We would like to thank Yoichi Miyahara for many helpful discussions and insights. MA and PG acknowledge funding from NSERC.

All procedures detailed below were approved by McGill University&#39;s Animal Care Committee and conformed to the guidelines of the Canadian Council of Animal Care.
1. Standardization of Neuronal Cultures Using Microfluidic Devices: Device Assembly
<ol>
	<li>Select a suitable microfluidic device for the desired experiment. To connect neurons within the same population, use the Neuro Devices (Figure 1) and to connect neurons in different populations use the Co-Culture Devices (Figure 6).</li>
	<li>Clean and prepare the desired number of sterile coverslips or glass bottom dishes. For best results on plastic surfaces use 35 mm dishes, on glass use 25 mm coverslips, or 35 mm glass bottom dishes. Select glass thickness based on the imaging system, for instance 0.15 mm.</li>
	<li>Coat the dishes or coverslips with 0.5-1 mL of 100 &#181;g/mL PDL for 2 h or overnight at room temperature. 
	Note: Protocol can be paused here and resumed the following day if desired. Furthemore, dishes can be coated with borate buffer-diluted PDL, Poly-L-Lysine (PLL), laminin or any other cell adhesion molecule.</li>
	<li>Wash the dishes twice with water (do not use phosphate-buffered saline (PBS) as salt crystals may block the channels), remove all the liquid and let it dry in a sterile environment such as a biosafety cabinet for 5-10 min or until the surface is completely dry. 
	Note: Be careful to ensure that the coverslips are absolutely dry, as any remaining liquid will interfere with the adherence of the microfluidic systems.</li>
	<li>Place microfluidic devices with patterns facing up under UV light in a sterile environment (biosafety cabinet) for 10 min. Be sure to follow sterile procedures when working in the biosafety cabinet10.</li>
	<li>Using tweezers place a microfluidic device with pattern facing down in contact with the clean coverslip/dish. Use the tweezers to softly press the device so that it adheres to the glass. 
	Note: The transparency of the adhered region will be visible when looking against the light. Make sure all corners are contacting the glass. Do this to all microfluidic devices. See Figure 1a .</li>
	<li>To fill the single population device with medium, point the pipette towards the channels and add 50 &#181;L of complete cell medium supplemented with serum-free B-27 (volume ratio 1:50) and 500 &#181;g/mL penicillin/streptomycin/glutamine (collectively called NBM) to the right upper well, and then add another 50 &#181;L to the well lying diagonally to it. Do this for all devices, making sure that the medium flows between wells. Next, add 50 &#181;L of medium to the remaining 2 wells. See Figure 2a .
	<ol>
		<li>To fill the multiple population device with medium, point the pipette towards the channels and add 30 &#181;L of complete NBM to the right wells, refer to Figure 6a. Do this for all devices, making sure that the medium flows between wells. Next, add 50 &#181;L of medium to the remaining 4 wells.</li>
	</ol>
	</li>
	<li>Place the devices in a bigger plate with an open dish with autoclaved water (wet chamber) and place in the incubator (37 &#176;C, 5% CO2 and 95% humidity) for 1-2 h while preparing the cell culture. See Figure 2b .</li>
</ol>
2. Plating Neurons in Microfluidic Systems
<ol>
	<li>Following the protocol outlined in Ref.8, obtain dissociated hippocampal or cortical neurons from Sprague Dawley rat embryos (either gender).</li>
	<li>Resuspend embryonic neurons in NBM at a concentration of 1-2 million neurons/mL. Verify cell concentrations in the microscope using a hemocytometer and following reference8. Adjust the cell concentration according to the desired cell density. To increase the chances of obtaining single hippocampal axons per channel, plate 10,000 neurons per device. To have multiple axons in the same channel, plate 60,000 neurons per device. 
	Note: These numbers vary according to the neuronal type used.</li>
	<li>Remove the medium from the microfluidic devices without emptying the wells. Leave approximately 5 &#181;L in each.</li>
	<li>To plate cells in the single population device, add 50 &#181;L of NBM to the lower right well. At this point the medium flows by itself to fill the other lower well. Add 20 &#181;L of the concentrate cell solution into the top right well of the microfluidic device, as indicated in Figure 1b .
	<ol>
		<li>To plate cells in the multiple population device add 20 &#181;L of the concentrate cell solution into each of the right wells in Figure 6a.</li>
	</ol>
	</li>
	<li>Check in the microscope if cells are inside the chambers and place the devices in the incubator for 15-30 min to promote cell attachment to the substrate.</li>
	<li>Check in the microscope if there are enough cells in the chambers. If more are needed, repeat steps 2.4 and 2.5.</li>
	<li>Add 50 &#181;L of NBM to the 2 top wells of the single population device and 20 &#181;L of NBM into the same well as the cells were injected in the multiple population device. The media protrudes slightly to form a positive meniscus giving the wells a muffin top aspect. Again, see Figure 2a.</li>
	<li>Maintain the cells at 37 &#176;C, 5% CO2 and 95% humidity.</li>
</ol>
3. Maintaining the Neuronal Cultures
<ol>
	<li>Remove NBM (roughly 30 &#181;L with a pipette) from the cells and apply new pre-warmed NBM the day following their introduction to the devices (that is 1 d after step 2).</li>
	<li>Check every 2 days if there is enough medium in each channel. If the muffin top is low just add more medium to the top wells.</li>
	<li>Culture cells for at least 7 d before removal of the microfluidic devices. The cells can survive in these devices for several weeks. Remove the devices 1-2 days before experiments are performed on samples.</li>
</ol>
4. Removal of Microfluidic Devices
<ol>
	<li>1 - 2 d before removal of microfluidic devices, add 2 mL of NBM prewarmed to 37 &#176;C to each sample dish, flooding the chambers, and maintain the devices in the incubator.</li>
	<li>Use sterile tweezers and one tip to remove the microfluidic devices from the coverslips leaving a patterned configuration of neurons. Use the tip to hold the coverslip in place and the tweezers to clasp the edge of the device at the bottom left corner of the well. Delicately apply torsion, raising the device up with the tweezers so that it peels off the coverslip. See Figure 2c-2d.</li>
	<li>Every 2-3 days, replace half the NBM until the sample is used for experiments.</li>
	<li>Before performing rewiring experiments on the sample, verify that neurites in the single population device channels and the neuronal populations in the multiple population device are isolated by examining the gaps between them in the microscope to ensure there are no filaments linking neuronal populations.</li>
</ol>
5. Preparing PDL-coated Beads
<ol>
	<li>Add 2 x 50 &#181;L drops of either 4, 10 or 20 &#181;m polystyrene beads diluted in water (1:500) to 1 mL of PDL (100 &#181;g/mL). Leave for at least 2 h at room temperature. 
	Note: Protocol can be paused here and resumed the following day.</li>
	<li>Centrifuge the solution at 8,820 x g for 1 min. Carefully remove supernatant without disturbing the beads accumulated at bottom of container.</li>
	<li>Wash the beads twice with 1 mL of sterile 10 mM HEPES pH 8.4 solution.</li>
	<li>Resuspend the PDL-coated beads in 200 mL of 10 mM HEPES pH 8.4 solution.</li>
</ol>
6. Preparing Micropipettes
<ol>
	<li>Prepare pipettes from glass capillary tubes (1 mm inner diameter, 1.5 mm outer diameter) using a horizontal electrode puller. Adjust settings so the outer tip of the pulled micropipette is ~ 2-5 &#181;m. Before pulling, ensure the glass tubes are clean.</li>
	<li>Fix pipettes to glass slides for storage, and ensure that the tip does not contact the surface of the slide as the tip is fragile. Store at room temperature in a covered container to protect from dust. Use pipettes the same day they are pulled.</li>
</ol>
7. PDL-bead Adhesion to Neurons
<ol>
	<li>Add 40-60 &#181;L of PDL-coated beads prepared in step 5 to a cell culture prepared in step 4. Center the pipette tip over the neurons, which are faintly visible on the coverslip, and add the beads (See Figure 3).</li>
	<li>Return the sample to the incubator for 1 h to promote the formation of synaptic contacts8,9.</li>
	<li>After the incubation, remove any un-adhered beads by gently washing the culture with pre-warmed NBM.</li>
</ol>
8. Preparing Physiological Saline Solution (for Room Temperature Experiments)
<ol>
	<li>Prepare physiological saline solution by combining the ingredients listed in references7,8. This is to regulate the cell environment outside the incubator.</li>
	<li>Verify osmolarity and pH levels as indicated in references7,8.</li>
	<li>Continuously infuse solution with O2 to minimize pH fluctuations while conducting experiments.</li>
	<li>Heat to room temperature.</li>
	<li>Set up the perfusion system by inserting one end of a plastic tube (optional dimensions) in O2-infused physiological solution and fixing the other end to a needle inserted in the sample holder. Place the tubing and solution higher than the sample (See Figure 4).
	<ol>
		<li>Disconnect the tube from the needle and connect it to a syringe. Use the syringe to exert pressure and draw liquid, filling the tube. Seal with a roller clamp and reconnect the needle.</li>
	</ol>
	</li>
</ol>
9. Bead Micromanipulation
<ol>
	<li>Install sample in an experimental set-up such that cells can be accessed from above by two micropipettes mounted in micromanipulators and accessed optically below, for instance with the 40X-phase objective (numerical aperture of 0.6) of an inverted optical microscope. In this configuration, mount a CCD camera for image capture on the side port of the microscope. Connect each pipette to 1 mL syringes via plastic tubing. At this step, replace NBM with physiological saline solution (1-2 mL) (See Figure 4).</li>
	<li>During experiments, continuously perfuse cells with the physiological saline solution prepared in step 8 at a rate of 0.5-1 mL/min.</li>
	<li>Select a PDL-bead NOT attached to a neuron in the field of view. Align the bead with a micropipette tip by focusing onto the bead then up to the micropipette. Bring the tip down as close as possible to the bead by monitoring it through the microscope.</li>
	<li>Apply negative pressure with the 1 mL syringe connected to the pipette to pick up the bead. Maintain negative pressure throughout the experiment.</li>
</ol>
10. Pulling Neurites
<ol>
	<li>Select a PDL-bead attached to a neuron in the field of view and attach it to the second micropipette using suction as described in steps 9.3-9.4.</li>
	<li>Pull the PDL-bead-neuron complex by slowly (~0.5 &#181;m/min) moving either the micromanipulator or the sample stage by 1 &#181;m and pause for 5 min to allow neurite initiation.</li>
	<li>Repeat step 10.2 twice. 
	Note: The first 3 &#181;m have to be pulled very slowly to guarantee experimental success, which occurs over 95% of the time.</li>
	<li>Pull the PDL-bead-neuron complex by slowly (~0.5 &#181;m/min) moving either the micromanipulator or the sample stage by 2 &#181;m and pause for 5 min to allow neurite elongation.</li>
	<li>After successful initiation and neurite extension for the first 5 &#181;m, pull the neurite at 20 &#181;m/min over millimeter-scale distances. 
	Note: Pulling can be performed continuously or in steps and at varying rates. See Figure 5b-5c .</li>
</ol>
11. Connecting Neurons
<ol>
	<li>Select a region rich in neurites and lower the PDL-bead-neurite complex so that it physically contacts it. Use other beads to gauge tip height above coverslip surface. See Figure 5d.</li>
	<li>Leave the PDL-bead-neurite complex in contact with the target neurite while manipulating the second micropipette. Lower the second pipette with the second PDL-bead on top of the newly formed neurite about 20 &#181;m form the first bead. Use the second PDL-bead to push the new neurite filament towards the target cell.</li>
	<li>Hold both beads in place for at least 1 h. Verify the absence of focal swelling, a thickening of the neurites contacting the bead, with the microscope16.</li>
	<li>During this time, use perfusion to slowly change the medium of the sample from physiological saline to pre-warmed, CO2-equilibrated NBM.</li>
	<li>Release the bead from the second pipette by releasing suction. If the new neurite remains attached, release the first bead as well. See Figure 5e .</li>
	<li>Gently remove saline solution and replace with NBM (~2 mL).</li>
	<li>Carefully place the sample back in the incubator to strengthen the neuronal connection for future experiments. This connection is stable for &#62;24 h7.</li>
</ol>
12. Verifying the Functionality of the New Connection via Whole-cell Paired Patch Clamp Recordings
<ol>
	<li>Follow references7,18,19. to assemble an electrophysiology set-up.</li>
	<li>Follow references7. to prepare the pre- and postsynaptic electrodes.</li>
	<li>Gather patch clamp data, again following references18,19.</li>
	<li>Compare the results to naturally occurring signals7 to determine the connection type.</li>
</ol>

