Inflammation-induced GluA1 trafficking and membrane insertion of Ca2+ permeable AMPA receptors in dorsal horn neurons is dependent on spinal tumor necrosis factor, PI3 kinase and protein kinase A.

Peripheral inflammation induces sensitization of nociceptive spinal cord neurons. Both spinal tumor necrosis factor (TNF) and neuronal membrane insertion of Ca2+ permeable AMPA receptor (AMPAr) contribute to spinal sensitization and resultant pain behavior, molecular mechanisms connecting these two events have not been studied in detail. Intrathecal (i.t.) injection of TNF-blockers attenuated paw carrageenan-induced mechanical and thermal hypersensitivity. Levels of GluA1 and GluA4 from dorsal spinal membrane fractions increased in carrageenan-injected rats compared to controls. In the same tissue, GluA2 levels were not altered. Inflammation-induced increases in membrane GluA1 were prevented by i.t. pre-treatment with antagonists to TNF, PI3K, PKA and NMDA. Interestingly, administration of TNF or PI3K inhibitors followed by carrageenan caused a marked reduction in plasma membrane GluA2 levels, despite the fact that membrane GluA2 levels were stable following inhibitor administration in the absence of carrageenan. TNF pre-incubation induced increased numbers of Co2+ labeled dorsal horn neurons, indicating more neurons with Ca2+ permeable AMPAr. In parallel to Western blot results, this increase was blocked by antagonism of PI3K and PKA. In addition, spinal slices from GluA1 transgenic mice, which had a single alanine replacement at GluA1 ser 845 or ser 831 that prevented phosphorylation, were resistant to TNF-induced increases in Co2+ labeling. However, behavioral responses following intraplantar carrageenan and formalin in the mutant mice were no different from littermate controls, suggesting a more complex regulation of nociception. Co-localization of GluA1, GluA2 and GluA4 with synaptophysin on identified spinoparabrachial neurons and their relative ratios were used to assess inflammation-induced trafficking of AMPAr to synapses. Inflammation induced an increase in synaptic GluA1, but not GluA2. Although total GluA4 also increased with inflammation, co-localization of GluA4 with synaptophysin, fell short of significance. Taken together these data suggest that peripheral inflammation induces a PI3K and PKA dependent TNFR1 activated pathway that culminates with trafficking of calcium permeable AMPAr into synapses of nociceptive dorsal horn projection neurons.

Enhancing VTA Cav1.3 L-type Ca2+ channel activity promotes cocaine and mood-related behaviors via overlapping AMPA receptor mechanisms in the nucleus accumbens.

Genetic factors significantly influence susceptibility for substance abuse and mood disorders. Rodent studies have begun to elucidate a role of Cav1.3 L-type Ca2+ channels in neuropsychiatric-related behaviors, such as addictive and depressive-like behaviors. Human studies have also linked the CACNA1D gene, which codes for the Cav1.3 protein, with bipolar disorder. However, the neurocircuitry and the molecular mechanisms underlying the role of Cav1.3 in neuropsychiatric phenotypes are not well established. In the present study, we directly manipulated Cav1.3 channels in Cav1.2 dihydropyridine insensitive mutant mice and found that ventral tegmental area (VTA) Cav1.3 channels mediate cocaine-related and depressive-like behavior through a common nucleus accumbens (NAc) shell calcium-permeable α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (CP-AMPAR) mechanism that requires GluA1 phosphorylation at S831. Selective activation of VTA Cav1.3 with (±)-BayK-8644 (BayK) enhanced cocaine conditioned place preference and cocaine psychomotor activity while inducing depressive-like behavior, an effect not observed in S831A phospho-mutant mice. Infusion of the CP-AMPAR-specific blocker Naspm into the NAc shell reversed the cocaine and depressive-like phenotypes. In addition, activation of VTA Cav1.3 channels resulted in social behavioral deficits. In contrast to the cocaine- and depression-related phenotypes, GluA1/A2 AMPARs in the NAc core mediated social deficits, independent of S831-GluA1 phosphorylation. Using a candidate gene analysis approach, we also identified single-nucleotide polymorphisms in the CACNA1D gene associated with cocaine dependence in human subjects. Together, our findings reveal novel, overlapping mechanisms through which VTA Cav1.3 mediates cocaine-related, depressive-like and social phenotypes, suggesting that Cav1.3 may serve as a target for the treatment of neuropsychiatric symptoms.

