Actin Cytoskeleton In Neurons Research Articles

Neuronal actin cytoskeleton gain of function in the human brain

While advancements in imaging techniques have led to major strides in deciphering the human brain, successful interventions are elusive and represent some of the most persistent translational gaps in medicine. Human restricted CHRFAM7A has been associated with neuropsychiatric disorders. The physiological role of CHRFAM7A in human brain is explored using multiomics approach on 600 post mortem human brain tissue samples. The emerging pathways and mechanistic hypotheses are tested and validated in an isogenic hiPSC model of CHRFAM7A knock-in medial ganglionic eminence progenitors and neurons. CHRFAM7A is identified as a modulator of intracellular calcium dynamics and an upstream regulator of Rac1. Rac1 activation re-designs the actin cytoskeleton leading to dynamic actin driven remodeling of membrane protrusion and a switch from filopodia to lamellipodia. The reinforced cytoskeleton leads to an advantage to tolerate stiffer mechanical properties of the extracellular environment. CHRFAM7A modifies the actin cytoskeleton to a more dynamic and stiffness resistant state in an α7nAChR dependent manner. CHRFAM7A may facilitate neuronal adaptation to changes in the brain environment in physiological and pathological conditions contributing to risk or recovery. Understanding how CHRFAM7A affects human brain requires human studies in the areas of memory formation and erasure, cognitive reserve, and neuronal plasticity. This work is supported in part by the Community Foundation for Greater Buffalo (Kinga Szigeti). Also, in part by the International Society for Neurochemistry (ISN) and The Company of Biologists (Nicolas Rosas). ROSMAP is supported by NIA grants P30AG10161, P30AG72975, R01AG15819, R01AG17917. U01AG46152, and U01AG61356.

Cell-autonomous reduction of CYFIP2 is insufficient to induce Alzheimer's disease-like pathologies in the hippocampal CA1 pyramidal neurons of aged mice

ABSTRACT Cytoplasmic FMR1-interacting protein 2 (CYFIP2) is an evolutionarily conserved multifunctional protein that regulates the neuronal actin cytoskeleton, mRNA translation and transport, and mitochondrial morphology and function. Supporting its critical roles in proper neuronal development and function, human genetic studies have repeatedly identified variants of the CYFIP2 gene in individuals diagnosed with neurodevelopmental disorders. Notably, a few recent studies have also suggested a mechanistic link between reduced CYFIP2 level and Alzheimer's disease (AD). Specifically, in the hippocampus of 12-month-old Cyfip2 heterozygous mice, several AD-like pathologies were identified, including increased levels of Tau phosphorylation and gliosis, and loss of dendritic spines in CA1 pyramidal neurons. However, detailed pathogenic mechanisms, such as cell types and their circuits where the pathologies originate, remain unknown for AD-like pathologies caused by CYFIP2 reduction. In this study, we aimed to address this issue by examining whether the cell-autonomous reduction of CYFIP2 in CA1 excitatory pyramidal neurons is sufficient to induce AD-like phenotypes in the hippocampus. We performed immunohistochemical, morphological, and biochemical analyses in 12-month-old Cyfip2 conditional knock-out mice, which have postnatally reduced CYFIP2 expression level in CA1, but not in CA3, excitatory pyramidal neurons of the hippocampus. Unexpectedly, we could not find any significant AD-like phenotype, suggesting that the CA1 excitatory neuron-specific reduction of CYFIP2 level is insufficient to lead to AD-like pathologies in the hippocampus. Therefore, we propose that CYFIP2 reduction in other neurons and/or their synaptic connections with CA1 pyramidal neurons may be critically involved in the hippocampal AD-like phenotypes of Cyfip2 heterozygous mice.

Cocaine and habit training cause dendritic spine rearrangement in the prelimbic cortex

Successfully navigating dynamic environments requires organisms to learn the consequences of their actions. The prelimbic prefrontal cortex (PL) formulates action-consequence memories and is modulated by addictive drugs like cocaine. We trained mice to obtain food rewards and then unexpectedly withheld reinforcement, triggering new action-consequence memory. New memory was disrupted by cocaine when delivered immediately following non-reinforcement, but not when delayed, suggesting that cocaine disrupted memory consolidation. Cocaine also rapidly inactivated cofilin, a primary regulator of the neuronal actin cytoskeleton. This observation led to the discovery that cocaine also within the time of memory consolidation elevated dendritic spine elimination and blunted spine formation rates on excitatory PL neurons, culminating in thin-type spine attrition. Training drug-naive mice to utilize inflexible response strategies also eliminated thin-type dendritic spines. Thus, cocaine may disrupt action-consequence memory, at least in part, by recapitulating neurobiological sequalae occurring in the formation of inflexible habits.

