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During early ischemic brain injury, glutamate receptor hyperactivation mediates neuronal death via osmotic cell swelling. Here we show that ischemia and excess NMDA receptor activation cause actin to rapidly and extensively reorganize within the somatodendritic compartment. Normally, F-actin is concentrated within dendritic spines. However,

Neuron navigator 1 (Nav1) is a cytoskeleton-associated protein expressed during brain development that is necessary for proper neuritogenesis, but the underlying mechanisms are poorly understood. Here we show that Nav1 is present in elongating axon tracts during mouse brain embryogenesis. We found that depletion of Nav1 in cultured neurons disrupts growth cone morphology and neurotrophin-stimulated neuritogenesis. In addition to regulating both F-actin and microtubule properties, Nav1 promotes actin-rich membrane ruffles in the growth cone and promotes macropinocytosis at those membrane ruffles, including internalization of the TrkB receptor for the neurotrophin brain-derived neurotropic factor (BDNF). Growth cone macropinocytosis is important for downstream signaling, neurite targeting, and membrane recycling, implicating Nav1 in one or more of these processes. Depletion of Nav1 also induces transient membrane blebbing via disruption of signaling in the Rho GTPase signaling pathway, supporting the novel role of Nav1 in dynamic actin-based membrane regulation at the cell periphery. These data demonstrate that Nav1 works at the interface of microtubules, actin, and plasma membrane to organize the cell periphery and promote uptake of growth and guidance cues to facilitate neural morphogenesis during development.

Dendritic spines are small, bulbous protrusions along the dendrites of neurons and are sites of excitatory postsynaptic activity. The morphology of spines has been implicated in their function in synaptic plasticity and their shapes have been well-characterized, but the potential mechanics underlying their shape development and maintenance have not yet been fully understood. In this work, we explore the mechanical principles that could underlie specific shapes using a minimal biophysical model of membrane-actin interactions. Using this model, we first identify the possible force regimes that give rise to the classic spine shapes-stubby, filopodia, thin, and mushroom-shaped spines. We also use this model to investigate how the spine neck might be stabilized using periodic rings of actin or associated proteins. Finally, we use this model to predict that the cooperation between force generation and ring structures can regulate the energy landscape of spine shapes across a wide range of tensions. Thus, our study provides insights into how mechanical aspects of actin-mediated force generation and tension can play critical roles in spine shape maintenance.

Dendritic spines are the primary excitatory postsynaptic sites that act as subcompartments of signaling. Ca2+ is often the first and most rapid signal in spines. Downstream of calcium, the cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA) pathway plays a critical role in the regulation of spine formation, morphological modifications, and ultimately, learning and memory. Although the dynamics of calcium are reasonably well-studied, calcium-induced cAMP/PKA dynamics, particularly with respect to frequency modulation, are not fully explored. In this study, we present a well-mixed model for the dynamics of calcium-induced cAMP/PKA dynamics in dendritic spines. The model is constrained using experimental observations in the literature. Further, we measured the calcium oscillation frequency in dendritic spines of cultured hippocampal CA1 neurons and used these dynamics as model inputs. Our model predicts that the various steps in this pathway act as frequency modulators for calcium, and the high frequency of calcium input is filtered by adenylyl cyclase 1 and phosphodiesterases in this pathway such that cAMP/PKA only responds to lower frequencies. This prediction has important implications for noise filtering and long-timescale signal transduction in dendritic spines. A companion manuscript presents a three-dimensional spatial model for the same pathway.

Neurons express three closely related dynamin genes. Dynamin 1 has long been implicated in the regulation of synaptic vesicle recycling in nerve terminals, and dynamins 2 and 3 were more recently shown also to contribute to synaptic vesicle recycling in specific and distinguishable ways. In cultured hippocampal neurons we found that chronic suppression of spontaneous network activity differentially regulated the targeting of endogenous dynamins 1 and 3 to nerve terminals, while dynamin 2 was unaffected. Specifically, when neural activity was chronically silenced for 1-2weeks by tetrodotoxin (TTX), the clustering of dynamin 1 at nerve terminals was reduced, while the clustering of dynamin 3 significantly increased. Moreover, dynamin 3 clustering was induced within hours by the sustained blockade of AMPA receptors, suggesting that AMPA receptors may function to prevent Dyn3 accumulation within nerve terminals. Clustering of dynamin 3 was induced by an antagonist of the calcium-dependent protein phosphatase calcineurin, but was not dependent upon intact actin filaments. TTX-induced clustering of Dyn3 occurred with a markedly slower time-course than the previously described clustering of synapsin 1. Potassium-induced depolarization rapidly de-clustered dynamin 3 from nerve terminals within minutes. These results, which have implications for homeostatic synapse restructuring, indicate that the three dynamins have evolved different regulatory mechanisms for trafficking to and from nerve terminals in response to changes in neural activity.

