Emotion enhances our ability to form vivid memories of even trivial events. Norepinephrine (NE), a neuromodulator released during emotional arousal, plays a central role in the emotional regulation of memory. However, the underlying molecular mechanism remains elusive. Toward this aim, we have examined the role of NE in contextual memory formation and in the synaptic delivery of GluR1-containing alpha-amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid (AMPA)-type glutamate receptors during long-term potentiation (LTP), a candidate synaptic mechanism for learning. We found that NE, as well as emotional stress, induces phosphorylation of GluR1 at sites critical for its synaptic delivery. Phosphorylation at these sites is necessary and sufficient to lower the threshold for GluR1 synaptic incorporation during LTP. In behavioral experiments, NE can lower the threshold for memory formation in wild-type mice but not in mice carrying mutations in the GluR1 phosphorylation sites. Our results indicate that NE-driven phosphorylation of GluR1 facilitates the synaptic delivery of GluR1-containing AMPARs, lowering the threshold for LTP, thereby providing a molecular mechanism for how emotion enhances learning and memory.

Incorporation of GluR1-containing AMPA receptors into synapses is essential to several forms of neural plasticity, including long-term potentiation (LTP). Numerous signaling pathways that trigger this process have been identified, but the direct modifications of GluR1 that control its incorporation into synapses are unclear. Here, we show that phosphorylation of GluR1 by PKC at a highly conserved serine 818 residue is increased during LTP and critical for LTP expression. GluR1 is phosphorylated by PKC at this site in vitro and in vivo. In addition, acute phosphorylation at GluR1 S818 by PKC, as well as a phosphomimetic mutation, promotes GluR1 synaptic incorporation. Conversely, preventing GluR1 S818 phosphorylation reduces LTP and blocks PKC-driven synaptic incorporation of GluR1. We conclude that the phosphorylation of GluR1 S818 by PKC is a critical event in the plasticity-driven synaptic incorporation of AMPA receptors.

PSD-95 is a major protein found in virtually all mature excitatory glutamatergic synapses in the brain. Here, we have addressed the role of PSD-95 in controlling glutamatergic synapse function by generating and characterizing a PSD-95 KO mouse. We found that the ␣-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) subtype of glutamate receptor (AMPAR)-mediated synaptic transmission was reduced in these mice. Two-photon (2P) uncaging of MNI-glutamate onto individual spines suggested that the decrease in AMPAR function in the PSD-95 KO mouse stems from an increase in the proportion of ''silent'' synapses i.e., synapses containing N-methyl-D-aspartate (NMDA) receptors (NMDARs) but no AMPARs. Unexpectedly, the silent synapses in the KO mouse were located onto morphologically mature spines. We also observed that a significant population of synapses appeared unaffected by PSD-95 gene deletion, suggesting that the functional role of PSD-95 displays synapse-specificity. In addition, we report that the decay of NMDAR-mediated current was slower in KO mice: The contribution of NR2B subunit containing receptors to the NMDARmediated synaptic current was greater in KO mice. The greater occurrence of silent synapses might be related to the greater magnitude of potentiation after long-term potentiation induction observed in these mice. Together, these results suggest a synapsespecific role for PSD-95 in controlling synaptic function that is independent of spine morphology.glutamate receptors ͉ hippocampus ͉ spines ͉ synaptic transmission ͉ two-photon uncaging G lutamate, the major excitatory neurotransmitter in the brain, activates ionotropic glutamate receptors of the ␣-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA), N-methyl-D-aspartate (NMDA), and kainate subtypes. There is considerable interest in elucidating the molecular mechanisms that controls synaptic targeting and trafficking of these receptors, in part because of their role in the induction and expression of various forms of synaptic plasticity (1). These receptors are imbedded in an electron-dense structure, the postsynaptic density (PSD), which is believed to contain key molecules involved in the regulation of glutamate receptor targeting and trafficking. PSD-95, a member of the membraneassociated guanylate kinase (MAGUK) superfamily of proteins, is a core component of the PSD and is thought to be important in the control of excitatory synapse function (2, 3). Because of its interaction with the cytoplasmic domains of NMDA receptor (NMDAR) subunits, it has long been suspected that PSD-95 might control the synaptic targeting of NMDARs (4, 5). However, more recent studies based on sustained or transient overexpression of PSD-95 in neurons have forced a reassessment of this view by suggesting that the primary role of PSD-95 is restricted to controlling AMPAR synaptic expression (6-10). Intriguingly, previous work on a PSD-95 KO mouse, reported no apparent changes in either AMPAR or NMDAR function (11). The interpretation of these data are, however, ...