Myosin XVI Regulates Actin Cytoskeleton Dynamics in Dendritic Spines of Purkinje Cells and Affects Presynaptic Organization.

The actin cytoskeleton is crucial for function and morphology of neuronal synapses. Moreover, altered regulation of the neuronal actin cytoskeleton has been implicated in neuropsychiatric diseases such as autism spectrum disorder (ASD). Myosin XVI is a neuronally expressed unconventional myosin known to bind the WAVE regulatory complex (WRC), a regulator of filamentous actin (F-actin) polymerization. Notably, the gene encoding the myosin’s heavy chain (MYO16) shows genetic association with neuropsychiatric disorders including ASD. Here, we investigated whether myosin XVI plays a role for actin cytoskeleton regulation in the dendritic spines of cerebellar Purkinje cells (PCs), a neuronal cell type crucial for motor learning, social cognition and vocalization. We provide evidence that both myosin XVI and the WRC component WAVE1 localize to PC spines. Fluorescence recovery after photobleaching (FRAP) analysis of GFP-actin in cultured PCs shows that Myo16 knockout as well as PC-specific Myo16 knockdown, lead to faster F-actin turnover in the dendritic spines of PCs. We also detect accelerated F-actin turnover upon interference with the WRC, and upon inhibition of Arp2/3 that drives formation of branched F-actin downstream of the WRC. In contrast, inhibition of formins that are responsible for polymerization of linear actin filaments does not cause faster F-actin turnover. Together, our data establish myosin XVI as a regulator of the postsynaptic actin cytoskeleton and suggest that it is an upstream activator of the WRC-Arp2/3 pathway in PC spines. Furthermore, ultra-structural and electrophysiological analyses of Myo16 knockout cerebellum reveals the presence of reduced numbers of synaptic vesicles at presynaptic terminals in the absence of the myosin. Therefore, we here define myosin XVI as an F-actin regulator important for presynaptic organization in the cerebellum.

Astrocyte-to-neuron communication through integrin-engaged Thy-1/CBP/Csk/Src complex triggers neurite retraction via the RhoA/ROCK pathway

Two key proteins for cellular communication between astrocytes and neurons are αvβ3 integrin and the receptor Thy-1. Binding of these molecules in the same (cis) or on adjacent (trans) cellular membranes induces Thy-1 clustering, triggering actin cytoskeleton remodeling. Molecular events that could explain how the Thy-1-αvβ3 integrin interaction signals have only been studied separately in different cell types, and the detailed transcellular communication and signal transduction pathways involved in neuronal cytoskeleton remodeling remain unresolved. Using biochemical and genetic approaches, single-molecule tracking, and high-resolution nanoscopy, we provide evidence that upon binding to αvβ3 integrin, Thy-1 mobility decreased while Thy-1 nanocluster size increased. This occurred concomitantly with inactivation and exclusion of the non-receptor tyrosine kinase Src from the Thy-1/C-terminal Src kinase (Csk)-binding protein (CBP)/Csk complex. The Src inactivation decreased the p190Rho GTPase activating protein phosphorylation, promoting RhoA activation, cofilin, and myosin light chain II phosphorylation and, consequently, neurite shortening. Finally, silencing the adaptor CBP demonstrated that this protein was a key transducer in the Thy-1 signaling cascade. In conclusion, these data support the hypothesis that the Thy-1-CBP-Csk-Src-RhoA-ROCK axis transmitted signals from astrocytic integrin-engaged Thy-1 (trans) to the neuronal actin cytoskeleton. Importantly, the β3 integrin in neurons (cis) was not found to be crucial for neurite shortening. This is the first study to detail the signaling pathway triggered by αvβ3, the endogenous Thy-1 ligand, highlighting the role of membrane-bound integrins as trans acting ligands in astrocyte-neuron communication.

Actin Tyrosine-53-Phosphorylation in Neuronal Maturation and Synaptic Plasticity.

