Microdevice for directional axodendritic connectivity between micro 3D neuronal cultures.
ABSTRACT: Neuronal cultures are widely used in neuroscience research. However, the randomness of circuits in conventional cultures prevents accurate in vitro modeling of cortical development and of the pathogenesis of neurological and psychiatric disorders. A basic feature of cortical circuits that is not captured in standard cultures of dissociated cortical cells is directional connectivity. In this work, a polydimethylsiloxane (PDMS)-based device that achieves directional connectivity between micro 3D cultures is demonstrated. The device consists of through-holes for micro three-dimensional (μ3D) clusters of cortical cells connected by microtrenches for axon and dendrite guidance. The design of the trenches relies in part on the concept of axonal edge guidance, as well as on the novel concept of specific dendrite targeting. This replicates dominant excitatory connectivity in the cortex, enables the guidance of the axon after it forms a synapse in passing (an "en passant" synapse), and ensures that directional selectivity is preserved over the lifetime of the culture. The directionality of connections was verified morphologically and functionally. Connections were dependent on glutamatergic synapses. The design of this device has the potential to serve as a building block for the reconstruction of more complex cortical circuits in vitro.
Project description:Engineering of neuronal network geometry by micropatterning technology is a key future technology for creating artificial brains on a chip. However, engineering of network geometry at the single-cell-level with functional morphology (axon/dendrite) and connectivity (synapses) is still challenging. Here, we describe a method for controlling the axon and dendrite morphology of single primary-cultured neurons and assembling a neural circuit using mobile microplates. The microplates enabled morphological control of neurons by their shapes and bringing their ends into contact caused the formation of physical connections. Functional synapse formation at the connection was indicated by immunostaining of synapse-related proteins and intracellular Ca<sup>2+</sup> imaging of neural activity. We believe that the method will be useful in engineering neural circuits with selected neurons and defined morphology.
Project description:Functional axon regeneration requires regenerating neurons to restore appropriate synaptic connectivity and circuit function. To model this process, we developed an assay in <i>Caenorhabditis elegans</i> that links axon and synapse regeneration of a single neuron to recovery of behavior. After axon injury and regeneration of the DA9 neuron, synapses reform at their pre-injury location. However, these regenerated synapses often lack key molecular components. Further, synaptic vesicles accumulate in the dendrite in response to axon injury. Dendritic vesicle release results in information misrouting that suppresses behavioral recovery. Dendritic synapse formation depends on dynein and <i>jnk-1</i>. But even when information transfer is corrected, axonal synapses fail to adequately transmit information. Our study reveals unexpected plasticity during functional regeneration. Regeneration of the axon is not sufficient for the reformation of correct neuronal circuits after injury. Rather, synapse reformation and function are also key variables, and manipulation of circuit reformation improves behavioral recovery.
Project description:Neural circuit assembly occurs with subcellular precision, yet the mechanisms underlying this precision remain largely unknown. Subcellular synaptic specificity could be achieved by molecularly distinct subcellular domains that locally regulate synapse formation, or by axon guidance cues restricting access to one of several acceptable targets. We address these models using two <i>Drosophila</i> neurons: the dbd sensory neuron and the A08a interneuron. In wild-type larvae, dbd synapses with the A08a medial dendrite but not the A08a lateral dendrite. dbd-specific overexpression of the guidance receptors Unc-5 or Robo-2 results in lateralization of the dbd axon, which forms anatomical and functional monosynaptic connections with the A08a lateral dendrite. We conclude that axon guidance cues, not molecularly distinct dendritic arbors, are a major determinant of dbd-A08a subcellular synapse specificity.
