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The development of selectively bred high and low alcohol-preferring mice (HAP and LAP, respectively) has allowed for an assessment of the polygenetic risk for pathological alcohol consumption and phenotypes associated with alcohol use disorder (AUD). Accumulating evidence indicates that the dorsal striatum (DS) is a central node in the neurocircuitry underlying addictive processes. Therefore, knowledge of differential gene, protein, and phosphorylated protein expression in the DS of HAP and LAP mice may foster new insights into how aberrant DS functioning may contribute to AUD-related phenotypes. To begin to elucidate these basal differences, a complementary and integrated analysis of DS tissue from alcohol-naïve male and female HAP and LAP mice was performed using RNA sequencing, quantitative proteomics, and phosphoproteomics. These datasets were subjected to a thorough analysis of gene ontology, pathway enrichment, and hub gene assessment. Analyses identified 2,108, 390, and 521 significant differentially expressed genes, proteins, and phosphopeptides, respectively between the two lines. Network analyses revealed an enrichment in the differential expression of genes, proteins, and phosphorylated proteins connected to cellular organization, cytoskeletal protein binding, and pathways involved in synaptic transmission and functioning. These findings suggest that the selective breeding to generate HAP and LAP mice may lead to a rearrangement of synaptic architecture which could alter DS neurotransmission and plasticity differentially between mouse lines. These rich data sets will serve as an excellent resource to inform future studies on how inherited differences in gene, protein, and phosphorylated protein expression contribute to AUD-related phenotypes.

Nav1.6 is the primary voltage-gated sodium channel isoform expressed in mature axon initial segments and nodes, making it critical for initiation and propagation of neuronal impulses. Thus, Nav1.6 modulation and dysfunction may have profound effects on input-output properties of neurons in normal and pathological conditions. Phosphorylation is a powerful and reversible mechanism regulating ion channel function. Because Nav1.6 and the multifunctional Ca(2+)/CaM-dependent protein kinase II (CaMKII) are independently linked to excitability disorders, we sought to investigate modulation of Nav1.6 function by CaMKII signaling. We show that inhibition of CaMKII, a Ser/Thr protein kinase associated with excitability, synaptic plasticity, and excitability disorders, with the CaMKII-specific peptide inhibitor CN21 reduces transient and persistent currents in Nav1.6-expressing Purkinje neurons by 87%. Using whole-cell voltage clamp of Nav1.6, we show that CaMKII inhibition in ND7/23 and HEK293 cells significantly reduces transient and persistent currents by 72% and produces a 5.8-mV depolarizing shift in the voltage dependence of activation. Immobilized peptide arrays and nanoflow LC-electrospray ionization/MS of Nav1.6 reveal potential sites of CaMKII phosphorylation, specifically Ser-561 and Ser-641/Thr-642 within the first intracellular loop of the channel. Using site-directed mutagenesis to test multiple potential sites of phosphorylation, we show that Ala substitutions of Ser-561 and Ser-641/Thr-642 recapitulate the depolarizing shift in activation and reduction in current density. Computational simulations to model effects of CaMKII inhibition on Nav1.6 function demonstrate dramatic reductions in spontaneous and evoked action potentials in a Purkinje cell model, suggesting that CaMKII modulation of Nav1.6 may be a powerful mechanism to regulate neuronal excitability.

Despite the rising prevalence of methadone treatment in pregnant women with opioid use disorder, the effects of methadone on neurobehavioral development remain unclear. We developed a translational mouse model of prenatal methadone exposure (PME) that resembles the typical pattern of opioid use by pregnant women who first use oxycodone then switch to methadone maintenance pharmacotherapy, and subsequently become pregnant while maintained on methadone. We investigated the effects of PME on physical development, sensorimotor behavior, and motor neuron properties using a multidisciplinary approach of physical, biochemical, and behavioral assessments along with brain slice electrophysiology and in vivo magnetic resonance imaging. Methadone accumulated in the placenta and fetal brain, but methadone levels in offspring dropped rapidly at birth which was associated with symptoms and behaviors consistent with neonatal opioid withdrawal. PME produced substantial impairments in offspring physical growth, activity in an open field, and sensorimotor milestone acquisition. Furthermore, these behavioral alterations were associated with reduced neuronal density in the motor cortex and a disruption in motor neuron intrinsic properties and local circuit connectivity. The present study adds to the limited body of work examining PME by providing a comprehensive, translationally relevant characterization of how PME disrupts offspring physical and neurobehavioral development., eLife digest The far-reaching opioid crisis extends to babies born to mothers who take prescription or illicit opioids during pregnancy. Opioids such as oxycodone and methadone can freely cross the placenta from mother to baby. With the rising misuse of and addiction to opioids, the number of babies born physically dependent on opioids has risen sharply over the last decade. Although these infants are only passively exposed to opioids in the womb, they can still experience withdrawal symptoms at birth. This withdrawal is characterized by irritability, excessive crying, body shakes, problems with feeding, fevers and diarrhea. While considerable attention has been given to treating opioid withdrawal in newborn babies, little is known about how these children develop in their first years of life. This is, in part, because it is difficult for researchers to separate drug-related effects from other factors in a child’s home environment that can also disrupt their development. In addition, the biological mechanisms underpinning opioid-related impairments to infant development also remain unclear. Animal models have been used to study the effects of opioid exposure during pregnancy (termed prenatal exposure) on infants. These models, however, could be improved to better replicate the typical pattern of opioid use among pregnant women. Recognizing this gap, Grecco et al. have developed a mouse model of prenatal methadone exposure where female mice that were previously dependent on oxycodone were treated with methadone throughout their pregnancy. Methadone is an opioid drug commonly prescribed for treating opioid use disorder in pregnant women and was found to accumulate at high levels in the fetal brain of mice, which fell quickly after birth. The offspring also experienced withdrawal symptoms. Grecco et al. then examined the physical, behavioral and brain development of mice born to opioid-treated mothers. These included assessments of the animals’ motor skills, sensory reflexes and behavior in their first four weeks of life. Additional experiments tested the properties of nerve cells in the brain to examine cell-level changes. The assessments showed that methadone exposure in the womb impaired the physical growth of offspring and this persisted into ‘adolescence’. Prenatal methadone exposure also delayed progress towards key developmental milestones and led to hyperactivity in three-week-old mice. Moreover, Grecco et al. found that these mice had reduced neuron density and cell-to-cell connectivity in the part of the brain which controls movement. These findings shed light on the potential consequences of prenatal methadone exposure on physical, behavioral and brain development in infants. This model could also be used to study new potential treatments or intervention strategies for offspring exposed to opioids during pregnancy.