Rapid reorganization and stabilization of the actin cytoskeleton in dendritic spines enables cellular processes underlying learning, such as long-term potentiation (LTP). Dendritic spines are enriched in exceptionally short and dynamic actin filaments, but the studies so far have not revealed the molecular mechanisms underlying the high actin dynamics in dendritic spines. Here, we show that actin in dendritic spines is dynamically phosphorylated at tyrosine-53 (Y53) in rat hippocampal and cortical neurons. Our findings show that actin phosphorylation increases the turnover rate of actin filaments and promotes the short-term dynamics of dendritic spines. During neuronal maturation, actin phosphorylation peaks at the first weeks of morphogenesis, when dendritic spines form, and the amount of Y53-phosphorylated actin decreases when spines mature and stabilize. Induction of LTP transiently increases the amount of phosphorylated actin and LTP induction is deficient in neurons expressing mutant actin that mimics phosphorylation. Actin phosphorylation provides a molecular mechanism to maintain the high actin dynamics in dendritic spines during neuronal development and to induce fast reorganization of the actin cytoskeleton in synaptic plasticity. In turn, dephosphorylation of actin is required for the stabilization of actin filaments that is necessary for proper dendritic spine maturation and LTP maintenance. Dendritic spines are small protrusions from neuronal dendrites where the postsynaptic components of most excitatory synapses reside. Precise control of dendritic spine morphology and density is critical for normal brain function. Accordingly, aberrant spine morphology is linked to many neurological diseases. The actin cytoskeleton is a structural element underlying the proper morphology of dendritic spines. Therefore, defects in the regulation of the actin cytoskeleton in neurons have been implicated in neurological diseases. Here, we revealed a novel mechanism for regulating neuronal actin cytoskeleton that explains the specific organization and dynamics of actin in spines. The better we understand the regulation of the dendritic spine morphology, the better we understand what goes wrong in neurological diseases.

The neuronal and actin commitment: Why do neurons need rings?

The role of the actin cytoskeleton in neurons has been extensively studied in actin-enriched compartments such as the growth cone and dendritic spines. The recent discovery of actin rings in the axon shaft and in dendrites, together with the identification of axon actin trails, has advanced our understanding on actin organization and dynamics in neurons. However, specifically in the case of actin rings, the mechanisms regulating their nucleation and assembly, and the functions that they may exert in axons and dendrites remain largely unexplored. Here we discuss the possible structural, mechanistic and functional properties of the subcortical neuronal cytoskeleton putting the current knowledge in perspective with the information available on actin rings formed in other biological contexts, and with the organization of actin-spectrin lattices in other cell types. The detailed analysis of these novel neuronal actin ring structures, together with the elucidation of the function of actin-binding proteins in neuron biology, has a large potential to uncover new mechanisms of neuronal function under normal conditions that may have impact in our understanding of axon degeneration and regeneration. © 2016 Wiley Periodicals, Inc.

KCC2 regulates actin dynamics in dendritic spines via interaction with β-PIX.

Chloride extrusion in mature neurons is largely mediated by the neuron-specific potassium-chloride cotransporter KCC2. In addition, independently of its chloride transport function, KCC2 regulates the development and morphology of dendritic spines through structural interactions with the actin cytoskeleton. The mechanism of this effect remains largely unknown. In this paper, we show a novel pathway for KCC2-mediated regulation of the actin cytoskeleton in neurons. We found that KCC2, through interaction with the b isoform of Rac/Cdc42 guanine nucleotide exchange factor β-PIX, regulates the activity of Rac1 GTPase and the phosphorylation of one of the major actin-regulating proteins, cofilin-1. KCC2-deficient neurons had abnormally high levels of phosphorylated cofilin-1. Consistently, dendritic spines of these neurons exhibited a large pool of stable actin, resulting in reduced spine motility and diminished density of functional synapses. In conclusion, we describe a novel signaling pathway that couples KCC2 to the cytoskeleton and regulates the formation of glutamatergic synapses.

Opposite effects of dexamethasone and retinoic acid on neuronal actin cytoskeleton

Opposite effects of dexamethasone and retinoic acid on neuronal actin cytoskeleton

Measuring F-actin properties in dendritic spines.