A novel gene (Cacng2; ␥ 2 ) encoding a protein similar to the voltage-activated Ca 2؉ channel ␥ 1 subunit was identified as the defective gene in the epileptic and ataxic mouse, stargazer. In this study, we analyzed the association of this novel neuronal ␥ 2 subunit with Ca 2؉ channels of rabbit brain, and the function of the ␥ 2 subunit in recombinant neuronal Ca 2؉ channels expressed in Xenopus oocytes. Our results showed that the ␥ 2 subunit and a closely related protein (called ␥ 3 ) co-sedimented and co-immunoprecipitated with neuronal Ca 2؉ channel subunits in vivo. Electrophysiological analyses showed that ␥ 2 co-expression caused a significant decrease in the current amplitude of both ␣ 1B (␣ 1 2.2)-class (36.8%) and ␣ 1A (␣ 1 2.1)-class (39.7%) Ca 2؉ channels (␣ 1 ␤ 3 ␣ 2 ␦). Interestingly, the inhibitory effects of the ␥ 2 subunit on current amplitude were dependent on the co-expression of the ␣ 2 ␦ subunit. In addition, co-expression of ␥ 2 or ␥ 1 also significantly decelerates the activation kinetics of ␣ 1B -class Ca 2؉ channels. Taken together, these results suggest that the ␥ 2 subunit is an important constituent of the neuronal Ca 2؉ channel complex and that it down-regulates neuronal Ca 2؉ channel activity. Furthermore, the ␥ 2 subunit likely contributes to the fine-tuning of neuronal Ca 2؉ channels by counterbalancing the effects of the ␣ 2 ␦ subunit.

AMPA receptors (AMPA-R) are major mediators of synaptic transmission and plasticity in the developing and adult central nervous system. Activity-dependent structural plasticity mediated by dynamic changes in the morphology of spines and dendrites is also essential for the formation and tuning of neuronal circuits. RhoA and Rac1 are known to play important roles in the regulation of spine and dendrite development in response to neuronal activity. These Rho GTPases are activated by guanine nucleotide exchange factors (GEFs). In this study, we identified GEF-H1/Lfc as a component of the AMPA-R complex in the brain. GEF-H1 is enriched in the postsynaptic density and is colocalized with GluR1 at spines. GEF-H1 activity negatively regulates spine density and length through a RhoA signaling cascade. In addition, AMPA-R-dependent changes in spine development are eliminated by down-regulation of GEF-H1. Altogether, these results strongly suggest that GEF-H1 is an important mediator of AMPA-R activity-dependent structural plasticity in neurons.glutamate receptor ͉ GTPase ͉ learning and memory ͉ structural plasticity ͉ synaptic plasticity