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The author Margaret H Magdesian is the CEO of Ananda Devices that produces instruments used in this Article.

Using standard micromanipulation and innovative microfluidic devices, a new technique was developed to rapidly initiate, elongate and precisely connect new functional neuronal circuits over large distances. Pipette micromanipulation is a common tool in most neuroscience labs4,13. The real challenge to achieving reproducible and reliable results was standardization of healthy, precisely positioned neuronal cultures for the duration of the experiment (which can be ...

Injuries to the adult central nervous system (CNS) may lead to permanent disability due to multiple mechanisms that limit axonal regrowth1. Following injury, many CNS axons do not form a new growth cone and fail to mount an effective regenerative response2. Furthermore, damage and scar tissue surrounding CNS lesions significantly inhibit axonal growth1,2,3. Current therapies to promote CNS regeneration after injury have focused on enhancing the intrinsic growth potential of the injured neuron and on masking the inhibitors of axonal extension associated with myelin debris and the glial scar1,3. Despite this, the capacity to regenerate long axons to distant targets and to form appropriate functional synapses remains severely limited4,5,6,7.
In the present work, microbeads, pipette micromanipulation, and microfluidic devices are used to rapidly initiate, elongate, and precisely connect new functional neuronal circuits over long distances. Previous work has shown that poly-D-lysine-coated beads (PDL-beads) induce membrane adhesion followed by the clustering of synaptic vesicle complexes and the formation of functional presynaptic boutons8. It was also shown that when the PDL-bead is mechanically pulled away after presynaptic differentiation, the synaptic protein cluster follows the bead, initiating a new neurite9. The following procedure exploits this fact along with the ability to culture embryonic hippocampal neurons of rats into organized regions on a coverslip using polydimethylsiloxane (PDMS) microfluidic devices to precisely rewire a neuronal circuit.
These PDMS microfluidic devices are non-toxic, optically transparent and consist of two chambers connected by a system of microchannels. Once assembled on a coverslip, each device serves as a mold to guide neuronal growth and maintain healthy neuronal cultures on precise patterns for longer than 4 weeks in vitro.
Here, a framework is presented in which to investigate the limits of extension and functionality of the new neurite. New, functional neurites are created and positioned to controllably (re)wire neuronal networks. The extension rates achieved are faster than 20 &#181;m/min over millimeter-scale distances and functional connections are established. These results show, unexpectedly, that the intrinsic capacity of these neurites for elongation is much faster than previously thought. This proposed mechanical approach bypasses slow chemical strategies and enables controlled connection to a specific target. This technique opens new avenues for the in vitro study of novel therapies to restore neuronal connectivity after injury. It also enables the manipulation and rewiring of neuronal networks to investigate fundamental aspects of neuronal signal processing and neuronal function in vitro.

<table><tbody><tr><td>Co-culture devices</td><td>Ananda Devices</td><td></td><td>Commercially available at http://www.anandadevices.com</td></tr><tr><td>Neuro devices</td><td>Ananda Devices</td><td></td><td>Commercially available at http://www.anandadevices.com</td></tr><tr><td>No. 1 Glass Coverslip 25 mm Round</td><td>Warner Instruments</td><td>64-0705</td><td></td></tr><tr><td>35 mm Glass Bottom Dishes #0, Uncoated, Gamma-Irradiated</td><td>MatTex Incorporation</td><td>P35G-0-20-C</td><td></td></tr><tr><td>35 mm cell culture dish, Non-Pyrogenic, Sterile</td><td>Corning Inc</td><td>430165</td><td></td></tr><tr><td>95 mm x 15 mm Petri Dish, Slippable Lid, Sterile Polystyrene</td><td>Fisherbrand</td><td>FB0875714G</td><td></td></tr><tr><td>50 mL Centrifuge tubes with printed graduations and flat caps</td><td>VWR</td><td>89039-656</td><td></td></tr><tr><td>15 mL Polypropylene Conical Tube, 17 x 120 mm style, Non Pyrogenic, Sterile</td><td>Falcon</td><td>352097</td><td></td></tr><tr><td>Neurobasal Medium</td><td>Life Technologies</td><td>21103-049</td><td>Extracellular solution</td></tr><tr><td>B-27 Supplement (50X), serum free</td><td>B-27 Supplement (50X), serum free</td><td>17504044</td><td>Extracellular solution</td></tr><tr><td>Pennicilin, Streptomyocin, Glutamine</td><td>Thermo Fisher Scientific&amp;nbsp;</td><td>11995-065</td><td>Extracellular solution</td></tr><tr><td>200 &amp;mu; L Pipettors</td><td>VWR</td><td>89079-458</td><td></td></tr><tr><td>2 - 20 &amp;mu;L Pipettors</td><td>Aerosol Resistant Tips</td><td>2149P</td><td></td></tr><tr><td>BD Falcon 3mL Transfer Pipettes [Non-sterile]</td><td>BD Falcon</td><td>357524</td><td></td></tr><tr><td>Glucose</td><td>Gibco</td><td>15023-021</td><td>Extracellular solution</td></tr><tr><td>HEPES</td><td>Sigma</td><td>7365-45-9</td><td>Extracellular solution/Beads</td></tr><tr><td>NaCl</td><td>Sigma-Aldrich</td><td>7647-14-5</td><td>Extracellular solution</td></tr><tr><td>KCl</td><td>Sigma-Aldrich</td><td>7447-40-7</td><td>Extracellular solution</td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>10043-52-4</td><td>Extracellular solution</td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>7786-30-3</td><td>Extracellular solution</td></tr><tr><td>#5 Dumont Dumostar Tweezers 11 cm</td><td>World Precision Instruments</td><td>500233</td><td></td></tr><tr><td>Dissection tools</td><td>Braun, Aesculap</td><td></td><td></td></tr><tr><td>Poly-D-lysine Hydrobromide</td><td>Sigma-Aldrich</td><td>P6407</td><td></td></tr><tr><td>Micro particles based on polystyrene, 10 &amp;mu;m</td><td>Sigma-Aldrich</td><td>72986</td><td></td></tr><tr><td>Borosilicate tubes</td><td>King Precision Glass, Inc.</td><td>14696-2</td><td></td></tr><tr><td>Horizontal Pipette Puller</td><td>Sutter Instruments</td><td>Brown-Flaming P-97</td><td></td></tr><tr><td>Micromanipulators, PCS-5000 Series</td><td>SD Instruments</td><td>MC7600R</td><td></td></tr><tr><td>1 mL Syringe</td><td>BD Luer-Lok</td><td>309628</td><td></td></tr><tr><td>Inverted Microscope</td><td>Olympus&amp;nbsp;</td><td>IX71</td><td></td></tr><tr><td>Objective</td><td>Olympus</td><td>UIS2, LUCPLFLN 40X</td><td></td></tr><tr><td>CCD Camera</td><td>Photometrics</td><td>Cascade II: 512</td><td></td></tr><tr><td>Leibovitz&#039;s (1x) L-15 Medium</td><td>Life Technologies</td><td>11415-064</td><td>Rat Dissection</td></tr><tr><td>Typsin-EDTA (0.05%), Phenol red</td><td>Life Technologies</td><td>25300054</td><td>Rat Dissection</td></tr><tr><td>DMEM (1x) Dulbecco&#039;s Modified Eagle Medium [+4.5 g/L D-Glucose, + L-Glutamine, + 110 mg/L Sodium Pyruvate]</td><td>Life Technologies</td><td>11995-065</td><td>Rat Dissection</td></tr><tr><td>HBSS (1x) Hank&#039;s Balanced Salt Solution [- Calcium Chloride, - Magnesium Chloride, - Magnesium Sulfate]</td><td>Life Technologies</td><td>14170-112</td><td>Rat Dissection</td></tr></tbody></table>

rewiring neuronal circuits: a new method for fast neurite extension and functional neuronal connection