Protein phosphorylation is a key mediator of signal transduction, allowing for dynamic regulation of substrate activity. Whereas protein kinases obtain substrate specificity by targeting specific amino acid sequences, serine/threonine phosphatase catalytic subunits are much more promiscuous in their ability to dephosphorylate substrates. To obtain substrate specificity, serine/threonine phosphatases utilize targeting proteins to regulate phosphatase subcellular localization and catalytic activity. Spinophilin and its homologue neurabin are two of the most abundant dendritic spine-localized protein phosphatase 1 (PP1) targeting proteins. The association between spinophilin and PP1 is increased in the striatum of animal models of Parkinson’s disease (PD). However, mechanisms that regulate the association of spinophilin and neurabin with PP1 are unclear. Here, we report that the association between spinophilin and PP1α or PP1γ1 was increased by CDK5 expression and activation in a heterologous cell system. This increased association is at least partially due to phosphorylation of PP1. Conversely, CDK5 expression and activation decreased the association of PP1 with neurabin. As with dopamine depletion, methamphetamine (METH) abuse causes persistent alterations in dopamine signaling which influence striatal medium spiny neuron function and biochemistry. Moreover, both METH toxicity and dopamine depletion are associated with deficits in motor control and motor learning. Pathologically, we observed a decreased association of spinophilin with PP1 in rat striatum evaluated one month following a binge METH paradigm. Behaviorally, we found that loss of spinophilin recapitulates rotarod pathology previously observed in dopamine-depleted and METH-treated animals. Together, these data have implications in multiple disease states associated with altered dopamine signaling such as PD and psychostimulant drug abuse and delineate a novel mechanism by which PP1 interactions with spinophilin and neurabin may be differentially regulated.

Early life trauma is a risk factor for a number of neuropsychiatric disorders, including schizophrenia (SZ). The current study assessed how an early life traumatic event, maternal deprivation (MD), alters cognition and brain function in rodents. Rats were maternally deprived in the early postnatal period and then recognition memory (RM) was tested in adulthood using the novel object recognition task. The expression of catechol-o-methyl transferase (COMT) and glutamic acid decarboxylase (GAD67) were quantified in the medial prefrontal cortex (mPFC), ventral striatum, and temporal cortex (TC). In addition, depth EEG recordings were obtained from the mPFC, vertex, and TC during a paired-click paradigm to assess the effects of MD on sensory gating. MD animals exhibited impaired RM, lower expression of COMT in the mPFC and TC, and lower expression of GAD67 in the TC. Increased bioelectric noise was observed at each recording site of MD animals. MD animals also exhibited altered information theoretic measures of stimulus encoding. These data indicate that a neurodevelopmental perturbation yields persistent alterations in cognition and brain function, and are consistent with human studies that identified relationships between allelic differences in COMT and GAD67 and bioelectric noise. These changes evoked by MD also lead to alterations in shared information between cognitive and primary sensory processing areas, which provides insight into how early life trauma confers a risk for neurodevelopmental disorders, such as SZ, later in life.

