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.

Involvement of phosphorylated Apis mellifera CREB in gating a honeybee's behavioral response to an external stimulus.

The transcription factor cAMP-response element-binding protein (CREB) is involved in neuronal plasticity. Phosphorylation activates CREB and an increased level of phosphorylated CREB is regarded as an indicator of CREB-dependent transcriptional activation. In honeybees(Apis mellifera)we recently demonstrated a particular high abundance of the phosphorylated honeybee CREB homolog (pAmCREB) in the central brain and in a subpopulation of mushroom body neurons. We hypothesize that these high pAmCREB levels are related to learning and memory formation. Here, we tested this hypothesis by analyzing brain pAmCREB levels in classically conditioned bees and bees experiencing unpaired presentations of conditioned stimulus (CS) and unconditioned stimulus (US). We demonstrate that both behavioral protocols display differences in memory formation but do not alter the level of pAmCREB in bee brains directly after training. Nevertheless, we report that bees responding to the CS during unpaired stimulus presentations exhibit higher levels of pAmCREB than nonresponding bees. In addition, Trichostatin A, a histone deacetylase inhibitor that is thought to enhance histone acetylation by CREB-binding protein, increases the bees' CS responsiveness. We conclude that pAmCREB is involved in gating a bee's behavioral response driven by an external stimulus.

Abundance of phosphorylated Apis mellifera CREB in the honeybee's mushroom body inner compact cells varies with age.

Hymenopteran eusociality has been proposed to be associated with the activity of the transcription factor CREB (cAMP-response element binding protein). The honeybee (Apis mellifera) is a eusocial insect displaying a pronounced age-dependent division of labor. In honeybee brains, CREB-dependent genes are regulated in an age-dependent manner, indicating that there might be a role for neuronal honeybee CREB (Apis mellifera CREB, or AmCREB) in the bee's division of labor. In this study, we further explore this hypothesis by asking where in the honeybee brain AmCREB-dependent processes might take place and whether they vary with age in these brain regions. CREB is activated following phosphorylation at a conserved serine residue. An increase of phosphorylated CREB is therefore regarded as an indicator of CREB-dependent transcriptional activation. Thus, we here examine the localization of phosphorylated AmCREB (pAmCREB) in the brain and its age-dependent variability. We report prominent pAmCREB staining in a subpopulation of intrinsic neurons of the mushroom bodies. In these neurons, the inner compact cells (IC), pAmCREB is located in the nuclei, axons, and dendrites. In the central bee brain, the IC somata and their dendritic region, we observed an age-dependent increase of pAmCREB. Our results demonstrate the IC to be candidate neurons involved in age-dependent division of labor. We hypothesize that the IC display a high level of CREB-dependent transcription that might be related to neuronal and behavioral plasticity underlying a bee's foraging behavior.

Behavioural pharmacology in classical conditioning of the proboscis extension response in honeybees (Apis mellifera).

Honeybees (Apis mellifera) are well known for their communication and orientation skills and for their impressive learning capability(1,2). Because the survival of a honeybee colony depends on the exploitation of food sources, forager bees learn and memorize variable flower sites as well as their profitability. Forager bees can be easily trained in natural settings where they forage at a feeding site and learn the related signals such as odor or color. Appetitive associative learning can also be studied under controlled conditions in the laboratory by conditioning the proboscis extension response (PER) of individually harnessed honeybees(3,4). This learning paradigm enables the study of the neuronal and molecular mechanisms that underlie learning and memory formation in a simple and highly reliable way(5-12). A behavioral pharmacology approach is used to study molecular mechanisms. Drugs are injected systemically to interfere with the function of specific molecules during or after learning and memory formation(13-16). Here we demonstrate how to train harnessed honeybees in PER conditioning and how to apply drugs systemically by injection into the bee flight muscle.