Mapping homeostatic synaptic plasticity using cable properties of dendrites.

When chronically silenced, cortical and hippocampal neurons homeostatically upregulate excitatory synaptic function. However, the subcellular position of such changes on the dendritic tree is not clear. We exploited the cable-filtering properties of dendrites to derive a parameter, the dendritic filtering index (DFI), to map the spatial distribution of synaptic currents. Our analysis indicates that young rat cortical neurons globally scale AMPA receptor-mediated currents, while mature hippocampal neurons do not, revealing distinct homeostatic strategies between brain regions and developmental stages. The DFI presents a useful tool for mapping the dendritic origin of synaptic currents and the location of synaptic plasticity changes.

Role for neonatal D-serine signaling: prevention of physiological and behavioral deficits in adult Pick1 knockout mice.

NMDA glutamate receptors have key roles in brain development, function and dysfunction. Regulatory roles of D-serine in NMDA receptor-mediated synaptic plasticity have been reported. Nonetheless, it is unclear whether and how neonatal deficits in NMDA-receptor-mediated neurotransmission affect adult brain functions and behavior. Likewise, the role of D-serine during development remains elusive. Here we report behavioral and electrophysiological deficits associated with the frontal cortex in Pick1 knockout mice, which show D-serine deficits in a neonatal- and forebrain-specific manner. The pathological manifestations observed in adult Pick1 mice are rescued by transient neonatal supplementation of D-serine, but not by a similar treatment in adulthood. These results indicate a role for D-serine in neurodevelopment and provide novel insights on how we interpret data of psychiatric genetics, indicating the involvement of genes associated with D-serine synthesis and degradation, as well as how we consider animal models with neonatal application of NMDA receptor antagonists.

The human language-associated gene SRPX2 regulates synapse formation and vocalization in mice.

Synapse formation in the developing brain depends on the coordinated activity of synaptogenic proteins, some of which have been implicated in a number of neurodevelopmental disorders. Here, we show that the sushi repeat-containing protein X-linked 2 (SRPX2) gene encodes a protein that promotes synaptogenesis in the cerebral cortex. In humans, SRPX2 is an epilepsy- and language-associated gene that is a target of the foxhead box protein P2 (FoxP2) transcription factor. We also show that FoxP2 modulates synapse formation through regulating SRPX2 levels and that SRPX2 reduction impairs development of ultrasonic vocalization in mice. Our results suggest FoxP2 modulates the development of neural circuits through regulating synaptogenesis and that SRPX2 is a synaptogenic factor that plays a role in the pathogenesis of language disorders.

Common exonic missense variants in the C2 domain of the human KIBRA protein modify lipid binding and cognitive performance.

The human KIBRA gene has been linked to human cognition through a lead intronic single-nucleotide polymorphism (SNP; rs17070145) that is associated with episodic memory performance and the risk to develop Alzheimer's disease. However, it remains unknown how this relates to the function of the KIBRA protein. Here, we identified two common missense SNPs (rs3822660G/T [M734I], rs3822659T/G [S735A]) in exon 15 of the human KIBRA gene to affect cognitive performance, and to be in almost complete linkage disequilibrium with rs17070145. The identified SNPs encode variants of the KIBRA C2 domain with distinct Ca(2+) dependent binding preferences for monophosphorylated phosphatidylinositols likely due to differences in the dynamics and folding of the lipid-binding pocket. Our results further implicate the KIBRA protein in higher brain function and provide direction to the cellular pathways involved.

Excitotoxicity through Ca2+-permeable AMPA receptors requires Ca2+-dependent JNK activation.

