Periodic Reporting for period 1 - NeuroContext (The impact of genetic background in the activity of single neurons)
Reporting period: 2021-09-01 to 2023-08-31
Recent advancements in technology and genetic engineering have significantly expedited our comprehension of the nervous system. Particularly, novel genetic tools allow the targeting of discrete neuronal types, facilitating the analysis and manipulation of their activity. This allows researchers to better decipher neuronal circuits responsible for driving behaviors.
For practical reasons, much of this research relies on standardized genotypes, wherein experiments are conducted in a single genetic background. Nonetheless, genetic variants found in wild populations dramatically affect the behavior of an individual. Consequently, we currently do not know how the genetic background affects the function of a particular neuron. This action, NeuroContext, was developed to fill this gap in our knowledge. Using the fruit fly’s powerful genetic tools, we harnessed the ability to manipulate neuronal activity along with an array of genetically diverse lines. Through this approach, we investigated the impact of genetic background on the role of individual neurons in a given behavior.
The findings of our study underscore the pivotal role played by the genetic context in determining a neuron's contribution to a behavior. Notably, when deactivating a neuron previously identified as crucial to a fly's response to threats, our study reveals that in certain genetic backgrounds, the activity of this neuron becomes dispensable. Furthermore, the magnitude of this effect varies depending on the specific genetic background.
Furthermore, by exploring the behavioral diversity given by genetic variation, future studies will better uncover the role of neurons in a more naturalistic setup. This will provide better understanding of the nervous system evolution and how certain neuron types are involved in physiology and disease.
For practical reasons, much of this research relies on standardized genotypes, wherein experiments are conducted in a single genetic background. Nonetheless, genetic variants found in wild populations dramatically affect the behavior of an individual. Consequently, we currently do not know how the genetic background affects the function of a particular neuron. This action, NeuroContext, was developed to fill this gap in our knowledge. Using the fruit fly’s powerful genetic tools, we harnessed the ability to manipulate neuronal activity along with an array of genetically diverse lines. Through this approach, we investigated the impact of genetic background on the role of individual neurons in a given behavior.
The findings of our study underscore the pivotal role played by the genetic context in determining a neuron's contribution to a behavior. Notably, when deactivating a neuron previously identified as crucial to a fly's response to threats, our study reveals that in certain genetic backgrounds, the activity of this neuron becomes dispensable. Furthermore, the magnitude of this effect varies depending on the specific genetic background.
Furthermore, by exploring the behavioral diversity given by genetic variation, future studies will better uncover the role of neurons in a more naturalistic setup. This will provide better understanding of the nervous system evolution and how certain neuron types are involved in physiology and disease.
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.
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.
The results from NeuroContext expand the available resources for neuroscientist to describe neuronal circuits that underlie behavior. We made use of common tools used in D.melanosgaster and expanded their potential to investigate neuronal circuits in a more naturalistic manner. Across many biological systems, collections of genetically diverse lines exist, each with minimal genetic variation within a line. They are often used to explore the genotype-phenotype map, i.e. describe which mutations that occur in natural populations affect the phenotype of study. We show that combining a set of these lines, the Drosophila genetic reference panel (DGRP), with other genetic tools it is possible to manipulate cell activity in different genetic backgrounds. Importantly, this approach maintains the diversity of behaviors influenced by the genetic background, as shown by the variation that we observe in flies’ responses to looming stimuli. While inhibiting the descending neuron DNp09 in different backgrounds, we show that the necessity of a neuron for a determined behavior depends on the genetic background.
This approach can now be applied in other paradigms in D.melanogaster and in other model systems. At its core, this expansion promises to enhance our comprehension of nervous system functionality within a naturalistic framework. Beyond its fundamental implications, this approach holds significant applied potential. It can serve as a powerful tool for screening and identifying optimal targets for medical research, shedding light on neuronal types that exhibit consistent effects across diverse genetic backgrounds.
This approach can now be applied in other paradigms in D.melanogaster and in other model systems. At its core, this expansion promises to enhance our comprehension of nervous system functionality within a naturalistic framework. Beyond its fundamental implications, this approach holds significant applied potential. It can serve as a powerful tool for screening and identifying optimal targets for medical research, shedding light on neuronal types that exhibit consistent effects across diverse genetic backgrounds.