During the last decade, numerous studies have demonstrated that the actin cytoskeleton plays a pivotal role in the control of dendritic spine shape. Synaptic stimulation rapidly changes the actin dynamics and many actin regulators have been shown to play roles in neuron functionality. Accordingly, defects in the regulation of the actin cytoskeleton in neurons have been implicated in memory disorders. Due to the small size of spines, it is difficult to detect changes in the actin structures in dendritic spines by conventional light microscopy imaging. Instead, to know how tightly actin filaments are bundled together, and how fast the filaments turnover, we need to use advanced microscopy techniques, such as fluorescence recovery after photobleaching (FRAP), photoactivatable green fluorescent protein (PAGFP) fluorescence decay and fluorescence anisotropy. Fluorescence anisotropy, which measures the Förster resonance energy transfer (FRET) between two GFP fluorophores, has been proposed as a method to measure the level of actin polymerization. Here, we propose a novel idea that fluorescence anisotropy could be more suitable to study the level of actin filament bundling instead of actin polymerization. We validate the method in U2OS cell line where the actin structures can be clearly distinguished and apply to analyze how actin filament organization in dendritic spines changes during neuronal maturation. In addition to fluorescence anisotropy validation, we take a critical look at the properties and limitations of FRAP and PAGFP fluorescence decay methods and offer our proposals for the analysis methods for these approaches. These three methods complement each other, each providing additional information about actin dynamics and organization in dendritic spines.

Forgetting Is Regulated via Musashi-Mediated Translational Control of the Arp2/3 Complex

A plastic nervous system requires the ability not only to acquire and store but also to forget. Here, we report that musashi (msi-1) is necessary for time-dependent memory loss in C. elegans. Tissue-specific rescue demonstrates that MSI-1 function is necessary in the AVA interneuron. Using RNA-binding protein immunoprecipitation (IP), we found that MSI-1 binds to mRNAs of three subunits of the Arp2/3 actin branching regulator complex in vivo and downregulates ARX-1, ARX-2, and ARX-3 translation upon associative learning. The role of msi-1 in forgetting is also reflected by the persistence of learning-induced GLR-1 synaptic size increase in msi-1 mutants. We demonstrate that memory length is regulated cooperatively through the activation of adducin (add-1) and by the inhibitory effect of msi-1. Thus, a GLR-1/MSI-1/Arp2/3 pathway induces forgetting and represents a novel mechanism of memory decay by linking translational control to the structure of the actin cytoskeleton in neurons.

Arg kinase signaling in dendrite and synapse stabilization pathways: Memory, cocaine sensitivity, and stress

The Abl2/Arg nonreceptor tyrosine kinase is enriched in dendritic spines where it is essential for maintaining dendrite and synapse stability in the postnatal mouse brain. Arg is activated downstream of integrin α3β1 receptors and it regulates the neuronal actin cytoskeleton by directly binding F-actin and via phosphorylation of substrates including p190RhoGAP and cortactin. Neurons in mice lacking Arg or integrin α3β1 develop normally through postnatal day 21 (P21), however by P42 mice exhibit major reductions in dendrite arbor size and complexity, and lose dendritic spines and synapses. As a result, mice with loss of Arg and Arg-dependent signaling pathways have impairments in memory tasks, heightened sensitivity to cocaine, and vulnerability to corticosteroid-induced neuronal remodeling. Therefore, understanding the molecular mechanisms of Arg regulation may lead to therapeutic approaches to treat human psychiatric and neurodegenerative diseases in which neuronal structure is destabilized.

Rho-Kinase Inhibitor, Fasudil, Prevents Neuronal Apoptosis via the Akt Activation and PTEN Inactivation in the Ischemic Penumbra of Rat Brain

Recently, some studies suggested that inhibition of Rho-kinase (ROCK) prevented cerebral ischemia injury through inhibiting inflammatory reaction, increasing cerebral blood flow, modulating the neuronal actin cytoskeleton polymerization, and preventing tau hyperphosphorylation and p25/CDK5 increase. However, there is little information regarding the effects of ROCK inhibitor on the neuronal apoptosis in ischemic brain injury. In this study, we determined whether ROCK inhibitor, fasudil, inhibited ischemic neuronal apoptosis through phosphatase and tensin homolog deleted on chromosome10 (PTEN)/Akt/signal pathway in vivo. Adult male Sprague-Dawley rats were subjected to permanent middle cerebral artery occlusion. Rats received ROCK inhibitor, fasudil (10 mg/kg), at 30 min before middle cerebral artery occlusion. The infarct area, neuronal apoptosis and caspase-3 activity was significantly decreased by fasudil with improvement of neurological deterioration. However, the beneficial effects of fasudil were attenuated by the co-application of LY294002 (PI3K inhibitor). Fasudil maintained postischemic Akt activity at relatively proper level and decreased the augmentation of PTEN and ROCK activity in the penumbra area. Furthermore, fasudil inhibited attenuation of GSK-β and Bad phosphorylation in the penumbra area. In conclusion, the findings provide another consideration that fasudil protects the brain against ischemia injury through decreasing neuronal apoptosis and reveals the link between the ROCK inhibition and the PTEN/Akt pathway.