The GluA4-containing Ca(2+)-permeable α-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid receptors (Ca-AMPARs) were previously shown to mediate excitotoxicity through mechanisms involving the activator protein-1 (AP-1), a c-Jun N-terminal kinase (JNK) substrate. To further investigate JNK involvement in excitotoxic pathways coupled to Ca-AMPARs we used HEK293 cells expressing GluA4-containing Ca-AMPARs (HEK-GluA4). Cell death induced by overstimulation of Ca-AMPARs was mediated, at least in part, by JNK. Importantly, JNK activation downstream of these receptors was dependent on the extracellular Ca(2+) concentration. In our quest for a molecular link between Ca-AMPARs and the JNK pathway we found that the JNK interacting protein-1 (JIP-1) interacts with the GluA4 subunit of AMPARs through the N-terminal domain. In vivo, the excitotoxin kainate promoted the association between GluA4 and JIP-1 in the rat hippocampus. Taken together, our results show that the JNK pathway is activated by Ca-AMPARs upon excitotoxic stimulation and suggest that JIP-1 may contribute to the propagation of the excitotoxic signal.

Burst firing induces postsynaptic LTD at developing mossy fibre-CA3 pyramid synapses.

Synaptic development is an activity-dependent process utilizing coordinated network activity to drive synaptogenesis and subsequent refinement of immature connections. Hippocampal CA3 pyramidal neurons (PYRs) exhibit intense burst firing (BF) early in development, concomitant with the period of mossy fibre (MF) development. However, whether developing MF-PYR synapses utilize PYR BF to promote MF synapse maturation remains unknown. Recently, we demonstrated that transient tonic depolarization of postsynaptic PYRs induces a persistent postsynaptic form of long-term depression (depolarization-induced long-term depression, DiLTD) at immature MF-PYR synapses. DiLTD induction is NMDAR independent but does require postsynaptic Ca(2+) influx through L-type voltage gated Ca(2+) channels (L-VGCCs), and is expressed as a reduction in AMPAR function through the loss of GluR2-lacking AMPARs present at immature MF-PYR synapses. Here we examined whether more physiologically relevant phasic L-VGCC activation by PYR action potential (AP) BF activity patterns can trigger DiLTD. Using combined electrophysiological and Ca(2+) imaging approaches we demonstrate that PYR BF effectively drives L-VGCC activation and that brief periods of repetitive PYR BF, produced by direct current injection or intrinsic network activity induces NMDAR-independent LTD by promoting Ca(2+) influx through the activated L-VGCCs. This BF induced LTD, just like DiLTD, is specific for developing MF-PYR synapses, is PICK1 dependent, and is expressed postsynaptically. Our results demonstrate that DiLTD can be induced by phasic L-VGCC activation driven by PYR BF, suggesting the engagement of natural PYR network activity patterns for MF synapse maturation.

Activity-dependent secretion of neuronal activity regulated pentraxin from vasopressin neurons into the systemic circulation.

Neuronal activity regulated pentraxin (Narp) is a secreted, synaptic protein that has been implicated in modulating synaptic transmission. However, it is unclear how Narp secretion is regulated. Since we noted prominent Narp immunostaining in vasopressin neurons of the hypothalamus and in the posterior pituitary, we assessed whether it, like vasopressin, is released into the systemic circulation in an activity-dependent fashion. Consistent with this hypothesis, electron microscopic studies of the posterior pituitary demonstrated that Narp is located in secretory vesicles containing vasopressin. Using affinity chromatography, we detected Narp in plasma and found that these levels are markedly decreased by hypophysectomy. In addition, we confirmed that injection of a viral Narp construct into the hypothalamus restores plasma Narp levels in Narp knockout mice. In checking for activity-dependent secretion of Narp from the posterior pituitary, we found that several stimuli known to trigger vasopressin release, i.e. hypovolemia, dehydration and endotoxin, elevate plasma Narp levels. Taken together, these findings provide compelling evidence that Narp is secreted from vasopressin neurons in an activity-dependent fashion.

Serine racemase binds to PICK1: potential relevance to schizophrenia.