Abstract Background To date, some of the most useful and physiologically relevant neuronal cell culture systems, such as high density co-cultures of astrocytes and primary hippocampal neurons, or differentiated stem cell-derived cultures, are characterized by high cell density and partially overlapping cellular structures. Efficient analytical strategies are required to enable rapid, reliable, quantitative analysis of neuronal morphology in these valuable model systems. Results Here we present the development and validation of a novel bioinformatics pipeline called NeuriteQuant. This tool enables fully automated morphological analysis of large-scale image data from neuronal cultures or brain sections that display a high degree of complexity and overlap of neuronal outgrowths. It also provides an efficient web-based tool to review and evaluate the analysis process. In addition to its built-in functionality, NeuriteQuant can be readily extended based on the rich toolset offered by ImageJ and its associated community of developers. As proof of concept we performed automated screens for modulators of neuronal development in cultures of primary neurons and neuronally differentiated P19 stem cells, which demonstrated specific dose-dependent effects on neuronal morphology. Conclusions NeuriteQuant is a freely available open-source tool for the automated analysis and effective review of large-scale high-content screens. It is especially well suited to quantify the effect of experimental manipulations on physiologically relevant neuronal cultures or brain sections that display a high degree of complexity and overlap among neurites or other cellular structures.

Abstract Background To date, some of the most useful and physiologically relevant neuronal cell culture systems, such as high density co-cultures of astrocytes and primary hippocampal neurons, or differentiated stem cell-derived cultures, are characterized by high cell density and partially overlapping cellular structures. Efficient analytical strategies are required to enable rapid, reliable, quantitative analysis of neuronal morphology in these valuable model systems. Results Here we present the development and validation of a novel bioinformatics pipeline called NeuriteQuant. This tool enables fully automated morphological analysis of large-scale image data from neuronal cultures or brain sections that display a high degree of complexity and overlap of neuronal outgrowths. It also provides an efficient web-based tool to review and evaluate the analysis process. In addition to its built-in functionality, NeuriteQuant can be readily extended based on the rich toolset offered by ImageJ and its associated community of developers. As proof of concept we performed automated screens for modulators of neuronal development in cultures of primary neurons and neuronally differentiated P19 stem cells, which demonstrated specific dose-dependent effects on neuronal morphology. Conclusions NeuriteQuant is a freely available open-source tool for the automated analysis and effective review of large-scale high-content screens. It is especially well suited to quantify the effect of experimental manipulations on physiologically relevant neuronal cultures or brain sections that display a high degree of complexity and overlap among neurites or other cellular structures.

The hypothesis that dynamic actin filaments participate in specific aspects of synaptic plasticity was investigated at the Schaffer-collateral-CA1 pyramidal cell synapse of mouse hippocampus. Low concentrations (0.01-1 [micro]M) of compounds that inhibit actin filament assembly were bath applied to hippocampal slices during extracellular recording of field excitatory postsynaptic potentials. Cytochalasin D, cytochalasin B, and latrunculin A all impaired the maintenance of LTP induced by brief high-frequency stimulation. This effect on LTP maintenance was specific, because none of the compounds affected basal synaptic transmission, paired-pulse facilitation, LTP induction, or post-tetanic potentiation. The effect of cytochalasin B was reversible. The results are consistent with a model in which dynamic actin filaments play an essential role in the molecular mechanisms underlying the early maintenance phase of LTP, such as growth of new synaptic connections or conversion of silent synapses. paired-pulse facilitation | cytochalasin | latrunculin | synapse

Myristoylated, alanine-rich C-kinase substrate (MARCKS) is an F-actin and phospholipid binding protein implicated in numerous cellular activities, including the regulation of morphology in neuronal dendrites and dendritic spines. MARCKS contains a lysine-rich effector domain that mediates its binding to plasma membrane phosphatidylinositol-4,5-biphosphate (PI(4,5)P 2 ) in a manner controlled by PKC and calcium/calmodulin. In neurons, manipulations of MARCKS concentration and membrane targeting strongly affect the numbers, shapes, and F-actin properties of dendritic spines, but the mechanisms remain unclear. Here, we tested the hypothesis that the effects of MARCKS on dendritic spine morphology are due to its capacity to regulate the availability of plasma membrane PI(4,5)P 2 . We observed that the concentration of free PI(4,5)P 2 on the dendritic plasma membrane was inversely proportional to the concentration of MARCKS. Endogenous PI(4,5)P 2 levels were increased or decreased, respectively, by acutely overexpressing either phosphatidylinositol-4-phosphate 5-kinase (PIP5K) or inositol polyphosphate 5-phosphatase (5ptase). PIP5K, like MARCKS depletion, induced severe spine shrinkage; 5ptase, like constitutively membrane-bound MARCKS, induced aberrant spine elongation. These phenotypes involved changes in actin properties driven by the F-actin severing protein cofilin. Collectively, these findings support a model in which neuronal activity regulates actin-dependent spine morphology through antagonistic interactions of MARCKS and PI(4,5)P 2 .