Unique Axon-to-Soma Signaling Pathways Mediate Dendritic Spine Loss and Hyper-Excitability Post-axotomy.

Axon damage may cause axon regeneration, retrograde synapse loss, and hyper-excitability, all of which affect recovery following acquired brain injury. While axon regeneration is studied extensively, less is known about signaling mediating retrograde synapse loss and hyper-excitability, especially in long projection pyramidal neurons. To investigate intrinsic injury signaling within neurons, we used an in vitro microfluidic platform that models dendritic spine loss and delayed hyper-excitability following remote axon injury. Our data show that sodium influx and reversal of sodium calcium exchangers (NCXs) at the site of axotomy, mediate dendritic spine loss following axotomy. In contrast, sodium influx and NCX reversal alone are insufficient to cause retrograde hyper-excitability. We found that calcium release from axonal ER is critical for the induction of hyper-excitability and inhibition loss. These data suggest that synapse loss and hyper-excitability are uncoupled responses following axon injury. Further, axonal ER may play a critical and underappreciated role in mediating retrograde hyper-excitability within the CNS.

Compartmentalization of Human Stem Cell-Derived Neurons within Pre-Assembled Plastic Microfluidic Chips.

Use of microfluidic devices to compartmentalize cultured neurons has become a standard method in neuroscience. This protocol shows how to use a pre-assembled multi-compartment chip made in a cyclic olefin copolymer (COC) to compartmentalize neurons differentiated from human stem cells. The footprint of these COC chips are the same as a standard microscope slide and are equally compatible with high resolution microscopy. Neurons are differentiated from human neural stem cells (NSCs) into glutamatergic neurons within the chip and maintained for 5 weeks, allowing sufficient time for these neurons to develop synapses and dendritic spines. Further, we demonstrate multiple common experimental procedures using these multi-compartment chips, including viral labeling, establishing microenvironments, axotomy, and immunocytochemistry.

Multi-compartment Microfluidic Device Geometry and Covalently Bound Poly-D-Lysine Influence Neuronal Maturation.

Multi-compartment microfluidic devices have become valuable tools for experimental neuroscientists, improving the organization of neurons and access to their distinct subcellular microenvironments for measurements and manipulations. While murine neurons are extensively used within these devices, there is a growing need to culture and maintain human neurons differentiated from stem cells within multi-compartment devices. Human neuron cultures have different metabolic demands and require longer culture times to achieve synaptic maturation. We tested different channel heights (100 µm, 400 µm, and open) to determine whether greater exposure to media for nutrient exchange might improve long-term growth of NIH-approved H9 embryonic stem cells differentiated into glutamatergic neurons. Our data showed an opposite result with both closed channel configurations having greater synaptic maturation compared to the open compartment configuration. These data suggest that restricted microenvironments surrounding neurons improve growth and maturation of neurons. We next tested whether covalently bound poly-D-lysine (PDL) might improve growth and maturation of these neurons as somata tend to cluster together on PDL adsorbed surfaces after long culture periods (>30 days). We found that covalently bound PDL greatly improved the differentiation and maturation of stem cell-derived neurons within the devices. Lastly, experimental paradigms using the multi-compartment platform show that axons of human stem cell derived neurons intrinsically regenerate in the absence of inhibitory cues and that isolated axons form presynaptic terminals when presented with synaptic targets.

Use of Pre-Assembled Plastic Microfluidic Chips for Compartmentalizing Primary Murine Neurons.

Microfabricated methods to compartmentalize neurons have become essential tools for many neuroscientists. This protocol describes the use of a commercially available pre-assembled plastic chip for compartmentalizing cultured primary rat hippocampal neurons. These plastic chips, contained within the footprint of a standard microscope slide, are compatible with high-resolution, live, and fluorescence imaging. This protocol demonstrates how to retrograde label neurons via isolated axons using a modified rabies virus encoding a fluorescent protein, create isolated microenvironments within one compartment, and perform axotomy and immunocytochemistry on-chip. Neurons are cultured for >3 weeks within the plastic chips, illustrating the compatibility of these chips for long-term neuronal cultures.

