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    • br Experimental methods br Acknowledgments br Introduction L

    2023-08-23


    Experimental methods
    Acknowledgments
    Introduction Long-term potentiation (LTP) and long-term depression (LTD) at excitatory synapses are thought to underlie experience-dependent learning and memory. These synaptic plasticity mechanisms are best characterized at hippocampal CA1 synapses, where they are frequently altered in animal models of human neurodevelopmental, neuropsychiatric, and neurological disorders. The most prevalent forms of LTP and LTD are induced by Ca2+ influx through postsynaptic NMDA receptors (NMDARs) and are expressed by long-lasting increases or decreases, respectively, in the synaptic localization and function of AMPA receptors (AMPARs) (Collingridge et al., 2010, Huganir and Nicoll, 2013). AMPARs are tetrameric assemblies of GluA1-GluA4 subunits, and except very early in postnatal development, the majority of receptors at CA1 synapses are composed of GluA1/2 or GluA2/3, with GluA2 decreasing conductance and preventing Ca2+ influx (Lu et al., 2009, Stubblefield and Benke, 2010). However, smaller numbers of high-conductance, Ca2+-permeable GluA1 homomeric AMPARs (CP-AMPARs) are also present, primarily in extrasynaptic and intracellular locations from where they can be recruited to synapses by some LTP-inducing stimuli (Guire et al., 2008, Lu et al., 2007, Plant et al., 2006, Qian et al., 2012, Rozov et al., 2012, Yang et al., 2010; but see Adesnik and Nicoll, 2007, Gray et al., 2007). CP-AMPARs are also recruited to hippocampal and cortical synapses during certain forms of homeostatic plasticity (Goel et al., 2011, Kim and Ziff, 2014, Soares et al., 2013, Sutton et al., 2006, Thiagarajan et al., 2005), as well as in response to seizures and ischemia (Liu and Zukin, 2007). In addition, CP-AMPARs are recruited to synapses in the amygdala during fear learning (Clem and Huganir, 2010) and in the nucleus accumbens and ventral tegmentum in models of drug addiction (Bellone et al., 2011, McCutcheon et al., 2011). Nonetheless, the signaling mechanisms regulating synaptic AMPAR subunit composition are still not well understood. Phosphorylation of S845 in the GluA1 C-terminal tail by the cyclic AMP (cAMP)-dependent protein kinase (PKA) can prime AMPARs for synaptic Cy5 NHS ester (non-sulfonated) during LTP in response to subsequent NMDAR-Ca2+ activation of Ca2+/calmodulin-dependent protein kinases I and II (CaMKII) and PKC (Esteban et al., 2003, Guire et al., 2008, Hu et al., 2007, Oh et al., 2006, Sun et al., 2005, Yang et al., 2008). In contrast, during LTD, the Ca2+-activated protein phosphatase-2B/calcineurin (CaN) dephosphorylates S845 and promotes AMPAR removal from synapses and endocytosis (Beattie et al., 2000, Ehlers, 2000, Lee et al., 1998, Lee et al., 2000, Mulkey et al., 1994, Sanderson et al., 2012). Previous studies also indicated that S845 phosphorylation plays a key role in regulating CP-AMPAR abundance, trafficking, and synaptic incorporation (Esteban et al., 2003, He et al., 2009, Man et al., 2007, Qian et al., 2012); GluA1 S845A knockin mice possess fewer GluA1 homomers, exhibit impaired PKA regulation of LTP, and have deficits in LTD in the hippocampus (He et al., 2009, Hu et al., 2007, Lee et al., 2003, Lee et al., 2010, Qian et al., 2012). In addition, recent studies have implicated CP-AMPARs, S845 phosphorylation, PKA, and CaN in homeostatic plasticity mechanisms that scale up synaptic strength in response to decreased neuronal firing (Diering et al., 2014, Goel et al., 2011, Kim and Ziff, 2014). Given all of the aforementioned studies implicating S845 in plasticity regulation, it may seem surprising that a recent biochemical study found that steady-state levels of S845 phosphorylation are very low, even in synaptic fractions (Hosokawa et al., 2015). However, an inherent limitation of even the most quantitative bulk phosphorylation measurements is that they cannot report rapid, localized changes in phosphorylation that occur within the confines of receptor-scaffolded kinase/phosphatase signaling complexes. In particular, PKA and CaN are targeted to GluA1 through binding to a common scaffold protein, A-kinase anchoring protein (AKAP) 79/150 (human79/rodent150; also known as AKAP5) (Woolfrey and Dell’Acqua, 2015). Inhibition of AKAP-PKA anchoring, like GluA1 S845A mutation, prevents PKA enhancement of LTP (Lu et al., 2007, Zhang et al., 2013). However, somewhat paradoxically, inhibition of AKAP-PKA signaling also interferes with LTD that relies on S845 dephosphorylation and AMPAR removal by AKAP-anchored CaN (Jurado et al., 2010, Kameyama et al., 1998, Lu et al., 2008, Sanderson et al., 2012, Snyder et al., 2005, Tunquist et al., 2008).