The fundamental premise of the NeuroContext action lies in the ability to manipulate the activity of a particular neuronal type in different genetic contexts. This requires consistent targeting of the neuron type with genetic tools to manipulate activity across different genetic backgrounds. An essential initial task in this action was to validate the feasibility of this approach. A potential strategy to assess the precision of a genetic tool's targeting involves tagged proteins to analyze the expression pattern of the functional genetic tool. For that reason, we started by developing a new genetic construct that combines the commonly used Tetanus toxin light chain (TNT) tagged with the reporter gene GFP. Unfortunately, we could not obtain transgenic animals with our plasmids. Consequently, we pursued an alternative approach, generating new recombinant D.melanogaster lines that combine, independently, TNT and GFP. This approach allowed us to check in the same animal where inactivation with TNT is being performed, by analyzing the expression pattern of GFP. Because the constructs are independent, there might be differences between the expression patterns. For this reason, we optimized an immunofluorescence staining to TNT to confirm the concordance between GFP and TNT expression. With this confirmation, we set out to explore if genetic background affects the correct expression of genetic tools. Remarkably, we observed a consistent and correct expression of the genetic tools targeting our neurons of interest across all tested genetic backgrounds.
We focused our work on DNp09, a descending neuron previously described in D. melanogaster to be involved in locomotion and freezing behavior. Like other animals, flies respond to a threat by fleeing, fighting or freezing. Freezing is state of total immobility that can last for seconds, thought to be adopted to avoid detection by predators. Inhibiting DNp09 reduced the probability of freezing upon presentation of looms, a stimulus that mimics a fast-approaching object. However, our findings unveiled a nuanced perspective, as in certain genotypes, the inactivation of DNp09 did not diminish the duration of freezing behavior upon looming stimulus presentation. This highlights the importance of considering the genetic background when manipulating neuronal activity. Comparing neuron morphology in the different genetic backgrounds with a quantitative approach, we show that, for DNp09, genetic background does not affect neuron morphology in the central brain. Thus, morphology does not explain the necessity of DNp09 for freezing behavior that is modulated genetic background. Currently, we are exploring the genetic basis to explain this phenotype, by first determining what chromosomes contain genetic variants that affect the necessity of DNp09 for freezing behavior.
The results from NeuroContext were presented in a European conference dedicated to new discoveries in fly neurobiology (NeuroFy 2022) and in a similar conference in the EUA (Neurobiology of Drosophila 2023). Internally, the results have been presented in lab meetings, internal seminar and scientific retreat. A research article is being prepared now, to be published in a peer-reviewed journal. Other levels of impact were achieved with this action. For science communication, I have participated in “Ciência di Noz Manera”, an initiative coordinated by RAISE, an European Union funded program to promote scientific knowledge in schools with reduced engagement with higher education. This involved, two phases, one day of science demonstrations, at Champalimaud foundation and a mentoring program at school Pedro D’Orey da Cunha. Moreover, demonstrations of scientific activities were performed to visiting schools at Champalimaud, further enhancing the initiative’s outreach.