Distal axotomy enhances retrograde presynaptic excitability onto injured pyramidal neurons via trans-synaptic signaling.

Injury of CNS nerve tracts remodels circuitry through dendritic spine loss and hyper-excitability, thus influencing recovery. Due to the complexity of the CNS, a mechanistic understanding of injury-induced synaptic remodeling remains unclear. Using microfluidic chambers to separate and injure distal axons, we show that axotomy causes retrograde dendritic spine loss at directly injured pyramidal neurons followed by retrograde presynaptic hyper-excitability. These remodeling events require activity at the site of injury, axon-to-soma signaling, and transcription. Similarly, directly injured corticospinal neurons in vivo also exhibit a specific increase in spiking following axon injury. Axotomy-induced hyper-excitability of cultured neurons coincides with elimination of inhibitory inputs onto injured neurons, including those formed onto dendritic spines. Netrin-1 downregulation occurs following axon injury and exogenous netrin-1 applied after injury normalizes spine density, presynaptic excitability, and inhibitory inputs at injured neurons. Our findings show that intrinsic signaling within damaged neurons regulates synaptic remodeling and involves netrin-1 signaling.Spinal cord injury can induce synaptic reorganization and remodeling in the brain. Here the authors study how severed distal axons signal back to the cell body to induce hyperexcitability, loss of inhibition and enhanced presynaptic release through netrin-1.

Messenger RNAs localized to distal projections of human stem cell derived neurons.

The identification of mRNAs in distal projections of model organisms has led to the discovery of multiple proteins that are locally synthesized for functional roles such as axon guidance, injury signaling and regeneration. The extent to which local protein synthesis is conserved in human neurons is unknown. Here we used compartmentalized microfluidic chambers to characterize the transcriptome of distal projections of human embryonic stem cells differentiated using a protocol which enriched for glutamatergic neurons (hESC-neurons). Using gene expression analysis, we identified mRNAs proportionally enriched in these projections, representing a functionally unique local transcriptome as compared to the human neuronal transcriptome inclusive of somata. Further, we found that the most abundant mRNAs within these hESC-neuron projections were functionally similar to the axonal transcriptome of rat cortical neurons. We confirmed the presence of two well characterized axonal mRNAs in model organisms, ß-actin and GAP43, within hESC-neuron projections using multiplexed single molecule RNA-FISH. Additionally, we report the novel finding that oxytocin mRNA localized to these human projections and confirmed its localization using RNA-FISH. This new evaluation of mRNA within human projections provides an important resource for studying local mRNA translation and has the potential to reveal both conserved and unique translation dependent mechanisms.

The E3 Ubiquitin Ligase TRIM9 Is a Filopodia Off Switch Required for Netrin-Dependent Axon Guidance.

Neuronal growth cone filopodia contain guidance receptors and contribute to axon guidance; however, the mechanism by which the guidance cue netrin increases filopodia density is unknown. Here, we demonstrate that TRIM9, an E3 ubiquitin ligase that localizes to filopodia tips and binds the netrin receptor DCC, interacts with and ubiquitinates the barbed-end polymerase VASP to modulate filopodial stability during netrin-dependent axon guidance. Studies with murine Trim9(+/+) and Trim9(-/-) cortical neurons, along with a non-ubiquitinatable VASP mutant, demonstrate that TRIM9-mediated ubiquitination of VASP reduces VASP filopodial tip localization, VASP dynamics at tips, and filopodial stability. Upon netrin treatment, VASP is deubiquitinated, which promotes VASP tip localization and filopodial stability. Trim9 deletion induces axon guidance defects in vitro and in vivo, whereas a gradient of deubiquitinase inhibition promotes axon turning in vitro. We conclude that a gradient of TRIM9-mediated ubiquitination of VASP creates a filopodial stability gradient during axon turning.

Magnetic Alignment of Microelements Containing Cultured Neuronal Networks for High-Throughput Screening.

