Age-associated increase of the active zone protein Bruchpilot within the honeybee mushroom body.

In honeybees, age-associated structural modifications can be observed in the mushroom bodies. Prominent examples are the synaptic complexes (microglomeruli, MG) in the mushroom body calyces, which were shown to alter their size and density with age. It is not known whether the amount of intracellular synaptic proteins in the MG is altered as well. The presynaptic protein Bruchpilot (BRP) is localized at active zones and is involved in regulating the probability of neurotransmitter release in the fruit fly, Drosophila melanogaster. Here, we explored the localization of the honeybee BRP (Apis mellifera BRP, AmBRP) in the bee brain and examined age-related changes in the AmBRP abundance in the central bee brain and in microglomeruli of the mushroom body calyces. We report predominant AmBRP localization near the membrane of presynaptic boutons within the mushroom body MG. The relative amount of AmBRP was increased in the central brain of two-week old bees whereas the amount of Synapsin, another presynaptic protein involved in the regulation of neurotransmitter release, shows an increase during the first two weeks followed by a decrease. In addition, we demonstrate an age-associated modulation of AmBRP located near the membrane of presynaptic boutons within MG located in mushroom body calyces where sensory input is conveyed to mushroom body intrinsic neurons. We discuss that the observed age-associated AmBRP modulation might be related to maturation processes or to homeostatic mechanisms that might help to maintain synaptic functionality in old animals

Differences in long-term memory stability and AmCREB level between forward and backward conditioned honeybees (Apis mellifera)

In classical conditioning a predictive relationship between a neutral stimulus (conditioned stimulus; CS) and a meaningful stimulus (unconditioned stimulus; US) is learned when the CS precedes the US. In backward conditioning the sequence of the stimuli is reversed. In this situation animals might learn that the CS signals the end or the absence of the US. In honeybees 30 min and 24 h following backward conditioning a memory for the excitatory and inhibitory properties of the CS could be retrieved, but it remains unclear whether a late long-term memory is formed that can be retrieved 72 h following backward conditioning. Here we examine this question by studying late long-term memory formation in forward and backward conditioning of the proboscis extension response (PER). We report a difference in the stability of memory formed upon forward and backward conditioning with the same number of conditioning trials. We demonstrate a transcription-dependent memory 72 h after forward conditioning but do not observe a 72 h memory after backward conditioning. Moreover we find that protein degradation is differentially involved in memory formation following these two conditioning protocols. We report differences in the level of a transcription factor, the cAMP response element binding protein (CREB) known to induce transcription underlying long-term memory formation, following forward and backward conditioning. Our results suggest that these alterations in CREB levels might be regulated by the proteasome. We propose that the differences observed are due to the sequence of stimulus presentation between forward and backward conditioning and not to differences in the strength of the association of both stimuli.13 page(s

Distribution of AmBRP and Synapsin in the honeybee central brain.

<p>Optical sections of a honeybee central brain incubated with the anti-BRP^last200 and anti-SYNORF1 to visualize the presynaptic proteins AmBRP and Synapsin. <b>a</b> Longitudinal section through a schematic honeybee brain showing ventrally located regions (nomenclature after Ito et al. (2014) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0175894#pone.0175894.ref034" target="_blank">34</a>]). <b>b-d</b> Distribution of BRP^last200 signals (b) and anti-SYNORF1 signals (c) in ventrally located brain regions of a 29-day-old bee. Both antibodies show staining in all brain regions with almost similar distribution (d). Prominent stained regions are the vertical lobes and the antennal lobes. <b>e</b> Longitudinal section through a schematic honeybee brain showing dorsally located regions (nomenclature after Ito et al. (2014) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0175894#pone.0175894.ref034" target="_blank">34</a>]).<b>f-h</b> Distribution of BRP^last200 signals (f) and anti-SYNORF1 signals (g) in dorsally located brain regions of a 29-day-old bee. Both antibodies show staining in all brain regions with similar distribution (h) Prominent stained regions are the peduncles, especially in the AmBRP staining. LCA, lateral calyx; MCA, medial calyx; VL, vertical lobe; PED, peduncle; ML, medial lobe; CB, central body; AL, antennal lobe; NA, neuraxis anterior; M, medial; NP, neuraxis posterior; L, lateral. Scale bars: 200 μm for b-d and f-h.</p

Primary antibodies used in this study.