Brain and spinal cord injury may lead to permanent disability and death because it is still not possible to regenerate neurons over long distances and accurately reconnect them with an appropriate target. Here a procedure is described to rapidly initiate, elongate, and precisely connect new functional neuronal circuits over long distances. The extension rates achieved reach over 1.2 mm/h, 30-60 times faster than the in vivo rates of the fastest growing axons from the peripheral nervous system (0.02 to 0.04 mm/h)28 and 10 times faster than previously reported for the same neuronal type at an earlier stage of development4. First, isolated populations of rat hippocampal neurons are grown for 2-3 weeks in microfluidic devices to precisely position the cells, enabling easy micromanipulation and experimental reproducibility. Next, beads coated with poly-D-lysine (PDL) are placed on neurites to form adhesive contacts and pipette micromanipulation is used to move the resulting bead-neurite complex. As the bead is moved, it pulls out a new neurite that can be extended over hundreds of micrometers and functionally connected to a target cell in less than 1 h. This process enables experimental reproducibility and ease of manipulation while bypassing slower chemical strategies to induce neurite growth. Preliminary measurements presented here demonstrate a neuronal growth rate far exceeding physiological ones. Combining these innovations allows for the precise establishment of neuronal networks in culture with an unprecedented degree of control. It is a novel method that opens the door to a plethora of information and insights into signal transmission and communication within the neuronal network as well as being a playground in which to explore the limits of neuronal growth. The potential applications and experiments are widespread with direct implications for therapies that aim to reconnect neuronal circuits after trauma or in neurodegenerative diseases.

Embryonic rat hippocampal neurons are cultured in microfluidic devices to enable precise positioning of cells, PDL-beads and micromanipulators. The first step is to properly assemble the microfluidic device on a glass coverslip or dish. It is essential that the microfluidic device be well attached to the substrate to avoid cells exiting the chambers and moving under the parts of the device that should be sealed (Figure 1a). To maintain healthy cultures for se...

Watch this Scientific Journal Video about Rewiring Neuronal Circuits: A New Method for Fast Neurite Extension and Functional Neuronal Connection at JoVE.com

Rewiring Neuronal Circuits: A New Method for Fast Neurite Extension and Functional Neuronal Connection

This procedure describes how to rapidly initiate, extend and connect neurites organized in microfluidic chambers using poly-D-lysine-coated beads fixed to micropipettes that guide neurite elongation.

Brain and spinal cord injury may lead to permanent disability and death because it is still not possible to regenerate neurons over long distances and ...

rewiring-neuronal-circuits-new-method-for-fast-neurite-extension

Research

JoVE Journal

Neuroscience

8.7K Views.  McGill University.  Deze procedure beschrijft hoe snel neurieten georganiseerd in microfluïdische kamers kunnen initiëren, verlengen en verbinden met behulp van poly-D-lysine beklede kralen die zijn bevestigd aan micropipetten die leiden tot neuriet verlenging. 

Video: Rewiring Neuronal Circuits: A New Method for Fast Neurite Extension and Functional Neuronal Connection

An Optogenetic Approach for Assessing Formation of Neuronal Connections in a Co-culture System

Here we describe a protocol to generate a co-culture consisting of 2 different neuronal populations. Induced pluripotent stem cells (iPSCs) are reprogrammed from human fibroblasts using episomal vectors. Colonies of iPSCs can be observed 30 days after initiation of fibroblast reprogramming. Pluripotent colonies are manually picked and grown in neural induction medium to permit differentiation into neural progenitor cells (NPCs). iPSCs rapidly convert into neuroepithelial cells within 1 week and retain the capability to self-renew when maintained at a high culture density. Primary mouse NPCs are differentiated into astrocytes by exposure to a serum-containing medium for 7 days and form a monolayer upon which embryonic day 18 (E18) rat cortical neurons (transfected with channelrhodopsin-2 (ChR2)) are added. Human NPCs tagged with the fluorescent protein, tandem dimer Tomato (tdTomato), are then seeded onto the astrocyte/cortical neuron culture the following day and allowed to differentiate for 28 to 35 days. We demonstrate that this system forms synaptic connections between iPSC-derived neurons and cortical neurons, evident from an increase in the frequency of synaptic currents upon photostimulation of the cortical neurons. This co-culture system provides a novel platform for evaluating the ability of iPSC-derived neurons to create synaptic connections with other neuronal populations.

A protocol to generate a co-culture system consisting of neurons derived from induced pluripotent stem cells (iPSCs), primary cortical neurons and astrocytes is described. This co-culture system allows detection of the formation of synaptic contacts and circuits between new, iPSC-derived neurons and pre-existing cortical neurons expressing channelrhodopsin-2.

Here we describe a protocol to generate a co-culture consisting of 2 different neuronal populations. Induced pluripotent stem cells (iPSCs) are ...