Accumulating evidence from both genetic and clinico-pharmacological studies suggests that D-serine, an endogenous coagonist to the NMDA subtype glutamate receptor, may be implicated in schizophrenia (SZ). Although an association of genes for D-serine degradation, such as D-amino acid oxidase and G72, has been reported, a role for D-serine in SZ has been unclear. In this study, we identify and characterize protein interacting with C-kinase (PICK1) as a protein interactor of the D-serine synthesizing enzyme, serine racemase (SR). The binding of endogenous PICK1 and SR requires the PDZ domain of PICK1. The gene coding for PICK1 is located at chromosome 22q13, a region frequently linked to SZ. In a case-control association study using well-characterized Japanese subjects, we observe an association of the PICK1 gene with SZ, which is more prominent in disorganized SZ. Our findings implicating PICK1 as a susceptibility gene for SZ are consistent with a role for D-serine in the disease.

Physiological and pathological caspase cleavage of the neuronal RasGEF GRASP-1 as detected using a cleavage site-specific antibody.

Caspases are proteases involved in various physiological and pathological processes in the nervous system, including development and pathogenesis. GRASP-1 is a recently identified neuronal substrate of caspase-3-subfamily caspases. It is a Ras-guanine nucleotide exchange factor (RasGEF) that interacts with the glutamate receptor interacting protein (GRIP). This alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) receptor/GRIP protein complex has been proposed to be involved in AMPA receptor synaptic targeting. The caspase-3 cleavage of GRASP-1 separates the N-terminal RasGEF catalytic domain from the C-terminal GRIP-interacting region, potentially disrupting regulation of the RasGEF activity by GRIP. To examine the regulation and regional distribution of the caspase-3 cleavage of GRASP-1 in vivo, we generated a cleavage site-specific antibody, termed CGP, against the cleaved N-terminal fragment of GRASP-1. Using this antibody, we have examined the caspase cleavage of GRASP-1 during postnatal development and following ischemia in mice. We found that caspase cleavage of GRASP-1 occurs in specific brain regions in a time-dependent manner during development and ischemia. This data provides an important account of the brain areas that might require caspase-3 activity in postnatal development and ischemic damage, which has not been documented. It also demonstrates that the CGP antibody is a powerful tool for studying neuronal activity of the caspase-3-subfamily caspases in vivo.

Identification of protein kinase C phosphorylation sites within the AMPA receptor GluR2 subunit.

Phosphorylation of AMPA receptor subunits is believed to regulate channel function and synaptic plasticity. Extensive biochemical and molecular studies have identified sites of PKA, PKC and CamKII phosphorylation in the C-termini of the GluR1 and 4 subunits. Recent studies have shown GluR1 phosphorylation to be bidirectionally altered during long-term potentiation (LTP) and long-term depression (LTD) in the hippocampus. The majority of AMPA receptors in the brain are believed to contain the GluR2 subunit that also contains potential sites for protein phosphorylation. Here we characterize PKC phosphorylation on the GluR2 subunit using biochemical and molecular techniques. Site-directed mutagenesis confirmed that this phosphorylation occurs on Serine 863 and Serine 880 of the GluR2 subunit C-terminus. Site identification allowed the generation of phosphorylation site-specific antibodies to facilitate the examination of GluR2 modification in primary neuronal culture. These studies confirmed that GluR2 is modified in response to the activation of PKC and suggests that phosphorylation of the ubiquitous GluR2 subunit may be important in the regulation of excitatory synaptic transmission.

Interaction of the AMPA receptor subunit GluR2/3 with PDZ domains regulates hippocampal long-term depression.

The interaction of PDZ domain-containing proteins with the C termini of alpha-amino-3-hydroxy-5-methylisoxazolepropionate (AMPA) receptors has been suggested to be important in the regulation of receptor targeting to excitatory synapses. Recent studies have shown that the rapid internalization of AMPA receptors at synapses may mediate, at least in part, the expression of long-term depression (LTD). We have previously shown that phosphorylation of Ser-880 on the AMPA receptor GluR2 subunit differentially regulated the interaction of GluR2 with the PDZ domain-containing proteins GRIP1 and PICK1. Here, we show that induction of LTD in hippocampal slices increases phosphorylation of Ser-880 within the GluR2 C-terminal PDZ ligand, suggesting that the modulation of GluR2 interaction with GRIP1 and PICK1 may regulate AMPA receptor internalization during LTD. Moreover, postsynaptic intracellular perfusion of GluR2 C-terminal peptides that disrupt GluR2 interaction with PICK1 inhibit the expression of hippocampal LTD. These results suggest that the interaction of GluR2 with PICK1 may play a regulatory role in the expression of LTD in the hippocampus.

