Functional and phosphoproteomic analysis of ß-adrenergic receptor signaling at excitatory synapses in the CA1 region of the ventral hippocampus.

Activation of ß-adrenergic receptors (ß-ARs) not only enhances learning and memory but also facilitates the induction of long-term potentiation (LTP), a form of synaptic plasticity involved in memory formation. To identify the mechanisms underlying ß-AR-dependent forms of LTP we examined the effects of the ß-AR agonist isoproterenol on LTP induction at excitatory synapses onto CA1 pyramidal cells in the ventral hippocampus. LTP induction at these synapses is inhibited by activation of SK-type K+ channels, suggesting that ß-AR activation might facilitate LTP induction by inhibiting SK channels. However, although the SK channel blocker apamin enhanced LTP induction, it did not fully mimic the effects of isoproterenol. We therefore searched for potential alternative mechanisms using liquid chromatography-tandem mass spectrometry to determine how ß-AR activation regulates phosphorylation of postsynaptic density (PSD) proteins. Strikingly, ß-AR activation regulated hundreds of phosphorylation sites in PSD proteins that have diverse roles in dendritic spine structure and function. Moreover, within the core scaffold machinery of the PSD, ß-AR activation increased phosphorylation at several sites previously shown to be phosphorylated after LTP induction. Together, our results suggest that ß-AR activation recruits a diverse set of signaling pathways that likely act in a concerted fashion to regulate LTP induction.

A brain atlas of synapse protein lifetime across the mouse lifespan.

The lifetime of proteins in synapses is important for their signaling, maintenance, and remodeling, and for memory duration. We quantified the lifetime of endogenous PSD95, an abundant postsynaptic protein in excitatory synapses, at single-synapse resolution across the mouse brain and lifespan, generating the Protein Lifetime Synaptome Atlas. Excitatory synapses have a wide range of PSD95 lifetimes extending from hours to several months, with distinct spatial distributions in dendrites, neurons, and brain regions. Synapses with short protein lifetimes are enriched in young animals and in brain regions controlling innate behaviors, whereas synapses with long protein lifetimes accumulate during development, are enriched in the cortex and CA1 where memories are stored, and are preferentially preserved in old age. Synapse protein lifetime increases throughout the brain in a mouse model of autism and schizophrenia. Protein lifetime adds a further layer to synapse diversity and enriches prevailing concepts in brain development, aging, and disease.

Behavioral Timescale Cooperativity and Competitive Synaptic Interactions Regulate the Induction of Complex Spike Burst-Dependent Long-Term Potentiation.

Although Hebbian LTP has an important role in memory formation, the properties of Hebbian LTP cannot fully account for, and in some cases seem incompatible with, fundamental properties of associative learning. Importantly, findings from computational and neurophysiological studies suggest that burst-dependent forms of plasticity, where dendritic spikes and bursts of action potentials provide the postsynaptic depolarization needed for LTP induction, may overcome some of the limitations of conventional Hebbian LTP. Thus, I investigated how excitatory synapses onto CA1 pyramidal cells interact during the induction of complex spike (CS) burst-dependent LTP in hippocampal slices from male mice. Consistent with previous findings, theta-frequency trains of synaptic stimulation induce a Hebbian form of plasticity where postsynaptic CS bursts provide the depolarization needed for NMDAR activation and LTP induction. However, in contrast to conventional Hebbian plasticity, where cooperative LTP induction requires coactivation of synapses on a timescale of tens of milliseconds, cooperative interactions between synapses activated several seconds apart can induce CS burst-dependent LTP. A novel, retroactive form of heterosynaptic plasticity, where activation of one group of synapses triggers LTP induction at other synapses that were active seconds earlier, also contributes to cooperativity in CS burst-dependent LTP. Moreover, competitive synaptic interactions that emerge during prolonged bouts of postsynaptic CS bursting potently regulate CS burst-dependent LTP. Together, the unusual properties of synaptic cooperativity and competition in CS burst-dependent LTP enable Hebbian synapses to operate and interact on behavioral timescales.SIGNIFICANCE STATEMENT While EPSP-evoked complex spike (CS) bursting induces LTP at excitatory synapses onto hippocampal CA1 pyramidal cells, the properties of synaptic interactions during the induction of CS burst-dependent LTP have not been investigated. Here I report that interactions between independent synaptic inputs during the induction of CS burst-dependent LTP exhibit a number of novel, computationally relevant properties. Unlike conventional Hebbian LTP, the induction of CS burst-dependent LTP is regulated by proactive and retroactive cooperative interactions between synapses activated several seconds apart. Moreover, activity-dependent, competitive interactions between synapses allow strongly activated synapses to suppress LTP induction at more weakly activated synapses. Thus, CS burst-dependent LTP exhibits a number of the unique properties that overcome significant limitations of standard Hebbian plasticity rules.