Developmental Biology

Perspectives on Neuroscience

As usual, the main cause for progress in natural sciences are methodological developments. Of course, conceptual developments follow and go in parallel, but I think the, the tremendous expansion of the field of neurosciences was triggered by a number of methodological breakthroughs at the lowest level. These were the progresses in the domain of molecular biology, genetic engineering, which provided model systems for the study of development for the study of certain diseases.These techniques also provided extremely valuable tools for the biochemical and molecular identification of neurons. We learned about a degree of diversification of morphologically seemingly identical neurons that are endowed with different molecular machineries. All this we owe to the development of molecular biology.A second important factor I think was the development of non-invasive imaging technologies. cause this allowed us to transfer a large number of concepts that have been developed in animal experimentation to investigations in human subjects. So this is the functional magnetic resonance imaging pository emission tomography.Also the introduction of the magneto ence holography, which allowed for much better spatial resolution for the analysis of electric activity in the brain led to a substantial expansion in the field. Then I think a non negligible factor was the availability, increasing availability of of massive computer power because as neurobiology started to apply multi-site recording techniques, which I consider essential for the analysis of neuron network properties, the data became increasingly complex and it became increasingly impossible to just evaluate the data by looking at them as in the classical times when I started in the field, there were no computers. We had to do cross correlation analysis by cutting long strips of paper and moving them over the floor.So the availability of of computing power has greatly advanced at least systems physiology. I think it had a major impact on these developments. And then last but not least, the availability of these computer facilities allowed us also to treat properties of neuronal networks at a theoretical level.The dynamics exhibited by neuronal networks are so complex that only modeling studies and theoretical approaches put us in a position to formulate educated hypothesis. Also, for the search of neuronal codes, we cannot proceed without these search tools. So I think altogether these methodological developments were responsible for the great expansion of the neurosciences, which I think is unprecedented in any Scientific discipline.As far as my own Research is concerned, there are two parts in my biography. Initially I studied developmental processes in the brain. I was particularly interested in developmental processes that are shaped by experience postnatally.And the rationale for this approach was that I felt that the organization of the mature brain is so complex that we better study the developmental principles and try to make sense out of them and try to extrapolate what these developmental processes will eventually lead to. But then an accidental finding that actually happened in this very laboratory where we are sitting changed my life because I discovered inadvertently that neurons in the primary visual cortex of kittens that were awake actually sitting on the table behind me watching a drifting grating, got entrained into highly synchronous oscillatory activity that I could not attribute neither to degrading to the stimulus nor to the main switch in Germany are 50 cycles because it was an oscillation in the 40 hertz range. And so for the first time I saw that spatially distributed neurons could coordinate their activity with very high precision in the millisecond range over fairly large distances.And this triggered the idea that maybe time is used as a coding space for the coordination of distributed neuronal responses. And I found this option so interesting that I decided to neglect the so much beloved field of developmental neurobiology and invest most of our efforts into the further analysis of this phenomenon. And this is now nearly 15 years ago or more.And since then we spent most of our time with multi-site recordings and the search for spatial temporal patents in distributed neural networks because it is our firm belief that information is not only encoded in the discharge rate of neurons as is classically assumed and confirmed, but that additional information is encoded in the precise temporary relations among the individual discharges of neurons whereby zero phase lake correlations. So precise coincidence is probably only the tip of the iceberg. But an important tip because synchronous activity is particularly interesting in neuronal networks because it propagates very easily synchronizing responses is a means to enhance the saliency of neuronal responses with great temporal position.It can be done on a spike by spike basis. And the initial hypothesis was that this synchronization is used at peripheral stages of sensory processing in order to establish relations between features that are processed by spatially distributed neurons. Since then, many more labs have engaged in search for the synchronization phenomena.It started I think in Europe with colleagues like RI who applied EEG techniques to assess synchronous oscillatory activity in the brain and relate these activities to cognitive functions. And since then the the role of oscillations and synchrony has been discussed in a very controversial way. Initial doubts that this phenomenon might exist, it all have now been countered I think, and we are now all engaged in the search for functional consequences.And there is a whole plethora of hypothesis of what that could mean. Does the accumulation Of this vast amount of data that we are confronted with nowadays and that we have very great difficulties to cope with reflect true progress. It certainly does reflect progress because we get to know more and more facts and details about the organization of neurons, neuron networks and the brain as a whole.And this is a necessary consequence of the classical analytical approaches that are applied in the natural sciences. One isolates a subsystem and then describes it in ever greater detail. But this is only one half of the scientific endeavor.What we also need is the inductive strategy. This the synthesis of all those data. And what we are now confronted with is that we have a huge amount of words.We have a tremendous vocabulary that is difficult to remember, but we are lacking the syntax to put those words together into meaningful sentences in many domains. This is the case in particular in systems neuroscience. Therefore we need, I think more theory because conditions have become so complex that near verbal descriptions are probably no longer sufficient to grasp or to accomplish this, this synthetic process.We need formal descriptions, we need analysis tools that allow us to generate models and this requires very powerful computations. And then we need of course synthetic thinkers. That's probably the the most important ingredient in the moment.People who can bundle a large amount of details into meaningful hypothesis or concepts. Now one can of course foster this process by providing the appropriate environment and worldwide one sees efforts to provide such environments by generating institutions that are usually called Institute for Computational Neuroscience or Institute for Theoretical Neuroscience. For the time being, many of these places are still existing in two great in isolation and are not sufficiently linked to experimental institutions.Ideally one would have, one would like to see a very close cooperation between theoreticians and experimentalists, as has been so fruitful in physics where theoretical physicists and experimental physicists very closely collaborate freely, exchange data ideas and do this without jealousy, which is still not the case in our field. But I think biology is now approaching a threshold where it is mature enough to support theoretical approaches in a meaningful way. And here in Frankfurt, we have founded the Frankfurt Institute of Advanced Studies exactly for that purpose because I experienced that physicists and mathematicians, theoreticians in general when embedded in the experimental institute, they tend to to be marginalized.They don't have enough colleagues to talk to because biologists usually are not trained in their language. And so the idea was let's create an institute in which several model systems, biological model systems that have in common to be multi-component systems that are tightly coupled like the moon system or the brain are studied by theoreticians, hoping that these theoreticians might discover common principles in these complex self-organizing systems and that they can share analytical tools, can share tools for model building. And I think it worked out because within about a year this institute grew from zero to 120 scientists.And the special structure of this institute, which is not surprising to colleagues in America, but this is quite exceptional in Europe, is that this institute is funded exclusively by donors private funds. It is not a university institute, it is linked to the university in Frankfurt, but it is finance in a different way for the simple reason that it was impossible to install such an interdisciplinary enterprise within the conventional faculties because what happens there is truly I the