<p>Primary antibodies used in this study.</p

AmBRP variants detected in the honeybee brain.

<p><b>a</b> Map of the BRP^last200 and the BRP^D2 antibodies’ epitopes in the large <i>Drosophila</i> BRP isoforms D (NP_724796). <b>b</b> Immunoblot of honeybee central brain and fruit fly head homogenate. The BRP^last200 antibody and the BRP^D2 antibody recognize two bands around 220 kDa in honeybee central brains (lane 2, 4), and two major bands and several light bands between 120 and 220 kDa in fruit fly heads (lane 3, 5).</p

The median number of anti-BRP^last200- and anti-SYNORF1-positive pixels per ROI varies with age in lip and collar.

<p><b>a</b> The median number of anti-BRP^last200-positive (anti-BRP) pixels per ROI in the collar is higher in 43-day-old bees compared with 1-, 8- and 15-day-old bees. <b>b</b> The median number of anti-SYNORF1-positive (anti-SYNORF1) pixels per ROI in the collar is lower in 43-day-old bees compared with 1- and 15-day-old bees. <b>c</b> The ratio of anti-BRP^last200-positive pixels to anti-SYNORF1-positive pixels per ROI in the collar is higher in 43-day-old bees compared with 1-, 8- and 15-day-old bees. <b>d</b>. 1-day- and 43-day-old bees differ in their median number of anti-BRP^last200-positive pixels per ROI in the collar. <b>e</b> The median number of anti-SYNORF1-positive pixels did not change with age in the lip. <b>f</b> The ratio of anti-BRP^last200-positive pixels to anti-SYNORF1-positive pixels per ROI in the lip is higher in 8-day-old bees compared with 1-day-old bees and in 43-day old bees compared with 15- and 29-day-old bees. Box blots show median, 25% and 75% quartiles and value range (min-max). (*) Significant differences (p < 0.05) detected with Mann-Whitney U test after Kruskal Wallis ANOVA.</p

AmBRP is predominantly located in the vicinity of the membrane of presynaptic boutons within microglomeruli in the mushroom body calyces.

<p><b>a-c</b> Confocal images of the medial calyx showing BRP^last200 staining (AmBRP, green, b) in combination with Phalloidin staining (F-actin, magenta, a) and an anti-SYNORF1 counterstaining (Synapsin, blue, a) to visualize pre- and postsynaptic structures in a 8-day-old bee. The calyx can be subdivided into three regions, lip, collar and basal ring. Experiments focused on the lip and the dense region of the collar (dCO). <b>d</b> Schematic representation of a microglomerulus (MG) (modified after [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0175894#pone.0175894.ref035" target="_blank">35</a>] showing already established pre- and postsynaptic marker (Synapsin, blue; F-actin, magenta). The bouton of a projection neuron is surrounded by spines from Kenyon cell dendrites. Anti-SYNORF1 labels the vesicle-associated protein Synapsin (blue) whereas Alexa Fluor 546 Phalloidin binds to F-actin located in dendritic spines (magenta). <b>e-k</b> Confocal images of MG in the dense collar region with labeled Synapsin (e), F-actin (f) and AmBRP (h). The F-actin signals form circles around Synapsin signals (g). AmBRP is located predominantly at the outer rim of the Synapsin-labeled signals and at the inner rim of the F-actin signals (i-k). The insets show a single, magnified MG from the corresponding image. LI, lip; CO, collar; BR, basal ring; dCO, dense collar; PN, projection neuron; KC, Kenyon cell.Scale bars: 20 μm for a-c, 2 μm for e-k, 1 μm for insets.</p