Chemogenetic Modulation and Single-Photon Calcium Imaging in Anterior Cingulate Cortex Reveal a Mechanism for Effort-Based Decisions.

The ACC is implicated in effort exertion and choices based on effort cost, but it is still unclear how it mediates this cost-benefit evaluation. Here, male rats were trained to exert effort for a high-value reward (sucrose pellets) in a progressive ratio lever-pressing task. Trained rats were then tested in two conditions: a no-choice condition where lever-pressing for sucrose was the only available food option, and a choice condition where a low-value reward (lab chow) was freely available as an alternative to pressing for sucrose. Disruption of ACC, via either chemogenetic inhibition or excitation, reduced lever-pressing in the choice, but not in the no-choice, condition. We next looked for value coding cells in ACC during effortful behavior and reward consumption phases during choice and no-choice conditions. For this, we used in vivo miniaturized fluorescence microscopy to reliably track responses of the same cells and compare how ACC neurons respond during the same effortful behavior where there was a choice versus when there was no-choice. We found that lever-press and sucrose-evoked responses were significantly weaker during choice compared with no-choice sessions, which may have rendered them more susceptible to chemogenetic disruption. Together, findings from our interference experiments and neural recordings suggest that a mechanism by which ACC mediates effortful decisions is in the discrimination of the utility of available options. ACC regulates these choices by providing a stable population code for the relative value of different options.SIGNIFICANCE STATEMENT The ACC is implicated in effort-based decision-making. Here, we used chemogenetics and in vivo calcium imaging to explore its mechanism. Rats were trained to lever press for a high-value reward and tested in two conditions: a no-choice condition where lever-pressing for the high-value reward was the only option, and a choice condition where a low-value reward was also available. Inhibition or excitation of ACC reduced effort toward the high-value option, but only in the choice condition. Neural responses in ACC were weaker in the choice compared with the no-choice condition. A mechanism by which ACC regulates effortful decisions is in providing a stable population code for the discrimination of the utility of available options.

Enhanced GABAergic Tonic Inhibition Reduces Intrinsic Excitability of Hippocampal CA1 Pyramidal Cells in Experimental Autoimmune Encephalomyelitis.

Cognitive impairment (CI), a debilitating and pervasive feature of multiple sclerosis (MS), is correlated with hippocampal atrophy. Findings from postmortem MS hippocampi indicate that expression of genes involved in both excitatory and inhibitory neurotransmission are altered in MS, and although deficits in excitatory neurotransmission have been reported in the MS model experimental autoimmune encephalomyelitis (EAE), the functional consequence of altered inhibitory neurotransmission remains poorly understood. In this study, we used electrophysiological and biochemical techniques to examine inhibitory neurotransmission in the CA1 region of the hippocampus in EAE. We find that tonic, GABAergic inhibition is enhanced in CA1 pyramidal cells from EAE mice. Although plasma membrane expression of the GABA transporter GAT-3 was decreased in the EAE hippocampus, an increased surface expression of α5 subunit-containing GABAA receptors appears to be primarily responsible for the increase in tonic inhibition during EAE. Enhanced tonic inhibition during EAE was associated with decreased CA1 pyramidal cell excitability and inhibition of α5 subunit-containing GABAA receptors with the negative allosteric modulator L-655,708 enhanced pyramidal cell excitability in EAE mice. Together, our results suggest that altered GABAergic neurotransmission may underlie deficits in hippocampus-dependent cognitive function in EAE and MS.

The Developmental Shift of NMDA Receptor Composition Proceeds Independently of GluN2 Subunit-Specific GluN2 C-Terminal Sequences.

The GluN2 subtype (2A versus 2B) determines biophysical properties and signaling of forebrain NMDA receptors (NMDARs). During development, GluN2A becomes incorporated into previously GluN2B-dominated NMDARs. This "switch" is proposed to be driven by distinct features of GluN2 cytoplasmic C-terminal domains (CTDs), including a unique CaMKII interaction site in GluN2B that drives removal from the synapse. However, these models remain untested in the context of endogenous NMDARs. We show that, although mutating the endogenous GluN2B CaMKII site has secondary effects on GluN2B CTD phosphorylation, the developmental changes in NMDAR composition occur normally and measures of plasticity and synaptogenesis are unaffected. Moreover, the switch proceeds normally in mice that have the GluN2A CTD replaced by that of GluN2B and commences without an observable decline in GluN2B levels but is impaired by GluN2A haploinsufficiency. Thus, GluN2A expression levels, and not GluN2 subtype-specific CTD-driven events, are the overriding factor in the developmental switch in NMDAR composition.

