather than in individual neurons. Because different neurons are located in different places, such a coding logic is often termed spatial, and can be visualized with optical imaging techniques. We employ in vivo calcium imaging in order to record odor‐evoked activity patterns in olfactory receptor neurons, different populations of local neurons in the antennal lobes, projection neurons linking antennal lobes to the mushroom bodies, and the intrinsic cells of the mushroom bodies themselves, the Kenyon cells. These studies confirm the combinatorial nature of coding at all of these stages. However, the transmission of odor‐evoked activity patterns from projection neuron dendrites via their axon terminals onto Kenyon cells is accompanied by a progressive sparsening of the population code. Activity patterns also show characteristic temporal properties. While a part of the temporal response properties reflect the physical sequence of odor filaments, another part is generated by local neuron networks. In honeybees, γ‐aminobutyric acid (GABA)‐ergic and histaminergic neurons both contribute inhibitory networks to the antennal lobe. Interestingly, temporal properties differ markedly in different brain areas. In particular, in the antennal lobe odor‐evoked activity develops over slow time courses, while responses in Kenyon cells are phasic and transient. The termination of an odor stimulus is reflected by a decrease in activity within most glomeruli of the antennal lobe and an off‐response in some glomeruli, while in the mushroom bodies about half of the odor‐activated Kenyon cells also exhibit off‐responses.

The Speed of Smell: Odor-Object Segregation within Milliseconds

Segregating objects from background, and determining which of many concurrent stimuli belong to the same object, remains one of the most challenging unsolved problems both in neuroscience and in technical applications. While this phenomenon has been investigated in depth in vision and audition it has hardly been investigated in olfaction. We found that for honeybees a 6-ms temporal difference in stimulus coherence is sufficient for odor-object segregation, showing that the temporal resolution of the olfactory system is much faster than previously thought.

Converging Circuits Mediate Temperature and Shock Aversive Olfactory Conditioning in Drosophila

BACKGROUND Drosophila learn to avoid odors that are paired with aversive stimuli. Electric shock is a potent aversive stimulus that acts via dopamine neurons to elicit avoidance of the associated odor. While dopamine signaling has been demonstrated to mediate olfactory electric shock conditioning, it remains unclear how this pathway is involved in other types of behavioral reinforcement, such as in learned avoidance of odors paired with increased temperature. RESULTS To better understand the neural mechanisms of distinct aversive reinforcement signals, we here established an olfactory temperature conditioning assay comparable to olfactory electric shock conditioning. We show that the AC neurons, which are internal thermal receptors expressing dTrpA1, are selectively required for odor-temperature but not for odor-shock memory. Furthermore, these separate sensory pathways for increased temperature and shock converge onto overlapping populations of dopamine neurons that signal aversive reinforcement. Temperature conditioning appears to require a subset of the dopamine neurons required for electric shock conditioning. CONCLUSIONS We conclude that dopamine neurons integrate different noxious signals into a general aversive reinforcement pathway.

Olfactory Trace Conditioning in Drosophila

The neural representation of a sensory stimulus evolves with time, and animals keep that representation even after stimulus cessation (i.e., a stimulus “trace”). To contrast the memories of an odor and an odor trace, we here establish a rigorous trace conditioning paradigm in the fruit fly, Drosophila melanogaster. We modify the olfactory associative learning paradigm, in which the odor and electric shock are presented with a temporal overlap (delay conditioning). Given a few-second temporal gap between the presentations of the odor and the shock in trace conditioning, the odor trace must be kept until the arrival of electric shock to form associative memory. We found that memories after trace and delay conditioning have striking similarities: both reached the same asymptotic learning level, although at different rates, and both kinds of memory have similar decay kinetics and highly correlated generalization profiles across odors. In search of the physiological correlate of the odor trace, we used in vivo calcium imaging to characterize the odor-evoked activity of the olfactory receptor neurons in the antennal lobe. After the offset of odor presentation, the receptor neurons showed persistent, odor-specific response patterns that lasted for a few seconds and were fundamentally different from the response patterns during the stimulation. Weak correlation between the behavioral odor generalization profile in trace conditioning and the physiological odor similarity profiles in the antennal lobe suggest that the odor trace used for associative learning may be encoded downstream of the olfactory receptor neurons.

Asymmetric neural coding revealed by in vivo calcium imaging in the honey bee brain.

Left–right asymmetries are common properties of nervous systems. Although lateralized sensory processing has been well studied, information is lacking about how asymmetries are represented at the level of neural coding. Using in vivo functional imaging, we identified a population-level left–right asymmetry in the honey bees primary olfactory centre, the antennal lobe (AL). When both antennae were stimulated via a frontal odour source, the inter-odour distances between neural response patterns were higher in the right than in the left AL. Behavioural data correlated with the brain imaging results: bees with only their right antenna were better in discriminating a target odour in a cross-adaptation paradigm. We hypothesize that the differences in neural odour representations in the two brain sides serve to increase coding capacity by parallel processing.

Millisecond Stimulus Onset-Asynchrony Enhances Information about Components in an Odor Mixture

Airborne odorants rarely occur as pure, isolated stimuli. In a natural environment, odorants that intermingle from multiple sources create mixtures in which the onset and offset of odor components are asynchronous. Odor mixtures are known to elicit interactions in both behavioral and physiological responses, changing the perceptive quality of mixtures compared with the components. However, relevant odors need to be segregated from a distractive background. Honeybees (Apis mellifera) can use stimulus onset asynchrony of as little as 6 ms to segregate learned odor components within a mixture. Using in vivo calcium imaging of projection neurons in the honeybee, we studied neuronal mechanisms of odor-background segregation based on stimulus onset asynchrony in the antennal lobe. We found that asynchronous mixtures elicit response patterns that are different from their synchronous counterpart: the responses to asynchronous mixtures contain more information about the constituent components. With longer onset shifts, more features of the components were present in the mixture response patterns. Moreover, we found that the processing of asynchronous mixtures activated more inhibitory interactions than the processing of synchronous mixtures. This study provides evidence of neuronal mechanisms that underlie odor-object segregation on a timescale much faster than found for mammals.