Voltage-dependent calcium channels selectively enable Ca 2؉ ion movement through cellular membranes. These multiprotein complexes are involved in a wide spectrum of biological processes such as signal transduction and cellular homeostasis. ␣ 1 is the membrane pore-forming subunit, whereas ␤ is an intracellular subunit that binds to ␣ 1 , facilitating and modulating channel function. We have expressed, purified, and characterized recombinant ␤ 3 and ␤ 2a using both biochemical and biophysical methods, including electrophysiology, to better understand the ␤ family's protein structural and functional correlates. Our results indicate that the ␤ protein is composed of two distinct domains that associate with one another in a stable manner. The data also suggest that the polypeptide regions outside these domains are not structured when ␤ is not in complex with the channel. In addition, the ␤ structural core, comprised of just these two domains without other sequences, binds tightly to the ␣ interaction domain (AID) motif, a sequence derived from the ␣ 1 subunit and the principal anchor site of ␤. Domain II is responsible for this binding, but domain I enhances it.Voltage-dependent calcium channels (VDCCs) 1 permit the flow of Ca 2ϩ ions through cellular membranes as a function of membrane potential. These protein complexes are central components in a variety of physiological systems of organisms, ranging from yeast to human. They play pivotal roles in signal transduction and homeostasis processes.Functional roles for these channels vary based on cell type. In muscle, both skeletal and cardiac, the predominant VDCCs (Ca V 1.1 and Ca V 1.2) cause release of Ca 2ϩ into the cytosol from intracellular stores, thereby initiating contraction (1, 2). In neurons and endocrine cells, neurotransmitter or hormone secretion requires VDCC activity. In addition, electrical activity, specifically the action potential in cardiac myocytes, is regulated by VDCCs. Finally, calcium influx and concentration controlled by VDCCs plays a significant role in neuronal gene expression (3). Pathways have been elucidated, where for one example, VDCC activity gives rise to phosphorylation of CREB, a transcription factor (4), thereby activating transcription of a myriad of target genes.The VDCC comprises four distinct polypeptides: ␣ 1 , ␣ 2 ␦, ␤, and ␥ (5). ␣ 1 is the membrane pore-forming subunit and numbers between 1800 and 2400 residues in length. Its sequence exhibits repeats comprising four transmembrane modules or domains, akin to the tetrameric architecture of potassium channels. Each module contains the canonical transmembrane arrangement for voltage-gated ion channels i.e. six transmembrane segments. Modules are connected by linkers that are located in the intracellular milieu, as are both the N and C termini. The high voltage-activated channel subunits, Ca V 1.x and Ca V 2.x (␣ 1 ), numbering seven in total, share a high degree of sequence similarity but nevertheless encode distinct electrophysiological activities.The ␤ subunit was firs...

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Sensitization of dorsal horn neurons (DHNs) in the spinal cord is dependent on pain-related synaptic plasticity and causes persistent pain. The DHN sensitization is mediated by a signal transduction pathway initiated by the activation of NMDA receptors (NMDA-Rs). Recent studies have shown that elevated levels of reactive oxygen species (ROS) and phosphorylation-dependent trafficking of GluA2 subunit of AMPA receptors (AMPA-Rs) are a part of the signaling pathway for DHN sensitization. However, the relationship between ROS and AMPA-R phosphorylation and trafficking is not known. Thus, this study investigated the effects of ROS scavengers on the phosphorylation and cell-surface localization of GluA1 and GluA2. Intrathecal NMDA- and intradermal capsaicin-induced hyperalgesic mice were used for this study since both pain models share the NMDA-R activation-dependent DHN sensitization in the spinal cord. Our behavioral, biochemical, and immunohistochemical analyses demonstrated that: 1) NMDA-R activation in vivo increased the phosphorylation of AMPA-Rs at GluA1 (S818, S831, and S845) and GluA2 (S880) subunits, 2) NMDA-R activation in vivo increased cell-surface localization of GluA1 but decreased that of GluA2, and 3) reduction of ROS levels by ROS scavengers PBN or TEMPOL reversed these changes in AMPA-Rs, as well as pain-related behavior. Given that AMPA-R trafficking to the cell surface and synapse is regulated by NMDA-R activation-dependent phosphorylation of GluA1 and GluA2, our study suggests that the ROS-dependent changes in the phosphorylation and cell-surface localization of AMPA-Rs are necessary for DHN sensitization and thus pain-related behavior. We further suggest that ROS reduction will ameliorate these molecular changes and pain.