Rapid homeostatic downregulation of LTP by extrasynaptic GluN2B receptors.

Although the activation of extrasynaptic GluN2B-containing N-methyl-d-aspartate (NMDA) receptors has been implicated in neurodegenerative diseases, such as Alzheimer's and Huntington's disease, their physiological function remains unknown. In this study, we found that extrasynaptic GluN2B receptors play a homeostatic role by antagonizing long-term potentiation (LTP) induction under conditions of prolonged synaptic stimulation. In particular, we have previously found that brief theta-pulse stimulation (5 Hz for 30 s) triggers robust LTP, whereas longer stimulation times (5 Hz for 3 min) have no effect on basal synaptic transmission in the hippocampal CA1 region. Here, we show that prolonged stimulation blocked LTP by activating extrasynaptic GluN2B receptors via glutamate spillover. In addition, we found that this homeostatic mechanism was absent in slices from the SAP102 knockout, providing evidence for a functional coupling between extrasynaptic GluN2B and the SAP102 scaffold protein. In conclusion, we uncovered a rapid homeostatic mechanism that antagonizes LTP induction via the activation of extrasynaptic GluN2B-containing NMDA receptors. NEW & NOTEWORTHY Although long-term potentiation (LTP) is an attractive model for memory storage, it tends to destabilize neuronal circuits because it drives synapses toward a maximum value. Unless opposed by homeostatic mechanisms operating through negative feedback rules, cumulative LTP could render synapses unable to encode additional information. In this study, we uncovered a rapid homeostatic mechanism that antagonizes LTP induction under conditions of prolonged synaptic stimulation via the activation of an extrasynaptic GluN2B-SAP102 complex.

Rbfox1 Regulates Synaptic Transmission through the Inhibitory Neuron-Specific vSNARE Vamp1.

Dysfunction of the neuronal RNA binding protein RBFOX1 has been linked to epilepsy and autism spectrum disorders. Rbfox1 loss in mice leads to neuronal hyper-excitability and seizures, but the physiological basis for this is unknown. We identify the vSNARE protein Vamp1 as a major Rbfox1 target. Vamp1 is strongly downregulated in Rbfox1 Nes-cKO mice due to loss of 3' UTR binding by RBFOX1. Cytoplasmic Rbfox1 stimulates Vamp1 expression in part by blocking microRNA-9. We find that Vamp1 is specifically expressed in inhibitory neurons, and that both Vamp1 knockdown and Rbfox1 loss lead to decreased inhibitory synaptic transmission and E/I imbalance. Re-expression of Vamp1 selectively within interneurons rescues the electrophysiological changes in the Rbfox1 cKO, indicating that Vamp1 loss is a major contributor to the Rbfox1 Nes-cKO phenotype. The regulation of interneuron-specific Vamp1 by Rbfox1 provides a paradigm for broadly expressed RNA-binding proteins performing specialized functions in defined neuronal subtypes.

Novel Ca2+-dependent mechanisms regulate spontaneous release at excitatory synapses onto CA1 pyramidal cells.

Although long thought to simply be a source of synaptic noise, spontaneous, action potential-independent release of neurotransmitter from presynaptic terminals has multiple roles in synaptic function. We explored whether and to what extent the two predominantly proposed mechanisms for explaining spontaneous release, stochastic activation of voltage-gated Ca2+ channels (VGCCs) or activation of Ca2+-sensing receptors (CaSRs) by extracellular Ca2+, played a role in the sensitivity of spontaneous release to the level of extracellular Ca2+ concentration at excitatory synapses at CA1 pyramidal cells of the adult male mouse hippocampus. Blocking VGCCs with Cd2+ had no effect on spontaneous release, ruling out stochastic activation of VGCCs. Although divalent cation agonists of CaSRs, Co2+ and Mg2+, dramatically enhanced miniature excitatory postsynaptic current (mEPSC) frequency, potent positive and negative allosteric modulators of CaSRs had no effect. Moreover, immunoblot analysis of hippocampal lysates failed to detect CaSR expression, ruling out the CaSR. Instead, the increase in mEPSC frequency induced by Co2+ and Mg2+ was mimicked by lowering postsynaptic Ca2+ levels with BAPTA. Together, our results suggest that a reduction in intracellular Ca2+ may trigger a homeostatic-like compensatory response that upregulates spontaneous transmission at excitatory synapses onto CA1 pyramidal cells in the adult hippocampus. NEW & NOTEWORTHY We show that the predominant theories for explaining the regulation of spontaneous, action potential-independent synaptic release do not explain the sensitivity of this type of synaptic transmission to external Ca2+ concentration at excitatory synapses onto hippocampal CA1 pyramidal cells. In addition, our data indicate that intracellular Ca2+ levels in CA1 pyramidal cells regulate spontaneous release, suggesting that excitatory synapses onto CA1 pyramidal cells may express a novel, rapid form of homeostatic plasticity.

