Multisite imaging of neural activity using a genetically encoded calcium sensor in the honey bee Apis mellifera

Background

How are complex social interactions encoded in the brain, what cognitive skills are required and how did the underlying neuronal circuits evolve? To tackle these questions, research needs access to brains from animals that naturally display a high degree of sociality and engage in sophisticated social behaviors. The most advanced form of sociality – eusociality – is particularly well described for insects^1,2. Among insects, hymenopterans present the largest number of eusocial species, including bees, ants and wasps. Bees live in colonies of up to 60,000 individuals, comprising the three adult casts: queen, males and workers ², the latter of which are again subdivided by specialized labor roles. For a colony to be successful, its members need to be able to communicate, understand social cues (e.g. olfactory cues such as pheromones³) and integrate them with higher cognitive abilities^4,5. While the understanding of neuronal circuits underlying behavior in other species has benefited from the development of neurogenetic tools in the last decades, the creation of transgenics in eusocial insects has proved particularly difficult⁶. In this preprint, Carcaud et al., generated a transgenic driver line as a tool to unlock the next level of investigating the social brain of the bees.

Findings

• The authors created the first pan-neuronal driver in a bee expressing a calcium sensor under the control of the synapsin promoter. To achieve this, they fused the promoter region of the gene encoding synapsin – a protein involved in transmitter release at chemical synapses throughout the brain – to the Calcium indicator GCaMP6f and introduced this whole expression cassette into the bee genome.
• Next, the researches verified the expression of the construct by using immunohistochemistry, labeling the GFP contained in the GCaMP6f construct. They found expression throughout the brain. Complementary immunostaining targeting the synapsin protein revealed general co-expression, but with some differences on the subcellular level. The synapsin protein localizes primarily to the presynapse, while the GCaMP6f construct could be detected in cell somata as well as neurites, a feature that could come in handy during functional imaging experiments.
• To prove its functionality, GCaMP6f expression was used to record neuronal responses upon odor stimulation in three olfaction-associated centers in the honey bee brain. Odors are detected by olfactory sensory neurons in the antennae, which send information to the antennal lobe. From there, projection neurons connect to higher-order processing centers, the mushroom bodies and the lateral horn³. The authors applied 16 aliphatic compounds with varying carbon chain lengths (from 6 to 9 carbons) belonging to different functional groups (primary and secondary alcohols, aldehydes, and ketones) as odor stimuli and recorded odor-evoked biphasic calcium transients in all three olfactory centers in the brain.
• Odor coding in the antennal lobe and the lateral horn was evaluated by comparing similarity relationships between response patterns. In the antennal lobe, odorants with similar chemical features (functional group/carbon chain length) had more similar response patterns, faithfully encoding the chemical structure. In the lateral horn however, this relationship was not as clear, suggesting signal transformation between the antennal lobe and lateral horn. In addition, response amplitudes in the antennal lobe positively correlated with the odor vapor pressure. In the lateral horn, this correlation was weaker. Response intensity in the lateral horn is thus not only dependent on how many odorant molecules are present in a given odor puff, suggesting some kind of control mechanism between these two olfactory centers.
• Finally, the authors used an appetitive conditioning experiment to test behavioral responses to the applied odorants. Odors that elicited a similar response pattern in the brain were also behaviorally treated as similar by the bees.

In summary, the preprint clearly demonstrates that the calcium indicator under a synapsin promoter is expressed throughout the brain and can be used in functional imaging experiments to simultaneously record from neuronal populations in different brain regions.

Why I chose to highlight this preprint

I chose this preprint because I believe that neuroscience has to leverage the diversity of species with their specific behaviors to gain further understanding of how the brain works and evolves. Therefore, it is good to see that neurogenetic tools are currently being developed for many different species. The GCaMP bee will definitely accelerate further discovery of neuronal circuits underlying the fascinating sociality and sophisticated behaviors of this species.

Questions to the authors

• Are you planning to investigate differences in odor coding strategies in the different social castes using calcium imaging? Which parts of the brain do you expect to be functionally most different between the different castes?
• Since bees are a prime example of eusociality, which experiments are you planning to investigate their social learning/communication utilizing the syn-GCaMP bees?
• Which other neurogenetic tools would you want to develop in the bees?

References

1. Wilson, E. O. The social conquest of earth. (Liveright Publishing Corporation, 2012).
2. Oster, G. F. & Wilson, E. O. Caste and ecology in the social insects. Monogr. Popul. Biol. (1978) doi:10.2307/2530130.
3. Paoli, M. & Galizia, G. C. Olfactory coding in honeybees. Cell and Tissue Research (2021) doi:10.1007/s00441-020-03385-5.
4. Cholé, H. et al. Social Contact Acts as Appetitive Reinforcement and Supports Associative Learning in Honeybees. Curr. Biol. (2019) doi:10.1016/j.cub.2019.03.025.
5. Barron, A. B. & Plath, J. A. The evolution of honey bee dance communication: A mechanistic perspective. Journal of Experimental Biology (2017) doi:10.1242/jeb.142778.
6. Schulte, C., Theilenberg, E., Müller-Borg, M., Gempe, T. & Beye, M. Highly efficient integration and expression of piggyBac-derived cassettes in the honeybee (Apis mellifera). Proc. Natl. Acad. Sci. U. S. A. (2014) doi:10.1073/pnas.1402341111.

Tags: antennal lobe, calcium imaging, eusocial insect, gcamp, honey bee, lateral horn, olfaction, panneuronal, transgenic

doi: https://doi.org/10.1242/prelights.32125

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