N-methyl-d-Aspartate receptors (NMDARs) are abundant postsynaptic proteins that are critical for normal synaptic communication. NMDAR channel function is regulated by multiple properties, including phosphorylation. Inhibition of protein phosphatase 1 (PP1) in hippocampal neurons increases NMDAR activity, an effect abrogated by loss of spinophilin, the major PP1-targeting protein in the postsynaptic density. However, how spinophilin regulates PP1-dependent NMDAR function is unclear. We hypothesize that spinophilin regulates PP1 binding to the NMDAR to alter NMDAR phosphorylation. Our data demonstrate that spinophilin interacts with the GluN2B subunit of the NMDAR. In human embryonic kidney 293 FT cells, activation and/or overexpression of protein kinase A increased the association between spinophilin and the GluN2B subunit of the NMDAR. Functionally, we found that spinophilin overexpression decreased PP1 binding to the GluN2B subunit of the NMDAR and attenuated the PP1-dependent dephosphorylation of GluN2B at Ser-1284. Moreover, in P28 hippocampal lysates isolated from spinophilin KO compared to WT mice, there was increased binding of GluN2B to PP1, decreased phosphorylation of GluN2B at Ser-1284, and altered GluN2B protein interactions with postsynaptic density-enriched proteins. Together, our data demonstrate that spinophilin decreases PP1 binding to GluN2B and concomitantly enhances the phosphorylation of GluN2B at Ser-1284. The putative consequences of these spinophilin-dependent alterations in GluN2B phosphorylation and interactions on synaptic GluN2B localization and function are discussed. Open Science: This manuscript was awarded with the Open Materials Badge For more information see: https://cos.io/our-services/open-science-badges/.

Normal neuronal communication and synaptic plasticity at glutamatergic synapses requires dynamic regulation of postsynaptic molecules. Protein expression and protein post-translational modifications regulate protein interactions that underlie this organization. In this Review, we highlight data obtained over the last 20 years that have used qualitative and quantitative proteomics-based approaches to identify postsynaptic protein complexes. Herein, we describe how these proteomics studies have helped lay the foundation for understanding synaptic physiology and perturbations in synaptic signaling observed in different pathologies. We also describe emerging technologies that can be useful in these analyses. We focus on protein complexes associated with the highly abundant and functionally critical proteins: calcium/calmodulin-dependent protein kinase II, the N-methyl-d-aspartate, and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid glutamate receptors, and postsynaptic density protein of 95 kDa.

G protein βγ subunits play essential roles in regulating cellular signaling cascades, yet little is known about their distribution in tissues or their subcellular localization. While previous studies have suggested specific isoforms may exhibit a wide range of distributions throughout the central nervous system, a thorough investigation of the expression patterns of both Gβ and Gγ isoforms within subcellular fractions has not been conducted. To address this, we applied a targeted proteomics approach known as multiple-reaction monitoring to analyze localization patterns of Gβ and Gγ isoforms in pre- and postsynaptic fractions isolated from cortex, cerebellum, hippocampus, and striatum. Particular Gβ and Gγ subunits were found to exhibit distinct regional and subcellular localization patterns throughout the brain. Significant differences in subcellular localization between pre- and postsynaptic fractions were observed within the striatum for most Gβ and Gγ isoforms, while others exhibited completely unique expression patterns in all four brain regions examined. Such differences are a prerequisite for understanding roles of individual subunits in regulating specific signaling pathways throughout the central nervous system.

The metabolism of the Xenopus laevis egg provides a cell survival signal. We found previously that increased carbon flux from glucose-6-phosphate (G6P) through the pentose phosphate pathway in egg extracts maintains NADPH levels and calcium/calmodulin regulated protein kinase II (CaMKII) activity to phosphorylate caspase 2 and suppress cell death pathways. Here we show that the addition of G6P to oocyte extracts inhibits the dephosphorylation/inactivation of CaMKII bound to caspase 2 by protein phosphatase 1. Thus, G6P sustains the phosphorylation of caspase 2 by CaMKII at Ser-135, preventing the induction of caspase 2-mediated apoptotic pathways. These findings expand our understanding of oocyte biology and clarify mechanisms underlying the metabolic regulation of CaMKII and apoptosis. Furthermore, these findings suggest novel approaches to disrupt the suppressive effects of the abnormal metabolism on cell death pathways.

Signaling changes that occur in the striatum following the loss of dopamine neurons in the Parkinson disease (PD) are poorly understood. While increases in the activity of kinases and decreases in the activity of phosphatases have been observed, the specific consequences of these changes are less well understood. Phosphatases, such as protein phosphatase 1 (PP1), are highly promiscuous and obtain substrate selectivity via targeting proteins. Spinophilin is the major PP1-targeting protein enriched in the postsynaptic density of striatal dendritic spines. Spinophilin association with PP1 is increased concurrent with decreases in PP1 activity in an animal model of PD. Using proteomic-based approaches, we observed dopamine depletion-induced decreases in spinophilin binding to multiple protein classes in the striatum. Specifically, there was a decrease in the association of spinophilin with neurofilament medium (NF-M) in dopamine-depleted striatum. Using a heterologous cell line, we determined that spinophilin binding to NF-M required overexpression of the catalytic subunit of protein kinase A and was decreased by cyclin-dependent protein kinase 5. Functionally, we demonstrate that spinophilin can decrease NF-M phosphorylation. Our data determine mechanisms that regulate, and putative consequences of, pathological changes in the association of spinophilin with NF-M that are observed in animal models of PD.