High-throughput screening (HTS) on neurons presents unique difficulties because they are postmitotic, limited in supply, and challenging to harvest from animals or generate from stem cells. These limitations have hindered neurological drug discovery, leaving an unmet need to develop cost-effective technology for HTS using neurons. Traditional screening methods use up to 20,000 neurons per well in 384-well plates. To increase throughput, we use "microraft" arrays, consisting of 1600 square, releasable, paramagnetic, polystyrene microelements (microrafts), each providing a culture surface for 500-700 neurons. These microrafts can be detached from the array and transferred to 384-well plates for HTS; however, they must be centered within wells for automated imaging. Here, we developed a magnet array plate, compatible with HTS fluid-handling systems, to center microrafts within wells. We used finite element analysis to select an effective size of the magnets and confirmed that adjacent magnetic fields do not interfere. We then experimentally tested the plate's centering ability and found a centering efficiency of 100%, compared with 4.35% using a flat magnet. We concluded that microrafts could be centered after settling randomly within the well, overcoming friction, and confirmed these results by centering microrafts containing hippocampal neurons cultured for 8 days.

Transferable neuronal mini-cultures to accelerate screening in primary and induced pluripotent stem cell-derived neurons.

The effort and cost of obtaining neurons for large-scale screens has limited drug discovery in neuroscience. To overcome these obstacles, we fabricated arrays of releasable polystyrene micro-rafts to generate thousands of uniform, mobile neuron mini-cultures. These mini-cultures sustain synaptically-active neurons which can be easily transferred, thus increasing screening throughput by >30-fold. Compared to conventional methods, micro-raft cultures exhibited significantly improved neuronal viability and sample-to-sample consistency. We validated the screening utility of these mini-cultures for both mouse neurons and human induced pluripotent stem cell-derived neurons by successfully detecting disease-related defects in synaptic transmission and identifying candidate small molecule therapeutics. This affordable high-throughput approach has the potential to transform drug discovery in neuroscience.

Axonal translation of ß-catenin regulates synaptic vesicle dynamics.

Many presynaptic transcripts have been observed in axons, yet their role in synapse development remains unknown. Using visually and pharmacologically isolated presynaptic terminals from dissociated rat hippocampal neurons, we found that ribosomes and ß-catenin mRNA preferentially localize to recently formed boutons. Locally translated ß-catenin accumulates at presynaptic terminals, where it regulates synaptic vesicle release dynamics. Thus, local translation of ß-catenin is a newly described mechanism for axons to independently functionalize nerve terminals at great distances from cellular somata.

Integration of pre-aligned liquid metal electrodes for neural stimulation within a user-friendly microfluidic platform.

Electrical stimulation of nervous tissue is used clinically for the treatment of multiple neurological disorders and experimentally for basic research. With the increase of optical probes to record neuronal activity, simple and user-friendly methods are desired to stimulate neurons and their subcellular compartments for biological experimentation. Here we describe the novel integration of liquid metal electrodes with microfluidic culture platforms to accomplish this goal. We integrated electrode and cell channels into a single poly(dimethylsiloxane) (PDMS) chip, eliminating entirely the need to align electrodes with microchannels. We designed the electrode channels such that the metal can be injected by hand and when the device is non-covalently bound to glass. We demonstrated the biocompatibility of the electrodes for long-term cultures (12 days) using hippocampal neurons. We demonstrated the use of these electrodes to depolarize neurons and recorded neuronal activity using the calcium indicator dye, Fluo-4. We established optimal stimulation parameters that induce neuronal spiking without inducing damage. We showed that the liquid metal electrode evoked larger calcium responses in somata than bath electrodes using the same stimulus parameters. Lastly we demonstrated the use of these liquid metal electrodes to target and depolarize axons. In summary, the integration of liquid metal electrodes with neuronal culture platforms provides a user-friendly and targeted method to stimulate neurons and their subcellular compartments, thus providing a novel tool for future biological investigations.