TY - JOUR T1 - Differential scaling of synaptic molecules within functional zones of an excitatory synapse during homeostatic plasticity JF - eneuro JO - eNeuro DO - 10.1523/ENEURO.0407-19.2020 SP - ENEURO.0407-19.2020 AU - Sridevi Venkatesan AU - Sandhya Subramaniam AU - Premchand Rajeev AU - Yukti Chopra AU - Mini Jose AU - Deepak Nair Y1 - 2020/03/12 UR - http://www.eneuro.org/content/early/2020/03/12/ENEURO.0407-19.2020.abstract N2 - Homeostatic scaling is a form of synaptic plasticity where individual synapses scale their strengths to compensate for global suppression or elevation of neuronal activity. This process can be studied by measuring miniature excitatory postsynaptic potential amplitudes and frequencies following the regulation of activity in neuronal cultures. Here we demonstrate a quantitative approach to characterize multiplicative synaptic scaling using immunolabelling of hippocampal neuronal cultures treated with tetrodotoxin or bicuculline to extract scaling factors for various synaptic proteins. This approach allowed us to directly examine the scaling of pre- and postsynaptic scaffolding molecules along with neurotransmitter receptors in primary cultures from mouse and rat hippocampal neurons. We show robust multiplicative scaling of synaptic scaffolding molecules namely, Shank2, PSD95, Bassoon, and AMPA receptor subunits and quantify their scaling factors. We use super-resolution microscopy to calculate scaling factors of surface expressed GluA2 within functional zones of the synapse and show that there is differential and correlated scaling of GluA2 levels within the spine, the postsynaptic density, and the perisynaptic regions. Our method opens a novel paradigm to quantify relative molecular changes of synaptic proteins within distinct subsynaptic compartments from a large number of synapses in response to alteration of neuronal activity, providing anatomical insights into the intricacies of variability in strength of individual synapses.Significance Statement Here we demonstrate a novel quantitative method based on rank ordered analysis to characterize multiplicative synaptic scaling from immunolabelling of hippocampal neuronal cultures after activity blockade. We show that along with glutamatergic receptors, several scaffolding molecules are scaled after blocking activity. This analysis paradigm can be generalized to any protein which alters its content post plasticity protocols. With the aid of conventional and super-resolution microscopy, we examine pools of AMPA type glutamatergic receptors, and confirm that they are scaled differentially within functional zones of synapses. Furthermore, we show that the AMPA receptor content within the postsynaptic density and perisynaptic compartments are altered differentially during homeostatic scaling, indicating a differential regulation of receptors within various subsynaptic compartments during homeostatic plasticity. ER -