Protein-protein interactions are thought to modulate the efficiency and specificity of Ca(2+)/calmodulin (CaM)-dependent protein kinase II (CaMKII) signaling in specific subcellular compartments. Here we show that the F-actin-binding protein α-actinin targets CaMKIIα to F-actin in cells by binding to the CaMKII regulatory domain, mimicking CaM. The interaction with α-actinin is blocked by CaMKII autophosphorylation at Thr-306, but not by autophosphorylation at Thr-305, whereas autophosphorylation at either site blocks Ca(2+)/CaM binding. The binding of α-actinin to CaMKII is Ca(2+)-independent and activates the phosphorylation of a subset of substrates in vitro. In intact cells, α-actinin selectively stabilizes CaMKII association with GluN2B-containing glutamate receptors and enhances phosphorylation of Ser-1303 in GluN2B, but inhibits CaMKII phosphorylation of Ser-831 in glutamate receptor GluA1 subunits by competing for activation by Ca(2+)/CaM. These data show that Ca(2+)-independent binding of α-actinin to CaMKII differentially modulates the phosphorylation of physiological targets that play key roles in long-term synaptic plasticity.

Chronic alcohol exposure can cause dramatic behavioral alterations, including increased anxiety-like behavior and depression. These alterations are proposed to be due in part to adaptations in the brain regions that regulate emotional behavior, including the bed nucleus of the stria terminalis (BNST), a principal output nucleus of the amygdala. However, to date there have been no studies that have examined the impact of in vivo alcohol exposure on synaptic function in the BNST. To better understand how alcohol can alter neuronal function, we examined the ability of in vivo alcohol exposure to alter glutamatergic transmission in the BNST using whole-cell voltage clamp recordings and biochemistry in brain slices obtained from C57Bl6 mice. Chronic intermittent, but not continuous, ethanol vapor exposure increased temporal summation of NMDA receptor (NMDAR)-mediated excitatory postsynaptic currents (EPSCs). Both electrophysiological and biochemical approaches suggest that this difference is not because of an alteration in glutamate release, but rather an increase in the levels of NR2B-containing NMDARs. Further, we found that ethanol modulation of NMDAR in the vBNST is altered after intermittent alcohol exposure. Our results support the hypothesis that NMDAR-mediated synaptic transmission is sensitized at key synapses in the extended amygdala and thus may be a suitable target for manipulation of the behavioral deficits associated with acute withdrawal from chronic alcohol exposure.

Repeated, high-dose methamphetamine (METH) administrations cause persistent dopaminergic deficits in rodents, nonhuman primates, and humans. In rats, this treatment also causes the formation of high-molecular mass (greater than approximately 120 kDa) dopamine transporter (DAT)-associated complexes, the loss of DAT monomer immunoreactivity, and a decrease in DAT function, as assessed in striatal synaptosomes prepared 24 h after METH treatment. The present study extends these findings by demonstrating the regional selectivity of DAT complex formation and monomer loss because these changes in DAT immunoreactivity were not observed in the nucleus accumbens. Furthermore, DAT complex formation was not a consequence limited to METH treatment because it was also caused by intrastriatal administration of 6-hydroxydopamine. Pretreatment with the D2 receptor antagonist, eticlopride [S-(-)-3-chloro-5-ethyl-N-[(1-ethyl-2-pyrrolidinyl)methyl]-6-hydroxy-2-methoxybenzamide hydrochloride], but not the D1 receptor antagonist, SCH23390 [R(+)-7-chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine hydrochloride], attenuated METH-induced DAT complex formation. Eticlopride pretreatment also attenuated METH-induced DAT monomer loss and decreases in DAT function; however, the attenuation was much less pronounced than the effect on DAT complex formation. Finally, results also revealed a negative correlation between METH-induced DAT complex formation and DAT activity. Taken together, these data further elucidate the underlying mechanisms and the functional consequences of repeated administrations of METH on the DAT protein. Furthermore, these data suggest a multifaceted role for D2 receptors in mediating METH-induced alterations of the DAT and its function.

Protein phosphatase 1 (PP1) catalytic subunits dephosphorylate specific substrates in discrete subcellular compartments to modulate many cellular processes. Canonical PP1-binding motifs (R/K-V/I-X-F) in a family of proteins mediate subcellular targeting, and the amino acids that form the binding pocket for the canonical motif are identical in all PP1 isoforms. However, PP1gamma1 but not PP1beta is selectively localized to F-actin-rich dendritic spines in neurons. Although the F-actin-binding proteins neurabin I and spinophilin (neurabin II) also bind PP1, their role in PP1 isoform selective targeting in intact cells is poorly understood. We show here that spinophilin selectively targets PP1gamma1, but not PP1beta, to F-actin-rich cortical regions of intact cells. Mutation of a PP1gamma1 selectivity determinant (N(464)EDYDRR(470) in spinophilin: conserved as residues 473-479 in neurabin) to VKDYDTW severely attenuated PP1gamma1 interactions with neurabins in vitro and in cells and disrupted PP1gamma1 targeting to F-actin. This domain is not involved in the weaker interactions of neurabins with PP1beta. In contrast, mutation of the canonical PP1-binding motif attenuated interactions of neurabins with both isoforms. Thus, selective targeting of PP1gamma1 to F-actin by neurabins in intact cells requires both the canonical PP1-binding motif and an auxiliary PP1gamma1-selectivity determinant.