EphA Signaling Promotes Actin-based Dendritic Spine Remodeling through Slingshot Phosphatase

Actin cytoskeletal remodeling plays a critical role in transforming the morphology of subcellular structures across various cell types. In the brain, restructuring of dendritic spines through actin cytoskeleletal reorganization is implicated in the regulation of synaptic efficacy and the storage of information in neural circuits. However, the upstream pathways that provoke actin-based spine changes remain only partly understood. Here we show that EphA receptor signaling remodels spines by triggering a sequence of events involving actin filament rearrangement and synapse/spine reorganization. Rapid EphA signaling over minutes activates the actin filament depolymerizing/severing factor cofilin, alters F-actin distribution in spines, and causes transient spine elongation through the phosphatases slingshot 1 (SSH1) and calcineurin/protein phosphatase 2B (PP2B). This early phase of spine extension is followed by synaptic reorganization events that take place over minutes to hours and involve the relocation of pre/postsynaptic components and ultimately spine retraction. Thus, EphA receptors utilize discrete cellular and molecular pathways to promote actin-based structural plasticity of excitatory synapses.

Myosin IIb Regulates Actin Dynamics during Synaptic Plasticity and Memory Formation

Reorganization of the actin cytoskeleton is essential for synaptic plasticity and memory formation. Presently, the mechanisms that trigger actin dynamics during these brain processes are poorly understood. In this study, we show that myosin II motor activity is downstream of LTP induction and is necessary for the emergence of specialized actin structures that stabilize an early phase of LTP. We also demonstrate that myosin II activity contributes importantly to an actin-dependent process that underlies memory consolidation. Pharmacological treatments that promote actin polymerization reversed the effects of a myosin II inhibitor on LTP and memory. We conclude that myosin II motors regulate plasticity by imparting mechanical forces onto the spine actin cytoskeleton in response to synaptic stimulation. These cytoskeletal forces trigger the emergence of actin structures that stabilize synaptic plasticity. Our studies provide a mechanical framework for understanding cytoskeletal dynamics associated with synaptic plasticity and memory formation.

Rho kinase inhibition protects CA1 cells in organotypic hippocampal slices during in vitro ischemia

The actin cytoskeleton is a dynamic superstructure that regulates multiple cellular functions and that has been implicated in cell death regulation. We investigated whether modulating the neuronal actin cytoskeleton polymerization by Rho-GTPase kinase (ROCK) inhibition influences cell death in hippocampal neuronal cultures and in murine organotypic hippocampal slice cultures subjected to in vitro ischemia (IVI). During IVI, spines on vehicle treated hippocampal neurons collapsed and large dendritic actin aggregates were formed. Following ROCK inhibition by Y27632, the actin aggregates were markedly smaller while large filopodia extended from the dendritic trunk. Y27632 also provided strong neuroprotection of hippocampal pyramidal CA1 neurons, which was of similar magnitude as protection by NMDA receptor blockade. Likewise, treatment with the F-actin depolymerizing agent latrunculin during IVI diminished actin aggregation and mitigated cell death following IVI. We propose that ROCK inhibition protects neurons against ischemic damage by disrupting actin polymerization thereby mitigating NMDA receptor induced toxicity and releasing ATP bound to actin for cellular energy use. We conclude that ROCK inhibitors abrogate multiple detrimental processes and could therefore be useful in stroke therapy.

