Transcriptional control of learning and memory

Understanding the interplay between gene expression and neuronal function is a major question in neuroscience. Activity regulated genes (ARGs) are characterised by rapid and transient increase in expression in response to neuronal activity. Many ARGs encode transcription factors regulating neuronal plasticity or memory. Although some ARGs have been extensively studied, little is known about how the expression of individual ARGs varies across cell types and physiological states, and how this influences downstream pathways, and ultimately behaviour. This project uses the simple brain of Drosophila as a model to investigate the function of ARGs in memory.

Drosophila offers a host of powerful genetic tools, together connectome and single-cell transcriptome data available. This and unparalleled knowledge about the anatomy and function of memory circuits provides a timely opportunity to address the function of ARGs, at a resolution that would be impossible to achieve in other organisms.

Flies learn to approach or avoid odours associated with rewards or punishments. The mushroom body (MB), their main memory centre, is made of axonal projections of neurons called Kenyon cells (KCs). The MB is further divided into compartments, each innervated by groups of reinforcing dopaminergic neurons representing distinct rewards or punishments. During olfactory learning, dopamine release on individual MB compartments depresses the connections between KCs and valence-encoding MB-output neurons (MBONs), leading to skewed behavioural responses towards learned odours.

Activation of dopaminergic neurons triggers ARG expression in KCs. Hr38, a gene encoding a highly conserved DNA-binding nuclear receptor-like protein, is the most strongly upregulated of these, and therefore a strong candidate for orchestrating expression of genes relevant for memory. However, the actual influence of Hr38 on the different memory phases, and the identity of the genes under its control remain unknown. I will address these questions, focusing on the three following aims:

1. Measuring Hr38 expression across memory stages. I will investigate how Hr38 expression changes throughout learning, consolidation, and memory retrieval. I will train flies to pair odours with rewards, punishments or optogenetic activation of selected dopaminergic neurons, and use transgenic reporters, immunohistochemistry and RNA FISH to visualise and quantify fluctuations of Hr38 expression. Differences between groups of KCs, or across dopaminergic and MBON populations, could reflect spatiotemporal requirements for Hr38. Comparisons with other ARGs will highlight differences in their requirements.

2. Requirement of Hr38 for memory. Based on the information gathered above, I will use RNA interference and CRISPR-generated conditional mutants to impair the expression of Hr38 and other ARGs in selected memory neurons. Measuring how these manipulations affect memory performance in a classical conditioning assay will identify where Hr38 action is required. Comparing short-term and long-term memory will reveal how Hr38 contributes to long-term plasticity and memory consolidation.

3. Hr38 DNA binding. A systems approach will be used to investigate which genes are regulated by Hr38. I will identify genes regulated by Hr38 using Dam-ID, which will highlight its interactions with DNA in KCs. Comparing trained with untrained flies will highlight learning-specific DNA-binding events. Gene regulatory network analysis based on these results will offer a broad picture of the molecular pathways required to enable plasticity. Analysis of available single-cell transcriptome data from Drosophila brains will confirm co-expression of identified candidates with Hr38 in specific cell types.