disciplinary, it crosses faculties and they seem to be realizable only by having a erect and an own building and gather people who come from very different disciplines under the same roof. Even though those Terms have been taken from physical systems initially, they still grasp some of the characteristics of of of the brain because the brain clearly is a self-organizing system.There's no conductor in the brain to coordinate all the distributed processes. The brain develops in a self organizing way through interaction between genes and environment. There is no instructor, there is no blueprint, a priori.And finally the term applies very well to what we observe in complex systems all the time, namely that complex systems tend to exhibit properties that cannot be described or inferred from the properties of the components. And the brain is a particularly nice example for this fact, which we know from all complex systems there's nothing special about the brain. Something that is special about the brain is that we have on the one side material processes, neuron interactions that can be described from a third person perspective as any other process in our environment.And that emergence from these neuronal interactions are mental phenomena, psychological phenomena such as emotions, thoughts, decisions. So there are realities which I would like to address as social realities which emerge or come into being by the interaction between brains that communicate with each other, mirror each other, ascribe properties to each other, could replace brains by persons, doesn't really matter because what makes the person is finally the brain in its in its bodily setting and due to these interactions phenomena emerge that are to be taken as real phenomena, they are realities, but they have to be described in a different language as the phenomena that bring them forth that are at their origin, namely neuronal interactions. And therefore I think that this is a classical case of emergence where something new is generated by a complex system, nobody would ever be able to infer from the knowledge, complete knowledge of neurons, and even from complete knowledge of neuron networks, that they are capable of developing value systems when they interact With each other.I think the main Problem in our efforts to repair brains, apart from the fact that neurons do not easily regenerate in the central nervous system, they don't like to extend processes once they are severed for reasons that we do not know. Maybe there was no Evo evolutionary pressure on the development of such mechanisms because brain damage that really leads to cell death in in a large scale was usually not compatible with survival. The great problem in restoring neuron networks that have been damaged is that it is difficult to recapitulate the developmental process.The mature brain is characterized by an extremely sophisticated circuitry that has developed step by step in a complex self-organizing process, guided by the dialogue between the genes and the increasingly more differentiated cellular environment that changes throughout development. And this process is a essentially bottom up process. Peripheral structures mature first, then they go through critical periods and the connectivity becomes fixed so that the, the higher stages can rely upon what has already been built and then they organize themselves as a function of what's already there.And this process goes on in human beings until age 20 and then the circuitry crystallizes and then we have to live with the brains that we have got at that point of time. There's further modification by learning and neuroplasticity, but this is limited. Now if one were to replace neurons at higher levels of processing, for example, by injection of stem cells or so these neurons we know already, they can differentiate, they the precursors can become neurons, but the problem is will they find the partners with which they have to interact?Will the information still be there That was available during development that guided the axon growth, that guided the formation of specific connections. And we know that to, to a large extent, this information is no longer available because the genes that one needs in order to express the necessary marker molecules, they get switched off once development comes to an end. And what is probably necessary is that one not only supplies cells that can differentiate into neurons and of course also ggl cells, but that one also switches on or helps the organism to switch on the genes that have been active during the developmental process that lead to the expression of all the molecular machinery that is required to tell the cells with whom they should connect, where they should connect.And what of course cannot be reinstalled easily is all the idiosyncratic wiring and the adjustment of synaptic game that is due to learning processes. The newly integrated neurons will of course have no souvenir, no recollection of the experience that has been imprinted into the morphology and the structure and the function of neurons that have disappeared. So there are certain limits to the ability to restore.And as we realize in the moment, there are certain places in the brain where this seems to pose less of a problem because nature has foreseen lifelong regeneration of neurons and apparently kept the machinery alive that is needed in order to promote this ongoing regeneration and insertion of neurons as in hippocampus and in the olfactory bulb. But the other structures where this is probably causing great problems, the first replacements that have been attempted and have had certain success even though it was not satisfactory yet, where the modulatory systems where topological specificity is not that much a problem. These systems are functioning more like endocrine or paracrine systems that liberate certain transmitters that are required in order to keep a certain tous of excitability.These systems are more easily replaceable because specificity is less of a problem, but the future will show how far we get there. Certainly very promising approaches in reinstalling oxon outgrowth in several spinal cords by inhibiting factors that inhibit outgrowth in the adult and by activating mechanisms that come to action during development and promote outgrowth. There are encouraging successes in this field, but one should also consider very seriously the option of neuronal prosthetics to record neuronal activity from places where it is still available and then through mechanical devices and computer interfaces activate actors that can translate this activity into action for the motor system.Or conversely, on the sensory side, cochlear implants already work admirably well. There are now attempts to develop retinal implants in order to reinstall rudimentary visual functions in people who have retinal degeneration. There's also a very promising option to insert light gated iron channels into retinal cells so that cells that are normally not light sensitive, which are the ganglion cells for example, or the bipolar cells become light sensitive so that one could in principle reins store some function that is lost when photoreceptors degenerate by simply transferring light sensitivity to second or third order neurons.This is already possible in animal experimentation. It might be another promising avenue to pursue. I think Europe has a very strong tradition in the development of concepts end of 19th century helm those seminal figures who have provided the grounds for later experimental neurobiology to some extent.They also are the grand grandfathers of a substantial number of systems physiologists who have been active in Europe. I think there is a good tradition in systems neuroscience in Europe has always been in the times when, when linear systems theory was still at its peak cybernetics, as it was called Europe, had a few very strong exponents such as rehad for example. Then the, there was something like, not iconic turn, but the molecular turn in Europe as well as anywhere else, molecular biology took over and replaced many of the systems oriented approaches.Partly because initially it had been cheaper than to work with whole animals, partly because non-invasive technology was not available and one couldn't do certain things in animals, which can now be done in human subjects. But I think there's a strong renaissance in systems physiology even though in Europe we have to fight a hard battle against animal protectionists concerning primary research, this is a real critical issue. The labs that can, they still get the license to work with primates become less and less and those who still have the license.This is the case with us where we have, we are exposed very much to public critique, but still systems neuroscience is having a renaissance. And I think there are good reasons for that because we will need it more and more in order to make sense out of the plethora of data that are collected by the properties of neurons and small circuits. And these data can only be integrated if we have concepts derived from systems neuroscience on the nature of the neuronal code, on the cooperation of areas in the brain, on global coordination of processes, on distributed processing.All those questions are heatedly debated in the moment and we just need more experiments along those lines. And I think Europe is not in a bad position to do this. Israel as well, one should not forget the contribution, also conceptual contribution of our colleagues in Israel, which is substantial.

