An analysis of candidate ionotropic receptors mutations that block synaptic homeostasis: PPK11 and PPK16 drive homeostatic synaptic plasticity.
- Author(s): Younger, Meg A.
- Advisor(s): Davis, Graeme W
- et al.
Homeostatic plasticity restricts changes in neural activity to allow for a stable yet plastic nervous system. At the synapse, homeostatic regulation maintains synaptic efficacy when there is a disruption of postsynaptic neurotransmitter sensitivity or excitability (Turrigiano, 2012; Davis, 2006). At the Drosophila neuromuscular junction (NMJ), when glutamate receptor sensitivity is decreased, either genetically or pharmacologically, the motoneuron potentiates neurotransmitter release to precisely offset changes in receptor sensitivity. This restores muscle excitation within minutes. The molecular mechanisms that underlie this form of synaptic homeostasis are beginning to be discovered, in large part due to an ongoing forward genetic screen for mutations that block synaptic homeostasis (Dickman and Davis, 2009; Müller et al., 2011). This thesis work focuses on the examination of two candidate mutations identified by this genetic screen. These were chosen for study because they completely blocked synaptic homeostasis and were likely to disrupt genes encoding ion channel subunits.
In chapter 2 we provide evidence that two Degenerin/Epithelial Sodium Channel (DEG/ENaC) subunits, pickpocket11 (ppk11) and pickpocket16 (ppk16), are necessary for synaptic homeostasis. These genes are required for both the rapid induction and sustained expression of synaptic homeostasis. Mutations in ppk11 and ppk16 do not disrupt baseline synaptic transmission or NMJ development. ppk11 and ppk16 reside in a single locus and are coregulated as an operon-like genetic unit, with increased transcription accompanying prolonged expression of synaptic homeostasis. We use pharmacology to show that DEG/ENaC channel conductance is continuously required for the potentiation of release during homeostasis. Lastly we show that DEG/ENaC channel function is necessary to enhance action-potential evoked presynaptic calcium influx specifically during synaptic homeostasis, but not baseline release. Taken together this identifies PPK11 and PPK16 as components of the molecular pathway that potentiates release during synaptic homeostasis and we present a model for how PPK11 and PPK16 enhance calcium influx during synaptic homeostasis.
In chapter 3 we examine a third gene encoding a DEG/ENaC channel subunit, pickpocket 18 (ppk18), which is in the same genetic locus as ppk11 and ppk16. We found that despite the proximity to ppk11 and ppk16, ppk18 is transcribed independently from the other two genes, and the quantity of ppk18 transcript is unchanged during the sustained expression of synaptic homeostasis. Furthermore, mutations in ppk18 do not block the rapid induction of synaptic homeostasis, indicating that it does not act in concert with ppk11 and ppk16 to potentiate release during synaptic homeostasis.
In chapter 4 we examine a separate mutation that was identified in the same forward genetic screen for mutations that block synaptic homeostasis. This mutation resides in a complicated locus containing four genes: Ionotropic Receptor 75a (IR75a), Ionotropic Receptor 75b (IR75b), Ionotropic Receptor 75c (IR75c), and GABA and glycine-like receptor of Drosophila (GRD). We use a combination of electrophysiology, quantitative PCR, immunostaining and genetics to examine the requirement of different genes in this locus for the homeostatic potentiation of release. We show that multiple mutations within this genomic region block synaptic homeostasis, and that the distribution of postsynaptic glutamate receptors is altered by a mutation within this locus, which may be an underlying cause. We are unable to identify the responsible gene, although our data points to GRD as the most promising candidate. This work provides a basis for future investigation into this locus.