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 longterm 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.extended amygdala | synaptic plasticity | excitatory transmission T he bed nucleus of the stria terminalis (BNST) is an area of the brain that underlies the negative reinforcing properties associated with drug/alcohol dependence, and has been shown in numerous studies to be critical for expression of stress-induced reinstatement of drug-seeking behavior (1-7). Although the molecular and physiological regulation of drug-related behaviors is not completely understood, numerous studies suggest a key role for synaptic plasticity in the long-term actions of alcohol and drugs of abuse. Thus, understanding how plasticity is regulated by ethanol and drugs of abuse in the BNST will be vital to the therapeutic development of treatments for alcohol and drug use disorders.A primary action of ethanol is inhibition of NMDA receptor (NMDAR) function (8-10). The NMDAR is a heterotetrameric complex composed of two obligatory GluN1 (formerly NR1) subunits and two GluN2 (formerly NR2) and/or GluN3 subunits (11). Numerous subunit combinations are possible, with eight different splice variants of GluN1 subunit and four distinct GluN2 subunit isoforms (A, B, C, and D). The predominant GluN2 subunits in adult forebrain are GluN2A and GluN2B, which dictate many channel properties, such as decay time, localization, intracellular signaling, and conductance (11,12).Exact mechanisms by which ethanol inhibits NMDAR function are not well-defined but fall into two categories: one involving direct interactions of ethanol with the NMDAR and/or the NMDAR-membrane interface, and another involving ethanoldriven posttranslational modifications of the NMDAR (13-17). It remains unclear whether there is subunit ...

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. In order 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 EPSCs. Both electrophysiological and biochemical approaches suggest that this difference is not due to 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 following 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.

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 (Ͼ170 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 ␣-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 ␤-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 formation in vivo and 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.

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.

Ca2+/calmodulin-dependent protein kinase IIα (CaMKIIα) autophosphorylation at Thr286 and Thr305/Thr306 regulates kinase activity, modulates subcellular targeting, and is critical for normal synaptic plasticity and learning and memory. Here, a mass spectrometry-based approach was used to identify Ca2+-dependent and -independent in vitro autophosphorylation sites in recombinant CaMKIIα and CaMKIIβ. CaMKII holoenzymes were then immunoprecipitated from subcellular fractions of forebrains isolated from either wildtype (WT) mice or mice with a Thr286 to Ala knock-in mutation of CaMKIIα (T286A-KI mice) and analyzed using the same approach in order to characterize in vivo phosphorylation sites in both CaMKII isoforms and identify CaMKII associated proteins (CaMKAPs). A total of 6 and 7 autophosphorylation sites in CaMKIIα and CaMKIIβ, respectively, were detected in WT mice. Thr286-phosphorylated CaMKIIα and Thr287-phosphorylated CaMKIIβ were selectively enriched in WT Triton-insoluble (synaptic) fractions compared to Triton-soluble (membrane) and cytosolic fractions. In contrast, Thr306-phosphorylated CaMKIIα and Ser315- and Thr320/Thr321-phosphorylated CaMKIIβ were selectively enriched in WT cytosolic fractions. The T286A-KI mutation significantly reduced levels of phosphorylation of CaMKIIα at Ser275 across all subcellular fractions, and of cytosolic CaMKIIβ at Ser315 and Thr320/Thr321. Significantly more CaMKAPs co-precipitated with WT CaMKII holoenzymes in the synaptic fraction compared to the membrane fraction, with functions including scaffolding, microtubule organization, actin organization, ribosomal function, vesicle trafficking, and others. The T286A-KI mutation altered the interactions of multiple CaMKAPs with CaMKII, including several proteins linked to autism spectrum disorders. These data identify CaMKII isoform phosphorylation sites and a network of synaptic protein interactions that are sensitive to the abrogation of Thr286 autophosphorylation of CaMKIIα, likely contributing to the diverse synaptic and behavioral deficits of T286A-KI mice.