Project description:Down syndrome cell adhesion molecule, or DSCAM, has been implicated in many neurodevelopmental processes including axon guidance, dendrite arborization, and synapse formation. Here we show that DSCAM plays an important role in regulating the morphogenesis of cortical pyramidal neurons in the mouse. We report that DSCAM expression is developmentally regulated and localizes to synaptic plasma membranes during a time of robust cortical dendrite arborization and spine formation. Analysis of mice that carry a spontaneous mutation in DSCAM (DSCAM(del17)) revealed gross morphological changes in brain size and shape in addition to subtle changes in cortical organization, volume, and lamination. Early postnatal mutant mice displayed a transient decrease in cortical thickness, but these reductions could not be attributed to changes in neuron production or cell death. DSCAM(del17) mutants showed temporary impairments in the branching of layer V pyramidal neuron dendrites at P10 and P17 that recovered to normal by adulthood. Defects in DSCAM(del17) dendrite branching correlated with a temporal increase in apical branch spine density and lasting changes in spine morphology. At P15 and P42, mutant mice displayed a decrease in the percentage of large, stable spines and an increase in the percentage of small, immature spines. Together, our findings suggest that DSCAM contributes to pyramidal neuron morphogenesis by regulating dendrite arborization and spine formation during cortical circuit development.
Project description:A deep understanding of the mechanisms behind neurite polarization and axon path-finding is important for interpreting how the human body guides neurite growth during development and response to injury. Further, it is of great clinical importance to identify diffusible chemical cues that promote neurite regeneration for nervous tissue repair. Despite the fast development of various types of concentration gradient generators, it has been challenging to fabricate neuron-friendly (i.e. shear-free and biocompatible for neuron growth and maturation) devices to create stable gradients, particularly for fast diffusing small molecules, which typically require high flow and shear rates. Here we present a finite element analysis for a polydimethylsiloxane/polyethylene glycol diacrylate (PDMS/PEG-DA) based gradient generator, describe the microfabrication process, and validate its use for neuronal axon polarization studies. This device provides a totally shear-free, biocompatible microenvironment with a linear and stable concentration gradient of small molecules such as forskolin. The gradient profile in this device can be customized by changing the composition or width of the PEG-DA barriers during direct UV photo-patterning within a permanently bonded PDMS device. Primary rat cortical neurons (embryonic E18) exposed to soluble forskolin gradients for 72 h exhibited statistically significant polarization and guidance of their axons. This device provides a useful platform for both chemotaxis and directional guidance studies, particularly for shear sensitive and non-adhesive cell cultures, while allowing fast new device design prototyping at a low cost.
Project description:Nystatin is a pharmacological agent commonly used for the treatment of oral, mucosal and cutaneous fungal infections. Nystatin has also been extensively applied to study the cellular function of cholesterol-enriched structures because of its ability to bind and extract cholesterol from mammalian membranes. In neurons, cholesterol level is tightly regulated, being essential for synapse and dendrite formation, and axonal guidance. However, the action of Nystatin on axon regeneration has been poorly evaluated. Here, we examine the effect of Nystatin on primary cultures of hippocampal neurons, showing how acute dose (minutes) of Nystatin increases the area of growth cones, and chronic treatment (days) enhances axon length, axon branching, and axon regeneration post-axotomy. We describe two alternative signaling pathways responsible for the observed effects and activated at different concentrations of Nystatin. At elevated concentrations, Nystatin promotes growth cone expansion through phosphorylation of Akt; whereas, at low concentrations, Nystatin enhances axon length and regrowth by increasing nitric oxide levels. Together, our findings indicate new signaling pathways of Nystatin and propose this compound as a novel regulator of axon regeneration.