N-methyl-D-aspartate receptors (NMDARs) are major targets of both acute and chronic alcohol, as well as regulators of plasticity in a number of brain regions. Aberrant plasticity may contribute to the treatment resistance and high relapse rates observed in alcoholics. Recent work suggests that chronic alcohol treatment preferentially modulates both the expression and subcellular localization of NMDARs containing the GluN2B subunit. Signaling through synaptic and extrasynaptic GluN2B-NMDARs has already been implicated in the pathophysiology of various other neurological disorders. NMDARs interact with a large number of proteins at the glutamate synapse, and a better understanding of how alcohol modulates this proteome is needed. We employed a discovery-based proteomic approach in subcellular fractions of hippocampal tissue from chronic intermittent alcohol (CIE) exposed C57Bl/6j mice to gain insight into alcohol-induced changes in GluN2B signaling complexes. Protein enrichment analyses revealed changes in the association of postsynaptic proteins, including scaffolding, glutamate receptor, and PDZ-domain binding proteins with GluN2B. In particular, GluN2B interaction with metabotropic glutamate (mGlu)1/5 receptor-dependent long-term depression (LTD) associated proteins such as Arc and Homer 1 was increased, while GluA2 was decreased. Accordingly, we found a lack of mGlu1/5 induced-LTD while α1-adrenergic receptor-induced LTD remained intact in hippocampal CA1 following CIE. These data suggest that CIE specifically disrupts mGlu1/5-LTD, representing a possible connection between NMDAR and mGlu receptor signaling. These studies not only demonstrate a new way in which alcohol can modulate plasticity in the hippocampus but emphasize the utility of this discovery-based proteomic approach to generate new hypotheses regarding alcohol-related mechanisms.

Studies described here tested the hypothesis that phosphodiesterase 10A inhibition by a selective antagonist, MP-10, activates the dopamine D2 receptor expressing medium spiny neurons to a greater extent than the D1 receptor expressing neurons. We used regional pattern of c-Fos induction in the neostriatal subregions of rodents and direct assessment of D1-positive and -negative neurons in the DRd1a-tdTomato mice for the purpose. MP-10 (1, 3, 10 or 30 mg/kg, PO) dose-dependently increased c-Fos immunopositive nuclei in all regions of neostriatum. However, the effect was statistically greater in the dorsolateral striatum, a region known to be activated preferentially by the D2 antagonism, than the D1-activated dorsomedial striatum. The D2 antagonist, haloperidol (0.3, 1, or 3 mg/kg, PO) produced an identical, regional pattern of c-Fos induction favoring the dorsolateral striatum of the rat. In contrast, the D1 agonist, SKF82958 (0.5, 1, or 2 mg/kg, PO), induced greater expression of c-Fos in the dorsomedial striatum. The C57Bl/6 mouse also showed regionally preferential c-Fos activation by haloperidol (2 mg/kg, IP) and SKF82858 (3 mg/kg, IP). In the Drd1a-tdTomato mice, MP-10 (3 or 10 mg/kg, IP) increased c-Fos immunoreactivity in both types of neurons, the induction was greater in the D1-negative neurons. Taken together, both the regional pattern of c-Fos induction in the striatal sub-regions and the greater induction of c-Fos in the D1-negative neurons indicate that PDE10A inhibition produces a small but significantly greater activation of the D2-containing striatopallidal pathway.

Multiple high-dose administrations of methamphetamine (METH) both rapidly (within hours) decrease plasmalemmal dopamine (DA) uptake and cause long-term deficits in DA transporter (DAT) levels and other dopaminergic parameters persisting weeks to months in rat striatum. In contrast, either a single administration of METH or multiple administrations of methylenedioxymethamphetamine (MDMA) cause less of an acute reduction in DA uptake and little or no persistent dopaminergic deficits. The long-term dopaminergic deficits caused by METH have been suggested, in part, to involve the DAT. Hence, this study assessed the impact of METH and MDMA administration on the DAT protein per se. Results revealed that multiple administrations of METH promoted formation of higher molecular weight (>170 kDa) DAT-associated protein complexes 24–48 hr after treatment. This increase was attenuated by either preventing hyperthermia or pretreatment with the tyrosine hydroxylase inhibitor α-methyl-p-tyrosine; notably, each of these manipulations has also been demonstrated previously to prevent the persistent deficits in dopaminergic function caused by METH treatment. In contrast, either a single injection of METH or multiple injections of MDMA caused little or no formation of these DAT complexes. The addition of the reducing agent β-mercaptoethanol to samples prepared from METH-treated rats diminished the intensity of these complexes. Taken together, these data are the first to demonstrate higher molecular weight DAT complex formationin vivoand that such formation can be altered by both pharmacological and physiological manipulations. The implications of this phenomenon with regard to the neurotoxic potential of these stimulants are discussed.