Activity-Dependent Regulation of Alternative Cleavage and Polyadenylation During Hippocampal Long-Term Potentiation.

Long-lasting forms of synaptic plasticity that underlie learning and memory require new transcription and translation for their persistence. The remarkable polarity and compartmentalization of neurons raises questions about the spatial and temporal regulation of gene expression within neurons. Alternative cleavage and polyadenylation (APA) generates mRNA isoforms with different 3' untranslated regions (3'UTRs) and/or coding sequences. Changes in the 3'UTR composition of mRNAs can alter gene expression by regulating transcript localization, stability and/or translation, while changes in the coding sequences lead to mRNAs encoding distinct proteins. Using specialized 3' end deep sequencing methods, we undertook a comprehensive analysis of APA following induction of long-term potentiation (LTP) of mouse hippocampal CA3-CA1 synapses. We identified extensive LTP-induced APA changes, including a general trend of 3'UTR shortening and activation of intronic APA isoforms. Comparison with transcriptome profiling indicated that most APA regulatory events were uncoupled from changes in transcript abundance. We further show that specific APA regulatory events can impact expression of two molecules with known functions during LTP, including 3'UTR APA of Notch1 and intronic APA of Creb1. Together, our results reveal that activity-dependent APA provides an important layer of gene regulation during learning and memory.

Mapping Gene Expression in Excitatory Neurons during Hippocampal Late-Phase Long-Term Potentiation.

The persistence of long-lasting changes in synaptic connectivity that underlie long-term memory require new RNA and protein synthesis. To elucidate the temporal pattern of gene expression that gives rise to long-lasting neuronal plasticity, we analyzed differentially-expressed (DE) RNAs in mouse hippocampal slices following induction of late phase long-term potentiation (L-LTP) specifically within pyramidal excitatory neurons using Translating Ribosome Affinity Purification RNA sequencing (TRAP-seq). We detected time-dependent changes in up- and down-regulated ribosome-associated mRNAs over 2 h following L-LTP induction, with minimal overlap of DE transcripts between time points. TRAP-seq revealed greater numbers of DE transcripts and magnitudes of LTP-induced changes than RNA-seq of all cell types in the hippocampus. Neuron-enriched transcripts had greater changes at the ribosome-loading level than the total RNA level, while RNA-seq identified many non-neuronal DE mRNAs. Our results highlight the importance of considering both time course and cell-type specificity in activity-dependent gene expression during memory formation.

Differential Regulation of NMDA Receptor-Mediated Transmission by SK Channels Underlies Dorsal-Ventral Differences in Dynamics of Schaffer Collateral Synaptic Function.

