Investigation of activity-regulated genes (ARGs) required for long-term memory (LTM)

For long-term memory (LTM) formation, neuronal activity needs to be modulated through experience-dependent changes at the synapse. This synaptic plasticity includes modulation of neurotransmitter release alongside receptor rearrangement on the post-synaptic membrane. These changes require rapid regulation of gene expression during memory formation and consolidation. Genes rapidly expressed upon neuronal activity are classed as activity-regulated genes (ARGs), which include transcription factors required for synaptic plasticity (e.g., c-fos). However, how these genes contribute to synaptic plasticity remains unclear. Recently, numerous ARGs were discovered in the fly Drosophila melanogaster, but many have not yet been characterised for a role in LTM. To understand the role of ARGs in LTM, we performed an RNAi knockdown screen of 16 ARGs in Kenyon cells using appetitive olfactory conditioning to measure LTM performance. We found no LTM phenotype with any ARGs, including Hr38, sr, kay and meng that have been reported to have an LTM phenotype in other conditioning assays. We performed a knockdown screen of known memory genes and found no LTM phenotype, likely due to ineffective RNAi knockdown lines. To confirm ARGs from the knockdown screen were expressed in α/β KCs and α′/β′ KCs, we constitutively activated these neurons for 1 h and isolated their nuclei for RNA-seq. We found most ARGs are expressed in either Kenyon cell type, including Hr38. To confirm that ARGs are upregulated upon appetitive learning, we performed appetitive conditioning and separately isolated nuclei of α/β and α′/β′ KC after 1 h post-training. We only found the ARG cbt expressed in α/β KCs upon training, but not in α′/β′ KCs. We found other upregulated genes in α/β and α′/β′ KCs that have not been identified as memory genes offering promising new candidates to validate. Overall, this research will help us further understand the molecular regulation of LTM.