Long-term memory leads to synaptic reorganization in the mushroom bodies: a memory trace in the insect brain?

Abstract

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.

Figure 1.

Spaced olfactory conditioning leads to transcription-dependent, odorant-specific, late long-term memory. A, Learning performances are measured as the percentage of conditioned responses (PER elicited by the CS). Over the 5 trials of conditioning, only bees from the paired groups (including those later treated with Actinomycin D: paired ActD group) formed the CS–US association. Almost no conditioned responses were produced when the CS and the US were temporally dissociated (unpaired) or absent (naive). B, Specific retention levels of the association assessed 72 h after conditioning, as the difference between responses to the learned odor (1-nonanol) and to a novel odor (1-hexanol). Bees from the paired group displayed significant levels of specific memory. Bees in which transcription was blocked after learning (paired ActD group) or those that had not learned (unpaired and naive groups) showed levels of specific responses not significantly different from 0. ***p < 0.001.

Figure 2.

Microglomerular density is increased in the mushroom body lips when an olfactory memory is formed. A, Microglomerular density was measured in each medial calyx (red frame), both in the lip—receiving olfactory input from the PNs exiting the AL—and in the collar—receiving visual input from the optic lobe (OL). ORNs, Olfactory receptor neurons. Microglomeruli were counted in 400 μm² circles placed in the medial lip (continuous outlines) and collar (dotted outlines) (see Materials and Methods). B, Estimated densities of microglomeruli averaged over 8 circles for both the lip and collar of each animal. Bees from the paired group showed a density significantly higher than that in the 3 control groups, in the lip only. *p < 0.05; **p < 0.01.

Figure 3.

The lip volume is not affected by associative olfactory long-term memory. A, The medial lips of the medial calyces (where microglomerular density was measured) were 3D-reconstructed and their volume estimated, as was the central body (CB), a nonolfactory center. B, No differences were found between the mean volumes of the lip and central body of the different groups of individuals. NS, Nonsignificant.