Distinct physiological stimuli are required for bidirectional synaptic plasticity in striatum and hippocampus, but differences in the underlying signaling mechanisms are poorly understood. We have begun to compare levels and interactions of key excitatory synaptic proteins in whole extracts and subcellular fractions isolated from micro-dissected striatum and hippocampus. Levels of multiple glutamate receptor subunits, calcium/calmodulin-dependent protein kinase II (CaMKII), a highly abundant serine/threonine kinase, and spinophilin, a F-actin and protein phosphatase 1 (PP1) binding protein, were significantly lower in striatal extracts, as well as in synaptic and/or extrasynaptic fractions, compared with similar hippocampal extracts/fractions. However, CaMKII interactions with spinophilin were more robust in striatum compared with hippocampus, and this enhanced association was restricted to the extrasynaptic fraction. NMDAR GluN2B subunits associate with both spinophilin and CaMKII, but spinophilin-GluN2B complexes were enriched in extrasynaptic fractions whereas CaMKII-GluN2B complexes were enriched in synaptic fractions. Notably, the association of GluN2B with both CaMKII and spinophilin was more robust in striatal extrasynaptic fractions compared with hippocampal extrasynaptic fractions. Selective differences in the assembly of synaptic and extrasynaptic signaling complexes may contribute to differential physiological regulation of excitatory transmission in striatum and hippocampus.

The bed nucleus of the stria terminalis (BNST) is a critical region for alcohol/drug-induced negative affect and stress-induced reinstatement. NMDA receptor (NMDAR)-dependent plasticity, such as long-term potentiation (LTP), has been postulated to play key roles in alcohol and drug addiction; yet, to date, little is understood regarding the mechanisms underlying LTP of the BNST, or its regulation by ethanol. Acute and chronic exposure to ethanol modulates glutamate transmission via actions on NMDARs. Despite intense investigation, tests of subunit specificity of ethanol actions on NMDARs using pharmacological approaches have produced mixed results. Thus, we use a conditional GluN2B KO mouse line to assess both basal and ethanol-dependent function of this subunit at glutamate synapses in the BNST. Deletion of GluN2B eliminated LTP, as well as actions of ethanol on NMDAR function. Further, we show that chronic ethanol exposure enhances LTP formation in the BNST. Using KO-validated pharmacological approaches with Ro25-6981 and memantine, we provide evidence suggesting that chronic ethanol exposure enhances LTP in the BNST via paradoxical extrasynaptic NMDAR involvement. These findings demonstrate that GluN2B is a key point of regulation for ethanol's actions and suggest a unique role of extrasynaptic GluN2B-containing receptors in facilitating LTP.

Mechanisms underlying age-dependent changes of dendritic spines on striatal medium spiny neurons are poorly understood. Spinophilin is an F-actin- and protein phosphatase 1 (PP1)-binding protein that targets PP1 to multiple downstream effectors to modulate dendritic spine morphology and function. We found that calcium/calmodulin-dependent protein kinase II (CaMKII) directly and indirectly associates with N- and C-terminal domains of spinophilin, but F-actin can displace CaMKII from the N-terminal domain. Spinophilin co-localizes PP1 with CaMKII on the F-actin cytoskeleton in heterologous cells, and spinophilin co-localizes with synaptic CaMKII in neuronal cultures. Thr286 autophosphorylation enhances the binding of CaMKII to spinophilin in vitro and in vivo. Although there is no change in total levels of Thr286 autophosphorylation, maturation from postnatal day 21 into adulthood robustly enhances the levels of CaMKII that co-immunoprecipitate with spinophilin from mouse striatal extracts. Moreover, N- and C-terminal domain fragments of spinophilin bind more CaMKII from adult vs. postnatal day 21 striatal lysates. Total levels of other proteins that interact with C-terminal domains of spinophilin decrease during maturation, perhaps reducing competition for CaMKII binding to the C-terminal domain. In contrast, total levels of α-internexin and binding of α-internexin to the spinophilin N-terminal domain increases with maturation, perhaps bridging an indirect interaction with CaMKII. Moreover, there is an increase in the levels of myosin Va, α-internexin, spinophilin, and PP1 in striatal CaMKII immune complexes isolated from adult and aged mice compared to those from postnatal day 21. These changes in spinophilin/CaMKII interactomes may contribute to changes in striatal dendritic spine density, morphology, and function during normal postnatal maturation and aging.