Activation of silent synapses by rapid activity-dependent synaptic recruitment of AMPA receptors.

Many recent studies have shown that excitatory synapses can contain NMDA receptor responses in the absence of functional AMPA receptors and are therefore postsynaptically silent at resting membrane potentials. The activation of silent synapses via the rapid acquisition of AMPA receptor responses may be important in synaptic plasticity and neuronal development. Our recent immunocytochemical studies that used cultured hippocampal neurons have provided evidence for "morphological silent synapses" that physically contain NMDA receptors but no AMPA receptors. Here we show that the activation of NMDA receptors by spontaneous synaptic activity results in the rapid recruitment of AMPA receptors into these morphological silent synapses within minutes. In parallel, we find a significant increase in the frequency of AMPA receptor-mediated miniature EPSCs (mEPSCs). NMDA receptor activation also results in a mobilization of calcium/calmodulin (CaM) kinase II to synapses and an increase in the phosphorylation of surface AMPA receptors on the major CaM kinase II phosphorylation site. These results demonstrate that AMPA receptors can be modified and recruited rapidly to silent synapses via the activation of NMDA receptors by spontaneous synaptic activity.

Coupling of agonist-induced AMPA receptor internalization with receptor recycling.

Excitatory post-synaptic currents in the CNS are primarily mediated by alpha-amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid (AMPA) receptors in response to glutamate. Internalization of cell-surface receptors has been shown to be one mechanism by which to control receptor function. To test for agonist control of AMPA receptor plasma membrane expression we used biochemical assays to study AMPA receptor internalization and insertion processes. In heterologous cells, we observed a slow constitutive internalization and a rapid agonist-induced internalization of AMPA receptors. To our surprise, however, agonist treatment had no effect on the steady-state levels of AMPA receptors on the cell surface. To examine whether this could be explained by an agonist-induced increase in the insertion rate of AMPA receptors into the plasma membrane we developed an assay to independently measure receptor insertion. Remarkably, agonist treatment of cells also dramatically increased AMPA receptor plasma membrane insertion rates. In addition, using an assay to measure recycling of internalized pools we found that internalized receptors are rapidly recycled to the cell surface. These results suggest that agonist-induced receptor internalization is coupled to increases in receptor recycling. This increase in receptor flux through intracellular pools may allow for rapid changes in receptor surface expression by independent regulatory control of internalization and insertion.

The neuronal Rho-GEF Kalirin-7 interacts with PDZ domain-containing proteins and regulates dendritic morphogenesis.

Spine function requires precise control of the actin cytoskeleton. Kalirin-7, a GDP/GTP exchange factor for Rac1, interacts with PDZ proteins such as PSD-95, colocalizing with PSD-95 at synapses of cultured hippocampal neurons. PSD-95 and Kalirin-7 interact in vivo and in heterologous expression systems. In primary cortical neurons, transfected Kalirin-7 is targeted to spines and increases the number and size of spine-like structures. A Kalirin-7 mutant unable to interact with PDZ proteins remains in the cell soma, inducing local formation of aberrant filopodial neurites. Kalirin-7 with an inactivated GEF domain reduces the number of spines below control levels. These results provide evidence that PDZ proteins target Kalirin-7 to the PSD, where it regulates dendritic morphogenesis through Rac1 signaling to the actin cytoskeleton.

Detection of protein phosphorylation in tissues and cells.