Biology

Rapid Neuronal Differentiation of Induced Pluripotent Stem Cells for Measuring Network Activity on Micro-electrode Arrays

Neurons derived from human induced Pluripotent Stem Cells (hiPSCs) provide a promising new tool for studying neurological disorders. In the past decade, many protocols for differentiating hiPSCs into neurons have been developed. However, these protocols are often slow with high variability, low reproducibility, and low efficiency. In addition, the neurons obtained with these protocols are often immature and lack adequate functional activity both at the single-cell and network levels unless the neurons are cultured for several months. Partially due to these limitations, the functional properties of hiPSC-derived neuronal networks are still not well characterized. Here, we adapt a recently published protocol that describes production of human neurons from hiPSCs by forced expression of the transcription factor neurogenin-212. This protocol is rapid (yielding mature neurons within 3 weeks) and efficient, with nearly 100% conversion efficiency of transduced cells (&#62;95% of DAPI-positive cells are MAP2 positive). Furthermore, the protocol yields a homogeneous population of excitatory neurons that would allow the investigation of cell-type specific contributions to neurological disorders. We modified the original protocol by generating stably transduced hiPSC cells, giving us explicit control over the total number of neurons. These cells are then used to generate hiPSC-derived neuronal networks on micro-electrode arrays. In this way, the spontaneous electrophysiological activity of hiPSC-derived neuronal networks can be measured and characterized, while retaining interexperimental consistency in terms of cell density. The presented protocol is broadly applicable, especially for mechanistic and pharmacological studies on human neuronal networks.

We modify and implement a previously published protocol describing the rapid, reproducible, and efficient differentiation of human&#160;induced&#160;Pluripotent Stem Cells (hiPSCs) into excitatory cortical neurons12. Specifically, our modification allows for control of neuronal cell density and use on micro-electrode arrays to measure electrophysiological properties at the network level.

Neurons derived from human induced Pluripotent Stem Cells (hiPSCs) provide a promising new tool for studying neurological disorders. In the past decade, ...