Project description:Synapse elimination and pruning of axon collaterals are crucial developmental events in the refinement of neuronal circuits. While a control of synapse formation by adhesion molecules is well established, the involvement of adhesion molecules in developmental synapse loss is poorly characterized. To investigate the consequences of mis-match expression of a homophilic synaptic adhesion molecule, we analysed an asymmetric, exclusively postsynaptic expression of N-cadherin. This was induced by transfecting individual neurons in cultures of N-cadherin knockout mouse neurons with a N-cadherin expression vector. 2 days after transfection, patch-clamp analysis of AMPA receptor-mediated miniature postsynaptic currents revealed an impaired synaptic function without a reduction in the number of presynaptic vesicle clusters. Long-term asymmetric expression of N-cadherin for 8 days subsequently led to synapse elimination as indicated by a loss of colocalization of presynaptic vesicles and postsynaptic PSD95 protein. We further studied long-term asymmetric N-cadherin expression by conditional, Cre-induced knockout of N-cadherin in individual neurons in cultures of N-cadherin expressing cortical mouse neurons. This resulted in a strong retraction of axonal processes in individual neurons that lacked N-cadherin protein. Moreover, an in vivo asymmetric expression of N-cadherin in the developmentally transient cortico-tectal projection was indicated by in-situ hybridization with layer V neurons lacking N-cadherin expression. Thus, mis-match expression of N-cadherin might contribute to selective synaptic connectivity.
Project description:In stereotyped neuronal networks, synaptic connectivity is dictated by cell surface proteins, which assign unique identities to neurons, and physically mediate axon guidance and synapse targeting. We recently identified two groups of immunoglobulin superfamily proteins in Drosophila, Dprs and DIPs, as strong candidates for synapse targeting functions. Here, we uncover the molecular basis of specificity in Dpr-DIP mediated cellular adhesions and neuronal connectivity. First, we present five crystal structures of Dpr-DIP and DIP-DIP complexes, highlighting the evolutionary and structural origins of diversification in Dpr and DIP proteins and their interactions. We further show that structures can be used to rationally engineer receptors with novel specificities or modified affinities, which can be used to study specific circuits that require Dpr-DIP interactions to help establish connectivity. We investigate one pair, engineered Dpr10 and DIP-?, for function in the neuromuscular circuit in flies, and reveal roles for homophilic and heterophilic binding in wiring.
Project description:The precise control of synaptic connectivity is essential for the development and function of neuronal circuits. While there have been significant advances in our understanding how cell adhesion molecules mediate axon guidance and synapse formation, the mechanisms controlling synapse maintenance or plasticity in vivo remain largely uncharacterized. In an unbiased RNAi screen we identified the Drosophila L1-type CAM Neuroglian (Nrg) as a central coordinator of synapse growth, function, and stability. We demonstrate that the extracellular Ig-domains and the intracellular Ankyrin-interaction motif are essential for synapse development and stability. Nrg binds to Ankyrin2 in vivo and mutations reducing the binding affinities to Ankyrin2 cause an increase in Nrg mobility in motoneurons. We then demonstrate that the Nrg-Ank2 interaction controls the balance of synapse growth and stability at the neuromuscular junction. In contrast, at a central synapse, transsynaptic interactions of pre- and postsynaptic Nrg require a dynamic, temporal and spatial, regulation of the intracellular Ankyrin-binding motif to coordinate pre- and postsynaptic development. Our study at two complementary model synapses identifies the regulation of the interaction between the L1-type CAM and Ankyrin as an important novel module enabling local control of synaptic connectivity and function while maintaining general neuronal circuit architecture.
Project description:Development of both dendrites and axons is important for the formation of neuronal circuits, because dendrites receive information and the axon is responsible for sending signals. In the past decade, extensive studies have revealed many molecules underlying axonal outgrowth and pathfinding. In contrast, much less is known about the molecular mechanisms that control dendrite development. Here we report the identification of an evolutionarily conserved Ig superfamily member, dendrite arborization and synapse maturation 1 (Dasm1), which plays a critical role in dendrite development. Dasm1 contains five Ig domains and two fibronectin III domains in the extracellular N terminus, a single transmembrane domain, and an intracellular C-terminal tail with a type I PDZ domain binding motif at the end. It is highly expressed in the brain and localized at the dendrites. Suppression of Dasm1 expression in hippocampal neurons via RNA interference or expression of Dasm1 without its cytoplasmic tail specifically impairs dendrite, but not axon, outgrowth. Together with its orthologues in other species, Dasm1 defines a family of molecules likely involved specifically in dendrite arborization.