In order to provide insight into in vivo roles of CaMKIIα autophosphorylation at Thr286 during postnatal development, behavioral, biochemical, and electrophysiological phenotypes of pre-adolescent Thr286 to Ala CaMKIIα knock-in (T286A-KI) and WT mice were examined. T286A-KI mice displayed cognitive deficits in a novel object recognition test and an anxiolytic phenotype in the elevated plus maze, suggesting disruption of normal developmental processes. At the molecular level, the ratio of total CaMKIIα to CaMKIIβ in hippocampal lysates was significantly decreased ≈2-fold in T286A-KI mice, and levels of both isoforms in synaptic subcellular fractions were decreased by ≈80%. Total levels of GluA1 AMPA-glutamate receptor subunits and phosphorylation of GluA1 at the CaMKII site (Ser831) in synaptic fractions were unaltered, as were the frequency and amplitude of AMPAR-mediated spontaneous excitatory postsynaptic currents at hippocampal CA3-CA1 synapses. Synaptic levels of NMDA-glutamate receptor GluN1, GluN2A and GluN2B subunits also were unaltered. However, the reduced ratio of CaMKII to NMDAR subunits in synaptic fractions was linked to increased synaptic NMDAR-mediated currents in T286A-KI mice, apparently due to increased functional contributions by GluN2B NMDARs (assessed by Ro 25-6981 sensitivity). Thus, disruption of CaMKII synaptic targeting caused by elimination of Thr286 autophosphorylation leads to synaptic and behavioral deficits during pre-adolescence.

Spinophilin regulates excitatory postsynaptic function and morphology during development by virtue of its interactions with filamentous actin, protein phosphatase 1, and a plethora of additional signaling proteins. To provide insight into the roles of spinophilin in mature brain, we characterized the spinophilin interactome in subcellular fractions solubilized from adult rodent striatum by using a shotgun proteomics approach to identify proteins in spinophilin immune complexes. Initial analyses of samples generated using a mouse spinophilin antibody detected 23 proteins that were not present in an IgG control sample; however, 12 of these proteins were detected in complexes isolated from spinophilin knock-out tissue. A second screen using two different spinophilin antibodies and either knock-out or IgG controls identified a total of 125 proteins. The probability of each protein being specifically associated with spinophilin in each sample was calculated, and proteins were ranked according to a chi(2) analysis of the probabilities from analyses of multiple samples. Spinophilin and the known associated proteins neurabin and multiple isoforms of protein phosphatase 1 were specifically detected. Multiple, novel, spinophilin-associated proteins (myosin Va, calcium/calmodulin-dependent protein kinase II, neurofilament light polypeptide, postsynaptic density 95, alpha-actinin, and densin) were then shown to interact with GST fusion proteins containing fragments of spinophilin. Additional biochemical and transfected cell imaging studies showed that alpha-actinin and densin directly interact with residues 151-300 and 446-817, respectively, of spinophilin. Taken together, we have developed a multi-antibody, shotgun proteomics approach to characterize protein interactomes in native tissues, delineating the importance of knock-out tissue controls and providing novel insights into the nature and function of the spinophilin interactome in mature striatum.

Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) promotes trafficking and activation of the GluR1 subunit of alpha-amino- 3-hydroxy-5-methyl-4-isoxazolepropionic acid-type glutamate receptors (AMPARs) during synaptic plasticity. GluR1 is also modulated in parallel by multiprotein complexes coordinated by synapse-associated protein 97 (SAP97) that contain A-kinase anchoring protein 79/150 (AKAP79/150), protein kinase A, and protein phosphatase 2B. Here we show that SAP97 is present in CaMKII immune complexes isolated from rodent brain as well as from HEK293 cells co-expressing CaMKIIalpha and SAP97. CaMKIIalpha phosphorylated recombinant SAP97 within immune complexes in vitro and in intact cells. Four alternative mRNA splice variants of SAP97 expressing combinations of four inserts (I2, I3, I4, I5) in the U5 region between Src homology 3 (SH3) and guanylyl kinase-like (GK) domains were identified in rat brain at postnatal day 21. CaMKIIalpha preferentially phosphorylated a full-length SAP97 and a glutathione S-transferase (GST) fusion protein containing the I3 and I5 inserts (SAP97-I3I5 and GST-SH3-I3I5-GK, respectively) and also specifically interacted with GST-SH3-I3I5-GK compared with GST proteins containing other naturally occurring insert combinations. AKAP79/150 also directly and specifically bound only to GST-SH3-I3I5-GK, but CaMKII phosphorylation of GST-SH3-I3I5-GK prevented this interaction. AKAP79-dependent down-regulation of GluR1 AMPAR currents was ablated by overexpression of SAP97-I2I5 (which does not bind AKAP79) or by infusion of active CaMKIIalpha. Collectively, the data suggest that CaMKIIalpha targets a specific SAP97 splice variant to disengage AKAP79/150 from regulating GluR1 AMPARs, providing new insight into protein-protein interactions and phosphorylation events that are required for normal regulation of glutamatergic synaptic transmission, learning, and memory.