An Enhanced Green Fluorescence Protein-based Assay for Studying Neurite Outgrowth in Primary Neurons

Neurite outgrowth is a fundamental event in the formation of the neural circuits during nervous system development. Severe neurite damage and synaptic dysfunction occur in various neurodegenerative diseases and age-related degeneration. Investigation of the mechanisms that regulate neurite outgrowth would not only shed valuable light on brain developmental processes but also on such neurological disorders. Due to the low transfection efficiency, it is currently challenging to study the effect of a specific protein on neurite outgrowth in primary mammalian neurons. Here, we describe a simple method for the investigation of neurite outgrowth by the co-transfection of primary rat cortical neurons with EGFP and a protein of interest (POI). This method allows the identification of POI transfected neurons through the EGFP signal, and thus the effect of the POI on neurite outgrowth can be determined precisely. This EGFP-based assay provides a convenient approach for the investigation of pathways regulating neurite outgrowth.

In this report, we describe a simple protocol for studying neurite outgrowth in embryonic rat cortical neurons by co-transfecting with EGFP and the protein of interest.

Neurite outgrowth is a fundamental event in the formation of the neural circuits during nervous system development. Severe neurite damage and synaptic ...

Inducing Plasticity of Astrocytic Receptors by Manipulation of Neuronal Firing Rates

Close to two decades of research has established that astrocytes in situ and in vivo express numerous G protein-coupled receptors (GPCRs) that can be stimulated by neuronally-released transmitter. However, the ability of astrocytic receptors to exhibit plasticity in response to changes in neuronal activity has received little attention. Here we describe a model system that can be used to globally scale up or down astrocytic group I metabotropic glutamate receptors (mGluRs) in acute brain slices. Included are methods on how to prepare parasagittal hippocampal slices, construct chambers suitable for long-term slice incubation, bidirectionally manipulate neuronal action potential frequency, load astrocytes and astrocyte processes with fluorescent Ca2+ indicator, and measure changes in astrocytic Gq GPCR activity by recording spontaneous and evoked astrocyte Ca2+ events using confocal microscopy. In essence, a &#8220;calcium roadmap&#8221; is provided for how to measure plasticity of astrocytic Gq GPCRs. Applications of the technique for study of astrocytes are discussed. Having an understanding of how astrocytic receptor signaling is affected by changes in neuronal activity has important implications for both normal synaptic function as well as processes underlying neurological disorders and neurodegenerative disease.

Here we describe an adaptation of protocols used to induce homeostatic plasticity in neurons for the study of plasticity of astrocytic G protein-coupled receptors. Recently used to examine changes in astrocytic group I mGluRs in juvenile mice, the method can be applied to measure scaling of various astrocytic GPCRs, in tissue from adult mice in situ and in vivo, and to gain a better appreciation of the sensitivity of astrocytic receptors to changes in neuronal activity.

Close to two decades of research has established that astrocytes in situ and in vivo express numerous G protein-coupled receptors (GPCRs) that can be ...

Electrophysiological and Morphological Characterization of Neuronal Microcircuits in Acute Brain Slices Using Paired Patch-Clamp Recordings

The combination of patch clamp recordings from two (or more) synaptically coupled neurons (paired recordings) in acute brain slice preparations with simultaneous intracellular biocytin filling allows a correlated analysis of their structural and functional properties. With this method it is possible to identify and characterize both pre- and postsynaptic neurons by their morphology and electrophysiological response pattern. Paired recordings allow studying the connectivity patterns between these neurons as well as the properties of both chemical and electrical synaptic transmission. Here, we give a step-by-step description of the procedures required to obtain reliable paired recordings together with an optimal recovery of the neuron morphology. We will describe how pairs of neurons connected via chemical synapses or gap junctions are identified in brain slice preparations. We will outline how neurons are reconstructed to obtain their 3D morphology of the dendritic and axonal domain and how synaptic contacts are identified and localized. We will also discuss the caveats and limitations of the paired recording technique, in particular those associated with dendritic and axonal truncations during the preparation of brain slices because these strongly affect connectivity estimates. However, because of the versatility of the paired recording approach it will remain a valuable tool in characterizing different aspects of synaptic transmission at identified neuronal microcircuits in the brain.

Patch-clamp recordings and simultaneous intracellular biocytin filling of synaptically coupled neurons in acute brain slices allow a correlated analysis of their structural and functional properties. The aim of this protocol is to describe the essential technical steps of electrophysiological recording from neuronal microcircuits and their subsequent morphological analysis.

The combination of patch clamp recordings from two (or more) synaptically coupled neurons (paired recordings) in acute brain slice preparations with ...

Three-dimensional Tissue Engineered Aligned Astrocyte Networks to Recapitulate Developmental Mechanisms and Facilitate Nervous System Regeneration

Neurotrauma and neurodegenerative disease often result in lasting neurological deficits due to the limited capacity of the central nervous system (CNS) to replace lost neurons and regenerate axonal pathways. However, during nervous system development, neuronal migration and axonal extension often occur along pathways formed by other cells, referred to as &#34;living scaffolds&#34;. Seeking to emulate these mechanisms and to design a strategy that circumvents the inhibitory environment of the CNS, this manuscript presents a protocol to fabricate tissue engineered astrocyte-based &#34;living scaffolds&#34;. To create these constructs, we employed a novel biomaterial encasement scheme to induce astrocytes to self-assemble into dense three-dimensional bundles of bipolar longitudinally-aligned somata and processes. First, hollow hydrogel micro-columns were assembled, and the inner lumen was coated with collagen extracellular-matrix. Dissociated cerebral cortical astrocytes were then delivered into the lumen of the cylindrical micro-column and, at a critical inner diameter of &#60;350 &#181;m, spontaneously self-aligned and contracted to produce long fiber-like cables consisting of dense bundles of astrocyte processes and collagen fibrils measuring &#60;150 &#181;m in diameter yet extending several cm in length. These engineered living scaffolds exhibited &#62;97% cell viability and were virtually exclusively comprised of astrocytes expressing a combination of the intermediate filament proteins glial-fibrillary acidic protein (GFAP), vimentin, and nestin. These aligned astrocyte networks were found to provide a permissive substrate for neuronal attachment and aligned neurite extension. Moreover, these constructs maintain integrity and alignment when extracted from the hydrogel encasement, making them suitable for CNS implantation. These preformed constructs structurally emulate key cytoarchitectural elements of naturally occurring glial-based &#34;living scaffolds&#34; in vivo. As such, these engineered living scaffolds may serve as test-beds to study neurodevelopmental mechanisms in vitro or facilitate neuroregeneration by directing neuronal migration and/or axonal pathfinding following CNS degeneration in vivo.

We showcase the development of self-assembled, three-dimensional bundles of longitudinally aligned astrocytic somata and processes within a novel biomaterial encasement. These engineered &#34;living scaffolds&#34;, exhibiting micron-scale diameter yet extending centimeters in length, may serve as test-beds to study neurodevelopmental mechanisms or facilitate neuroregeneration by directing neuronal migration and/or axonal pathfinding.

Neurotrauma and neurodegenerative disease often result in lasting neurological deficits due to the limited capacity of the central nervous system (CNS) to ...

