The COVID-19 pandemic made progress in the last year particularly difficult. We were locked down and unable to do experiments for around 6 months. Although we returned on a rotational and ‘spaced’ arrangement we were unable to order many reagents, and services such as single-cell library preparation and sequencing, and ordering genetically-engineered flies were unavailable. We have tried our best to catch up with things and hope we can still accomplish most of what we initially proposed. Despite the COVID-imposed difficulties we have nevertheless made great progress.
The primary objectives of the SCCMMI project were/are:
1. Discover how forming persistent memory alters gene expression within engram neurons.
2. Delineate molecular mechanisms linking appetitive motivation and reinforcement.
1.1 Complete a Drop-seq single cell representation of the entire midbrain of the fly.
Since the initial proposal we transitioned to using the improved technology of 10X Genomics. We have collected >300,000 cells from a combination of experiments which provides about 6X coverage of the entire Drosophila midbrain. This number is adequate to include representation and classification of rare classes of neuron.
We for example have sufficient computational power to cluster dopaminergic neurons into the different subtypes that innervate unique compartments of the mushroom body. More cells may permit further classification of dopaminergic neurons within a mushroom body compartment, as expected from our ultrastructural connectomic studies (Otto et al., 2020).
We also collected 26,000 cells from the Drosophila ventral nerve cord (the equivalent of the spinal cord in insects) providing the first single-cell transcriptomic atlas of this part of the nervous system (Allen et al, 2020)
2.1. Discover genes whose expression is induced by hunger and thirst.
Our single-cell experiments of thirst have been spectacular, and we believe we have made a fundamental and truly novel discovery. Although we initially predicted a response in peptidergic neurons (Senapati et al., 2019), we found robust changes in gene expression in water-deprived (thirsty) flies in specific glial cells. Importantly, the glial expression of some of these thirst-induced genes returns to baseline with thirst quenching, suggesting that they report a physiologically-relevant change in the state of the brain. Using glial-cell-type specific expression of RNA interference we found that loss-of-function of a gene called aay reduces drinking in thirsty flies, whereas over-expression in glia increases drinking. aay encodes a phosphoserine phosphatase, which catalyses the last step of D-serine synthesis. D-serine is a known co-agonist of neuronally expressed NMDA-type glutamate receptors and it is believed to be released from glia, by activity-induced ‘gliotransmission’. To our knowledge, our work provides the first compelling link for D-serine mediated neuromodulation to a specific element of state-dependent behaviour - a clear link between D-serine gliotransmission and thirst-directed drinking behaviour. This work will be submitted for publication shortly.
2.2. Investigate neural mechanisms and genes linked to persistent plasticity and compulsive behaviour.
We have collected single-cell sequencing data from flies conditioned to form long-term olfactory memories reinforced with sucrose, and from those trained by pairing odour exposure with artificial activation of all the rewarding dopaminergic neurons. We have established that we have a model for compulsive behaviour. We can generate flies that will forego food when hungry and will also run across 120V of electric shocks to seek an illusory reward. Seeking reward at cost is a defining feature of a compulsive state. Most excitingly, our single-cell sequencing of these flies in the compulsive condition has revealed selective upregulation of a key memory-relevant kinase within the mushroom body KCs. We are now testing whether the induced expression of this kinase is a casual factor in the induction of the compulsive condition.
2.3. What is the relevance of transposon expression in the brain?
Our prior work established that certain transposable elements (TE) appear to be over-expressed in the KCs of the mushroom body. We have discovered that cell-specific TE expression arises because pieces of certain transposons are captured by splicing into the mature mRNAs of cellular genes (Treiber and Waddell, 2020). If a particular gene houses a transposon the gene frequently produces additional mRNAs that contain TE sequence. Importantly, many of these change the open reading frame of the mRNA and therefore presumably give rise to additional protein isoforms. Given extensive polymorphism of TEs even between individual flies, these results show that each fly has its own unique transcriptome. We are currently testing whether flies with TEs in genes with known neural roles, have altered behaviour. The most exciting corollary of these findings is that TEs contribute to individual differences in animal behaviour, or personality.
