Synapses and circuits rely on neuroplasticity to adjust output and meet physiological needs. Forms of homeostatic synaptic plasticity impart stability at synapses by countering destabilizing perturbations. The Drosophila melanogaster larval neuromuscular junction (NMJ) is a model synapse with robust expression of homeostatic plasticity. At the NMJ, a homeostatic system detects impaired postsynaptic sensitivity to neurotransmitter and activates a retrograde signal that restores synaptic function by adjusting neurotransmitter release. This process has been separated into temporally distinct phases, induction and maintenance. One prevailing hypothesis is that a shared mechanism governs both phases. Here, we show the two phases are separable. Combining genetics, pharmacology, and electrophysiology, we find that a signaling system consisting of PLCβ, inositol triphosphate (IP3), IP3 receptors, and Ryanodine receptors is required only for the maintenance of homeostatic plasticity. We also find that the NMJ is capable of inducing homeostatic signaling even when its sustained maintenance process is absent.Editorial note: This article has been through an editorial process in which the authors decide how to respond to the issues raised during peer review. The Reviewing Editor's assessment is that all the issues have been addressed (<xref ref-type="decision-letter" rid="SA1">see decision letter</xref>).

23Synapses and circuits rely on neuroplasticity to adjust output and meet physiological needs. 24Forms of homeostatic synaptic plasticity impart stability at synapses by countering destabilizing 25 perturbations. The Drosophila melanogaster larval neuromuscular junction (NMJ) is a model syn-26 apse with robust expression of homeostatic plasticity. At the NMJ, a homeostatic system detects 27 impaired postsynaptic sensitivity to neurotransmitter and activates a retrograde signal that re-28 stores synaptic function by adjusting neurotransmitter release. This process has been separated 29 into temporally distinct phases, induction and maintenance. One prevailing hypothesis is that a 30 shared mechanism governs both phases. Here we show the two phases are separable. Combin-31 ing genetics, pharmacology, and electrophysiology, we find that a signaling system consisting of 32PLCb, inositol triphosphate (IP3), IP3 receptors, and Ryanodine receptors is required only for the 33 maintenance of homeostatic plasticity. We also find that the NMJ is capable of inducing homeo-34 static signaling even when its sustained maintenance process is absent. 35 cascade that induces increased neurotransmitter vesicle release, or quantal content (QC). As a 63 result, the NMJ maintains a normal postsynaptic response level (Frank et al., 2006; Petersen et 64 al., 1997). Mechanistically, this increase in QC depends upon the successful execution of discrete 65 presynaptic events, such as increases in neuronal Ca 2+ influx and an increase in the size of the 66 readily releasable pool (RRP) of synaptic vesicles (Frank et al., 2006; Müller and Davis, 2012; 67 Müller et al., 2012). The field has termed this compensatory signaling process as presynaptic 68 homeostatic potentiation (PHP) (Delvendahl and Müller, 2019). Two factors that govern the ex-69 pression of PHP are the nature of the NMJ synaptic challenge and the amount of time elapsed 70 after presentation of the challenge. Acute pharmacological inhibition of postsynaptic glutamate 71 receptors initiates a rapid induction of PHP that restores synaptic output in minutes (Frank et al., 72 2006). By contrast, genetic lesions and other long-term reductions of NMJ sensitivity to neuro-73 transmitter induce PHP in a way that is sustained throughout life (Brusich et al.

Homeostasis is a vital mode of biological self-regulation. The hallmarks of homeostasis for any biological system are a baseline set point of physiological activity, detection of unacceptable deviations from the set point, and effective corrective measures to counteract deviations. Homeostatic synaptic plasticity (HSP) is a form of neuroplasticity in which neurons and circuits resist environmental perturbations and stabilize levels of activity. One assumption is that if a perturbation triggers homeostatic corrective changes in neuronal properties, those corrective measures should be reversed upon removal of the perturbation. We test the reversibility and limits of HSP at the well-studied Drosophila melanogaster neuromuscular junction (NMJ). At the Drosophila NMJ, impairment of glutamate receptors causes a decrease in quantal size, which is offset by a corrective, homeostatic increase in the number of vesicles released per evoked presynaptic stimulus, or quantal content. This process has been termed presynaptic homeostatic potentiation (PHP). Taking advantage of the GAL4/GAL80TS/UAS expression system, we triggered PHP by expressing a dominant-negative glutamate receptor subunit at the NMJ. We then reversed PHP by halting expression of the dominant-negative receptor. Our data show that PHP is fully reversible over a time course of 48–72 h after the dominant-negative glutamate receptor stops being genetically expressed. As an extension of these experiments, we find that when glutamate receptors are impaired, neither PHP nor NMJ growth is reliably sustained at high culturing temperatures (30–32°C). These data suggest that a limitation of homeostatic signaling at high temperatures could stem from the synapse facing a combination of challenges simultaneously.

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