Bioengineering

Design, Surface Treatment, Cellular Plating, and Culturing of Modular Neuronal Networks Composed of Functionally Inter-connected Circuits

The brain operates through the coordinated activation and the dynamic communication of neuronal assemblies. A major open question is how a vast repertoire of dynamical motifs, which underlie most diverse brain functions, can emerge out of a fixed topological and modular organization of brain circuits. Compared to in vivo studies of neuronal circuits which present intrinsic experimental difficulties, in vitro preparations offer a much larger possibility to manipulate and probe the structural, dynamical and chemical properties of experimental neuronal systems. This work describes an in vitro experimental methodology which allows growing of modular networks composed by spatially distinct, functionally interconnected neuronal assemblies. The protocol allows controlling the two-dimensional (2D) architecture of the neuronal network at different levels of topological complexity.
A desired network patterning can be achieved both on regular cover slips and substrate embedded micro electrode arrays. Micromachined structures are embossed on a silicon wafer and used to create biocompatible polymeric stencils, which incorporate the negative features of the desired network architecture. The stencils are placed on the culturing substrates during the surface coating procedure with a molecular layer for promoting cellular adhesion. After removal of the stencils, neurons are plated and they spontaneously redirected to the coated areas. By decreasing the inter-compartment distance, it is possible to obtain either isolated or interconnected neuronal circuits. To promote cell survival, cells are co-cultured with a supporting neuronal network which is located at the periphery of the culture dish. Electrophysiological and optical recordings of the activity of modular networks obtained respectively by using substrate embedded micro electrode arrays and calcium imaging are presented. While each module shows spontaneous global synchronizations, the occurrence of inter-module synchronization is regulated by the density of connection among the circuits.

This manuscript describes a protocol to grow in vitro modular networks consisting of spatially confined, functionally inter-connected neuronal circuits. A polymeric mask is used to pattern a protein layer to promote cellular adhesion over the culturing substrate. Plated neurons grow on coated areas establishing spontaneous connections and exhibiting electrophysiological activity.

The brain operates through the coordinated activation and the dynamic communication of neuronal assemblies. A major open question is how a vast repertoire ...

Anatomically Inspired Three-dimensional Micro-tissue Engineered Neural Networks for Nervous System Reconstruction, Modulation, and Modeling

Functional recovery rarely occurs&#160;following injury or disease-induced degeneration within the central nervous system (CNS)&#160;due to the inhibitory environment and the limited capacity for neurogenesis. We are developing&#160;a strategy to simultaneously address&#160;neuronal and axonal pathway loss within the damaged CNS. This manuscript presents the fabrication protocol for micro-tissue engineered neural networks (micro-TENNs), implantable constructs consisting of neurons and aligned axonal tracts spanning the extracellular matrix (ECM) lumen of a preformed hydrogel cylinder hundreds of microns in diameter that may extend centimeters in length. Neuronal aggregates are delimited to the extremes of the three-dimensional encasement and are spanned by axonal projections. Micro-TENNs are uniquely poised as a strategy for CNS reconstruction, emulating aspects of brain connectome cytoarchitecture and potentially providing means for network replacement. The neuronal aggregates may synapse with host tissue to form new functional relays to restore and/or modulate missing or damaged circuitry. These constructs may also act as pro-regenerative&#160;&#34;living scaffolds&#34; capable of exploiting developmental mechanisms for cell migration and axonal pathfinding, providing synergistic structural and soluble cues based on the state of regeneration. Micro-TENNs are fabricated by pouring liquid hydrogel into a cylindrical mold containing a longitudinally centered needle. Once the hydrogel has gelled, the needle is removed, leaving a hollow micro-column. An ECM solution is added to the lumen to provide an environment suitable for neuronal adhesion and axonal outgrowth. Dissociated neurons are mechanically aggregated for precise seeding within one or both ends of the micro-column. This methodology reliably produces self-contained miniature constructs with long-projecting axonal tracts that may recapitulate features of brain neuroanatomy. Synaptic immunolabeling and genetically encoded calcium indicators suggest that micro-TENNs possess extensive synaptic distribution and intrinsic electrical activity. Consequently, micro-TENNs represent a promising strategy for targeted neurosurgical reconstruction of brain pathways and may also be applied as biofidelic models to study neurobiological phenomena in vitro.

This manuscript details the fabrication of micro-tissue engineered neural networks: three-dimensional micron-sized constructs comprised of long aligned axonal tracts spanning aggregated neuronal population(s) encased in a tubular hydrogel. These living scaffolds can serve as functional relays to reconstruct or modulate neural circuitry or as biofidelic test-beds mimicking gray-white matter neuroanatomy.

Functional recovery rarely occurs&#160;following injury or disease-induced degeneration within the central nervous system (CNS)&#160;due to the inhibitory ...

Advanced Live-Imaging Techniques for Accurate Neurite Outgrowth Measurement

Standardization of a Novel Semi-Automatic Software for Neurite Outgrowth Measurement

Effective live-imaging techniques are crucial to assess neuronal morphology in order to measure neurite outgrowth in real time. The proper measurement of neurite outgrowth has been a long-standing challenge over the years in the neuroscience research field. This parameter serves as a cornerstone in numerous in vitro experimental setups, ranging from dissociated cultures and organotypic cultures to cell lines. By quantifying the neurite length, it is possible to determine if a specific treatment worked or if axonal regeneration is enhanced in different experimental groups. In this study, the aim is to demonstrate the robustness and accuracy of the Incucyte Neurotrack neurite outgrowth analysis software. This semi-automatic software is available in a time-lapse microscopy system which offers several advantages over commonly used methodologies in the quantification of the neurite length in phase contrast images. The algorithm masks and quantifies several parameters in each image and returns neuronal cell metrics, including neurite length, branch points, cell-body clusters, and cell-body cluster areas. Firstly, we validated the robustness and accuracy of the software by correlating its values with those of the manual NeuronJ, a Fiji plug-in. Secondly, we used the algorithm which is able to work both on phase contrast images as well as on immunocytochemistry images. Using specific neuronal markers, we validated the feasibility of the fluorescence-based neurite outgrowth analysis on sensory neurons in vitro cultures. Additionally, this software can measure neurite length across various seeding conditions, ranging from individual cells to complex neuronal nets. In conclusion, the software provides an innovative and time-effective platform for neurite outgrowth assays, paving the way for faster and more reliable quantifications.

Author Spotlight: Unveiling the Molecular Basis of Pain Perception and Neuropathic Pain

Neurite outgrowth assays provide a quantitative value about regenerative neuronal processes. The advantage of this semi-automatic software is that it segments cell bodies and neurites separately by creating a mask and measures various parameters such as neurite length, number of branch points, cell-body cluster area, and number of cell clusters.

Effective live-imaging techniques are crucial to assess neuronal morphology in order to measure neurite outgrowth in real time. The proper measurement of ...

Rewiring Neuronal Circuits: A New Method for Fast Neurite Extension and Functional Neuronal Connection

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