Phosphorylation is one of the principal regulatory mechanisms in the nervous system. Several different procedures used to characterize the phosphorylation state of neuronal proteins are described in this unit, including analysis of phosphorylation in situ, phosphoamino acid analysis, and phosphopeptide map analysis. In addition, there is a protocol describing in vitro phosphorylation of fusion proteins. These methods are often combined to provide a comprehensive evaluation of the phosphorylation state of a particular protein.

Regulation of AMPA receptor GluR1 subunit surface expression by a 4. 1N-linked actin cytoskeletal association.

The synaptic localization, clustering, and immobilization of neurotransmitter receptors and ion channels play important roles in synapse formation and synaptic transmission. Although several proteins have been identified that interact with AMPA receptors and that may regulate their synaptic targeting, little is known about the interaction of AMPA receptors with the cytoskeleton. In studies examining the interaction of the AMPA receptor GluR1 subunit with neuronal proteins, we determined that GluR1 interacts with the 4.1G and 4.1N proteins, homologs of the erythrocyte membrane cytoskeletal protein 4.1. Using the yeast two-hybrid system and a heterologous cell system, we demonstrated that both 4.1G and 4.1N bind to a membrane proximal region of the GluR1 C terminus, and that a region within the C-terminal domain of 4.1G or 4.1N is sufficient to mediate the interaction. We also found that 4.1N can associate with GluR1 in vivo and colocalizes with AMPA receptors at excitatory synapses. Disruption of the interaction of GluR1 with 4.1N or disruption of actin filaments decreased the surface expression of GluR1 in heterologous cells. Moreover, disruption of actin filaments in cultured cortical neurons dramatically reduced the level of surface AMPA receptors. These results suggest that protein 4.1N may link AMPA receptors to the actin cytoskeleton.

Phosphorylation of the AMPA receptor subunit GluR2 differentially regulates its interaction with PDZ domain-containing proteins.

PSD-95, DLG, ZO-1 (PDZ) domain-mediated protein interactions have been shown to play important roles in the regulation of glutamate receptor function at excitatory synapses. Recent studies demonstrating the rapid regulation of AMPA receptor function during synaptic plasticity have suggested that AMPA receptor interaction with PDZ domain-containing proteins may be dynamically modulated. Here we show that PKC phosphorylation of the AMPA receptor GluR2 subunit differentially modulates its interaction with the PDZ domain-containing proteins GRIP1 and PICK1. The serine residue [serine-880 (Ser880)] in the GluR2 C-terminal sequence (IESVKI) critical for PDZ domain binding is a substrate of PKC and is phosphorylated in vivo. In vitro binding and coimmunoprecipitation studies show that phosphorylation of serine-880 within the GluR2 PDZ ligand significantly decreases GluR2 binding to GRIP1 but not to PICK1. Immunostaining of cultured hippocampal neurons demonstrates that the Ser880-phosphorylated GluR2 subunits are enriched and colocalized with PICK1 in the dendrites, with very little staining observed at excitatory synapses. Interestingly, PKC activation in neurons increases the Ser880 phosphorylation of GluR2 subunits and recruits PICK1 to excitatory synapses. Moreover, PKC stimulation in neurons results in rapid internalization of surface GluR2 subunits. These results suggest that GluR2 phosphorylation of serine-880 may be important in the regulation of the AMPA receptor internalization during synaptic plasticity.

Targeting of PKA to glutamate receptors through a MAGUK-AKAP complex.

Compartmentalization of glutamate receptors with the signaling enzymes that regulate their activity supports synaptic transmission. Two classes of binding proteins organize these complexes: the MAGUK proteins that cluster glutamate receptors and AKAPs that anchor kinases and phosphatases. In this report, we demonstrate that glutamate receptors and PKA are recruited into a macromolecular signaling complex through direct interaction between the MAGUK proteins, PSD-95 and SAP97, and AKAP79/150. The SH3 and GK regions of the MAGUKs mediate binding to the AKAP. Cell-based studies indicate that phosphorylation of AMPA receptors is enhanced by a SAP97-AKAP79 complex that directs PKA to GluR1 via a PDZ domain interaction. As AMPA receptor phosphorylation is implicated in regulating synaptic plasticity, these data suggest that a MAGUK-AKAP complex may be centrally involved.