Sustained nigrostriatal dopamine depletion increases the serine/threonine phosphorylation of multiple striatal proteins that play a role in corticostriatal synaptic plasticity, including Thr(286) phosphorylation of calcium/calmodulin-dependent protein kinase IIalpha (CaMKIIalpha). Mechanisms underlying these changes are unclear, but protein phosphatases play a critical role in the acute modulation of striatal protein phosphorylation. Here we show that dopamine depletion for periods ranging from 3 weeks to 10 months significantly reduces the total activity of protein phosphatase (PP) 1, but not of PP2A, in whole lysates of rat striatum, as measured using multiple substrates, including Thr(286)-autophosphorylated CaMKIIalpha. Striatal PP1 activity is partially inhibited by a fragment of the PP1-binding protein neurabin-I, Nb-(146-493), because of the selective inhibition of the PP1gamma(1) isoform. The fraction of PP1 activity that is insensitive to Nb-(146-493) was unaffected by dopamine depletion, demonstrating that dopamine depletion specifically reduces the activity of PP1 isoforms that are sensitive to Nb-(146-493) (i.e. PP1gamma(1)). However, total striatal levels of PP1gamma(1) or any other PP1 isoform were unaffected by dopamine depletion, and our previous studies showed that total levels of the PP1 regulatory/targeting proteins DARPP-32, spinophilin, and neurabin were also unchanged. Rather, co-immunoprecipitation experiments demonstrated that dopamine depletion increases the association of PP1gamma(1) with spinophilin in striatal extracts. In combination, these data demonstrate that striatal dopamine depletion inhibits a specific synaptic phosphatase by increasing PP1gamma(1) interaction with spinophilin, perhaps contributing to hyperphosphorylation of synaptic proteins and disruptions of synaptic plasticity and/or dendritic morphology.

Conantokin G (Con G), derived from the venom of Conus geographus, is the most characterized natural peptide antagonist targeted to N-methyl-d-aspartate (NMDA) receptors. Although Con G is known to bind to the glutamate binding site on the NR2 subunit of the receptor, it is unclear whether it can allosterically modulate the function of the receptor through the glycine binding site on the NR1 subunit. This study was designed to evaluate the action of Con G on NMDA receptor–mediated spontaneous excitatory postsynaptic currents (sEPSCs) and its modulation by glycine in cultured cortical neurons (13–19 days in vitro) using the whole cell patch-clamp technique. Con G inhibited NMDA receptor–mediated sEPSCs in a concentration-dependent manner. Also, the potency of Con G decreased as a function of time in culture. The inhibition of EPSCs observed after application of Con G in the presence of high (10 μM) and nominal (no added) concentrations of glycine was not different at 13 days in vitro (DIV). Furthermore, similar results were obtained with experiments on Con G–induced inhibition of NMDA-evoked whole cell currents. These results indicate that glycine concentrations do not have a direct effect on Con G–induced inhibition of NMDA currents. In addition, age dependency in the action of Con G on cortical neurons in vitro suggests that this model system would be useful in examining the effects of different agonists/antagonists on native synaptic NMDA receptors.

Repeated high-dose injections of methamphetamine (METH) rapidly decrease dopamine uptake by the vesicular monoamine transporter-2 (VMAT-2) associated with dopaminergic nerve terminals, as assessed in nonmembrane-associated vesicles purified from striata of treated rats. The purpose of this study was to determine whether METH similarly affects vesicular uptake in the hippocampus; a region innervated by both serotonergic and noradrenergic neurons and profoundly affected by METH treatment. Results revealed that repeated high-dose METH administrations rapidly (within 1 h) reduced hippocampal vesicular dopamine uptake, as assessed in vesicles purified from treated rats. This reduction was likely associated with serotonergic nerve terminals because METH did not further reduce vesicular monoamine uptake in para-chloroamphetamine-lesioned animals. Pretreatment with the serotonin transporter inhibitor fluoxetine blocked both this acute effect on VMAT-2 and the decrease in serotonin content observed 7 days after METH treatment. In contrast, there was no conclusive evidence that METH affected vesicular dopamine uptake in noradrenergic neurons or caused persistent noradrenergic deficits. These findings suggest a link between METH-induced alterations in serotonergic hippocampal vesicular uptake and the persistent hippocampal serotonergic deficits induced by the stimulant.

This study examined the stress protein response (HSP70 family) of reproductively inactive oysters fed 0.7 g algal paste containing 0, 0.35 and 3.5 micrograms polychlorinated biphenyls (PCBs) daily. A second set of treatment groups investigated the combined effect of PCBs and sediments (0.3 g sediments daily per oyster) on HSP70 response. After 8 weeks of PCB exposure, oyster tissues (mantle and gill) were sampled and analyzed for HSP70. Preliminary results did not show a significant effect in HSP70 response in oysters fed PCB sorbed to algal paste, albeit PCBs accumulated up to 1342 ng/g dry weight in the mantle, and up to 180 ng/g dry weight in gill tissues. However, the addition of sediments caused a significant increase in HSP70 levels of gills and mantle, although the mantle was less sensitive to the sediments.