The insect mushroom bodies (MBs) are paired brain centers which, like the mammalian hippocampus, have a prominent function in learning and memory. Despite convergent evidence for their crucial role in the formation and storage of associative memories, little is known about the mechanisms underlying such storage. In mammals and other species, the consolidation of stable memories is accompanied by structural plasticity involving variations in synapse number and/or size. Here, we address the question of whether the formation of olfactory long-term memory (LTM) could be associated with changes in the synaptic architecture of the MB networks. For this, we took advantage of the modular architecture of the honeybee MB neuropil, where synaptic contacts between olfactory input and MB neurons are segregated into discrete units (microglomeruli) which can be easily visualized and counted. We show that the density in microglomeruli increases as a specific olfactory LTM is formed, while the volume of the neuropil remains constant. Such variation is reproducible and is clearly correlated with memory consolidation, as it requires gene transcription. Thus stable structural synaptic rearrangements, including the growth of new synapses, seem to be a common property of insect and mammalian brain networks involved in the storage of stable memory traces.

The storage of stable memories is generally considered to rely on changes in the functional properties and/or the synaptic connectivity of neural networks. However, these changes are not easily tractable given the complexity of the learning procedures and brain circuits studied. Such a search can be narrowed down by studying memories of specific stimuli in a given sensory modality and by working on networks with a modular and relatively simple organization. We have therefore focused on associative memories of individual odors and the possible related changes in the honeybee primary olfactory center, the antennal lobe (AL). As this brain structure is organized in well-identified morpho-functional units, the glomeruli, we looked for evidence of structural and functional plasticity in these units in relation with the bees' ability to store long-term memories (LTMs) of specific odors. Restrained bees were trained to form an odor-specific LTM in an appetitive Pavlovian conditioning protocol. The stability and specificity of this memory was tested behaviorally 3 d after conditioning. At that time, we performed both a structural and a functional analysis on a subset of 17 identified glomeruli by measuring glomerular volume under confocal microscopy, and odor-evoked activity, using in vivo calcium imaging. We show that long-term olfactory memory for a given odor is associated with volume increases in a subset of glomeruli. Independent of these structural changes, odor-evoked activity was not modified. Lastly, we show that structural glomerular plasticity can be predicted based on a putative model of interglomerular connections.[Supplemental material is available online at http: //www.learnmem.org.]In nature, animals' survival relies on their capacity to adapt to changes in the environment by constantly learning and memorizing novel information and modifying their behavior accordingly. This capacity relies on the long-term storage of learned information involving specific stable modifications of neural networks, both in their connectivity and in the strength of synaptic transmission (Matsuzaki 2007). For such long-term memory (LTM) to be specific for particular stimuli (for instance, an odor), they must rely on specific neural traces of these stimuli in the brain. A crucial question is whether such specific traces can be tracked down to individual neural units, given the complexity of the neuronal networks usually involved in the formation of LTM. Brain regions constituted of relatively few neurons and organized in clearly identified modules offer an excellent opportunity to answer this question. Among these is the insect antennal lobe (AL), an olfactory center that shares many similarities with the vertebrate olfactory bulb, but provides numerical simplicity. In the honeybee, the AL contains ;160 interconnected neuropil subunits, the glomeruli, which can be unambiguously identified across individuals (Galizia et al. 1999a). Experiments using in vivo calcium imaging have shown that glomeruli are functional units for odor...

Storage of information into long-term memory (LTM) usually requires at least two waves of transcription in many species. However, there is no clear evidence of this phenomenon in insects, which are influential models for memory studies. We measured retention in honeybees after injecting a transcription inhibitor at different times before and after conditioning. We identified two separate time windows during which the transcription blockade impairs memory quantitatively and qualitatively, suggesting the occurrence of an early transcription wave (triggered during conditioning) and a later one (starting several hours after learning). Hence insects, like other species, would require two transcription waves for LTM formation.