Proinflammatory cytokines provoke oxidative damage to actin in neuronal cells mediated by Rac1 and NADPH oxidase

The proinflammatory cytokines TNFα and Il-1β orchestrate the progression of CNS inflammation, which substantially contributes to neurodegeneration in many CNS pathologies. TNFα and Il-1β stimulate actin filament reorganization in non-neuronal cells often accompanied by the formation of reactive oxygen species (ROS). Actin filament dynamics is vital for cellular plasticity, mitochondrial function, and gene expression despite being highly susceptible to oxidative damage. We demonstrated that, in neuronal cells, TNFα and Il-1β stimulate a transient, redox-dependent reorganization of the actin cytoskeleton into lamellipodia under the regulation of Rac1 and a neuronal NADPH oxidase as the source of ROS. The persistent presence of intracellular ROS provoked oxidative damage (carbonylation) to actin coinciding with the loss of lamellipodia and arrest of cellular plasticity. Inhibition of NADPH oxidase activity or Rac1 abolished the adverse effects of cytokines. These findings suggest that oxidative damage to the neuronal actin cytoskeleton could represent a key step in CNS neurodegeneration.

Evaluation of the effects of propylisopropylacetic acid (PIA) on neuronal growth cone morphology

Propylisopropylacetic acid (PIA) is a constitutional isomer of valproic acid (VPA). It has previously been found to be a weak antiepileptic, but in common with mood stabilizers, causes inositol depletion and growth cone spreading, suggesting the basis of a new series of mood stabilizers. To assess this possibility, we have compared the effects of racemic (R,S)-PIA and its individual enantiomers to those of the mood stabilizers lithium (Li+), VPA and carbamazepine (CBZ). Unlike Li+ and VPA, but in common with CBZ and (R,S)-PIA, neither (R)-PIA nor (S)-PIA enantiomer induces T-cell factor (TCF)-mediated gene expression. However, as seen for other mood stabilizers, both enantiomers are potent inducers of growth cone spreading. To investigate the mechanism for these effects, we examined changes in the actin cytoskeleton following drug treatment with Li+, VPA, CBZ, (R,S)-PIA or its individual enantiomers. All exhibit a re-distribution of F-actin to the growth cone periphery, a feature of spread growth cones. (R,S)-PIA has the strongest effect as it also elevates F-actin polymerization at the cell periphery. This change in the actin cytoskeleton is associated with a substantial increase in F-actin-rich protrusions on the surface of the growth cone and in its close vicinity. These results demonstrate an effect of (R,S)-PIA on the neuronal actin cytoskeleton shared in common with other mood stabilizers, and suggest a potential to induce structural changes within the CNS.

Autonomous functions for the Sec14p/spectrin-repeat region of Kalirin

Kalirin is a GDP/GTP exchange factor (GEF) for Rho proteins that modulates the actin cytoskeleton in neurons. Alternative splicing generates Δ-isoforms, which encode the RhoGEF domain, but lack the N-terminal Sec14p domain and first 4 spectrin-like repeats of the full-length isoforms. Splicing has functional consequences, with Kal7 but not ΔKal7 causing formation of dendritic spines. Cells lacking endogenous Kalirin were used to explore differences between these splice variants. Expression of ΔKal7 in this system induces extensive lamellipodial sheets, while expression of Kal7 induces formation of adherent compact, round cells with abundant cortical actin. Based on in vitro and cell-based assays, Kal7 and ΔKal7 are equally active GEFs, suggesting that other domains are involved in controlling cell morphology. Catalytically inactive Kal7 and a Kalirin fragment which includes only Sec14p and spectrin-like domains retain the ability to produce compact, round cells and fractionate as high molecular weight complexes. Separating the Sec14p domain from the spectrin-like repeats eliminates the ability of Kal7 to cause this response. The isolated Sec14p domain binds PI(3,5)P2 and PI3P, but does not alter cell morphology. We conclude that the Sec14p and N-terminal spectrin-like domains of Kalirin play critical roles in distinguishing the actions of full-length and Δ-Kalirin proteins.

Increased Ethanol Resistance and Consumption in Eps8 Knockout Mice Correlates with Altered Actin Dynamics

Dynamic modulation of the actin cytoskeleton is critical for synaptic plasticity, abnormalities of which are thought to contribute to mental illness and addiction. Here we report that mice lacking Eps8, a regulator of actin dynamics, are resistant to some acute intoxicating effects of ethanol and show increased ethanol consumption. In the brain, the N-methyl-D-aspartate (NMDA) receptor is a major target of ethanol. We show that Eps8 is localized to postsynaptic structures and is part of the NMDA receptor complex. Moreover, in Eps8 null mice, NMDA receptor currents and their sensitivity to inhibition by ethanol are abnormal. In addition, Eps8 null neurons are resistant to the actin-remodeling activities of NMDA and ethanol. We propose that proper regulation of the actin cytoskeleton is a key determinant of cellular and behavioral responses to ethanol.