Behavioral, physiological, and anatomical evidence indicates that the dorsal and ventral zones of the hippocampus have distinct roles in cognition. How the unique functions of these zones might depend on differences in synaptic and neuronal function arising from the strikingly different gene expression profiles exhibited by dorsal and ventral CA1 pyramidal cells is unclear. To begin to address this question, we investigated the mechanisms underlying differences in synaptic transmission and plasticity at dorsal and ventral Schaffer collateral (SC) synapses in the mouse hippocampus. We find that, although basal synaptic transmission is similar, SC synapses in the dorsal and ventral hippocampus exhibit markedly different responses to θ frequency patterns of stimulation. In contrast to dorsal hippocampus, θ frequency stimulation fails to elicit postsynaptic complex-spike bursting and does not induce LTP at ventral SC synapses. Moreover, EPSP-spike coupling, a process that strongly influences information transfer at synapses, is weaker in ventral pyramidal cells. Our results indicate that all these differences in postsynaptic function are due to an enhanced activation of SK-type K+ channels that suppresses NMDAR-dependent EPSP amplification at ventral SC synapses. Consistent with this, mRNA levels for the SK3 subunit of SK channels are significantly higher in ventral CA1 pyramidal cells. Together, our findings indicate that a dorsal-ventral difference in SK channel regulation of NMDAR activation has a profound effect on the transmission, processing, and storage of information at SC synapses and thus likely contributes to the distinct roles of the dorsal and ventral hippocampus in different behaviors.SIGNIFICANCE STATEMENT Differences in short- and long-term plasticity at Schaffer collateral (SC) synapses in the dorsal and ventral hippocampus likely contribute importantly to the distinct roles of these regions in cognition and behavior. Although dorsal and ventral CA1 pyramidal cells exhibit markedly different gene expression profiles, how these differences influence plasticity at SC synapses is unclear. Here we report that increased mRNA levels for the SK3 subunit of SK-type K+ channels in ventral pyramidal cells is associated with an enhanced activation of SK channels that strongly suppresses NMDAR activation at ventral SC synapses. This leads to striking differences in multiple aspects of synaptic transmission at dorsal and ventral SC synapses and underlies the reduced ability of ventral SC synapses to undergo LTP.

Long-term potentiation modulates synaptic phosphorylation networks and reshapes the structure of the postsynaptic interactome.

The postsynaptic site of neurons is composed of more than 1500 proteins arranged in protein-protein interaction complexes, the composition of which is modulated by protein phosphorylation through the actions of complex signaling networks. Components of these networks function as key regulators of synaptic plasticity, in particular hippocampal long-term potentiation (LTP). The postsynaptic density (PSD) is a complex multicomponent structure that includes receptors, enzymes, scaffold proteins, and structural proteins. We triggered LTP in the mouse hippocampus CA1 region and then performed large-scale analyses to identify phosphorylation-mediated events in the PSD and changes in the protein-protein interactome of the PSD that were associated with LTP induction. Our data indicated LTP-induced reorganization of the PSD. The dynamic reorganization of the PSD links glutamate receptor signaling to kinases (writers) and phosphatases (erasers), as well as the target proteins that are modulated by protein phosphorylation and the proteins that recognize the phosphorylation status of their binding partners (readers). Protein phosphorylation and protein interaction networks converged at highly connected nodes within the PSD network. Furthermore, the LTP-regulated phosphoproteins, which included the scaffold proteins Shank3, Syngap1, Dlgap1, and Dlg4, represented the "PSD risk" for schizophrenia and autism spectrum disorder, such that without these proteins in the analysis, the association with the PSD and these two psychiatric diseases was not present. These data are a rich resource for future studies of LTP and suggest that the PSD holds the keys to understanding the molecular events that contribute to complex neurological disorders that affect synaptic plasticity.

NMDA receptors are selectively partitioned into complexes and supercomplexes during synapse maturation.

How neuronal proteomes self-organize is poorly understood because of their inherent molecular and cellular complexity. Here, focusing on mammalian synapses we use blue-native PAGE and 'gene-tagging' of GluN1 to report the first biochemical purification of endogenous NMDA receptors (NMDARs) directly from adult mouse brain. We show that NMDARs partition between two discrete populations of receptor complexes and ∼1.5 MDa supercomplexes. We tested the assembly mechanism with six mouse mutants, which indicates a tripartite requirement of GluN2B, PSD93 and PSD95 gate the incorporation of receptors into ∼1.5 MDa supercomplexes, independent of either canonical PDZ-ligands or GluN2A. Supporting the essential role of GluN2B, quantitative gene-tagging revealed a fourfold molar excess of GluN2B over GluN2A in adult forebrain. NMDAR supercomplexes are assembled late in postnatal development and triggered by synapse maturation involving epigenetic and activity-dependent mechanisms. Finally, screening the quaternary organization of 60 native proteins identified numerous discrete supercomplexes that populate the mammalian synapse.

Basal levels of AMPA receptor GluA1 subunit phosphorylation at threonine 840 and serine 845 in hippocampal neurons.

Dephosphorylation of AMPA receptor (AMPAR) GluA1 subunits at two sites, serine 845 (S845) and threonine 840 (T840), is thought to be involved in NMDA receptor-dependent forms of long-term depression (LTD). Importantly, the notion that dephosphorylation of these sites contributes to LTD assumes that a significant fraction of GluA1 subunits are basally phosphorylated at these sites. To examine this question, we used immunoprecipitation/depletion assays to estimate the proportion of GluA1 subunits basally phosphorylated at S845 and T840. Although dephosphorylation of S845 is thought to have a key role in LTD, our results indicate that few GluA1 subunits in hippocampal neurons are phosphorylated at this site. In contrast, ∼50% of GluA1 subunits are basally phosphorylated at T840, suggesting that dephosphorylation of this site can contribute to the down-regulation of AMPAR-mediated synaptic transmission in LTD.

Misaligned feeding impairs memories.

Robust sleep/wake rhythms are important for health and cognitive function. Unfortunately, many people are living in an environment where their circadian system is challenged by inappropriate meal- or work-times. Here we scheduled food access to the sleep time and examined the impact on learning and memory in mice. Under these conditions, we demonstrate that the molecular clock in the master pacemaker, the suprachiasmatic nucleus (SCN), is unaltered while the molecular clock in the hippocampus is synchronized by the timing of food availability. This chronic circadian misalignment causes reduced hippocampal long term potentiation and total CREB expression. Importantly this mis-timed feeding resulted in dramatic deficits in hippocampal-dependent learning and memory. Our findings suggest that the timing of meals have far-reaching effects on hippocampal physiology and learned behaviour.

ß-Adrenergic receptor signaling and modulation of long-term potentiation in the mammalian hippocampus.

Encoding new information in the brain requires changes in synaptic strength. Neuromodulatory transmitters can facilitate synaptic plasticity by modifying the actions and expression of specific signaling cascades, transmitter receptors and their associated signaling complexes, genes, and effector proteins. One critical neuromodulator in the mammalian brain is norepinephrine (NE), which regulates multiple brain functions such as attention, perception, arousal, sleep, learning, and memory. The mammalian hippocampus receives noradrenergic innervation and hippocampal neurons express ß-adrenergic receptors, which are known to play important roles in gating the induction of long-lasting forms of synaptic potentiation. These forms of long-term potentiation (LTP) are believed to importantly contribute to long-term storage of spatial and contextual memories in the brain. In this review, we highlight the contributions of noradrenergic signaling in general and ß-adrenergic receptors in particular, toward modulating hippocampal LTP. We focus on the roles of NE and ß-adrenergic receptors in altering the efficacies of specific signaling molecules such as NMDA and AMPA receptors, protein phosphatases, and translation initiation factors. Also, the roles of ß-adrenergic receptors in regulating synaptic "tagging" and "capture" of LTP within synaptic networks of the hippocampus are reviewed. Understanding the molecular and cellular bases of noradrenergic signaling will enrich our grasp of how the brain makes new, enduring memories, and may shed light on credible strategies for improving mental health through treatment of specific disorders linked to perturbed memory processing and dysfunctional noradrenergic synaptic transmission.

Inhibitory interactions between phosphorylation sites in the C terminus of α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid-type glutamate receptor GluA1 subunits.

The C terminus of AMPA-type glutamate receptor (AMPAR) GluA1 subunits contains several phosphorylation sites that regulate AMPAR activity and trafficking at excitatory synapses. Although many of these sites have been extensively studied, little is known about the signaling mechanisms regulating GluA1 phosphorylation at Thr-840. Here, we report that neuronal depolarization in hippocampal slices induces a calcium and protein phosphatase 1/2A-dependent dephosphorylation of GluA1 at Thr-840 and a nearby site at Ser-845. Despite these similarities, inhibitors of NMDA-type glutamate receptors and protein phosphatase 2B prevented depolarization-induced Ser-845 dephosphorylation but had no effect on Thr-840 dephosphorylation. Instead, depolarization-induced Thr-840 dephosphorylation was prevented by blocking voltage-gated calcium channels, indicating that distinct Ca(2+) sources converge to regulate GluA1 dephosphorylation at Thr-840 and Ser-845 in separable ways. Results from immunoprecipitation/depletion assays indicate that Thr-840 phosphorylation inhibits protein kinase A (PKA)-mediated increases in Ser-845 phosphorylation. Consistent with this, PKA-mediated increases in AMPAR currents, which are dependent on Ser-845 phosphorylation, were inhibited in HEK-293 cells expressing a Thr-840 phosphomimetic version of GluA1. Conversely, mimicking Ser-845 phosphorylation inhibited protein kinase C phosphorylation of Thr-840 in vitro, and PKA activation inhibited Thr-840 phosphorylation in hippocampal slices. Together, the regulation of Thr-840 and Ser-845 phosphorylation by distinct sources of Ca(2+) influx and the presence of inhibitory interactions between these sites highlight a novel mechanism for conditional regulation of AMPAR phosphorylation and function.